Genetic Modifiers of Neurofibromatosis Type 1-Associated Café-au-Lait Macule Count Identified Using Multi-platform Analysis

PubMed Central

Pemov, Alexander; Sung, Heejong; Hyland, Paula L.; Sloan, Jennifer L.; Ruppert, Sarah L.; Baldwin, Andrea M.; Boland, Joseph F.; Bass, Sara E.; Lee, Hyo Jung; Jones, Kristine M.; Zhang, Xijun; Mullikin, James C.; Widemann, Brigitte C.; Wilson, Alexander F.; Stewart, Douglas R.

2014-01-01

Neurofibromatosis type 1 (NF1) is an autosomal dominant, monogenic disorder of dysregulated neurocutaneous tissue growth. Pleiotropy, variable expressivity and few NF1 genotype-phenotype correlates limit clinical prognostication in NF1. Phenotype complexity in NF1 is hypothesized to derive in part from genetic modifiers unlinked to the NF1 locus. In this study, we hypothesized that normal variation in germline gene expression confers risk for certain phenotypes in NF1. In a set of 79 individuals with NF1, we examined the association between gene expression in lymphoblastoid cell lines with NF1-associated phenotypes and sequenced select genes with significant phenotype/expression correlations. In a discovery cohort of 89 self-reported European-Americans with NF1 we examined the association between germline sequence variants of these genes with café-au-lait macule (CALM) count, a tractable, tumor-like phenotype in NF1. Two correlated, common SNPs (rs4660761 and rs7161) between DPH2 and ATP6V0B were significantly associated with the CALM count. Analysis with tiled regression also identified SNP rs4660761 as significantly associated with CALM count. SNP rs1800934 and 12 rare variants in the mismatch repair gene MSH6 were also associated with CALM count. Both SNPs rs7161 and rs4660761 (DPH2 and ATP6V0B) were highly significant in a mega-analysis in a combined cohort of 180 self-reported European-Americans; SNP rs1800934 (MSH6) was near-significant in a meta-analysis assuming dominant effect of the minor allele. SNP rs4660761 is predicted to regulate ATP6V0B, a gene associated with melanosome biology. Individuals with homozygous mutations in MSH6 can develop an NF1-like phenotype, including multiple CALMs. Through a multi-platform approach, we identified variants that influence NF1 CALM count. PMID:25329635

Phenotypic and Genotypic Characterization of Enterobacteriaceae Producing Oxacillinase-48–Like Carbapenemases, United States

PubMed Central

Zhu, Wenming; de Man, Tom J.B.; Avillan, Johannetsy J.; Anderson, Karen F.; Lonsway, David R.; Rowe, Lori A.; Batra, Dhwani; Rasheed, J. Kamile; Limbago, Brandi M.

2018-01-01

Oxacillinase (OXA)–48–like carbapenemases remain relatively uncommon in the United States. We performed phenotypic and genotypic characterization of 30 Enterobacteriaceae producing OXA-48–like carbapenemases that were recovered from patients during 2010–2014. Isolates were collected from 12 states and not associated with outbreaks, although we could not exclude limited local transmission. The alleles β-lactamase OXA-181 (blaOXA-181) (43%), blaOXA-232 (33%), and blaOXA-48 (23%) were found. All isolates were resistant to ertapenem and showed positive results for the ertapenem and meropenem modified Hodge test and the modified carbapenem inactivation method; 73% showed a positive result for the Carba Nordmann–Poirel test. Whole-genome sequencing identified extended-spectrum β-lactamase genes in 93% of isolates. In all blaOXA-232 isolates, the gene was on a ColKP3 plasmid. A total of 12 of 13 isolates harboring blaOXA-181 contained the insertion sequence ΔISEcp1. In all isolates with blaOXA-48, the gene was located on a TN1999 transposon; these isolates also carried IncL/M plasmids. PMID:29553324

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS1[OPEN

PubMed Central

2018-01-01

Virus-induced gene silencing (VIGS) is used extensively for gene function studies in plants. VIGS is inexpensive and rapid compared with silencing conducted through stable transformation, but many virus-silencing vectors, especially in grasses, induce only transient silencing phenotypes. A major reason for transient phenotypes is the instability of the foreign gene fragment (insert) in the vector during VIGS. Here, we report the development of a Brome mosaic virus (BMV)-based vector that better maintains inserts through modification of the original BMV vector RNA sequence. Modification of the BMV RNA3 sequence yielded a vector, BMVCP5, that better maintained phytoene desaturase and heat shock protein70-1 (HSP70-1) inserts in Nicotiana benthamiana and maize (Zea mays). Longer maintenance of inserts was correlated with greater target gene silencing and more extensive visible silencing phenotypes displaying greater tissue penetration and involving more leaves. The modified vector accumulated similarly to the original vector in N. benthamiana after agroinfiltration, thus maintaining a high titer of virus in this intermediate host used to produce virus inoculum for grass hosts. For HSP70, silencing one family member led to a large increase in the expression of another family member, an increase likely related to the target gene knockdown and not a general effect of virus infection. The cause of the increased insert stability in the modified vector is discussed in relationship to its recombination and accumulation potential. The modified vector will improve functional genomic studies in grasses, and the conceptual methods used to improve the vector may be applied to other VIGS vectors. PMID:29127260

Characterizing visible and invisible cell wall mutant phenotypes.

PubMed

Carpita, Nicholas C; McCann, Maureen C

2015-07-01

About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with 'invisible' phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basis of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Phenotypic comparison of individuals with homozygous or heterozygous mutation of NOTCH3 in a large CADASIL family.

PubMed

Abou Al-Shaar, Hussam; Qadi, Najeeb; Al-Hamed, Mohamed H; Meyer, Brian F; Bohlega, Saeed

2016-08-15

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary microangiopathy caused by mutations in NOTCH3, very rarely homoallelic. To describe the clinical, radiological, and neuropsychological features in an extended CADASIL family including members with either a homozygous or heterozygous NOTCH3 R1231C mutation. The pedigree included 3 generations of a family with 13 affected individuals. The patients were examined clinically and radiologically. Neuropsychological testing was performed on the proband. Sequencing of the entire coding DNA sequence (CDS) and flanking regions of NOTCH3 was undertaken using PCR amplification and direct Sanger sequencing. Homozygous C3769T mutation, predicting R1231C in exon 22 of NOTCH3 was found in 7 family members. Six other family members harbored the same in the heterozygous state. Homozygous individuals showed a slightly more severe clinical and radiological phenotype of earlier onset compared to their heterozygous counterparts. This study reports the largest number of patients with homozygous NOTCH3 mutation. The phenotype and imaging features of homozygous individuals is within the spectrum of CADASIL, although slightly at the severe end when compared to heterozygotes carrying the same mutation. Both genetic modifiers and environmental factors may play an essential role in modification and alteration of the clinical phenotype and white matter changes among CADASIL patients. Copyright © 2016 Elsevier B.V. All rights reserved.

A Modified Protocol with Improved Detection Rate for Mis-Matched Donor HLA from Low Quantities of DNA in Urine Samples from Kidney Graft Recipients.

PubMed

Kwok, Janette; Choi, Leo C W; Ho, Jenny C Y; Chan, Gavin S W; Mok, Maggie M Y; Lam, Man-Fei; Chak, Wai-Leung; Cheuk, Au; Chau, Ka-Foon; Tong, Matthew; Chan, Kwok-Wah; Chan, Tak-Mao

2016-01-01

Urine from kidney transplant recipient has proven to be a viable source for donor DNA. However, an optimized protocol would be required to determine mis-matched donor HLA specificities in view of the scarcity of DNA obtained in some cases. In this study, fresh early morning urine specimens were obtained from 155 kidney transplant recipients with known donor HLA phenotype. DNA was extracted and typing of HLA-A, B and DRB1 loci by polymerase chain reaction-specific sequence primers was performed using tailor-made condition according to the concentration of extracted DNA. HLA typing of DNA extracted from urine revealed both recipient and donor HLA phenotypes, allowing the deduction of the unknown donor HLA and hence the degree of HLA mis-match. By adopting the modified procedures, mis-matched donor HLA phenotypes were successfully deduced in all of 35 tested urine samples at DNA quantities spanning the range of 620-24,000 ng. This urine-based method offers a promising and reliable non-invasive means for the identification of mis-matched donor HLA antigens in kidney transplant recipients with unknown donor HLA phenotype or otherwise inadequate donor information.

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS.

PubMed

Ding, Xin Shun; Mannas, Stephen W; Bishop, Bethany A; Rao, Xiaolan; Lecoultre, Mitchell; Kwon, Soonil; Nelson, Richard S

2018-01-01

Virus-induced gene silencing (VIGS) is used extensively for gene function studies in plants. VIGS is inexpensive and rapid compared with silencing conducted through stable transformation, but many virus-silencing vectors, especially in grasses, induce only transient silencing phenotypes. A major reason for transient phenotypes is the instability of the foreign gene fragment (insert) in the vector during VIGS. Here, we report the development of a Brome mosaic virus (BMV)-based vector that better maintains inserts through modification of the original BMV vector RNA sequence. Modification of the BMV RNA3 sequence yielded a vector, BMVCP5, that better maintained phytoene desaturase and heat shock protein70-1 ( HSP70-1 ) inserts in Nicotiana benthamiana and maize ( Zea mays ). Longer maintenance of inserts was correlated with greater target gene silencing and more extensive visible silencing phenotypes displaying greater tissue penetration and involving more leaves. The modified vector accumulated similarly to the original vector in N. benthamiana after agroinfiltration, thus maintaining a high titer of virus in this intermediate host used to produce virus inoculum for grass hosts. For HSP70 , silencing one family member led to a large increase in the expression of another family member, an increase likely related to the target gene knockdown and not a general effect of virus infection. The cause of the increased insert stability in the modified vector is discussed in relationship to its recombination and accumulation potential. The modified vector will improve functional genomic studies in grasses, and the conceptual methods used to improve the vector may be applied to other VIGS vectors. © 2018 American Society of Plant Biologists. All Rights Reserved.

CLC-2 single nucleotide polymorphisms (SNPs) as potential modifiers of cystic fibrosis disease severity

PubMed Central

Blaisdell, Carol J; Howard, Timothy D; Stern, Augustus; Bamford, Penelope; Bleecker, Eugene R; Stine, O Colin

2004-01-01

Background Cystic fibrosis (CF) lung disease manifest by impaired chloride secretion leads to eventual respiratory failure. Candidate genes that may modify CF lung disease severity include alternative chloride channels. The objectives of this study are to identify single nucleotide polymorphisms (SNPs) in the airway epithelial chloride channel, CLC-2, and correlate these polymorphisms with CF lung disease. Methods The CLC-2 promoter, intron 1 and exon 20 were examined for SNPs in adult CF dF508/dF508 homozygotes with mild and severe lung disease (forced expiratory volume at one second (FEV1) > 70% and < 40%). Results PCR amplification of genomic CLC-2 and sequence analysis revealed 1 polymorphism in the hClC -2 promoter, 4 in intron 1, and none in exon 20. Fisher's analysis within this data set, did not demonstrate a significant relationship between the severity of lung disease and SNPs in the CLC-2 gene. Conclusions CLC-2 is not a key modifier gene of CF lung phenotype. Further studies evaluating other phenotypes associated with CF may be useful in the future to assess the ability of CLC-2 to modify CF disease severity. PMID:15507145

Silent polymorphisms in the RYR1 gene do not modify the phenotype of the p.4898 I>T pathogenic mutation in central core disease: a case report

PubMed Central

2014-01-01

Background Central core disease is a congenital myopathy, characterized by presence of central core-like areas in muscle fibers. Patients have mild or moderate weakness, hypotonia and motor developmental delay. The disease is caused by mutations in the human ryanodine receptor gene (RYR1), which encodes a calcium-release channel. Since the RYR1 gene is huge, containing 106 exons, mutation screening has been limited to three ‘hot spots’, with particular attention to the C-terminal region. Recent next- generation sequencing methods are now identifying multiple numbers of variants in patients, in which interpretation and phenotype prevision is difficult. Case presentation In a Brazilian Caucasian family, clinical, histopathological and molecular analysis identified a new case of central core disease in a 48-year female. Sanger sequencing of the C-terminal region of the RYR1 gene identified two different missense mutations: c.14256 A > C polymorphism in exon 98 and c.14693 T > C in exon 102, which have already been described as pathogenic. Trans-position of the 2 mutations was confirmed because patient’s daughter, mother and sister carried only the exon 98’s mutation, a synonymous variant that was subsequently found in the frequency of 013–0,05 of alleles. Further next generation sequencing study of the whole RYR1 gene in the patient revealed the presence of additional 5 common silent polymorphisms in homozygosis and 8 polymorphisms in heterozygosis. Conclusions Considering that patient’s relatives showed no pathologic phenotype, and the phenotype presented by the patient is within the range observed in other central core disease patients with the same mutation, it was concluded that the c.14256 A > C polymorphism alone is not responsible for disease, and the associated additional silent polymorphisms are not acting as modifiers of the primary pathogenic mutation in the affected patient. The case described above illustrates the present reality where new methods for wide genome screening are becoming more accessible and able to identify a great variety of mutations and polymorphisms of unknown function in patients and their families. PMID:25084811

Phenotypic approaches to drought in cassava: review

PubMed Central

Okogbenin, Emmanuel; Setter, Tim L.; Ferguson, Morag; Mutegi, Rose; Ceballos, Hernan; Olasanmi, Bunmi; Fregene, Martin

2012-01-01

Cassava is an important crop in Africa, Asia, Latin America, and the Caribbean. Cassava can be produced adequately in drought conditions making it the ideal food security crop in marginal environments. Although cassava can tolerate drought stress, it can be genetically improved to enhance productivity in such environments. Drought adaptation studies in over three decades in cassava have identified relevant mechanisms which have been explored in conventional breeding. Drought is a quantitative trait and its multigenic nature makes it very challenging to effectively manipulate and combine genes in breeding for rapid genetic gain and selection process. Cassava has a long growth cycle of 12–18 months which invariably contributes to a long breeding scheme for the crop. Modern breeding using advances in genomics and improved genotyping, is facilitating the dissection and genetic analysis of complex traits including drought tolerance, thus helping to better elucidate and understand the genetic basis of such traits. A beneficial goal of new innovative breeding strategies is to shorten the breeding cycle using minimized, efficient or fast phenotyping protocols. While high throughput genotyping have been achieved, this is rarely the case for phenotyping for drought adaptation. Some of the storage root phenotyping in cassava are often done very late in the evaluation cycle making selection process very slow. This paper highlights some modified traits suitable for early-growth phase phenotyping that may be used to reduce drought phenotyping cycle in cassava. Such modified traits can significantly complement the high throughput genotyping procedures to fast track breeding of improved drought tolerant varieties. The need for metabolite profiling, improved phenomics to take advantage of next generation sequencing technologies and high throughput phenotyping are basic steps for future direction to improve genetic gain and maximize speed for drought tolerance breeding. PMID:23717282

Histones: Controlling Tumor Signaling Circuitry

PubMed Central

Martins, Manoela D.; Castilho, Rogerio M.

2014-01-01

Epigenetic modifications constitute the next frontier in tumor biology research. Post-translation modification of histones dynamically influences gene expression independent of alterations to the DNA sequence. These mechanisms are often mediated by histone linkers or by proteins associated with the recruitment of DNA-binding proteins, HDAC I and II interacting proteins and transcriptional activators, coactivators or corepressors. Early evidence suggested that histones and their modifiers are involved in sophisticated processes that modulate tumor behavior and cellular phenotype. In this review, we discuss how recent discoveries about chromatin modifications, particularly histone acetylation, are shaping our knowledge of cell biology and our understanding of the molecular circuitry governing tumor progression and consider whether recent insights may extend to novel therapeutic approaches. Furthermore, we discuss the latest oncogenomic findings in Head and Neck Squamous Cell Carcinoma (HNSCC) from studies using Next Generation Sequencing (NGS) technology and highlight the impact of mutations identified in histones and their modifiers. PMID:25177526

A novel dominant GJB2 (DFNA3) mutation in a Chinese family

NASA Astrophysics Data System (ADS)

Wang, Hongyang; Wu, Kaiwen; Yu, Lan; Xie, Linyi; Xiong, Wenping; Wang, Dayong; Guan, Jing; Wang, Qiuju

2017-01-01

To decipher the phenotype and genotype of a Chinese family with autosomal dominant non-syndromic hearing loss (ADNSHL) and a novel dominant missense mutation in the GJB2 gene (DFNA3), mutation screening of GJB2 was performed on the propositus from a five-generation ADNSHL family through polymerase chain reaction amplification and Sanger sequencing. The candidate variation and the co-segregation of the phenotype were verified in all ascertained family members. Targeted genes capture and next-generation sequencing (NGS) were performed to explore additional genetic variations. We identified the novel GJB2 mutation c.524C > A (p.P175H), which segregated with high frequency and was involved in progressive sensorineural hearing loss. One subject with an additional c.235delC mutation showed a more severe phenotype than did the other members with single GJB2 dominant variations. Four patients diagnosed with noise-induced hearing loss did not carry this mutation. No other pathogenic variations or modifier genes were identified by NGS. In conclusion, a novel missense mutation in GJB2 (DFNA3), affecting the second extracellular domain of the protein, was identified in a family with ADNSHL.

Whole genome sequencing of extreme phenotypes identifies variants in CD101 and UBE2V1 associated with increased risk of sexually acquired HIV-1

PubMed Central

Buckingham, Kati J.; Shively, Kathryn; Mugo, Nelly R.; Mullins, James I.; McElrath, M. Juliana; Baeten, Jared M.; Celum, Connie

2017-01-01

Host genetic variation modifying HIV-1 acquisition risk can inform development of HIV-1 prevention strategies. However, associations between rare or intermediate-frequency variants and HIV-1 acquisition are not well studied. We tested for the association between variation in genic regions and extreme HIV-1 acquisition phenotypes in 100 sub-Saharan Africans with whole genome sequencing data. Missense variants in immunoglobulin-like regions of CD101 and, among women, one missense/5’ UTR variant in UBE2V1, were associated with increased HIV-1 acquisition risk (p = 1.9x10-4 and p = 3.7x10-3, respectively, for replication). Both of these genes are known to impact host inflammatory pathways. Effect sizes increased with exposure to HIV-1 after adjusting for the independent effect of increasing exposure on acquisition risk. Trial registration: ClinicalTrials.gov NCT00194519; NCT00557245 PMID:29108000

Growth hormone deficiency with advanced bone age: phenotypic interaction between GHRH receptor and CYP21A2 mutations diagnosed by sanger and whole exome sequencing.

PubMed

Correa, Fernanda A; França, Marcela M; Fang, Qing; Ma, Qianyi; Bachega, Tania A; Rodrigues, Andresa; Ozel, Bilge A; Li, Jun Z; Mendonca, Berenice B; Jorge, Alexander A L; Carvalho, Luciani R; Camper, Sally A; Arnhold, Ivo J P

2017-12-01

Isolated growth hormone deficiency (IGHD) is the most common pituitary hormone deficiency and, clinically, patients have delayed bone age. High sequence similarity between CYP21A2 gene and CYP21A1P pseudogene poses difficulties for exome sequencing interpretation. A 7.5 year-old boy born to second-degree cousins presented with severe short stature (height SDS -3.7) and bone age of 6 years. Clonidine and combined pituitary stimulation tests revealed GH deficiency. Pituitary MRI was normal. The patient was successfully treated with rGH. Surprisingly, at 10.8 years, his bone age had advanced to 13 years, but physical exam, LH and testosterone levels remained prepubertal. An ACTH stimulation test disclosed a non-classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency explaining the bone age advancement and, therefore, treatment with cortisone acetate was added. The genetic diagnosis of a homozygous mutation in GHRHR (p.Leu144His), a homozygous CYP21A2 mutation (p.Val282Leu) and CYP21A1P pseudogene duplication was established by Sanger sequencing, MLPA and whole-exome sequencing. We report the unusual clinical presentation of a patient born to consanguineous parents with two recessive endocrine diseases: non-classic congenital adrenal hyperplasia modifying the classical GH deficiency phenotype. We used a method of paired read mapping aided by neighbouring mis-matches to overcome the challenges of exome-sequencing in the presence of a pseudogene.

Artificial selection increased body weight but induced increase of runs of homozygosity in Hanwoo cattle

PubMed Central

Kim, Kwondo; Jung, Jaehoon; Caetano-Anollés, Kelsey; Sung, Samsun; Yoo, DongAhn; Choi, Bong-Hwan; Kim, Hyung-Chul; Jeong, Jin-Young; Cho, Yong-Min; Park, Eung-Woo; Choi, Tae-Jeong; Park, Byoungho; Lim, Dajeong

2018-01-01

Artificial selection has been demonstrated to have a rapid and significant effect on the phenotype and genome of an organism. However, most previous studies on artificial selection have focused solely on genomic sequences modified by artificial selection or genomic sequences associated with a specific trait. In this study, we generated whole genome sequencing data of 126 cattle under artificial selection, and 24,973,862 single nucleotide variants to investigate the relationship among artificial selection, genomic sequences and trait. Using runs of homozygosity detected by the variants, we showed increase of inbreeding for decades, and at the same time demonstrated a little influence of recent inbreeding on body weight. Also, we could identify ~0.2 Mb runs of homozygosity segment which may be created by recent artificial selection. This approach may aid in development of genetic markers directly influenced by artificial selection, and provide insight into the process of artificial selection. PMID:29561881

Modeling Structure-Function Relationships in Synthetic DNA Sequences using Attribute Grammars

PubMed Central

Cai, Yizhi; Lux, Matthew W.; Adam, Laura; Peccoud, Jean

2009-01-01

Recognizing that certain biological functions can be associated with specific DNA sequences has led various fields of biology to adopt the notion of the genetic part. This concept provides a finer level of granularity than the traditional notion of the gene. However, a method of formally relating how a set of parts relates to a function has not yet emerged. Synthetic biology both demands such a formalism and provides an ideal setting for testing hypotheses about relationships between DNA sequences and phenotypes beyond the gene-centric methods used in genetics. Attribute grammars are used in computer science to translate the text of a program source code into the computational operations it represents. By associating attributes with parts, modifying the value of these attributes using rules that describe the structure of DNA sequences, and using a multi-pass compilation process, it is possible to translate DNA sequences into molecular interaction network models. These capabilities are illustrated by simple example grammars expressing how gene expression rates are dependent upon single or multiple parts. The translation process is validated by systematically generating, translating, and simulating the phenotype of all the sequences in the design space generated by a small library of genetic parts. Attribute grammars represent a flexible framework connecting parts with models of biological function. They will be instrumental for building mathematical models of libraries of genetic constructs synthesized to characterize the function of genetic parts. This formalism is also expected to provide a solid foundation for the development of computer assisted design applications for synthetic biology. PMID:19816554

Comparison of traditional phenotypic identification methods with partial 5' 16S rRNA gene sequencing for species-level identification of nonfermenting Gram-negative bacilli.

PubMed

Cloud, Joann L; Harmsen, Dag; Iwen, Peter C; Dunn, James J; Hall, Gerri; Lasala, Paul Rocco; Hoggan, Karen; Wilson, Deborah; Woods, Gail L; Mellmann, Alexander

2010-04-01

Correct identification of nonfermenting Gram-negative bacilli (NFB) is crucial for patient management. We compared phenotypic identifications of 96 clinical NFB isolates with identifications obtained by 5' 16S rRNA gene sequencing. Sequencing identified 88 isolates (91.7%) with >99% similarity to a sequence from the assigned species; 61.5% of sequencing results were concordant with phenotypic results, indicating the usability of sequencing to identify NFB.

EPIGENETIC TRANSGENERATIONAL ACTIONS OF ENDOCRINE DISRUPTORS

PubMed Central

Skinner, Michael K.; Manikkam, Mohan; Guerrero-Bosagna, Carlos

2010-01-01

Environmental factors have a significant impact on biology. Therefore, environmental toxicants through similar mechanisms can modulate biological systems to influence physiology and promote disease states. The majority of environmental toxicants do not have the capacity to modulate DNA sequence, but can alter the epigenome. In the event an environmental toxicant such as an endocrine disruptor modifies the epigenome of a somatic cell, this may promote disease in the individual exposed, but not be transmitted to the next generation. In the event a toxicant modifies the epigenome of the germ line permanently, then the disease promoted can become transgenerationaly transmitted to subsequent progeny. The current review focuses on the ability of environmental factors such as endocrine disruptors to promote transgenerational phenotypes. PMID:21055462

A challenge to the striking genotypic heterogeneity of retinitis pigmentosa: a better understanding of the pathophysiology using the newest genetic strategies

PubMed Central

Sorrentino, F S; Gallenga, C E; Bonifazzi, C; Perri, P

2016-01-01

Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterized by a complex association between tremendous genotypic multiplicity and great phenotypic heterogeneity. The severity of the clinical manifestation depends on penetrance and expressivity of the disease-gene. Also, various interactions between gene expression and environmental factors have been hypothesized. More than 250 genes with ~4500 causative mutations have been reported to be involved in different RP-related mechanisms. Nowadays, not more than the 50% of RPs are attributable to identified genes, whereas the rest of molecular defects are still undetectable, especially in populations where few genetic screenings have been performed. Therefore, new genetic strategies can be a remarkably useful tool to aid clinical diagnosis, potentially modifying treatment options, and family counseling. Genome-wide analytical techniques (array comparative genomic hybridization and single-nucleotide polymorphism genotyping) and DNA sequencing strategies (arrayed primer extension, Sanger sequencing, and ultra high-throughput sequencing) are successfully used to early make molecular diagnosis detecting single or multiple mutations in the huge heterogeneity of RPs. To date, further research needs to be carried out to better investigate the genotype/phenotype correlation, putting together genetic and clinical findings to provide detailed information concerning the risk of RP development and novel effective treatments. PMID:27564722

Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases

PubMed Central

Butler, Nathaniel M.; Baltes, Nicholas J.; Voytas, Daniel F.; Douches, David S.

2016-01-01

Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species. PMID:27493650

PHENOTYPIC VARIABILITY IN INDIVIDUALS WITH TYPE V OSTEOGENESIS IMPERFECTA WITH IDENTICAL IFITM5 MUTATIONS

PubMed Central

Fitzgerald, Jamie; Holden, Paul; Wright, Hollis; Wilmot, Beth; Hata, Abigail; Steiner, Robert D.; Basel, Don

2016-01-01

Background Osteogenesis imperfecta (OI) type V is a dominantly inherited skeletal dysplasia characterized by fractures and progressive deformity of long bones. In addition, patients often present with radial head dislocation, hyperplastic callus, and calcification of the forearm interosseous membrane. Recently, a specific mutation in the IFITM5 gene was found to be responsible for OI type V. This mutation, a C to T transition 14 nucleotides upstream from the endogenous start codon, creates a new start methionine that appears to be preferentially used by the translational machinery. However, the mechanism by which the lengthened protein results in a dominant type of OI is unknown. Methods and Results We report 7 ethnically diverse (African-American, Caucasian, Hispanic, and African) individuals with OI type V from 2 families and 2 sporadic cases. Exome sequencing failed to identify a causative mutation. Using Sanger sequencing, we found that all affected individuals in our cohort possess the c.−14 IFITM5 variant, further supporting the notion that OI type V is caused by a single, discrete mutation. Our patient cohort demonstrated inter-and intrafamilial phenotypic variability, including a father with classic OI type V whose daughter had a phenotype similar to OI type I. This clinical variability suggests that modifier genes influence the OI type V phenotype. We also confirm that the mutation creates an aberrant IFITM5 protein containing an additional 5 amino acids at the N-terminus. Conclusions The variable clinical signs in these cases illustrate the significant variability of the OI type V phenotype caused by the c.−14 IFITM5 mutation. The affected individuals are more ethnically diverse than previously reported. PMID:28824928

The evolutionary capacitor HSP90 buffers the regulatory effects of mammalian endogenous retroviruses.

PubMed

Hummel, Barbara; Hansen, Erik C; Yoveva, Aneliya; Aprile-Garcia, Fernando; Hussong, Rebecca; Sawarkar, Ritwick

2017-03-01

Understanding how genotypes are linked to phenotypes is important in biomedical and evolutionary studies. The chaperone heat-shock protein 90 (HSP90) buffers genetic variation by stabilizing proteins with variant sequences, thereby uncoupling phenotypes from genotypes. Here we report an unexpected role of HSP90 in buffering cis-regulatory variation affecting gene expression. By using the tripartite-motif-containing 28 (TRIM28; also known as KAP1)-mediated epigenetic pathway, HSP90 represses the regulatory influence of endogenous retroviruses (ERVs) on neighboring genes that are critical for mouse development. Our data based on natural variations in the mouse genome show that genes respond to HSP90 inhibition in a manner dependent on their genomic location with regard to strain-specific ERV-insertion sites. The evolutionary-capacitor function of HSP90 may thus have facilitated the exaptation of ERVs as key modifiers of gene expression and morphological diversification. Our findings add a new regulatory layer through which HSP90 uncouples phenotypic outcomes from individual genotypes.

Characterization of the modified Hodge test-positive isolates of Enterobacteriaceae in Taiwan.

PubMed

Hung, Kuei-Hsiang; Yan, Jing-Jou; Lu, Jang-Jih; Chen, Hung-Mo; Wu, Jiunn-Jong

2013-02-01

The modified Hodge test is a phenotypic test to detect KPC-type carbapenemase producers among Enterobacteriaceae, as recommended by the Clinical Laboratory Standards Institute. However, false positive results were reported. In this study, we aimed to large-scale investigate the characterization of the modified Hodge test-positive isolates of Enterobacteriaceae collected between 2006 and 2010 in Taiwan. Fifty-six isolates, including 24 Enterobacter cloacae, 17 Escherichia coli, 10 Klebsiella pneumoniae, and 5 Citrobacter freundii, tested positive with the modified Hodge test. The in vitro activities of 10 antimicrobial agents were determined by the agar dilution method. Boronic acid combined-disk test was used to further confirm the KPC producers. Phenotype of ESBL, AmpC, class B carbapenemases, and profile of outer membrane proteins were investigated by the confirmatory test, boronic acid disk method, 2-mercaptopropionic acid double-disk method, and urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. β-lactamase genes were examined by PCR and sequencing. These isolates were resistant to ceftazidime (100%), aztreonam (82.1%), ertapenem (64.3%), gentamicin (53.6%), ciprofloxacin (50%), levofloxacin (48.2%), cefepime (19.6%), imipenem (16.1%), meropenem (12.5%), and amikacin (8.9%). Phenotypic testing among isolates revealed the production of ESBLs, metallo-β-lactamases (MBLs), and AmpC in 10 (17.9%), 16 (28.6%), and 12 (44.4%) isolates, respectively. Carbapenemase and non-carbapenemase β-lactamase genes bla(TEM-1), bla(SHV), bla(CTX-M), bla(IMP-8), bla(CMY-2), and bla(DHA-1) were found in 32 (57.1%), 19 (33.9%), 4 (7.1%), 16 (28.6%), 14 (25%), and 5 (8.9%) of the strains, respectively. No class A and D carbapenemase genes were detected. Outer membrane protein profile showed obviously decreased expression in 49 (87.5%) isolates with positive result of modified Hodge test. Our data show that ESBLs, AmpC, and imipenemase-8 (IMP-8) carbapenemase coupled with decreased expression of outer membrane protein were prevalent in Enterobacteriaceae isolates testing positive for the modified Hodge test in Taiwan. Copyright © 2012. Published by Elsevier B.V.

Quality Control Test for Sequence-Phenotype Assignments

PubMed Central

Ortiz, Maria Teresa Lara; Rosario, Pablo Benjamín Leon; Luna-Nevarez, Pablo; Gamez, Alba Savin; Martínez-del Campo, Ana; Del Rio, Gabriel

2015-01-01

Relating a gene mutation to a phenotype is a common task in different disciplines such as protein biochemistry. In this endeavour, it is common to find false relationships arising from mutations introduced by cells that may be depurated using a phenotypic assay; yet, such phenotypic assays may introduce additional false relationships arising from experimental errors. Here we introduce the use of high-throughput DNA sequencers and statistical analysis aimed to identify incorrect DNA sequence-phenotype assignments and observed that 10–20% of these false assignments are expected in large screenings aimed to identify critical residues for protein function. We further show that this level of incorrect DNA sequence-phenotype assignments may significantly alter our understanding about the structure-function relationship of proteins. We have made available an implementation of our method at http://bis.ifc.unam.mx/en/software/chispas. PMID:25700273

Utility of 16S rDNA Sequencing for Identification of Rare Pathogenic Bacteria.

PubMed

Loong, Shih Keng; Khor, Chee Sieng; Jafar, Faizatul Lela; AbuBakar, Sazaly

2016-11-01

Phenotypic identification systems are established methods for laboratory identification of bacteria causing human infections. Here, the utility of phenotypic identification systems was compared against 16S rDNA identification method on clinical isolates obtained during a 5-year study period, with special emphasis on isolates that gave unsatisfactory identification. One hundred and eighty-seven clinical bacteria isolates were tested with commercial phenotypic identification systems and 16S rDNA sequencing. Isolate identities determined using phenotypic identification systems and 16S rDNA sequencing were compared for similarity at genus and species level, with 16S rDNA sequencing as the reference method. Phenotypic identification systems identified ~46% (86/187) of the isolates with identity similar to that identified using 16S rDNA sequencing. Approximately 39% (73/187) and ~15% (28/187) of the isolates showed different genus identity and could not be identified using the phenotypic identification systems, respectively. Both methods succeeded in determining the species identities of 55 isolates; however, only ~69% (38/55) of the isolates matched at species level. 16S rDNA sequencing could not determine the species of ~20% (37/187) of the isolates. The 16S rDNA sequencing is a useful method over the phenotypic identification systems for the identification of rare and difficult to identify bacteria species. The 16S rDNA sequencing method, however, does have limitation for species-level identification of some bacteria highlighting the need for better bacterial pathogen identification tools. © 2016 Wiley Periodicals, Inc.

Whole genome sequencing and integrative genomic analysis approach on two 22q11.2 deletion syndrome family trios for genotype to phenotype correlations

PubMed Central

Chung, Jonathan H.; Cai, Jinlu; Suskin, Barrie G.; Zhang, Zhengdong; Coleman, Karlene

2015-01-01

The 22q11.2 deletion syndrome (22q11DS) affects 1:4000 live births and presents with highly variable phenotype expressivity. In this study, we developed an analytical approach utilizing whole genome sequencing and integrative analysis to discover genetic modifiers. Our pipeline combined available tools in order to prioritize rare, predicted deleterious, coding and non-coding single nucleotide variants (SNVs) and insertion/deletions (INDELs) from whole genome sequencing (WGS). We sequenced two unrelated probands with 22q11DS, with contrasting clinical findings, and their unaffected parents. Proband P1 had cognitive impairment, psychotic episodes, anxiety, and tetralogy of Fallot (TOF); while proband P2 had juvenile rheumatoid arthritis but no other major clinical findings. In P1, we identified common variants in COMT and PRODH on 22q11.2 as well as rare potentially deleterious DNA variants in other behavioral/neurocognitive genes. We also identified a de novo SNV in ADNP2 (NM_014913.3:c.2243G>C), encoding a neuroprotective protein that may be involved in behavioral disorders. In P2, we identified a novel non-synonymous SNV in ZFPM2 (NM_012082.3:c.1576C>T), a known causative gene for TOF, which may act as a protective variant downstream of TBX1, haploinsufficiency of which is responsible for congenital heart disease in individuals with 22q11DS. PMID:25981510

Genotyping in 46 patients with tentative diagnosis of Treacher Collins syndrome revealed unexpected phenotypic variation.

PubMed

Teber, Ozge Altug; Gillessen-Kaesbach, Gabriele; Fischer, Sven; Böhringer, Stefan; Albrecht, Beate; Albert, Angelika; Arslan-Kirchner, Mine; Haan, Eric; Hagedorn-Greiwe, Monika; Hammans, Christof; Henn, Wolfram; Hinkel, Georg Klaus; König, Rainer; Kunstmann, Erdmute; Kunze, Jürgen; Neumann, Luitgard M; Prott, Eva-Christina; Rauch, Anita; Rott, Hans-Dieter; Seidel, Heide; Spranger, Stephanie; Sprengel, Martin; Zoll, Barbara; Lohmann, Dietmar R; Wieczorek, Dagmar

2004-11-01

To define the range of phenotypic expression in Treacher Collins syndrome (TCS; Franceschetti-Klein syndrome), we performed mutation analysis in the TCOF1 gene in 46 patients with tentative diagnosis of TCS and evaluated the clinical data, including a scoring system. A total of 27 coding exons of TCOF1 and adjacent splice junctions were analysed by direct sequencing. In 36 patients with a clinically unequivocal diagnosis of TCS, we detected 28 pathogenic mutations, including 25 novel alterations. No mutation was identified in the remaining eight patients with unequivocal diagnosis of TCS and 10 further patients, in whom the referring diagnosis of TCS was clinically doubtful. There is no overt genotype-phenotype correlation except that conductive deafness is significantly less frequent in patients with mutations in the 3' part of the open reading frame. Inter- and intrafamilial variation is wide. Some mutation carriers, parents of typically affected patients, are so mildly affected that the diagnosis might be overlooked clinically. This suggests that modifying factors are important for phenotypic expression. Based on these findings, minimal diagnostic criteria were defined: downward slanting palpebral fissures and hypoplasia of the zygomatic arch. The difficulties in genetic counselling, especially diagnosis of family members with a mild phenotype, are described.

Exome sequencing identifies DYNC2H1 mutations as a common cause of asphyxiating thoracic dystrophy (Jeune syndrome) without major polydactyly, renal or retinal involvement

PubMed Central

Schmidts, Miriam; Arts, Heleen H; Bongers, Ernie M H F; Yap, Zhimin; Oud, Machteld M; Antony, Dinu; Duijkers, Lonneke; Emes, Richard D; Stalker, Jim; Yntema, Jan-Bart L; Plagnol, Vincent; Hoischen, Alexander; Gilissen, Christian; Forsythe, Elisabeth; Lausch, Ekkehart; Veltman, Joris A; Roeleveld, Nel; Superti-Furga, Andrea; Kutkowska-Kazmierczak, Anna; Kamsteeg, Erik-Jan; Elçioğlu, Nursel; van Maarle, Merel C; Graul-Neumann, Luitgard M; Devriendt, Koenraad; Smithson, Sarah F; Wellesley, Diana; Verbeek, Nienke E; Hennekam, Raoul C M; Kayserili, Hulya; Scambler, Peter J; Beales, Philip L; Knoers, Nine VAM; Roepman, Ronald; Mitchison, Hannah M

2013-01-01

Background Jeune asphyxiating thoracic dystrophy (JATD) is a rare, often lethal, recessively inherited chondrodysplasia characterised by shortened ribs and long bones, sometimes accompanied by polydactyly, and renal, liver and retinal disease. Mutations in intraflagellar transport (IFT) genes cause JATD, including the IFT dynein-2 motor subunit gene DYNC2H1. Genetic heterogeneity and the large DYNC2H1 gene size have hindered JATD genetic diagnosis. Aims and methods To determine the contribution to JATD we screened DYNC2H1 in 71 JATD patients JATD patients combining SNP mapping, Sanger sequencing and exome sequencing. Results and conclusions We detected 34 DYNC2H1 mutations in 29/71 (41%) patients from 19/57 families (33%), showing it as a major cause of JATD especially in Northern European patients. This included 13 early protein termination mutations (nonsense/frameshift, deletion, splice site) but no patients carried these in combination, suggesting the human phenotype is at least partly hypomorphic. In addition, 21 missense mutations were distributed across DYNC2H1 and these showed some clustering to functional domains, especially the ATP motor domain. DYNC2H1 patients largely lacked significant extra-skeletal involvement, demonstrating an important genotype–phenotype correlation in JATD. Significant variability exists in the course and severity of the thoracic phenotype, both between affected siblings with identical DYNC2H1 alleles and among individuals with different alleles, which suggests the DYNC2H1 phenotype might be subject to modifier alleles, non-genetic or epigenetic factors. Assessment of fibroblasts from patients showed accumulation of anterograde IFT proteins in the ciliary tips, confirming defects similar to patients with other retrograde IFT machinery mutations, which may be of undervalued potential for diagnostic purposes. PMID:23456818

Chm-1 gene-modified bone marrow mesenchymal stem cells maintain the chondrogenic phenotype of tissue-engineered cartilage.

PubMed

Chen, Zhuoyue; Wei, Jing; Zhu, Jun; Liu, Wei; Cui, Jihong; Li, Hongmin; Chen, Fulin

2016-05-05

Marrow mesenchymal stem cells (MSCs) can differentiate into specific phenotypes, including chondrocytes, and have been widely used for cartilage tissue engineering. However, cartilage grafts from MSCs exhibit phenotypic alternations after implantation, including matrix calcification and vascular ingrowth. We compared chondromodulin-1 (Chm-1) expression between chondrocytes and MSCs. We found that chondrocytes expressed a high level of Chm-1. We then adenovirally transduced MSCs with Chm-1 and applied modified cells to engineer cartilage in vivo. A gross inspection and histological observation indicated that the chondrogenic phenotype of the tissue-engineered cartilage graft was well maintained, and the stable expression of Chm-1 was detected by immunohistological staining in the cartilage graft derived from the Chm-1 gene-modified MSCs. Our findings defined an essential role for Chm-1 in maintaining chondrogenic phenotype and demonstrated that Chm-1 gene-modified MSCs may be used in cartilage tissue engineering.

Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts.

PubMed

Ding, Xin Shun; Schneider, William L; Chaluvadi, Srinivasa Rao; Mian, M A Rouf; Nelson, Richard S

2006-11-01

Virus-induced gene silencing (VIGS) is used to analyze gene function in dicotyledonous plants but less so in monocotyledonous plants (particularly rice and corn), partially due to the limited number of virus expression vectors available. Here, we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb. (tall fescue) that caused systemic mosaic symptoms on barley, rice, and a specific cultivar of maize (Va35) under greenhouse conditions. Through sequencing, the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca), and genetic determinants that controlled the systemic infection of rice were mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Coinoculation of RNAs 1 and 2 from F-BMV and RNA 3 from R-BMV expressing a portion of a plant gene to leaves of barley, rice, and maize plants resulted in visual silencing-like phenotypes. The visual phenotypes were correlated with decreased target host transcript levels in the corresponding leaves. The VIGS visual phenotype varied from maintained during silencing of actin 1 transcript expression to transient with incomplete penetration through affected tissue during silencing of phytoene desaturase expression. F-BMV RNA 3 was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector C-BMV(A/G) (C for chimeric) was shown to be useful for VIGS. These BMV vectors will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.

The expression of preaxial polydactyly is influenced by modifying genetic elements and is not maintained by chromosomal inversion in an avian biomedical model.

PubMed

Robb, E A; Delany, M E

2012-01-01

Polydactyly (Po) is a common mutation found in many vertebrates. The UCD-Po.003 congenic chicken line was previously characterized for Po inheritance (autosomal dominant) and the mutation was mapped to chromosome 2p. Here, we describe for the first time the range and variability of the phenotype in this congenic line. Further, we studied the hypothesis that a chromosomal inversion was responsible for the maintenance of a large (6.3 Mb) candidate gene region. Fluorescence in situ hybridization employing BACs encompassing a 10.7-Mb region of GGA2p showed that the Po chromosome was normal, i.e. exhibits the wild-type BAC order. Continued fine-mapping along with a change in breeding strategy reduced the size of the causative region to 1.43 Mb. Recent research indicates that the cause of preaxial Po resides within a 794-bp highly conserved zone of polarizing activity regulatory sequence (ZRS) element located in intron 5 of the LMBR1 gene; however, the ZRS polymorphism of interest is found in some but not all breeds of polydactylous chicken. Therefore, we sequenced the ZRS in 101 heterozygous and 30 unaffected (wild-type) individuals to establish the relevance of this region to the Po condition in the UCD-Po.003 congenic line. A single point mutation (C/A at coordinate GGA2p: 8,414,121) within the ZRS segregated with carrier status. The polydactylous UCD-Silkie line also maintains this SNP in addition to a single base deletion. An inheritance analysis of the phenotypic variation in UCD-Po.003 suggests recessive epistasis as the mode of inheritance for the additional modifying genetic elements, residing outside the ZRS, to impact the preaxial polydactyl phenotype. These results contribute to our understanding of the cause of Po in an important vertebrate model. Copyright © 2012 S. Karger AG, Basel.

Detecting Chemically Modified DNA Bases Using Surface Enhanced Raman Spectroscopy

PubMed Central

Barhoumi, Aoune; Halas, Naomi J.

2013-01-01

Post-translational modifications of DNA- changes in the chemical structure of individual bases that occur without changes in the DNA sequence- are known to alter gene expression. They are believed to result in frequently deleterious phenotypic changes, such as cancer. Methylation of adenine, methylation and hydroxymethylation of cytosine, and guanine oxidation are the primary DNA base modifications identified to date. Here we show it is possible to use surface enhanced Raman spectroscopy (SERS) to detect these primary DNA base modifications. SERS detection of modified DNA bases is label-free and requires minimal additional sample preparation, reducing the possibility of additional chemical modifications induced prior to measurement. This approach shows the feasibility of DNA base modification assessment as a potentially routine analysis that may be further developed for clinical diagnostics. PMID:24427449

Detecting Chemically Modified DNA Bases Using Surface Enhanced Raman Spectroscopy.

PubMed

Barhoumi, Aoune; Halas, Naomi J

2011-12-15

Post-translational modifications of DNA- changes in the chemical structure of individual bases that occur without changes in the DNA sequence- are known to alter gene expression. They are believed to result in frequently deleterious phenotypic changes, such as cancer. Methylation of adenine, methylation and hydroxymethylation of cytosine, and guanine oxidation are the primary DNA base modifications identified to date. Here we show it is possible to use surface enhanced Raman spectroscopy (SERS) to detect these primary DNA base modifications. SERS detection of modified DNA bases is label-free and requires minimal additional sample preparation, reducing the possibility of additional chemical modifications induced prior to measurement. This approach shows the feasibility of DNA base modification assessment as a potentially routine analysis that may be further developed for clinical diagnostics.

FAT1 Gene Alteration in Facioscapulohumeral Muscular Dystrophy Type 1.

PubMed

Park, Hyung Jun; Lee, Wookjae; Kim, Se Hoon; Lee, Jung Hwan; Shin, Ha Young; Kim, Seung Min; Park, Kee Duk; Lee, Ji Hyun; Choi, Young Chul

2018-03-01

Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array. Recent studies revealed that the FAT1 expression is associated with disease activity of FSHD, and the FAT1 alterations result in myopathy with a FSHD-like phenotype. We describe a 59-year-old woman with both contracted D4Z4 repeat units and a FAT1 mutation. Shoulder girdle muscle weakness developed at the age of 56 years, and was followed by proximal leg weakness. When we examined her at 59 years of age, she displayed asymmetric and predominant weakness of facial and proximal muscles. Muscle biopsy showed increased variation in fiber size and multifocal degenerating fibers with lymphocytic infiltration. Southern blot analysis revealed 8 D4Z4 repeat units, and targeted sequencing of modifier genes demonstrated the c.10331 A>G variant in the FAT1 gene. This FAT1 variant has previously been reported as pathogenic variant in a patient with FSHD-like phenotype. Our study is the first report of a FAT1 mutation in a FSHD1 patient, and suggests that FAT1 alterations might work as a genetic modifier. © Copyright: Yonsei University College of Medicine 2018.

Dissemination of Genes Encoding Aminoglycoside-Modifying Enzymes and armA Among Enterobacteriaceae Isolates in Northwest Iran.

PubMed

Ghotaslou, Reza; Yeganeh Sefidan, Fatemeh; Akhi, Mohammad Taghi; Asgharzadeh, Mohammad; Mohammadzadeh Asl, Yalda

2017-10-01

Enzymatic inactivation is one of the most important mechanisms of resistance to aminoglycosides. The aim of this study was to investigate the prevalence of armA and diversity of the genes encoding aminoglycoside-modifying enzymes (AMEs) and their associations with resistance phenotypes in Enterobacteriaceae isolates. Three hundred and seven Enterobacteriaceae isolates were collected from five hospitals in northwest Iran. The disk diffusion method for amikacin, gentamicin, tobramycin, kanamycin, and streptomycin, as well as the minimum inhibitory concentration for amikacin, gentamicin, tobramycin, and kanamycin were done for susceptibility testing. Thirteen AME genes and armA methylase were screened using the PCR and sequencing assays. Two hundred and twenty (71.7%) of isolates were resistant to aminoglycosides and 155 (70.5%) of them were positive for aminoglycoside resistance genes. The most prevalent AME genes were ant(3″)-Ia and aph(3″)-Ib with the frequency 35.9% and 30.5%, respectively. Also, 21 (9.5%) of resistant isolates were positive for armA methylase gene. The prevalence of resistance to aminoglycoside is high and AME genes frequently are disseminated in Enterobacteriaceae isolates. There is an association between phenotypic resistance and the presence of some aminoglycoside genes.

Sequencing of disease-modifying therapies for relapsing-remitting multiple sclerosis: a theoretical approach to optimizing treatment.

PubMed

Grand'Maison, Francois; Yeung, Michael; Morrow, Sarah A; Lee, Liesly; Emond, Francois; Ward, Brian J; Laneuville, Pierre; Schecter, Robyn

2018-04-18

Multiple sclerosis (MS) is a chronic disease which usually begins in young adulthood and is a lifelong condition. Individuals with MS experience physical and cognitive disability resulting from inflammation and demyelination in the central nervous system. Over the past decade, several disease-modifying therapies (DMTs) have been approved for the management of relapsing-remitting MS (RRMS), which is the most prevalent phenotype. The chronic nature of the disease and the multiple treatment options make benefit-risk-based sequencing of therapy essential to ensure optimal care. The efficacy and short- and long-term risks of treatment differ for each DMT due to their different mechanism of action on the immune system. While transitioning between DMTs, in addition to immune system effects, factors such as age, disease duration and severity, disability status, monitoring requirements, preference for the route of administration, and family planning play an important role. Determining a treatment strategy is therefore challenging as it requires careful consideration of the differences in efficacy, safety and tolerability, while at the same time minimizing risks of immune modulation. In this review, we discuss a sequencing approach for treating RRMS, with importance given to the long-term risks and individual preference when devising a treatment plan. Evidence-based strategies to counter breakthrough disease are also addressed.

Novel PAX3 mutations causing Waardenburg syndrome type 1 in Tunisian patients.

PubMed

Trabelsi, Mediha; Nouira, Malek; Maazoul, Faouzi; Kraoua, Lilia; Meddeb, Rim; Ouertani, Ines; Chelly, Imen; Benoit, Valérie; Besbes, Ghazi; Mrad, Ridha

2017-12-01

Waardenburg syndrome (WS) is an auditory-pigmentary disease characterized by a clinical and genetic variability. WS is classified into four types depending on the presence or absence of additional symptoms: WS1, WS2, WS3 and WS4. Type 1 and 3 are mostly caused by PAX3 mutations, while type 2 and type 4 are genetically heterogeneous. The aims of this study are to confirm the diagnostic of WS1 by the sequencing of PAX3 gene and to evaluate the genotype phenotype correlation. A clinical classification was established for 14 patients WS, as proposed by the Waardenburg Consortium, and noted a predominance of type 1 and type 2 with 6 patients WS1, 7 patients WS2 and 1 patient WS3. A significant inter and intra-familial clinical heterogeneity was also observed. A sequencing of PAX3 gene in the 6 patients WS1 confirmed the diagnosis in 4 of them by revealing three novel mutations that modify two functional domains of the protein: the c.942delC; the c.933_936dupTTAC and the c.164delTCCGCCACA. These three variations are most likely responsible for the phenotype, however their pathogenic effects need to be confirmed by functional studies. The MLPA analysis of the 2 patients who were sequence negative for PAX3 gene revealed, in one of them, a heterozygous deletion of exons 5 to 9 confirming the WS1 diagnosis. Both clinical and molecular approaches led to the conclusion that there is a lack of genotype-phenotype correlation in WS1, an element that must be taken into account in genetic counseling. The absence of PAX3 mutation in one patient WS1 highlights the fact that the clinical classification is sometimes insufficient to distinguish WS1 from other types WS hence the interest of sequencing the other WS genes in this patient. Copyright © 2017 Elsevier B.V. All rights reserved.

Genome-wide association study to identify potential genetic modifiers in a canine model for Duchenne muscular dystrophy.

PubMed

Brinkmeyer-Langford, Candice; Balog-Alvarez, Cynthia; Cai, James J; Davis, Brian W; Kornegay, Joe N

2016-08-22

Duchenne muscular dystrophy (DMD) causes progressive muscle degeneration, cardiomyopathy and respiratory failure in approximately 1/5,000 boys. Golden Retriever muscular dystrophy (GRMD) resembles DMD both clinically and pathologically. Like DMD, GRMD exhibits remarkable phenotypic variation among affected dogs, suggesting the influence of modifiers. Understanding the role(s) of genetic modifiers of GRMD may identify genes and pathways that also modify phenotypes in DMD and reveal novel therapies. Therefore, our objective in this study was to identify genetic modifiers that affect discrete GRMD phenotypes. We performed a linear mixed-model (LMM) analysis using 16 variably-affected dogs from our GRMD colony (8 dystrophic, 8 non-dystrophic). All of these dogs were either full or half-siblings, and phenotyped for 19 objective, quantitative biomarkers at ages 6 and 12 months. Each biomarker was individually assessed. Gene expression profiles of 59 possible candidate genes were generated for two muscle types: the cranial tibialis and medial head of the gastrocnemius. SNPs significantly associated with GRMD biomarkers were identified on multiple chromosomes (including the X chromosome). Gene expression levels for candidate genes located near these SNPs correlated with biomarker values, suggesting possible roles as GRMD modifiers. The results of this study enhance our understanding of GRMD pathology and represent a first step toward the characterization of GRMD modifiers that may be relevant to DMD pathology. Such modifiers are likely to be useful for DMD treatment development based on their relationships to GRMD phenotypes.

An experimental phylogeny to benchmark ancestral sequence reconstruction

PubMed Central

Randall, Ryan N.; Radford, Caelan E.; Roof, Kelsey A.; Natarajan, Divya K.; Gaucher, Eric A.

2016-01-01

Ancestral sequence reconstruction (ASR) is a still-burgeoning method that has revealed many key mechanisms of molecular evolution. One criticism of the approach is an inability to validate its algorithms within a biological context as opposed to a computer simulation. Here we build an experimental phylogeny using the gene of a single red fluorescent protein to address this criticism. The evolved phylogeny consists of 19 operational taxonomic units (leaves) and 17 ancestral bifurcations (nodes) that display a wide variety of fluorescent phenotypes. The 19 leaves then serve as ‘modern' sequences that we subject to ASR analyses using various algorithms and to benchmark against the known ancestral genotypes and ancestral phenotypes. We confirm computer simulations that show all algorithms infer ancient sequences with high accuracy, yet we also reveal wide variation in the phenotypes encoded by incorrectly inferred sequences. Specifically, Bayesian methods incorporating rate variation significantly outperform the maximum parsimony criterion in phenotypic accuracy. Subsampling of extant sequences had minor effect on the inference of ancestral sequences. PMID:27628687

Weakener of White (Wow), a Gene That Modifies the Expression of the White Eye Color Locus and That Suppresses Position Effect Variegation in Drosophila Melanogaster

PubMed Central

Birchler, J. A.; Bhadra, U.; Rabinow, L.; Linsk, R.; Nguyen-Huynh, A. T.

1994-01-01

A locus is described in Drosophila melanogaster that modifies the expression of the white eye color gene. This trans-acting modifier reduces the expression of the white gene in the eye, but elevates the expression in other adult tissues. Because of the eye phenotype in which the expression of white is lessened but not eliminated, the newly described locus is called the Weakener of white (Wow). Northern analysis reveals that Wow can exert an inverse or direct modifying effect depending upon the developmental stage. Two related genes, brown and scarlet, that are coordinately expressed with white, are also affected by Wow. In addition, Wow modulates the steady state RNA level of the retrotransposon, copia. When tested with a white promoter-Alcohol dehydrogenase reporter, Wow confers the modifying effect to the reporter, suggesting a requirement of the white regulatory sequences for mediating the response. In addition to being a dosage sensitive regulator of white, brown, scarlet and copia, Wow acts as a suppressor of position effect variegation. There are many dosage sensitive suppressors of position effect variegation and many dosage-sensitive modifiers of gene expression. The Wow mutations provide evidence for an overlap between the two types of modifiers. PMID:7982560

Phylogeny of the ammonia-producing ruminal bacteria Peptostreptococcus anaerobius, Clostridium sticklandii, and Clostridium aminophilum sp. nov

NASA Technical Reports Server (NTRS)

Paster, B. J.; Russell, J. B.; Yang, C. M.; Chow, J. M.; Woese, C. R.; Tanner, R.

1993-01-01

In previous studies, gram-positive bacteria which grew rapidly with peptides or an amino acid as the sole energy source were isolated from bovine rumina. Three isolates, strains C, FT (T = type strain), and SR, were considered to be ecologically important since they produced up to 20-fold more ammonia than other ammonia-producing ruminal bacteria. On the basis of phenotypic criteria, the taxonomic position of these new isolates was uncertain. In this study, the 16S rRNA sequences of these isolates and related bacteria were determined to establish the phylogenetic positions of the organisms. The sequences of strains C, FT, and SR and reference strains of Peptostreptococcus anaerobius, Clostridium sticklandii, Clostridium coccoides, Clostridium aminovalericum, Acetomaculum ruminis, Clostridium leptum, Clostridium lituseburense, Clostridium acidiurici, and Clostridium barkeri were determined by using a modified Sanger dideoxy chain termination method. Strain C, a large coccus purported to belong to the genus Peptostreptococcus, was closely related to P. anaerobius, with a level of sequence similarity of 99.6%. Strain SR, a heat-resistant, short, rod-shaped organism, was closely related to C. sticklandii, with a level of sequence similarity of 99.9%. However, strain FT, a heat-resistant, pleomorphic, rod-shaped organism, was only distantly related to some clostridial species and P. anaerobius. On the basis of the sequence data, it was clear that strain FT warranted designation as a separate species. The closest known relative of strain FT was C. coccoides (level of similarity, only 90.6%). Additional strains that are phenotypically similar to strain FT were isolated in this study.(ABSTRACT TRUNCATED AT 250 WORDS).

Bridging epigenomics and complex disease: the basics.

PubMed

Teperino, Raffaele; Lempradl, Adelheid; Pospisilik, J Andrew

2013-05-01

The DNA sequence largely defines gene expression and phenotype. However, it is becoming increasingly clear that an additional chromatin-based regulatory network imparts both stability and plasticity to genome output, modifying phenotype independently of the genetic blueprint. Indeed, alterations in this "epigenetic" control layer underlie, at least in part, the reason for monozygotic twins being discordant for disease. Functionally, this regulatory layer comprises post-translational modifications of DNA and histones, as well as small and large noncoding RNAs. Together these regulate gene expression by changing chromatin organization and DNA accessibility. Successive technological advances over the past decade have enabled researchers to map the chromatin state with increasing accuracy and comprehensiveness, catapulting genetic research into a genome-wide era. Here, aiming particularly at the genomics/epigenomics newcomer, we review the epigenetic basis that has helped drive the technological shift and how this progress is shaping our understanding of complex disease.

Cftr gene targeting in mouse embryonic stem cells mediated by Small Fragment Homologous Replacement (SFHR).

PubMed

Sangiuolo, Federica; Scaldaferri, Maria Lucia; Filareto, Antonio; Spitalieri, Paola; Guerra, Lorenzo; Favia, Maria; Caroppo, Rosa; Mango, Ruggiero; Bruscia, Emanuela; Gruenert, Dieter C; Casavola, Valeria; De Felici, Massimo; Novelli, Giuseppe

2008-01-01

Different gene targeting approaches have been developed to modify endogenous genomic DNA in both human and mouse cells. Briefly, the process involves the targeting of a specific mutation in situ leading to the gene correction and the restoration of a normal gene function. Most of these protocols with therapeutic potential are oligonucleotide based, and rely on endogenous enzymatic pathways. One gene targeting approach, "Small Fragment Homologous Replacement (SFHR)", has been found to be effective in modifying genomic DNA. This approach uses small DNA fragments (SDF) to target specific genomic loci and induce sequence and subsequent phenotypic alterations. This study shows that SFHR can stably introduce a 3-bp deletion (deltaF508, the most frequent cystic fibrosis (CF) mutation) into the Cftr (CF Transmembrane Conductance Regulator) locus in the mouse embryonic stem (ES) cell genome. After transfection of deltaF508-SDF into murine ES cells, SFHR-mediated modification was evaluated at the molecular levels on DNA and mRNA obtained from transfected ES cells. About 12% of transcript corresponding to deleted allele was detected, while 60% of the electroporated cells completely lost any measurable CFTR-dependent chloride efflux. The data indicate that the SFHR technique can be used to effectively target and modify genomic sequences in ES cells. Once the SFHR-modified ES cells differentiate into different cell lineages they can be useful for elucidating tissue-specific gene function and for the development of transplantation-based cellular and therapeutic protocols.

Serratia marcescens harbouring SME-type class A carbapenemases in Canada and the presence of blaSME on a novel genomic island, SmarGI1-1.

PubMed

Mataseje, L F; Boyd, D A; Delport, J; Hoang, L; Imperial, M; Lefebvre, B; Kuhn, M; Van Caeseele, P; Willey, B M; Mulvey, M R

2014-07-01

An increasing prevalence since 2010 of Serratia marcescens harbouring the Ambler class A carbapenemase SME prompted us to further characterize these isolates. Isolates harbouring bla(SME) were identified by PCR and sequencing. Phenotypic analysis for carbapenemase activity was carried out by a modified Hodge test and a modified Carba NP test. Antimicrobial susceptibilities were determined by Etest and Vitek 2. Typing was by PFGE of macrorestriction digests. Whole-genome sequencing of three isolates was carried out to characterize the genomic region harbouring the bla(SME)-type genes. All S. marcescens harbouring SME-type enzymes could be detected using a modified Carba NP test. Isolates harbouring bla(SME) were resistant to penicillins and carbapenems, but remained susceptible to third-generation cephalosporins, as well as fluoroquinolones and trimethoprim/sulfamethoxazole. Isolates exhibited diverse genetic backgrounds, though 57% of isolates were found in three clusters. Analysis of whole-genome sequence data from three isolates revealed that the bla(SME) gene occurred in a novel cryptic prophage genomic island, SmarGI1-1. There has been an increasing occurrence of S. marcescens harbouring bla(SME) in Canada since 2010. The bla(SME) gene was found on a genomic island, SmarGI1-1, that can be excised and circularized, which probably contributes to its dissemination amongst S. marcescens. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Topological Structure of the Space of Phenotypes: The Case of RNA Neutral Networks

PubMed Central

Aguirre, Jacobo; Buldú, Javier M.; Stich, Michael; Manrubia, Susanna C.

2011-01-01

The evolution and adaptation of molecular populations is constrained by the diversity accessible through mutational processes. RNA is a paradigmatic example of biopolymer where genotype (sequence) and phenotype (approximated by the secondary structure fold) are identified in a single molecule. The extreme redundancy of the genotype-phenotype map leads to large ensembles of RNA sequences that fold into the same secondary structure and can be connected through single-point mutations. These ensembles define neutral networks of phenotypes in sequence space. Here we analyze the topological properties of neutral networks formed by 12-nucleotides RNA sequences, obtained through the exhaustive folding of sequence space. A total of 412 sequences fragments into 645 subnetworks that correspond to 57 different secondary structures. The topological analysis reveals that each subnetwork is far from being random: it has a degree distribution with a well-defined average and a small dispersion, a high clustering coefficient, and an average shortest path between nodes close to its minimum possible value, i.e. the Hamming distance between sequences. RNA neutral networks are assortative due to the correlation in the composition of neighboring sequences, a feature that together with the symmetries inherent to the folding process explains the existence of communities. Several topological relationships can be analytically derived attending to structural restrictions and generic properties of the folding process. The average degree of these phenotypic networks grows logarithmically with their size, such that abundant phenotypes have the additional advantage of being more robust to mutations. This property prevents fragmentation of neutral networks and thus enhances the navigability of sequence space. In summary, RNA neutral networks show unique topological properties, unknown to other networks previously described. PMID:22028856

Distribution of genotype network sizes in sequence-to-structure genotype-phenotype maps.

PubMed

Manrubia, Susanna; Cuesta, José A

2017-04-01

An essential quantity to ensure evolvability of populations is the navigability of the genotype space. Navigability, understood as the ease with which alternative phenotypes are reached, relies on the existence of sufficiently large and mutually attainable genotype networks. The size of genotype networks (e.g. the number of RNA sequences folding into a particular secondary structure or the number of DNA sequences coding for the same protein structure) is astronomically large in all functional molecules investigated: an exhaustive experimental or computational study of all RNA folds or all protein structures becomes impossible even for moderately long sequences. Here, we analytically derive the distribution of genotype network sizes for a hierarchy of models which successively incorporate features of increasingly realistic sequence-to-structure genotype-phenotype maps. The main feature of these models relies on the characterization of each phenotype through a prototypical sequence whose sites admit a variable fraction of letters of the alphabet. Our models interpolate between two limit distributions: a power-law distribution, when the ordering of sites in the prototypical sequence is strongly constrained, and a lognormal distribution, as suggested for RNA, when different orderings of the same set of sites yield different phenotypes. Our main result is the qualitative and quantitative identification of those features of sequence-to-structure maps that lead to different distributions of genotype network sizes. © 2017 The Author(s).

MC1R diversity in Northern Island Melanesia has not been constrained by strong purifying selection and cannot explain pigmentation phenotype variation in the region.

PubMed

Norton, Heather L; Werren, Elizabeth; Friedlaender, Jonathan

2015-10-19

Variation in human skin pigmentation evolved in response to the selective pressure of ultra-violet radiation (UVR). Selection to maintain darker skin in high UVR environments is expected to constrain pigmentation phenotype and variation in pigmentation loci. Consistent with this hypothesis, the gene MC1R exhibits reduced diversity in African populations from high UVR regions compared to low-UVR non-African populations. However, MC1R diversity in non-African populations that have evolved under high-UVR conditions is not well characterized. In order to test the hypothesis that MC1R variation has been constrained in Melanesians the coding region of the MC1R gene was sequenced in 188 individuals from Northern Island Melanesia. The role of purifying selection was assessed using a modified McDonald Kreitman's test. Pairwise FST was calculated between Melanesian populations and populations from the 1000 Genomes Project. The SNP rs2228479 was genotyped in a larger sample (n = 635) of Melanesians and tested for associations with skin and hair pigmentation. We observe three nonsynonymous and two synonymous mutations. A modified McDonald Kreitman's test failed to detect a significant signal of purifying selection. Pairwise FST values calculated between the four islands sampled here indicate little regional substructure in MC1R. When compared to African, European, East and South Asian populations, Melanesians do not exhibit reduced population divergence (measured as FST) or a high proportion of haplotype sharing with Africans, as one might expect if ancestral haplotypes were conserved across high UVR populations in and out of Africa. The only common nonsynonymous polymorphism observed, rs2228479, is not significantly associated with skin or hair pigmentation in a larger sample of Melanesians. The pattern of sequence diversity here does not support a model of strong selective constraint on MC1R in Northern Island Melanesia This absence of strong constraint, as well as the recent population history of the region, may explain the observed frequencies of the derived rs2228479 allele. These results emphasize the complex genetic architecture of pigmentation phenotypes, which are controlled by multiple, possibly interacting loci. They also highlight the role that population history can play in influencing phenotypic diversity in the absence of strong natural selection.

SigniSite: Identification of residue-level genotype-phenotype correlations in protein multiple sequence alignments.

PubMed

Jessen, Leon Eyrich; Hoof, Ilka; Lund, Ole; Nielsen, Morten

2013-07-01

Identifying which mutation(s) within a given genotype is responsible for an observable phenotype is important in many aspects of molecular biology. Here, we present SigniSite, an online application for subgroup-free residue-level genotype-phenotype correlation. In contrast to similar methods, SigniSite does not require any pre-definition of subgroups or binary classification. Input is a set of protein sequences where each sequence has an associated real number, quantifying a given phenotype. SigniSite will then identify which amino acid residues are significantly associated with the data set phenotype. As output, SigniSite displays a sequence logo, depicting the strength of the phenotype association of each residue and a heat-map identifying 'hot' or 'cold' regions. SigniSite was benchmarked against SPEER, a state-of-the-art method for the prediction of specificity determining positions (SDP) using a set of human immunodeficiency virus protease-inhibitor genotype-phenotype data and corresponding resistance mutation scores from the Stanford University HIV Drug Resistance Database, and a data set of protein families with experimentally annotated SDPs. For both data sets, SigniSite was found to outperform SPEER. SigniSite is available at: http://www.cbs.dtu.dk/services/SigniSite/.

A functional U-statistic method for association analysis of sequencing data.

PubMed

Jadhav, Sneha; Tong, Xiaoran; Lu, Qing

2017-11-01

Although sequencing studies hold great promise for uncovering novel variants predisposing to human diseases, the high dimensionality of the sequencing data brings tremendous challenges to data analysis. Moreover, for many complex diseases (e.g., psychiatric disorders) multiple related phenotypes are collected. These phenotypes can be different measurements of an underlying disease, or measurements characterizing multiple related diseases for studying common genetic mechanism. Although jointly analyzing these phenotypes could potentially increase the power of identifying disease-associated genes, the different types of phenotypes pose challenges for association analysis. To address these challenges, we propose a nonparametric method, functional U-statistic method (FU), for multivariate analysis of sequencing data. It first constructs smooth functions from individuals' sequencing data, and then tests the association of these functions with multiple phenotypes by using a U-statistic. The method provides a general framework for analyzing various types of phenotypes (e.g., binary and continuous phenotypes) with unknown distributions. Fitting the genetic variants within a gene using a smoothing function also allows us to capture complexities of gene structure (e.g., linkage disequilibrium, LD), which could potentially increase the power of association analysis. Through simulations, we compared our method to the multivariate outcome score test (MOST), and found that our test attained better performance than MOST. In a real data application, we apply our method to the sequencing data from Minnesota Twin Study (MTS) and found potential associations of several nicotine receptor subunit (CHRN) genes, including CHRNB3, associated with nicotine dependence and/or alcohol dependence. © 2017 WILEY PERIODICALS, INC.

Anonymization of electronic medical records for validating genome-wide association studies

PubMed Central

Loukides, Grigorios; Gkoulalas-Divanis, Aris; Malin, Bradley

2010-01-01

Genome-wide association studies (GWAS) facilitate the discovery of genotype–phenotype relations from population-based sequence databases, which is an integral facet of personalized medicine. The increasing adoption of electronic medical records allows large amounts of patients’ standardized clinical features to be combined with the genomic sequences of these patients and shared to support validation of GWAS findings and to enable novel discoveries. However, disseminating these data “as is” may lead to patient reidentification when genomic sequences are linked to resources that contain the corresponding patients’ identity information based on standardized clinical features. This work proposes an approach that provably prevents this type of data linkage and furnishes a result that helps support GWAS. Our approach automatically extracts potentially linkable clinical features and modifies them in a way that they can no longer be used to link a genomic sequence to a small number of patients, while preserving the associations between genomic sequences and specific sets of clinical features corresponding to GWAS-related diseases. Extensive experiments with real patient data derived from the Vanderbilt's University Medical Center verify that our approach generates data that eliminate the threat of individual reidentification, while supporting GWAS validation and clinical case analysis tasks. PMID:20385806

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

PubMed

Wills, Quin F; Mellado-Gomez, Esther; Nolan, Rory; Warner, Damien; Sharma, Eshita; Broxholme, John; Wright, Benjamin; Lockstone, Helen; James, William; Lynch, Mark; Gonzales, Michael; West, Jay; Leyrat, Anne; Padilla-Parra, Sergi; Filippi, Sarah; Holmes, Chris; Moore, Michael D; Bowden, Rory

2017-01-07

Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

Variation of clinical expression in patients with Stargardt dystrophy and sequence variations in the ABCR gene.

PubMed

Fishman, G A; Stone, E M; Grover, S; Derlacki, D J; Haines, H L; Hockey, R R

1999-04-01

To report the spectrum of ophthalmic findings in patients with Stargardt dystrophy or fundus flavimaculatus who have a specific sequence variation in the ABCR gene. Twenty-nine patients with Stargardt dystrophy or fundus flavimaculatus from different pedigrees were identified with possible disease-causing sequence variations in the ABCR gene from a group of 66 patients who were screened for sequence variations in this gene. Patients underwent a routine ocular examination, including slitlamp biomicroscopy and a dilated fundus examination. Fluorescein angiography was performed on 22 patients, and electroretinographic measurements were obtained on 24 of 29 patients. Kinetic visual fields were measured with a Goldmann perimeter in 26 patients. Single-strand conformation polymorphism analysis and DNA sequencing were used to identify variations in coding sequences of the ABCR gene. Three clinical phenotypes were observed among these 29 patients. In phenotype I, 9 of 12 patients had a sequence change in exon 42 of the ABCR gene in which the amino acid glutamic acid was substituted for glycine (Gly1961Glu). In only 4 of these 9 patients was a second possible disease-causing mutation found on the other ABCR allele. In addition to an atrophic-appearing macular lesion, phenotype I was characterized by localized perifoveal yellowish white flecks, the absence of a dark choroid, and normal electroretinographic amplitudes. Phenotype II consisted of 10 patients who showed a dark choroid and more diffuse yellowish white flecks in the fundus. None exhibited the Gly1961Glu change. Phenotype III consisted of 7 patients who showed extensive atrophic-appearing changes of the retinal pigment epithelium. Electroretinographic cone and rod amplitudes were reduced. One patient showed the Gly1961Glu change. A wide variation in clinical phenotype can occur in patients with sequence changes in the ABCR gene. In individual patients, a certain phenotype seems to be associated with the presence of a Gly1961Glu change in exon 42 of the ABCR gene. The identification of correlations between specific mutations in the ABCR gene and clinical phenotypes will better facilitate the counseling of patients on their visual prognosis. This information will also likely be important for future therapeutic trials in patients with Stargardt dystrophy.

Long-term treatment with green tea polyphenols modifies the gut microbiome of female sprague-dawley rats.

PubMed

Wang, Jincheng; Tang, Lili; Zhou, Hongyuan; Zhou, Jun; Glenn, Travis C; Shen, Chwan-Li; Wang, Jia-Sheng

2018-06-01

Green tea polyphenols (GTP) have been shown to exert a spectrum of health benefits to animals and humans. It is plausible that the beneficial effects of GTP are a result of its interaction with the gut microbiota. This study evaluated the effect of long-term treatment with GTP on the gut microbiota of experimental rats and the potential linkage between changes of the gut microbiota with the beneficial effects of GTP. Six-month-old Sprague-Dawley rats were randomly allocated into three dosing regimens (0, 0.5%, and 1.5% of GTP) and followed for 6 months. At the end of month 3 or month 6, half of the animals from each group were sacrificed and their colon contents were collected for microbiome analysis using 16S ribosomal RNA and shotgun metagenomic community sequencing. GTP treatment significantly decreased the biodiversity and modified the microbial community in a dose-dependent manner; similar patterns were observed at both sampling times. Multiple operational taxonomic units and phylotypes were modified: the phylotypes Bacteroidetes and Oscillospira, previously linked to the lean phenotype in human and animal studies, were enriched; and Peptostreptococcaceae previously linked to colorectal cancer phenotype was depleted in GTP treated groups in a dose-dependent manner. Several microbial gene orthologs were modified, among which genes related to energy production and conversion were consistently enriched in samples from month 6 in a dose-dependent manner. This study showed that long-term treatment with GTP induced a dose-dependent modification of the gut microbiome in experimental rats, which might be linked to beneficial effects of GTP. Copyright © 2018 Elsevier Inc. All rights reserved.

BMP15 c.-9C>G promoter sequence variant may contribute to the cause of non-syndromic premature ovarian failure.

PubMed

Fonseca, Dora Janeth; Ortega-Recalde, Oscar; Esteban-Perez, Clara; Moreno-Ortiz, Harold; Patiño, Liliana Catherine; Bermúdez, Olga María; Ortiz, Angela María; Restrepo, Carlos M; Lucena, Elkin; Laissue, Paul

2014-11-01

BMP15 has drawn particular attention in the pathophysiology of reproduction, as its mutations in mammalian species have been related to different reproductive phenotypes. In humans, BMP15 coding regions have been sequenced in large panels of women with premature ovarian failure (POF), but only some mutations have been definitely validated as causing the phenotype. A functional association between the BMP15 c.-9C>G promoter polymorphism and cause of POF have been reported. The aim of this study was to determine the potential functional effect of this sequence variant on specific BMP15 promoter transactivation disturbances. Bioinformatics was used to identify transcription factor binding sites located on the promoter region of BMP15. Reverse transcription polymerase chain reaction was used to study specific gene expression in ovarian tissue. Luciferase reporter assays were used to establish transactivation disturbances caused by the BMP15 c.-9C>G variant. The c.-9C>G variant was found to modify the PITX1 transcription factor binding site. PITX1 and BMP15 co-expressed in human and mouse ovarian tissue, and PITX1 transactivated both BMP15 promoter versions (-9C and -9G). It was found that the BMP15 c.-9G allele was related to BMP15 increased transcription, supporting c.-9C>G as a causal agent of POF. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Nine known and five novel mutations in the erythroid transcription factor KLF1 gene and phenotypic expression of fetal hemoglobin in hemoglobin E disorder.

PubMed

Tepakhan, Wanicha; Yamsri, Supawadee; Sanchaisuriya, Kanokwan; Fucharoen, Goonnapa; Xu, Xiangmin; Fucharoen, Supan

2016-07-01

Hemoglobin E is the most common Hb variant found in South East Asia. Variation of Hb F expression in Hb E syndrome is associated with several genetic modifiers. We report several single nucleotide polymorphisms (SNPs), including nine known and five novel mutations of the Krüppel-like factor 1 (KLF1; an erythroid specific transcription factor) gene and determine their associations with phenotypic expression of Hb F in Hb E disorders. KLF1 mutations were examined using high resolution melting (HRM) assay and DNA sequencing in 575 homozygous Hb E, 278 heterozygous Hb E and 100 normal subjects. Fourteen mutations were mostly observed in subjects with elevated Hb F, including nine known mutations (G176AfsX179, T334R, R238H, -154 (C>T), A298P, S270W, R301H, -148 (G>A) and G335R and five novel mutations (Q217X, Q223X, Y290_S293del, K307N, and M358I). None of them, but the -148 (G>A), were observed in normal controls to have Hb F <1%. Combined KLF1 mutations with other SNPs including (G)γ-XmnI, BCL11A and HBS1L-MYB were associated with higher Hb F levels. KLF1 is therefore an important genetic factor associated with increased Hb F and in combination with other modifying factors could explain the phenotypic variation of Hb F expression in this common hemoglobinopathy. Copyright © 2016 Elsevier Inc. All rights reserved.

Complete Genome Sequence of the Cystic Fibrosis Pathogen Achromobacter xylosoxidans NH44784-1996 Complies with Important Pathogenic Phenotypes

PubMed Central

Jakobsen, Tim Holm; Hansen, Martin Asser; Jensen, Peter Østrup; Hansen, Lars; Riber, Leise; Cockburn, April; Kolpen, Mette; Rønne Hansen, Christine; Ridderberg, Winnie; Eickhardt, Steffen; Hansen, Marlene; Kerpedjiev, Peter; Alhede, Morten; Qvortrup, Klaus; Burmølle, Mette; Moser, Claus; Kühl, Michael; Ciofu, Oana; Givskov, Michael; Sørensen, Søren J.; Høiby, Niels; Bjarnsholt, Thomas

2013-01-01

Achromobacter xylosoxidans is an environmental opportunistic pathogen, which infects an increasing number of immunocompromised patients. In this study we combined genomic analysis of a clinical isolated A. xylosoxidans strain with phenotypic investigations of its important pathogenic features. We present a complete assembly of the genome of A. xylosoxidans NH44784-1996, an isolate from a cystic fibrosis patient obtained in 1996. The genome of A. xylosoxidans NH44784-1996 contains approximately 7 million base pairs with 6390 potential protein-coding sequences. We identified several features that render it an opportunistic human pathogen, We found genes involved in anaerobic growth and the pgaABCD operon encoding the biofilm adhesin poly-β-1,6-N-acetyl-D-glucosamin. Furthermore, the genome contains a range of antibiotic resistance genes coding efflux pump systems and antibiotic modifying enzymes. In vitro studies of A. xylosoxidans NH44784-1996 confirmed the genomic evidence for its ability to form biofilms, anaerobic growth via denitrification, and resistance to a broad range of antibiotics. Our investigation enables further studies of the functionality of important identified genes contributing to the pathogenicity of A. xylosoxidans and thereby improves our understanding and ability to treat this emerging pathogen. PMID:23894309

Rapid gene identification in sugar beet using deep sequencing of DNA from phenotypic pools selected from breeding panels.

PubMed

Ries, David; Holtgräwe, Daniela; Viehöver, Prisca; Weisshaar, Bernd

2016-03-15

The combination of bulk segregant analysis (BSA) and next generation sequencing (NGS), also known as mapping by sequencing (MBS), has been shown to significantly accelerate the identification of causal mutations for species with a reference genome sequence. The usual approach is to cross homozygous parents that differ for the monogenic trait to address, to perform deep sequencing of DNA from F2 plants pooled according to their phenotype, and subsequently to analyze the allele frequency distribution based on a marker table for the parents studied. The method has been successfully applied for EMS induced mutations as well as natural variation. Here, we show that pooling genetically diverse breeding lines according to a contrasting phenotype also allows high resolution mapping of the causal gene in a crop species. The test case was the monogenic locus causing red vs. green hypocotyl color in Beta vulgaris (R locus). We determined the allele frequencies of polymorphic sequences using sequence data from two diverging phenotypic pools of 180 B. vulgaris accessions each. A single interval of about 31 kbp among the nine chromosomes was identified which indeed contained the causative mutation. By applying a variation of the mapping by sequencing approach, we demonstrated that phenotype-based pooling of diverse accessions from breeding panels and subsequent direct determination of the allele frequency distribution can be successfully applied for gene identification in a crop species. Our approach made it possible to identify a small interval around the causative gene. Sequencing of parents or individual lines was not necessary. Whenever the appropriate plant material is available, the approach described saves time compared to the generation of an F2 population. In addition, we provide clues for planning similar experiments with regard to pool size and the sequencing depth required.

Comprehensive detection of genes causing a phenotype using phenotype sequencing and pathway analysis.

PubMed

Harper, Marc; Gronenberg, Luisa; Liao, James; Lee, Christopher

2014-01-01

Discovering all the genetic causes of a phenotype is an important goal in functional genomics. We combine an experimental design for detecting independent genetic causes of a phenotype with a high-throughput sequencing analysis that maximizes sensitivity for comprehensively identifying them. Testing this approach on a set of 24 mutant strains generated for a metabolic phenotype with many known genetic causes, we show that this pathway-based phenotype sequencing analysis greatly improves sensitivity of detection compared with previous methods, and reveals a wide range of pathways that can cause this phenotype. We demonstrate our approach on a metabolic re-engineering phenotype, the PEP/OAA metabolic node in E. coli, which is crucial to a substantial number of metabolic pathways and under renewed interest for biofuel research. Out of 2157 mutations in these strains, pathway-phenoseq discriminated just five gene groups (12 genes) as statistically significant causes of the phenotype. Experimentally, these five gene groups, and the next two high-scoring pathway-phenoseq groups, either have a clear connection to the PEP metabolite level or offer an alternative path of producing oxaloacetate (OAA), and thus clearly explain the phenotype. These high-scoring gene groups also show strong evidence of positive selection pressure, compared with strictly neutral selection in the rest of the genome.

A comprehensive molecular study on Coffin-Siris and Nicolaides-Baraitser syndromes identifies a broad molecular and clinical spectrum converging on altered chromatin remodeling.

PubMed

Wieczorek, Dagmar; Bögershausen, Nina; Beleggia, Filippo; Steiner-Haldenstätt, Sabine; Pohl, Esther; Li, Yun; Milz, Esther; Martin, Marcel; Thiele, Holger; Altmüller, Janine; Alanay, Yasemin; Kayserili, Hülya; Klein-Hitpass, Ludger; Böhringer, Stefan; Wollstein, Andreas; Albrecht, Beate; Boduroglu, Koray; Caliebe, Almuth; Chrzanowska, Krystyna; Cogulu, Ozgur; Cristofoli, Francesca; Czeschik, Johanna Christina; Devriendt, Koenraad; Dotti, Maria Teresa; Elcioglu, Nursel; Gener, Blanca; Goecke, Timm O; Krajewska-Walasek, Malgorzata; Guillén-Navarro, Encarnación; Hayek, Joussef; Houge, Gunnar; Kilic, Esra; Simsek-Kiper, Pelin Özlem; López-González, Vanesa; Kuechler, Alma; Lyonnet, Stanislas; Mari, Francesca; Marozza, Annabella; Mathieu Dramard, Michèle; Mikat, Barbara; Morin, Gilles; Morice-Picard, Fanny; Ozkinay, Ferda; Rauch, Anita; Renieri, Alessandra; Tinschert, Sigrid; Utine, G Eda; Vilain, Catheline; Vivarelli, Rossella; Zweier, Christiane; Nürnberg, Peter; Rahmann, Sven; Vermeesch, Joris; Lüdecke, Hermann-Josef; Zeschnigk, Michael; Wollnik, Bernd

2013-12-20

Chromatin remodeling complexes are known to modify chemical marks on histones or to induce conformational changes in the chromatin in order to regulate transcription. De novo dominant mutations in different members of the SWI/SNF chromatin remodeling complex have recently been described in individuals with Coffin-Siris (CSS) and Nicolaides-Baraitser (NCBRS) syndromes. Using a combination of whole-exome sequencing, NGS-based sequencing of 23 SWI/SNF complex genes, and molecular karyotyping in 46 previously undescribed individuals with CSS and NCBRS, we identified a de novo 1-bp deletion (c.677delG, p.Gly226Glufs*53) and a de novo missense mutation (c.914G>T, p.Cys305Phe) in PHF6 in two individuals diagnosed with CSS. PHF6 interacts with the nucleosome remodeling and deacetylation (NuRD) complex implicating dysfunction of a second chromatin remodeling complex in the pathogenesis of CSS-like phenotypes. Altogether, we identified mutations in 60% of the studied individuals (28/46), located in the genes ARID1A, ARID1B, SMARCB1, SMARCE1, SMARCA2, and PHF6. We show that mutations in ARID1B are the main cause of CSS, accounting for 76% of identified mutations. ARID1B and SMARCB1 mutations were also found in individuals with the initial diagnosis of NCBRS. These individuals apparently belong to a small subset who display an intermediate CSS/NCBRS phenotype. Our proposed genotype-phenotype correlations are important for molecular screening strategies.

PhenoVar: a phenotype-driven approach in clinical genomics for the diagnosis of polymalformative syndromes

PubMed Central

2014-01-01

Background We propose a phenotype-driven analysis of encrypted exome data to facilitate the widespread implementation of exome sequencing as a clinical genetic screening test. Twenty test-patients with varied syndromes were selected from the literature. For each patient, the mutation, phenotypic data, and genetic diagnosis were available. Next, control exome-files, each modified to include one of these twenty mutations, were assigned to the corresponding test-patients. These data were used by a geneticist blinded to the diagnoses to test the efficiency of our software, PhenoVar. The score assigned by PhenoVar to any genetic diagnosis listed in OMIM (Online Mendelian Inheritance in Man) took into consideration both the patient’s phenotype and all variations present in the corresponding exome. The physician did not have access to the individual mutations. PhenoVar filtered the search using a cut-off phenotypic match threshold to prevent undesired discovery of incidental findings and ranked the OMIM entries according to diagnostic score. Results When assigning the same weight to all variants in the exome, PhenoVar predicted the correct diagnosis in 10/20 patients, while in 15/20 the correct diagnosis was among the 4 highest ranked diagnoses. When assigning a higher weight to variants known, or bioinformatically predicted, to cause disease, PhenoVar’s yield increased to 14/20 (18/20 in top 4). No incidental findings were identified using our cut-off phenotypic threshold. Conclusion The phenotype-driven approach described could render widespread use of ES more practical, ethical and clinically useful. The implications about novel disease identification, advancement of complex diseases and personalized medicine are discussed. PMID:24884844

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii.

PubMed

Shen, Bang; Powell, Robin H; Behnke, Michael S

2017-06-22

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

PubMed Central

Shen, Bang; Powell, Robin H.; Behnke, Michael S.

2017-01-01

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites. PMID:28671645

GenomeRNAi: a database for cell-based RNAi phenotypes.

PubMed

Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

2007-01-01

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at http://rnai.dkfz.de.

GenomeRNAi: a database for cell-based RNAi phenotypes

PubMed Central

Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

2007-01-01

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at PMID:17135194

Transplantation of osteoporotic bone marrow stromal cells rejuvenated by the overexpression of SATB2 prevents alveolar bone loss in ovariectomized rats.

PubMed

Xu, Rongyao; Fu, Zongyun; Liu, Xue; Xiao, Tao; Zhang, Ping; Du, Yifei; Yuan, Hua; Cheng, Jie; Jiang, Hongbing

2016-11-01

Estrogen-deficient osteoporosis is an aging-related disease with high morbidity that not only significantly increases a woman's risk of fragility fracture but is also associated with tooth and bone loss in the supporting alveolar bone of the jaw. Emerging evidence suggests that the aging of bone marrow stromal cells (BMSCs) contributes to the development of osteoporosis. In this study, we aimed to investigate the role of the special AT-rich sequence-binding protein 2 (SATB2), a stemness and senescence regulator of craniofacial BMSCs, in rat ovariectomy-induced alveolar osteoporosis. We also sought to determine whether transplantation of SATB2-modified BMSCs could ameliorate estrogen deficient alveolar bone loss. Our data revealed that BMSCs from ovariectomy-induced alveolar bone exhibited typical senescence phenotypes such as diminished stemness and osteogenic capacity, increased expression of senescence or osteoclastic markers and enhanced adipogenic potential. These phenotypic changes are a result of SATB2-mediated senescence dysregulation as evidenced by nuclear γH2AX foci formation. Moreover, overexpression of SATB2 significantly alleviated the senescence of osteoporotic BMSCs in vitro. Importantly, transplantation of SATB2-modified BMSCs significantly attenuated ovariectomy-induced alveolar bone loss in vivo. Together, our results revealed that SATB2 is a critical regulator of alveolar BMSC senescence, and its overexpression decreases these senescent changes both in vitro and in vivo. SATB2-modified BMSC delivery could be a viable and promising therapeutic strategy for alveolar bone loss induced by estrogen-deficient osteoporosis. Copyright © 2016 Elsevier Inc. All rights reserved.

The usefulness of DNA sequencing after extraction by Whatman FTA filter matrix technology and phenotypic tests for differentiation of Candida albicans and Candida dubliniensis.

PubMed

Kiraz, Nuri; Oz, Yasemin; Aslan, Huseyin; Muslumanoglu, Hamza

2014-02-01

Since C. dubliniensis is similar to C. albicans phenotypically, it can be misidentified as C. albicans. We aimed to investigate the prevalence of C. dubliniensis among isolates previously identified as C. albicans in our stocks and to compare the phenotypic methods and DNA sequencing of D1/D2 region on the ribosomal large subunit (rLSU) gene. A total of 850 isolates included in this study. Phenotypic identification was performed based on germ tube formation, chlamydospore production, colony colors on chromogenic agar, inability of growth at 45 °C and growth on hypertonic Sabouraud dextrose agar. Eighty isolates compatible with C. dubliniensis by at least one phenotypic test were included in the sequence analysis. Nested PCR amplification of D1/D2 region of the rLSU gene was performed after the fungal DNA extraction by Whatman FTA filter paper technology. The sequencing analysis of PCR products carried out by an automated capillary gel electrophoresis device. The rate of C. dubliniensis was 2.35 % (n = 20) among isolates previously described as C. albicans. Consequently, none of the phenotypic tests provided satisfactory performance alone in our study, and molecular methods required special equipment and high cost. Thus, at least two phenotypic methods can be used for identification of C. dubliniensis, and molecular methods can be used for confirmation.

SAM syndrome is characterized by extensive phenotypic heterogeneity.

PubMed

Taiber, Shahar; Samuelov, Liat; Mohamad, Janan; Cohen Barak, Eran; Sarig, Ofer; Shalev, Stavit Allon; Lestringant, Gilles; Sprecher, Eli

2018-03-31

Severe skin dermatitis, multiple allergies and metabolic wasting (SAM) syndrome is a rare life-threatening inherited condition caused by bi-allelic mutations in DSG1 encoding desmoglein 1. The disease was initially reported to manifest with severe erythroderma, failure to thrive, atopic manifestations, recurrent infections, hypotrichosis and palmoplantar keratoderma. We present 3 new cases of SAM syndrome in 2 families and review the cases published so far. Whole exome and direct sequencing were used to identify SAM syndrome-causing mutations. Consistent with previous data, SAM syndrome was found in all 3 patients to result from homozygous mutations in DSG1 predicted to result in premature termination of translation. In contrast, as compared with patients previously reported, the present cases were found to display a wide range of clinical presentations of variable degrees of severity. The present data emphasizes the fact that SAM syndrome is characterized by extensive phenotypic heterogeneity, suggesting the existence of potent modifier traits. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Severe vascular calcification and tumoral calcinosis in a family with hyperphosphatemia: a fibroblast growth factor 23 mutation identified by exome sequencing

PubMed Central

Shah, Anuja; Miller, Clinton J.; Nast, Cynthia C.; Adams, Mark D.; Truitt, Barbara; Tayek, John A.; Tong, Lili; Mehtani, Parag; Monteon, Francisco; Sedor, John R.; Clinkenbeard, Erica L.; White, Kenneth; Mehrotra, Rajnish; LaPage, Janine; Dickson, Patricia; Adler, Sharon G.; Iyengar, Sudha K.

2014-01-01

Background Tumoral calcinosis is an autosomal recessive disorder characterized by ectopic calcification and hyperphosphatemia. Methods We describe a family with tumoral calcinosis requiring amputations. The predominant metabolic anomaly identified in three affected family members was hyperphosphatemia. Biochemical and phenotypic analysis of 13 kindred members, together with exome analysis of 6 members, was performed. Results We identified a novel Q67K mutation in fibroblast growth factor 23 (FGF23), segregating with a null (deletion) allele on the other FGF23 homologue in three affected members. Affected siblings had high circulating plasma C-terminal FGF23 levels, but undetectable intact FGF23 or N-terminal FGF23, leading to loss of FGF23 function. Conclusions This suggests that in human, as in experimental models, severe prolonged hyperphosphatemia may be sufficient to produce bone differentiation proteins in vascular cells, and vascular calcification severe enough to require amputation. Genetic modifiers may contribute to the phenotypic variation within and between families. PMID:25378588

From genotype to phenotype; clinical variability in Lesch-Nyhan disease. The role of epigenetics.

PubMed

Trigueros Genao, M; Torres, R J

2014-11-01

Lesch-Nyhan disease is a rare genetic disease characterized by a deficiency in the function of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Patients affected by this disease experience hyperuricemia, motor disorders, mental retardation and, in the most severe cases, self-mutilation. Its clinical manifestations depend on the enzymatic activity of HGPRT, which is classically linked to the type of alteration in the HGPRT gene. More than 400 mutations of this gene have been found. At present, one of the controversial aspects of the disease is the relationship between the genotype and phenotype; cases have been described lacking a mutation, such as the patient presented in this article, as well as families who despite sharing the same genetic defect show disorders with differing severity. Epigenetic processes, which modify the genetic expression without changing the sequence of the deoxyribonucleic acid (DNA), could explain the clinical variability observed in this disease. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

Epigenomic alterations define lethal CIMP-positive ependymomas of infancy

PubMed Central

Mack, S. C.; Witt, H.; Piro, R. M.; Gu, L.; Zuyderduyn, S.; Stütz, A. M.; Wang, X.; Gallo, M.; Garzia, L.; Zayne, K.; Zhang, X.; Ramaswamy, V.; Jäger, N.; Jones, D. T. W.; Sill, M.; Pugh, T. J.; Ryzhova, M.; Wani, K. M.; Shih, D. J. H.; Head, R.; Remke, M.; Bailey, S. D.; Zichner, T.; Faria, C. C.; Barszczyk, M.; Stark, S.; Seker-Cin, H.; Hutter, S.; Johann, P.; Bender, S.; Hovestadt, V.; Tzaridis, T.; Dubuc, A. M.; Northcott, P. A.; Peacock, J.; Bertrand, K. C.; Agnihotri, S.; Cavalli, F. M. G.; Clarke, I.; Nethery-Brokx, K.; Creasy, C. L.; Verma, S. K.; Koster, J.; Wu, X.; Yao, Y.; Milde, T.; Sin-Chan, P.; Zuccaro, J.; Lau, L.; Pereira, S.; Castelo-Branco, P.; Hirst, M.; Marra, M. A.; Roberts, S. S.; Fults, D.; Massimi, L.; Cho, Y. J.; Van Meter, T.; Grajkowska, W.; Lach, B.; Kulozik, A. E.; von Deimling, A.; Witt, O.; Scherer, S. W.; Fan, X.; Muraszko, K. M.; Kool, M.; Pomeroy, S. L.; Gupta, N.; Phillips, J.; Huang, A.; Tabori, U.; Hawkins, C.; Malkin, D.; Kongkham, P. N.; Weiss, W. A.; Jabado, N.; Rutka, J. T.; Bouffet, E.; Korbel, J. O.; Lupien, M.; Aldape, K. D.; Bader, G. D.; Eils, R.; Lichter, P.; Dirks, P. B.; Pfister, S. M.; Korshunov, A.; Taylor, M. D.

2014-01-01

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland. PMID:24553142

Epigenomic alterations define lethal CIMP-positive ependymomas of infancy.

PubMed

Mack, S C; Witt, H; Piro, R M; Gu, L; Zuyderduyn, S; Stütz, A M; Wang, X; Gallo, M; Garzia, L; Zayne, K; Zhang, X; Ramaswamy, V; Jäger, N; Jones, D T W; Sill, M; Pugh, T J; Ryzhova, M; Wani, K M; Shih, D J H; Head, R; Remke, M; Bailey, S D; Zichner, T; Faria, C C; Barszczyk, M; Stark, S; Seker-Cin, H; Hutter, S; Johann, P; Bender, S; Hovestadt, V; Tzaridis, T; Dubuc, A M; Northcott, P A; Peacock, J; Bertrand, K C; Agnihotri, S; Cavalli, F M G; Clarke, I; Nethery-Brokx, K; Creasy, C L; Verma, S K; Koster, J; Wu, X; Yao, Y; Milde, T; Sin-Chan, P; Zuccaro, J; Lau, L; Pereira, S; Castelo-Branco, P; Hirst, M; Marra, M A; Roberts, S S; Fults, D; Massimi, L; Cho, Y J; Van Meter, T; Grajkowska, W; Lach, B; Kulozik, A E; von Deimling, A; Witt, O; Scherer, S W; Fan, X; Muraszko, K M; Kool, M; Pomeroy, S L; Gupta, N; Phillips, J; Huang, A; Tabori, U; Hawkins, C; Malkin, D; Kongkham, P N; Weiss, W A; Jabado, N; Rutka, J T; Bouffet, E; Korbel, J O; Lupien, M; Aldape, K D; Bader, G D; Eils, R; Lichter, P; Dirks, P B; Pfister, S M; Korshunov, A; Taylor, M D

2014-02-27

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.

Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells.

PubMed

Wu, Ting-Hsiang; Sagullo, Enrico; Case, Dana; Zheng, Xin; Li, Yanjing; Hong, Jason S; TeSlaa, Tara; Patananan, Alexander N; McCaffery, J Michael; Niazi, Kayvan; Braas, Daniel; Koehler, Carla M; Graeber, Thomas G; Chiou, Pei-Yu; Teitell, Michael A

2016-05-10

mtDNA sequence alterations are challenging to generate but desirable for basic studies and potential correction of mtDNA diseases. Here, we report a new method for transferring isolated mitochondria into somatic mammalian cells using a photothermal nanoblade, which bypasses endocytosis and cell fusion. The nanoblade rescued the pyrimidine auxotroph phenotype and respiration of ρ0 cells that lack mtDNA. Three stable isogenic nanoblade-rescued clones grown in uridine-free medium showed distinct bioenergetics profiles. Rescue lines 1 and 3 reestablished nucleus-encoded anapleurotic and catapleurotic enzyme gene expression patterns and had metabolite profiles similar to the parent cells from which the ρ0 recipient cells were derived. By contrast, rescue line 2 retained a ρ0 cell metabolic phenotype despite growth in uridine-free selection. The known influence of metabolite levels on cellular processes, including epigenome modifications and gene expression, suggests metabolite profiling can help assess the quality and function of mtDNA-modified cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of genetic background on the dystrophic phenotype in mdx mice

PubMed Central

Coley, William D.; Bogdanik, Laurent; Vila, Maria Candida; Yu, Qing; Van Der Meulen, Jack H.; Rayavarapu, Sree; Novak, James S.; Nearing, Marie; Quinn, James L.; Saunders, Allison; Dolan, Connor; Andrews, Whitney; Lammert, Catherine; Austin, Andrew; Partridge, Terence A.; Cox, Gregory A.; Lutz, Cathleen; Nagaraju, Kanneboyina

2016-01-01

Genetic background significantly affects phenotype in multiple mouse models of human diseases, including muscular dystrophy. This phenotypic variability is partly attributed to genetic modifiers that regulate the disease process. Studies have demonstrated that introduction of the γ-sarcoglycan-null allele onto the DBA/2J background confers a more severe muscular dystrophy phenotype than the original strain, demonstrating the presence of genetic modifier loci in the DBA/2J background. To characterize the phenotype of dystrophin deficiency on the DBA/2J background, we created and phenotyped DBA/2J-congenic Dmdmdx mice (D2-mdx) and compared them with the original, C57BL/10ScSn-Dmdmdx (B10-mdx) model. These strains were compared with their respective control strains at multiple time points between 6 and 52 weeks of age. Skeletal and cardiac muscle function, inflammation, regeneration, histology and biochemistry were characterized. We found that D2-mdx mice showed significantly reduced skeletal muscle function as early as 7 weeks and reduced cardiac function by 28 weeks, suggesting that the disease phenotype is more severe than in B10-mdx mice. In addition, D2-mdx mice showed fewer central myonuclei and increased calcifications in the skeletal muscle, heart and diaphragm at 7 weeks, suggesting that their pathology is different from the B10-mdx mice. The new D2-mdx model with an earlier onset and more pronounced dystrophy phenotype may be useful for evaluating therapies that target cardiac and skeletal muscle function in dystrophin-deficient mice. Our data align the D2-mdx with Duchenne muscular dystrophy patients with the LTBP4 genetic modifier, making it one of the few instances of cross-species genetic modifiers of monogenic traits. PMID:26566673

Molecular analysis of the XLRS1 gene in 4 females affected with X-linked juvenile retinoschisis.

PubMed

Saleheen, Danish; Ali, Azam; Khanum, Shaheen; Ozair, Mohammad Z; Zaidi, Moazzam; Sethi, Muhammad J; Khan, Nadir; Frossard, Philippe

2008-10-01

X-linked juvenile retinoschisis (XLRS) is the most common cause of juvenile macular degeneration in males. Because of its X-linked mode of transmission, the disease is rare in females. In this article, we describe a mutation screen conducted on a family in which 4 female patients affected with XLRS presented with an unusually severe phenotype. DNA was extracted from peripheral blood, and the XLRS1 gene was amplified on DNA samples of all the available family members. The mutation screen was conducted by performing direct DNA sequencing using an MJ Research PTC-225 Peltier Thermal Cycler. A novel mutation, 588-593ins.C, was identified in exon 6 of the gene. The affected father was found to be heterozygous for the mutation, whereas all the female patients were homozygous for this mutation. The homozygosity of the mutation in the affected females led to severe phenotypes. The defective allele was expressed in infancy in 1 patient, whereas the disease manifested itself at variable ages in the other patients, reflecting a variation in the phenotype. This report describes a novel mutation in a family in which consanguinity has led to XLRS in 4 females. A variation in the phenotype of the disease is consistent with the published literature and suggests the involvement of genetic modifiers or environmental factors in influencing the clinical severity of the disease.

Frequent phosphodiesterase 11A gene (PDE11A) defects in patients with Carney complex (CNC) caused by PRKAR1A mutations: PDE11A may contribute to adrenal and testicular tumors in CNC as a modifier of the phenotype.

PubMed

Libé, Rossella; Horvath, Anelia; Vezzosi, Delphine; Fratticci, Amato; Coste, Joel; Perlemoine, Karine; Ragazzon, Bruno; Guillaud-Bataille, Marine; Groussin, Lionel; Clauser, Eric; Raffin-Sanson, Marie-Laure; Siegel, Jennifer; Moran, Jason; Drori-Herishanu, Limor; Faucz, Fabio Rueda; Lodish, Maya; Nesterova, Maria; Bertagna, Xavier; Bertherat, Jerome; Stratakis, Constantine A

2011-01-01

Carney complex (CNC) is an autosomal dominant multiple neoplasia, caused mostly by inactivating mutations of the regulatory subunit 1A of the protein kinase A (PRKAR1A). Primary pigmented nodular adrenocortical disease (PPNAD) is the most frequent endocrine manifestation of CNC with a great inter-individual variability. Germline, protein-truncating mutations of phosphodiesterase type 11A (PDE11A) have been described to predispose to a variety of endocrine tumors, including adrenal and testicular tumors. Our objective was to investigate the role of PDE11A as a possible gene modifier of the phenotype in a series of 150 patients with CNC. A higher frequency of PDE11A variants in patients with CNC compared with healthy controls was found (25.3 vs. 6.8%, P < 0.0001). Among CNC patients, those with PPNAD were significantly more frequently carriers of PDE11A variants compared with patients without PPNAD (30.8 vs. 13%, P = 0.025). Furthermore, men with PPNAD were significantly more frequently carriers of PDE11A sequence variants (40.7%) than women with PPNAD (27.3%) (P < 0.001). A higher frequency of PDE11A sequence variants was also found in patients with large-cell calcifying Sertoli cell tumors (LCCSCT) compared with those without LCCSCT (50 vs. 10%, P = 0.0056). PDE11A variants were significantly associated with the copresence of PPNAD and LCCSCT in men: 81 vs. 20%, P < 0.004). The simultaneous inactivation of PRKAR1A and PDE11A by small inhibitory RNA led to an increase in cAMP-regulatory element-mediated transcriptional activity under basal conditions and after stimulation by forskolin. We demonstrate, in a large cohort of CNC patients, a high frequency of PDE11A variants, suggesting that PDE11A is a genetic modifying factor for the development of testicular and adrenal tumors in patients with germline PRKAR1A mutation.

Phenotype/genotype correlation in a case series of Stargardt's patients identifies novel mutations in the ABCA4 gene.

PubMed

Gemenetzi, M; Lotery, A J

2013-11-01

To investigate phenotypic variability in terms of best-corrected visual acuity (BCVA) in patients with Stargardt disease (STGD) and confirmed ABCA4 mutations. Entire coding region analysis of the ABCA4 gene by direct sequencing of seven patients with clinical findings of STGD seen in the Retina Clinics of Southampton Eye Unit between 2002 and 2011.Phenotypic variables recorded were BCVA, fluorescein angiographic appearance, electrophysiology, and visual fields. All patients had heterozygous amino acid-changing variants (missense mutations) in the ABCA4 gene. A splice sequence change was found in a 30-year-old patient with severly affected vision. Two novel sequence changes were identified: a missense mutation in a mildly affected 44-year-old patient and a frameshift mutation in a severly affected 34-year-old patient. The identified ABCA4 mutations were compatible with the resulting phenotypes in terms of BCVA. Higher BCVAs were recorded in patients with missense mutations. Sequence changes, predicted to have more deleterious effect on protein function, resulted in a more severe phenotype. This case series of STGD patients demonstrates novel genotype/phenotype correlations, which may be useful to counselling of patients. This information may prove useful in selection of candidates for clinical trials in ABCA4 disease.

Genome-wide ENU mutagenesis for the discovery of novel male fertility regulators.

PubMed

Jamsai, Duangporn; O'Bryan, Moira K

2010-06-01

The completion of genome sequencing projects has provided an extensive knowledge of the contents of the genomes of human, mouse, and many other organisms. Despite this, the function of most of the estimated 25,000 human genes remains largely unknown. Attention has now turned to elucidating gene function and identifying biological pathways that contribute to human diseases, including male infertility. Our understanding of the genetic regulation of male fertility has been accelerated through the use of genetically modified mouse models including knockout, knock-in, gene-trapped, and transgenic mice. Such reverse genetic approaches however, require some fore-knowledge of a gene's function and, as such, bias against the discovery of completely novel genes and biological pathways. To facilitate high throughput gene discovery, genome-wide mouse mutagenesis via the use of a potent chemical mutagen, N-ethyl-N-nitrosourea (ENU), has been developed over the past decade. This forward genetic, or phenotype-driven, approach relies upon observing a phenotype first, then subsequently defining the underlining genetic defect. Mutations are randomly introduced into the mouse genome via ENU exposure. Through a controlled breeding scheme, mutations causing a phenotype of interest (e.g., male infertility) are then identified by linkage analysis and candidate gene sequencing. This approach allows for the possibility of revealing comprehensive phenotype-genotype relationships for a range of genes and pathways i.e. in addition to null alleles, mice containing partial loss of function or gain-of-function mutations, can be recovered. Such point mutations are likely to be more reflective of those that occur within the human population. Many research groups have successfully used this approach to generate infertile mouse lines and some novel male fertility genes have been revealed. In this review, we focus on the utility of ENU mutagenesis for the discovery of novel male fertility regulators.

Phenotypic Changes in Transgenic Tobacco Plants Overexpressing Vacuole-Targeted Thermotoga maritima BglB Related to Elevated Levels of Liberated Hormones.

PubMed

Nguyen, Quynh Anh; Lee, Dae-Seok; Jung, Jakyun; Bae, Hyeun-Jong

2015-01-01

The hyperthermostable β-glucosidase BglB of Thermotoga maritima was modified by adding a short C-terminal tetrapeptide (AFVY, which transports phaseolin to the vacuole, to its C-terminal sequence). The modified β-glucosidase BglB was transformed into tobacco (Nicotiana tabacum L.) plants. We observed a range of significant phenotypic changes in the transgenic plants compared to the wild-type (WT) plants. The transgenic plants had faster stem growth, earlier flowering, enhanced root systems development, an increased biomass biosynthesis rate, and higher salt stress tolerance in young plants compared to WT. In addition, programed cell death was enhanced in mature plants. Furthermore, the C-terminal AFVY tetrapeptide efficiently sorted T. maritima BglB into the vacuole, which was maintained in an active form and could perform its glycoside hydrolysis function on hormone conjugates, leading to elevated hormone [abscisic acid (ABA), indole 3-acetic acid (IAA), and cytokinin] levels that likely contributed to the phenotypic changes in the transgenic plants. The elevation of cytokinin led to upregulation of the transcription factor WUSCHELL, a homeodomain factor that regulates the development, division, and reproduction of stem cells in the shoot apical meristems. Elevation of IAA led to enhanced root development, and the elevation of ABA contributed to enhanced tolerance to salt stress and programed cell death. These results suggest that overexpressing vacuole-targeted T. maritima BglB may have several advantages for molecular farming technology to improve multiple targets, including enhanced production of the β-glucosidase BglB, increased biomass, and shortened developmental stages, that could play pivotal roles in bioenergy and biofuel production.

Mutation Spectrum of the LRP5, NDP, and TSPAN12 Genes in Chinese Patients With Familial Exudative Vitreoretinopathy.

PubMed

Tang, Miao; Sun, Limei; Hu, Andina; Yuan, Miner; Yang, Yu; Peng, Xuening; Ding, Xiaoyan

2017-11-01

LRP5, NDP, and TSPAN12 are known to be associated with familial exudative vitreoretinopathy (FEVR). In this study, a comprehensive mutation screening for the three genes was performed in patients with a clinical diagnosis of FEVR in Han Chinese. Genomic DNA and clinical data were collected from 100 probands and their family members. Sanger sequencing was performed to screen for LRP5, NDP, and TSPAN12 mutations and phenotype-genotype correlation was analyzed. There were 23 causative mutations identified in 23 unrelated probands (10/23 in LRP5, 8/23 in TSPAN12, and 5/23 in NDP). Apart from NDP mutations, only two LRP5 mutations inherited in an autosomal recessive manner. Among the 23 causative mutations, 13 were novel variants (4/10 in LRP5, 6/8 in TSPAN12, and 3/5 in NDP). According to the modified classification system, statistical significance was observed in the distribution of mutated genes (P = 0.049). None of the causative mutations was found in group I FEVR. Probands with LRP5 or NDP mutations were mainly categorized into group III and IV, TSPAN12 mutations were mainly observed in probands with group IV and V FEVR. The detection rate for mutations in the three known genes was 23%. Mutations in LRP5 and TSPAN12 were more frequent, accounting for 10% and 8%, respectively. The NDP mutations were only identified in 6% in this cohort. There were 13 novel variants found, which provided a deeper understanding of this disease. Potential phenotype-genotype correlation was observed in the modified system. TSPAN12 mutations might lead to the most severe phenotype.

Phenotypic Changes in Transgenic Tobacco Plants Overexpressing Vacuole-Targeted Thermotoga maritima BglB Related to Elevated Levels of Liberated Hormones

PubMed Central

Nguyen, Quynh Anh; Lee, Dae-Seok; Jung, Jakyun; Bae, Hyeun-Jong

2015-01-01

The hyperthermostable β-glucosidase BglB of Thermotoga maritima was modified by adding a short C-terminal tetrapeptide (AFVY, which transports phaseolin to the vacuole, to its C-terminal sequence). The modified β-glucosidase BglB was transformed into tobacco (Nicotiana tabacum L.) plants. We observed a range of significant phenotypic changes in the transgenic plants compared to the wild-type (WT) plants. The transgenic plants had faster stem growth, earlier flowering, enhanced root systems development, an increased biomass biosynthesis rate, and higher salt stress tolerance in young plants compared to WT. In addition, programed cell death was enhanced in mature plants. Furthermore, the C-terminal AFVY tetrapeptide efficiently sorted T. maritima BglB into the vacuole, which was maintained in an active form and could perform its glycoside hydrolysis function on hormone conjugates, leading to elevated hormone [abscisic acid (ABA), indole 3-acetic acid (IAA), and cytokinin] levels that likely contributed to the phenotypic changes in the transgenic plants. The elevation of cytokinin led to upregulation of the transcription factor WUSCHELL, a homeodomain factor that regulates the development, division, and reproduction of stem cells in the shoot apical meristems. Elevation of IAA led to enhanced root development, and the elevation of ABA contributed to enhanced tolerance to salt stress and programed cell death. These results suggest that overexpressing vacuole-targeted T. maritima BglB may have several advantages for molecular farming technology to improve multiple targets, including enhanced production of the β-glucosidase BglB, increased biomass, and shortened developmental stages, that could play pivotal roles in bioenergy and biofuel production. PMID:26618153

Autozygome Sequencing Expands the Horizon of Human Knockout Research and Provides Novel Insights into Human Phenotypic Variation

PubMed Central

Anazi, Shamsa; Alshamekh, Shomoukh; Alkuraya, Fowzan S.

2013-01-01

The use of autozygosity as a mapping tool in the search for autosomal recessive disease genes is well established. We hypothesized that autozygosity not only unmasks the recessiveness of disease causing variants, but can also reveal natural knockouts of genes with less obvious phenotypic consequences. To test this hypothesis, we exome sequenced 77 well phenotyped individuals born to first cousin parents in search of genes that are biallelically inactivated. Using a very conservative estimate, we show that each of these individuals carries biallelic inactivation of 22.8 genes on average. For many of the 169 genes that appear to be biallelically inactivated, available data support involvement in modulating metabolism, immunity, perception, external appearance and other phenotypic aspects, and appear therefore to contribute to human phenotypic variation. Other genes with biallelic inactivation may contribute in yet unknown mechanisms or may be on their way to conversion into pseudogenes due to true recent dispensability. We conclude that sequencing the autozygome is an efficient way to map the contribution of genes to human phenotypic variation that goes beyond the classical definition of disease. PMID:24367280

G-STRATEGY: Optimal Selection of Individuals for Sequencing in Genetic Association Studies

PubMed Central

Wang, Miaoyan; Jakobsdottir, Johanna; Smith, Albert V.; McPeek, Mary Sara

2017-01-01

In a large-scale genetic association study, the number of phenotyped individuals available for sequencing may, in some cases, be greater than the study’s sequencing budget will allow. In that case, it can be important to prioritize individuals for sequencing in a way that optimizes power for association with the trait. Suppose a cohort of phenotyped individuals is available, with some subset of them possibly already sequenced, and one wants to choose an additional fixed-size subset of individuals to sequence in such a way that the power to detect association is maximized. When the phenotyped sample includes related individuals, power for association can be gained by including partial information, such as phenotype data of ungenotyped relatives, in the analysis, and this should be taken into account when assessing whom to sequence. We propose G-STRATEGY, which uses simulated annealing to choose a subset of individuals for sequencing that maximizes the expected power for association. In simulations, G-STRATEGY performs extremely well for a range of complex disease models and outperforms other strategies with, in many cases, relative power increases of 20–40% over the next best strategy, while maintaining correct type 1 error. G-STRATEGY is computationally feasible even for large datasets and complex pedigrees. We apply G-STRATEGY to data on HDL and LDL from the AGES-Reykjavik and REFINE-Reykjavik studies, in which G-STRATEGY is able to closely-approximate the power of sequencing the full sample by selecting for sequencing a only small subset of the individuals. PMID:27256766

Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution

PubMed Central

Cottinet, Denis; Condamine, Florence; Bremond, Nicolas; Griffiths, Andrew D.; Rainey, Paul B.; de Visser, J. Arjan G. M.; Baudry, Jean; Bibette, Jérôme

2016-01-01

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes–via growth–over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology. PMID:27077662

Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution.

PubMed

Cottinet, Denis; Condamine, Florence; Bremond, Nicolas; Griffiths, Andrew D; Rainey, Paul B; de Visser, J Arjan G M; Baudry, Jean; Bibette, Jérôme

2016-01-01

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes-via growth-over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology.

GATA2 null mutation associated with incomplete penetrance in a family with Emberger syndrome.

PubMed

Brambila-Tapia, Aniel Jessica Leticia; García-Ortiz, José Elías; Brouillard, Pascal; Nguyen, Ha-Long; Vikkula, Miikka; Ríos-González, Blanca Estela; Sandoval-Muñiz, Roberto de Jesús; Sandoval-Talamantes, Ana Karen; Bobadilla-Morales, Lucina; Corona-Rivera, Jorge Román; Arnaud-Lopez, Lisette

2017-09-01

GATA2 mutations are associated with several conditions, including Emberger syndrome which is the association of primary lymphedema with hematological anomalies and an increased risk for myelodysplasia and leukemia. To describe a family with Emberger syndrome with incomplete penetrance. A DNA sequencing of GATA2 gene was performed in the parents and offspring (five individuals in total). The family consisted of 5 individuals with a GATA2 null mutation (c.130G>T, p.Glu44*); three of them were affected (two of which were deceased) while two remained unaffected at the age of 40 and 13 years old. The three affected siblings (two boys and one girl) presented with lymphedema of the lower limbs, recurrent warts, epistaxis and recurrent infections. Two died due to hematological abnormalities (AML and pancytopenia). In contrast, the two other family members who carry the same mutation (the mother and one brother) have not presented any symptoms and their blood tests remain normal. Incomplete penetrance may indicate that GATA2 haploinsufficiency is not enough to produce the phenotype of Emberger syndrome. It could be useful to perform whole exome or genome sequencing, in cases where incomplete penetrance or high variable expressivity is described, in order to probably identify specific gene interactions that drastically modify the phenotype. In addition, skewed gene expression by an epigenetic mechanism of gene regulation should also be considered.

MTO1 mutations are associated with hypertrophic cardiomyopathy and lactic acidosis and cause respiratory chain deficiency in humans and yeast.

PubMed

Baruffini, Enrico; Dallabona, Cristina; Invernizzi, Federica; Yarham, John W; Melchionda, Laura; Blakely, Emma L; Lamantea, Eleonora; Donnini, Claudia; Santra, Saikat; Vijayaraghavan, Suresh; Roper, Helen P; Burlina, Alberto; Kopajtich, Robert; Walther, Anett; Strom, Tim M; Haack, Tobias B; Prokisch, Holger; Taylor, Robert W; Ferrero, Ileana; Zeviani, Massimo; Ghezzi, Daniele

2013-11-01

We report three families presenting with hypertrophic cardiomyopathy, lactic acidosis, and multiple defects of mitochondrial respiratory chain (MRC) activities. By direct sequencing of the candidate gene MTO1, encoding the mitochondrial-tRNA modifier 1, or whole exome sequencing analysis, we identified novel missense mutations. All MTO1 mutations were predicted to be deleterious on MTO1 function. Their pathogenic role was experimentally validated in a recombinant yeast model, by assessing oxidative growth, respiratory activity, mitochondrial protein synthesis, and complex IV activity. In one case, we also demonstrated that expression of wt MTO1 could rescue the respiratory defect in mutant fibroblasts. The severity of the yeast respiratory phenotypes partly correlated with the different clinical presentations observed in MTO1 mutant patients, although the clinical outcome was highly variable in patients with the same mutation and seemed also to depend on timely start of pharmacological treatment, centered on the control of lactic acidosis by dichloroacetate. Our results indicate that MTO1 mutations are commonly associated with a presentation of hypertrophic cardiomyopathy, lactic acidosis, and MRC deficiency, and that ad hoc recombinant yeast models represent a useful system to test the pathogenic potential of uncommon variants, and provide insight into their effects on the expression of a biochemical phenotype. © 2013 The Authors. *Human Mutation published by Wiley Periodicals, Inc.

Whole exome sequencing identifies novel genes for fetal hemoglobin response to hydroxyurea in children with sickle cell anemia.

PubMed

Sheehan, Vivien A; Crosby, Jacy R; Sabo, Aniko; Mortier, Nicole A; Howard, Thad A; Muzny, Donna M; Dugan-Perez, Shannon; Aygun, Banu; Nottage, Kerri A; Boerwinkle, Eric; Gibbs, Richard A; Ware, Russell E; Flanagan, Jonathan M

2014-01-01

Hydroxyurea has proven efficacy in children and adults with sickle cell anemia (SCA), but with considerable inter-individual variability in the amount of fetal hemoglobin (HbF) produced. Sibling and twin studies indicate that some of that drug response variation is heritable. To test the hypothesis that genetic modifiers influence pharmacological induction of HbF, we investigated phenotype-genotype associations using whole exome sequencing of children with SCA treated prospectively with hydroxyurea to maximum tolerated dose (MTD). We analyzed 171 unrelated patients enrolled in two prospective clinical trials, all treated with dose escalation to MTD. We examined two MTD drug response phenotypes: HbF (final %HbF minus baseline %HbF), and final %HbF. Analyzing individual genetic variants, we identified multiple low frequency and common variants associated with HbF induction by hydroxyurea. A validation cohort of 130 pediatric sickle cell patients treated to MTD with hydroxyurea was genotyped for 13 non-synonymous variants with the strongest association with HbF response to hydroxyurea in the discovery cohort. A coding variant in Spalt-like transcription factor, or SALL2, was associated with higher final HbF in this second independent replication sample and SALL2 represents an outstanding novel candidate gene for further investigation. These findings may help focus future functional studies and provide new insights into the pharmacological HbF upregulation by hydroxyurea in patients with SCA.

A Novel Lung Disease Phenotype Adjusted for Mortality Attrition for Cystic Fibrosis Genetic Modifier Studies

PubMed Central

Taylor, Chelsea; Commander, Clayton W.; Collaco, Joseph M.; Strug, Lisa J.; Li, Weili; Wright, Fred A.; Webel, Aaron D.; Pace, Rhonda G.; Stonebraker, Jaclyn R.; Naughton, Kathleen; Dorfman, Ruslan; Sandford, Andrew; Blackman, Scott M.; Berthiaume, Yves; Paré, Peter; Drumm, Mitchell L.; Zielenski, Julian; Durie, Peter; Cutting, Garry R.; Knowles, Michael R.; Corey, Mary

2011-01-01

SUMMARY Genetic studies of lung disease in Cystic Fibrosis are hampered by the lack of a severity measure that accounts for chronic disease progression and mortality attrition. Further, combining analyses across studies requires common phenotypes that are robust to study design and patient ascertainment. Using data from the North American Cystic Fibrosis Modifier Consortium (Canadian Consortium for CF Genetic Studies, Johns Hopkins University CF Twin and Sibling Study, and University of North Carolina/Case Western Reserve University Gene Modifier Study), the authors calculated age-specific CF percentile values of FEV1 which were adjusted for CF age-specific mortality data. The phenotype was computed for 2061 patients representing the Canadian CF population, 1137 extreme phenotype patients in the UNC/Case Western study, and 1323 patients from multiple CF sib families in the CF Twin and Sibling Study. Despite differences in ascertainment and median age, our phenotype score was distributed in all three samples in a manner consistent with ascertainment differences, reflecting the lung disease severity of each individual in the underlying population. The new phenotype score was highly correlated with the previously recommended complex phenotype, but the new phenotype is more robust for shorter follow-up and for extreme ages. A disease progression and mortality adjusted phenotype reduces the need for stratification or additional covariates, increasing statistical power and avoiding possible distortions. This approach will facilitate large scale genetic and environmental epidemiological studies which will provide targeted therapeutic pathways for the clinical benefit of patients with CF. PMID:21462361

IDEPI: rapid prediction of HIV-1 antibody epitopes and other phenotypic features from sequence data using a flexible machine learning platform.

PubMed

Hepler, N Lance; Scheffler, Konrad; Weaver, Steven; Murrell, Ben; Richman, Douglas D; Burton, Dennis R; Poignard, Pascal; Smith, Davey M; Kosakovsky Pond, Sergei L

2014-09-01

Since its identification in 1983, HIV-1 has been the focus of a research effort unprecedented in scope and difficulty, whose ultimate goals--a cure and a vaccine--remain elusive. One of the fundamental challenges in accomplishing these goals is the tremendous genetic variability of the virus, with some genes differing at as many as 40% of nucleotide positions among circulating strains. Because of this, the genetic bases of many viral phenotypes, most notably the susceptibility to neutralization by a particular antibody, are difficult to identify computationally. Drawing upon open-source general-purpose machine learning algorithms and libraries, we have developed a software package IDEPI (IDentify EPItopes) for learning genotype-to-phenotype predictive models from sequences with known phenotypes. IDEPI can apply learned models to classify sequences of unknown phenotypes, and also identify specific sequence features which contribute to a particular phenotype. We demonstrate that IDEPI achieves performance similar to or better than that of previously published approaches on four well-studied problems: finding the epitopes of broadly neutralizing antibodies (bNab), determining coreceptor tropism of the virus, identifying compartment-specific genetic signatures of the virus, and deducing drug-resistance associated mutations. The cross-platform Python source code (released under the GPL 3.0 license), documentation, issue tracking, and a pre-configured virtual machine for IDEPI can be found at https://github.com/veg/idepi.

Lights, camera, action: high-throughput plant phenotyping is ready for a close-up

USDA-ARS?s Scientific Manuscript database

Modern techniques for crop improvement rely on both DNA sequencing and accurate quantification of plant traits to identify genes and germplasm of interest. With rapid advances in DNA sequencing technologies, plant phenotyping is now a bottleneck in advancing crop yields [1,2]. Furthermore, the envir...

Ectopic expression of the Drosophila Cdk1 inhibitory kinases, Wee1 and Myt1, interferes with the second mitotic wave and disrupts pattern formation during eye development.

PubMed Central

Price, Donald M; Jin, Zhigang; Rabinovitch, Simon; Campbell, Shelagh D

2002-01-01

Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it in response to DNA damage or developmental signals during G2. Unlike yeast, metazoans have two distinct Wee1-like kinases, a nuclear protein (Wee1) and a cytoplasmic protein (Myt1). We have isolated the genes encoding Drosophila Wee1 and Myt1 and are using genetic approaches to dissect their functions during normal development. Overexpression of Dwee1 or Dmyt1 during eye development generates a rough adult eye phenotype. The phenotype can be modified by altering the gene dosage of known regulators of the G2/M transition, suggesting that we could use these transgenic strains in modifier screens to identify potential regulators of Wee1 and Myt1. To confirm this idea, we tested a collection of deletions for loci that can modify the eye overexpression phenotypes and identified several loci as dominant modifiers. Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting that Myt1 is potentially a downstream target for Notch activity during eye development. We also observed interactions with p53, which suggest that Wee1 and Myt1 activity can block apoptosis. PMID:12072468

High frequency of C9orf72 hexanucleotide repeat expansion in amyotrophic lateral sclerosis patients from two founder populations sharing the same risk haplotype.

PubMed

Goldstein, Orly; Gana-Weisz, Mali; Nefussy, Beatrice; Vainer, Batel; Nayshool, Omri; Bar-Shira, Anat; Traynor, Bryan J; Drory, Vivian E; Orr-Urtreger, Avi

2018-04-01

We characterized the C9orf72 hexanucleotide repeat expansion (RE) mutation in amyotrophic lateral sclerosis (ALS) patients of 2 distinct origins, Ashkenazi and North Africa Jews (AJ, NAJ), its frequency, and genotype-phenotype correlations. In AJ, 80% of familial ALS (fALS) and 11% of sporadic ALS carried the RE, a total of 12.9% of all AJ-ALS compared to 0.3% in AJ controls (odds ratio [OR] = 44.3, p < 0.0001). In NAJ, 10% of fALS and 9% of sporadic ALS carried the RE, a total of 9.1% of all NAJ-ALS compared to 1% in controls (OR = 9.9, p = 0.0006). We identified a risk haplotype shared among all ALS patients, although an association with age at disease onset, fALS, and dementia were observed only in AJ. Variations were identified downstream the repeats. The risk haplotype and these polymorphisms were at high frequencies in alleles with 8 repeats or more, suggesting sequence instability. The different genotype-phenotype correlations and OR, together with the large range in age at onset, suggest that other modifiers and risk factors may affect penetrance and phenotype in ALS. Copyright © 2017 Elsevier Inc. All rights reserved.

Computational evaluation of exome sequence data using human and model organism phenotypes improves diagnostic efficiency

PubMed Central

Bone, William P.; Washington, Nicole L.; Buske, Orion J.; Adams, David R.; Davis, Joie; Draper, David; Flynn, Elise D.; Girdea, Marta; Godfrey, Rena; Golas, Gretchen; Groden, Catherine; Jacobsen, Julius; Köhler, Sebastian; Lee, Elizabeth M. J.; Links, Amanda E.; Markello, Thomas C.; Mungall, Christopher J.; Nehrebecky, Michele; Robinson, Peter N.; Sincan, Murat; Soldatos, Ariane G.; Tifft, Cynthia J.; Toro, Camilo; Trang, Heather; Valkanas, Elise; Vasilevsky, Nicole; Wahl, Colleen; Wolfe, Lynne A.; Boerkoel, Cornelius F.; Brudno, Michael; Haendel, Melissa A.; Gahl, William A.; Smedley, Damian

2016-01-01

Purpose: Medical diagnosis and molecular or biochemical confirmation typically rely on the knowledge of the clinician. Although this is very difficult in extremely rare diseases, we hypothesized that the recording of patient phenotypes in Human Phenotype Ontology (HPO) terms and computationally ranking putative disease-associated sequence variants improves diagnosis, particularly for patients with atypical clinical profiles. Genet Med 18 6, 608–617. Methods: Using simulated exomes and the National Institutes of Health Undiagnosed Diseases Program (UDP) patient cohort and associated exome sequence, we tested our hypothesis using Exomiser. Exomiser ranks candidate variants based on patient phenotype similarity to (i) known disease–gene phenotypes, (ii) model organism phenotypes of candidate orthologs, and (iii) phenotypes of protein–protein association neighbors. Genet Med 18 6, 608–617. Results: Benchmarking showed Exomiser ranked the causal variant as the top hit in 97% of known disease–gene associations and ranked the correct seeded variant in up to 87% when detectable disease–gene associations were unavailable. Using UDP data, Exomiser ranked the causative variant(s) within the top 10 variants for 11 previously diagnosed variants and achieved a diagnosis for 4 of 23 cases undiagnosed by clinical evaluation. Genet Med 18 6, 608–617. Conclusion: Structured phenotyping of patients and computational analysis are effective adjuncts for diagnosing patients with genetic disorders. Genet Med 18 6, 608–617. PMID:26562225

A Murine Hypertrophic Cardiomyopathy Model: The DBA/2J Strain.

PubMed

Zhao, Wenyuan; Zhao, Tieqiang; Chen, Yuanjian; Zhao, Fengbo; Gu, Qingqing; Williams, Robert W; Bhattacharya, Syamal K; Lu, Lu; Sun, Yao

2015-01-01

Familial hypertrophic cardiomyopathy (HCM) is attributed to mutations in genes that encode for the sarcomere proteins, especially Mybpc3 and Myh7. Genotype-phenotype correlation studies show significant variability in HCM phenotypes among affected individuals with identical causal mutations. Morphological changes and clinical expression of HCM are the result of interactions with modifier genes. With the exceptions of angiotensin converting enzyme, these modifiers have not been identified. Although mouse models have been used to investigate the genetics of many complex diseases, natural murine models for HCM are still lacking. In this study we show that the DBA/2J (D2) strain of mouse has sequence variants in Mybpc3 and Myh7, relative to widely used C57BL/6J (B6) reference strain and the key features of human HCM. Four-month-old of male D2 mice exhibit hallmarks of HCM including increased heart weight and cardiomyocyte size relative to B6 mice, as well as elevated markers for cardiac hypertrophy including β-myosin heavy chain (MHC), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and skeletal muscle alpha actin (α1-actin). Furthermore, cardiac interstitial fibrosis, another feature of HCM, is also evident in the D2 strain, and is accompanied by up-regulation of type I collagen and α-smooth muscle actin (SMA)-markers of fibrosis. Of great interest, blood pressure and cardiac function are within the normal range in the D2 strain, demonstrating that cardiac hypertrophy and fibrosis are not secondary to hypertension, myocardial infarction, or heart failure. Because D2 and B6 strains have been used to generate a large family of recombinant inbred strains, the BXD cohort, the D2 model can be effectively exploited for in-depth genetic analysis of HCM susceptibility and modifier screens.

The food and environmental safety of Bt crops.

PubMed

Koch, Michael S; Ward, Jason M; Levine, Steven L; Baum, James A; Vicini, John L; Hammond, Bruce G

2015-01-01

Bacillus thuringiensis (Bt) microbial pesticides have a 50-year history of safety in agriculture. Cry proteins are among the active insecticidal ingredients in these pesticides, and genes coding for Cry proteins have been introduced into agricultural crops using modern biotechnology. The Cry gene sequences are often modified to enable effective expression in planta and several Cry proteins have been modified to increase biological activity against the target pest(s). Additionally, the domains of different but structurally conserved Cry proteins can be combined to produce chimeric proteins with enhanced insecticidal properties. Environmental studies are performed and include invertebrates, mammals, and avian species. Mammalian studies used to support the food and feed safety assessment are also used to support the wild mammal assessment. In addition to the NTO assessment, the environmental assessment includes a comparative assessment between the Bt crop and the appropriate conventional control that is genetically similar but lacks the introduced trait to address unintended effects. Specific phenotypic, agronomic, and ecological characteristics are measured in the Bt crop and the conventional control to evaluate whether the introduction of the insect resistance has resulted in any changes that might cause ecological harm in terms of altered weed characteristics, susceptibility to pests, or adverse environmental impact. Additionally, environmental interaction data are collected in field experiments for Bt crop to evaluate potential adverse effects. Further to the agronomic and phenotypic evaluation, potential movement of transgenes from a genetically modified crop plants into wild relatives is assessed for a new pest resistance gene in a new crop. This review summarizes the evidence for safety of crops containing Cry proteins for humans, livestock, and other non-target organisms.

The food and environmental safety of Bt crops

PubMed Central

Koch, Michael S.; Ward, Jason M.; Levine, Steven L.; Baum, James A.; Vicini, John L.; Hammond, Bruce G.

2015-01-01

Bacillus thuringiensis (Bt) microbial pesticides have a 50-year history of safety in agriculture. Cry proteins are among the active insecticidal ingredients in these pesticides, and genes coding for Cry proteins have been introduced into agricultural crops using modern biotechnology. The Cry gene sequences are often modified to enable effective expression in planta and several Cry proteins have been modified to increase biological activity against the target pest(s). Additionally, the domains of different but structurally conserved Cry proteins can be combined to produce chimeric proteins with enhanced insecticidal properties. Environmental studies are performed and include invertebrates, mammals, and avian species. Mammalian studies used to support the food and feed safety assessment are also used to support the wild mammal assessment. In addition to the NTO assessment, the environmental assessment includes a comparative assessment between the Bt crop and the appropriate conventional control that is genetically similar but lacks the introduced trait to address unintended effects. Specific phenotypic, agronomic, and ecological characteristics are measured in the Bt crop and the conventional control to evaluate whether the introduction of the insect resistance has resulted in any changes that might cause ecological harm in terms of altered weed characteristics, susceptibility to pests, or adverse environmental impact. Additionally, environmental interaction data are collected in field experiments for Bt crop to evaluate potential adverse effects. Further to the agronomic and phenotypic evaluation, potential movement of transgenes from a genetically modified crop plants into wild relatives is assessed for a new pest resistance gene in a new crop. This review summarizes the evidence for safety of crops containing Cry proteins for humans, livestock, and other non-target organisms. PMID:25972882

Determining which phenotypes underlie a pleiotropic signal

PubMed Central

Majumdar, Arunabha; Haldar, Tanushree; Witte, John S.

2016-01-01

Discovering pleiotropic loci is important to understand the biological basis of seemingly distinct phenotypes. Most methods for assessing pleiotropy only test for the overall association between genetic variants and multiple phenotypes. To determine which specific traits are pleiotropic, we evaluate via simulation and application three different strategies. The first is model selection techniques based on the inverse regression of genotype on phenotypes. The second is a subset-based meta-analysis ASSET [Bhattacharjee et al., 2012], which provides an optimal subset of non-null traits. And the third is a modified Benjamini-Hochberg (B-H) procedure of controlling the expected false discovery rate [Benjamini and Hochberg, 1995] in the framework of phenome-wide association study. From our simulations we see that an inverse regression based approach MultiPhen [O’Reilly et al., 2012] is more powerful than ASSET for detecting overall pleiotropic association, except for when all the phenotypes are associated and have genetic effects in the same direction. For determining which specific traits are pleiotropic, the modified B-H procedure performs consistently better than the other two methods. The inverse regression based selection methods perform competitively with the modified B-H procedure only when the phenotypes are weakly correlated. The efficiency of ASSET is observed to lie below and in between the efficiency of the other two methods when the traits are weakly and strongly correlated, respectively. In our application to a large GWAS, we find that the modified B-H procedure also performs well, indicating that this may be an optimal approach for determining the traits underlying a pleiotropic signal. PMID:27238845

Joubert syndrome: A model for untangling recessive disorders with extreme genetic heterogeneity

PubMed Central

R, Bachmann-Gagescu; JC, Dempsey; IG, Phelps; BJ, O’Roak; DM, Knutzen; TC, Rue; GE, Ishak; CR, Isabella; N, Gorden; J, Adkins; EA, Boyle; N, de Lacy; D, O’Day; A, Alswaid; AR, Devi; L, Lingappa; C, Lourenço; L, Martorell; À, Garcia-Cazorla; H, Ozyürek; G, Haliloğlu; B, Tuysuz; M, Topçu; P, Chance; MA, Parisi; I, Glass; J, Shendure; D, Doherty

2016-01-01

Background Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by hypotonia, ataxia, cognitive impairment, abnormal eye movements, respiratory control disturbances, and a distinctive mid-hindbrain malformation. JS demonstrates substantial phenotypic variability and genetic heterogeneity. This study provides a comprehensive view of the current genetic basis, phenotypic range and gene-phenotype associations in JS. Methods We sequenced 27 JS-associated genes in 440 affected individuals (375 families) from a cohort of 532 individuals (440 families) with JS, using molecular inversion probe-based targeted capture and next generation sequencing. Variant pathogenicity was defined using the Combined Annotation Dependent Depletion (CADD) algorithm with an optimized score cut-off. Results We identified presumed causal variants in 62% of pedigrees, including the first B9D2 mutations associated with JS. 253 different mutations in 23 genes highlight the extreme genetic heterogeneity of JS. Phenotypic analysis revealed that only 34% of individuals have a “pure JS” phenotype. Retinal disease is present in 30% of individuals, renal disease in 25%, coloboma in 17%, polydactyly in 15%, liver fibrosis in 14% and encephalocele in 8%. Loss of CEP290 function is associated with retinal dystrophy, while loss of TMEM67 function is associated with liver fibrosis and coloboma, but we observe no clear-cut distinction between JS-subtypes. Conclusion This work illustrates how combining advanced sequencing techniques with phenotypic data addresses extreme genetic heterogeneity to provide diagnostic and carrier testing, guide medical monitoring for progressive complications, facilitate interpretation of genome-wide sequencing results in individuals with a variety of phenotypes, and enable gene-specific treatments in the future. PMID:26092869

Phenotypic detection of broad-spectrum beta-lactamases in microbiological practice.

PubMed

Htoutou Sedlakova, Miroslava; Hanulik, Vojtech; Chroma, Magdalena; Hricova, Kristyna; Kolar, Milan; Latal, Tomas; Schaumann, Reiner; Rodloff, Arne C

2011-05-01

Enterobacteriaceae producing ESBL and AmpC enzymes can be associated with failure of antibiotic therapy and related morbidity and mortality. Their routine detection in microbiology laboratories is still a problem. The aim of this study was to compare the sensitivity of selected phenotypic methods. A total of 106 strains of the Enterobacteriaceae family were tested, in which molecular biology methods confirmed the presence of genes encoding ESBL or AmpC. In ESBL-positive strains, the sensitivity of the ESBL Etest (AB Biodisk) and a modified double-disk synergy test (DDST) were evaluated. AmpC strains were tested by a modified AmpC disk method using 3-aminophenylboronic acid. For simultaneous detection of ESBL and AmpC, the microdilution method with a modified set of antimicrobial agents was used. The sensitivity of the ESBL Etest was 95%; the modified DDST yielded 100% sensitivity for ESBL producers and the AmpC test correctly detected 95% of AmpC-positive strains. The sensitivity of the modified microdilution method was 87% and 95% for ESBL and AmpC beta lactamases, respectively. The detection of ESBL and AmpC beta lactamases should be based on specific phenotypic methods such as the modified DDST, ESBL Etest, AmpC disk test and the modified microdilution method.

Phenotypic detection of broad-spectrum beta-lactamases in microbiological practice

PubMed Central

Sedlakova, Miroslava Htoutou; Hanulik, Vojtech; Chroma, Magdalena; Hricova, Kristyna; Kolar, Milan; Latal, Tomas; Schaumann, Reiner; Rodloff, Arne C.

2011-01-01

Summary Background Enterobacteriaceae producing ESBL and AmpC enzymes can be associated with failure of antibiotic therapy and related morbidity and mortality. Their routine detection in microbiology laboratories is still a problem. The aim of this study was to compare the sensitivity of selected phenotypic methods. Material/Methods A total of 106 strains of the Enterobacteriaceae family were tested, in which molecular biology methods confirmed the presence of genes encoding ESBL or AmpC. In ESBL-positive strains, the sensitivity of the ESBL Etest (AB Biodisk) and a modified double-disk synergy test (DDST) were evaluated. AmpC strains were tested by a modified AmpC disk method using 3-aminophenylboronic acid. For simultaneous detection of ESBL and AmpC, the microdilution method with a modified set of antimicrobial agents was used. Results The sensitivity of the ESBL Etest was 95%; the modified DDST yielded 100% sensitivity for ESBL producers and the AmpC test correctly detected 95% of AmpC-positive strains. The sensitivity of the modified microdilution method was 87% and 95% for ESBL and AmpC beta lactamases, respectively. Conclusions The detection of ESBL and AmpC beta lactamases should be based on specific phenotypic methods such as the modified DDST, ESBL Etest, AmpC disk test and the modified microdilution method. PMID:21525803

Phenotype-information-phenotype cycle for deconvolution of combinatorial antibody libraries selected against complex systems.

PubMed

Zhang, Hongkai; Torkamani, Ali; Jones, Teresa M; Ruiz, Diana I; Pons, Jaume; Lerner, Richard A

2011-08-16

Use of large combinatorial antibody libraries and next-generation sequencing of nucleic acids are two of the most powerful methods in modern molecular biology. The libraries are screened using the principles of evolutionary selection, albeit in real time, to enrich for members with a particular phenotype. This selective process necessarily results in the loss of information about less-fit molecules. On the other hand, sequencing of the library, by itself, gives information that is mostly unrelated to phenotype. If the two methods could be combined, the full potential of very large molecular libraries could be realized. Here we report the implementation of a phenotype-information-phenotype cycle that integrates information and gene recovery. After selection for phage-encoded antibodies that bind to targets expressed on the surface of Escherichia coli, the information content of the selected pool is obtained by pyrosequencing. Sequences that encode specific antibodies are identified by a bioinformatic analysis and recovered by a stringent affinity method that is uniquely suited for gene isolation from a highly degenerate collection of nucleic acids. This approach can be generalized for selection of antibodies against targets that are present as minor components of complex systems.

Draft Genome Sequences of Phenotypically Distinct Janthinobacterium sp. Isolates Cultured from the Hudson Valley Watershed

PubMed Central

Bettina, Alexandra M.; Doing, Georgia; O’Brien, Kelsey

2018-01-01

ABSTRACT Investigation of the Hudson Valley watershed reveals many violacein-producing bacteria. These are of interest for their biotherapeutic potential in treating chytrid infections of amphibians. The draft whole-genome sequences for seven Janthinobacterium isolates with a variety of phenotypes are provided in this study. PMID:29348334

Integrated rare variant-based risk gene prioritization in disease case-control sequencing studies.

PubMed

Lin, Jhih-Rong; Zhang, Quanwei; Cai, Ying; Morrow, Bernice E; Zhang, Zhengdong D

2017-12-01

Rare variants of major effect play an important role in human complex diseases and can be discovered by sequencing-based genome-wide association studies. Here, we introduce an integrated approach that combines the rare variant association test with gene network and phenotype information to identify risk genes implicated by rare variants for human complex diseases. Our data integration method follows a 'discovery-driven' strategy without relying on prior knowledge about the disease and thus maintains the unbiased character of genome-wide association studies. Simulations reveal that our method can outperform a widely-used rare variant association test method by 2 to 3 times. In a case study of a small disease cohort, we uncovered putative risk genes and the corresponding rare variants that may act as genetic modifiers of congenital heart disease in 22q11.2 deletion syndrome patients. These variants were missed by a conventional approach that relied on the rare variant association test alone.

MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

PubMed Central

Venken, Koen J. T.; Schulze, Karen L.; Haelterman, Nele A.; Pan, Hongling; He, Yuchun; Evans-Holm, Martha; Carlson, Joseph W.; Levis, Robert W.; Spradling, Allan C.; Hoskins, Roger A.; Bellen, Hugo J.

2011-01-01

We demonstrate the versatility of a collection of insertions of the transposon Minos mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 attP sites. MiMIC integrates almost at random in the genome to create sites for DNA manipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp system. Insertions within coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the Drosophila melanogaster toolkit. PMID:21985007

Relationships between functional genes in Lactobacillus delbrueckii ssp. bulgaricus isolates and phenotypic characteristics associated with fermentation time and flavor production in yogurt elucidated using multilocus sequence typing.

PubMed

Liu, Wenjun; Yu, Jie; Sun, Zhihong; Song, Yuqin; Wang, Xueni; Wang, Hongmei; Wuren, Tuoya; Zha, Musu; Menghe, Bilige; Heping, Zhang

2016-01-01

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is well known for its worldwide application in yogurt production. Flavor production and acid producing are considered as the most important characteristics for starter culture screening. To our knowledge this is the first study applying functional gene sequence multilocus sequence typing technology to predict the fermentation and flavor-producing characteristics of yogurt-producing bacteria. In the present study, phenotypic characteristics of 35 L. bulgaricus strains were quantified during the fermentation of milk to yogurt and during its subsequent storage; these included fermentation time, acidification rate, pH, titratable acidity, and flavor characteristics (acetaldehyde concentration). Furthermore, multilocus sequence typing analysis of 7 functional genes associated with fermentation time, acid production, and flavor formation was done to elucidate the phylogeny and genetic evolution of the same L. bulgaricus isolates. The results showed that strains significantly differed in fermentation time, acidification rate, and acetaldehyde production. Combining functional gene sequence analysis with phenotypic characteristics demonstrated that groups of strains established using genotype data were consistent with groups identified based on their phenotypic traits. This study has established an efficient and rapid molecular genotyping method to identify strains with good fermentation traits; this has the potential to replace time-consuming conventional methods based on direct measurement of phenotypic traits. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A modifier of Huntington's disease onset at the MLH1 locus.

PubMed

Lee, Jong-Min; Chao, Michael J; Harold, Denise; Abu Elneel, Kawther; Gillis, Tammy; Holmans, Peter; Jones, Lesley; Orth, Michael; Myers, Richard H; Kwak, Seung; Wheeler, Vanessa C; MacDonald, Marcy E; Gusella, James F

2017-10-01

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expanded CAG repeat in HTT. Many clinical characteristics of HD such as age at motor onset are determined largely by the size of HTT CAG repeat. However, emerging evidence strongly supports a role for other genetic factors in modifying the disease pathogenesis driven by mutant huntingtin. A recent genome-wide association analysis to discover genetic modifiers of HD onset age provided initial evidence for modifier loci on chromosomes 8 and 15 and suggestive evidence for a locus on chromosome 3. Here, genotyping of candidate single nucleotide polymorphisms in a cohort of 3,314 additional HD subjects yields independent confirmation of the former two loci and moves the third to genome-wide significance at MLH1, a locus whose mouse orthologue modifies CAG length-dependent phenotypes in a Htt-knock-in mouse model of HD. Both quantitative and dichotomous association analyses implicate a functional variant on ∼32% of chromosomes with the beneficial modifier effect that delays HD motor onset by 0.7 years/allele. Genomic DNA capture and sequencing of a modifier haplotype localize the functional variation to a 78 kb region spanning the 3'end of MLH1 and the 5'end of the neighboring LRRFIP2, and marked by an isoleucine-valine missense variant in MLH1. Analysis of expression Quantitative Trait Loci (eQTLs) provides modest support for altered regulation of MLH1 and LRRFIP2, raising the possibility that the modifier affects regulation of both genes. Finally, polygenic modification score and heritability analyses suggest the existence of additional genetic modifiers, supporting expanded, comprehensive genetic analysis of larger HD datasets. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Revisiting Robustness and Evolvability: Evolution in Weighted Genotype Spaces

PubMed Central

Partha, Raghavendran; Raman, Karthik

2014-01-01

Robustness and evolvability are highly intertwined properties of biological systems. The relationship between these properties determines how biological systems are able to withstand mutations and show variation in response to them. Computational studies have explored the relationship between these two properties using neutral networks of RNA sequences (genotype) and their secondary structures (phenotype) as a model system. However, these studies have assumed every mutation to a sequence to be equally likely; the differences in the likelihood of the occurrence of various mutations, and the consequence of probabilistic nature of the mutations in such a system have previously been ignored. Associating probabilities to mutations essentially results in the weighting of genotype space. We here perform a comparative analysis of weighted and unweighted neutral networks of RNA sequences, and subsequently explore the relationship between robustness and evolvability. We show that assuming an equal likelihood for all mutations (as in an unweighted network), underestimates robustness and overestimates evolvability of a system. In spite of discarding this assumption, we observe that a negative correlation between sequence (genotype) robustness and sequence evolvability persists, and also that structure (phenotype) robustness promotes structure evolvability, as observed in earlier studies using unweighted networks. We also study the effects of base composition bias on robustness and evolvability. Particularly, we explore the association between robustness and evolvability in a sequence space that is AU-rich – sequences with an AU content of 80% or higher, compared to a normal (unbiased) sequence space. We find that evolvability of both sequences and structures in an AU-rich space is lesser compared to the normal space, and robustness higher. We also observe that AU-rich populations evolving on neutral networks of phenotypes, can access less phenotypic variation compared to normal populations evolving on neutral networks. PMID:25390641

A versatile phenotyping system and analytics platform reveals diverse temporal responses to water availability in Setaria

USDA-ARS?s Scientific Manuscript database

With rapid advances in DNA sequencing, phenotyping has become the rate-limiting step in using large-scale genomic data to understand and improve agricultural crops. Here, the Bellwether Phenotyping platform for controlled-environment plant growth and automated, multimodal phenotyping is described. T...

Network Analysis of Sequence-Function Relationships and Exploration of Sequence Space of TEM β-Lactamases.

PubMed

Zeil, Catharina; Widmann, Michael; Fademrecht, Silvia; Vogel, Constantin; Pleiss, Jürgen

2016-05-01

The Lactamase Engineering Database (www.LacED.uni-stuttgart.de) was developed to facilitate the classification and analysis of TEM β-lactamases. The current version contains 474 TEM variants. Two hundred fifty-nine variants form a large scale-free network of highly connected point mutants. The network was divided into three subnetworks which were enriched by single phenotypes: one network with predominantly 2be and two networks with 2br phenotypes. Fifteen positions were found to be highly variable, contributing to the majority of the observed variants. Since it is expected that a considerable fraction of the theoretical sequence space is functional, the currently sequenced 474 variants represent only the tip of the iceberg of functional TEM β-lactamase variants which form a huge natural reservoir of highly interconnected variants. Almost 50% of the variants are part of a quartet. Thus, two single mutations that result in functional enzymes can be combined into a functional protein. Most of these quartets consist of the same phenotype, or the mutations are additive with respect to the phenotype. By predicting quartets from triplets, 3,916 unknown variants were constructed. Eighty-seven variants complement multiple quartets and therefore have a high probability of being functional. The construction of a TEM β-lactamase network and subsequent analyses by clustering and quartet prediction are valuable tools to gain new insights into the viable sequence space of TEM β-lactamases and to predict their phenotype. The highly connected sequence space of TEM β-lactamases is ideally suited to network analysis and demonstrates the strengths of network analysis over tree reconstruction methods. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A High Proportion of Chromosome 21 Promoter Polymorphisms Influence Transcriptional Activity

PubMed Central

Buckland, Paul R.; Coleman, Sharol L.; Hoogendoorn, Bastiaan; Guy, Carol; Smith, S. Kaye; O’Donovan, Michael C.

2004-01-01

We have sought to obtain an unbiased estimate of the proportion of polymorphisms in promoters of human genes that have functional effects. We carried out polymorphism discovery on a randomly selected group of 51 gene promoters mapping to human chromosome 21 and successfully analyzed the effect on transcription of 38 of the sequence variants. To achieve this, a total of 53 different haplotypes from 20 promoters were cloned into a modified pGL3 luciferase reporter gene vector and were tested for their abilities to promote transcription in HEK293t and JEG-3 cells. Up to seven (18%) of the 38 tested variants altered transcription by 1.5-fold, confirming that a surprisingly high proportion of promoter region polymorphisms are likely to be functionally important. The functional variants were distributed across the promoters of CRYAA, IFNAR1, KCNJ15, NCAM2, IGSF5, and B3GALT5. Three of the genes (NCAM2, IFNAR1, and CRYAA) have been previously associated with human phenotypes and the polymorphisms we describe here may therefore play a role in those phenotypes. PMID:15200235

Nucleotide sequence analysis of the recA gene and discrimination of the three isolates of urease-positive thermophilic Campylobacter (UPTC) isolated from seagulls (Larus spp.) in Northern Ireland.

PubMed

Matsuda, M; Tai, K; Moore, J E; Millar, B C; Murayama, O

2004-01-01

Nucleotide sequencing after TA cloning of the amplicon of the almost-full length recA gene from three strains of UPTC (A1, A2, and A3) isolated from seagulls in Northern Ireland, the phenotypical and genotypical characteristics of which have been demonstrated to be indistinguishable, clarified nucleotide differences at three nucleotide positions among the three strains. In conclusion, the nucleotide sequences of the recA gene were found to discriminate among the three strains of UPTC, A1, A2, and A3, which are indistinguishable phenotypically and genotypically. Thus, the present study strongly suggests that nucleotide sequence data of the amplicon of a suitable gene or region could aid in discriminating among isolates of the UPTC group, which are indistinguishable phenotypically and genotypically. Copyright 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

RoboOligo: software for mass spectrometry data to support manual and de novo sequencing of post-transcriptionally modified ribonucleic acids

PubMed Central

Sample, Paul J.; Gaston, Kirk W.; Alfonzo, Juan D.; Limbach, Patrick A.

2015-01-01

Ribosomal ribonucleic acid (RNA), transfer RNA and other biological or synthetic RNA polymers can contain nucleotides that have been modified by the addition of chemical groups. Traditional Sanger sequencing methods cannot establish the chemical nature and sequence of these modified-nucleotide containing oligomers. Mass spectrometry (MS) has become the conventional approach for determining the nucleotide composition, modification status and sequence of modified RNAs. Modified RNAs are analyzed by MS using collision-induced dissociation tandem mass spectrometry (CID MS/MS), which produces a complex dataset of oligomeric fragments that must be interpreted to identify and place modified nucleosides within the RNA sequence. Here we report the development of RoboOligo, an interactive software program for the robust analysis of data generated by CID MS/MS of RNA oligomers. There are three main functions of RoboOligo: (i) automated de novo sequencing via the local search paradigm. (ii) Manual sequencing with real-time spectrum labeling and cumulative intensity scoring. (iii) A hybrid approach, coined ‘variable sequencing’, which combines the user intuition of manual sequencing with the high-throughput sampling of automated de novo sequencing. PMID:25820423

SSTAR, a Stand-Alone Easy-To-Use Antimicrobial Resistance Gene Predictor.

PubMed

de Man, Tom J B; Limbago, Brandi M

2016-01-01

We present the easy-to-use Sequence Search Tool for Antimicrobial Resistance, SSTAR. It combines a locally executed BLASTN search against a customizable database with an intuitive graphical user interface for identifying antimicrobial resistance (AR) genes from genomic data. Although the database is initially populated from a public repository of acquired resistance determinants (i.e., ARG-ANNOT), it can be customized for particular pathogen groups and resistance mechanisms. For instance, outer membrane porin sequences associated with carbapenem resistance phenotypes can be added, and known intrinsic mechanisms can be included. Unique about this tool is the ability to easily detect putative new alleles and truncated versions of existing AR genes. Variants and potential new alleles are brought to the attention of the user for further investigation. For instance, SSTAR is able to identify modified or truncated versions of porins, which may be of great importance in carbapenemase-negative carbapenem-resistant Enterobacteriaceae. SSTAR is written in Java and is therefore platform independent and compatible with both Windows and Unix operating systems. SSTAR and its manual, which includes a simple installation guide, are freely available from https://github.com/tomdeman-bio/Sequence-Search-Tool-for-Antimicrobial-Resistance-SSTAR-. IMPORTANCE Whole-genome sequencing (WGS) is quickly becoming a routine method for identifying genes associated with antimicrobial resistance (AR). However, for many microbiologists, the use and analysis of WGS data present a substantial challenge. We developed SSTAR, software with a graphical user interface that enables the identification of known AR genes from WGS and has the unique capacity to easily detect new variants of known AR genes, including truncated protein variants. Current software solutions do not notify the user when genes are truncated and, therefore, likely nonfunctional, which makes phenotype predictions less accurate. SSTAR users can apply any AR database of interest as a reference comparator and can manually add genes that impact resistance, even if such genes are not resistance determinants per se (e.g., porins and efflux pumps).

Elucidation of the full genetic information of Japanese rubella vaccines and the genetic changes associated with in vitro and in vivo vaccine virus phenotypes.

PubMed

Otsuki, Noriyuki; Abo, Hitoshi; Kubota, Toru; Mori, Yoshio; Umino, Yukiko; Okamoto, Kiyoko; Takeda, Makoto; Komase, Katsuhiro

2011-02-24

Rubella is a mild disease characterized by low-grade fever, and a morbilliform rash, but causes congenital defects in neonates born from mothers who suffered from rubella during the pregnancy. After many passages of wild-type rubella virus strains in various types of cultured cells, five live attenuated rubella vaccines were developed in Japan. An inability to elicit anti-rubella virus antibodies in experimentally infected animals was used as an in vivo marker phenotype of Japanese rubella vaccines. All Japanese rubella vaccine viruses exhibit a temperature-sensitive (ts) phenotype, and replicate very poorly at a high temperature. We determined the entire genome sequences of three Japanese rubella vaccines (Matsuba, TCRB19, and Matsuura), thereby completing the sequencing of all five Japanese rubella vaccines. In addition, the entire genome sequences of three vaccine progenitors were determined. Comparative nucleotide sequence analyses revealed mutations that were introduced into the genomes of the TO-336 and Matsuura vaccines during their production by laboratory passaging. Analyses involving cellular expression of viral P150 nonstructural protein-derived peptides revealed that the N1159S mutation conferred the ts phenotype on the TO-336 vaccine, and that reduced thermal stability of the P150 protease domain was a cause of the ts phenotype of some rubella vaccine viruses. The ts phenotype of vaccine viruses was not necessarily correlated with their inability to elicit humoral immune responses in animals. Therefore, the molecular mechanisms underlying the inability of these vaccines to elicit humoral responses in animals are more complicated than the previously considered mechanism involving the ts phenotype as the major cause. Copyright © 2011 Elsevier Ltd. All rights reserved.

Mitochondrial C4375T mutation might be a molecular risk factor in a maternal Chinese hypertensive family under haplotype C.

PubMed

Chen, Hong; Sun, Min; Fan, Zhen; Tong, Maoqing; Chen, Guodong; Li, Danhui; Ye, Jihui; Yang, Yumin; Zhu, Yongding; Zhu, Jianhua

2017-12-04

Here, we reported a Han Chinese essential hypertensive pedigree based on clinical hereditary and molecular data. To know the molecular basis on this family, mitochondrial genome of one proband from the family was identified through direct sequencing analysis. The age of onset year and affected degree of patients are different in this family. And matrilineal family members carrying C4375T mutation and belong to Eastern Asian halopgroup C. Phylogenetic analysis shows 4375C is highly conservative in 17 species. It is suggested that these mutations might participate in the development of hypertension in this family. And halopgroup C might play a modifying role on the phenotype in this Chinese hypertensive family.

Peruvian Maca (Lepidium peruvianum) – III: The Effects of Cultivation Altitude on Phytochemical and Genetic Differences in the Four Prime Maca Phenotypes

PubMed Central

Meissner, Henry O.; Mscisz, Alina; Baraniak, Marek; Piatkowska, Ewa; Pisulewski, Pawel; Mrozikiewicz, Mieczyslaw; Bobkiewicz-Kozlowska, Teresa

2017-01-01

In two trials, dietary and Glucosinolates’ characteristics in four Maca phenotypes have been examined with an extension into the determination of DNA sequences. Hypocotyls of the four prime phenotypes of Peruvian Maca - Lepidium peruvianum Chacon, labelled as “Yellow”, “Black”, “Red” and “Purple” were separated from mixed Maca crops cultivated in four geographically-distant locations in the Peruvian Andes at altitudes between 2,800m and 4,300 m a.s.l. It was found that at higher altitudes where Red and Purple Maca phenotypes were grown, the significantly higher (P<0.05) Glucosinolates’ concentrations, adopted as the marker of Maca physiological activity, were observed with the Purple phenotype showing the highest Glucosinolates’ content at 4,300m a.s.l., followed by the Red-coloured hypocotyls. Black Maca showed a reversal, but also a significant (P<0.05) trend, while the Yellow phenotype showed no visible altitude-inflicted response (P>0.05) and has consistently the lowest Glucosinolates content. Thus, it is reasonable to assume that the altitude at which Red, Purple and Black phenotypes of L. peruvianum are grown, may be responsible for the variation in physiologic functionalities, leading to different than expected specific therapeutic and health benefits induced by Maca phenotypes grown at diverse altitudes. Although promising, insufficiently precise differences in DNA sequences failed to distinguish, without any reasonable doubt, four Maca phenotypes cultivated either in the same or geographically-distant locations, and harvested at different altitudes a.s.l. Further research on DNA sequences is needed, with more primers and larger number of Maca phenotypes, considering biosynthesis of secondary metabolites and adaptation pathways induced by harsh environment at altitudes where Maca is cultivated. PMID:28824342

Peruvian Maca (Lepidium peruvianum) - III: The Effects of Cultivation Altitude on Phytochemical and Genetic Differences in the Four Prime Maca Phenotypes.

PubMed

Meissner, Henry O; Mscisz, Alina; Baraniak, Marek; Piatkowska, Ewa; Pisulewski, Pawel; Mrozikiewicz, Mieczyslaw; Bobkiewicz-Kozlowska, Teresa

2017-06-01

In two trials, dietary and Glucosinolates' characteristics in four Maca phenotypes have been examined with an extension into the determination of DNA sequences. Hypocotyls of the four prime phenotypes of Peruvian Maca - Lepidium peruvianum Chacon, labelled as "Yellow", "Black", "Red" and "Purple" were separated from mixed Maca crops cultivated in four geographically-distant locations in the Peruvian Andes at altitudes between 2,800m and 4,300 m a.s.l. It was found that at higher altitudes where Red and Purple Maca phenotypes were grown, the significantly higher ( P <0.05) Glucosinolates' concentrations, adopted as the marker of Maca physiological activity, were observed with the Purple phenotype showing the highest Glucosinolates' content at 4,300m a.s.l., followed by the Red-coloured hypocotyls. Black Maca showed a reversal, but also a significant ( P <0.05) trend, while the Yellow phenotype showed no visible altitude-inflicted response ( P >0.05) and has consistently the lowest Glucosinolates content. Thus, it is reasonable to assume that the altitude at which Red, Purple and Black phenotypes of L. peruvianum are grown, may be responsible for the variation in physiologic functionalities, leading to different than expected specific therapeutic and health benefits induced by Maca phenotypes grown at diverse altitudes. Although promising, insufficiently precise differences in DNA sequences failed to distinguish, without any reasonable doubt, four Maca phenotypes cultivated either in the same or geographically-distant locations, and harvested at different altitudes a.s.l. Further research on DNA sequences is needed, with more primers and larger number of Maca phenotypes, considering biosynthesis of secondary metabolites and adaptation pathways induced by harsh environment at altitudes where Maca is cultivated.

A weighted U-statistic for genetic association analyses of sequencing data.

PubMed

Wei, Changshuai; Li, Ming; He, Zihuai; Vsevolozhskaya, Olga; Schaid, Daniel J; Lu, Qing

2014-12-01

With advancements in next-generation sequencing technology, a massive amount of sequencing data is generated, which offers a great opportunity to comprehensively investigate the role of rare variants in the genetic etiology of complex diseases. Nevertheless, the high-dimensional sequencing data poses a great challenge for statistical analysis. The association analyses based on traditional statistical methods suffer substantial power loss because of the low frequency of genetic variants and the extremely high dimensionality of the data. We developed a Weighted U Sequencing test, referred to as WU-SEQ, for the high-dimensional association analysis of sequencing data. Based on a nonparametric U-statistic, WU-SEQ makes no assumption of the underlying disease model and phenotype distribution, and can be applied to a variety of phenotypes. Through simulation studies and an empirical study, we showed that WU-SEQ outperformed a commonly used sequence kernel association test (SKAT) method when the underlying assumptions were violated (e.g., the phenotype followed a heavy-tailed distribution). Even when the assumptions were satisfied, WU-SEQ still attained comparable performance to SKAT. Finally, we applied WU-SEQ to sequencing data from the Dallas Heart Study (DHS), and detected an association between ANGPTL 4 and very low density lipoprotein cholesterol. © 2014 WILEY PERIODICALS, INC.

A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.

PubMed

Agrotis, Alexander; Ketteler, Robin

2015-01-01

CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

Phenotypic instability between the near isogenic substrains BALB/cJ and BALB/cByJ.

PubMed

Sittig, Laura J; Jeong, Choongwon; Tixier, Emily; Davis, Joe; Barrios-Camacho, Camila M; Palmer, Abraham A

2014-12-01

Closely related substrains of inbred mice often show phenotypic differences that are presumed to be caused by recent mutations. The substrains BALB/cJ and BALB/cByJ, which were separated in 1935, have been reported to show numerous highly significant behavioral and morphological differences. In an effort to identify some of the causal mutations, we phenotyped BALB/cJ and BALB/cByJ mice as well as their F1, F2, and N2 progeny for behavioral and morphological phenotypes. We also generated whole-genome sequence data for both inbred strains (~3.5× coverage) with the intention of identifying polymorphic markers to be used for linkage analysis. We observed significant differences in body weight, the weight of the heart, liver, spleen and brain, and corpus callosum length between the two substrains. We also observed that BALB/cJ animals showed greater anxiety-like behavior in the open field test, less depression-like behavior in the tail suspension test, and reduced aggression compared to BALB/cByJ mice. Some but not all of these physiological and behavioral results were inconsistent with prior publications. These inconsistencies led us to suspect that the differences were due to, or modified by, non-genetic factors. Thus, we did not perform linkage analysis. We provide a comprehensive summary of the prior literature about phenotypic differences between these substrains as well as our current findings. We conclude that many differences between these strains are unstable and therefore ill-suited to linkage analysis; the source of this instability is unclear. We discuss the broader implications of these observations for the design of future studies.

Phenotypic instability between the near isogenic substrains BALB/cJ and BALB/cByJ

PubMed Central

Sittig, Laura J.; Jeong, Choongwon; Tixier, Emily; Davis, Joe; Barrios Camacho, Camila M.; Palmer, Abraham A.

2014-01-01

Closely related substrains of inbred mice often show phenotypic difzferences that are presumed to be caused by recent mutations. The substrains BALB/cJ and BALB/cByJ, which were separated in 1935, have been reported to show numerous highly significant behavioral and morphological differences. In an effort to identify some of the causal mutations, we phenotyped BALB/cJ and BALB/cByJ mice as well as their F1, F2, and N2 progeny for behavioral and morphological phenotypes. We also generated whole genome sequence data for both inbred strains (∼3.5× coverage) with the intention of identifying polymorphic markers to be used for linkage analysis. We observed significant differences in body weight, the weight of the heart, liver, spleen and brain, and corpus callosum length between the two substrains. We also observed that BALB/cJ animals showed greater anxiety-like behavior in the open field test, less depression-like behavior in the tail suspension test, and reduced aggression compared to BALB/cByJ mice. Some but not all of these physiological and behavioral results were inconsistent with prior publications. These inconsistencies led us to suspect that the differences were due to, or modified by, non-genetic factors. Thus, we did not perform linkage analysis. We provide a comprehensive summary of the prior literature about phenotypic differences between these substrains as well as our current findings. We conclude that many differences between these strains are unstable and therefore ill-suited to linkage analysis; the source of this instability is unclear. We discuss the broader implications of these observations for the design of future studies. PMID:24997021

p.H1069Q mutation in ATP7B and biochemical parameters of copper metabolism and clinical manifestation of Wilson's disease.

PubMed

Gromadzka, Graznya; Schmidt, Harmut H J; Genschel, Janine; Bochow, Bettina; Rodo, M; Tarnacka, Beatek; Litwin, Thomas; Chabik, Grzegorz; Członkowska, Anna

2006-02-01

We compared the effect of the p.H1069Q mutation and other non-p.H1069Q mutations in ATP7B on the phenotypic expression of Wilson's disease (WD), and assessed whether the clinical phenotype of WD in compound heterozygotes depends on the type of mutation coexisting with the p.H1069Q. One hundred forty-two patients with clinically, biochemically, and genetically diagnosed WD were studied. The mutational analysis of ATP7B was performed by direct sequencing. A total number of 26 mutations in ATP7B were identified. The p.His1069Gln was the most common mutation (allelic frequency: 72%). Seventy-three patients were homozygous for this mutation. Of compound heterozygotes, 37 had frameshift/nonsense mutation, and 20 had other missense mutation on one of their ATP7B alleles. Twelve patients had two non-p.H1069Q mutations. Patients homozygous for the p.H1069Q mutation had the less severe disturbances of copper metabolism and the latest presentation of first WD symptoms. The most severely disturbed copper metabolism and the earliest age at initial disease manifestation was noticed in non-p.H1069Q patients. In compound heterozygotes, the type of mutation coexisting with the p.H1069Q to a small extent influenced WD phenotype. The phenotype of WD varied considerably among patients with the same genotype. The p.H1069Q mutation is associated with late WD manifestation and with a mild disruption of copper metabolism. In compound heterozygotes, the phenotype of WD to a small extent depends on the type of mutation coexisting with the p.H1069Q. Besides genotype, additional modifying factors seem to determine WD manifestations. Copyright (c) 2005 Movement Disorder Society.

Epigenetics in Comparative Biology: Why We Should Pay Attention

PubMed Central

Burggren, Warren W.; Crews, David

2014-01-01

The past decade has seen an explosion of articles in scientific journals involving non-genetic influences on phenotype through modulation of gene function without changes in gene sequence. The excitement in modern molecular biology surrounding the impact exerted by the environment on development of the phenotype is focused largely on mechanism and has not incorporated questions asked (and answers provided) by early philosophers, biologists, and psychologists. As such, this emergence of epigenetic studies is somewhat “old wine in new bottles” and represents a reformulation of the old debate of preformationism versus epigenesis—one resolved in the 1800s. Indeed, this tendency to always look forward, with minimal concern or regard of what has gone before, has led to the present situation in which “true” epigenetic studies are believed to consist of one of two schools. The first is primarily medically based and views epigenetic mechanisms as pathways for disease (e.g., “the epigenetics of cancer”). The second is primarily from the basic sciences, particularly molecular genetics, and regards epigenetics as a potentially important mechanism for organisms exposed to variable environments across multiple generations. There is, however, a third, and separate, school based on the historical literature and debates and regards epigenetics as more of a perspective than a phenomenon. Against this backdrop, comparative integrative biologists are particularly well-suited to understand epigenetic phenomena as a way for organisms to respond rapidly with modified phenotypes (relative to natural selection) to changes in the environment. Using evolutionary principles, it is also possible to interpret “sunsetting” of modified phenotypes when environmental conditions result in a disappearance of the epigenetic modification of gene regulation. Comparative integrative biologists also recognize epigenetics as a potentially confounding source of variation in their data. Epigenetic modification of phenotype (molecular, cellular, morphological, physiological, and behavioral) can be highly variable depending upon ancestral environmental exposure and can contribute to apparent “random” noise in collected datasets. Thus, future research should go beyond the study of epigenetic mechanisms at the level of the gene and devote additional investigation of epigenetic outcomes at the level of both the individual organism and how it affects the evolution of populations. This review is the first of seven in this special issue of Integrative and Comparative Biology that addresses in detail these and other key topics in the study of epigenetics. PMID:24722321

Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype.

PubMed

Barnidge, David R; Lundström, Susanna L; Zhang, Bo; Dasari, Surendra; Murray, David L; Zubarev, Roman A

2015-12-04

In our previous work, we showed that electrospray ionization of intact polyclonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.

The evolution of transcriptional regulation in eukaryotes

NASA Technical Reports Server (NTRS)

Wray, Gregory A.; Hahn, Matthew W.; Abouheif, Ehab; Balhoff, James P.; Pizer, Margaret; Rockman, Matthew V.; Romano, Laura A.

2003-01-01

Gene expression is central to the genotype-phenotype relationship in all organisms, and it is an important component of the genetic basis for evolutionary change in diverse aspects of phenotype. However, the evolution of transcriptional regulation remains understudied and poorly understood. Here we review the evolutionary dynamics of promoter, or cis-regulatory, sequences and the evolutionary mechanisms that shape them. Existing evidence indicates that populations harbor extensive genetic variation in promoter sequences, that a substantial fraction of this variation has consequences for both biochemical and organismal phenotype, and that some of this functional variation is sorted by selection. As with protein-coding sequences, rates and patterns of promoter sequence evolution differ considerably among loci and among clades for reasons that are not well understood. Studying the evolution of transcriptional regulation poses empirical and conceptual challenges beyond those typically encountered in analyses of coding sequence evolution: promoter organization is much less regular than that of coding sequences, and sequences required for the transcription of each locus reside at multiple other loci in the genome. Because of the strong context-dependence of transcriptional regulation, sequence inspection alone provides limited information about promoter function. Understanding the functional consequences of sequence differences among promoters generally requires biochemical and in vivo functional assays. Despite these challenges, important insights have already been gained into the evolution of transcriptional regulation, and the pace of discovery is accelerating.

Whole Exome Sequencing Identifies Novel Genes for Fetal Hemoglobin Response to Hydroxyurea in Children with Sickle Cell Anemia

PubMed Central

Sheehan, Vivien A.; Crosby, Jacy R.; Sabo, Aniko; Mortier, Nicole A.; Howard, Thad A.; Muzny, Donna M.; Dugan-Perez, Shannon; Aygun, Banu; Nottage, Kerri A.; Boerwinkle, Eric; Gibbs, Richard A.; Ware, Russell E.; Flanagan, Jonathan M.

2014-01-01

Hydroxyurea has proven efficacy in children and adults with sickle cell anemia (SCA), but with considerable inter-individual variability in the amount of fetal hemoglobin (HbF) produced. Sibling and twin studies indicate that some of that drug response variation is heritable. To test the hypothesis that genetic modifiers influence pharmacological induction of HbF, we investigated phenotype-genotype associations using whole exome sequencing of children with SCA treated prospectively with hydroxyurea to maximum tolerated dose (MTD). We analyzed 171 unrelated patients enrolled in two prospective clinical trials, all treated with dose escalation to MTD. We examined two MTD drug response phenotypes: HbF (final %HbF minus baseline %HbF), and final %HbF. Analyzing individual genetic variants, we identified multiple low frequency and common variants associated with HbF induction by hydroxyurea. A validation cohort of 130 pediatric sickle cell patients treated to MTD with hydroxyurea was genotyped for 13 non-synonymous variants with the strongest association with HbF response to hydroxyurea in the discovery cohort. A coding variant in Spalt-like transcription factor, or SALL2, was associated with higher final HbF in this second independent replication sample and SALL2 represents an outstanding novel candidate gene for further investigation. These findings may help focus future functional studies and provide new insights into the pharmacological HbF upregulation by hydroxyurea in patients with SCA. PMID:25360671

Hereditary spastic paraplegias: identification of a novel SPG57 variant affecting TFG oligomerization and description of HSP subtypes in Sudan.

PubMed

Elsayed, Liena E O; Mohammed, Inaam N; Hamed, Ahlam A A; Elseed, Maha A; Johnson, Adam; Mairey, Mathilde; Mohamed, Hassab Elrasoul S A; Idris, Mohamed N; Salih, Mustafa A M; El-Sadig, Sarah M; Koko, Mahmoud E; Mohamed, Ashraf Y O; Raymond, Laure; Coutelier, Marie; Darios, Frédéric; Siddig, Rayan A; Ahmed, Ahmed K M A; Babai, Arwa M A; Malik, Hiba M O; Omer, Zulfa M B M; Mohamed, Eman O E; Eltahir, Hanan B; Magboul, Nasr Aldin A; Bushara, Elfatih E; Elnour, Abdelrahman; Rahim, Salah M Abdel; Alattaya, Abdelmoneim; Elbashir, Mustafa I; Ibrahim, Muntaser E; Durr, Alexandra; Audhya, Anjon; Brice, Alexis; Ahmed, Ammar E; Stevanin, Giovanni

2016-01-01

Hereditary spastic paraplegias (HSP) are the second most common type of motor neuron disease recognized worldwide. We investigated a total of 25 consanguineous families from Sudan. We used next-generation sequencing to screen 74 HSP-related genes in 23 families. Linkage analysis and candidate gene sequencing was performed in two other families. We established a genetic diagnosis in six families with autosomal recessive HSP (SPG11 in three families and TFG/SPG57, SACS and ALS2 in one family each). A heterozygous mutation in a gene involved in an autosomal dominant HSP (ATL1/SPG3A) was also identified in one additional family. Six out of seven identified variants were novel. The c.64C>T (p.(Arg22Trp)) TFG/SPG57 variant (PB1 domain) is the second identified that underlies HSP, and we demonstrated its impact on TFG oligomerization in vitro. Patients did not present with visual impairment as observed in a previously reported SPG57 family (c.316C>T (p.(Arg106Cys)) in coiled-coil domain), suggesting unique contributions of the PB1 and coiled-coil domains in TFG complex formation/function and a possible phenotype correlation to variant location. Some families manifested marked phenotypic variations implying the possibility of modifier factors complicated by high inbreeding. Finally, additional genetic heterogeneity is expected in HSP Sudanese families. The remaining families might unravel new genes or uncommon modes of inheritance.

Hereditary spastic paraplegias: identification of a novel SPG57 variant affecting TFG oligomerization and description of HSP subtypes in Sudan

PubMed Central

Elsayed, Liena E O; Mohammed, Inaam N; Hamed, Ahlam A A; Elseed, Maha A; Johnson, Adam; Mairey, Mathilde; Mohamed, Hassab Elrasoul S A; Idris, Mohamed N; Salih, Mustafa A M; El-sadig, Sarah M; Koko, Mahmoud E; Mohamed, Ashraf Y O; Raymond, Laure; Coutelier, Marie; Darios, Frédéric; Siddig, Rayan A; Ahmed, Ahmed K M A; Babai, Arwa M A; Malik, Hiba M O; Omer, Zulfa M B M; Mohamed, Eman O E; Eltahir, Hanan B; Magboul, Nasr Aldin A; Bushara, Elfatih E; Elnour, Abdelrahman; Rahim, Salah M Abdel; Alattaya, Abdelmoneim; Elbashir, Mustafa I; Ibrahim, Muntaser E; Durr, Alexandra; Audhya, Anjon; Brice, Alexis; Ahmed, Ammar E; Stevanin, Giovanni

2017-01-01

Hereditary spastic paraplegias (HSP) are the second most common type of motor neuron disease recognized worldwide. We investigated a total of 25 consanguineous families from Sudan. We used next-generation sequencing to screen 74 HSP-related genes in 23 families. Linkage analysis and candidate gene sequencing was performed in two other families. We established a genetic diagnosis in six families with autosomal recessive HSP (SPG11 in three families and TFG/SPG57, SACS and ALS2 in one family each). A heterozygous mutation in a gene involved in an autosomal dominant HSP (ATL1/SPG3A) was also identified in one additional family. Six out of seven identified variants were novel. The c.64C>T (p.(Arg22Trp)) TFG/SPG57 variant (PB1 domain) is the second identified that underlies HSP, and we demonstrated its impact on TFG oligomerization in vitro. Patients did not present with visual impairment as observed in a previously reported SPG57 family (c.316C>T (p.(Arg106Cys)) in coiled-coil domain), suggesting unique contributions of the PB1 and coiled-coil domains in TFG complex formation/function and a possible phenotype correlation to variant location. Some families manifested marked phenotypic variations implying the possibility of modifier factors complicated by high inbreeding. Finally, additional genetic heterogeneity is expected in HSP Sudanese families. The remaining families might unravel new genes or uncommon modes of inheritance. PMID:27601211

Cuticular hydrocarbon phenotypes do not indicate cryptic species in fungus-growing termites (Isoptera: Macrotermitinae).

PubMed

Marten, Andreas; Kaib, Manfred; Brandl, Roland

2009-05-01

In several termite species, distinct differences in the composition of cuticular hydrocarbons among colonies correspond to high genetic divergence of mitochondrial DNA sequences. These observations suggest that hydrocarbon phenotypes represent cryptic species. Different cuticular hydrocarbon phenotypes also are found among colonies of fungus-growing termites of the genus Macrotermes. To determine if these hydrocarbon differences in Macrotermes also indicate cryptic species, we sequenced the mitochondrial CO I gene from species in West and East Africa. Among individuals of a supposed species but belonging to different cuticular hydrocarbon phenotypes, the genetic distances are much smaller than distances between species. Unlike what has been observed in other termites, Macrotermes hydrocarbon phenotypes do not represent cryptic species. Our findings suggest fundamental differences in the evolution and/or function of cuticular hydrocarbons among different termite lineages.

Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy.

PubMed

Yu, Hong-Hao; Zhao, Heng; Qing, Yu-Bo; Pan, Wei-Rong; Jia, Bao-Yu; Zhao, Hong-Ye; Huang, Xing-Xu; Wei, Hong-Jiang

2016-10-09

Dystrophinopathy, including Duchenne muscle dystrophy (DMD) and Becker muscle dystrophy (BMD) is an incurable X-linked hereditary muscle dystrophy caused by a mutation in the DMD gene in coding dystrophin. Advances in further understanding DMD/BMD for therapy are expected. Studies on mdx mice and dogs with muscle dystrophy provide limited insight into DMD disease mechanisms and therapeutic testing because of the different pathological manifestations. Miniature pigs share similar physiology and anatomy with humans and are thus an excellent animal model of human disease. Here, we successfully achieved precise DMD targeting in Chinese Diannan miniature pigs by co-injecting zygotes with Cas9 mRNA and sgRNA targeting DMD . Two piglets were obtained after embryo transfer, one of piglets was identified as DMD -modified individual via traditional cloning, sequencing and T7EN1 cleavage assay. An examination of targeting rates in the DMD -modified piglet revealed that sgRNA:Cas9-mediated on-target mosaic mutations were 70% and 60% of dystrophin alleles in skeletal and smooth muscle, respectively. Meanwhile, no detectable off-target mutations were found, highlighting the high specificity of genetic modification using CRISPR/Cas9. The DMD -modified piglet exhibited degenerative and disordered phenotypes in skeletal and cardiac muscle, and declining thickness of smooth muscle in the stomach and intestine. In conclusion, we successfully generated myopathy animal model by modifying the DMD via CRISPR/Cas9 system in a miniature pig.

Identification of five novel modifier loci of ApcMin harbored in the BXH14 recombinant inbred strain

PubMed Central

Siracusa, Linda D.

2012-01-01

Every year thousands of people in the USA are diagnosed with small intestine and colorectal cancers (CRC). Although environmental factors affect disease etiology, uncovering underlying genetic factors is imperative for risk assessment and developing preventative therapies. Familial adenomatous polyposis is a heritable genetic disorder in which individuals carry germ-line mutations in the adenomatous polyposis coli (APC) gene that predisposes them to CRC. The Apc Min mouse model carries a point mutation in the Apc gene and develops polyps along the intestinal tract. Inbred strain background influences polyp phenotypes in Apc Min mice. Several Modifier of Min (Mom) loci that alter tumor phenotypes associated with the Apc Min mutation have been identified to date. We screened BXH recombinant inbred (RI) strains by crossing BXH RI females with C57BL/6J (B6) Apc Min males and quantitating tumor phenotypes in backcross progeny. We found that the BXH14 RI strain harbors five modifier loci that decrease polyp multiplicity. Furthermore, we show that resistance is determined by varying combinations of these modifier loci. Gene interaction network analysis shows that there are multiple networks with proven gene–gene interactions, which contain genes from all five modifier loci. We discuss the implications of this result for studies that define susceptibility loci, namely that multiple networks may be acting concurrently to alter tumor phenotypes. Thus, the significance of this work resides not only with the modifier loci we identified but also with the combinations of loci needed to get maximal protection against polyposis and the impact of this finding on human disease studies. Abbreviations:APCadenomatous polyposis coliGWASgenome-wide association studiesQTLquantitative trait lociSNPsingle-nucleotide polymorphism. PMID:22637734

A coarse-grained biophysical model of sequence evolution and the population size dependence of the speciation rate

PubMed Central

Khatri, Bhavin S.; Goldstein, Richard A.

2015-01-01

Speciation is fundamental to understanding the huge diversity of life on Earth. Although still controversial, empirical evidence suggests that the rate of speciation is larger for smaller populations. Here, we explore a biophysical model of speciation by developing a simple coarse-grained theory of transcription factor-DNA binding and how their co-evolution in two geographically isolated lineages leads to incompatibilities. To develop a tractable analytical theory, we derive a Smoluchowski equation for the dynamics of binding energy evolution that accounts for the fact that natural selection acts on phenotypes, but variation arises from mutations in sequences; the Smoluchowski equation includes selection due to both gradients in fitness and gradients in sequence entropy, which is the logarithm of the number of sequences that correspond to a particular binding energy. This simple consideration predicts that smaller populations develop incompatibilities more quickly in the weak mutation regime; this trend arises as sequence entropy poises smaller populations closer to incompatible regions of phenotype space. These results suggest a generic coarse-grained approach to evolutionary stochastic dynamics, allowing realistic modelling at the phenotypic level. PMID:25936759

Rare Variant Association Test with Multiple Phenotypes

PubMed Central

Lee, Selyeong; Won, Sungho; Kim, Young Jin; Kim, Yongkang; Kim, Bong-Jo; Park, Taesung

2016-01-01

Although genome-wide association studies (GWAS) have now discovered thousands of genetic variants associated with common traits, such variants cannot explain the large degree of “missing heritability,” likely due to rare variants. The advent of next generation sequencing technology has allowed rare variant detection and association with common traits, often by investigating specific genomic regions for rare variant effects on a trait. Although multiply correlated phenotypes are often concurrently observed in GWAS, most studies analyze only single phenotypes, which may lessen statistical power. To increase power, multivariate analyses, which consider correlations between multiple phenotypes, can be used. However, few existing multi-variant analyses can identify rare variants for assessing multiple phenotypes. Here, we propose Multivariate Association Analysis using Score Statistics (MAAUSS), to identify rare variants associated with multiple phenotypes, based on the widely used Sequence Kernel Association Test (SKAT) for a single phenotype. We applied MAAUSS to Whole Exome Sequencing (WES) data from a Korean population of 1,058 subjects, to discover genes associated with multiple traits of liver function. We then assessed validation of those genes by a replication study, using an independent dataset of 3,445 individuals. Notably, we detected the gene ZNF620 among five significant genes. We then performed a simulation study to compare MAAUSS's performance with existing methods. Overall, MAAUSS successfully conserved type 1 error rates and in many cases, had a higher power than the existing methods. This study illustrates a feasible and straightforward approach for identifying rare variants correlated with multiple phenotypes, with likely relevance to missing heritability. PMID:28039885

Mutational robustness accelerates the origin of novel RNA phenotypes through phenotypic plasticity.

PubMed

Wagner, Andreas

2014-02-18

Novel phenotypes can originate either through mutations in existing genotypes or through phenotypic plasticity, the ability of one genotype to form multiple phenotypes. From molecules to organisms, plasticity is a ubiquitous feature of life, and a potential source of exaptations, adaptive traits that originated for nonadaptive reasons. Another ubiquitous feature is robustness to mutations, although it is unknown whether such robustness helps or hinders the origin of new phenotypes through plasticity. RNA is ideal to address this question, because it shows extensive plasticity in its secondary structure phenotypes, a consequence of their continual folding and unfolding, and these phenotypes have important biological functions. Moreover, RNA is to some extent robust to mutations. This robustness structures RNA genotype space into myriad connected networks of genotypes with the same phenotype, and it influences the dynamics of evolving populations on a genotype network. In this study I show that both effects help accelerate the exploration of novel phenotypes through plasticity. My observations are based on many RNA molecules sampled at random from RNA sequence space, and on 30 biological RNA molecules. They are thus not only a generic feature of RNA sequence space but are relevant for the molecular evolution of biological RNA. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Deciphering the distance to antibiotic resistance for the pneumococcus using genome sequencing data

PubMed Central

Mobegi, Fredrick M.; Cremers, Amelieke J. H.; de Jonge, Marien I.; Bentley, Stephen D.; van Hijum, Sacha A. F. T.; Zomer, Aldert

2017-01-01

Advances in genome sequencing technologies and genome-wide association studies (GWAS) have provided unprecedented insights into the molecular basis of microbial phenotypes and enabled the identification of the underlying genetic variants in real populations. However, utilization of genome sequencing in clinical phenotyping of bacteria is challenging due to the lack of reliable and accurate approaches. Here, we report a method for predicting microbial resistance patterns using genome sequencing data. We analyzed whole genome sequences of 1,680 Streptococcus pneumoniae isolates from four independent populations using GWAS and identified probable hotspots of genetic variation which correlate with phenotypes of resistance to essential classes of antibiotics. With the premise that accumulation of putative resistance-conferring SNPs, potentially in combination with specific resistance genes, precedes full resistance, we retrogressively surveyed the hotspot loci and quantified the number of SNPs and/or genes, which if accumulated would confer full resistance to an otherwise susceptible strain. We name this approach the ‘distance to resistance’. It can be used to identify the creep towards complete antibiotics resistance in bacteria using genome sequencing. This approach serves as a basis for the development of future sequencing-based methods for predicting resistance profiles of bacterial strains in hospital microbiology and public health settings. PMID:28205635

Looking beyond the exome: a phenotype-first approach to molecular diagnostic resolution in rare and undiagnosed diseases.

PubMed

Pena, Loren D M; Jiang, Yong-Hui; Schoch, Kelly; Spillmann, Rebecca C; Walley, Nicole; Stong, Nicholas; Rapisardo Horn, Sarah; Sullivan, Jennifer A; McConkie-Rosell, Allyn; Kansagra, Sujay; Smith, Edward C; El-Dairi, Mays; Bellet, Jane; Keels, Martha Ann; Jasien, Joan; Kranz, Peter G; Noel, Richard; Nagaraj, Shashi K; Lark, Robert K; Wechsler, Daniel S G; Del Gaudio, Daniela; Leung, Marco L; Hendon, Laura G; Parker, Collette C; Jones, Kelly L; Goldstein, David B; Shashi, Vandana

2018-04-01

PurposeTo describe examples of missed pathogenic variants on whole-exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing.MethodsGuided by phenotypic information, three children with negative WES underwent targeted single-gene testing.ResultsIndividual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and a next-generation sequencing (NGS)-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the noncoding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity, and magnetic resonance image changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, probably missed owing to failure of alignment.ConclusionThese cases illustrate potential pitfalls of WES/NGS testing and the importance of phenotype-guided molecular testing in yielding diagnoses.

Herpes Simplex Virus 2 Latency-Associated Transcript (LAT) region mutations do not identify a role for LAT-Associated Micro RNAs in viral reactivation in the Guinea Pig Genital Model.

PubMed

Kawamura, Yoshiki; Bosch-Marce, Marta; Tang, Shuang; Patel, Amita; Krause, Philip R

2018-05-02

Despite the long-standing observation that herpes simplex virus (HSV) Latency-Associated Transcript (LAT) promoter-deletion viruses show impaired recurrence phenotypes in relevant animal models, the mechanism by which these sequences exert this phenotypic effect is unknown. We constructed and evaluated four mutant HSV-2 viruses with targeted mutations in the LAT promoter and LAT-associated miRNAs affecting (1) the LAT TATA box, (2) the LAT ICP4-binding site, (3) miR-I and miR-II (miR-I/II), which both target ICP34.5, and (4) miR-III, which targets ICP0. While the LAT-TATA box mutant caused milder acute infections than wild-type (WT), there was no difference in recurrence phenotype between these viruses. LAT and miRNA expression during latency were not impaired by this mutation, suggesting that other promoter elements may be more important for latent HSV-2 LAT expression. Mutation of the LAT ICP4-binding site also did not cause an in vivo phenotypic difference between mutant and WT viruses. Acute infection and reactivation from latency of the miR-I/II mutant was similar to that of its rescuant, although slightly reduced in severity relative to the wild-type virus. The miR-III mutant also exhibited WT phenotypes in acute and recurrent phases of infection. While not ruling out an effect of these elements in human latency or reactivation, these findings do not identify a specific role for LAT or LAT-associated miRNAs in the HSV-2 LAT promoter deletion phenotype in guinea pigs. Thus, other sequences in this region may play a more important role in the long-studied LAT-associated phenotype in animals. IMPORTANCE While it has been known for several decades that specific HSV-1 and HSV-2 sequences near the LAT promoter are required for efficient viral reactivation in animal models, the mechanism is still not known. We constructed four mutant viruses with the goal of identifying critical sequence elements and of specifically testing the hypothesis that microRNAs that are expressed during latency play a role. Determination that specific LAT promoter sequences and miRNA sequences do not influence viral reactivation of HSV-2 helps to narrow down the search for the mechanism by which the virus controls its latency and recurrence phenotype. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Smoothness within ruggedness: the role of neutrality in adaptation.

PubMed Central

Huynen, M A; Stadler, P F; Fontana, W

1996-01-01

RNA secondary structure folding algorithms predict the existence of connected networks of RNA sequences with identical structure. On such networks, evolving populations split into subpopulations, which diffuse independently in sequence space. This demands a distinction between two mutation thresholds: one at which genotypic information is lost and one at which phenotypic information is lost. In between, diffusion enables the search of vast areas in genotype space while still preserving the dominant phenotype. By this dynamic the success of phenotypic adaptation becomes much less sensitive to the initial conditions in genotype space. Images Fig. 2 PMID:8552647

Genomes by design

PubMed Central

Haimovich, Adrian D.; Muir, Paul; Isaacs, Farren J.

2016-01-01

Next-generation DNA sequencing has revealed the complete genome sequences of numerous organisms, establishing a fundamental and growing understanding of genetic variation and phenotypic diversity. Engineering at the gene, network and whole-genome scale aims to introduce targeted genetic changes both to explore emergent phenotypes and to introduce new functionalities. Expansion of these approaches into massively parallel platforms establishes the ability to generate targeted genome modifications, elucidating causal links between genotype and phenotype, as well as the ability to design and reprogramme organisms. In this Review, we explore techniques and applications in genome engineering, outlining key advances and defining challenges. PMID:26260262

Development of resources and tools for mapping genetic sources of phenotypic variation

USDA-ARS?s Scientific Manuscript database

Commercial and experimental genetic resources were established and investigated for a range of reproductive and disease susceptibility phenotypes. The phenotyping efforts were accompanied with RNA and whole genome sequencing and novel assemblies of the swine genome. The efforts were complemented wit...

Phenotypic and genotypic diversity of Lactobacillus buchneri strains isolated from spoiled, fermented cucumber.

PubMed

Daughtry, Katheryne V; Johanningsmeier, Suzanne D; Sanozky-Dawes, Rosemary; Klaenhammer, Todd R; Barrangou, Rodolphe

2018-09-02

Lactobacillus buchneri is a Gram-positive, obligate heterofermentative, facultative anaerobe commonly affiliated with spoilage of food products. Notably, L. buchneri is able to metabolize lactic acid into acetic acid and 1,2-propanediol. Although beneficial to the silage industry, this metabolic capability is detrimental to preservation of cucumbers by fermentation. The objective of this study was to characterize isolates of L. buchneri purified from both industrial and experimental fermented cucumber after the onset of secondary fermentation. Genotypic and phenotypic characterization included 16S rRNA sequencing, DiversiLab® rep-PCR, colony morphology, API 50 CH carbohydrate analysis, and ability to degrade lactic acid in modified MRS and fermented cucumber media. Distinct groups of isolates were identified with differing colony morphologies that varied in color (translucent white to opaque yellow), diameter (1 mm-11 mm), and shape (umbonate, flat, circular or irregular). Growth rates in MRS revealed strain differences, and a wide spectrum of carbon source utilization was observed. Some strains were able to ferment as many as 21 of 49 tested carbon sources, including inulin, fucose, gentiobiose, lactose, mannitol, potassium ketogluconate, saccharose, raffinose, galactose, and xylose, while others metabolized as few as eight carbohydrates as the sole source of carbon. All isolates degraded lactic acid in both fermented cucumber medium and modified MRS, but exhibited differences in the rate and extent of lactate degradation. Isolates clustered into eight distinct groups based on rep-PCR fingerprints with 20 of 36 of the isolates exhibiting >97% similarity. Although isolated from similar environmental niches, significant phenotypic and genotypic diversity was found among the L. buchneri cultures. A collection of unique L. buchneri strains was identified and characterized, providing the basis for further analysis of metabolic and genomic capabilities of this species to enable control of lactic acid degradation in fermented plant materials. Published by Elsevier B.V.

Analysis of aberrant pre-messenger RNA splicing resulting from mutations in ATP8B1 and efficient in vitro rescue by adapted U1 small nuclear RNA.

PubMed

van der Woerd, Wendy L; Mulder, Johanna; Pagani, Franco; Beuers, Ulrich; Houwen, Roderick H J; van de Graaf, Stan F J

2015-04-01

ATP8B1 deficiency is a severe autosomal recessive liver disease resulting from mutations in the ATP8B1 gene characterized by a continuous phenotypical spectrum from intermittent (benign recurrent intrahepatic cholestasis; BRIC) to progressive familial intrahepatic cholestasis (PFIC). Current therapeutic options are insufficient, and elucidating the molecular consequences of mutations could lead to personalized mutation-specific therapies. We investigated the effect on pre-messenger RNA splicing of 14 ATP8B1 mutations at exon-intron boundaries using an in vitro minigene system. Eleven mutations, mostly associated with a PFIC phenotype, resulted in aberrant splicing and a complete absence of correctly spliced product. In contrast, three mutations led to partially correct splicing and were associated with a BRIC phenotype. These findings indicate an inverse correlation between the level of correctly spliced product and disease severity. Expression of modified U1 small nuclear RNAs (snRNA) complementary to the splice donor sites strongly improved or completely rescued splicing for several ATP8B1 mutations located at donor, as well as acceptor, splice sites. In one case, we also evaluated exon-specific U1 snRNAs that, by targeting nonconserved intronic sequences, might reduce possible off-target events. Although very effective in correcting exon skipping, they also induced retention of the short downstream intron. We systematically characterized the molecular consequences of 14 ATP8B1 mutations at exon-intron boundaries associated with ATP8B1 deficiency and found that the majority resulted in total exon skipping. The amount of correctly spliced product inversely correlated with disease severity. Compensatory modified U1 snRNAs, complementary to mutated donor splice sites, were able to improve exon definition very efficiently and could be a novel therapeutic strategy in ATP8B1 deficiency as well as other genetic diseases. © 2014 by the American Association for the Study of Liver Diseases.

Phenotypic and genotypic detection of Candida albicans and Candida dubliniensis strains isolated from oral mucosa of AIDS pediatric patients

PubMed Central

Livério, Harisson Oliveira; Ruiz, Luciana da Silva; de Freitas, Roseli Santos; Nishikaku, Angela; de Souza, Ana Clara; Paula, Claudete Rodrigues; Domaneschi, Carina

2017-01-01

ABSTRACT The aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species. PMID:28423089

Phenotypic and genotypic detection of Candida albicans and Candida dubliniensis strains isolated from oral mucosa of AIDS pediatric patients.

PubMed

Livério, Harisson Oliveira; Ruiz, Luciana da Silva; Freitas, Roseli Santos de; Nishikaku, Angela; Souza, Ana Clara de; Paula, Claudete Rodrigues; Domaneschi, Carina

2017-04-13

The aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species.

Flavitrack: an annotated database of flavivirus sequences

PubMed Central

Misra, Milind

2009-01-01

Motivation Properly annotated sequence data for flaviviruses, which cause diseases, such as tick-borne encephalitis (TBE), dengue fever (DF), West Nile (WN) and yellow fever (YF), can aid in the design of antiviral drugs and vaccines to prevent their spread. Flavitrack was designed to help identify conserved sequence motifs, interpret mutational and structural data and track evolution of phenotypic properties. Summary Flavitrack contains over 590 complete flavivirus genome/protein sequences and information on known mutations and literature references. Each sequence has been manually annotated according to its date and place of isolation, phenotype and lethality. Internal tools are provided to rapidly determine relationships between viruses in Flavitrack and sequences provided by the user. Availability http://carnot.utmb.edu/flavitrack Contact chschein@utmb.edu Supplementary information http://carnot.utmb.edu/flavitrack/B1S1.html PMID:17660525

Rational Protein Engineering Guided by Deep Mutational Scanning

PubMed Central

Shin, HyeonSeok; Cho, Byung-Kwan

2015-01-01

Sequence–function relationship in a protein is commonly determined by the three-dimensional protein structure followed by various biochemical experiments. However, with the explosive increase in the number of genome sequences, facilitated by recent advances in sequencing technology, the gap between protein sequences available and three-dimensional structures is rapidly widening. A recently developed method termed deep mutational scanning explores the functional phenotype of thousands of mutants via massive sequencing. Coupled with a highly efficient screening system, this approach assesses the phenotypic changes made by the substitution of each amino acid sequence that constitutes a protein. Such an informational resource provides the functional role of each amino acid sequence, thereby providing sufficient rationale for selecting target residues for protein engineering. Here, we discuss the current applications of deep mutational scanning and consider experimental design. PMID:26404267

Effects of a Transposable Element Insertion on Alcohol Dehydrogenase Expression in Drosophila Melanogaster

PubMed Central

Dunn, R. C.; Laurie, C. C.

1995-01-01

Variation in the DNA sequence and level of alcohol dehydrogenase (Adh) gene expression in Drosophila melanogaster have been studied to determine what types of DNA polymorphisms contribute to phenotypic variation in natural populations. The Adh gene, like many others, shows a high level of variability in both DNA sequence and quantitative level of expression. A number of transposable element insertions occur in the Adh region and one of these, a copia insertion in the 5' flanking region, is associated with unusually low Adh expression. To determine whether this insertion (called RI42) causes the low expression level, the insertion was excised from the cloned RI42 Adh gene and the effect was assessed by P-element transformation. Removal of this insertion causes a threefold increase in the level of ADH, clearly showing that it contributes to the naturally occurring variation in expression at this locus. Removal of all but one LTR also causes a threefold increase, indicating that the mechanism is not a simple sequence disruption. Furthermore, this copia insertion, which is located between the two Adh promoters and their upstream enhancer sequences, has differential effects on the levels of proximal and distal transcripts. Finally, a test for the possible modifying effects of two suppressor loci, su(w(a)) and su(f), on this insertional mutation was negative, in contrast to a previous report in the literature. PMID:7498745

Latency-associated transcript (LAT) exon 1 controls herpes simplex virus species-specific phenotypes: reactivation in the guinea pig genital model and neuron subtype-specific latent expression of LAT.

PubMed

Bertke, Andrea S; Patel, Amita; Imai, Yumi; Apakupakul, Kathleen; Margolis, Todd P; Krause, Philip R

2009-10-01

Herpes simplex virus 1 (HSV-1) and HSV-2 cause similar acute infections but differ in their abilities to reactivate from trigeminal and lumbosacral dorsal root ganglia. During latency, HSV-1 and HSV-2 also preferentially express their latency-associated transcripts (LATs) in different sensory neuronal subtypes that are positive for A5 and KH10 markers, respectively. Chimeric virus studies showed that LAT region sequences influence both of these viral species-specific phenotypes. To further map the LAT region sequences responsible for these phenotypes, we constructed the chimeric virus HSV2-LAT-E1, in which exon 1 (from the LAT TATA to the intron splice site) was replaced by the corresponding sequence from HSV-1 LAT. In intravaginally infected guinea pigs, HSV2-LAT-E1 reactivated inefficiently relative to the efficiency of its rescuant and wild-type HSV-2, but it yielded similar levels of viral DNA, LAT, and ICP0 during acute and latent infection. HSV2-LAT-E1 preferentially expressed the LAT in A5+ neurons (as does HSV-1), while the chimeric viruses HSV2-LAT-P1 (LAT promoter swap) and HSV2-LAT-S1 (LAT sequence swap downstream of the promoter) exhibited neuron subtype-specific latent LAT expression phenotypes more similar to that of HSV-2 than that of HSV-1. Rescuant viruses displayed the wild-type HSV-2 phenotypes of efficient reactivation in the guinea pig genital model and a tendency to express LAT in KH10+ neurons. The region that is critical for HSV species-specific differences in latency and reactivation thus lies between the LAT TATA and the intron splice site, and minor differences in the 5' ends of chimeric sequences in HSV2-LAT-E1 and HSV2-LAT-S1 point to sequences immediately downstream of the LAT TATA.

Latency-Associated Transcript (LAT) Exon 1 Controls Herpes Simplex Virus Species-Specific Phenotypes: Reactivation in the Guinea Pig Genital Model and Neuron Subtype-Specific Latent Expression of LAT▿

PubMed Central

Bertke, Andrea S.; Patel, Amita; Imai, Yumi; Apakupakul, Kathleen; Margolis, Todd P.; Krause, Philip R.

2009-01-01

Herpes simplex virus 1 (HSV-1) and HSV-2 cause similar acute infections but differ in their abilities to reactivate from trigeminal and lumbosacral dorsal root ganglia. During latency, HSV-1 and HSV-2 also preferentially express their latency-associated transcripts (LATs) in different sensory neuronal subtypes that are positive for A5 and KH10 markers, respectively. Chimeric virus studies showed that LAT region sequences influence both of these viral species-specific phenotypes. To further map the LAT region sequences responsible for these phenotypes, we constructed the chimeric virus HSV2-LAT-E1, in which exon 1 (from the LAT TATA to the intron splice site) was replaced by the corresponding sequence from HSV-1 LAT. In intravaginally infected guinea pigs, HSV2-LAT-E1 reactivated inefficiently relative to the efficiency of its rescuant and wild-type HSV-2, but it yielded similar levels of viral DNA, LAT, and ICP0 during acute and latent infection. HSV2-LAT-E1 preferentially expressed the LAT in A5+ neurons (as does HSV-1), while the chimeric viruses HSV2-LAT-P1 (LAT promoter swap) and HSV2-LAT-S1 (LAT sequence swap downstream of the promoter) exhibited neuron subtype-specific latent LAT expression phenotypes more similar to that of HSV-2 than that of HSV-1. Rescuant viruses displayed the wild-type HSV-2 phenotypes of efficient reactivation in the guinea pig genital model and a tendency to express LAT in KH10+ neurons. The region that is critical for HSV species-specific differences in latency and reactivation thus lies between the LAT TATA and the intron splice site, and minor differences in the 5′ ends of chimeric sequences in HSV2-LAT-E1 and HSV2-LAT-S1 point to sequences immediately downstream of the LAT TATA. PMID:19641003

Phenotypic and molecular identification of Fonsecaea pedrosoi strains isolated from chromoblastomycosis patients in Mexico and Venezuela.

PubMed

Carolina Rojas, O; León-Cachón, Rafael B R; Pérez-Maya, Antonio Alí; Aguirre-Garza, Marcelino; Moreno-Treviño, María G; González, Gloria M

2015-05-01

Chromoblastomycosis is a chronic granulomatous disease caused frequently by fungi of the Fonsecaea genus. The objective of this study was the phenotypic and molecular identification of F. pedrosoi strains isolated from chromoblastomycosis patients in Mexico and Venezuela. Ten strains were included in this study. For phenotypic identification, we used macroscopic and microscopic morphologies, carbohydrate assimilation test, urea hydrolysis, cixcloheximide tolerance, proteolitic activity and the thermotolerance test. The antifungal activity of five drugs was evaluated against the isolates. Molecular identification was performed by sequencing the internal transcribed spacer (ITS) ribosomal DNA regions of the isolated strains. The physiological analysis and morphological features were variable and the precise identification was not possible. All isolates were susceptible to itraconazole, terbinafine, voriconazole and posaconazole. Amphotericin B was the least effective drug. The alignment of the 559-nucleotide ITS sequences from our strains compared with sequences of GenBank revealed high homology with F. pedrosoi (EU285266.1). In this study, all patients were from rural areas, six from Mexico and four from Venezuela. Ten isolates were identified by phenotypic and molecular analysis, using ITS sequence and demonstrated that nine isolates from Mexico and Venezuela were 100% homologous and one isolate showed a small genetic distance. © 2015 Blackwell Verlag GmbH.

Short-Sequence DNA Repeats in Prokaryotic Genomes

PubMed Central

van Belkum, Alex; Scherer, Stewart; van Alphen, Loek; Verbrugh, Henri

1998-01-01

Short-sequence DNA repeat (SSR) loci can be identified in all eukaryotic and many prokaryotic genomes. These loci harbor short or long stretches of repeated nucleotide sequence motifs. DNA sequence motifs in a single locus can be identical and/or heterogeneous. SSRs are encountered in many different branches of the prokaryote kingdom. They are found in genes encoding products as diverse as microbial surface components recognizing adhesive matrix molecules and specific bacterial virulence factors such as lipopolysaccharide-modifying enzymes or adhesins. SSRs enable genetic and consequently phenotypic flexibility. SSRs function at various levels of gene expression regulation. Variations in the number of repeat units per locus or changes in the nature of the individual repeat sequences may result from recombination processes or polymerase inadequacy such as slipped-strand mispairing (SSM), either alone or in combination with DNA repair deficiencies. These rather complex phenomena can occur with relative ease, with SSM approaching a frequency of 10−4 per bacterial cell division and allowing high-frequency genetic switching. Bacteria use this random strategy to adapt their genetic repertoire in response to selective environmental pressure. SSR-mediated variation has important implications for bacterial pathogenesis and evolutionary fitness. Molecular analysis of changes in SSRs allows epidemiological studies on the spread of pathogenic bacteria. The occurrence, evolution and function of SSRs, and the molecular methods used to analyze them are discussed in the context of responsiveness to environmental factors, bacterial pathogenicity, epidemiology, and the availability of full-genome sequences for increasing numbers of microorganisms, especially those that are medically relevant. PMID:9618442

Large-Scale Comparative Phenotypic and Genomic Analyses Reveal Ecological Preferences of Shewanella Species and Identify Metabolic Pathways Conserved at the Genus Level ▿ †

PubMed Central

Rodrigues, Jorge L. M.; Serres, Margrethe H.; Tiedje, James M.

2011-01-01

The use of comparative genomics for the study of different microbiological species has increased substantially as sequence technologies become more affordable. However, efforts to fully link a genotype to its phenotype remain limited to the development of one mutant at a time. In this study, we provided a high-throughput alternative to this limiting step by coupling comparative genomics to the use of phenotype arrays for five sequenced Shewanella strains. Positive phenotypes were obtained for 441 nutrients (C, N, P, and S sources), with N-based compounds being the most utilized for all strains. Many genes and pathways predicted by genome analyses were confirmed with the comparative phenotype assay, and three degradation pathways believed to be missing in Shewanella were confirmed as missing. A number of previously unknown gene products were predicted to be parts of pathways or to have a function, expanding the number of gene targets for future genetic analyses. Ecologically, the comparative high-throughput phenotype analysis provided insights into niche specialization among the five different strains. For example, Shewanella amazonensis strain SB2B, isolated from the Amazon River delta, was capable of utilizing 60 C compounds, whereas Shewanella sp. strain W3-18-1, isolated from deep marine sediment, utilized only 25 of them. In spite of the large number of nutrient sources yielding positive results, our study indicated that except for the N sources, they were not sufficiently informative to predict growth phenotypes from increasing evolutionary distances. Our results indicate the importance of phenotypic evaluation for confirming genome predictions. This strategy will accelerate the functional discovery of genes and provide an ecological framework for microbial genome sequencing projects. PMID:21642407

Haptoglobin Phenotype Modifies Serum Iron Levels and the Effect of Smoking on Parkinson Disease Risk

PubMed Central

Costa-Mallen, Paola; Zabetian, Cyrus P.; Agarwal, Pinky; Hu, Shu-Ching; Yearout, Dora; Samii, Ali; Leverenz, James B.; Roberts, John W.; Checkoway, Harvey

2015-01-01

Introduction Haptoglobin is a hemoglobin-binding protein that exists in three functionally different phenotypes, and haptoglobin phenotype 2-1 has previously been associated with Parkinson disease (PD) risk, with mechanisms not elucidated. Some evidence is emerging that low levels of serum iron may increase PD risk. In this study we investigated whether PD patients have lower serum iron and ferritin than controls, and whether this is dependent on haptoglobin phenotype. We also investigated the effect of Hp phenotype as a modifier of the effect of smoking on PD risk. Methods The study population consisted of 128 PD patients and 226 controls. Serum iron, ferritin, and haptoglobin phenotype were determined, and compared between PD cases and controls. Stratified analysis by haptoglobin phenotype was performed to determine effect of haptoglobin phenotype on serum iron parameter differences between PD cases and controls and to investigate its role in the protective effect of smoking on PD risk. Results PD cases had lower serum iron than controls (83.28 ug/100ml vs 94.00 ug/100 ml, p 0.006), and in particular among subjects with phenotype 2-1. The protective effect of smoking on PD risk resulted stronger in subjects with phenotype 1-1 and 2-2, and weakest among subjects with phenotype 2-1. Ferritin levels were higher in PD cases than controls among subjects of White ethnicity. Conclusions Our results report for the first time that the haptoglobin phenotype may be a contributor of iron levels abnormalities in PD patients. The mechanisms for these haptoglobin-phenotype specific effects will have to be further elucidated. PMID:26228081

Huntington's Disease: Relationship Between Phenotype and Genotype.

PubMed

Sun, Yi-Min; Zhang, Yan-Bin; Wu, Zhi-Ying

2017-01-01

Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disease with the typical manifestations of involuntary movements, psychiatric and behavior disorders, and cognitive impairment. It is caused by the dynamic mutation in CAG triplet repeat number in exon 1 of huntingtin (HTT) gene. The symptoms of HD especially the age at onset are related to the genetic characteristics, both the CAG triplet repeat and the modified factors. Here, we reviewed the recent advancement on the genotype-phenotype relationship of HD, mainly focus on the characteristics of different expanded CAG repeat number, genetic modifiers, and CCG repeat number in the 3' end of CAG triplet repeat and their effects on the phenotype. We also reviewed the special forms of HD (juvenile HD, atypical onset HD, and homozygous HD) and their phenotype-genotype correlations. The review will aid clinicians to predict the onset age and disease course of HD, give the genetic counseling, and accelerate research into the HD mechanism.

Phenotype analysis of congenital and neurodevelopmental disorders in the next generation sequencing era.

PubMed

Carey, John C

2017-09-01

The designation, phenotype, was proposed as a term by Wilhelm Johannsen in 1909. The word is derived from the Greek, phano (showing) and typo (type), phanotypos. Phenotype has become a widely recognized term, even outside of the genetics community, in recent years with the ongoing identification of human disease genes. The term has been defined as the observable constitution of an organism, but sometimes refers to a condition when a person has a particular clinical presentation. Analysis of phenotype is a timely theme because advances in the understanding of the genetic basis of human disease and the emergence of next generation sequencing have spurred a renewed interest in phenotype and the proposal to establish a "Human Phenome Project." This article summarizes the principles of phenotype analysis that are important in medical genetics and describes approaches to comprehensive phenotype analysis in the investigation of patients with human disorders. I discuss the various elements related to disease phenotypes and highlight neurofibromatosis type 1 and the Elements of Morphology Project as illustrations of the principles. In recent years, the notion of "deep phenotyping" has emerged. Currently there are now a number of proposed strategies and resources to approach this concept. Not since the 1960s and 1970s has there been such an exciting time in the history of medicine surrounding the analysis of phenotype in genetic disorders. © 2017 Wiley Periodicals, Inc.

Interpreting a sequenced genome: toward a cosmid transgenic library of Caenorhabditis elegans.

PubMed

Janke, D L; Schein, J E; Ha, T; Franz, N W; O'Neil, N J; Vatcher, G P; Stewart, H I; Kuervers, L M; Baillie, D L; Rose, A M

1997-10-01

We have generated a library of transgenic Caenorhabditis elegans strains that carry sequenced cosmids from the genome of the nematode. Each strain carries an extrachromosomal array containing a single cosmid, sequenced by the C. elegans Genome Sequencing Consortium, and a dominate Rol-6 marker. More than 500 transgenic strains representing 250 cosmids have been constructed. Collectively, these strains contain approximately 8 Mb of sequence data, or approximately 8% of the C. elegans genome. The transgenic strains are being used to rescue mutant phenotypes, resulting in a high-resolution map alignment of the genetic, physical, and DNA sequence maps of the nematode. We have chosen the region of chromosome III deleted by sDf127 and not covered by the duplication sDp8(III;I) as a starting point for a systematic correlation of mutant phenotypes with nucleotide sequence. In this defined region, we have identified 10 new essential genes whose mutant phenotypes range from developmental arrest at early larva, to maternal effect lethal. To date, 8 of these 10 essential genes have been rescued. In this region, these rescues represent approximately 10% of the genes predicted by GENEFINDER and considerably enhance the map alignment. Furthermore, this alignment facilitates future efforts to physically position and clone other genes in the region. [Updated information about the Transgenic Library is available via the Internet at http://darwin.mbb.sfu.ca/imbb/dbaillie/cos mid.html.

Inherited Paediatric Motor Neuron Disorders: Beyond Spinal Muscular Atrophy

PubMed Central

Sampaio, Hugo; Mowat, David; Roscioli, Tony

2017-01-01

Paediatric motor neuron diseases encompass a group of neurodegenerative diseases characterised by the onset of muscle weakness and atrophy before the age of 18 years, attributable to motor neuron loss across various neuronal networks in the brain and spinal cord. While the genetic underpinnings are diverse, advances in next generation sequencing have transformed diagnostic paradigms. This has reinforced the clinical phenotyping and molecular genetic expertise required to navigate the complexities of such diagnoses. In turn, improved genetic technology and subsequent gene identification have enabled further insights into the mechanisms of motor neuron degeneration and how these diseases form part of a neurodegenerative disorder spectrum. Common pathophysiologies include abnormalities in axonal architecture and function, RNA processing, and protein quality control. This review incorporates an overview of the clinical manifestations, genetics, and pathophysiology of inherited paediatric motor neuron disorders beyond classic SMN1-related spinal muscular atrophy and describes recent advances in next generation sequencing and its clinical application. Specific disease-modifying treatment is becoming a clinical reality in some disorders of the motor neuron highlighting the importance of a timely and specific diagnosis. PMID:28634552

Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

NASA Astrophysics Data System (ADS)

Travella, Silvia; Keller, Beat

Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial Virus Prefusion F Protein Expressed by a Vaccine Candidate

PubMed Central

Liang, Bo; Surman, Sonja; Amaro-Carambot, Emerito; Kabatova, Barbora; Mackow, Natalie; Lingemann, Matthias; Yang, Lijuan; McLellan, Jason S.; Graham, Barney S.; Kwong, Peter D.; Schaap-Nutt, Anne; Collins, Peter L.

2015-01-01

ABSTRACT Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are the first and second leading viral agents of severe respiratory tract disease in infants and young children worldwide. Vaccines are not available, and an RSV vaccine is particularly needed. A live attenuated chimeric recombinant bovine/human PIV3 (rB/HPIV3) vector expressing the RSV fusion (F) glycoprotein from an added gene has been under development as a bivalent vaccine against RSV and HPIV3. Previous clinical evaluation of this vaccine candidate suggested that increased genetic stability and immunogenicity of the RSV F insert were needed. This was investigated in the present study. RSV F expression was enhanced 5-fold by codon optimization and by modifying the amino acid sequence to be identical to that of an early passage of the original clinical isolate. This conferred a hypofusogenic phenotype that presumably reflects the original isolate. We then compared vectors expressing stabilized prefusion and postfusion versions of RSV F. In a hamster model, prefusion F induced increased quantity and quality of RSV-neutralizing serum antibodies and increased protection against wild-type (wt) RSV challenge. In contrast, a vector expressing the postfusion F was less immunogenic and protective. The genetic stability of the RSV F insert was high and was not affected by enhanced expression or the prefusion or postfusion conformation of RSV F. These studies provide an improved version of the previously well-tolerated rB/HPIV3-RSV F vaccine candidate that induces a superior RSV-neutralizing serum antibody response. IMPORTANCE Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are two major causes of pediatric pneumonia and bronchiolitis. The rB/HPIV3 vector expressing RSV F protein is a candidate bivalent live vaccine against HPIV3 and RSV. Previous clinical evaluation indicated the need to increase the immunogenicity and genetic stability of the RSV F insert. Here, we increased RSV F expression by codon optimization and by modifying the RSV F amino acid sequence to conform to that of an early passage of the original isolate. This resulted in a hypofusogenic phenotype, which likely represents the original phenotype before adaptation to cell culture. We also included stabilized versions of prefusion and postfusion RSV F protein. Prefusion RSV F induced a larger quantity and higher quality of RSV-neutralizing serum antibodies and was highly protective. This provides an improved candidate for further clinical evaluation. PMID:26157122

Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial Virus Prefusion F Protein Expressed by a Vaccine Candidate.

PubMed

Liang, Bo; Surman, Sonja; Amaro-Carambot, Emerito; Kabatova, Barbora; Mackow, Natalie; Lingemann, Matthias; Yang, Lijuan; McLellan, Jason S; Graham, Barney S; Kwong, Peter D; Schaap-Nutt, Anne; Collins, Peter L; Munir, Shirin

2015-09-01

Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are the first and second leading viral agents of severe respiratory tract disease in infants and young children worldwide. Vaccines are not available, and an RSV vaccine is particularly needed. A live attenuated chimeric recombinant bovine/human PIV3 (rB/HPIV3) vector expressing the RSV fusion (F) glycoprotein from an added gene has been under development as a bivalent vaccine against RSV and HPIV3. Previous clinical evaluation of this vaccine candidate suggested that increased genetic stability and immunogenicity of the RSV F insert were needed. This was investigated in the present study. RSV F expression was enhanced 5-fold by codon optimization and by modifying the amino acid sequence to be identical to that of an early passage of the original clinical isolate. This conferred a hypofusogenic phenotype that presumably reflects the original isolate. We then compared vectors expressing stabilized prefusion and postfusion versions of RSV F. In a hamster model, prefusion F induced increased quantity and quality of RSV-neutralizing serum antibodies and increased protection against wild-type (wt) RSV challenge. In contrast, a vector expressing the postfusion F was less immunogenic and protective. The genetic stability of the RSV F insert was high and was not affected by enhanced expression or the prefusion or postfusion conformation of RSV F. These studies provide an improved version of the previously well-tolerated rB/HPIV3-RSV F vaccine candidate that induces a superior RSV-neutralizing serum antibody response. Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are two major causes of pediatric pneumonia and bronchiolitis. The rB/HPIV3 vector expressing RSV F protein is a candidate bivalent live vaccine against HPIV3 and RSV. Previous clinical evaluation indicated the need to increase the immunogenicity and genetic stability of the RSV F insert. Here, we increased RSV F expression by codon optimization and by modifying the RSV F amino acid sequence to conform to that of an early passage of the original isolate. This resulted in a hypofusogenic phenotype, which likely represents the original phenotype before adaptation to cell culture. We also included stabilized versions of prefusion and postfusion RSV F protein. Prefusion RSV F induced a larger quantity and higher quality of RSV-neutralizing serum antibodies and was highly protective. This provides an improved candidate for further clinical evaluation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Role of 16S rRNA Gene Sequencing in Identification of Microorganisms Misidentified by Conventional Methods

PubMed Central

Petti, C. A.; Polage, C. R.; Schreckenberger, P.

2005-01-01

Traditional methods for microbial identification require the recognition of differences in morphology, growth, enzymatic activity, and metabolism to define genera and species. Full and partial 16S rRNA gene sequencing methods have emerged as useful tools for identifying phenotypically aberrant microorganisms. We report on three bacterial blood isolates from three different College of American Pathologists-certified laboratories that were referred to ARUP Laboratories for definitive identification. Because phenotypic identification suggested unusual organisms not typically associated with the submitted clinical diagnosis, consultation with the Medical Director was sought and further testing was performed including partial 16S rRNA gene sequencing. All three patients had endocarditis, and conventional methods identified isolates from patients A, B, and C as a Facklamia sp., Eubacterium tenue, and a Bifidobacterium sp. 16S rRNA gene sequencing identified the isolates as Enterococcus faecalis, Cardiobacterium valvarum, and Streptococcus mutans, respectively. We conclude that the initial identifications of these three isolates were erroneous, may have misled clinicians, and potentially impacted patient care. 16S rRNA gene sequencing is a more objective identification tool, unaffected by phenotypic variation or technologist bias, and has the potential to reduce laboratory errors. PMID:16333109

Effects of phenotypes in heterocyclic aromatic amine (HCA) metabolism-related genes on the association of HCA intake with the risk of colorectal adenomas.

PubMed

Barbir, Aline; Linseisen, Jakob; Hermann, Silke; Kaaks, Rudolf; Teucher, Birgit; Eichholzer, Monika; Rohrmann, Sabine

2012-09-01

Heterocyclic aromatic amines (HCA), formed by high-temperature cooking of meat, are well-known risk factors for colorectal cancer (CRC). Enzymes metabolizing HCAs may influence the risk of CRC depending on the enzyme activity level. We aimed to assess effect modification by polymorphisms in the HCA-metabolizing genes on the association of HCA intake with colorectal adenoma (CRA) risk, which are precursors of CRC. A case-control study nested in the EPIC-Heidelberg cohort was conducted. Between 1994 and 2005, 413 adenoma cases were identified and 796 controls were matched to cases. Genotypes were determined and used to predict phenotypes (i.e., enzyme activities). Odds ratios (OR) and corresponding 95 % confidence intervals (CI) were calculated by logistic regression analysis. CRA risk was positively associated with PhIP, MeIQx, and DiMeIQx (p trend = 0.006, 0.022, and 0.045, respectively) intake. SULT1A1 phenotypes modified the effect of MeIQx on CRA risk (p (Interaction) > 0.01) such that the association of MeIQx intake with CRA was stronger for slow than for normal phenotypes. Other modifying effects by phenotypes did not reach statistical significance. HCA intake is positively associated with CRA risk, regardless of phenotypes involved in the metabolizing process. Due to the number of comparisons made in the analysis, the modifying effect of SULT1A1 on the association of HCA intake with CRA risk may be due to chance.

Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

DOE PAGES

Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; ...

2015-05-12

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with anymore » transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.« less

Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with anymore » transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.« less

The coexistence of mitochondrial ND6 T14484C and 12S rRNA A1555G mutations in a Chinese family with Leber's hereditary optic neuropathy and hearing loss

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei Qiping; Zhou Xiangtian; Yang Li

2007-06-15

We report here the clinical, genetic and molecular characterization of one three-generation Han Chinese family with Leber's hereditary optic neuropathy (LHON) and hearing loss. Four of 14 matrilineal relatives exhibited the moderate central vision loss at the average age of 12.5 years. Of these, one subject exhibited both LHON and mild hearing impairment. Sequence analysis of the complete mitochondrial genomes in the pedigree showed the presence of homoplasmic LHON-associated ND6 T14484C mutation, deafness-associated 12S rRNA A1555 mutation and 47 other variants belonging to Eastern Asian haplogroup H2. None of other mitochondrial variants was evolutionarily conserved and functional significance. Therefore, themore » coexistence of the A1555G mutation and T14484C mutations in this Chinese family indicate that the A1555G mutation may play a synergistic role in the phenotypic manifestation of LHON associated ND6 T14484C mutation. However, the incomplete penetrance of vision and hearing loss suggests the involvement of nuclear modifier genes and environmental factors in the phenotypic expression of these mtDNA mutations.« less

Mannosyltransferase is required for cell wall biosynthesis, morphology and control of asexual development in Neurospora crassa.

PubMed

Bowman, Shaun M; Piwowar, Amy; Ciocca, Maria; Free, Stephen J

2005-01-01

Two Neurospora mutants with a phenotype that includes a tight colonial growth pattern, an inability to form conidia and an inability to form protoperithecia have been isolated and characterized. The relevant mutations were mapped to the same locus on the sequenced Neurospora genome. The mutations responsible for the mutant phenotype then were identified by examining likely candidate genes from the mutant genomes at the mapped locus with PCR amplification and a sequencing assay. The results demonstrate that a map and sequence strategy is a feasible way to identify mutant genes in Neurospora. The gene responsible for the phenotype is a putative alpha-1,2-mannosyltransferase gene. The mutant cell wall has an altered composition demonstrating that the gene functions in cell wall biosynthesis. The results demonstrate that the mnt-1 gene is required for normal cell wall biosynthesis, morphology and for the regulation of asexual development.

A public platform for the verification of the phenotypic effect of candidate genes for resistance to aflatoxin accumulation and Aspergillus flavus infection in maize.

PubMed

Warburton, Marilyn L; Williams, William Paul; Hawkins, Leigh; Bridges, Susan; Gresham, Cathy; Harper, Jonathan; Ozkan, Seval; Mylroie, J Erik; Shan, Xueyan

2011-07-01

A public candidate gene testing pipeline for resistance to aflatoxin accumulation or Aspergillus flavus infection in maize is presented here. The pipeline consists of steps for identifying, testing, and verifying the association of selected maize gene sequences with resistance under field conditions. Resources include a database of genetic and protein sequences associated with the reduction in aflatoxin contamination from previous studies; eight diverse inbred maize lines for polymorphism identification within any maize gene sequence; four Quantitative Trait Loci (QTL) mapping populations and one association mapping panel, all phenotyped for aflatoxin accumulation resistance and associated phenotypes; and capacity for Insertion/Deletion (InDel) and SNP genotyping in the population(s) for mapping. To date, ten genes have been identified as possible candidate genes and put through the candidate gene testing pipeline, and results are presented here to demonstrate the utility of the pipeline.

Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations

PubMed Central

Siggs, Owen M.; Miosge, Lisa A.; Roots, Carla M.; Enders, Anselm; Bertram, Edward M.; Crockford, Tanya L.; Whittle, Belinda; Potter, Paul K.; Simon, Michelle M.; Mallon, Ann-Marie; Brown, Steve D. M.; Beutler, Bruce; Goodnow, Christopher C.; Lunter, Gerton; Cornall, Richard J.

2013-01-01

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis. PMID:23382690

Exome sequencing reveals a de novo POLD1 mutation causing phenotypic variability in mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL).

PubMed

Elouej, Sahar; Beleza-Meireles, Ana; Caswell, Richard; Colclough, Kevin; Ellard, Sian; Desvignes, Jean Pierre; Béroud, Christophe; Lévy, Nicolas; Mohammed, Shehla; De Sandre-Giovannoli, Annachiara

2017-06-01

Mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL) is an autosomal dominant systemic disorder characterized by prominent loss of subcutaneous fat, a characteristic facial appearance and metabolic abnormalities. This syndrome is caused by heterozygous de novo mutations in the POLD1 gene. To date, 19 patients with MDPL have been reported in the literature and among them 14 patients have been characterized at the molecular level. Twelve unrelated patients carried a recurrent in-frame deletion of a single codon (p.Ser605del) and two other patients carried a novel heterozygous mutation in exon 13 (p.Arg507Cys). Additionally and interestingly, germline mutations of the same gene have been involved in familial polyposis and colorectal cancer (CRC) predisposition. We describe a male and a female patient with MDPL respectively affected with mild and severe phenotypes. Both of them showed mandibular hypoplasia, a beaked nose with bird-like facies, prominent eyes, a small mouth, growth retardation, muscle and skin atrophy, but the female patient showed such a severe and early phenotype that a first working diagnosis of Hutchinson-Gilford Progeria was made. The exploration was performed by direct sequencing of POLD1 gene exon 15 in the male patient with a classical MDPL phenotype and by whole exome sequencing in the female patient and her unaffected parents. Exome sequencing identified in the latter patient a de novo heterozygous undescribed mutation in the POLD1 gene (NM_002691.3: c.3209T>A), predicted to cause the missense change p.Ile1070Asn in the ZnF2 (Zinc Finger 2) domain of the protein. This mutation was not reported in the 1000 Genome Project, dbSNP and Exome sequencing databases. Furthermore, the Isoleucine1070 residue of POLD1 is highly conserved among various species, suggesting that this substitution may cause a major impairment of POLD1 activity. For the second patient, affected with a typical MDPL phenotype, direct sequencing of POLD1 exon 15 revealed the recurrent in-frame deletion (c.1812_1814del, p.S605del). Our work highlights that mutations in different POLD1 domains can lead to phenotypic variability, ranging from dominantly inherited cancer predisposition syndromes, to mild MDPL phenotypes without lifespan reduction, to very severe MDPL syndromes with major premature aging features. These results also suggest that POLD1 gene testing should be considered in patients presenting with severe progeroid features. Copyright © 2017 Elsevier Inc. All rights reserved.

Multidimensional structure-function relationships in human β-cardiac myosin from population-scale genetic variation

PubMed Central

Homburger, Julian R.; Green, Eric M.; Caleshu, Colleen; Sunitha, Margaret S.; Taylor, Rebecca E.; Ruppel, Kathleen M.; Metpally, Raghu Prasad Rao; Colan, Steven D.; Michels, Michelle; Day, Sharlene M.; Olivotto, Iacopo; Bustamante, Carlos D.; Dewey, Frederick E.; Ho, Carolyn Y.; Spudich, James A.; Ashley, Euan A.

2016-01-01

Myosin motors are the fundamental force-generating elements of muscle contraction. Variation in the human β-cardiac myosin heavy chain gene (MYH7) can lead to hypertrophic cardiomyopathy (HCM), a heritable disease characterized by cardiac hypertrophy, heart failure, and sudden cardiac death. How specific myosin variants alter motor function or clinical expression of disease remains incompletely understood. Here, we combine structural models of myosin from multiple stages of its chemomechanical cycle, exome sequencing data from two population cohorts of 60,706 and 42,930 individuals, and genetic and phenotypic data from 2,913 patients with HCM to identify regions of disease enrichment within β-cardiac myosin. We first developed computational models of the human β-cardiac myosin protein before and after the myosin power stroke. Then, using a spatial scan statistic modified to analyze genetic variation in protein 3D space, we found significant enrichment of disease-associated variants in the converter, a kinetic domain that transduces force from the catalytic domain to the lever arm to accomplish the power stroke. Focusing our analysis on surface-exposed residues, we identified a larger region significantly enriched for disease-associated variants that contains both the converter domain and residues on a single flat surface on the myosin head described as the myosin mesa. Notably, patients with HCM with variants in the enriched regions have earlier disease onset than patients who have HCM with variants elsewhere. Our study provides a model for integrating protein structure, large-scale genetic sequencing, and detailed phenotypic data to reveal insight into time-shifted protein structures and genetic disease. PMID:27247418

Use of whole-genome sequencing for Campylobacter surveillance from NARMS retail poultry in the United States in 2015.

PubMed

Whitehouse, Chris A; Young, Shenia; Li, Cong; Hsu, Chih-Hao; Martin, Gordon; Zhao, Shaohua

2018-08-01

Whole genome sequencing (WGS) has become a rapid and affordable tool for public health surveillance and outbreak detection. In this study, we used the Illuminia MiSeq ® to sequence 589 Campylobacter isolates obtained in 2015 from retail poultry meats as part of the National Antimicrobial Resistance Monitoring System (NARMS). WGS data were used to identify the Campylobacter species and to compare the concordance between resistance genotypes and phenotypes. WGS accurately identified 386 C. jejuni and 203 C. coli using gyrA sequence information. Ten resistance genes, including tetO, bla OXA-61 , aph(2″)-Ic, aph(2″)-If, aph(2″)-Ig, aph(3')-III, ant(6)-1a, aadE, aph(3")-VIIa, and Inu(C), plus mutations in housekeeping genes (gyrA at position 86, 23S rRNA at position 2074 and 2075), were identified by WGS analysis. Overall, there was a high concordance between phenotypic resistance to a given drug and the presence of known resistance genes. Concordance between both resistance and susceptible phenotypes and genotype was 100% for ciprofloxacin, nalidixic acid, gentamicin, azithromycin, and florfenicol. A few discrepancies were observed for tetracycline, clindamycin, and telithromycin. The concordance between resistance phenotype and genotype ranged from 67.9% to 100%; whereas, the concordance between susceptible phenotype and genotype ranged from 98.0% to 99.6%. Our study demonstrates that WGS can correctly identify Campylobacter species and predict antimicrobial resistance with a high degree of accuracy. Published by Elsevier Ltd.

Genotypic and phenotypic evaluation of off-type grasses in hybrid Bermudagrass [Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt-Davy] putting greens using genotyping-by-sequencing and morphological characterization.

PubMed

Reasor, Eric H; Brosnan, James T; Staton, Margaret E; Lane, Thomas; Trigiano, Robert N; Wadl, Phillip A; Conner, Joann A; Schwartz, Brian M

2018-01-01

Interspecific hybrid bermudagrass [ Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt-Davy] is one of the most widely used grasses on golf courses, with cultivars derived from 'Tifgreen' or 'Tifdwarf' particularly used for putting greens. Many bermudagrass cultivars established for putting greens can be genetically unstable and lead to the occurrence of undesirable off-type grasses that vary in phenotype. The objective of this research was to genetically and phenotypically differentiate off-type grasses and hybrid cultivars. Beginning in 2013, off-type and desirable hybrid bermudagrass samples were collected from golf course putting greens in the southeastern United States and genetically and phenotypically characterized using genotyping-by-sequencing and morphology. Genotyping-by-sequencing determined that 11% (5) of off-type and desirable samples from putting greens were genetically divergent from standard cultivars such as Champion, MiniVerde, Tifdwarf, TifEagle, and Tifgreen. In addition, genotyping-by-sequencing was unable to genetically distinguish all standard cultivars from one another due to their similar origin and clonal propagation; however, over 90,000 potentially informative nucleotide variants were identified among the triploid hybrid cultivars. Although few genetic differences were found in this research, samples harvested from golf course putting greens had variable morphology and were clustered into three distinct phenotypic groups. The majority of off-type grasses in hybrid bermudagrass putting greens were genetically similar with variable morphological traits. Off-type grasses within golf course putting greens have the potential to compromise putting surface functionality and aesthetics.

Unraveling Genetic Modifiers in the Gria4 Mouse Model of Absence Epilepsy

PubMed Central

Frankel, Wayne N.; Mahaffey, Connie L.; McGarr, Tracy C.; Beyer, Barbara J.; Letts, Verity A.

2014-01-01

Absence epilepsy (AE) is a common type of genetic generalized epilepsy (GGE), particularly in children. AE and GGE are complex genetic diseases with few causal variants identified to date. Gria4 deficient mice provide a model of AE, one for which the common laboratory inbred strain C3H/HeJ (HeJ) harbors a natural IAP retrotransposon insertion in Gria4 that reduces its expression 8-fold. Between C3H and non-seizing strains such as C57BL/6, genetic modifiers alter disease severity. Even C3H substrains have surprising variation in the duration and incidence of spike-wave discharges (SWD), the characteristic electroencephalographic feature of absence seizures. Here we discovered extensive IAP retrotransposition in the C3H substrain, and identified a HeJ-private IAP in the Pcnxl2 gene, which encodes a putative multi-transmembrane protein of unknown function, resulting in decreased expression. By creating new Pcnxl2 frameshift alleles using TALEN mutagenesis, we show that Pcnxl2 deficiency is responsible for mitigating the seizure phenotype – making Pcnxl2 the first known modifier gene for absence seizures in any species. This finding gave us a handle on genetic complexity between strains, directing us to use another C3H substrain to map additional modifiers including validation of a Chr 15 locus that profoundly affects the severity of SWD episodes. Together these new findings expand our knowledge of how natural variation modulates seizures, and highlights the feasibility of characterizing and validating modifiers in mouse strains and substrains in the post-genome sequence era. PMID:25010494

Application of whole genome shotgun sequencing for detection and characterization of genetically modified organisms and derived products.

PubMed

Holst-Jensen, Arne; Spilsberg, Bjørn; Arulandhu, Alfred J; Kok, Esther; Shi, Jianxin; Zel, Jana

2016-07-01

The emergence of high-throughput, massive or next-generation sequencing technologies has created a completely new foundation for molecular analyses. Various selective enrichment processes are commonly applied to facilitate detection of predefined (known) targets. Such approaches, however, inevitably introduce a bias and are prone to miss unknown targets. Here we review the application of high-throughput sequencing technologies and the preparation of fit-for-purpose whole genome shotgun sequencing libraries for the detection and characterization of genetically modified and derived products. The potential impact of these new sequencing technologies for the characterization, breeding selection, risk assessment, and traceability of genetically modified organisms and genetically modified products is yet to be fully acknowledged. The published literature is reviewed, and the prospects for future developments and use of the new sequencing technologies for these purposes are discussed.

Genome sequencing reveals loci under artificial selection that underlie disease phenotypes in the laboratory rat.

PubMed

Atanur, Santosh S; Diaz, Ana Garcia; Maratou, Klio; Sarkis, Allison; Rotival, Maxime; Game, Laurence; Tschannen, Michael R; Kaisaki, Pamela J; Otto, Georg W; Ma, Man Chun John; Keane, Thomas M; Hummel, Oliver; Saar, Kathrin; Chen, Wei; Guryev, Victor; Gopalakrishnan, Kathirvel; Garrett, Michael R; Joe, Bina; Citterio, Lorena; Bianchi, Giuseppe; McBride, Martin; Dominiczak, Anna; Adams, David J; Serikawa, Tadao; Flicek, Paul; Cuppen, Edwin; Hubner, Norbert; Petretto, Enrico; Gauguier, Dominique; Kwitek, Anne; Jacob, Howard; Aitman, Timothy J

2013-08-01

Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Genome Sequencing Reveals Loci under Artificial Selection that Underlie Disease Phenotypes in the Laboratory Rat

PubMed Central

Atanur, Santosh S.; Diaz, Ana Garcia; Maratou, Klio; Sarkis, Allison; Rotival, Maxime; Game, Laurence; Tschannen, Michael R.; Kaisaki, Pamela J.; Otto, Georg W.; Ma, Man Chun John; Keane, Thomas M.; Hummel, Oliver; Saar, Kathrin; Chen, Wei; Guryev, Victor; Gopalakrishnan, Kathirvel; Garrett, Michael R.; Joe, Bina; Citterio, Lorena; Bianchi, Giuseppe; McBride, Martin; Dominiczak, Anna; Adams, David J.; Serikawa, Tadao; Flicek, Paul; Cuppen, Edwin; Hubner, Norbert; Petretto, Enrico; Gauguier, Dominique; Kwitek, Anne; Jacob, Howard; Aitman, Timothy J.

2013-01-01

Summary Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models. PaperClip PMID:23890820

The Genetic Basis of Mendelian Phenotypes: Discoveries, Challenges, and Opportunities

PubMed Central

Chong, Jessica X.; Buckingham, Kati J.; Jhangiani, Shalini N.; Boehm, Corinne; Sobreira, Nara; Smith, Joshua D.; Harrell, Tanya M.; McMillin, Margaret J.; Wiszniewski, Wojciech; Gambin, Tomasz; Coban Akdemir, Zeynep H.; Doheny, Kimberly; Scott, Alan F.; Avramopoulos, Dimitri; Chakravarti, Aravinda; Hoover-Fong, Julie; Mathews, Debra; Witmer, P. Dane; Ling, Hua; Hetrick, Kurt; Watkins, Lee; Patterson, Karynne E.; Reinier, Frederic; Blue, Elizabeth; Muzny, Donna; Kircher, Martin; Bilguvar, Kaya; López-Giráldez, Francesc; Sutton, V. Reid; Tabor, Holly K.; Leal, Suzanne M.; Gunel, Murat; Mane, Shrikant; Gibbs, Richard A.; Boerwinkle, Eric; Hamosh, Ada; Shendure, Jay; Lupski, James R.; Lifton, Richard P.; Valle, David; Nickerson, Deborah A.; Bamshad, Michael J.

2015-01-01

Discovering the genetic basis of a Mendelian phenotype establishes a causal link between genotype and phenotype, making possible carrier and population screening and direct diagnosis. Such discoveries also contribute to our knowledge of gene function, gene regulation, development, and biological mechanisms that can be used for developing new therapeutics. As of February 2015, 2,937 genes underlying 4,163 Mendelian phenotypes have been discovered, but the genes underlying ∼50% (i.e., 3,152) of all known Mendelian phenotypes are still unknown, and many more Mendelian conditions have yet to be recognized. This is a formidable gap in biomedical knowledge. Accordingly, in December 2011, the NIH established the Centers for Mendelian Genomics (CMGs) to provide the collaborative framework and infrastructure necessary for undertaking large-scale whole-exome sequencing and discovery of the genetic variants responsible for Mendelian phenotypes. In partnership with 529 investigators from 261 institutions in 36 countries, the CMGs assessed 18,863 samples from 8,838 families representing 579 known and 470 novel Mendelian phenotypes as of January 2015. This collaborative effort has identified 956 genes, including 375 not previously associated with human health, that underlie a Mendelian phenotype. These results provide insight into study design and analytical strategies, identify novel mechanisms of disease, and reveal the extensive clinical variability of Mendelian phenotypes. Discovering the gene underlying every Mendelian phenotype will require tackling challenges such as worldwide ascertainment and phenotypic characterization of families affected by Mendelian conditions, improvement in sequencing and analytical techniques, and pervasive sharing of phenotypic and genomic data among researchers, clinicians, and families. PMID:26166479

Genotype to Phenotype Mapping of the E. coli lac Promoter

NASA Astrophysics Data System (ADS)

Otwinowski, Jakub; Nemenman, Ilya

2014-03-01

Genotype-to-phenotype maps and the related fitness landscapes that include epistatic interactions are difficult to measure because of their high dimensional structure. Here we construct such a map using the recently collected corpora of high-throughput sequence data from the 75 base pairs long mutagenized E. coli lac promoter region, where each sequence is associated with induced transcriptional activity measured by a fluorescent reporter. We find that the additive (non-epistatic) contributions of individual mutations account for about two-thirds of the explainable phenotype variance, while pairwise epistasis explains about 7% of the variance for the full mutagenized sequence and about 15% for the subsequence associated with protein binding sites. Surprisingly, there is no evidence for third order epistatic contributions, and our inferred fitness landscape is essentially single peaked, with a small amount of antagonistic epistasis. We identify transcription factor (CRP) and RNA polymerase binding sites in the promotor region and their interactions. We conclude with a cautionary note that inferred properties of fitness landscapes may be severely influenced by biases in the sequence data. Funded in part by HFSP and James S. McDonnell Foundation.

Application of single-cell sequencing in human cancer.

PubMed

Rantalainen, Mattias

2017-11-02

Precision medicine is emerging as a cornerstone of future cancer care with the objective of providing targeted therapies based on the molecular phenotype of each individual patient. Traditional bulk-level molecular phenotyping of tumours leads to significant information loss, as the molecular profile represents an average phenotype over large numbers of cells, while cancer is a disease with inherent intra-tumour heterogeneity at the cellular level caused by several factors, including clonal evolution, tissue hierarchies, rare cells and dynamic cell states. Single-cell sequencing provides means to characterize heterogeneity in a large population of cells and opens up opportunity to determine key molecular properties that influence clinical outcomes, including prognosis and probability of treatment response. Single-cell sequencing methods are now reliable enough to be used in many research laboratories, and we are starting to see applications of these technologies for characterization of human primary cancer cells. In this review, we provide an overview of studies that have applied single-cell sequencing to characterize human cancers at the single-cell level, and we discuss some of the current challenges in the field. © The Author 2017. Published by Oxford University Press.

Holistic and component plant phenotyping using temporal image sequence.

PubMed

Das Choudhury, Sruti; Bashyam, Srinidhi; Qiu, Yumou; Samal, Ashok; Awada, Tala

2018-01-01

Image-based plant phenotyping facilitates the extraction of traits noninvasively by analyzing large number of plants in a relatively short period of time. It has the potential to compute advanced phenotypes by considering the whole plant as a single object (holistic phenotypes) or as individual components, i.e., leaves and the stem (component phenotypes), to investigate the biophysical characteristics of the plants. The emergence timing, total number of leaves present at any point of time and the growth of individual leaves during vegetative stage life cycle of the maize plants are significant phenotypic expressions that best contribute to assess the plant vigor. However, image-based automated solution to this novel problem is yet to be explored. A set of new holistic and component phenotypes are introduced in this paper. To compute the component phenotypes, it is essential to detect the individual leaves and the stem. Thus, the paper introduces a novel method to reliably detect the leaves and the stem of the maize plants by analyzing 2-dimensional visible light image sequences captured from the side using a graph based approach. The total number of leaves are counted and the length of each leaf is measured for all images in the sequence to monitor leaf growth. To evaluate the performance of the proposed algorithm, we introduce University of Nebraska-Lincoln Component Plant Phenotyping Dataset (UNL-CPPD) and provide ground truth to facilitate new algorithm development and uniform comparison. The temporal variation of the component phenotypes regulated by genotypes and environment (i.e., greenhouse) are experimentally demonstrated for the maize plants on UNL-CPPD. Statistical models are applied to analyze the greenhouse environment impact and demonstrate the genetic regulation of the temporal variation of the holistic phenotypes on the public dataset called Panicoid Phenomap-1. The central contribution of the paper is a novel computer vision based algorithm for automated detection of individual leaves and the stem to compute new component phenotypes along with a public release of a benchmark dataset, i.e., UNL-CPPD. Detailed experimental analyses are performed to demonstrate the temporal variation of the holistic and component phenotypes in maize regulated by environment and genetic variation with a discussion on their significance in the context of plant science.

The origin of human complex diversity: Stochastic epistatic modules and the intrinsic compatibility between distributional robustness and phenotypic changeability.

PubMed

Ijichi, Shinji; Ijichi, Naomi; Ijichi, Yukina; Imamura, Chikako; Sameshima, Hisami; Kawaike, Yoichi; Morioka, Hirofumi

2018-01-01

The continuing prevalence of a highly heritable and hypo-reproductive extreme tail of a human neurobehavioral quantitative diversity suggests the possibility that the reproductive majority retains the genetic mechanism for the extremes. From the perspective of stochastic epistasis, the effect of an epistatic modifier variant can randomly vary in both phenotypic value and effect direction among the careers depending on the genetic individuality, and the modifier careers are ubiquitous in the population distribution. The neutrality of the mean genetic effect in the careers warrants the survival of the variant under selection pressures. Functionally or metabolically related modifier variants make an epistatic network module and dozens of modules may be involved in the phenotype. To assess the significance of stochastic epistasis, a simplified module-based model was employed. The individual repertoire of the modifier variants in a module also participates in the genetic individuality which determines the genetic contribution of each modifier in the career. Because the entire contribution of a module to the phenotypic outcome is consequently unpredictable in the model, the module effect represents the total contribution of the related modifiers as a stochastic unit in the simulations. As a result, the intrinsic compatibility between distributional robustness and quantitative changeability could mathematically be simulated using the model. The artificial normal distribution shape in large-sized simulations was preserved in each generation even if the lowest fitness tail was un-reproductive. The robustness of normality beyond generations is analogous to the real situations of human complex diversity including neurodevelopmental conditions. The repeated regeneration of the un-reproductive extreme tail may be inevitable for the reproductive majority's competence to survive and change, suggesting implications of the extremes for others. Further model-simulations to illustrate how the fitness of extreme individuals can be low through generations may be warranted to increase the credibility of this stochastic epistasis model.

Investigation of timing effects in modified composite quadrupolar echo pulse sequences by mean of average Hamiltonian theory

NASA Astrophysics Data System (ADS)

Mananga, Eugene Stephane

2018-01-01

The utility of the average Hamiltonian theory and its antecedent the Magnus expansion is presented. We assessed the concept of convergence of the Magnus expansion in quadrupolar spectroscopy of spin-1 via the square of the magnitude of the average Hamiltonian. We investigated this approach for two specific modified composite pulse sequences: COM-Im and COM-IVm. It is demonstrated that the size of the square of the magnitude of zero order average Hamiltonian obtained on the appropriated basis is a viable approach to study the convergence of the Magnus expansion. The approach turns to be efficient in studying pulse sequences in general and can be very useful to investigate coherent averaging in the development of high resolution NMR technique in solids. This approach allows comparing theoretically the two modified composite pulse sequences COM-Im and COM-IVm. We also compare theoretically the current modified composite sequences (COM-Im and COM-IVm) to the recently published modified composite pulse sequences (MCOM-I, MCOM-IV, MCOM-I_d, MCOM-IV_d).

Dystonia: an update on phenomenology, classification, pathogenesis and treatment.

PubMed

Balint, Bettina; Bhatia, Kailash P

2014-08-01

This article will highlight recent advances in dystonia with focus on clinical aspects such as the new classification, syndromic approach, new gene discoveries and genotype-phenotype correlations. Broadening of phenotype of some of the previously described hereditary dystonias and environmental risk factors and trends in treatment will be covered. Based on phenomenology, a new consensus update on the definition, phenomenology and classification of dystonia and a syndromic approach to guide diagnosis have been proposed. Terminology has changed and 'isolated dystonia' is used wherein dystonia is the only motor feature apart from tremor, and the previously called heredodegenerative dystonias and dystonia plus syndromes are now subsumed under 'combined dystonia'. The recently discovered genes ANO3, GNAL and CIZ1 appear not to be a common cause of adult-onset cervical dystonia. Clinical and genetic heterogeneity underlie myoclonus-dystonia, dopa-responsive dystonia and deafness-dystonia syndrome. ALS2 gene mutations are a newly recognized cause for combined dystonia. The phenotypic and genotypic spectra of ATP1A3 mutations have considerably broadened. Two new genome-wide association studies identified new candidate genes. A retrospective analysis suggested complicated vaginal delivery as a modifying risk factor in DYT1. Recent studies confirm lasting therapeutic effects of deep brain stimulation in isolated dystonia, good treatment response in myoclonus-dystonia, and suggest that early treatment correlates with a better outcome. Phenotypic classification continues to be important to recognize particular forms of dystonia and this includes syndromic associations. There are a number of genes underlying isolated or combined dystonia and there will be further new discoveries with the advances in genetic technologies such as exome and whole-genome sequencing. The identification of new genes will facilitate better elucidation of pathogenetic mechanisms and possible corrective therapies.

New PAH gene promoter KLF1 and 3'-region C/EBPalpha motifs influence transcription in vitro.

PubMed

Klaassen, Kristel; Stankovic, Biljana; Kotur, Nikola; Djordjevic, Maja; Zukic, Branka; Nikcevic, Gordana; Ugrin, Milena; Spasovski, Vesna; Srzentic, Sanja; Pavlovic, Sonja; Stojiljkovic, Maja

2017-02-01

Phenylketonuria (PKU) is a metabolic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. Although the PAH genotype remains the main determinant of PKU phenotype severity, genotype-phenotype inconsistencies have been reported. In this study, we focused on unanalysed sequences in non-coding PAH gene regions to assess their possible influence on the PKU phenotype. We transiently transfected HepG2 cells with various chloramphenicol acetyl transferase (CAT) reporter constructs which included PAH gene non-coding regions. Selected non-coding regions were indicated by in silico prediction to contain transcription factor binding sites. Furthermore, electrophoretic mobility shift assay (EMSA) and supershift assays were performed to identify which transcriptional factors were engaged in the interaction. We found novel KLF1 motif in the PAH promoter, which decreases CAT activity by 50 % in comparison to basal transcription in vitro. The cytosine at the c.-170 promoter position creates an additional binding site for the protein complex involving KLF1 transcription factor. Moreover, we assessed for the first time the role of a multivariant variable number tandem repeat (VNTR) region located in the 3'-region of the PAH gene. We found that the VNTR3, VNTR7 and VNTR8 constructs had approximately 60 % of CAT activity. The regulation is mediated by the C/EBPalpha transcription factor, present in protein complex binding to VNTR3. Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the PAH gene which decrease transcription in vitro and, thus, could be considered as PAH expression modifiers. New transcription motifs in non-coding regions will contribute to better understanding of the PKU phenotype complexity and may become important for the optimisation of PKU treatment.

Epigenetics in comparative biology: why we should pay attention.

PubMed

Burggren, Warren W; Crews, David

2014-07-01

The past decade has seen an explosion of articles in scientific journals involving non-genetic influences on phenotype through modulation of gene function without changes in gene sequence. The excitement in modern molecular biology surrounding the impact exerted by the environment on development of the phenotype is focused largely on mechanism and has not incorporated questions asked (and answers provided) by early philosophers, biologists, and psychologists. As such, this emergence of epigenetic studies is somewhat "old wine in new bottles" and represents a reformulation of the old debate of preformationism versus epigenesis-one resolved in the 1800s. Indeed, this tendency to always look forward, with minimal concern or regard of what has gone before, has led to the present situation in which "true" epigenetic studies are believed to consist of one of two schools. The first is primarily medically based and views epigenetic mechanisms as pathways for disease (e.g., "the epigenetics of cancer"). The second is primarily from the basic sciences, particularly molecular genetics, and regards epigenetics as a potentially important mechanism for organisms exposed to variable environments across multiple generations. There is, however, a third, and separate, school based on the historical literature and debates and regards epigenetics as more of a perspective than a phenomenon. Against this backdrop, comparative integrative biologists are particularly well-suited to understand epigenetic phenomena as a way for organisms to respond rapidly with modified phenotypes (relative to natural selection) to changes in the environment. Using evolutionary principles, it is also possible to interpret "sunsetting" of modified phenotypes when environmental conditions result in a disappearance of the epigenetic modification of gene regulation. Comparative integrative biologists also recognize epigenetics as a potentially confounding source of variation in their data. Epigenetic modification of phenotype (molecular, cellular, morphological, physiological, and behavioral) can be highly variable depending upon ancestral environmental exposure and can contribute to apparent "random" noise in collected datasets. Thus, future research should go beyond the study of epigenetic mechanisms at the level of the gene and devote additional investigation of epigenetic outcomes at the level of both the individual organism and how it affects the evolution of populations. This review is the first of seven in this special issue of Integrative and Comparative Biology that addresses in detail these and other key topics in the study of epigenetics. © The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Looking beyond the exome: a phenotype-first approach to molecular diagnostic resolution in rare and undiagnosed diseases

PubMed Central

Pena, Loren DM; Jiang, Yong-Hui; Schoch, Kelly; Spillmann, Rebecca C.; Walley, Nicole; Stong, Nicholas; Horn, Sarah Rapisardo; Sullivan, Jennifer A.; McConkie-Rosell, Allyn; Kansagra, Sujay; Smith, Edward C.; El-Dairi, Mays; Bellet, Jane; Ann Keels, Martha; Jasien, Joan; Kranz, Peter G.; Noel, Richard; Nagaraj, Shashi K.; Lark, Robert K.; Wechsler, Daniel SG; del Gaudio, Daniela; Leung, Marco L.; Hendon, Laura G.; Parker, Collette C.; Jones, Kelly L.; Goldstein, David B.; Shashi, Vandana

2017-01-01

Purpose To describe examples of missed pathogenic variants on whole exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing. Methods Guided by phenotypic information, three children with negative WES underwent targeted single gene testing. Results Individual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and an NGS-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the non-coding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity and MRI changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, likely missed due to failure of alignment. Conclusions These cases illustrate potential pitfalls of WES/NGS testing, and the importance of phenotype-guided molecular testing in yielding diagnoses. PMID:28914269

Present Day Biology seen in the Looking Glass of Physics of Complexity

NASA Astrophysics Data System (ADS)

Schuster, P.

Darwin's theory of variation and selection in its simplest form is directly applicable to RNA evolution in vitro as well as to virus evolution, and it allows for quantitative predictions. Understanding evolution at the molecular level is ultimately related to the central paradigm of structural biology: sequence⇒ structure ⇒ function. We elaborate on the state of the art in modeling and understanding evolution of RNA driven by reproduction and mutation. The focus will be laid on the landscape concept—originally introduced by Sewall Wright—and its application to problems in biology. The relation between genotypes and phenotypes is the result of two consecutive mappings from a space of genotypes called sequence space onto a space of phenotypes or structures, and fitness is the result of a mapping from phenotype space into non-negative real numbers. Realistic landscapes as derived from folding of RNA sequences into structures are characterized by two properties: (i) they are rugged in the sense that sequences lying nearby in sequence space may have very different fitness values and (ii) they are characterized by an appreciable degree of neutrality implying that a certain fraction of genotypes and/or phenotypes cannot be distinguished in the selection process. Evolutionary dynamics on realistic landscapes will be studied as a function of the mutation rate, and the role of neutrality in the selection process will be discussed.

A targeted genetic modifier screen links the SWI2/SNF2 protein domino to growth and autophagy genes in Drosophila melanogaster.

PubMed

Kwon, Matt Hyoung; Callaway, Heather; Zhong, Jim; Yedvobnick, Barry

2013-05-20

Targeted genetic studies can facilitate phenotypic analyses and provide important insights into development and other complex processes. The SWI2/SNF2 DNA-dependent ATPase Domino (Dom) of Drosophila melanogaster, a component of the Tip60 acetyltransferase complex, has been associated with a wide spectrum of cellular processes at multiple developmental stages. These include hematopoiesis, cell proliferation, homeotic gene regulation, histone exchange during DNA repair, and Notch signaling. To explore the wider gene network associated with Dom action, we used RNAi directed against domino (dom) to mediate loss-of-function at the wing margin, a tissue that is readily scored for phenotypic changes. Dom RNAi driven through GAL4-UAS elicited dominant wing nicking that responded phenotypically to the dose of dom and other loci known to function with dom. We screened for phenotypic modifiers of this wing phenotype among 2500 transpositions of the EP P element and found both enhancers and suppressors. Several classes of modifier were obtained, including those encoding transcription factors, RNA regulatory proteins, and factors that regulate cell growth, proliferation and autophagy, a lysosomal degradation pathway that affects cell growth under conditions of starvation and stress. Our analysis is consistent with prior studies, suggesting that Dom acts pleiotropically as a positive effector of Notch signaling and a repressor of proliferation. This genetic system should facilitate screens for additional loci associated with Dom function, and complement biochemical approaches to their regulatory activity.

Genotype and cardiovascular phenotype correlations with TBX1 in 1,022 velo-cardio- facial/DiGeorge/22q11.2 deletion syndrome patients

PubMed Central

Guo, Tingwei; McGinn, Donna McDonald; Blonska, Anna; Shanske, Alan; Bassett, Anne; Chow, Eva; Bowser, Mark; Sheridan, Molly; Beemer, Frits; Devriendt, Koen; Swillen, Ann; Breckpot, Jeroen; Digilio, M. Cristina; Marino, Bruno; Dallapiccola, Bruno; Carpenter, Courtney; Zheng, Xin; Johnson, Jacob; Chung, Jonathan; Higgins, Anne Marie; Philip, Nicole; Simon, Tony J.; Coleman, Karlene; Heine-Suner, Damian; Rosell, Jordi; Kates, Wendy; Devoto, Marcella; Goldmuntz, Elizabeth; Zackai, Elaine; Wang, Tao; Shprintzen, Robert; Emanuel, Beverly; Morrow, Bernice

2011-01-01

Haploinsufficiency of TBX1, encoding a T-box transcription factor, is largely responsible for the physical malformations in velo-cardio-facial/DiGeorge/22q11.2 deletion syndrome (22q11DS) patients. Cardiovascular malformations in these patients are highly variable, raising the question as to whether DNA variations in the TBX1 locus on the remaining allele of 22q11.2, could be responsible. To test this, a large sample size is needed. The TBX1 gene was sequenced in 360 consecutive 22q11DS patients. Rare and common variations were identified. We did not detect enrichment in rare SNP number in those with or without a congenital heart defect. One exception was that there was increased number of very rare SNPs between those with normal heart anatomy compared to those with right-sided aortic arch or persistent truncus arteriosus, suggesting potentially protective roles in the SNPs for these phenotype enrichment groups. Nine common SNPs (MAF >0.05) were chosen and used to genotype the entire cohort of 1,022 22q11DS subjects. We did not find a correlation between common SNPs or haplotypes and cardiovascular phenotype. This work demonstrates that common DNA variations in TBX1 do not explain variable cardiovascular expression in 22q11DS patients, implicating existence of modifiers in other genes on 22q11.2 or elsewhere in the genome. PMID:21796729

Using Mouse Models to Explore Genotype-Phenotype Relationship in Down Syndrome

ERIC Educational Resources Information Center

Salehi, Ahmad; Faizi, Mehrdad; Belichenko, Pavel V.; Mobley, William C.

2007-01-01

Down Syndrome (DS) caused by trisomy 21 is characterized by a variety of phenotypes and involves multiple organs. Sequencing of human chromosome 21 (HSA21) and subsequently of its orthologues on mouse chromosome 16 have created an unprecedented opportunity to explore the complex relationship between various DS phenotypes and the extra copy of…

Myopathy With SQSTM1 and TIA1 Variants: Clinical and Pathological Features.

PubMed

Niu, Zhiyv; Pontifex, Carly Sabine; Berini, Sarah; Hamilton, Leslie E; Naddaf, Elie; Wieben, Eric; Aleff, Ross A; Martens, Kristina; Gruber, Angela; Engel, Andrew G; Pfeffer, Gerald; Milone, Margherita

2018-01-01

The aim of this study is to identify the molecular defect of three unrelated individuals with late-onset predominant distal myopathy; to describe the spectrum of phenotype resulting from the contributing role of two variants in genes located on two different chromosomes; and to highlight the underappreciated complex forms of genetic myopathies. Clinical and laboratory data of three unrelated probands with predominantly distal weakness manifesting in the sixth-seventh decade of life, and available affected and unaffected family members were reviewed. Next-generation sequencing panel, whole exome sequencing, and targeted analyses of family members were performed to elucidate the genetic etiology of the myopathy. Genetic analyses detected two contributing variants located on different chromosomes in three unrelated probands: a heterozygous pathogenic mutation in SQSTM1 (c.1175C>T, p.Pro392Leu) and a heterozygous variant in TIA1 (c.1070A>G, p.Asn357Ser). The affected fraternal twin of one proband also carries both variants, while the unaffected family members harbor one or none. Two unrelated probands (family 1, II.3, and family 3, II.1) have a distal myopathy with rimmed vacuoles that manifested with index extensor weakness; the other proband (family 2, I.1) has myofibrillar myopathy manifesting with hypercapnic respiratory insufficiency and distal weakness. The findings indicate that all the affected individuals have a myopathy associated with both variants in SQSTM1 and TIA1 , respectively, suggesting that the two variants determine the phenotype and likely functionally interact. We speculate that the TIA1 variant is a modifier of the SQSTM1 mutation. We identify the combination of SQSTM1 and TIA1 variants as a novel genetic defect associated with myofibrillar myopathy and suggest to consider sequencing both genes in the molecular investigation of myopathy with rimmed vacuoles and myofibrillar myopathy although additional studies are needed to investigate the digenic nature of the disease.

Pitfalls in setting up genetic studies on preeclampsia.

PubMed

Laivuori, Hannele

2013-04-01

This presentation will consider approaches to discover susceptibility genes for a complex genetic disorder such as preeclampsia. The clinical disease presumably results from the additive effects of multiple sequence variants from the mother and the foetus together with environmental factors. Disease heterogeneity and underpowered study designs are likely to be behind non-reproducible results in candidate gene association studies. To avoid spurious findings, sample size and characteristics of the study populations as well as replication studies in an independent study population should be an essential part of a study design. In family-based linkage studies relationship with genotype and phenotype may be modified by a variety of factors. The large number of families needed in discovering genetic variants with modest effect sizes is difficult to attain. Moreover, the identification of underlying mutations has proven difficult. When pooling data or performing meta-analyses from different populations, disease and locus heterogeneity may become a major issue. First genome-wide association studies (GWAS) have identified risk loci for preeclampsia. Adequately powered replication studies are critical in order to replicate the initial GWAS findings. This approach requires rigorous multiple testing correction. The expected effect sizes of individual sequence variants on preeclampsia are small, but this approach is likely to decipher new clues to the pathogenesis. The rare variants, gene-gene and gene-environmental interactions as well as noncoding genetic variations and epigenetics are expected to explain the missing heritability. Next-generation sequencing technologies will make large amount of data on genomes and transcriptomes available. Complexity of the data poses a challenge. Different depths of coverage might be chosen depending on the design of the study, and validation of the results by different methods is mandatory. In order to minimize disease heterogeneity in genetic studies of preeclampsia, identification of subtypes and intermediate phenotypes would be highly desirable. Copyright © 2013. Published by Elsevier B.V.

Clinical phenotype-based gene prioritization: an initial study using semantic similarity and the human phenotype ontology.

PubMed

Masino, Aaron J; Dechene, Elizabeth T; Dulik, Matthew C; Wilkens, Alisha; Spinner, Nancy B; Krantz, Ian D; Pennington, Jeffrey W; Robinson, Peter N; White, Peter S

2014-07-21

Exome sequencing is a promising method for diagnosing patients with a complex phenotype. However, variant interpretation relative to patient phenotype can be challenging in some scenarios, particularly clinical assessment of rare complex phenotypes. Each patient's sequence reveals many possibly damaging variants that must be individually assessed to establish clear association with patient phenotype. To assist interpretation, we implemented an algorithm that ranks a given set of genes relative to patient phenotype. The algorithm orders genes by the semantic similarity computed between phenotypic descriptors associated with each gene and those describing the patient. Phenotypic descriptor terms are taken from the Human Phenotype Ontology (HPO) and semantic similarity is derived from each term's information content. Model validation was performed via simulation and with clinical data. We simulated 33 Mendelian diseases with 100 patients per disease. We modeled clinical conditions by adding noise and imprecision, i.e. phenotypic terms unrelated to the disease and terms less specific than the actual disease terms. We ranked the causative gene against all 2488 HPO annotated genes. The median causative gene rank was 1 for the optimal and noise cases, 12 for the imprecision case, and 60 for the imprecision with noise case. Additionally, we examined a clinical cohort of subjects with hearing impairment. The disease gene median rank was 22. However, when also considering the patient's exome data and filtering non-exomic and common variants, the median rank improved to 3. Semantic similarity can rank a causative gene highly within a gene list relative to patient phenotype characteristics, provided that imprecision is mitigated. The clinical case results suggest that phenotype rank combined with variant analysis provides significant improvement over the individual approaches. We expect that this combined prioritization approach may increase accuracy and decrease effort for clinical genetic diagnosis.

Asthma phenotypes modify the impact of environmetnal factors on lung function

EPA Science Inventory

Previous studies have examined the role of childhood asthma phenotypes based on clinical history on asthma severity and symptom aggravation by environmental risk factors. The current study focuses on the associations between lung function in childhood and environmental factors an...

SuperPhy: predictive genomics for the bacterial pathogen Escherichia coli.

PubMed

Whiteside, Matthew D; Laing, Chad R; Manji, Akiff; Kruczkiewicz, Peter; Taboada, Eduardo N; Gannon, Victor P J

2016-04-12

Predictive genomics is the translation of raw genome sequence data into a phenotypic assessment of the organism. For bacterial pathogens, these phenotypes can range from environmental survivability, to the severity of human disease. Significant progress has been made in the development of generic tools for genomic analyses that are broadly applicable to all microorganisms; however, a fundamental missing component is the ability to analyze genomic data in the context of organism-specific phenotypic knowledge, which has been accumulated from decades of research and can provide a meaningful interpretation of genome sequence data. In this study, we present SuperPhy, an online predictive genomics platform ( http://lfz.corefacility.ca/superphy/ ) for Escherichia coli. The platform integrates the analytical tools and genome sequence data for all publicly available E. coli genomes and facilitates the upload of new genome sequences from users under public or private settings. SuperPhy provides real-time analyses of thousands of genome sequences with results that are understandable and useful to a wide community, including those in the fields of clinical medicine, epidemiology, ecology, and evolution. SuperPhy includes identification of: 1) virulence and antimicrobial resistance determinants 2) statistical associations between genotypes, biomarkers, geospatial distribution, host, source, and phylogenetic clade; 3) the identification of biomarkers for groups of genomes on the based presence/absence of specific genomic regions and single-nucleotide polymorphisms and 4) in silico Shiga-toxin subtype. SuperPhy is a predictive genomics platform that attempts to provide an essential link between the vast amounts of genome information currently being generated and phenotypic knowledge in an organism-specific context.

Genetic Basis and Genetic Modifiers of β-Thalassemia and Sickle Cell Disease.

PubMed

Thein, Swee Lay

2017-01-01

β-thalassemia and sickle cell disease (SCD) are prototypical Mendelian single gene disorders, both caused by mutations affecting the adult β-globin gene. Despite the apparent genetic simplicity, both disorders display a remarkable spectrum of phenotypic severity and share two major genetic modifiers-α-globin genotype and innate ability to produce fetal hemoglobin (HbF, α 2 γ 2 ).This article provides an overview of the genetic basis for SCD and β-thalassemia, and genetic modifiers identified through phenotype correlation studies. Identification of the genetic variants modifying HbF production in combination with α-globin genotype provide some prediction of disease severity for β-thalassemia and SCD but generation of a personalized genetic risk score to inform prognosis and guide management requires a larger panel of genetic modifiers yet to be discovered.Nonetheless, genetic studies have been successful in characterizing some of the key variants and pathways involved in HbF regulation, providing new therapeutic targets for HbF reactivation.

A novel p.Gly603Arg mutation in CACNA1F causes Åland island eye disease and incomplete congenital stationary night blindness phenotypes in a family

PubMed Central

Vincent, Ajoy; Wright, Tom; Day, Megan A.; Westall, Carol A.

2011-01-01

Purpose To report, for the first time, that X-linked incomplete congenital stationary night blindness (CSNB2A) and Åland island eye disease (AIED) phenotypes coexist in a molecularly confirmed pedigree and to present novel phenotypic characteristics of calcium channel alpha-1F subunit gene (CACNA1F)-related disease. Methods Two affected subjects (the proband and his maternal grandfather) and an unaffected obligate carrier (the proband’s mother) underwent detailed ophthalmological evaluation, fundus autofluorescence imaging, and spectral-domain optical coherence tomography. Goldmann visual field assessment and full-field electroretinogram (ERG) were performed in the two affected subjects, and multichannel flash visual evoked potential was performed on the proband. Scotopic 15 Hz flicker ERG series were performed in both affected subjects to evaluate the function of the slow and fast rod pathways. Haplotype analysis using polymorphic microsatellite markers flanking CACNA1F was performed in all three family members. The proband’s DNA was sequenced for mutations in the coding sequence of CACNA1F and nyctalopin (NYX) genes. Segregation analysis was performed in the family. Results Both affected subjects had symptoms of nonprogressive nyctalopia since childhood, while the proband also had photophobia. Both cases had a distance visual acuity of 20/50 or better in each eye, normal contrast sensitivity, and an incomplete type of Schubert-Bornschein ERGs. The proband also had high myopia, a mild red-green color deficit, hypopigmented fundus, and foveal hypoplasia with no evidence of chiasmal misrouting. Spectral-domain optical coherence tomography confirmed the presence of foveal hypoplasia in the proband. The clinical phenotype of the proband and his maternal grandfather fit the clinical description of AIED and CSNB2A, respectively. The fundus autofluorescence and the visual fields were normal in both cases; the scotopic 15 Hz flicker ERG demonstrated only fast rod pathway activity in both. Both affected cases shared the same haplotype across CACNA1F. The proband carried a novel hemizygous c.1807G>C mutation (p.G603R) in the CACNA1F gene. The change segregated with the disease phenotypes and was not identified in 360 control chromosomes. No mutations were identified in NYX. Conclusions This report of a missense mutation in CACNA1F causing AIED and CSNB2A phenotypes in a family confirms that both diseases are allelic and that other genetic or environmental modifiers influence the expression of CACNA1F. This is the first report to suggest that in CACNA1F-related disease, the rod system activity is predominantly from the fast rod pathways. PMID:22194652

Estimating rice yield related traits and quantitative trait loci analysis under different nitrogen treatments using a simple tower-based field phenotyping system with modified single-lens reflex cameras

NASA Astrophysics Data System (ADS)

Naito, Hiroki; Ogawa, Satoshi; Valencia, Milton Orlando; Mohri, Hiroki; Urano, Yutaka; Hosoi, Fumiki; Shimizu, Yo; Chavez, Alba Lucia; Ishitani, Manabu; Selvaraj, Michael Gomez; Omasa, Kenji

2017-03-01

Application of field based high-throughput phenotyping (FB-HTP) methods for monitoring plant performance in real field conditions has a high potential to accelerate the breeding process. In this paper, we discuss the use of a simple tower based remote sensing platform using modified single-lens reflex cameras for phenotyping yield traits in rice under different nitrogen (N) treatments over three years. This tower based phenotyping platform has the advantages of simplicity, ease and stability in terms of introduction, maintenance and continual operation under field conditions. Out of six phenological stages of rice analyzed, the flowering stage was the most useful in the estimation of yield performance under field conditions. We found a high correlation between several vegetation indices (simple ratio (SR), normalized difference vegetation index (NDVI), transformed vegetation index (TVI), corrected transformed vegetation index (CTVI), soil-adjusted vegetation index (SAVI) and modified soil-adjusted vegetation index (MSAVI)) and multiple yield traits (panicle number, grain weight and shoot biomass) across a three trials. Among all of the indices studied, SR exhibited the best performance in regards to the estimation of grain weight (R2 = 0.80). Under our tower-based field phenotyping system (TBFPS), we identified quantitative trait loci (QTL) for yield related traits using a mapping population of chromosome segment substitution lines (CSSLs) and a single nucleotide polymorphism data set. Our findings suggest the TBFPS can be useful for the estimation of yield performance during early crop development. This can be a major opportunity for rice breeders whom desire high throughput phenotypic selection for yield performance traits.

Novel Phenotype-Genotype Correlations of Restrictive Cardiomyopathy With Myosin-Binding Protein C (MYBPC3) Gene Mutations Tested by Next-Generation Sequencing.

PubMed

Wu, Wei; Lu, Chao-Xia; Wang, Yi-Ning; Liu, Fang; Chen, Wei; Liu, Yong-Tai; Han, Ye-Chen; Cao, Jian; Zhang, Shu-Yang; Zhang, Xue

2015-07-10

MYBPC3 dysfunctions have been proven to induce dilated cardiomyopathy, hypertrophic cardiomyopathy, and/or left ventricular noncompaction; however, the genotype-phenotype correlation between MYBPC3 and restrictive cardiomyopathy (RCM) has not been established. The newly developed next-generation sequencing method is capable of broad genomic DNA sequencing with high throughput and can help explore novel correlations between genetic variants and cardiomyopathies. A proband from a multigenerational family with 3 live patients and 1 unrelated patient with clinical diagnoses of RCM underwent a next-generation sequencing workflow based on a custom AmpliSeq panel, including 64 candidate pathogenic genes for cardiomyopathies, on the Ion Personal Genome Machine high-throughput sequencing benchtop instrument. The selected panel contained a total of 64 genes that were reportedly associated with inherited cardiomyopathies. All patients fulfilled strict criteria for RCM with clinical characteristics, echocardiography, and/or cardiac magnetic resonance findings. The multigenerational family with 3 adult RCM patients carried an identical nonsense MYBPC3 mutation, and the unrelated patient carried a missense mutation in the MYBPC3 gene. All of these results were confirmed by the Sanger sequencing method. This study demonstrated that MYBPC3 gene mutations, revealed by next-generation sequencing, were associated with familial and sporadic RCM patients. It is suggested that the next-generation sequencing platform with a selected panel provides a highly efficient approach for molecular diagnosis of hereditary and idiopathic RCM and helps build new genotype-phenotype correlations. © 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Linked genetic variants on chromosome 10 control ear morphology and body mass among dog breeds.

PubMed

Webster, Matthew T; Kamgari, Nona; Perloski, Michele; Hoeppner, Marc P; Axelsson, Erik; Hedhammar, Åke; Pielberg, Gerli; Lindblad-Toh, Kerstin

2015-06-23

The domestic dog is a rich resource for mapping the genetic components of phenotypic variation due to its unique population history involving strong artificial selection. Genome-wide association studies have revealed a number of chromosomal regions where genetic variation associates with morphological characters that typify dog breeds. A region on chromosome 10 is among those with the highest levels of genetic differentiation between dog breeds and is associated with body mass and ear morphology, a common motif of animal domestication. We characterised variation in this region to uncover haplotype structure and identify candidate functional variants. We first identified SNPs that strongly associate with body mass and ear type by comparing sequence variation in a 3 Mb region between 19 breeds with a variety of phenotypes. We next genotyped a subset of 123 candidate SNPs in 288 samples from 46 breeds to identify the variants most highly associated with phenotype and infer haplotype structure. A cluster of SNPs that associate strongly with the drop ear phenotype is located within a narrow interval downstream of the gene MSRB3, which is involved in human hearing. These SNPs are in strong genetic linkage with another set of variants that correlate with body mass within the gene HMGA2, which affects human height. In addition we find evidence that this region has been under selection during dog domestication, and identify a cluster of SNPs within MSRB3 that are highly differentiated between dogs and wolves. We characterise genetically linked variants that potentially influence ear type and body mass in dog breeds, both key traits that have been modified by selective breeding that may also be important for domestication. The finding that variants on long haplotypes have effects on more than one trait suggests that genetic linkage can be an important determinant of the phenotypic response to selection in domestic animals.

Identifying Genetic Sources of Phenotypic Heterogeneity in Orofacial Clefts by Targeted Sequencing.

PubMed

Carlson, Jenna C; Taub, Margaret A; Feingold, Eleanor; Beaty, Terri H; Murray, Jeffrey C; Marazita, Mary L; Leslie, Elizabeth J

2017-07-17

Orofacial clefts (OFCs), including nonsyndromic cleft lip with or without cleft palate (NSCL/P), are common birth defects. NSCL/P is highly heterogeneous with multiple phenotypic presentations. Two common subtypes of NSCL/P are cleft lip (CL) and cleft lip with cleft palate (CLP) which have different population prevalence. Similarly, NSCL/P can be divided into bilateral and unilateral clefts, with unilateral being the most common. Individuals with unilateral NSCL/P are more likely to be affected on the left side of the upper lip, but right side affection also occurs. Moreover, NSCL/P is twice as common in males as in females. The goal of this study is to discover genetic variants that have different effects in case subgroups. We conducted both common variant and rare variant analyses in 1034 individuals of Asian ancestry with NSCL/P, examining four sources of heterogeneity within CL/P: cleft type, sex, laterality, and side. We identified several regions associated with subtype differentiation: cleft type differences in 8q24 (p = 1.00 × 10 -4 ), laterality differences in IRF6, a gene previously implicated with wound healing (p = 2.166 × 10 -4 ), sex differences and side of unilateral CL differences in FGFR2 (p = 3.00 × 10 -4 ; p = 6.00 × 10 -4 ), and sex differences in VAX1 (p < 1.00 × 10 -4 ) among others. Many of the regions associated with phenotypic modification were either adjacent to or overlapping functional elements based on ENCODE chromatin marks and published craniofacial enhancers. We have identified multiple common and rare variants as potential phenotypic modifiers of NSCL/P, and suggest plausible elements responsible for phenotypic heterogeneity, further elucidating the complex genetic architecture of OFCs. Birth Defects Research 109:1030-1038, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The emerging CHO systems biology era: harnessing the 'omics revolution for biotechnology.

PubMed

Kildegaard, Helene Faustrup; Baycin-Hizal, Deniz; Lewis, Nathan E; Betenbaugh, Michael J

2013-12-01

Chinese hamster ovary (CHO) cells are the primary factories for biopharmaceuticals because of their capacity to correctly fold and post-translationally modify recombinant proteins compatible with humans. New opportunities are arising to enhance these cell factories, especially since the CHO-K1 cell line was recently sequenced. Now, the CHO systems biology era is underway. Critical 'omics data sets, including proteomics, transcriptomics, metabolomics, fluxomics, and glycomics, are emerging, allowing the elucidation of the molecular basis of CHO cell physiology. The incorporation of these data sets into mathematical models that describe CHO phenotypes will provide crucial biotechnology insights. As 'omics technologies and computational systems biology mature, genome-scale approaches will lead to major innovations in cell line development and metabolic engineering, thereby improving protein production and bioprocessing. Copyright © 2013 Elsevier Ltd. All rights reserved.

SATB2-associated syndrome presenting with Rett-like phenotypes.

PubMed

Lee, J S; Yoo, Y; Lim, B C; Kim, K J; Choi, M; Chae, J-H

2016-06-01

The SATB2-associated syndrome (SAS) was proposed recently, after the SATB2 gene was initially discovered to be associated with isolated cleft palate. This syndrome is characterized by intellectual disability with delayed speech development, facial dysmorphism, cleft or high-arched palate, and dentition problems. Here, we describe two novel SATB2 sequence variants in two unrelated patients presenting with Rett-like phenotypes. We performed trio-based whole-exome sequencing in a 17-month-old girl presenting with severe retardation and Rett-like phenotypes, which revealed a de novo missense variant in SATB2 (p.Glu396Gln). Moreover, targeted sequencing of the SATB2 gene was performed in a 2-year-old girl with severe psychomotor retardation, facial hypotonia, and cleft palate who also exhibited some features of Rett syndrome. A nonsense variant in SATB2 was identified in this patient (p.Arg459*). This study expanded the clinical and genetic spectrum of SAS. SATB2 variants should be considered in cases with psychomotor retardation alone or in any cases with Rett-like phenotypes, regardless of the typical features of SAS such as cleft palate. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Novel FAM20A mutation causes autosomal recessive amelogenesis imperfecta.

PubMed

Volodarsky, Michael; Zilberman, Uri; Birk, Ohad S

2015-06-01

To relate the peculiar phenotype of amelogenesis imperfecta in a large Bedouin family to the genotype determined by whole genome linkage analysis. Amelogenesis imperfecta (AI) is a broad group of inherited pathologies affecting enamel formation, characterized by variability in phenotypes, causing mutations and modes of inheritance. Autosomal recessive or compound heterozygous mutations in FAM20A, encoding sequence similarity 20, member A, have been shown to cause several AI phenotypes. Five members from a large consanguineous Bedouin family presented with hypoplastic amelogenesis imperfecta with unerupted and resorbed permanent molars. Following Soroka Medical Center IRB approval and informed consent, blood samples were obtained from six affected offspring, five obligatory carriers and two unaffected siblings. Whole genome linkage analysis was performed followed by Sanger sequencing of FAM20A. The sequencing unravelled a novel homozygous deletion mutation in exon 11 (c.1523delC), predicted to insert a premature stop codon (p.Thr508Lysfs*6). We provide an interesting case of novel mutation in this rare disorder, in which the affected kindred is unique in the large number of family members sharing a similar phenotype. Copyright © 2015 Elsevier Ltd. All rights reserved.

Early-onset encephalopathy with paroxysmal movement disorders and epileptic seizures without hemiplegic attacks: About three children with novel ATP1A3 mutations.

PubMed

Marzin, Pauline; Mignot, Cyril; Dorison, Nathalie; Dufour, Louis; Ville, Dorothée; Kaminska, Anna; Panagiotakaki, Eleni; Dienpendaele, Anne-Sophie; Penniello, Marie-José; Nougues, Marie-Christine; Keren, Boris; Depienne, Christel; Nava, Caroline; Milh, Mathieu; Villard, Laurent; Richelme, Christian; Rivier, Clotilde; Whalen, Sandra; Heron, Delphine; Lesca, Gaëtan; Doummar, Diane

2018-05-31

Heterozygous mutations in the ATP1A3 gene are responsible for various neurological disorders, ranging from early-onset alternating hemiplegia of childhood to adult-onset dystonia-parkinsonism. Next generation sequencing allowed the description of other phenotypes, including early-onset epileptic encephalopathy in two patients. We report on three more patients carrying ATP1A3 mutations with a close phenotype and discuss the relationship of this phenotype to alternating hemiplegia of childhood. The patients' DNA underwent next generation sequencing. A retrospective analysis of clinical case records is reported. Each of the three patients had an unreported heterozygous de novo sequence variant in ATP1A3. These patients shared a similar phenotype characterized by early-onset attacks of movement disorders, some of which proved to be epileptic, and severe developmental delay. (Hemi)plegic attacks had not been considered before genetic testing. Together with the two previously reported cases, our patients confirm that ATP1A3 mutations are associated with a phenotype combining features of early-onset encephalopathy, epilepsy and dystonic fits, as in the most severe forms of alternating hemiplegia of childhood, but in which (hemi)plegic attacks are absent or only suspected retrospectively. Copyright © 2018 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Modeling kinetic rate variation in third generation DNA sequencing data to detect putative modifications to DNA bases

PubMed Central

Schadt, Eric E.; Banerjee, Onureena; Fang, Gang; Feng, Zhixing; Wong, Wing H.; Zhang, Xuegong; Kislyuk, Andrey; Clark, Tyson A.; Luong, Khai; Keren-Paz, Alona; Chess, Andrew; Kumar, Vipin; Chen-Plotkin, Alice; Sondheimer, Neal; Korlach, Jonas; Kasarskis, Andrew

2013-01-01

Current generation DNA sequencing instruments are moving closer to seamlessly sequencing genomes of entire populations as a routine part of scientific investigation. However, while significant inroads have been made identifying small nucleotide variation and structural variations in DNA that impact phenotypes of interest, progress has not been as dramatic regarding epigenetic changes and base-level damage to DNA, largely due to technological limitations in assaying all known and unknown types of modifications at genome scale. Recently, single-molecule real time (SMRT) sequencing has been reported to identify kinetic variation (KV) events that have been demonstrated to reflect epigenetic changes of every known type, providing a path forward for detecting base modifications as a routine part of sequencing. However, to date no statistical framework has been proposed to enhance the power to detect these events while also controlling for false-positive events. By modeling enzyme kinetics in the neighborhood of an arbitrary location in a genomic region of interest as a conditional random field, we provide a statistical framework for incorporating kinetic information at a test position of interest as well as at neighboring sites that help enhance the power to detect KV events. The performance of this and related models is explored, with the best-performing model applied to plasmid DNA isolated from Escherichia coli and mitochondrial DNA isolated from human brain tissue. We highlight widespread kinetic variation events, some of which strongly associate with known modification events, while others represent putative chemically modified sites of unknown types. PMID:23093720

Modeling kinetic rate variation in third generation DNA sequencing data to detect putative modifications to DNA bases.

PubMed

Schadt, Eric E; Banerjee, Onureena; Fang, Gang; Feng, Zhixing; Wong, Wing H; Zhang, Xuegong; Kislyuk, Andrey; Clark, Tyson A; Luong, Khai; Keren-Paz, Alona; Chess, Andrew; Kumar, Vipin; Chen-Plotkin, Alice; Sondheimer, Neal; Korlach, Jonas; Kasarskis, Andrew

2013-01-01

Current generation DNA sequencing instruments are moving closer to seamlessly sequencing genomes of entire populations as a routine part of scientific investigation. However, while significant inroads have been made identifying small nucleotide variation and structural variations in DNA that impact phenotypes of interest, progress has not been as dramatic regarding epigenetic changes and base-level damage to DNA, largely due to technological limitations in assaying all known and unknown types of modifications at genome scale. Recently, single-molecule real time (SMRT) sequencing has been reported to identify kinetic variation (KV) events that have been demonstrated to reflect epigenetic changes of every known type, providing a path forward for detecting base modifications as a routine part of sequencing. However, to date no statistical framework has been proposed to enhance the power to detect these events while also controlling for false-positive events. By modeling enzyme kinetics in the neighborhood of an arbitrary location in a genomic region of interest as a conditional random field, we provide a statistical framework for incorporating kinetic information at a test position of interest as well as at neighboring sites that help enhance the power to detect KV events. The performance of this and related models is explored, with the best-performing model applied to plasmid DNA isolated from Escherichia coli and mitochondrial DNA isolated from human brain tissue. We highlight widespread kinetic variation events, some of which strongly associate with known modification events, while others represent putative chemically modified sites of unknown types.

Identification of a novel 15.5 kb SHOX deletion associated with marked intrafamilial phenotypic variability and analysis of its molecular origin.

PubMed

Alexandrou, Angelos; Papaevripidou, Ioannis; Tsangaras, Kyriakos; Alexandrou, Ioanna; Tryfonidis, Marios; Christophidou-Anastasiadou, Violetta; Zamba-Papanicolaou, Eleni; Koumbaris, George; Neocleous, Vassos; Phylactou, Leonidas A; Skordis, Nicos; Tanteles, George A; Sismani, Carolina

2016-12-01

Haploinsufficiency of the short stature homeobox contaning SHOX gene has been shown to result in a spectrum of phenotypes ranging from Leri-Weill dyschondrosteosis (LWD) at the more severe end to SHOX-related short stature at the milder end of the spectrum. Most alterations are whole gene deletions, point mutations within the coding region, or microdeletions in its flanking sequences. Here, we present the clinical and molecular data as well as the potential molecular mechanism underlying a novel microdeletion, causing a variable SHOX-related haploinsufficiency disorder in a three-generation family. The phenotype resembles that of LWD in females, in males, however, the phenotypic expression is milder. The 15523-bp SHOX intragenic deletion, encompassing exons 3-6, was initially detected by array-CGH, followed by MLPA analysis. Sequencing of the breakpoints indicated an Alu recombination-mediated deletion (ARMD) as the potential causative mechanism.

Phenotype classification of single cells using SRS microscopy, RNA sequencing, and microfluidics (Conference Presentation)

NASA Astrophysics Data System (ADS)

Streets, Aaron M.; Cao, Chen; Zhang, Xiannian; Huang, Yanyi

2016-03-01

Phenotype classification of single cells reveals biological variation that is masked in ensemble measurement. This heterogeneity is found in gene and protein expression as well as in cell morphology. Many techniques are available to probe phenotypic heterogeneity at the single cell level, for example quantitative imaging and single-cell RNA sequencing, but it is difficult to perform multiple assays on the same single cell. In order to directly track correlation between morphology and gene expression at the single cell level, we developed a microfluidic platform for quantitative coherent Raman imaging and immediate RNA sequencing (RNA-Seq) of single cells. With this device we actively sort and trap cells for analysis with stimulated Raman scattering microscopy (SRS). The cells are then processed in parallel pipelines for lysis, and preparation of cDNA for high-throughput transcriptome sequencing. SRS microscopy offers three-dimensional imaging with chemical specificity for quantitative analysis of protein and lipid distribution in single cells. Meanwhile, the microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved RNA-Seq detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. By combining coherent Raman microscopy with RNA sequencing, we can better understand the relationship between cellular morphology and gene expression at the single-cell level.

Integrating mRNA and Protein Sequencing Enables the Detection and Quantitative Profiling of Natural Protein Sequence Variants of Populus trichocarpa.

PubMed

Abraham, Paul E; Wang, Xiaojing; Ranjan, Priya; Nookaew, Intawat; Zhang, Bing; Tuskan, Gerald A; Hettich, Robert L

2015-12-04

Next-generation sequencing has transformed the ability to link genotypes to phenotypes and facilitates the dissection of genetic contribution to complex traits. However, it is challenging to link genetic variants with the perturbed functional effects on proteins encoded by such genes. Here we show how RNA sequencing can be exploited to construct genotype-specific protein sequence databases to assess natural variation in proteins, providing information about the molecular toolbox driving cellular processes. For this study, we used two natural genotypes selected from a recent genome-wide association study of Populus trichocarpa, an obligate outcrosser with tremendous phenotypic variation across the natural population. This strategy allowed us to comprehensively catalogue proteins containing single amino acid polymorphisms (SAAPs), as well as insertions and deletions. We profiled the frequency of 128 types of naturally occurring amino acid substitutions, including both expected (neutral) and unexpected (non-neutral) SAAPs, with a subset occurring in regions of the genome having strong polymorphism patterns consistent with recent positive and/or divergent selection. By zeroing in on the molecular signatures of these important regions that might have previously been uncharacterized, we now provide a high-resolution molecular inventory that should improve accessibility and subsequent identification of natural protein variants in future genotype-to-phenotype studies.

How Reliable Are the Reported Genetic Associations in Disc Degeneration?: The Influence of Phenotypes, Age, Population Size, and Inclusion Sequence in 809 Patients.

PubMed

Rajasekaran, S; Kanna, Rishi Mugesh; Reddy, Ranjani Raja; Natesan, Senthil; Raveendran, Muthuraja; Cheung, Kenneth M C; Chan, Danny; Kao, Patrick Y P; Yee, Anita; Shetty, Ajoy Prasad

2016-11-01

Prospective genetic association study. The aim of this study was to document the variations in the genetic associations, when different magnetic resonance imaging (MRI) phenotypes, age stratification, cohort size, and sequence of cohort inclusion are varied in the same study population. Genetic associations with disc degeneration have shown high inconsistency, generally attributed to hereditary factors and ethnic variations. However, the effect of different phenotypes, size of the study population, age of the cohort, etc have not been documented clearly. Seventy-one single-nucleotide polymorphisms (SNPs) of 41 candidate genes were correlated to six MRI markers of disc degeneration (annular tears, Pfirmann grading, Schmorl nodes, Modic changes, Total Endplate Damage score, and disc bulge) in 809 patients with back pain and/or sciatica. In the same study group, the correlations were then retested for different age groups, different sample, size and sequence of subject inclusion (first 404 and the second 405) and the differences documented. The mean age of population (M: 455, F: 354) was 36.7 ± 10.8 years. Different genetic associations were found with different phenotypes: disc bulge with three SNPs of CILP; annular tears with rs2249350 of ADAMTS5 and rs11247361 IGF1R; modic changes with VDR and MMP20; Pfirmann grading with three SNPs of MMP20 and Schmorl node with SNPs of CALM1 and FN1 and none with Total End Plate Score.Subgroup analysis based on three age groups and dividing the total population into two groups also completely changed the associations for all the six radiographic parameters. In the same study population, SNP associations completely change with different phenotypes. Variations in age, inclusion sequence, and sample size resulted in change of genetic associations. Our study questions the validity of previous studies and necessitates the need for standardizing the description of disc degeneration, phenotype selection, study sample size, age, and other variables in future studies. 4.

Whole-Genome Sequencing Analysis Accurately Predicts Antimicrobial Resistance Phenotypes in Campylobacter spp.

PubMed Central

Tyson, G. H.; Chen, Y.; Li, C.; Mukherjee, S.; Young, S.; Lam, C.; Folster, J. P.; Whichard, J. M.; McDermott, P. F.

2015-01-01

The objectives of this study were to identify antimicrobial resistance genotypes for Campylobacter and to evaluate the correlation between resistance phenotypes and genotypes using in vitro antimicrobial susceptibility testing and whole-genome sequencing (WGS). A total of 114 Campylobacter species isolates (82 C. coli and 32 C. jejuni) obtained from 2000 to 2013 from humans, retail meats, and cecal samples from food production animals in the United States as part of the National Antimicrobial Resistance Monitoring System were selected for study. Resistance phenotypes were determined using broth microdilution of nine antimicrobials. Genomic DNA was sequenced using the Illumina MiSeq platform, and resistance genotypes were identified using assembled WGS sequences through blastx analysis. Eighteen resistance genes, including tet(O), blaOXA-61, catA, lnu(C), aph(2″)-Ib, aph(2″)-Ic, aph(2′)-If, aph(2″)-Ig, aph(2″)-Ih, aac(6′)-Ie-aph(2″)-Ia, aac(6′)-Ie-aph(2″)-If, aac(6′)-Im, aadE, sat4, ant(6′), aad9, aph(3′)-Ic, and aph(3′)-IIIa, and mutations in two housekeeping genes (gyrA and 23S rRNA) were identified. There was a high degree of correlation between phenotypic resistance to a given drug and the presence of one or more corresponding resistance genes. Phenotypic and genotypic correlation was 100% for tetracycline, ciprofloxacin/nalidixic acid, and erythromycin, and correlations ranged from 95.4% to 98.7% for gentamicin, azithromycin, clindamycin, and telithromycin. All isolates were susceptible to florfenicol, and no genes associated with florfenicol resistance were detected. There was a strong correlation (99.2%) between resistance genotypes and phenotypes, suggesting that WGS is a reliable indicator of resistance to the nine antimicrobial agents assayed in this study. WGS has the potential to be a powerful tool for antimicrobial resistance surveillance programs. PMID:26519386

Whole-Genome Sequencing Analysis Accurately Predicts Antimicrobial Resistance Phenotypes in Campylobacter spp.

PubMed

Zhao, S; Tyson, G H; Chen, Y; Li, C; Mukherjee, S; Young, S; Lam, C; Folster, J P; Whichard, J M; McDermott, P F

2016-01-15

The objectives of this study were to identify antimicrobial resistance genotypes for Campylobacter and to evaluate the correlation between resistance phenotypes and genotypes using in vitro antimicrobial susceptibility testing and whole-genome sequencing (WGS). A total of 114 Campylobacter species isolates (82 C. coli and 32 C. jejuni) obtained from 2000 to 2013 from humans, retail meats, and cecal samples from food production animals in the United States as part of the National Antimicrobial Resistance Monitoring System were selected for study. Resistance phenotypes were determined using broth microdilution of nine antimicrobials. Genomic DNA was sequenced using the Illumina MiSeq platform, and resistance genotypes were identified using assembled WGS sequences through blastx analysis. Eighteen resistance genes, including tet(O), blaOXA-61, catA, lnu(C), aph(2″)-Ib, aph(2″)-Ic, aph(2')-If, aph(2″)-Ig, aph(2″)-Ih, aac(6')-Ie-aph(2″)-Ia, aac(6')-Ie-aph(2″)-If, aac(6')-Im, aadE, sat4, ant(6'), aad9, aph(3')-Ic, and aph(3')-IIIa, and mutations in two housekeeping genes (gyrA and 23S rRNA) were identified. There was a high degree of correlation between phenotypic resistance to a given drug and the presence of one or more corresponding resistance genes. Phenotypic and genotypic correlation was 100% for tetracycline, ciprofloxacin/nalidixic acid, and erythromycin, and correlations ranged from 95.4% to 98.7% for gentamicin, azithromycin, clindamycin, and telithromycin. All isolates were susceptible to florfenicol, and no genes associated with florfenicol resistance were detected. There was a strong correlation (99.2%) between resistance genotypes and phenotypes, suggesting that WGS is a reliable indicator of resistance to the nine antimicrobial agents assayed in this study. WGS has the potential to be a powerful tool for antimicrobial resistance surveillance programs. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Genome-Wide Identification of Regulatory Sequences Undergoing Accelerated Evolution in the Human Genome

PubMed Central

Dong, Xinran; Wang, Xiao; Zhang, Feng; Tian, Weidong

2016-01-01

Accelerated evolution of regulatory sequence can alter the expression pattern of target genes, and cause phenotypic changes. In this study, we used DNase I hypersensitive sites (DHSs) to annotate putative regulatory sequences in the human genome, and conducted a genome-wide analysis of the effects of accelerated evolution on regulatory sequences. Working under the assumption that local ancient repeat elements of DHSs are under neutral evolution, we discovered that ∼0.44% of DHSs are under accelerated evolution (ace-DHSs). We found that ace-DHSs tend to be more active than background DHSs, and are strongly associated with epigenetic marks of active transcription. The target genes of ace-DHSs are significantly enriched in neuron-related functions, and their expression levels are positively selected in the human brain. Thus, these lines of evidences strongly suggest that accelerated evolution on regulatory sequences plays important role in the evolution of human-specific phenotypes. PMID:27401230

Genome-scale investigation of phenotypically distinct but nearly clonal Trichoderma strains

PubMed Central

Weld, Richard J.; Cox, Murray P.; Bradshaw, Rosie E.; McLean, Kirstin L.; Stewart, Alison; Steyaert, Johanna M.

2016-01-01

Biological control agents (BCA) are beneficial organisms that are applied to protect plants from pests. Many fungi of the genus Trichoderma are successful BCAs but the underlying mechanisms are not yet fully understood. Trichoderma cf. atroviride strain LU132 is a remarkably effective BCA compared to T. cf. atroviride strain LU140 but these strains were found to be highly similar at the DNA sequence level. This unusual combination of phenotypic variability and high DNA sequence similarity between separately isolated strains prompted us to undertake a genome comparison study in order to identify DNA polymorphisms. We further investigated if the polymorphisms had functional effects on the phenotypes. The two strains were clearly identified as individuals, exhibiting different growth rates, conidiation and metabolism. Superior pathogen control demonstrated by LU132 depended on its faster growth, which is a prerequisite for successful distribution and competition. Genome sequencing identified only one non-synonymous single nucleotide polymorphism (SNP) between the strains. Based on this SNP, we successfully designed and validated an RFLP protocol that can be used to differentiate LU132 from LU140 and other Trichoderma strains. This SNP changed the amino acid sequence of SERF, encoded by the previously undescribed single copy gene “small EDRK-rich factor” (serf). A deletion of serf in the two strains did not lead to identical phenotypes, suggesting that, in addition to the single functional SNP between the nearly clonal Trichoderma cf. atroviride strains, other non-genomic factors contribute to their phenotypic variation. This finding is significant as it shows that genomics is an extremely useful but not exhaustive tool for the study of biocontrol complexity and for strain typing. PMID:27190719

Enhanced Fluorescent Siderophore Biosynthesis and Loss of Phenazine-1-Carboxamide in Phenotypic Variant of Pseudomonas chlororaphis HT66.

PubMed

Liu, Yang; Wang, Zheng; Bilal, Muhammad; Hu, Hongbo; Wang, Wei; Huang, Xianqing; Peng, Huasong; Zhang, Xuehong

2018-01-01

Pseudomonas chlororaphis HT66 is a plant-beneficial bacterium that exhibits wider antagonistic spectrum against a variety of plant pathogenic fungi due to its main secondary metabolite, i.e., phenazine-1-carboxamide (PCN). In the present study, a spontaneous phenotypic variant designated as HT66-FLUO was isolated from the fermentation process of wild-type HT66 strain. The newly isolated phenotypic variant was morphologically distinct from the wild-type strain such as larger cell size, semi-transparent, non-production of PCN (Green or yellow crystals) and enhanced fluorescence under UV light. The whole-genome, RNA-sequencing, and phenotypic assays were performed to identify the reason of phenotypic variation in HT66-FLUO as compared to the HT66. Transcriptomic analysis revealed that 1,418 genes, representing approximately 22% of the 6393 open reading frames (ORFs) had undergone substantial reprogramming of gene expression in the HT66-FLUO. The whole-genome sequence indicated no gene alteration in HT66-FLUO as compared to HT66 according to the known reference sequence. The levels of global regulatory factor gacA and gacS expression were not significantly different between HT66 and HT66-FLUO. It was observed that overexpressing gacS rather than gacA in HT66-FLUO can recover switching of the variant to HT66. The β-galactosidase ( LacZ ) activity and qRT-PCR results indicate the downregulated expression of rsmX, rsmY , and rsmZ in HT66-FLUO as compared to HT66. Overexpressing three small RNAs in HT66-FLUO can revert switching of colony phenotype toward wild-type HT66 up to a certain degree, restore partial PCN production and reduces the fluorescent siderophores yield. However, the origin of the spontaneous phenotypic variant was difficult to be determined. In conclusion, this study helps to understand the gene regulatory effect in the spontaneous phenotypic variant.

Enhanced Fluorescent Siderophore Biosynthesis and Loss of Phenazine-1-Carboxamide in Phenotypic Variant of Pseudomonas chlororaphis HT66

PubMed Central

Liu, Yang; Wang, Zheng; Bilal, Muhammad; Hu, Hongbo; Wang, Wei; Huang, Xianqing; Peng, Huasong; Zhang, Xuehong

2018-01-01

Pseudomonas chlororaphis HT66 is a plant-beneficial bacterium that exhibits wider antagonistic spectrum against a variety of plant pathogenic fungi due to its main secondary metabolite, i.e., phenazine-1-carboxamide (PCN). In the present study, a spontaneous phenotypic variant designated as HT66-FLUO was isolated from the fermentation process of wild-type HT66 strain. The newly isolated phenotypic variant was morphologically distinct from the wild-type strain such as larger cell size, semi-transparent, non-production of PCN (Green or yellow crystals) and enhanced fluorescence under UV light. The whole-genome, RNA-sequencing, and phenotypic assays were performed to identify the reason of phenotypic variation in HT66-FLUO as compared to the HT66. Transcriptomic analysis revealed that 1,418 genes, representing approximately 22% of the 6393 open reading frames (ORFs) had undergone substantial reprogramming of gene expression in the HT66-FLUO. The whole-genome sequence indicated no gene alteration in HT66-FLUO as compared to HT66 according to the known reference sequence. The levels of global regulatory factor gacA and gacS expression were not significantly different between HT66 and HT66-FLUO. It was observed that overexpressing gacS rather than gacA in HT66-FLUO can recover switching of the variant to HT66. The β-galactosidase (LacZ) activity and qRT-PCR results indicate the downregulated expression of rsmX, rsmY, and rsmZ in HT66-FLUO as compared to HT66. Overexpressing three small RNAs in HT66-FLUO can revert switching of colony phenotype toward wild-type HT66 up to a certain degree, restore partial PCN production and reduces the fluorescent siderophores yield. However, the origin of the spontaneous phenotypic variant was difficult to be determined. In conclusion, this study helps to understand the gene regulatory effect in the spontaneous phenotypic variant. PMID:29740409

Non-coding variants contribute to the clinical heterogeneity of TTR amyloidosis.

PubMed

Iorio, Andrea; De Lillo, Antonella; De Angelis, Flavio; Di Girolamo, Marco; Luigetti, Marco; Sabatelli, Mario; Pradotto, Luca; Mauro, Alessandro; Mazzeo, Anna; Stancanelli, Claudia; Perfetto, Federico; Frusconi, Sabrina; My, Filomena; Manfellotto, Dario; Fuciarelli, Maria; Polimanti, Renato

2017-09-01

Coding mutations in TTR gene cause a rare hereditary form of systemic amyloidosis, which has a complex genotype-phenotype correlation. We investigated the role of non-coding variants in regulating TTR gene expression and consequently amyloidosis symptoms. We evaluated the genotype-phenotype correlation considering the clinical information of 129 Italian patients with TTR amyloidosis. Then, we conducted a re-sequencing of TTR gene to investigate how non-coding variants affect TTR expression and, consequently, phenotypic presentation in carriers of amyloidogenic mutations. Polygenic scores for genetically determined TTR expression were constructed using data from our re-sequencing analysis and the GTEx (Genotype-Tissue Expression) project. We confirmed a strong phenotypic heterogeneity across coding mutations causing TTR amyloidosis. Considering the effects of non-coding variants on TTR expression, we identified three patient clusters with specific expression patterns associated with certain phenotypic presentations, including late onset, autonomic neurological involvement, and gastrointestinal symptoms. This study provides novel data regarding the role of non-coding variation and the gene expression profiles in patients affected by TTR amyloidosis, also putting forth an approach that could be used to investigate the mechanisms at the basis of the genotype-phenotype correlation of the disease.

De novo truncating mutations in ASXL3 are associated with a novel clinical phenotype with similarities to Bohring-Opitz syndrome

PubMed Central

2013-01-01

Background Molecular diagnostics can resolve locus heterogeneity underlying clinical phenotypes that may otherwise be co-assigned as a specific syndrome based on shared clinical features, and can associate phenotypically diverse diseases to a single locus through allelic affinity. Here we describe an apparently novel syndrome, likely caused by de novo truncating mutations in ASXL3, which shares characteristics with Bohring-Opitz syndrome, a disease associated with de novo truncating mutations in ASXL1. Methods We used whole-genome and whole-exome sequencing to interrogate the genomes of four subjects with an undiagnosed syndrome. Results Using genome-wide sequencing, we identified heterozygous, de novo truncating mutations in ASXL3, a transcriptional repressor related to ASXL1, in four unrelated probands. We found that these probands shared similar phenotypes, including severe feeding difficulties, failure to thrive, and neurologic abnormalities with significant developmental delay. Further, they showed less phenotypic overlap with patients who had de novo truncating mutations in ASXL1. Conclusion We have identified truncating mutations in ASXL3 as the likely cause of a novel syndrome with phenotypic overlap with Bohring-Opitz syndrome. PMID:23383720

Systematic internal transcribed spacer sequence analysis for identification of clinical mold isolates in diagnostic mycology: a 5-year study.

PubMed

Ciardo, Diana E; Lucke, Katja; Imhof, Alex; Bloemberg, Guido V; Böttger, Erik C

2010-08-01

The implementation of internal transcribed spacer (ITS) sequencing for routine identification of molds in the diagnostic mycology laboratory was analyzed in a 5-year study. All mold isolates (n = 6,900) recovered in our laboratory from 2005 to 2009 were included in this study. According to a defined work flow, which in addition to troublesome phenotypic identification takes clinical relevance into account, 233 isolates were subjected to ITS sequence analysis. Sequencing resulted in successful identification for 78.6% of the analyzed isolates (57.1% at species level, 21.5% at genus level). In comparison, extended in-depth phenotypic characterization of the isolates subjected to sequencing achieved taxonomic assignment for 47.6% of these, with a mere 13.3% at species level. Optimization of DNA extraction further improved the efficacy of molecular identification. This study is the first of its kind to testify to the systematic implementation of sequence-based identification procedures in the routine workup of mold isolates in the diagnostic mycology laboratory.

Zone of Polarizing Activity Regulatory Sequence Mutations/Duplications with Preaxial Polydactyly and Longitudinal Preaxial Ray Deficiency in the Phenotype: A Review of Human Cases, Animal Models, and Insights Regarding the Pathogenesis

PubMed Central

2018-01-01

Clinicians and scientists interested in developmental biology have viewed preaxial polydactyly (PPD) and longitudinal preaxial ray deficiency (LPAD) as two different entities. Point mutations and duplications in the zone of polarizing activity regulatory sequence (ZRS) are associated with anterior ectopic expression of Sonic Hedgehog (SHH) in the limb bud and usually result in a PPD phenotype. However, some of these mutations/duplications also have LPAD in the phenotype. This unusual PPD-LPAD association in ZRS mutations/duplications has not been specifically reviewed in the literature. The author reviews this unusual entity and gives insights regarding its pathogenesis. PMID:29651423

Impact of exogenous sequences on the characteristics of an epidemic type 2 recombinant vaccine-derived poliovirus.

PubMed

Riquet, Franck B; Blanchard, Claire; Jegouic, Sophie; Balanant, Jean; Guillot, Sophie; Vibet, Marie-Anne; Rakoto-Andrianarivelo, Mala; Delpeyroux, Francis

2008-09-01

Pathogenic circulating vaccine-derived polioviruses (cVDPVs) have become a major obstacle to the successful completion of the global polio eradication program. Most cVDPVs are recombinant between the oral poliovirus vaccine (OPV) and human enterovirus species C (HEV-C). To study the role of HEV-C sequences in the phenotype of cVDPVs, we generated a series of recombinants between a Madagascar cVDPV isolate and its parental OPV type 2 strain. Results indicated that the HEV-C sequences present in this cVDPV contribute to its characteristics, including pathogenicity, suggesting that interspecific recombination contributes to the phenotypic biodiversity of polioviruses and may favor the emergence of cVDPVs.

FGFR2 mutation in 46,XY sex reversal with craniosynostosis

PubMed Central

Bagheri-Fam, Stefan; Ono, Makoto; Li, Li; Zhao, Liang; Ryan, Janelle; Lai, Raymond; Katsura, Yukako; Rossello, Fernando J.; Koopman, Peter; Scherer, Gerd; Bartsch, Oliver; Eswarakumar, Jacob V.P.; Harley, Vincent R.

2015-01-01

Patients with 46,XY gonadal dysgenesis (GD) exhibit genital anomalies, which range from hypospadias to complete male-to-female sex reversal. However, a molecular diagnosis is made in only 30% of cases. Heterozygous mutations in the human FGFR2 gene cause various craniosynostosis syndromes including Crouzon and Pfeiffer, but testicular defects were not reported. Here, we describe a patient whose features we would suggest represent a new FGFR2-related syndrome, craniosynostosis with XY male-to-female sex reversal or CSR. The craniosynostosis patient was chromosomally XY, but presented as a phenotypic female due to complete GD. DNA sequencing identified the FGFR2c heterozygous missense mutation, c.1025G>C (p.Cys342Ser). Substitution of Cys342 by Ser or other amino acids (Arg/Phe/Try/Tyr) has been previously reported in Crouzon and Pfeiffer syndrome. We show that the ‘knock-in’ Crouzon mouse model Fgfr2cC342Y/C342Y carrying a Cys342Tyr substitution displays XY gonadal sex reversal with variable expressivity. We also show that despite FGFR2c-Cys342Tyr being widely considered a gain-of-function mutation, Cys342Tyr substitution in the gonad leads to loss of function, as demonstrated by sex reversal in Fgfr2cC342Y/− mice carrying the knock-in allele on a null background. The rarity of our patient suggests the influence of modifier genes which exacerbated the testicular phenotype. Indeed, patient whole exome analysis revealed several potential modifiers expressed in Sertoli cells at the time of testis determination in mice. In summary, this study identifies the first FGFR2 mutation in a 46,XY GD patient. We conclude that, in certain rare genetic contexts, maintaining normal levels of FGFR2 signaling is important for human testis determination. PMID:26362256

From classical mutagenesis to nuclease-based breeding - directing natural DNA repair for a natural end-product.

PubMed

Pacher, Michael; Puchta, Holger

2017-05-01

Production of mutants of crop plants by the use of chemical or physical genotoxins has a long tradition. These factors induce the natural DNA repair machinery to repair damage in an error-prone way. In the case of radiation, multiple double-strand breaks (DSBs) are induced randomly in the genome, leading in very rare cases to a desirable phenotype. In recent years the use of synthetic, site-directed nucleases (SDNs) - also referred to as sequence-specific nucleases - like the CRISPR/Cas system has enabled scientists to use exactly the same naturally occurring DNA repair mechanisms for the controlled induction of genomic changes at pre-defined sites in plant genomes. As these changes are not necessarily associated with the permanent integration of foreign DNA, the obtained organisms per se cannot be regarded as genetically modified as there is no way to distinguish them from natural variants. This applies to changes induced by DSBs as well as single-strand breaks, and involves repair by non-homologous end-joining and homologous recombination. The recent development of SDN-based 'DNA-free' approaches makes mutagenesis strategies in classical breeding indistinguishable from SDN-derived targeted genome modifications, even in regard to current regulatory rules. With the advent of new SDN technologies, much faster and more precise genome editing becomes available at reasonable cost, and potentially without requiring time-consuming deregulation of newly created phenotypes. This review will focus on classical mutagenesis breeding and the application of newly developed SDNs in order to emphasize similarities in the context of the regulatory situation for genetically modified crop plants. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Unraveling the intrafamilial correlations and heritability of tumor types in MEN1: a Groupe d'étude des Tumeurs Endocrines study.

PubMed

Thevenon, J; Bourredjem, A; Faivre, L; Cardot-Bauters, C; Calender, A; Le Bras, M; Giraud, S; Niccoli, P; Odou, M F; Borson-Chazot, F; Barlier, A; Lombard-Bohas, C; Clauser, E; Tabarin, A; Pasmant, E; Chabre, O; Castermans, E; Ruszniewski, P; Bertherat, J; Delemer, B; Christin-Maitre, S; Beckers, A; Guilhem, I; Rohmer, V; Goichot, B; Caron, P; Baudin, E; Chanson, P; Groussin, L; Du Boullay, H; Weryha, G; Lecomte, P; Schillo, F; Bihan, H; Archambeaud, F; Kerlan, V; Bourcigaux, N; Kuhn, J M; Vergès, B; Rodier, M; Renard, M; Sadoul, J L; Binquet, C; Goudet, P

2015-12-01

MEN1, which is secondary to the mutation of the MEN1 gene, is a rare autosomal-dominant disease that predisposes mutation carriers to endocrine tumors. Most studies demonstrated the absence of direct genotype-phenotype correlations. The existence of a higher risk of death in the Groupe d'étude des Tumeurs Endocrines-cohort associated with a mutation in the JunD interacting domain suggests heterogeneity across families in disease expressivity. This study aims to assess the existence of modifying genetic factors by estimating the intrafamilial correlations and heritability of the six main tumor types in MEN1. The study included 797 patients from 265 kindred and studied seven phenotypic criteria: parathyroid and pancreatic neuroendocrine tumors (NETs) and pituitary, adrenal, bronchial, and thymic (thNET) tumors and the presence of metastasis. Intrafamilial correlations and heritability estimates were calculated from family tree data using specific validated statistical analysis software. Intrafamilial correlations were significant and decreased along parental degrees distance for pituitary, adrenal and thNETs. The heritability of these three tumor types was consistently strong and significant with 64% (s.e.m.=0.13; P<0.001) for pituitary tumor, 65% (s.e.m.=0.21; P<0.001) for adrenal tumors, and 97% (s.e.m.=0.41; P=0.006) for thNETs. The present study shows the existence of modifying genetic factors for thymus, adrenal, and pituitary MEN1 tumor types. The identification of at-risk subgroups of individuals within cohorts is the first step toward personalization of care. Next generation sequencing on this subset of tumors will help identify the molecular basis of MEN1 variable genetic expressivity. © 2015 European Society of Endocrinology.

Effects of Tributyltin Chloride on Cybrids with or without an ATP Synthase Pathologic Mutation

PubMed Central

López-Gallardo, Ester; Llobet, Laura; Emperador, Sonia; Montoya, Julio; Ruiz-Pesini, Eduardo

2016-01-01

Background: The oxidative phosphorylation system (OXPHOS) includes nuclear chromosome (nDNA)– and mitochondrial DNA (mtDNA)–encoded polypeptides. Many rare OXPHOS disorders, such as striatal necrosis syndromes, are caused by genetic mutations. Despite important advances in sequencing procedures, causative mutations remain undetected in some patients. It is possible that etiologic factors, such as environmental toxins, are the cause of these cases. Indeed, the inhibition of a particular enzyme by a poison could imitate the biochemical effects of pathological mutations in that enzyme. Moreover, environmental factors can modify the penetrance or expressivity of pathological mutations. Objectives: We studied the interaction between mitochondrially encoded ATP synthase 6 (p.MT-ATP6) subunit and an environmental exposure that may contribute phenotypic differences between healthy individuals and patients suffering from striatal necrosis syndromes or other mitochondriopathies. Methods: We analyzed the effects of the ATP synthase inhibitor tributyltin chloride (TBTC), a widely distributed environmental factor that contaminates human food and water, on transmitochondrial cell lines with or without an ATP synthase mutation that causes striatal necrosis syndrome. Doses were selected based on TBTC concentrations previously reported in human whole blood samples. Results: TBTC modified the phenotypic effects caused by a pathological mtDNA mutation. Interestingly, wild-type cells treated with this xenobiotic showed similar bioenergetics when compared with the untreated mutated cells. Conclusions: In addition to the known genetic causes, our findings suggest that environmental exposure to TBTC might contribute to the etiology of striatal necrosis syndromes. Citation: López-Gallardo E, Llobet L, Emperador S, Montoya J, Ruiz-Pesini E. 2016. Effects of tributyltin chloride on cybrids with or without an ATP synthase pathologic mutation. Environ Health Perspect 124:1399–1405; http://dx.doi.org/10.1289/EHP182 PMID:27129022

Effects of Tributyltin Chloride on Cybrids with or without an ATP Synthase Pathologic Mutation.

PubMed

López-Gallardo, Ester; Llobet, Laura; Emperador, Sonia; Montoya, Julio; Ruiz-Pesini, Eduardo

2016-09-01

The oxidative phosphorylation system (OXPHOS) includes nuclear chromosome (nDNA)- and mitochondrial DNA (mtDNA)-encoded polypeptides. Many rare OXPHOS disorders, such as striatal necrosis syndromes, are caused by genetic mutations. Despite important advances in sequencing procedures, causative mutations remain undetected in some patients. It is possible that etiologic factors, such as environmental toxins, are the cause of these cases. Indeed, the inhibition of a particular enzyme by a poison could imitate the biochemical effects of pathological mutations in that enzyme. Moreover, environmental factors can modify the penetrance or expressivity of pathological mutations. We studied the interaction between mitochondrially encoded ATP synthase 6 (p.MT-ATP6) subunit and an environmental exposure that may contribute phenotypic differences between healthy individuals and patients suffering from striatal necrosis syndromes or other mitochondriopathies. We analyzed the effects of the ATP synthase inhibitor tributyltin chloride (TBTC), a widely distributed environmental factor that contaminates human food and water, on transmitochondrial cell lines with or without an ATP synthase mutation that causes striatal necrosis syndrome. Doses were selected based on TBTC concentrations previously reported in human whole blood samples. TBTC modified the phenotypic effects caused by a pathological mtDNA mutation. Interestingly, wild-type cells treated with this xenobiotic showed similar bioenergetics when compared with the untreated mutated cells. In addition to the known genetic causes, our findings suggest that environmental exposure to TBTC might contribute to the etiology of striatal necrosis syndromes. López-Gallardo E, Llobet L, Emperador S, Montoya J, Ruiz-Pesini E. 2016. Effects of tributyltin chloride on cybrids with or without an ATP synthase pathologic mutation. Environ Health Perspect 124:1399-1405; http://dx.doi.org/10.1289/EHP182.

Modified Carbapenem Inactivation Method for Phenotypic Detection of Carbapenemase Production among Enterobacteriaceae

PubMed Central

Simner, Patricia J.; Lonsway, David R.; Roe-Carpenter, Darcie E.; Johnson, J. Kristie; Brasso, William B.; Bobenchik, April M.; Lockett, Zabrina C.; Charnot-Katsikas, Angella; Ferraro, Mary Jane; Thomson, Richard B.; Jenkins, Stephen G.; Limbago, Brandi M.; Das, Sanchita

2017-01-01

ABSTRACT The ability of clinical microbiology laboratories to reliably detect carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) is an important element of the effort to prevent and contain the spread of these pathogens and an integral part of antimicrobial stewardship. All existing methods have limitations. A new, straightforward, inexpensive, and specific phenotypic method for the detection of carbapenemase production, the carbapenem inactivation method (CIM), was recently described. Here we describe a two-stage evaluation of a modified carbapenem inactivation method (mCIM), in which tryptic soy broth was substituted for water during the inactivation step and the length of this incubation was extended. A validation study was performed in a single clinical laboratory to determine the accuracy of the mCIM, followed by a nine-laboratory study to verify the reproducibility of these results and define the zone size cutoff that best discriminated between CP-CRE and members of the family Enterobacteriaceae that do not produce carbapenemases. Bacterial isolates previously characterized through whole-genome sequencing or targeted PCR as to the presence or absence of carbapenemase genes were tested for carbapenemase production using the mCIM; isolates with Ambler class A, B, and D carbapenemases, non-CP-CRE isolates, and carbapenem-susceptible isolates were included. The sensitivity of the mCIM observed in the validation study was 99% (95% confidence interval [95% CI], 93% to 100%), and the specificity was 100% (95% CI, 82% to 100%). In the second stage of the study, the range of sensitivities observed across nine laboratories was 93% to 100%, with a mean of 97%; the range of specificities was 97% to 100%, with a mean of 99%. The mCIM was easy to perform and interpret for Enterobacteriaceae, with results in less than 24 h and excellent reproducibility across laboratories. PMID:28381609

Overexpression of Nrp/b (nuclear restrict protein in brain) suppresses the malignant phenotype in the C6/ST1 glioma cell line.

PubMed

Degaki, Theri Leica; Demasi, Marcos Angelo Almeida; Sogayar, Mari Cleide

2009-11-01

Upon searching for glucocorticoid-regulated cDNA sequences associated with the transformed to normal phenotypic reversion of C6/ST1 rat glioma cells, we identified Nrp/b (nuclear restrict protein in brain) as a novel rat gene. Here we report on the identification and functional characterization of the complete sequence encoding the rat NRP/B protein. The cloned cDNA presented a 1767 nucleotides open-reading frame encoding a 589 amino acids residues sequence containing a BTB/POZ (broad complex Tramtrack bric-a-brac/Pox virus and zinc finger) domain in its N-terminal region and kelch motifs in its C-terminal region. Sequence analysis indicates that the rat Nrp/b displays a high level of identity with the equivalent gene orthologs from other organisms. Among rat tissues, Nrp/b expression is more pronounced in brain tissue. We show that overexpression of the Nrp/b cDNA in C6/ST1 cells suppresses anchorage independence in vitro and tumorigenicity in vivo, altering their malignant nature towards a more benign phenotype. Therefore, Nrp/b may be postulated as a novel tumor suppressor gene, with possible relevance for glioblastoma therapy.

Mitochondrial DNA sequence data reveals association of haplogroup U with psychosis in bipolar disorder.

PubMed

Frye, Mark A; Ryu, Euijung; Nassan, Malik; Jenkins, Gregory D; Andreazza, Ana C; Evans, Jared M; McElroy, Susan L; Oglesbee, Devin; Highsmith, W Edward; Biernacka, Joanna M

2017-01-01

Converging genetic, postmortem gene-expression, cellular, and neuroimaging data implicate mitochondrial dysfunction in bipolar disorder. This study was conducted to investigate whether mitochondrial DNA (mtDNA) haplogroups and single nucleotide variants (SNVs) are associated with sub-phenotypes of bipolar disorder. MtDNA from 224 patients with Bipolar I disorder (BPI) was sequenced, and association of sequence variations with 3 sub-phenotypes (psychosis, rapid cycling, and adolescent illness onset) was evaluated. Gene-level tests were performed to evaluate overall burden of minor alleles for each phenotype. The haplogroup U was associated with a higher risk of psychosis. Secondary analyses of SNVs provided nominal evidence for association of psychosis with variants in the tRNA, ND4 and ND5 genes. The association of psychosis with ND4 (gene that encodes NADH dehydrogenase 4) was further supported by gene-level analysis. Preliminary analysis of mtDNA sequence data suggests a higher risk of psychosis with the U haplogroup and variation in the ND4 gene implicated in electron transport chain energy regulation. Further investigation of the functional consequences of this mtDNA variation is encouraged. Copyright © 2016. Published by Elsevier Ltd.

Predictive genomics: a cancer hallmark network framework for predicting tumor clinical phenotypes using genome sequencing data.

PubMed

Wang, Edwin; Zaman, Naif; Mcgee, Shauna; Milanese, Jean-Sébastien; Masoudi-Nejad, Ali; O'Connor-McCourt, Maureen

2015-02-01

Tumor genome sequencing leads to documenting thousands of DNA mutations and other genomic alterations. At present, these data cannot be analyzed adequately to aid in the understanding of tumorigenesis and its evolution. Moreover, we have little insight into how to use these data to predict clinical phenotypes and tumor progression to better design patient treatment. To meet these challenges, we discuss a cancer hallmark network framework for modeling genome sequencing data to predict cancer clonal evolution and associated clinical phenotypes. The framework includes: (1) cancer hallmarks that can be represented by a few molecular/signaling networks. 'Network operational signatures' which represent gene regulatory logics/strengths enable to quantify state transitions and measures of hallmark traits. Thus, sets of genomic alterations which are associated with network operational signatures could be linked to the state/measure of hallmark traits. The network operational signature transforms genotypic data (i.e., genomic alterations) to regulatory phenotypic profiles (i.e., regulatory logics/strengths), to cellular phenotypic profiles (i.e., hallmark traits) which lead to clinical phenotypic profiles (i.e., a collection of hallmark traits). Furthermore, the framework considers regulatory logics of the hallmark networks under tumor evolutionary dynamics and therefore also includes: (2) a self-promoting positive feedback loop that is dominated by a genomic instability network and a cell survival/proliferation network is the main driver of tumor clonal evolution. Surrounding tumor stroma and its host immune systems shape the evolutionary paths; (3) cell motility initiating metastasis is a byproduct of the above self-promoting loop activity during tumorigenesis; (4) an emerging hallmark network which triggers genome duplication dominates a feed-forward loop which in turn could act as a rate-limiting step for tumor formation; (5) mutations and other genomic alterations have specific patterns and tissue-specificity, which are driven by aging and other cancer-inducing agents. This framework represents the logics of complex cancer biology as a myriad of phenotypic complexities governed by a limited set of underlying organizing principles. It therefore adds to our understanding of tumor evolution and tumorigenesis, and moreover, potential usefulness of predicting tumors' evolutionary paths and clinical phenotypes. Strategies of using this framework in conjunction with genome sequencing data in an attempt to predict personalized drug targets, drug resistance, and metastasis for cancer patients, as well as cancer risks for healthy individuals are discussed. Accurate prediction of cancer clonal evolution and clinical phenotypes will have substantial impact on timely diagnosis, personalized treatment and personalized prevention of cancer. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

A speculated ribozyme site in the herpes simplex virus type 1 latency-associated transcript gene is not essential for a wild-type reactivation phenotype

PubMed Central

Carpenter, Dale; Singh, Sukhpreet; Osorio, Nelson; Hsiang, Chinhui; Jiang, Xianzhi; Jin, Ling; Jones, Clinton; Wechsler, Steven L

2010-01-01

During herpes simplex virus-1 (HSV-1) latency in sensory neurons, LAT (latency-associated transcript) is the only abundantly expressed viral gene. LAT plays an important role in the HSV-1 latency-reactivation cycle, because LAT deletion mutants have a significantly decreased reactivation phenotype. Based solely on sequence analysis, it was speculated that LAT encodes a ribozyme that plays an important role in how LAT enhances the virus’ reactivation phenotype. Because LAT ribozyme activity has never been reported, we decided to test the converse hypothesis, namely, that this region of LAT does not encode a ribozyme function important for LAT’s ability to enhance the reactivation phenotype. We constructed a viral mutant (LAT-Rz) in which the speculated ribozyme consensus sequence was altered such that no ribozyme was encoded. We report here that LAT-Rz had a wild-type reactivation phenotype in mice, confirming the hypothesis that the speculated LAT ribozyme is not a dominant factor in stimulating the latency-reactivation cycle in mice. PMID:18982533

Cyclic neutropenia and severe congenital neutropenia in patients with a shared ELANE mutation and paternal haplotype: evidence for phenotype determination by modifying genes.

PubMed

Newburger, Peter E; Pindyck, Talia N; Zhu, Zhiqing; Bolyard, Audrey Anna; Aprikyan, Andrew A G; Dale, David C; Smith, Gary D; Boxer, Laurence A

2010-08-01

Cyclic neutropenia (CN) and severe congenital neutropenia (SCN) are disorders of neutrophil production that differ markedly in disease severity. Mutations of the ELANE gene (the symbol recently replacing ELA2) are considered largely responsible for most cases of CN and SCN, but specific mutations are typically associated with one or the other. We performed ELANE genotyping on all individuals and paternal sperm in an SCN kindred with eight SCN progeny of a sperm donor and six different mothers. One patient with CN had the same S97L ELANE mutation as seven patients with the SCN phenotype. The mutant allele was detected in the donor's spermatozoa, representing 18% of the ELANE gene pool, but not in DNA from his lymphocytes, neutrophils, or buccal mucosa, indicating gonadal mosaicism. The coexistence of CN and SCN phenotypes in this kindred with a shared paternal haplotype strongly suggests both a role for modifying genes in determination of congenital neutropenia disease phenotypes, and the classification of CN and SCN within a spectrum of phenotypes expressing varying degrees of the same disease process. (c) 2010 Wiley-Liss, Inc.

Exome sequencing-driven discovery of coding polymorphisms associated with common metabolic phenotypes.

PubMed

Albrechtsen, A; Grarup, N; Li, Y; Sparsø, T; Tian, G; Cao, H; Jiang, T; Kim, S Y; Korneliussen, T; Li, Q; Nie, C; Wu, R; Skotte, L; Morris, A P; Ladenvall, C; Cauchi, S; Stančáková, A; Andersen, G; Astrup, A; Banasik, K; Bennett, A J; Bolund, L; Charpentier, G; Chen, Y; Dekker, J M; Doney, A S F; Dorkhan, M; Forsen, T; Frayling, T M; Groves, C J; Gui, Y; Hallmans, G; Hattersley, A T; He, K; Hitman, G A; Holmkvist, J; Huang, S; Jiang, H; Jin, X; Justesen, J M; Kristiansen, K; Kuusisto, J; Lajer, M; Lantieri, O; Li, W; Liang, H; Liao, Q; Liu, X; Ma, T; Ma, X; Manijak, M P; Marre, M; Mokrosiński, J; Morris, A D; Mu, B; Nielsen, A A; Nijpels, G; Nilsson, P; Palmer, C N A; Rayner, N W; Renström, F; Ribel-Madsen, R; Robertson, N; Rolandsson, O; Rossing, P; Schwartz, T W; Slagboom, P E; Sterner, M; Tang, M; Tarnow, L; Tuomi, T; van't Riet, E; van Leeuwen, N; Varga, T V; Vestmar, M A; Walker, M; Wang, B; Wang, Y; Wu, H; Xi, F; Yengo, L; Yu, C; Zhang, X; Zhang, J; Zhang, Q; Zhang, W; Zheng, H; Zhou, Y; Altshuler, D; 't Hart, L M; Franks, P W; Balkau, B; Froguel, P; McCarthy, M I; Laakso, M; Groop, L; Christensen, C; Brandslund, I; Lauritzen, T; Witte, D R; Linneberg, A; Jørgensen, T; Hansen, T; Wang, J; Nielsen, R; Pedersen, O

2013-02-01

Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m(2) and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case-control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans. Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10(-14)), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10(-11)) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10(-10)). We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits.

Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease

PubMed Central

Owen, Joseph R.; Noyes, Noelle; Young, Amy E.; Prince, Daniel J.; Blanchard, Patricia C.; Lehenbauer, Terry W.; Aly, Sharif S.; Davis, Jessica H.; O’Rourke, Sean M.; Abdo, Zaid; Belk, Keith; Miller, Michael R.; Morley, Paul; Van Eenennaam, Alison L.

2017-01-01

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease–associated bacterial isolates (Histophilus somni, Mycoplasma bovis, Mannheimia haemolytica, and Pasteurella multocida) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis. While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 H. somni, M. haemolytica, and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% (P < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease treatment and morbidity/mortality outcomes. PMID:28739600

Massively Parallel Sequencing Reveals the Complex Structure of an Irradiated Human Chromosome on a Mouse Background in the Tc1 Model of Down Syndrome

PubMed Central

Clayton, Stephen; Prigmore, Elena; Langley, Elizabeth; Yang, Fengtang; Maguire, Sean; Fu, Beiyuan; Rajan, Diana; Sheppard, Olivia; Scott, Carol; Hauser, Heidi; Stephens, Philip J.; Stebbings, Lucy A.; Ng, Bee Ling; Fitzgerald, Tomas; Quail, Michael A.; Banerjee, Ruby; Rothkamm, Kai; Tybulewicz, Victor L. J.; Fisher, Elizabeth M. C.; Carter, Nigel P.

2013-01-01

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype – phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439. PMID:23596509

Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease.

PubMed

Owen, Joseph R; Noyes, Noelle; Young, Amy E; Prince, Daniel J; Blanchard, Patricia C; Lehenbauer, Terry W; Aly, Sharif S; Davis, Jessica H; O'Rourke, Sean M; Abdo, Zaid; Belk, Keith; Miller, Michael R; Morley, Paul; Van Eenennaam, Alison L

2017-09-07

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease-associated bacterial isolates ( Histophilus somni , Mycoplasma bovis , Mannheimia haemolytica , and Pasteurella multocida ) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 H. somni , M. haemolytica , and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% ( P < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease treatment and morbidity/mortality outcomes. Copyright © 2017 Owen et al.

Translational genomics for plant breeding with the genome sequence explosion.

PubMed

Kang, Yang Jae; Lee, Taeyoung; Lee, Jayern; Shim, Sangrea; Jeong, Haneul; Satyawan, Dani; Kim, Moon Young; Lee, Suk-Ha

2016-04-01

The use of next-generation sequencers and advanced genotyping technologies has propelled the field of plant genomics in model crops and plants and enhanced the discovery of hidden bridges between genotypes and phenotypes. The newly generated reference sequences of unstudied minor plants can be annotated by the knowledge of model plants via translational genomics approaches. Here, we reviewed the strategies of translational genomics and suggested perspectives on the current databases of genomic resources and the database structures of translated information on the new genome. As a draft picture of phenotypic annotation, translational genomics on newly sequenced plants will provide valuable assistance for breeders and researchers who are interested in genetic studies. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Fastidious Gram-Negatives: Identification by the Vitek 2 Neisseria-Haemophilus Card and by Partial 16S rRNA Gene Sequencing Analysis.

PubMed

Sönksen, Ute Wolff; Christensen, Jens Jørgen; Nielsen, Lisbeth; Hesselbjerg, Annemarie; Hansen, Dennis Schrøder; Bruun, Brita

2010-12-31

Taxonomy and identification of fastidious Gram negatives are evolving and challenging. We compared identifications achieved with the Vitek 2 Neisseria-Haemophilus (NH) card and partial 16S rRNA gene sequence (526 bp stretch) analysis with identifications obtained with extensive phenotypic characterization using 100 fastidious Gram negative bacteria. Seventy-five strains represented 21 of the 26 taxa included in the Vitek 2 NH database and 25 strains represented related species not included in the database. Of the 100 strains, 31 were the type strains of the species. Vitek 2 NH identification results: 48 of 75 database strains were correctly identified, 11 strains gave `low discrimination´, seven strains were unidentified, and nine strains were misidentified. Identification of 25 non-database strains resulted in 14 strains incorrectly identified as belonging to species in the database. Partial 16S rRNA gene sequence analysis results: For 76 strains phenotypic and sequencing identifications were identical, for 23 strains the sequencing identifications were either probable or possible, and for one strain only the genus was confirmed. Thus, the Vitek 2 NH system identifies most of the commonly occurring species included in the database. Some strains of rarely occurring species and strains of non-database species closely related to database species cause problems. Partial 16S rRNA gene sequence analysis performs well, but does not always suffice, additional phenotypical characterization being useful for final identification.

Fastidious Gram-Negatives: Identification by the Vitek 2 Neisseria-Haemophilus Card and by Partial 16S rRNA Gene Sequencing Analysis

PubMed Central

Sönksen, Ute Wolff; Christensen, Jens Jørgen; Nielsen, Lisbeth; Hesselbjerg, Annemarie; Hansen, Dennis Schrøder; Bruun, Brita

2010-01-01

Taxonomy and identification of fastidious Gram negatives are evolving and challenging. We compared identifications achieved with the Vitek 2 Neisseria-Haemophilus (NH) card and partial 16S rRNA gene sequence (526 bp stretch) analysis with identifications obtained with extensive phenotypic characterization using 100 fastidious Gram negative bacteria. Seventy-five strains represented 21 of the 26 taxa included in the Vitek 2 NH database and 25 strains represented related species not included in the database. Of the 100 strains, 31 were the type strains of the species. Vitek 2 NH identification results: 48 of 75 database strains were correctly identified, 11 strains gave `low discrimination´, seven strains were unidentified, and nine strains were misidentified. Identification of 25 non-database strains resulted in 14 strains incorrectly identified as belonging to species in the database. Partial 16S rRNA gene sequence analysis results: For 76 strains phenotypic and sequencing identifications were identical, for 23 strains the sequencing identifications were either probable or possible, and for one strain only the genus was confirmed. Thus, the Vitek 2 NH system identifies most of the commonly occurring species included in the database. Some strains of rarely occurring species and strains of non-database species closely related to database species cause problems. Partial 16S rRNA gene sequence analysis performs well, but does not always suffice, additional phenotypical characterization being useful for final identification. PMID:21347215

Sequenced sorghum mutant library- an efficient platform for discovery of causal gene mutations

USDA-ARS?s Scientific Manuscript database

Ethyl methanesulfonate (EMS) efficiently generates high-density mutations in genomes. We applied whole-genome sequencing to 256 phenotyped mutant lines of sorghum (Sorghum bicolor L. Moench) to 16x coverage. Comparisons with the reference sequence revealed >1.8 million canonical EMS-induced G/C to A...

Phenotypic mutant library: potential for gene discovery

USDA-ARS?s Scientific Manuscript database

The rapid development of high throughput and affordable Next- Generation Sequencing (NGS) techniques has renewed interest in gene discovery using forward genetics. The conventional forward genetic approach starts with isolation of mutants with a phenotype of interest, mapping the mutation within a s...

Progranulin mutation causes frontotemporal dementia in the Swedish Karolinska family.

PubMed

Chiang, Huei-Hsin; Rosvall, Lina; Brohede, Jesper; Axelman, Karin; Björk, Behnosh F; Nennesmo, Inger; Robins, Tiina; Graff, Caroline

2008-11-01

Frontotemporal dementia (FTD) is a neurodegenerative disease characterized by cognitive impairment, language dysfunction, and/or changes in personality. Recently it has been shown that progranulin (GRN) mutations can cause FTD as well as other neurodegenerative phenotypes. DNA from 30 family members, of whom seven were diagnosed with FTD, in the Karolinska family was available for GRN sequencing. Fibroblast cell mRNA from one affected family member and six control individuals was available for relative quantitative real-time polymerase chain reaction to investigate the effect of the mutation. Furthermore, the cDNA of an affected individual was sequenced. Clinical and neuropathologic findings of a previously undescribed family branch are presented. A frameshift mutation in GRN (g.102delC) was detected in all affected family members and absent in four unaffected family members older than 70 years. Real-time polymerase chain reaction data showed an approximately 50% reduction of GRN fibroblast mRNA in an affected individual. The mutated mRNA transcripts were undetectable by cDNA sequencing. Segregation and RNA analyses showed that the g.102delC mutation, previously reported, causes FTD in the Karolinska family. Our findings add further support to the significance of GRN in FTD etiology and the presence of modifying genes, which emphasize the need for further studies into the mechanisms of clinical heterogeneity. However, the results already call for attention to the complexity of predictive genetic testing of GRN mutations.

High-resolution melting analysis (HRM) for mutational screening of Dnajc17 gene in patients affected by thyroid dysgenesis.

PubMed

Nettore, I C; Desiderio, S; De Nisco, E; Cacace, V; Albano, L; Improda, N; Ungaro, P; Salerno, M; Colao, A; Macchia, P E

2018-06-01

Congenital hypothyroidism is a frequent disease occurring with an incidence of about 1/1500 newborns/year. In about 75% of the cases, CH is caused by alterations in thyroid morphogenesis, defined "thyroid dysgenesis" (TD). TD is generally a sporadic disease but in about 5% of the cases a genetic origin has been demonstrated. Previous studies indicate that Dnajc17 as a candidate modifier gene for hypothyroidism, since it is expressed in the thyroid bud, interacts with NKX2.1 and PAX8 and it has been associated to the hypothyroid phenotype in mice carrying a single Nkx2.1 and Pax8 genes (double heterozygous knock-out). The work evaluates the possible involvement of DNAJC17 in the pathogenesis of TD. High-resolution DNA melting analysis (HRM) and direct sequencing have been used to screen for mutations in the DNAJC17 coding sequence in 89 patients with TD. Two mutations have been identified in the coding sequence of DNAJC17 gene, one in exon 5 (c.350A>C; rs79709714) and one in exon 9 (c.610G>C; rs117485355). The last one is a rare variant, while the rs79709714 is a polymorphism. Both are present in databases and the frequency of the alleles is not different between TD patients and controls. DNAJC17 mutations are not frequently present in patients with TD.

Epigenetics of sex determination and gonadogenesis.

PubMed

Piferrer, Francesc

2013-04-01

Epigenetics is commonly defined as the study of heritable changes in gene function that cannot be explained by changes in DNA sequence. The three major epigenetic mechanisms for gene expression regulation include DNA methylation, histone modifications, and non-coding RNAs. Epigenetic mechanisms provide organisms with the ability to integrate genomic and environmental information to modify the activity of their genes for generating a particular phenotype. During development, cells differentiate, acquire, and maintain identity through changes in gene expression. This is crucial for sex determination and differentiation, which are among the most important developmental processes for the proper functioning and perpetuation of species. This review summarizes studies showing how epigenetic regulatory mechanisms contribute to sex determination and reproductive organ formation in plants, invertebrates, and vertebrates. Further progress will be made by integrating several approaches, including genomics and Next Generation Sequencing to create epigenetic maps related to different aspects of sex determination and gonadogenesis. Epigenetics will also contribute to understand the etiology of several disorders of sexual development. It also might play a significant role in the control of reproduction in animal farm production and will aid in recognizing the environmental versus genetic influences on sex determination of sensitive species in a global change scenario. Copyright © 2013 Wiley Periodicals, Inc.

Sequence and immunogenicity of a clinically approved novel measles virus vaccine vector

PubMed Central

Zuniga, Amando; Liniger, Mathias; Morin, Teldja Neige Azzouz; Marty, René R.; Wiegand, Marian; Ilter, Orhan; Weibel, Sara; Billeter, Martin A.; Knuchel, Marlyse C.; Naim, Hussein Y.

2013-01-01

The measles virus vaccine (MVbv) is a clinically certified and well-tolerated vaccine strain that has been given both parenterally and mucosally. It has been extensively used in children and has proven to be safe and effective in eliciting protective immunity. This specific strain was therefore chosen to generate a measles viral vector. The genome of the commercial MVbv vaccine strain was isolated, sequenced and a plasmid, p(+)MVb, enabling transcription of the viral antigenome and rescue of MVb, was constructed. Phylogenic and phenotypic analysis revealed that MVbv and the rescued MVb constitute another evolutionary branch within the hitherto classified measles vaccines. Plasmid p(+)MVb was modified by insertion of artificial MV-type transcription units (ATUs) for the generation of recombinant viruses (rMVb) expressing additional proteins. Replication characteristics and immunogenicity of rMVb vectors were similar to the parental MVbv and to other vaccine strains. The expression of the additional proteins was stable over 10 serial virus transfers, which corresponds to an amplification greater than 1020. The excellent safety record and its efficient application as aerosol may add to the usefulness of the derived vectors. PMID:23324616

The alternatively-included 11a sequence modifies the effects of Mena on actin cytoskeletal organization and cell behavior

PubMed Central

Balsamo, Michele; Mondal, Chandrani; Carmona, Guillaume; McClain, Leslie M.; Riquelme, Daisy N.; Tadros, Jenny; Ma, Duan; Vasile, Eliza; Condeelis, John S.; Lauffenburger, Douglas A.; Gertler, Frank B.

2016-01-01

During tumor progression, alternative splicing gives rise to different Mena protein isoforms. We analyzed how Mena11a, an isoform enriched in epithelia and epithelial-like cells, affects Mena-dependent regulation of actin dynamics and cell behavior. While other Mena isoforms promote actin polymerization and drive membrane protrusion, we find that Mena11a decreases actin polymerization and growth factor-stimulated membrane protrusion at lamellipodia. Ectopic Mena11a expression slows mesenchymal-like cell motility, while isoform-specific depletion of endogenous Mena11a in epithelial-like tumor cells perturbs cell:cell junctions and increases membrane protrusion and overall cell motility. Mena11a can dampen membrane protrusion and reduce actin polymerization in the absence of other Mena isoforms, indicating that it is not simply an inactive Mena isoform. We identify a phosphorylation site within 11a that is required for some Mena11a-specific functions. RNA-seq data analysis from patient cohorts demonstrates that the difference between mRNAs encoding constitutive Mena sequences and those containing the 11a exon correlates with metastasis in colorectal cancer, suggesting that 11a exon exclusion contributes to invasive phenotypes and leads to poor clinical outcomes. PMID:27748415

The alternatively-included 11a sequence modifies the effects of Mena on actin cytoskeletal organization and cell behavior.

PubMed

Balsamo, Michele; Mondal, Chandrani; Carmona, Guillaume; McClain, Leslie M; Riquelme, Daisy N; Tadros, Jenny; Ma, Duan; Vasile, Eliza; Condeelis, John S; Lauffenburger, Douglas A; Gertler, Frank B

2016-10-17

During tumor progression, alternative splicing gives rise to different Mena protein isoforms. We analyzed how Mena11a, an isoform enriched in epithelia and epithelial-like cells, affects Mena-dependent regulation of actin dynamics and cell behavior. While other Mena isoforms promote actin polymerization and drive membrane protrusion, we find that Mena11a decreases actin polymerization and growth factor-stimulated membrane protrusion at lamellipodia. Ectopic Mena11a expression slows mesenchymal-like cell motility, while isoform-specific depletion of endogenous Mena11a in epithelial-like tumor cells perturbs cell:cell junctions and increases membrane protrusion and overall cell motility. Mena11a can dampen membrane protrusion and reduce actin polymerization in the absence of other Mena isoforms, indicating that it is not simply an inactive Mena isoform. We identify a phosphorylation site within 11a that is required for some Mena11a-specific functions. RNA-seq data analysis from patient cohorts demonstrates that the difference between mRNAs encoding constitutive Mena sequences and those containing the 11a exon correlates with metastasis in colorectal cancer, suggesting that 11a exon exclusion contributes to invasive phenotypes and leads to poor clinical outcomes.

Carbapenem-resistant Gram-negative bacteria associated with catheter-related bloodstream infections in three intensive care units in Egypt.

PubMed

Abdulall, Abeer K; Tawfick, Mahmoud M; El Manakhly, Arwa R; El Kholy, Amani

2018-06-23

We aimed to identify the carbapenem-resistant Gram-negative bacteria (GNB) causing catheter-related bloodstream infections (CRBSI) in intensive care units (ICU) in a tertiary care Egyptian hospital, to study their resistance mechanisms by phenotypic and genetic tests, and to use ERIC-PCR for assessing their relatedness. The study was conducted over 2 years in three ICUs in a tertiary care hospital in Egypt during 2015-2016. We identified 194 bloodstream infections (BSIs); 130 (67.01%) were caused by GNB, of which 57 were isolated from CRBSI patients (73.84%). Identification of isolates was performed using conventional methods and MALDI-TOF MS. Antimicrobial susceptibility testing (AST) was done by disc diffusion following CLSI guidelines. Phenotypic detection of carbapenemases enzymes activity was by modified Hodge test and the Carba-NP method. Isolates were investigated for the most common carbapenemases encoding genes bla KPC , bla NDM , and bla OXA-48 using multiplex PCR. Molecular typing of carbapenem-resistant isolates was done by ERIC-PCR followed by sequencing of common resistance genes. The overall rate of CRBSI in our study was 3.6 per 1000 central venous catheter (CVC) days. Among 57 Gram-negative CRBSI isolates, Klebsiella pneumoniae (K. pneumoniae) was the most frequently isolated (27/57; 47.4%), of which more than 70% were resistant to Meropenem. Phenotypic tests for carbapenemases showed that 37.9% of isolates were positive by modified Hodge test and 63.8% by Carba-NP detection. Multiplex PCR assay detected the bla NDM in 28.6% of the isolates and bla KPC in 26.8%, bla NDM and bla KPC were detected together in the same isolate in 5.6%, while bla OXA-48 -like were not detected. ERIC-PCR detected limited genetic relatedness between K. pneumoniae isolates. Elevated resistance rates were observed to all antibiotics including carbapenems among K. pneumoniae isolates causing CRBSI. ERIC-PCR showed that the resistant isolates were mainly polyclonal. Our results call for reinforcement of antimicrobial stewardship and measures to prevent CRBSI.

Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.)

PubMed Central

Urasaki, Naoya; Goeku, Satoko; Kaneshima, Risa; Takamine, Tomonori; Tarora, Kazuhiko; Takeuchi, Makoto; Moromizato, Chie; Yonamine, Kaname; Hosaka, Fumiko; Terakami, Shingo; Matsumura, Hideo; Yamamoto, Toshiya; Shoda, Moriyuki

2015-01-01

To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a ‘piping-leaf-type’ cultivar, ‘Yugafu’, and a ‘spiny-tip-leaf-type’ variety, ‘Yonekura’. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the ‘spiny-leaf type’ as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding. PMID:26175625

Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.).

PubMed

Urasaki, Naoya; Goeku, Satoko; Kaneshima, Risa; Takamine, Tomonori; Tarora, Kazuhiko; Takeuchi, Makoto; Moromizato, Chie; Yonamine, Kaname; Hosaka, Fumiko; Terakami, Shingo; Matsumura, Hideo; Yamamoto, Toshiya; Shoda, Moriyuki

2015-06-01

To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a 'piping-leaf-type' cultivar, 'Yugafu', and a 'spiny-tip-leaf-type' variety, 'Yonekura'. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the 'spiny-leaf type' as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding.

Duffy blood group phenotype-genotype correlations using high-resolution melting analysis PCR and microarray reveal complex cases including a new null FY*A allele: the role for sequencing in genotyping algorithms.

PubMed

Lopez, G H; Morrison, J; Condon, J A; Wilson, B; Martin, J R; Liew, Y-W; Flower, R L; Hyland, C A

2015-10-01

Duffy blood group phenotypes can be predicted by genotyping for single nucleotide polymorphisms (SNPs) responsible for the Fy(a) /Fy(b) polymorphism, for weak Fy(b) antigen, and for the red cell null Fy(a-b-) phenotype. This study correlates Duffy phenotype predictions with serotyping to assess the most reliable procedure for typing. Samples, n = 155 (135 donors and 20 patients), were genotyped by high-resolution melt PCR and by microarray. Samples were in three serology groups: 1) Duffy patterns expected n = 79, 2) weak and equivocal Fy(b) patterns n = 29 and 3) Fy(a-b-) n = 47 (one with anti-Fy3 antibody). Discrepancies were observed for five samples. For two, SNP genotyping predicted weak Fy(b) expression discrepant with Fy(b-) (Group 1 and 3). For three, SNP genotyping predicted Fy(a) , discrepant with Fy(a-b-) (Group 3). DNA sequencing identified silencing mutations in these FY*A alleles. One was a novel FY*A 719delG. One, the sample with the anti-Fy3, was homozygous for a 14-bp deletion (FY*01N.02); a true null. Both the high-resolution melting analysis and SNP microarray assays were concordant and showed genotyping, as well as phenotyping, is essential to ensure 100% accuracy for Duffy blood group assignments. Sequencing is important to resolve phenotype/genotype conflicts which here identified alleles, one novel, that carry silencing mutations. The risk of alloimmunisation may be dependent on this zygosity status. © 2015 International Society of Blood Transfusion.

Phenotypic and genotypic analysis of Borrelia burgdorferi isolates from various sources.

PubMed Central

Adam, T; Gassmann, G S; Rasiah, C; Göbel, U B

1991-01-01

A total of 17 B. burgdorferi isolates from various sources were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, restriction enzyme analysis, Southern hybridization with probes complementary to unique regions of evolutionarily conserved genes (16S rRNA and fla), and direct sequencing of in vitro polymerase chain reaction-amplified fragments of the 16S rRNA gene. Three groups were distinguished on the basis of phenotypic and genotypic traits, the latter traced to the nucleotide sequence level. Images PMID:1649797

Overexpression of α3/α5/β4 nicotinic receptor subunits modifies impulsive-like behavior.

PubMed

Viñals, Xavier; Molas, Susanna; Gallego, Xavier; Fernández-Montes, Rubén D; Robledo, Patricia; Dierssen, Mara; Maldonado, Rafael

2012-05-01

Recent studies have revealed that sequence variants in genes encoding the α3/α5/β4 nicotinic acetylcholine receptor subunits are associated with nicotine dependence. In this study, we evaluated two specific aspects of executive functioning related to drug addiction (impulsivity and working memory) in transgenic mice over expressing α3/α5/β4 nicotinic receptor subunits. Impulsivity and working memory were evaluated in an operant delayed alternation task, where mice must inhibit responding between 2 and 8s in order to receive food reinforcement. Working memory was also evaluated in a spontaneous alternation task in an open field. Transgenic mice showed less impulsive-like behavior than wild-type controls, and this behavioral phenotype was related to the number of copies of the transgene. Thus, transgenic Line 22 (16-28 copies) showed a more pronounced phenotype than Line 30 (4-5 copies). Overexpression of these subunits in Line 22 reduced spontaneous alternation behavior suggesting deficits in working memory processing in this particular paradigm. These results reveal the involvement of α3/α5/β4 nicotinic receptor subunits in working memory and impulsivity, two behavioral traits directly related to the vulnerability to develop nicotine dependence. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Environmental Impact on DNA Methylation in the Germline: State of the Art and Gaps of Knowledge

PubMed Central

Pacchierotti, Francesca; Spanò, Marcello

2015-01-01

The epigenome consists of chemical changes in DNA and chromatin that without modifying the DNA sequence modulate gene expression and cellular phenotype. The epigenome is highly plastic and reacts to changing external conditions with modifications that can be inherited to daughter cells and across generations. Whereas this innate plasticity allows for adaptation to a changing environment, it also implies the potential of epigenetic derailment leading to so-called epimutations. DNA methylation is the most studied epigenetic mark. DNA methylation changes have been associated with cancer, infertility, cardiovascular, respiratory, metabolic, immunologic, and neurodegenerative pathologies. Experiments in rodents demonstrate that exposure to a variety of chemical stressors, occurring during the prenatal or the adult life, may induce DNA methylation changes in germ cells, which may be transmitted across generations with phenotypic consequences. An increasing number of human biomonitoring studies show environmentally related DNA methylation changes mainly in blood leukocytes, whereas very few data have been so far collected on possible epigenetic changes induced in the germline, even by the analysis of easily accessible sperm. In this paper, we review the state of the art on factors impinging on DNA methylation in the germline, highlight gaps of knowledge, and propose priorities for future studies. PMID:26339587

PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

PubMed Central

2013-01-01

Background Saccharomyces cerevisiae is extensively used in bio-industries. However, its genetic engineering to introduce new metabolism pathways can cause unexpected phenotypic alterations. For example, humanisation of the glycosylation pathways is a high priority pharmaceutical industry goal for production of therapeutic glycoproteins in yeast. Genomic modifications can lead to several described physiological changes: biomass yields decrease, temperature sensitivity or cell wall structure modifications. We have observed that deletion of several N-mannosyltransferases in Saccharomyces cerevisiae, results in strains that can no longer be analyzed by classical PCR on yeast colonies. Findings In order to validate our glyco-engineered Saccharomyces cerevisiae strains, we developed a new protocol to carry out PCR directly on genetically modified yeast colonies. A liquid culture phase, combined with the use of a Hot Start DNA polymerase, allows a 3-fold improvement of PCR efficiency. The results obtained are repeatable and independent of the targeted sequence; as such the protocol is well adapted for intensive screening applications. Conclusions The developed protocol enables by-passing of many of the difficulties associated with PCR caused by phenotypic modifications brought about by humanisation of the glycosylation in yeast and allows rapid validation of glyco-engineered Saccharomyces cerevisiae cells. It has the potential to be extended to other yeast strains presenting cell wall structure modifications. PMID:23688076

Epigenetic regulation of vascular smooth muscle cell function in atherosclerosis.

PubMed

Findeisen, Hannes M; Kahles, Florian K; Bruemmer, Dennis

2013-04-01

Epigenetics involve heritable and acquired changes in gene transcription that occur independently of the DNA sequence. Epigenetic mechanisms constitute a hierarchic upper-level of transcriptional control through complex modifications of chromosomal components and nuclear structures. These modifications include, for example, DNA methylation or post-translational modifications of core histones; they are mediated by various chromatin-modifying enzymes; and ultimately they define the accessibility of a transcriptional complex to its target DNA. Integrating epigenetic mechanisms into the pathophysiologic concept of complex and multifactorial diseases such as atherosclerosis may significantly enhance our understanding of related mechanisms and provide promising therapeutic approaches. Although still in its infancy, intriguing scientific progress has begun to elucidate the role of epigenetic mechanisms in vascular biology, particularly in the control of smooth muscle cell phenotypes. In this review, we will summarize epigenetic pathways in smooth muscle cells, focusing on mechanisms involved in the regulation of vascular remodeling.

Epigenetic regulation of vascular smooth muscle cell function in atherosclerosis.

PubMed

Findeisen, Hannes M; Kahles, Florian K; Bruemmer, Dennis

2013-05-01

Epigenetics involve heritable and acquired changes in gene transcription that occur independently of the DNA sequence. Epigenetic mechanisms constitute a hierarchic upper-level of transcriptional control through complex modifications of chromosomal components and nuclear structures. These modifications include, for example, DNA methylation or post-translational modifications of core histones; they are mediated by various chromatin-modifying enzymes; and ultimately they define the accessibility of a transcriptional complex to its target DNA. Integrating epigenetic mechanisms into the pathophysiologic concept of complex and multifactorial diseases such as atherosclerosis may significantly enhance our understanding of related mechanisms and provide promising therapeutic approaches. Although still in its infancy, intriguing scientific progress has begun to elucidate the role of epigenetic mechanisms in vascular biology, particularly in the control of smooth muscle cell phenotypes. In this review, we will summarize epigenetic pathways in smooth muscle cells, focusing on mechanisms involved in the regulation of vascular remodeling.

Of mice and men: molecular genetics of congenital heart disease.

PubMed

Andersen, Troels Askhøj; Troelsen, Karin de Linde Lind; Larsen, Lars Allan

2014-04-01

Congenital heart disease (CHD) affects nearly 1 % of the population. It is a complex disease, which may be caused by multiple genetic and environmental factors. Studies in human genetics have led to the identification of more than 50 human genes, involved in isolated CHD or genetic syndromes, where CHD is part of the phenotype. Furthermore, mapping of genomic copy number variants and exome sequencing of CHD patients have led to the identification of a large number of candidate disease genes. Experiments in animal models, particularly in mice, have been used to verify human disease genes and to gain further insight into the molecular pathology behind CHD. The picture emerging from these studies suggest that genetic lesions associated with CHD affect a broad range of cellular signaling components, from ligands and receptors, across down-stream effector molecules to transcription factors and co-factors, including chromatin modifiers.

Accuracy of different bioinformatics methods in detecting antibiotic resistance and virulence factors from Staphylococcus aureus whole genome sequences.

PubMed

Mason, Amy; Foster, Dona; Bradley, Phelim; Golubchik, Tanya; Doumith, Michel; Gordon, N Claire; Pichon, Bruno; Iqbal, Zamin; Staves, Peter; Crook, Derrick; Walker, A Sarah; Kearns, Angela; Peto, Tim

2018-06-20

Background : In principle, whole genome sequencing (WGS) can predict phenotypic resistance directly from genotype, replacing laboratory-based tests. However, the contribution of different bioinformatics methods to genotype-phenotype discrepancies has not been systematically explored to date. Methods : We compared three WGS-based bioinformatics methods (Genefinder (read-based), Mykrobe (de Bruijn graph-based) and Typewriter (BLAST-based)) for predicting presence/absence of 83 different resistance determinants and virulence genes, and overall antimicrobial susceptibility, in 1379 Staphylococcus aureus isolates previously characterised by standard laboratory methods (disc diffusion, broth and/or agar dilution and PCR). Results : 99.5% (113830/114457) of individual resistance-determinant/virulence gene predictions were identical between all three methods, with only 627 (0.5%) discordant predictions, demonstrating high overall agreement (Fliess-Kappa=0.98, p<0.0001). Discrepancies when identified were in only one of the three methods for all genes except the cassette recombinase, ccrC(b ). Genotypic antimicrobial susceptibility prediction matched laboratory phenotype in 98.3% (14224/14464) cases (2720 (18.8%) resistant, 11504 (79.5%) susceptible). There was greater disagreement between the laboratory phenotypes and the combined genotypic predictions (97 (0.7%) phenotypically-susceptible but all bioinformatic methods reported resistance; 89 (0.6%) phenotypically-resistant, but all bioinformatics methods reported susceptible) than within the three bioinformatics methods (54 (0.4%) cases, 16 phenotypically-resistant, 38 phenotypically-susceptible). However, in 36/54 (67%), the consensus genotype matched the laboratory phenotype. Conclusions : In this study, the choice between these three specific bioinformatic methods to identify resistance-determinants or other genes in S. aureus did not prove critical, with all demonstrating high concordance with each other and phenotypic/molecular methods. However, each has some limitations and therefore consensus methods provide some assurance. Copyright © 2018 American Society for Microbiology.

A zebra-band phenotype in maize can be suppressed in constant light, and results from mutation of a PPOXlike gene (protophorphyrinogen oxidase IX-like) for porphyrin biosynthesis

USDA-ARS?s Scientific Manuscript database

A zebra-band phenotype was identified in a maize population of transposon-tagged mutants (UniformMu, searchable by sequence at MaizeGDB.org). Genotype-phenotype analysis of an F2 family showed that the zebra stripes co-segregated with a single Mu insertion in the second exon of a Protoporphyrinogen ...

Distinct subspecies or phenotypic plasticity? Genetic and morphological differentiation of mountain honey bees in East Africa.

PubMed

Gruber, Karl; Schöning, Caspar; Otte, Marianne; Kinuthia, Wanja; Hasselmann, Martin

2013-09-01

Identifying the forces shaping intraspecific phenotypic and genotypic divergence are of key importance in evolutionary biology. Phenotypic divergence may result from local adaptation or, especially in species with strong gene flow, from pronounced phenotypic plasticity. Here, we examine morphological and genetic divergence among populations of the western honey bee Apis mellifera in the topographically heterogeneous East African region. The currently accepted "mountain refugia hypothesis" states that populations living in disjunct montane forests belong to a different lineage than those in savanna habitats surrounding these forests. We obtained microsatellite data, mitochondrial sequences, and morphometric data from worker honey bees collected from feral colonies in three montane forests and corresponding neighboring savanna regions in Kenya. Honey bee colonies from montane forests showed distinct worker morphology compared with colonies in savanna areas. Mitochondrial sequence data did not support the existence of the two currently accepted subspecies. Furthermore, analyses of the microsatellite data with a Bayesian clustering method did not support the existence of two source populations as it would be expected under the mountain refugia scenario. Our findings suggest that phenotypic plasticity rather than distinct ancestry is the leading cause behind the phenotypic divergence observed between montane forest and savanna honey bees. Our study thus corroborates the idea that high gene flow may select for increased plasticity.

Contingency, convergence and hyper-astronomical numbers in biological evolution.

PubMed

Louis, Ard A

2016-08-01

Counterfactual questions such as "what would happen if you re-run the tape of life?" turn on the nature of the landscape of biological possibilities. Since the number of potential sequences that store genetic information grows exponentially with length, genetic possibility spaces can be so unimaginably vast that commentators frequently reach of hyper-astronomical metaphors that compare their size to that of the universe. Re-run the tape of life and the likelihood of encountering the same sequences in such hyper-astronomically large spaces is infinitesimally small, suggesting that evolutionary outcomes are highly contingent. On the other hand, the wide-spread occurrence of evolutionary convergence implies that similar phenotypes can be found again with relative ease. How can this be? Part of the solution to this conundrum must lie in the manner that genotypes map to phenotypes. By studying simple genotype-phenotype maps, where the counterfactual space of all possible phenotypes can be enumerated, it is shown that strong bias in the arrival of variation may explain why certain phenotypes are (repeatedly) observed in nature, while others never appear. This biased variation provides a non-selective cause for certain types of convergence. It illustrates how the role of randomness and contingency may differ significantly between genetic and phenotype spaces. Copyright © 2016 Elsevier Ltd. All rights reserved.

Chorea-acanthocytosis

PubMed Central

Walker, Susan; Dad, Rubina; Thiruvahindrapuram, Bhooma; Ullah, Muhammed Ikram; Ahmad, Arsalan; Hassan, Muhammad Jawad; Scherer, Stephen W.

2018-01-01

Objective To determine a molecular diagnosis for a large multigenerational family of South Asian ancestry with seizures, hyperactivity, and episodes of tongue biting. Methods Two affected individuals from the family were analyzed by whole-genome sequencing on the Illumina HiSeq X platform, and rare variants were prioritized for interpretation with respect to the phenotype. Results A previously undescribed, 1-kb homozygous deletion was identified in both individuals sequenced, which spanned 2 exons of the VPS13A gene, and was found to segregate in other family members. Conclusions VPS13A is associated with autosomal recessive chorea-acanthocytosis, a diagnosis consistent with the phenotype observed in this family. Whole-genome sequencing presents a comprehensive and agnostic approach for detecting diagnostic mutations in families with rare neurologic disorders. PMID:29845114

Mapping Second Chromosome Mutations to Defined Genomic Regions in Drosophila melanogaster

PubMed Central

Kahsai, Lily; Cook, Kevin R.

2017-01-01

Hundreds of Drosophila melanogaster stocks are currently maintained at the Bloomington Drosophila Stock Center with mutations that have not been associated with sequence-defined genes. They have been preserved because they have interesting loss-of-function phenotypes. The experimental value of these mutations would be increased by tying them to specific genomic intervals so that geneticists can more easily associate them with annotated genes. Here, we report the mapping of 85 second chromosome complementation groups in the Bloomington collection to specific, small clusters of contiguous genes or individual genes in the sequenced genome. This information should prove valuable to Drosophila geneticists interested in processes associated with particular phenotypes and those searching for mutations affecting specific sequence-defined genes. PMID:29066472

Absence of BiP co-chaperone DNAJC3 causes diabetes mellitus and multisystemic neurodegeneration.

PubMed

Synofzik, Matthis; Haack, Tobias B; Kopajtich, Robert; Gorza, Matteo; Rapaport, Doron; Greiner, Markus; Schönfeld, Caroline; Freiberg, Clemens; Schorr, Stefan; Holl, Reinhard W; Gonzalez, Michael A; Fritsche, Andreas; Fallier-Becker, Petra; Zimmermann, Richard; Strom, Tim M; Meitinger, Thomas; Züchner, Stephan; Schüle, Rebecca; Schöls, Ludger; Prokisch, Holger

2014-12-04

Diabetes mellitus and neurodegeneration are common diseases for which shared genetic factors are still only partly known. Here, we show that loss of the BiP (immunoglobulin heavy-chain binding protein) co-chaperone DNAJC3 leads to diabetes mellitus and widespread neurodegeneration. We investigated three siblings with juvenile-onset diabetes and central and peripheral neurodegeneration, including ataxia, upper-motor-neuron damage, peripheral neuropathy, hearing loss, and cerebral atrophy. Exome sequencing identified a homozygous stop mutation in DNAJC3. Screening of a diabetes database with 226,194 individuals yielded eight phenotypically similar individuals and one family carrying a homozygous DNAJC3 deletion. DNAJC3 was absent in fibroblasts from all affected subjects in both families. To delineate the phenotypic and mutational spectrum and the genetic variability of DNAJC3, we analyzed 8,603 exomes, including 506 from families affected by diabetes, ataxia, upper-motor-neuron damage, peripheral neuropathy, or hearing loss. This analysis revealed only one further loss-of-function allele in DNAJC3 and no further associations in subjects with only a subset of the features of the main phenotype. Our findings demonstrate that loss-of-function DNAJC3 mutations lead to a monogenic, recessive form of diabetes mellitus in humans. Moreover, they present a common denominator for diabetes and widespread neurodegeneration. This complements findings from mice in which knockout of Dnajc3 leads to diabetes and modifies disease in a neurodegenerative model of Marinesco-Sjögren syndrome. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Novel mutations of RPGR in Chinese retinitis pigmentosa patients and the genotype-phenotype correlation.

PubMed

Yang, Liping; Yin, Xiaobei; Feng, Lina; You, Debo; Wu, Lemeng; Chen, Ningning; Li, Aijun; Li, Genlin; Ma, Zhizhong

2014-01-01

X-linked Retinitis Pigmentosa (XLRP) accounts for 10-20% of all RP cases, and represents the most severe subtype of this disease. Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene are the most common causes of XLRP, accounting for over 70-75% of all XLRP cases. In this work, we analyzed all the exons of RPGR gene with Sanger sequencing in seven Chinese XLRP families, two of these with a provisional diagnosis of adRP but without male-to-male transmission. Three novel deletions (c.2233_34delAG; c.2236_37delGA and c.2403_04delAG) and two known nonsense mutations (c.851C→G and c.2260G→T) were identified in five families. Two novel deletions (c.2233_34delAG and c.2236_37delGA) resulted in the same frame shift (p.E746RfsX22), created similar phenotype in Family 3 and 4. The novel deletion (c.2403_04delAG; p.E802GfsX31) resulted in both XLRP and x-linked cone-rod dystrophy within the male patients of family 5, which suggested the presence of either genetic or environmental modifiers, or both, play a substantial role in disease expression. Genotype-phenotype correlation analysis suggested that (1) both patients and female carriers with mutation in Exon 8 (Family 1) manifest more severe disease than did those with ORF15 mutations (Family 2&3&4); (2) mutation close to downstream of ORF15 (Family 5) demonstrate the early preferential loss of cone function with moderate loss of rod function.

Linking Genes to Cardiovascular Diseases: Gene Action and Gene–Environment Interactions

PubMed Central

2016-01-01

A unique myocardial characteristic is its ability to grow/remodel in order to adapt; this is determined partly by genes and partly by the environment and the milieu intérieur. In the “post-genomic” era, a need is emerging to elucidate the physiologic functions of myocardial genes, as well as potential adaptive and maladaptive modulations induced by environmental/epigenetic factors. Genome sequencing and analysis advances have become exponential lately, with escalation of our knowledge concerning sometimes controversial genetic underpinnings of cardiovascular diseases. Current technologies can identify candidate genes variously involved in diverse normal/abnormal morphomechanical phenotypes, and offer insights into multiple genetic factors implicated in complex cardiovascular syndromes. The expression profiles of thousands of genes are regularly ascertained under diverse conditions. Global analyses of gene expression levels are useful for cataloging genes and correlated phenotypes, and for elucidating the role of genes in maladies. Comparative expression of gene networks coupled to complex disorders can contribute insights as to how “modifier genes” influence the expressed phenotypes. Increasingly, a more comprehensive and detailed systematic understanding of genetic abnormalities underlying, for example, various genetic cardiomyopathies is emerging. Implementing genomic findings in cardiology practice may well lead directly to better diagnosing and therapeutics. There is currently evolving a strong appreciation for the value of studying gene anomalies, and doing so in a non-disjointed, cohesive manner. However, it is challenging for many—practitioners and investigators—to comprehend, interpret, and utilize the clinically increasingly accessible and affordable cardiovascular genomics studies. This survey addresses the need for fundamental understanding in this vital area. PMID:26545598

Genome-Wide Identification of Regulatory Sequences Undergoing Accelerated Evolution in the Human Genome.

PubMed

Dong, Xinran; Wang, Xiao; Zhang, Feng; Tian, Weidong

2016-10-01

Accelerated evolution of regulatory sequence can alter the expression pattern of target genes, and cause phenotypic changes. In this study, we used DNase I hypersensitive sites (DHSs) to annotate putative regulatory sequences in the human genome, and conducted a genome-wide analysis of the effects of accelerated evolution on regulatory sequences. Working under the assumption that local ancient repeat elements of DHSs are under neutral evolution, we discovered that ∼0.44% of DHSs are under accelerated evolution (ace-DHSs). We found that ace-DHSs tend to be more active than background DHSs, and are strongly associated with epigenetic marks of active transcription. The target genes of ace-DHSs are significantly enriched in neuron-related functions, and their expression levels are positively selected in the human brain. Thus, these lines of evidences strongly suggest that accelerated evolution on regulatory sequences plays important role in the evolution of human-specific phenotypes. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Sequence heterogeneity of cannabidiolic- and tetrahydrocannabinolic acid-synthase in Cannabis sativa L. and its relationship with chemical phenotype.

PubMed

Onofri, Chiara; de Meijer, Etienne P M; Mandolino, Giuseppe

2015-08-01

Sequence variants of THCA- and CBDA-synthases were isolated from different Cannabis sativa L. strains expressing various wild-type and mutant chemical phenotypes (chemotypes). Expressed and complete sequences were obtained from mature inflorescences. Each strain was shown to have a different specificity and/or ability to convert the precursor CBGA into CBDA and/or THCA type products. The comparison of the expressed sequences led to the identification of different mutations, all of them due to SNPs. These SNPs were found to relate to the cannabinoid composition of the inflorescence at maturity and are therefore proposed to have a functional significance. The amount of variation was found to be higher within the CBDAS sequence family than in the THCAS family, suggesting a more recent evolution of THCA-forming enzymes from the CBDAS group. We therefore consider CBDAS as the ancestral type of these synthases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Polymorphisms within the canine MLPH gene are associated with dilute coat color in dogs

PubMed Central

Philipp, Ute; Hamann, Henning; Mecklenburg, Lars; Nishino, Seiji; Mignot, Emmanuel; Günzel-Apel, Anne-Rose; Schmutz, Sheila M; Leeb, Tosso

2005-01-01

Background Pinschers and other dogs with coat color dilution show a characteristic pigmentation phenotype. The fur colors are a lighter shade, e.g. silvery grey (blue) instead of black and a sandy color (Isabella fawn) instead of red or brown. In some dogs the coat color dilution is sometimes accompanied by hair loss and recurrent skin inflammation, the so called color dilution alopecia (CDA) or black hair follicular dysplasia (BHFD). In humans and mice a comparable pigmentation phenotype without any documented hair loss is caused by mutations within the melanophilin gene (MLPH). Results We sequenced the canine MLPH gene and performed a mutation analysis of the MLPH exons in 6 Doberman Pinschers and 5 German Pinschers. A total of 48 sequence variations was identified within and between the breeds. Three families of dogs showed co-segregation for at least one polymorphism in an MLPH exon and the dilute phenotype. No single polymorphism was identified in the coding sequences or at splice sites that is likely to be causative for the dilute phenotype of all dogs examined. In 18 German Pinschers a mutation in exon 7 (R199H) was consistently associated with the dilute phenotype. However, as this mutation was present in homozygous state in four dogs of other breeds with wildtype pigmentation, it seems unlikely that this mutation is truly causative for coat color dilution. In Doberman Pinschers as well as in Large Munsterlanders with BHFD, a set of single nucleotide polymorphisms (SNPs) around exon 2 was identified that show a highly significant association to the dilute phenotype. Conclusion This study provides evidence that coat color dilution is caused by one or more mutations within or near the MLPH gene in several dog breeds. The data on polymorphisms that are strongly associated with the dilute phenotype will allow the genetic testing of Pinschers to facilitate the breeding of dogs with defined coat colors and to select against Large Munsterlanders carrying BHFD. PMID:15960853

Polymorphisms within the canine MLPH gene are associated with dilute coat color in dogs.

PubMed

Philipp, Ute; Hamann, Henning; Mecklenburg, Lars; Nishino, Seiji; Mignot, Emmanuel; Günzel-Apel, Anne-Rose; Schmutz, Sheila M; Leeb, Tosso

2005-06-16

Pinschers and other dogs with coat color dilution show a characteristic pigmentation phenotype. The fur colors are a lighter shade, e.g. silvery grey (blue) instead of black and a sandy color (Isabella fawn) instead of red or brown. In some dogs the coat color dilution is sometimes accompanied by hair loss and recurrent skin inflammation, the so called color dilution alopecia (CDA) or black hair follicular dysplasia (BHFD). In humans and mice a comparable pigmentation phenotype without any documented hair loss is caused by mutations within the melanophilin gene (MLPH). We sequenced the canine MLPH gene and performed a mutation analysis of the MLPH exons in 6 Doberman Pinschers and 5 German Pinschers. A total of 48 sequence variations was identified within and between the breeds. Three families of dogs showed co-segregation for at least one polymorphism in an MLPH exon and the dilute phenotype. No single polymorphism was identified in the coding sequences or at splice sites that is likely to be causative for the dilute phenotype of all dogs examined. In 18 German Pinschers a mutation in exon 7 (R199H) was consistently associated with the dilute phenotype. However, as this mutation was present in homozygous state in four dogs of other breeds with wildtype pigmentation, it seems unlikely that this mutation is truly causative for coat color dilution. In Doberman Pinschers as well as in Large Munsterlanders with BHFD, a set of single nucleotide polymorphisms (SNPs) around exon 2 was identified that show a highly significant association to the dilute phenotype. This study provides evidence that coat color dilution is caused by one or more mutations within or near the MLPH gene in several dog breeds. The data on polymorphisms that are strongly associated with the dilute phenotype will allow the genetic testing of Pinschers to facilitate the breeding of dogs with defined coat colors and to select against Large Munsterlanders carrying BHFD.

Changes in Muscle Metabolism are Associated with Phenotypic Variability in Golden Retriever Muscular Dystrophy




PubMed Central

Nghiem, Peter P.; Bello, Luca; Stoughton, William B.; López, Sara Mata; Vidal, Alexander H.; Hernandez, Briana V.; Hulbert, Katherine N.; Gourley, Taylor R.; Bettis, Amanda K.; Balog-Alvarez, Cynthia J.; Heath-Barnett, Heather; Kornegay, Joe N.

2017-01-01

Duchenne muscular dystrophy (DMD) is an X-chromosome-linked disorder and the most common monogenic disease in people. Affected boys are diagnosed at a young age, become non-ambulatory by their early teens, and succumb to cardiorespiratory failure by their thirties. Despite being a monogenic condition resulting from mutations in the DMD gene, affected boys have noteworthy phenotypic variability. Efforts have identified genetic modifiers that could modify disease progression and be pharmacologic targets. Dogs affected with golden retriever muscular dystrophy (GRMD) have absent dystrophin and demonstrate phenotypic variability at the functional, histopathological, and molecular level. Our laboratory is particularly interested in muscle metabolism changes in dystrophin-deficient muscle. We identified several metabolic alterations, including myofiber type switching from fast (type II) to slow (type I), reduced glycolytic enzyme expression, reduced and morphologically abnormal mitochondria, and differential AMP-kinase phosphorylation (activation) between hypertrophied and wasted muscle. We hypothesize that muscle metabolism changes are, in part, responsible for phenotypic variability in GRMD. Pharmacological therapies aimed at modulating muscle metabolism can be tested in GRMD dogs for efficacy. PMID:28955176

Phenotype-Based Screening of Small Molecules to Modify Plant Cell Walls Using BY-2 Cells.

PubMed

Okubo-Kurihara, Emiko; Matsui, Minami

2018-01-01

The plant cell wall is an important and abundant biomass with great potential for use as a modern recyclable resource. For effective utilization of this cellulosic biomass, its ability to degrade efficiently is key point. With the aim of modifying the cell wall to allow easy decomposition, we used chemical biological technology to alter its structure. As a first step toward evaluating the chemicals in the cell wall we employed a phenotype-based approach using high-throughput screening. As the plant cell wall is essential in determining cell morphology, phenotype-based screening is particularly effective in identifying compounds that bring about alterations in the cell wall. For rapid and reproducible screening, tobacco BY-2 cell is an excellent system in which to observe cell morphology. In this chapter, we provide a detailed chemical biological methodology for studying cell morphology using tobacco BY-2 cells.

Machine-learned analysis of the association of next-generation sequencing-based human TRPV1 and TRPA1 genotypes with the sensitivity to heat stimuli and topically applied capsaicin.

PubMed

Kringel, Dario; Geisslinger, Gerd; Resch, Eduard; Oertel, Bruno G; Thrun, Michael C; Heinemann, Sarah; Lötsch, Jörn

2018-03-27

Heat pain and its modulation by capsaicin varies among subjects in experimental and clinical settings. A plausible cause is a genetic component, of which TRPV1 ion channels, by their response to both heat and capsaicin, are primary candidates. However, TRPA1 channels can heterodimerize with TRPV1 channels and carry genetic variants reported to modulate heat pain sensitivity. To address the role of these candidate genes in capsaicin-induced hypersensitization to heat, pain thresholds acquired before and after topical application of capsaicin and TRPA1/TRPV1 exomic sequences derived by next-generation sequencing were assessed in n = 75 healthy volunteers and the genetic information comprised 278 loci. Gaussian mixture modeling indicated 2 phenotype groups with high or low capsaicin-induced hypersensitization to heat. Unsupervised machine learning implemented as swarm-based clustering hinted at differences in the genetic pattern between these phenotype groups. Several methods of supervised machine learning implemented as random forests, adaptive boosting, k-nearest neighbors, naive Bayes, support vector machines, and for comparison, binary logistic regression predicted the phenotype group association consistently better when based on the observed genotypes than when using a random permutation of the exomic sequences. Of note, TRPA1 variants were more important for correct phenotype group association than TRPV1 variants. This indicates a role of the TRPA1 and TRPV1 next-generation sequencing-based genetic pattern in the modulation of the individual response to heat-related pain phenotypes. When considering earlier evidence that topical capsaicin can induce neuropathy-like quantitative sensory testing patterns in healthy subjects, implications for future analgesic treatments with transient receptor potential inhibitors arise.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Genome-wide copy number variation in the bovine genome detected using low coverage sequence of popular beef breeds

USDA-ARS?s Scientific Manuscript database

Genomic structural variations are an important source of genetic diversity. Copy number variations (CNVs), gains and losses of large regions of genomic sequence between individuals of a species, are known to be associated with both diseases and phenotypic traits. Deeply sequenced genomes are often u...

A screen for E3 ubiquitination ligases that genetically interact with the adaptor protein Cindr during Drosophila eye patterning

PubMed Central

Ketosugbo, Kwami F.; Bushnell, Henry L.

2017-01-01

Ubiquitination is a crucial post-translational modification that can target proteins for degradation. The E3 ubiquitin ligases are responsible for recognizing substrate proteins for ubiquitination, hence providing specificity to the process of protein degradation. Here, we describe a genetic modifier screen that identified E3 ligases that modified the rough-eye phenotype generated by expression of cindrRNAi transgenes during Drosophila eye development. In total, we identified 36 E3 ligases, as well as 4 Cullins, that modified the mild cindrRNA mis-patterning phenotype. This indicates possible roles for these E3s/Cullins in processes that require Cindr function, including cytoskeletal regulation, cell adhesion, cell signaling and cell survival. Three E3 ligases identified in our screen had previously been linked to regulating JNK signaling. PMID:29117266

Whole exome sequencing: a state-of-the-art approach for defining (and exploring!) genetic landscapes in pediatric nephrology.

PubMed

Gulati, Ashima; Somlo, Stefan

2018-05-01

The genesis of whole exome sequencing as a powerful tool for detailing the protein coding sequence of the human genome was conceptualized based on the availability of next-generation sequencing technology and knowledge of the human reference genome. The field of pediatric nephrology enriched with molecularly unsolved phenotypes is allowing the clinical and research application of whole exome sequencing to enable novel gene discovery and provide amendment of phenotypic misclassification. Recent studies in the field have informed us that newer high-throughput sequencing techniques are likely to be of high yield when applied in conjunction with conventional genomic approaches such as linkage analysis and other strategies used to focus subsequent analysis. They have also emphasized the need for the validation of novel genetic findings in large collaborative cohorts and the production of robust corroborative biological data. The well-structured application of comprehensive genomic testing in clinical and research arenas will hopefully continue to advance patient care and precision medicine, but does call for attention to be paid to its integrated challenges.

Systematic Internal Transcribed Spacer Sequence Analysis for Identification of Clinical Mold Isolates in Diagnostic Mycology: a 5-Year Study▿ †

PubMed Central

Ciardo, Diana E.; Lucke, Katja; Imhof, Alex; Bloemberg, Guido V.; Böttger, Erik C.

2010-01-01

The implementation of internal transcribed spacer (ITS) sequencing for routine identification of molds in the diagnostic mycology laboratory was analyzed in a 5-year study. All mold isolates (n = 6,900) recovered in our laboratory from 2005 to 2009 were included in this study. According to a defined work flow, which in addition to troublesome phenotypic identification takes clinical relevance into account, 233 isolates were subjected to ITS sequence analysis. Sequencing resulted in successful identification for 78.6% of the analyzed isolates (57.1% at species level, 21.5% at genus level). In comparison, extended in-depth phenotypic characterization of the isolates subjected to sequencing achieved taxonomic assignment for 47.6% of these, with a mere 13.3% at species level. Optimization of DNA extraction further improved the efficacy of molecular identification. This study is the first of its kind to testify to the systematic implementation of sequence-based identification procedures in the routine workup of mold isolates in the diagnostic mycology laboratory. PMID:20573873

Calcaneal Scoring as an Adjunct to Modified Oxford Hip Scores: Prediction of Contralateral Slipped Capital Femoral Epiphysis.

PubMed

Nicholson, Allen D; Huez, Coridon M; Sanders, James O; Liu, Raymond W; Cooperman, Daniel R

2016-03-01

In 2 recent studies, modified Oxford hip scores of 16 through 18 have been shown to predict an extremely high risk of contralateral slipping in unilateral slipped capital femoral epiphysis (SCFE). However, the modified Oxford system is not widely used. This may be due, in part, to the complexity of the scoring system, difficulty in viewing all 5 radiographic features on a single x-ray and phenotypic variation in the features. Ossification of the calcaneal apophysis provides an osteologic marker of skeletal maturation in relation to peak height velocity and has been described previously. We examine the value of the calcaneal apophyseal ossification sequence for predicting modified Oxford hip scores. We examined 279 pelvis and matching foot x-rays that were taken at the same session from 94 healthy children aged 3 to 18 years. A fellowship-trained pediatric orthopaedist determined the modified Oxford hip score for each hip radiograph. The calcaneal x-rays had been previously graded. Modified Oxford hip scores were compared with calcaneal scores for each set of matched hip and calcaneal x-rays. Stage 0 to 2 calcanei had 94% of corresponding hip radiographs rated as modified Oxford scores of 16 to 18. Stage 3 calcanei had 54% rated as 16 to 18 and 31% rated as scores 19 to 21. Stage 4 calcanei had 31% rated as scores 19 to 21, and 68% rated as scores 22 to 26. Stage 5 calcanei had 100% rated as 22 to 26. Using data from Popejoy and colleagues' study, the weighted risk of contralateral SCFE was 94% for calcaneal stage 0, 86.5% for calcaneal stage 1, 90.3% for calcaneal stage 2, 55.8% for calcaneal stage 3, 6.1% for calcaneal stage 4, and 0 for calcaneal stage 5. Calcaneal stages 0 to 3 correspond entirely to modified Oxford scores indicating elevated risk of contralateral SCFE. The calcaneal scoring system has potential for adjunctive use with the modified Oxford score for prediction of contralateral SCFE.

Progression of regional grey matter atrophy in multiple sclerosis

PubMed Central

Marinescu, Razvan V; Young, Alexandra L; Firth, Nicholas C; Jorge Cardoso, M; Tur, Carmen; De Angelis, Floriana; Cawley, Niamh; Brownlee, Wallace J; De Stefano, Nicola; Laura Stromillo, M; Battaglini, Marco; Ruggieri, Serena; Gasperini, Claudio; Filippi, Massimo; Rocca, Maria A; Rovira, Alex; Sastre-Garriga, Jaume; Geurts, Jeroen J G; Vrenken, Hugo; Wottschel, Viktor; Leurs, Cyra E; Uitdehaag, Bernard; Pirpamer, Lukas; Enzinger, Christian; Ourselin, Sebastien; Gandini Wheeler-Kingshott, Claudia A; Chard, Declan; Thompson, Alan J; Barkhof, Frederik; Alexander, Daniel C; Ciccarelli, Olga

2018-01-01

Abstract See Stankoff and Louapre (doi:10.1093/brain/awy114) for a scientific commentary on this article. Grey matter atrophy is present from the earliest stages of multiple sclerosis, but its temporal ordering is poorly understood. We aimed to determine the sequence in which grey matter regions become atrophic in multiple sclerosis and its association with disability accumulation. In this longitudinal study, we included 1417 subjects: 253 with clinically isolated syndrome, 708 with relapsing-remitting multiple sclerosis, 128 with secondary-progressive multiple sclerosis, 125 with primary-progressive multiple sclerosis, and 203 healthy control subjects from seven European centres. Subjects underwent repeated MRI (total number of scans 3604); the mean follow-up for patients was 2.41 years (standard deviation = 1.97). Disability was scored using the Expanded Disability Status Scale. We calculated the volume of brain grey matter regions and brainstem using an unbiased within-subject template and used an established data-driven event-based model to determine the sequence of occurrence of atrophy and its uncertainty. We assigned each subject to a specific event-based model stage, based on the number of their atrophic regions. Linear mixed-effects models were used to explore associations between the rate of increase in event-based model stages, and T2 lesion load, disease-modifying treatments, comorbidity, disease duration and disability accumulation. The first regions to become atrophic in patients with clinically isolated syndrome and relapse-onset multiple sclerosis were the posterior cingulate cortex and precuneus, followed by the middle cingulate cortex, brainstem and thalamus. A similar sequence of atrophy was detected in primary-progressive multiple sclerosis with the involvement of the thalamus, cuneus, precuneus, and pallidum, followed by the brainstem and posterior cingulate cortex. The cerebellum, caudate and putamen showed early atrophy in relapse-onset multiple sclerosis and late atrophy in primary-progressive multiple sclerosis. Patients with secondary-progressive multiple sclerosis showed the highest event-based model stage (the highest number of atrophic regions, P < 0.001) at the study entry. All multiple sclerosis phenotypes, but clinically isolated syndrome, showed a faster rate of increase in the event-based model stage than healthy controls. T2 lesion load and disease duration in all patients were associated with increased event-based model stage, but no effects of disease-modifying treatments and comorbidity on event-based model stage were observed. The annualized rate of event-based model stage was associated with the disability accumulation in relapsing-remitting multiple sclerosis, independent of disease duration (P < 0.0001). The data-driven staging of atrophy progression in a large multiple sclerosis sample demonstrates that grey matter atrophy spreads to involve more regions over time. The sequence in which regions become atrophic is reasonably consistent across multiple sclerosis phenotypes. The spread of atrophy was associated with disease duration and with disability accumulation over time in relapsing-remitting multiple sclerosis. PMID:29741648

Progression of regional grey matter atrophy in multiple sclerosis.

PubMed

Eshaghi, Arman; Marinescu, Razvan V; Young, Alexandra L; Firth, Nicholas C; Prados, Ferran; Jorge Cardoso, M; Tur, Carmen; De Angelis, Floriana; Cawley, Niamh; Brownlee, Wallace J; De Stefano, Nicola; Laura Stromillo, M; Battaglini, Marco; Ruggieri, Serena; Gasperini, Claudio; Filippi, Massimo; Rocca, Maria A; Rovira, Alex; Sastre-Garriga, Jaume; Geurts, Jeroen J G; Vrenken, Hugo; Wottschel, Viktor; Leurs, Cyra E; Uitdehaag, Bernard; Pirpamer, Lukas; Enzinger, Christian; Ourselin, Sebastien; Gandini Wheeler-Kingshott, Claudia A; Chard, Declan; Thompson, Alan J; Barkhof, Frederik; Alexander, Daniel C; Ciccarelli, Olga

2018-06-01

See Stankoff and Louapre (doi:10.1093/brain/awy114) for a scientific commentary on this article.Grey matter atrophy is present from the earliest stages of multiple sclerosis, but its temporal ordering is poorly understood. We aimed to determine the sequence in which grey matter regions become atrophic in multiple sclerosis and its association with disability accumulation. In this longitudinal study, we included 1417 subjects: 253 with clinically isolated syndrome, 708 with relapsing-remitting multiple sclerosis, 128 with secondary-progressive multiple sclerosis, 125 with primary-progressive multiple sclerosis, and 203 healthy control subjects from seven European centres. Subjects underwent repeated MRI (total number of scans 3604); the mean follow-up for patients was 2.41 years (standard deviation = 1.97). Disability was scored using the Expanded Disability Status Scale. We calculated the volume of brain grey matter regions and brainstem using an unbiased within-subject template and used an established data-driven event-based model to determine the sequence of occurrence of atrophy and its uncertainty. We assigned each subject to a specific event-based model stage, based on the number of their atrophic regions. Linear mixed-effects models were used to explore associations between the rate of increase in event-based model stages, and T2 lesion load, disease-modifying treatments, comorbidity, disease duration and disability accumulation. The first regions to become atrophic in patients with clinically isolated syndrome and relapse-onset multiple sclerosis were the posterior cingulate cortex and precuneus, followed by the middle cingulate cortex, brainstem and thalamus. A similar sequence of atrophy was detected in primary-progressive multiple sclerosis with the involvement of the thalamus, cuneus, precuneus, and pallidum, followed by the brainstem and posterior cingulate cortex. The cerebellum, caudate and putamen showed early atrophy in relapse-onset multiple sclerosis and late atrophy in primary-progressive multiple sclerosis. Patients with secondary-progressive multiple sclerosis showed the highest event-based model stage (the highest number of atrophic regions, P < 0.001) at the study entry. All multiple sclerosis phenotypes, but clinically isolated syndrome, showed a faster rate of increase in the event-based model stage than healthy controls. T2 lesion load and disease duration in all patients were associated with increased event-based model stage, but no effects of disease-modifying treatments and comorbidity on event-based model stage were observed. The annualized rate of event-based model stage was associated with the disability accumulation in relapsing-remitting multiple sclerosis, independent of disease duration (P < 0.0001). The data-driven staging of atrophy progression in a large multiple sclerosis sample demonstrates that grey matter atrophy spreads to involve more regions over time. The sequence in which regions become atrophic is reasonably consistent across multiple sclerosis phenotypes. The spread of atrophy was associated with disease duration and with disability accumulation over time in relapsing-remitting multiple sclerosis.

Silencing Effect of Hominoid Highly Conserved Noncoding Sequences on Embryonic Brain Development

PubMed Central

Mahmoudi Saber, Morteza

2017-01-01

Abstract Superfamily Hominoidea, which consists of Hominidae (humans and great apes) and Hylobatidae (gibbons), is well-known for sharing human-like characteristics, however, the genomic origins of these shared unique phenotypes have mainly remained elusive. To decipher the underlying genomic basis of Hominoidea-restricted phenotypes, we identified and characterized Hominoidea-restricted highly conserved noncoding sequences (HCNSs) that are a class of potential regulatory elements which may be involved in evolution of lineage-specific phenotypes. We discovered 679 such HCNSs from human, chimpanzee, gorilla, orangutan and gibbon genomes. These HCNSs were demonstrated to be under purifying selection but with lineage-restricted characteristics different from old CNSs. A significant proportion of their ancestral sequences had accelerated rates of nucleotide substitutions, insertions and deletions during the evolution of common ancestor of Hominoidea, suggesting the intervention of positive Darwinian selection for creating those HCNSs. In contrary to enhancer elements and similar to silencer sequences, these Hominoidea-restricted HCNSs are located in close proximity of transcription start sites. Their target genes are enriched in the nervous system, development and transcription, and they tend to be remotely located from the nearest coding gene. Chip-seq signals and gene expression patterns suggest that Hominoidea-restricted HCNSs are likely to be functional regulatory elements by imposing silencing effects on their target genes in a tissue-restricted manner during fetal brain development. These HCNSs, emerged through adaptive evolution and conserved through purifying selection, represent a set of promising targets for future functional studies of the evolution of Hominoidea-restricted phenotypes. PMID:28633494

Evolutionary Analysis Predicts Sensitive Positions of MMP20 and Validates Newly- and Previously-Identified MMP20 Mutations Causing Amelogenesis Imperfecta

PubMed Central

Gasse, Barbara; Prasad, Megana; Delgado, Sidney; Huckert, Mathilde; Kawczynski, Marzena; Garret-Bernardin, Annelyse; Lopez-Cazaux, Serena; Bailleul-Forestier, Isabelle; Manière, Marie-Cécile; Stoetzel, Corinne; Bloch-Zupan, Agnès; Sire, Jean-Yves

2017-01-01

Amelogenesis imperfecta (AI) designates a group of genetic diseases characterized by a large range of enamel disorders causing important social and health problems. These defects can result from mutations in enamel matrix proteins or protease encoding genes. A range of mutations in the enamel cleavage enzyme matrix metalloproteinase-20 gene (MMP20) produce enamel defects of varying severity. To address how various alterations produce a range of AI phenotypes, we performed a targeted analysis to find MMP20 mutations in French patients diagnosed with non-syndromic AI. Genomic DNA was isolated from saliva and MMP20 exons and exon-intron boundaries sequenced. We identified several homozygous or heterozygous mutations, putatively involved in the AI phenotypes. To validate missense mutations and predict sensitive positions in the MMP20 sequence, we evolutionarily compared 75 sequences extracted from the public databases using the Datamonkey webserver. These sequences were representative of mammalian lineages, covering more than 150 million years of evolution. This analysis allowed us to find 324 sensitive positions (out of the 483 MMP20 residues), pinpoint functionally important domains, and build an evolutionary chart of important conserved MMP20 regions. This is an efficient tool to identify new- and previously-identified mutations. We thus identified six functional MMP20 mutations in unrelated families, finding two novel mutated sites. The genotypes and phenotypes of these six mutations are described and compared. To date, 13 MMP20 mutations causing AI have been reported, making these genotypes and associated hypomature enamel phenotypes the most frequent in AI. PMID:28659819

Evolutionary Analysis Predicts Sensitive Positions of MMP20 and Validates Newly- and Previously-Identified MMP20 Mutations Causing Amelogenesis Imperfecta.

PubMed

Gasse, Barbara; Prasad, Megana; Delgado, Sidney; Huckert, Mathilde; Kawczynski, Marzena; Garret-Bernardin, Annelyse; Lopez-Cazaux, Serena; Bailleul-Forestier, Isabelle; Manière, Marie-Cécile; Stoetzel, Corinne; Bloch-Zupan, Agnès; Sire, Jean-Yves

2017-01-01

Amelogenesis imperfecta (AI) designates a group of genetic diseases characterized by a large range of enamel disorders causing important social and health problems. These defects can result from mutations in enamel matrix proteins or protease encoding genes. A range of mutations in the enamel cleavage enzyme matrix metalloproteinase-20 gene ( MMP20 ) produce enamel defects of varying severity. To address how various alterations produce a range of AI phenotypes, we performed a targeted analysis to find MMP20 mutations in French patients diagnosed with non-syndromic AI. Genomic DNA was isolated from saliva and MMP20 exons and exon-intron boundaries sequenced. We identified several homozygous or heterozygous mutations, putatively involved in the AI phenotypes. To validate missense mutations and predict sensitive positions in the MMP20 sequence, we evolutionarily compared 75 sequences extracted from the public databases using the Datamonkey webserver. These sequences were representative of mammalian lineages, covering more than 150 million years of evolution. This analysis allowed us to find 324 sensitive positions (out of the 483 MMP20 residues), pinpoint functionally important domains, and build an evolutionary chart of important conserved MMP20 regions. This is an efficient tool to identify new- and previously-identified mutations. We thus identified six functional MMP20 mutations in unrelated families, finding two novel mutated sites. The genotypes and phenotypes of these six mutations are described and compared. To date, 13 MMP20 mutations causing AI have been reported, making these genotypes and associated hypomature enamel phenotypes the most frequent in AI.

Identification of Novel Growth Regulators in Plant Populations Expressing Random Peptides1[OPEN

PubMed Central

Bao, Zhilong; Clancy, Maureen A.

2017-01-01

The use of chemical genomics approaches allows the identification of small molecules that integrate into biological systems, thereby changing discrete processes that influence growth, development, or metabolism. Libraries of chemicals are applied to living systems, and changes in phenotype are observed, potentially leading to the identification of new growth regulators. This work describes an approach that is the nexus of chemical genomics and synthetic biology. Here, each plant in an extensive population synthesizes a unique small peptide arising from a transgene composed of a randomized nucleic acid sequence core flanked by translational start, stop, and cysteine-encoding (for disulfide cyclization) sequences. Ten and 16 amino acid sequences, bearing a core of six and 12 random amino acids, have been synthesized in Arabidopsis (Arabidopsis thaliana) plants. Populations were screened for phenotypes from the seedling stage through senescence. Dozens of phenotypes were observed in over 2,000 plants analyzed. Ten conspicuous phenotypes were verified through separate transformation and analysis of multiple independent lines. The results indicate that these populations contain sequences that often influence discrete aspects of plant biology. Novel peptides that affect photosynthesis, flowering, and red light response are described. The challenge now is to identify the mechanistic integrations of these peptides into biochemical processes. These populations serve as a new tool to identify small molecules that modulate discrete plant functions that could be produced later in transgenic plants or potentially applied exogenously to impart their effects. These findings could usher in a new generation of agricultural growth regulators, herbicides, or defense compounds. PMID:28807931

Molecular and Biochemical Analysis of the Galactose Phenotype of Dairy Streptococcus thermophilus Strains Reveals Four Different Fermentation Profiles

PubMed Central

de Vin, Filip; Rådström, Peter; Herman, Lieve; De Vuyst, Luc

2005-01-01

Lactose-limited fermentations of 49 dairy Streptococcus thermophilus strains revealed four distinct fermentation profiles with respect to galactose consumption after lactose depletion. All the strains excreted galactose into the medium during growth on lactose, except for strain IMDOST40, which also displayed extremely high galactokinase (GalK) activity. Among this strain collection eight galactose-positive phenotypes sensu stricto were found and their fermentation characteristics and Leloir enzyme activities were measured. As the gal promoter seems to play an important role in the galactose phenotype, the galR-galK intergenic region was sequenced for all strains yielding eight different nucleotide sequences (NS1 to NS8). The gal promoter played an important role in the Gal-positive phenotype but did not determine it exclusively. Although GalT and GalE activities were detected for all Gal-positive strains, GalK activity could only be detected for two out of eight Gal-positive strains. This finding suggests that the other six S. thermophilus strains metabolize galactose via an alternative route. For each type of fermentation profile obtained, a representative strain was chosen and four complete Leloir gene clusters were sequenced. It turned out that Gal-positive strains contained more amino acid differences within their gal genes than Gal-negative strains. Finally, the biodiversity regarding lactose-galactose utilization among the different S. thermophilus strains used in this study was shown by RAPD-PCR. Five Gal-positive strains that contain nucleotide sequence NS2 in their galR-galK intergenic region were closely related. PMID:16000774

From Genomes to Phenotypes: Traitar, the Microbial Trait Analyzer.

PubMed

Weimann, Aaron; Mooren, Kyra; Frank, Jeremy; Pope, Phillip B; Bremges, Andreas; McHardy, Alice C

2016-01-01

The number of sequenced genomes is growing exponentially, profoundly shifting the bottleneck from data generation to genome interpretation. Traits are often used to characterize and distinguish bacteria and are likely a driving factor in microbial community composition, yet little is known about the traits of most microbes. We describe Traitar, the microbial trait analyzer, which is a fully automated software package for deriving phenotypes from a genome sequence. Traitar provides phenotype classifiers to predict 67 traits related to the use of various substrates as carbon and energy sources, oxygen requirement, morphology, antibiotic susceptibility, proteolysis, and enzymatic activities. Furthermore, it suggests protein families associated with the presence of particular phenotypes. Our method uses L1-regularized L2-loss support vector machines for phenotype assignments based on phyletic patterns of protein families and their evolutionary histories across a diverse set of microbial species. We demonstrate reliable phenotype assignment for Traitar to bacterial genomes from 572 species of eight phyla, also based on incomplete single-cell genomes and simulated draft genomes. We also showcase its application in metagenomics by verifying and complementing a manual metabolic reconstruction of two novel Clostridiales species based on draft genomes recovered from commercial biogas reactors. Traitar is available at https://github.com/hzi-bifo/traitar. IMPORTANCE Bacteria are ubiquitous in our ecosystem and have a major impact on human health, e.g., by supporting digestion in the human gut. Bacterial communities can also aid in biotechnological processes such as wastewater treatment or decontamination of polluted soils. Diverse bacteria contribute with their unique capabilities to the functioning of such ecosystems, but lab experiments to investigate those capabilities are labor-intensive. Major advances in sequencing techniques open up the opportunity to study bacteria by their genome sequences. For this purpose, we have developed Traitar, software that predicts traits of bacteria on the basis of their genomes. It is applicable to studies with tens or hundreds of bacterial genomes. Traitar may help researchers in microbiology to pinpoint the traits of interest, reducing the amount of wet lab work required.

Genotypic tropism testing by massively parallel sequencing: qualitative and quantitative analysis.

PubMed

Däumer, Martin; Kaiser, Rolf; Klein, Rolf; Lengauer, Thomas; Thiele, Bernhard; Thielen, Alexander

2011-05-13

Inferring viral tropism from genotype is a fast and inexpensive alternative to phenotypic testing. While being highly predictive when performed on clonal samples, sensitivity of predicting CXCR4-using (X4) variants drops substantially in clinical isolates. This is mainly attributed to minor variants not detected by standard bulk-sequencing. Massively parallel sequencing (MPS) detects single clones thereby being much more sensitive. Using this technology we wanted to improve genotypic prediction of coreceptor usage. Plasma samples from 55 antiretroviral-treated patients tested for coreceptor usage with the Monogram Trofile Assay were sequenced with standard population-based approaches. Fourteen of these samples were selected for further analysis with MPS. Tropism was predicted from each sequence with geno2pheno[coreceptor]. Prediction based on bulk-sequencing yielded 59.1% sensitivity and 90.9% specificity compared to the trofile assay. With MPS, 7600 reads were generated on average per isolate. Minorities of sequences with high confidence in CXCR4-usage were found in all samples, irrespective of phenotype. When using the default false-positive-rate of geno2pheno[coreceptor] (10%), and defining a minority cutoff of 5%, the results were concordant in all but one isolate. The combination of MPS and coreceptor usage prediction results in a fast and accurate alternative to phenotypic assays. The detection of X4-viruses in all isolates suggests that coreceptor usage as well as fitness of minorities is important for therapy outcome. The high sensitivity of this technology in combination with a quantitative description of the viral population may allow implementing meaningful cutoffs for predicting response to CCR5-antagonists in the presence of X4-minorities.

The phosphotransferase system-dependent sucrose utilization regulon in enteropathogenic Escherichia coli strains is located in a variable chromosomal region containing iap sequences.

PubMed

Treviño-Quintanilla, Luis Gerardo; Escalante, Adelfo; Caro, Alma Delia; Martínez, Alfredo; González, Ricardo; Puente, José Luis; Bolívar, Francisco; Gosset, Guillermo

2007-01-01

The capacity to utilize sucrose as a carbon and energy source (Scr(+) phenotype) is a highly variable trait among Escherichia coli strains. In this study, seven enteropathogenic E. coli (EPEC) strains from different sources were studied for their capacity to grow using sucrose. Liquid media cultures showed that all analyzed strains have the Scr(+) phenotype and two distinct groups were defined: one of five and another of two strains displaying doubling times of 67 and 125 min, respectively. The genes conferring the Scr(+) phenotype in one of the fast-growing strains (T19) were cloned and sequenced. Comparative sequence analysis revealed that this strain possesses the scr regulon genes scrKYABR, encoding phosphoenolpyruvate:phosphotransferase system-dependent sucrose transport and utilization activities. Transcript level quantification revealed sucrose-dependent induction of scrK and scrR genes in fast-growing strains, whereas no transcripts were detected in slow-growing strains. Sequence comparison analysis revealed that the scr genes in strain T19 are almost identical to those present in the scr regulon of prototype EPEC E2348/69 and in both strains, the scr genes are inserted in the chromosomal intergenic region of hypothetical genes ygcE and ygcF. Comparison of the ygcE-ygcF intergenic region sequence of strains MG1655, enterohemorrhagic EDL933, uropathogenic ECFT073 and EPEC T19-E2348/69 revealed that the number of extragenic highly repeated iap sequences corresponded to nine, four, two and none, respectively. These results show that the iap sequence-containing chromosomal ygcE-ygcF intergenic region is highly variable in E. coli. Copyright (c) 2007 S. Karger AG, Basel.

Multi-source and ontology-based retrieval engine for maize mutant phenotypes

USDA-ARS?s Scientific Manuscript database

In the midst of this genomics era, major plant genome databases are collecting massive amounts of heterogeneous information, including sequence data, gene product information, images of mutant phenotypes, etc., as well as textual descriptions of many of these entities. While basic browsing and sear...

A survey of copy number variation in the porcine genome detected from whole-genome sequence

USDA-ARS?s Scientific Manuscript database

An important challenge to post-genomic biology is relating observed phenotypic variation to the underlying genotypic variation. Genome-wide association studies (GWAS) have made thousands of connections between single nucleotide polymorphisms (SNPs) and phenotypes, implicating regions of the genome t...

Medical subject heading (MeSH) annotations illuminate maize genetics and evolution

USDA-ARS?s Scientific Manuscript database

In the modern era, high-density marker panels and/or whole-genome sequencing,coupled with advanced phenotyping pipelines and sophisticated statistical methods, have dramatically increased our ability to generate lists of candidate genes or regions that are putatively associated with phenotypes or pr...

Comparative functional pan-genome analyses to build connections between genomic dynamics and phenotypic evolution in polycyclic aromatic hydrocarbon metabolism in the genus Mycobacterium.

PubMed

Kweon, Ohgew; Kim, Seong-Jae; Blom, Jochen; Kim, Sung-Kwan; Kim, Bong-Soo; Baek, Dong-Heon; Park, Su Inn; Sutherland, John B; Cerniglia, Carl E

2015-02-14

The bacterial genus Mycobacterium is of great interest in the medical and biotechnological fields. Despite a flood of genome sequencing and functional genomics data, significant gaps in knowledge between genome and phenome seriously hinder efforts toward the treatment of mycobacterial diseases and practical biotechnological applications. In this study, we propose the use of systematic, comparative functional pan-genomic analysis to build connections between genomic dynamics and phenotypic evolution in polycyclic aromatic hydrocarbon (PAH) metabolism in the genus Mycobacterium. Phylogenetic, phenotypic, and genomic information for 27 completely genome-sequenced mycobacteria was systematically integrated to reconstruct a mycobacterial phenotype network (MPN) with a pan-genomic concept at a network level. In the MPN, mycobacterial phenotypes show typical scale-free relationships. PAH degradation is an isolated phenotype with the lowest connection degree, consistent with phylogenetic and environmental isolation of PAH degraders. A series of functional pan-genomic analyses provide conserved and unique types of genomic evidence for strong epistatic and pleiotropic impacts on evolutionary trajectories of the PAH-degrading phenotype. Under strong natural selection, the detailed gene gain/loss patterns from horizontal gene transfer (HGT)/deletion events hypothesize a plausible evolutionary path, an epistasis-based birth and pleiotropy-dependent death, for PAH metabolism in the genus Mycobacterium. This study generated a practical mycobacterial compendium of phenotypic and genomic changes, focusing on the PAH-degrading phenotype, with a pan-genomic perspective of the evolutionary events and the environmental challenges. Our findings suggest that when selection acts on PAH metabolism, only a small fraction of possible trajectories is likely to be observed, owing mainly to a combination of the ambiguous phenotypic effects of PAHs and the corresponding pleiotropy- and epistasis-dependent evolutionary adaptation. Evolutionary constraints on the selection of trajectories, like those seen in PAH-degrading phenotypes, are likely to apply to the evolution of other phenotypes in the genus Mycobacterium.

Two genetic loci control syllable sequences of ultrasonic courtship vocalizations in inbred mice

PubMed Central

2011-01-01

Background The ultrasonic vocalizations (USV) of courting male mice are known to possess a phonetic structure with a complex combination of several syllables. The genetic mechanisms underlying the syllable sequence organization were investigated. Results This study compared syllable sequence organization in two inbred strains of mice, 129S4/SvJae (129) and C57BL6J (B6), and demonstrated that they possessed two mutually exclusive phenotypes. The 129S4/SvJae (129) strain frequently exhibited a "chevron-wave" USV pattern, which was characterized by the repetition of chevron-type syllables. The C57BL/6J strain produced a "staccato" USV pattern, which was characterized by the repetition of short-type syllables. An F1 strain obtained by crossing the 129S4/SvJae and C57BL/6J strains produced only the staccato phenotype. The chevron-wave and staccato phenotypes reappeared in the F2 generations, following the Mendelian law of independent assortment. Conclusions These results suggest that two genetic loci control the organization of syllable sequences. These loci were occupied by the staccato and chevron-wave alleles in the B6 and 129 mouse strains, respectively. Recombination of these alleles might lead to the diversity of USV patterns produced by mice. PMID:22018021

Identification of a novel MYO7A mutation in Usher syndrome type 1.

PubMed

Cheng, Ling; Yu, Hongsong; Jiang, Yan; He, Juan; Pu, Sisi; Li, Xin; Zhang, Li

2018-01-05

Usher syndrome (USH) is an autosomal recessive disease characterized by deafness and retinitis pigmentosa. In view of the high phenotypic and genetic heterogeneity in USH, performing genetic screening with traditional methods is impractical. In the present study, we carried out targeted next-generation sequencing (NGS) to uncover the underlying gene in an USH family (2 USH patients and 15 unaffected relatives). One hundred and thirty-five genes associated with inherited retinal degeneration were selected for deep exome sequencing. Subsequently, variant analysis, Sanger validation and segregation tests were utilized to identify the disease-causing mutations in this family. All affected individuals had a classic USH type I (USH1) phenotype which included deafness, vestibular dysfunction and retinitis pigmentosa. Targeted NGS and Sanger sequencing validation suggested that USH1 patients carried an unreported splice site mutation, c.5168+1G>A, as a compound heterozygous mutation with c.6070C>T (p.R2024X) in the MYO7A gene. A functional study revealed decreased expression of the MYO7A gene in the individuals carrying heterozygous mutations. In conclusion, targeted next-generation sequencing provided a comprehensive and efficient diagnosis for USH1. This study revealed the genetic defects in the MYO7A gene and expanded the spectrum of clinical phenotypes associated with USH1 mutations.

Phenotypic and genomic analysis of serotype 3 Sabin poliovirus vaccine produced in MRC-5 cell substrate.

PubMed

Alirezaie, Behnam; Taqavian, Mohammad; Aghaiypour, Khosrow; Esna-Ashari, Fatemeh; Shafyi, Abbas

2011-05-01

The cell substrate has a pivotal role in live virus vaccines production. It is necessary to evaluate the effects of the cell substrate on the properties of the propagated viruses, especially in the case of viruses which are unstable genetically such as polioviruses, by monitoring the molecular and phenotypical characteristics of harvested viruses. To investigate the presence/absence of mutation(s), the near full-length genomic sequence of different harvests of the type 3 Sabin strain of poliovirus propagated in MRC-5 cells were determined. The sequences were compared with genomic sequences of different virus seeds, vaccines, and OPV-like isolates. Nearly complete genomic sequencing results, however, revealed no detectable mutations throughout the genome RNA-plaque purified (RSO)-derived monopool of type 3 OPVs manufactured in MRC-5. Thirty-six years of experience in OPV production, trend analysis, and vaccine surveillance also suggest that: (i) different monopools of serotype 3 OPV produced in MRC-5 retained their phenotypic characteristics (temperature sensitivity and neuroattenuation), (ii) MRC-5 cells support the production of acceptable virus yields, (iii) OPV replicated in the MRC-5 cell substrate is a highly efficient and safe vaccine. These results confirm previous reports that MRC-5 is a desirable cell substrate for the production of OPV. Copyright © 2011 Wiley-Liss, Inc.

Whole-genome sequencing in patients with ciliopathies uncovers a novel recurrent tandem duplication in IFT140.

PubMed

Geoffroy, Véronique; Stoetzel, Corinne; Scheidecker, Sophie; Schaefer, Elise; Perrault, Isabelle; Bär, Séverine; Kröll, Ariane; Delbarre, Marion; Antin, Manuela; Leuvrey, Anne-Sophie; Henry, Charline; Blanché, Hélène; Decker, Eva; Kloth, Katja; Klaus, Günter; Mache, Christoph; Martin-Coignard, Dominique; McGinn, Steven; Boland, Anne; Deleuze, Jean-François; Friant, Sylvie; Saunier, Sophie; Rozet, Jean-Michel; Bergmann, Carsten; Dollfus, Hélène; Muller, Jean

2018-04-24

Ciliopathies represent a wide spectrum of rare diseases with overlapping phenotypes and a high genetic heterogeneity. Among those, IFT140 is implicated in a variety of phenotypes ranging from isolated retinis pigmentosa to more syndromic cases. Using whole-genome sequencing in patients with uncharacterized ciliopathies, we identified a novel recurrent tandem duplication of exon 27-30 (6.7 kb) in IFT140, c.3454-488_4182+2588dup p.(Tyr1152_Thr1394dup), missed by whole-exome sequencing. Pathogenicity of the mutation was assessed on the patients' skin fibroblasts. Several hundreds of patients with a ciliopathy phenotype were screened and biallelic mutations were identified in 11 families representing 12 pathogenic variants of which seven are novel. Among those unrelated families especially with a Mainzer-Saldino syndrome, eight carried the same tandem duplication (two at the homozygous state and six at the heterozygous state). In conclusion, we demonstrated the implication of structural variations in IFT140-related diseases expanding its mutation spectrum. We also provide evidences for a unique genomic event mediated by an Alu-Alu recombination occurring on a shared haplotype. We confirm that whole-genome sequencing can be instrumental in the ability to detect structural variants for genomic disorders. © 2018 Wiley Periodicals, Inc.

The whole genome sequences and experimentally phased haplotypes of over 100 personal genomes.

PubMed

Mao, Qing; Ciotlos, Serban; Zhang, Rebecca Yu; Ball, Madeleine P; Chin, Robert; Carnevali, Paolo; Barua, Nina; Nguyen, Staci; Agarwal, Misha R; Clegg, Tom; Connelly, Abram; Vandewege, Ward; Zaranek, Alexander Wait; Estep, Preston W; Church, George M; Drmanac, Radoje; Peters, Brock A

2016-10-11

Since the completion of the Human Genome Project in 2003, it is estimated that more than 200,000 individual whole human genomes have been sequenced. A stunning accomplishment in such a short period of time. However, most of these were sequenced without experimental haplotype data and are therefore missing an important aspect of genome biology. In addition, much of the genomic data is not available to the public and lacks phenotypic information. As part of the Personal Genome Project, blood samples from 184 participants were collected and processed using Complete Genomics' Long Fragment Read technology. Here, we present the experimental whole genome haplotyping and sequencing of these samples to an average read coverage depth of 100X. This is approximately three-fold higher than the read coverage applied to most whole human genome assemblies and ensures the highest quality results. Currently, 114 genomes from this dataset are freely available in the GigaDB repository and are associated with rich phenotypic data; the remaining 70 should be added in the near future as they are approved through the PGP data release process. For reproducibility analyses, 20 genomes were sequenced at least twice using independent LFR barcoded libraries. Seven genomes were also sequenced using Complete Genomics' standard non-barcoded library process. In addition, we report 2.6 million high-quality, rare variants not previously identified in the Single Nucleotide Polymorphisms database or the 1000 Genomes Project Phase 3 data. These genomes represent a unique source of haplotype and phenotype data for the scientific community and should help to expand our understanding of human genome evolution and function.

Prediction of BRCA1 and BRCA2 mutation status using post-irradiation assays of lymphoblastoid cell lines is compromised by inter-cell-line phenotypic variability.

PubMed

Lovelock, Paul K; Wong, Ee Ming; Sprung, Carl N; Marsh, Anna; Hobson, Karen; French, Juliet D; Southey, Melissa; Sculley, Tom; Pandeya, Nirmala; Brown, Melissa A; Chenevix-Trench, Georgia; Spurdle, Amanda B; McKay, Michael J

2007-09-01

Assays to determine the pathogenicity of unclassified sequence variants in disease-associated genes include the analysis of lymphoblastoid cell lines (LCLs). We assessed the ability of several assays of LCLs to distinguish carriers of germline BRCA1 and BRCA2 gene mutations from mutation-negative controls to determine their utility for use in a diagnostic setting. Post-ionising radiation cell viability and micronucleus formation, and telomere length were assayed in LCLs carrying BRCA1 or BRCA2 mutations, and in unaffected mutation-negative controls. Post-irradiation cell viability and micronucleus induction assays of LCLs from individuals carrying pathogenic BRCA1 mutations, unclassified BRCA1 sequence variants or wildtype BRCA1 sequence showed significant phenotypic heterogeneity within each group. Responses were not consistent with predicted functional consequences of known pathogenic or normal sequences. Telomere length was also highly heterogeneous within groups of LCLs carrying pathogenic BRCA1 or BRCA2 mutations, and normal BRCA1 sequences, and was not predictive of mutation status. Given the significant degree of phenotypic heterogeneity of LCLs after gamma-irradiation, and the lack of association with BRCA1 or BRCA2 mutation status, we conclude that the assays evaluated in this study should not be used as a means of differentiating pathogenic and non-pathogenic sequence variants for clinical application. We suggest that a range of normal controls must be included in any functional assays of LCLs to ensure that any observed differences between samples reflect the genotype under investigation rather than generic inter-individual variation.

The expanding spectrum of COL2A1 gene variants IN 136 patients with a skeletal dysplasia phenotype

PubMed Central

Barat-Houari, Mouna; Dumont, Bruno; Fabre, Aurélie; Them, Frédéric TM; Alembik, Yves; Alessandri, Jean-Luc; Amiel, Jeanne; Audebert, Séverine; Baumann-Morel, Clarisse; Blanchet, Patricia; Bieth, Eric; Brechard, Marie; Busa, Tiffany; Calvas, Patrick; Capri, Yline; Cartault, François; Chassaing, Nicolas; Ciorca, Vidrica; Coubes, Christine; David, Albert; Delezoide, Anne-Lise; Dupin-Deguine, Delphine; El Chehadeh, Salima; Faivre, Laurence; Giuliano, Fabienne; Goldenberg, Alice; Isidor, Bertrand; Jacquemont, Marie-Line; Julia, Sophie; Kaplan, Josseline; Lacombe, Didier; Lebrun, Marine; Marlin, Sandrine; Martin-Coignard, Dominique; Martinovic, Jelena; Masurel, Alice; Melki, Judith; Mozelle-Nivoix, Monique; Nguyen, Karine; Odent, Sylvie; Philip, Nicole; Pinson, Lucile; Plessis, Ghislaine; Quélin, Chloé; Shaeffer, Elise; Sigaudy, Sabine; Thauvin, Christel; Till, Marianne; Touraine, Renaud; Vigneron, Jacqueline; Baujat, Geneviève; Cormier-Daire, Valérie; Le Merrer, Martine; Geneviève, David; Touitou, Isabelle

2016-01-01

Heterozygous COL2A1 variants cause a wide spectrum of skeletal dysplasia termed type II collagenopathies. We assessed the impact of this gene in our French series. A decision tree was applied to select 136 probands (71 Stickler cases, 21 Spondyloepiphyseal dysplasia congenita cases, 11 Kniest dysplasia cases, and 34 other dysplasia cases) before molecular diagnosis by Sanger sequencing. We identified 66 different variants among the 71 positive patients. Among those patients, 18 belonged to multiplex families and 53 were sporadic. Most variants (38/44, 86%) were located in the triple helical domain of the collagen chain and glycine substitutions were mainly observed in severe phenotypes, whereas arginine to cysteine changes were more often encountered in moderate phenotypes. This series of skeletal dysplasia is one of the largest reported so far, adding 44 novel variants (15%) to published data. We have confirmed that about half of our Stickler patients (46%) carried a COL2A1 variant, and that the molecular spectrum was different across the phenotypes. To further address the question of genotype–phenotype correlation, we plan to screen our patients for other candidate genes using a targeted next-generation sequencing approach. PMID:26626311

The expanding spectrum of COL2A1 gene variants IN 136 patients with a skeletal dysplasia phenotype.

PubMed

Barat-Houari, Mouna; Dumont, Bruno; Fabre, Aurélie; Them, Frédéric Tm; Alembik, Yves; Alessandri, Jean-Luc; Amiel, Jeanne; Audebert, Séverine; Baumann-Morel, Clarisse; Blanchet, Patricia; Bieth, Eric; Brechard, Marie; Busa, Tiffany; Calvas, Patrick; Capri, Yline; Cartault, François; Chassaing, Nicolas; Ciorca, Vidrica; Coubes, Christine; David, Albert; Delezoide, Anne-Lise; Dupin-Deguine, Delphine; El Chehadeh, Salima; Faivre, Laurence; Giuliano, Fabienne; Goldenberg, Alice; Isidor, Bertrand; Jacquemont, Marie-Line; Julia, Sophie; Kaplan, Josseline; Lacombe, Didier; Lebrun, Marine; Marlin, Sandrine; Martin-Coignard, Dominique; Martinovic, Jelena; Masurel, Alice; Melki, Judith; Mozelle-Nivoix, Monique; Nguyen, Karine; Odent, Sylvie; Philip, Nicole; Pinson, Lucile; Plessis, Ghislaine; Quélin, Chloé; Shaeffer, Elise; Sigaudy, Sabine; Thauvin, Christel; Till, Marianne; Touraine, Renaud; Vigneron, Jacqueline; Baujat, Geneviève; Cormier-Daire, Valérie; Le Merrer, Martine; Geneviève, David; Touitou, Isabelle

2016-07-01

Heterozygous COL2A1 variants cause a wide spectrum of skeletal dysplasia termed type II collagenopathies. We assessed the impact of this gene in our French series. A decision tree was applied to select 136 probands (71 Stickler cases, 21 Spondyloepiphyseal dysplasia congenita cases, 11 Kniest dysplasia cases, and 34 other dysplasia cases) before molecular diagnosis by Sanger sequencing. We identified 66 different variants among the 71 positive patients. Among those patients, 18 belonged to multiplex families and 53 were sporadic. Most variants (38/44, 86%) were located in the triple helical domain of the collagen chain and glycine substitutions were mainly observed in severe phenotypes, whereas arginine to cysteine changes were more often encountered in moderate phenotypes. This series of skeletal dysplasia is one of the largest reported so far, adding 44 novel variants (15%) to published data. We have confirmed that about half of our Stickler patients (46%) carried a COL2A1 variant, and that the molecular spectrum was different across the phenotypes. To further address the question of genotype-phenotype correlation, we plan to screen our patients for other candidate genes using a targeted next-generation sequencing approach.

Characterization of Lactobacillus from Algerian Goat’S Milk Based on Phenotypic, 16S rDNA Sequencing and their Technological Properties

PubMed Central

Marroki, Ahmed; Zúñiga, Manuel; Kihal, Mabrouk; Pérez- Martínez, Gaspar

2011-01-01

Nineteen strains of Lactobacillus isolated from goat’s milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility) was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21) and one strain of L. rhamnosus (LbMF25) have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria. PMID:24031617

A new era in clinical genetic testing for hypertrophic cardiomyopathy.

PubMed

Wheeler, Matthew; Pavlovic, Aleksandra; DeGoma, Emil; Salisbury, Heidi; Brown, Colleen; Ashley, Euan A

2009-12-01

Building on seminal studies of the last 20 years, genetic testing for hypertrophic cardiomyopathy (HCM) has become a clinical reality in the form of targeted exonic sequencing of known disease-causing genes. This has been driven primarily by the decreasing cost of sequencing, but the high profile of genome-wide association studies, the launch of direct-to-consumer genetic testing, and new legislative protection have also played important roles. In the clinical management of hypertrophic cardiomyopathy, genetic testing is primarily used for family screening. An increasing role is recognized, however, in diagnostic settings: in the differential diagnosis of HCM; in the differentiation of HCM from hypertensive or athlete's heart; and more rarely in preimplantation genetic diagnosis. Aside from diagnostic clarification and family screening, use of the genetic test for guiding therapy remains controversial, with data currently too limited to derive a reliable mutation risk prediction from within the phenotypic noise of different modifying genomes. Meanwhile, the power of genetic testing derives from the confidence with which a mutation can be called present or absent in a given individual. This confidence contrasts with our more limited ability to judge the significance of mutations for which co-segregation has not been demonstrated. These variants of "unknown" significance represent the greatest challenge to the wider adoption of genetic testing in HCM. Looking forward, next-generation sequencing technologies promise to revolutionize the current approach as whole genome sequencing will soon be available for the cost of today's targeted panel. In summary, our future will be characterized not by lack of genetic information but by our ability to effectively parse it.

HIV drug resistance testing among patients failing second line antiretroviral therapy. Comparison of in-house and commercial sequencing.

PubMed

Chimukangara, Benjamin; Varyani, Bhavini; Shamu, Tinei; Mutsvangwa, Junior; Manasa, Justen; White, Elizabeth; Chimbetete, Cleophas; Luethy, Ruedi; Katzenstein, David

2017-05-01

HIV genotyping is often unavailable in low and middle-income countries due to infrastructure requirements and cost. We compared genotype resistance testing in patients with virologic failure, by amplification of HIV pol gene, followed by "in-house" sequencing and commercial sequencing. Remnant plasma samples from adults and children failing second-line ART were amplified and sequenced using in-house and commercial di-deoxysequencing, and analyzed in Harare, Zimbabwe and at Stanford, U.S.A, respectively. HIV drug resistance mutations were determined using the Stanford HIV drug resistance database. Twenty-six of 28 samples were amplified and 25 were successfully genotyped. Comparison of average percent nucleotide and amino acid identities between 23 pairs sequenced in both laboratories were 99.51 (±0.56) and 99.11 (±0.95), respectively. All pairs clustered together in phylogenetic analysis. Sequencing analysis identified 6/23 pairs with mutation discordances resulting in differences in phenotype, but these did not impact future regimens. The results demonstrate our ability to produce good quality drug resistance data in-house. Despite discordant mutations in some sequence pairs, the phenotypic predictions were not clinically significant. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular Findings Among Patients Referred for Clinical Whole-Exome Sequencing

PubMed Central

Yang, Yaping; Muzny, Donna M.; Xia, Fan; Niu, Zhiyv; Person, Richard; Ding, Yan; Ward, Patricia; Braxton, Alicia; Wang, Min; Buhay, Christian; Veeraraghavan, Narayanan; Hawes, Alicia; Chiang, Theodore; Leduc, Magalie; Beuten, Joke; Zhang, Jing; He, Weimin; Scull, Jennifer; Willis, Alecia; Landsverk, Megan; Craigen, William J.; Bekheirnia, Mir Reza; Stray-Pedersen, Asbjorg; Liu, Pengfei; Wen, Shu; Alcaraz, Wendy; Cui, Hong; Walkiewicz, Magdalena; Reid, Jeffrey; Bainbridge, Matthew; Patel, Ankita; Boerwinkle, Eric; Beaudet, Arthur L.; Lupski, James R.; Plon, Sharon E.; Gibbs, Richard A.; Eng, Christine M.

2015-01-01

IMPORTANCE Clinical whole-exome sequencing is increasingly used for diagnostic evaluation of patients with suspected genetic disorders. OBJECTIVE To perform clinical whole-exome sequencing and report (1) the rate of molecular diagnosis among phenotypic groups, (2) the spectrum of genetic alterations contributing to disease, and (3) the prevalence of medically actionable incidental findings such as FBN1 mutations causing Marfan syndrome. DESIGN, SETTING, AND PATIENTS Observational study of 2000 consecutive patients with clinical whole-exome sequencing analyzed between June 2012 and August 2014. Whole-exome sequencing tests were performed at a clinical genetics laboratory in the United States. Results were reported by clinical molecular geneticists certified by the American Board of Medical Genetics and Genomics. Tests were ordered by the patient’s physician. The patients were primarily pediatric (1756 [88%]; mean age, 6 years; 888 females [44%], 1101 males [55%], and 11 fetuses [1% gender unknown]), demonstrating diverse clinical manifestations most often including nervous system dysfunction such as developmental delay. MAIN OUTCOMES AND MEASURES Whole-exome sequencing diagnosis rate overall and by phenotypic category, mode of inheritance, spectrum of genetic events, and reporting of incidental findings. RESULTS A molecular diagnosis was reported for 504 patients (25.2%) with 58% of the diagnostic mutations not previously reported. Molecular diagnosis rates for each phenotypic category were 143/526 (27.2%; 95% CI, 23.5%–31.2%) for the neurological group, 282/1147 (24.6%; 95% CI, 22.1%–27.2%) for the neurological plus other organ systems group, 30/83 (36.1%; 95% CI, 26.1%–47.5%) for the specific neurological group, and 49/244 (20.1%; 95% CI, 15.6%–25.8%) for the nonneurological group. The Mendelian disease patterns of the 527 molecular diagnoses included 280 (53.1%) autosomal dominant, 181 (34.3%) autosomal recessive (including 5 with uniparental disomy), 65 (12.3%) X-linked, and 1 (0.2%) mitochondrial. Of 504 patients with a molecular diagnosis, 23 (4.6%) had blended phenotypes resulting from 2 single gene defects. About 30% of the positive cases harbored mutations in disease genes reported since 2011. There were 95 medically actionable incidental findings in genes unrelated to the phenotype but with immediate implications for management in 92 patients (4.6%), including 59 patients (3%) with mutations in genes recommended for reporting by the American College of Medical Genetics and Genomics. CONCLUSIONS AND RELEVANCE Whole-exome sequencing provided a potential molecular diagnosis for 25% of a large cohort of patients referred for evaluation of suspected genetic conditions, including detection of rare genetic events and new mutations contributing to disease. The yield of whole-exome sequencing may offer advantages over traditional molecular diagnostic approaches in certain patients. PMID:25326635

Comparative genomics of 28 Salmonella enterica isolates: evidence for CRISPR-mediated adaptive sublineage evolution.

PubMed

Fricke, W Florian; Mammel, Mark K; McDermott, Patrick F; Tartera, Carmen; White, David G; Leclerc, J Eugene; Ravel, Jacques; Cebula, Thomas A

2011-07-01

Despite extensive surveillance, food-borne Salmonella enterica infections continue to be a significant burden on public health systems worldwide. As the S. enterica species comprises sublineages that differ greatly in antigenic representation, virulence, and antimicrobial resistance phenotypes, a better understanding of the species' evolution is critical for the prediction and prevention of future outbreaks. The roles that virulence and resistance phenotype acquisition, exchange, and loss play in the evolution of S. enterica sublineages, which to a certain extent are represented by serotypes, remains mostly uncharacterized. Here, we compare 17 newly sequenced and phenotypically characterized nontyphoidal S. enterica strains to 11 previously sequenced S. enterica genomes to carry out the most comprehensive comparative analysis of this species so far. These phenotypic and genotypic data comparisons in the phylogenetic species context suggest that the evolution of known S. enterica sublineages is mediated mostly by two mechanisms, (i) the loss of coding sequences with known metabolic functions, which leads to functional reduction, and (ii) the acquisition of horizontally transferred phage and plasmid DNA, which provides virulence and resistance functions and leads to increasing specialization. Matches between S. enterica clustered regularly interspaced short palindromic repeats (CRISPR), part of a defense mechanism against invading plasmid and phage DNA, and plasmid and prophage regions suggest that CRISPR-mediated immunity could control short-term phenotype changes and mediate long-term sublineage evolution. CRISPR analysis could therefore be critical in assessing the evolutionary potential of S. enterica sublineages and aid in the prediction and prevention of future S. enterica outbreaks.

Comparative Genomics of 28 Salmonella enterica Isolates: Evidence for CRISPR-Mediated Adaptive Sublineage Evolution ▿†

PubMed Central

Fricke, W. Florian; Mammel, Mark K.; McDermott, Patrick F.; Tartera, Carmen; White, David G.; LeClerc, J. Eugene; Ravel, Jacques; Cebula, Thomas A.

2011-01-01

Despite extensive surveillance, food-borne Salmonella enterica infections continue to be a significant burden on public health systems worldwide. As the S. enterica species comprises sublineages that differ greatly in antigenic representation, virulence, and antimicrobial resistance phenotypes, a better understanding of the species' evolution is critical for the prediction and prevention of future outbreaks. The roles that virulence and resistance phenotype acquisition, exchange, and loss play in the evolution of S. enterica sublineages, which to a certain extent are represented by serotypes, remains mostly uncharacterized. Here, we compare 17 newly sequenced and phenotypically characterized nontyphoidal S. enterica strains to 11 previously sequenced S. enterica genomes to carry out the most comprehensive comparative analysis of this species so far. These phenotypic and genotypic data comparisons in the phylogenetic species context suggest that the evolution of known S. enterica sublineages is mediated mostly by two mechanisms, (i) the loss of coding sequences with known metabolic functions, which leads to functional reduction, and (ii) the acquisition of horizontally transferred phage and plasmid DNA, which provides virulence and resistance functions and leads to increasing specialization. Matches between S. enterica clustered regularly interspaced short palindromic repeats (CRISPR), part of a defense mechanism against invading plasmid and phage DNA, and plasmid and prophage regions suggest that CRISPR-mediated immunity could control short-term phenotype changes and mediate long-term sublineage evolution. CRISPR analysis could therefore be critical in assessing the evolutionary potential of S. enterica sublineages and aid in the prediction and prevention of future S. enterica outbreaks. PMID:21602358

Genome-wide annotation of mutations in a phenotyped mutant library provides an efficient platform for discovery of casual gene mutations

USDA-ARS?s Scientific Manuscript database

Ethyl methanesulfonate (EMS) efficiently generates high-density mutations in genomes. Conventionally, these mutations are identified by techniques that can detect single-nucleotide mismatches in heteroduplexes of individual PCR amplicons. We applied whole-genome sequencing to 256-phenotyped mutant l...

Phylogenetic distribution of phenotypic traits in bacillus thuringiensis analyzed by multilocus sequence typing

USDA-ARS?s Scientific Manuscript database

Strains from a collection of 3,639 diverse Bacillus thuringiensis isolates were classified based on phenotypic profiles resulting from six biochemical tests, including production of amylase (T), lecithinase (L), urease (U), acid from sucrose (S) and salicin (A), and the hydrolysis of esculin (E). St...

Deciphering the molecular mechanisms underlying the binding of the TWIST1/E12 complex to regulatory E-box sequences

PubMed Central

Bouard, Charlotte; Terreux, Raphael; Honorat, Mylène; Manship, Brigitte; Ansieau, Stéphane; Vigneron, Arnaud M.; Puisieux, Alain; Payen, Léa

2016-01-01

Abstract The TWIST1 bHLH transcription factor controls embryonic development and cancer processes. Although molecular and genetic analyses have provided a wealth of data on the role of bHLH transcription factors, very little is known on the molecular mechanisms underlying their binding affinity to the E-box sequence of the promoter. Here, we used an in silico model of the TWIST1/E12 (TE) heterocomplex and performed molecular dynamics (MD) simulations of its binding to specific (TE-box) and modified E-box sequences. We focused on (i) active E-box and inactive E-box sequences, on (ii) modified active E-box sequences, as well as on (iii) two box sequences with modified adjacent bases the AT- and TA-boxes. Our in silico models were supported by functional in vitro binding assays. This exploration highlighted the predominant role of protein side-chain residues, close to the heart of the complex, at anchoring the dimer to DNA sequences, and unveiled a shift towards adjacent ((-1) and (-1*)) bases and conserved bases of modified E-box sequences. In conclusion, our study provides proof of the predictive value of these MD simulations, which may contribute to the characterization of specific inhibitors by docking approaches, and their use in pharmacological therapies by blocking the tumoral TWIST1/E12 function in cancers. PMID:27151200

Application of Modified Spin-Echo–based Sequences for Hepatic MR Elastography: Evaluation, Comparison with the Conventional Gradient-Echo Sequence, and Preliminary Clinical Experience

PubMed Central

Mariappan, Yogesh K.; Dzyubak, Bogdan; Glaser, Kevin J.; Venkatesh, Sudhakar K.; Sirlin, Claude B.; Hooker, Jonathan; McGee, Kiaran P.

2017-01-01

Purpose To (a) evaluate modified spin-echo (SE) magnetic resonance (MR) elastographic sequences for acquiring MR images with improved signal-to-noise ratio (SNR) in patients in whom the standard gradient-echo (GRE) MR elastographic sequence yields low hepatic signal intensity and (b) compare the stiffness values obtained with these sequences with those obtained with the conventional GRE sequence. Materials and Methods This HIPAA-compliant retrospective study was approved by the institutional review board; the requirement to obtain informed consent was waived. Data obtained with modified SE and SE echo-planar imaging (EPI) MR elastographic pulse sequences with short echo times were compared with those obtained with the conventional GRE MR elastographic sequence in two patient cohorts, one that exhibited adequate liver signal intensity and one that exhibited low liver signal intensity. Shear stiffness values obtained with the three sequences in 130 patients with successful GRE-based examinations were retrospectively tested for statistical equivalence by using a 5% margin. In 47 patients in whom GRE examinations were considered to have failed because of low SNR, the SNR and confidence level with the SE-based sequences were compared with those with the GRE sequence. Results The results of this study helped confirm the equivalence of SE MR elastography and SE-EPI MR elastography to GRE MR elastography (P = .0212 and P = .0001, respectively). The SE and SE-EPI MR elastographic sequences provided substantially improved SNR and stiffness inversion confidence level in 47 patients in whom GRE MR elastography had failed. Conclusion Modified SE-based MR elastographic sequences provide higher SNR MR elastographic data and reliable stiffness measurements; thus, they enable quantification of stiffness in patients in whom the conventional GRE MR elastographic sequence failed owing to low signal intensity. The equivalence of the three sequences indicates that the current diagnostic thresholds are applicable to SE MR elastographic sequences for assessing liver fibrosis. © RSNA, 2016 PMID:27509543

Genome Sequence of Saccharomyces cerevisiae Double-Stranded RNA Virus L-A-28.

PubMed

Konovalovas, Aleksandras; Serviené, Elena; Serva, Saulius

2016-06-16

We cloned and sequenced the complete genome of the L-A-28 virus from the Saccharomyces cerevisiae K28 killer strain. This sequence completes the set of currently identified L-A helper viruses required for expression of double-stranded RNA-originated killer phenotypes in baking yeast. Copyright © 2016 Konovalovas et al.

Functional Regression Models for Epistasis Analysis of Multiple Quantitative Traits.

PubMed

Zhang, Futao; Xie, Dan; Liang, Meimei; Xiong, Momiao

2016-04-01

To date, most genetic analyses of phenotypes have focused on analyzing single traits or analyzing each phenotype independently. However, joint epistasis analysis of multiple complementary traits will increase statistical power and improve our understanding of the complicated genetic structure of the complex diseases. Despite their importance in uncovering the genetic structure of complex traits, the statistical methods for identifying epistasis in multiple phenotypes remains fundamentally unexplored. To fill this gap, we formulate a test for interaction between two genes in multiple quantitative trait analysis as a multiple functional regression (MFRG) in which the genotype functions (genetic variant profiles) are defined as a function of the genomic position of the genetic variants. We use large-scale simulations to calculate Type I error rates for testing interaction between two genes with multiple phenotypes and to compare the power with multivariate pairwise interaction analysis and single trait interaction analysis by a single variate functional regression model. To further evaluate performance, the MFRG for epistasis analysis is applied to five phenotypes of exome sequence data from the NHLBI's Exome Sequencing Project (ESP) to detect pleiotropic epistasis. A total of 267 pairs of genes that formed a genetic interaction network showed significant evidence of epistasis influencing five traits. The results demonstrate that the joint interaction analysis of multiple phenotypes has a much higher power to detect interaction than the interaction analysis of a single trait and may open a new direction to fully uncovering the genetic structure of multiple phenotypes.

A rare CYP21A2 mutation in a congenital adrenal hyperplasia kindred displaying genotype-phenotype nonconcordance.

PubMed

Khattab, Ahmed; Yuen, Tony; Al-Malki, Sultan; Yau, Mabel; Kazmi, Diya; Sun, Li; Harbison, Madeleine; Haider, Shozeb; Zaidi, Mone; New, Maria I

2016-01-01

Congenital adrenal hyperplasia (CAH) owing to 21-hydroxylase deficiency is caused by the autosomal recessive inheritance of mutations in the gene CYP21A2. CYP21A2 mutations lead to variable impairment of the 21-hydroxylase enzyme, which, in turn, is associated with three clinical phenotypes, namely, salt wasting, simple virilizing, and nonclassical CAH. However, it is known that a given mutation can associate with different clinical phenotypes, resulting in a high rate of genotype-phenotype nonconcordance. We aimed to study the genotype-phenotype nonconcordance in a family with three siblings affected with nonclassical CAH. All had hormonal evidence of nonclassical CAH, but this phenotype could not be explained by the genotype obtained from commercial CYP21A2 genetic testing, which revealed heterozygosity for the maternal 30 kb deletion mutation. We performed Sanger sequencing of the entire CYP21A2 gene in this family to search for a rare mutation that was not covered by commercial testing and found in the three siblings a second, rare c.1097G>A (p.R366H) mutation in exon 8. Computational modeling confirmed that this was a mild mutation consistent with nonclassical CAH. We recommend that sequencing of entire genes for rare mutations should be carried out when genotype-phenotype nonconcordance is observed in patients with autosomal recessive monogenic disorders, including CAH. © 2015 New York Academy of Sciences.

Massively Parallel Sequencing of Patients with Intellectual Disability, Congenital Anomalies and/or Autism Spectrum Disorders with a Targeted Gene Panel

PubMed Central

Brett, Maggie; McPherson, John; Zang, Zhi Jiang; Lai, Angeline; Tan, Ee-Shien; Ng, Ivy; Ong, Lai-Choo; Cham, Breana; Tan, Patrick; Rozen, Steve; Tan, Ene-Choo

2014-01-01

Developmental delay and/or intellectual disability (DD/ID) affects 1–3% of all children. At least half of these are thought to have a genetic etiology. Recent studies have shown that massively parallel sequencing (MPS) using a targeted gene panel is particularly suited for diagnostic testing for genetically heterogeneous conditions. We report on our experiences with using massively parallel sequencing of a targeted gene panel of 355 genes for investigating the genetic etiology of eight patients with a wide range of phenotypes including DD/ID, congenital anomalies and/or autism spectrum disorder. Targeted sequence enrichment was performed using the Agilent SureSelect Target Enrichment Kit and sequenced on the Illumina HiSeq2000 using paired-end reads. For all eight patients, 81–84% of the targeted regions achieved read depths of at least 20×, with average read depths overlapping targets ranging from 322× to 798×. Causative variants were successfully identified in two of the eight patients: a nonsense mutation in the ATRX gene and a canonical splice site mutation in the L1CAM gene. In a third patient, a canonical splice site variant in the USP9X gene could likely explain all or some of her clinical phenotypes. These results confirm the value of targeted MPS for investigating DD/ID in children for diagnostic purposes. However, targeted gene MPS was less likely to provide a genetic diagnosis for children whose phenotype includes autism. PMID:24690944

Phenotypic Checkpoints Regulate Neuronal Development

PubMed Central

Ben-Ari, Yehezkel; Spitzer, Nicholas C.

2010-01-01

Nervous system development proceeds by sequential gene expression mediated by cascades of transcription factors in parallel with sequences of patterned network activity driven by receptors and ion channels. These sequences are cell type- and developmental stage-dependent and modulated by paracrine actions of substances released by neurons and glia. How and to what extent these sequences interact to enable neuronal network development is not understood. Recent evidence demonstrates that CNS development requires intermediate stages of differentiation providing functional feedback that influences gene expression. We suggest that embryonic neuronal functions constitute a series of phenotypic checkpoint signatures; neurons failing to express these functions are delayed or developmentally arrested. Such checkpoints are likely to be a general feature of neuronal development and may constitute presymptomatic signatures of neurological disorders when they go awry. PMID:20864191

The near demise and subsequent revival of classical genetics for investigating Caenorhabditis elegans embryogenesis: RNAi meets next-generation DNA sequencing.

PubMed

Bowerman, Bruce

2011-10-01

Molecular genetic investigation of the early Caenorhabditis elegans embryo has contributed substantially to the discovery and general understanding of the genes, pathways, and mechanisms that regulate and execute developmental and cell biological processes. Initially, worm geneticists relied exclusively on a classical genetics approach, isolating mutants with interesting phenotypes after mutagenesis and then determining the identity of the affected genes. Subsequently, the discovery of RNA interference (RNAi) led to a much greater reliance on a reverse genetics approach: reducing the function of known genes with RNAi and then observing the phenotypic consequences. Now the advent of next-generation DNA sequencing technologies and the ensuing ease and affordability of whole-genome sequencing are reviving the use of classical genetics to investigate early C. elegans embryogenesis.

Identification of Mucorales From Clinical Specimens: A 4-Year Experience in a Single Institution

PubMed Central

Yang, Mina; Lee, Jang Ho; Kim, Young-Kwon; Ki, Chang-Seok

2016-01-01

Mucormycosis, a fatal opportunistic infection in immunocompromised hosts, is caused by fungi belonging to the order Mucorales. Early diagnosis based on exact identification and multidisciplinary treatments is critical. However, identification of Mucorales fungi is difficult and often delayed, resulting in poor prognosis. This study aimed to compare the results of phenotypic and molecular identification of 12 Mucorales isolates collected from 4-yr-accumulated data. All isolates were identified on the basis of phenotypic characteristics such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing were performed to target internal transcribed spacer (ITS) and/or D1/D2 regions. Target DNA sequencing identified five Lichtheimia isolates, two Rhizopus microsporus isolates, two Rhizomucor pusillus isolates, one Cunninghamella bertholletiae isolate, one Mucor fragilis isolate, and one Syncephalastrum racemosum isolate. Five of the 12 (41.7%) isolates were incorrectly identified on the basis of phenotypic identification. DNA sequencing showed that of these five isolates, two were Lichtheimia isolates, one was Mucor isolate, one was Rhizomucor isolate, and one was Rhizopus microspores. All the isolates were identified at the species level by ITS and/or D1/D2 analyses. Phenotypic differentiation and identification of Mucorales is difficult because different Mucorales share similar morphology. Our results indicate that the molecular methods employed in this study are valuable for identifying Mucorales. PMID:26522761

Identification of mucorales from clinical specimens: a 4-year experience in a single institution.

PubMed

Yang, Mina; Lee, Jang Ho; Kim, Young Kwon; Ki, Chang Seok; Huh, Hee Jae; Lee, Nam Yong

2016-01-01

Mucormycosis, a fatal opportunistic infection in immunocompromised hosts, is caused by fungi belonging to the order Mucorales. Early diagnosis based on exact identification and multidisciplinary treatments is critical. However, identification of Mucorales fungi is difficult and often delayed, resulting in poor prognosis. This study aimed to compare the results of phenotypic and molecular identification of 12 Mucorales isolates collected from 4-yr-accumulated data. All isolates were identified on the basis of phenotypic characteristics such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing were performed to target internal transcribed spacer (ITS) and/or D1/D2 regions. Target DNA sequencing identified five Lichtheimia isolates, two Rhizopus microsporus isolates, two Rhizomucor pusillus isolates, one Cunninghamella bertholletiae isolate, one Mucor fragilis isolate, and one Syncephalastrum racemosum isolate. Five of the 12 (41.7%) isolates were incorrectly identified on the basis of phenotypic identification. DNA sequencing showed that of these five isolates, two were Lichtheimia isolates, one was Mucor isolate, one was Rhizomucor isolate, and one was Rhizopus microspores. All the isolates were identified at the species level by ITS and/or D1/D2 analyses. Phenotypic differentiation and identification of Mucorales is difficult because different Mucorales share similar morphology. Our results indicate that the molecular methods employed in this study are valuable for identifying Mucorales.

Experimental evolution reveals genome-wide spectrum and dynamics of mutations in the rice blast fungus, Magnaporthe oryzae.

PubMed

Jeon, Junhyun; Choi, Jaeyoung; Lee, Gir-Won; Dean, Ralph A; Lee, Yong-Hwan

2013-01-01

Knowledge on mutation processes is central to interpreting genetic analysis data as well as understanding the underlying nature of almost all evolutionary phenomena. However, studies on genome-wide mutational spectrum and dynamics in fungal pathogens are scarce, hindering our understanding of their evolution and biology. Here, we explored changes in the phenotypes and genome sequences of the rice blast fungus Magnaporthe oryzae during the forced in vitro evolution by weekly transfer of cultures on artificial media. Through combination of experimental evolution with high throughput sequencing technology, we found that mutations accumulate rapidly prior to visible phenotypic changes and that both genetic drift and selection seem to contribute to shaping mutational landscape, suggesting the buffering capacity of fungal genome against mutations. Inference of mutational effects on phenotypes through the use of T-DNA insertion mutants suggested that at least some of the DNA sequence mutations are likely associated with the observed phenotypic changes. Furthermore, our data suggest oxidative damages and UV as major sources of mutation during subcultures. Taken together, our work revealed important properties of original source of variation in the genome of the rice blast fungus. We believe that these results provide not only insights into stability of pathogenicity and genome evolution in plant pathogenic fungi but also a model in which evolution of fungal pathogens in natura can be comparatively investigated.

PAH mutation spectrum and correlation with PKU manifestation in north Jiangsu province population.

PubMed

Wang, Zhen-Wen; Jiang, Shi-Wen; Zhou, Bao-Cheng

2018-02-01

Phenylketonuria (PKU) is a common autosomal recessive disorder of phenylalanine metabolism and mainly results a deficiency of phenylalanine hydroxylase gene (PAH). The incidence of various PAH mutations have race and ethnicity differences. We report a spectrum of PAH mutations complied from 35 PKU children who are all Chinese Han population from north Jiangsu in this study. All 13 exons and their flanking intron sequences of PAH were determined by Ion Torrent PGM™ sequencing. The relationship of genotype and phenotype was analyzed based on the sum of the arbitrary value (AV) values of the two alleles. We identified 61 mutations, with a frequency of 87.14%, among 70 alleles of 35 patients. The most prevalent mutations were R243Q (26.23%), R241C (9.84%) and V399V (8.20%). Furthermore, the consistency between prediction of the biochemical phenotype and the observed phenotype was 81.25%, with the highest consistency observed in classic PKU (87.50%). A significant correlation was found between pretreatment levels of phenylalanine and AV sum (r = -0.87, P < 0.05). Finally, our study constructs PAH mutation spectrum by next generation sequencing (NGS), and reveals that the PAH genotypes and biochemical phenotypes were significantly correlated. These offers facilitate the provision of appropriate genetic counseling for PKU patients. Copyright © 2017. Published by Elsevier Taiwan.

A generalized association test based on U statistics.

PubMed

Wei, Changshuai; Lu, Qing

2017-07-01

Second generation sequencing technologies are being increasingly used for genetic association studies, where the main research interest is to identify sets of genetic variants that contribute to various phenotypes. The phenotype can be univariate disease status, multivariate responses and even high-dimensional outcomes. Considering the genotype and phenotype as two complex objects, this also poses a general statistical problem of testing association between complex objects. We here proposed a similarity-based test, generalized similarity U (GSU), that can test the association between complex objects. We first studied the theoretical properties of the test in a general setting and then focused on the application of the test to sequencing association studies. Based on theoretical analysis, we proposed to use Laplacian Kernel-based similarity for GSU to boost power and enhance robustness. Through simulation, we found that GSU did have advantages over existing methods in terms of power and robustness. We further performed a whole genome sequencing (WGS) scan for Alzherimer's disease neuroimaging initiative data, identifying three genes, APOE , APOC1 and TOMM40 , associated with imaging phenotype. We developed a C ++ package for analysis of WGS data using GSU. The source codes can be downloaded at https://github.com/changshuaiwei/gsu . weichangshuai@gmail.com ; qlu@epi.msu.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Front acceleration by dynamic selection in Fisher population waves

NASA Astrophysics Data System (ADS)

Bénichou, O.; Calvez, V.; Meunier, N.; Voituriez, R.

2012-10-01

We introduce a minimal model of population range expansion in which the phenotypes of individuals present no selective advantage and differ only in their diffusion rate. We show that such neutral phenotypic variability (i.e., that does not modify the growth rate) alone can yield phenotype segregation at the front edge, even in absence of genetic noise, and significantly impact the dynamical properties of the expansion wave. We present an exact asymptotic traveling wave solution and show analytically that phenotype segregation accelerates the front propagation. The results are compatible with field observations such as invasions of cane toads in Australia or bush crickets in Britain.

High-throughput discovery of novel developmental phenotypes.

PubMed

Dickinson, Mary E; Flenniken, Ann M; Ji, Xiao; Teboul, Lydia; Wong, Michael D; White, Jacqueline K; Meehan, Terrence F; Weninger, Wolfgang J; Westerberg, Henrik; Adissu, Hibret; Baker, Candice N; Bower, Lynette; Brown, James M; Caddle, L Brianna; Chiani, Francesco; Clary, Dave; Cleak, James; Daly, Mark J; Denegre, James M; Doe, Brendan; Dolan, Mary E; Edie, Sarah M; Fuchs, Helmut; Gailus-Durner, Valerie; Galli, Antonella; Gambadoro, Alessia; Gallegos, Juan; Guo, Shiying; Horner, Neil R; Hsu, Chih-Wei; Johnson, Sara J; Kalaga, Sowmya; Keith, Lance C; Lanoue, Louise; Lawson, Thomas N; Lek, Monkol; Mark, Manuel; Marschall, Susan; Mason, Jeremy; McElwee, Melissa L; Newbigging, Susan; Nutter, Lauryl M J; Peterson, Kevin A; Ramirez-Solis, Ramiro; Rowland, Douglas J; Ryder, Edward; Samocha, Kaitlin E; Seavitt, John R; Selloum, Mohammed; Szoke-Kovacs, Zsombor; Tamura, Masaru; Trainor, Amanda G; Tudose, Ilinca; Wakana, Shigeharu; Warren, Jonathan; Wendling, Olivia; West, David B; Wong, Leeyean; Yoshiki, Atsushi; MacArthur, Daniel G; Tocchini-Valentini, Glauco P; Gao, Xiang; Flicek, Paul; Bradley, Allan; Skarnes, William C; Justice, Monica J; Parkinson, Helen E; Moore, Mark; Wells, Sara; Braun, Robert E; Svenson, Karen L; de Angelis, Martin Hrabe; Herault, Yann; Mohun, Tim; Mallon, Ann-Marie; Henkelman, R Mark; Brown, Steve D M; Adams, David J; Lloyd, K C Kent; McKerlie, Colin; Beaudet, Arthur L; Bućan, Maja; Murray, Stephen A

2016-09-22

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

High-throughput discovery of novel developmental phenotypes

PubMed Central

Dickinson, Mary E.; Flenniken, Ann M.; Ji, Xiao; Teboul, Lydia; Wong, Michael D.; White, Jacqueline K.; Meehan, Terrence F.; Weninger, Wolfgang J.; Westerberg, Henrik; Adissu, Hibret; Baker, Candice N.; Bower, Lynette; Brown, James M.; Caddle, L. Brianna; Chiani, Francesco; Clary, Dave; Cleak, James; Daly, Mark J.; Denegre, James M.; Doe, Brendan; Dolan, Mary E.; Edie, Sarah M.; Fuchs, Helmut; Gailus-Durner, Valerie; Galli, Antonella; Gambadoro, Alessia; Gallegos, Juan; Guo, Shiying; Horner, Neil R.; Hsu, Chih-wei; Johnson, Sara J.; Kalaga, Sowmya; Keith, Lance C.; Lanoue, Louise; Lawson, Thomas N.; Lek, Monkol; Mark, Manuel; Marschall, Susan; Mason, Jeremy; McElwee, Melissa L.; Newbigging, Susan; Nutter, Lauryl M.J.; Peterson, Kevin A.; Ramirez-Solis, Ramiro; Rowland, Douglas J.; Ryder, Edward; Samocha, Kaitlin E.; Seavitt, John R.; Selloum, Mohammed; Szoke-Kovacs, Zsombor; Tamura, Masaru; Trainor, Amanda G; Tudose, Ilinca; Wakana, Shigeharu; Warren, Jonathan; Wendling, Olivia; West, David B.; Wong, Leeyean; Yoshiki, Atsushi; MacArthur, Daniel G.; Tocchini-Valentini, Glauco P.; Gao, Xiang; Flicek, Paul; Bradley, Allan; Skarnes, William C.; Justice, Monica J.; Parkinson, Helen E.; Moore, Mark; Wells, Sara; Braun, Robert E.; Svenson, Karen L.; de Angelis, Martin Hrabe; Herault, Yann; Mohun, Tim; Mallon, Ann-Marie; Henkelman, R. Mark; Brown, Steve D.M.; Adams, David J.; Lloyd, K.C. Kent; McKerlie, Colin; Beaudet, Arthur L.; Bucan, Maja; Murray, Stephen A.

2016-01-01

Approximately one third of all mammalian genes are essential for life. Phenotypes resulting from mouse knockouts of these genes have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5000 knockout mouse lines, we have identified 410 lethal genes during the production of the first 1751 unique gene knockouts. Using a standardised phenotyping platform that incorporates high-resolution 3D imaging, we identified novel phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes identified in our screen, thus providing a novel dataset that facilitates prioritization and validation of mutations identified in clinical sequencing efforts. PMID:27626380

Macrophage phenotype in the epigallocatechin-3-gallate (EGCG)-modified collagen determines foreign body reaction.

PubMed

Chu, Chenyu; Liu, Li; Wang, Yufei; Wei, Shimin; Wang, Yuanjing; Man, Yi; Qu, Yili

2018-04-28

Collagen has been widely used in guided bone regeneration, and the implantation of collagen membranes will elicit the foreign body reaction (FBR). The imbalance of FBR often leads to failure of dental implants. Therefore, modulation of the FBR after implantation of collagen membranes becomes increasingly important. Macrophages, pivotal in FBR, have been distinguished into pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. Epigallocatechin-3-gallate (EGCG)-modified collagen membranes have been previously shown to regulate secretion of inflammatory factors. In this study, immunohistochemistry of CD31 showed that areas of blood vessels were significantly enlarged after implantation of EGCG-modified collagen membranes compared with those treated with pure collagen membranes. Besides, haematoxylin-eosin staining and immunofluorescence showed an increased number of M2 macrophages after implantation of EGCG-modified collagen membranes. In addition, quantitative real-time polymerase chain reaction showed that after implantation of EGCG-modified collagen membranes, expression of CXCL1 (predominant chemoattractants to neutrophils and inflammation promotors) was significantly downregulated, whereas expressions of STAB1, CCR2, CCR3, CCL2, and CCL3 (related to M2 macrophages) were significantly upregulated. From these findings, we conclude that EGCG-modified collagen membranes were able to regulate the recruitment and polarization of macrophages, so that ameliorate FBR. Copyright © 2018 John Wiley & Sons, Ltd.

37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data.

Code of Federal Regulations, 2014 CFR

2014-07-01

... base or modified or unusual amino acid may be presented in a given sequence as the corresponding unmodified base or amino acid if the modified base or modified or unusual amino acid is one of those listed... the Feature section. Otherwise, each occurrence of a base or amino acid not appearing in WIPO Standard...

Cancer Deep Phenotyping Extraction from Electronic Medical Records | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

As sequencing costs continue to decline, a torrent of cancer genomic data is looming. Very soon, our ability to deeply investigate the cancer genome will outpace our ability to correlate these changes with the phenotypes that they produce. We propose the advanced development and extension of a software platform for performing deep phenotype extraction directly from clinical text of cancer patients, with the goal of enabling translational cancer research and precision medicine.

Distinct subspecies or phenotypic plasticity? Genetic and morphological differentiation of mountain honey bees in East Africa

PubMed Central

Gruber, Karl; Schöning, Caspar; Otte, Marianne; Kinuthia, Wanja; Hasselmann, Martin

2013-01-01

Identifying the forces shaping intraspecific phenotypic and genotypic divergence are of key importance in evolutionary biology. Phenotypic divergence may result from local adaptation or, especially in species with strong gene flow, from pronounced phenotypic plasticity. Here, we examine morphological and genetic divergence among populations of the western honey bee Apis mellifera in the topographically heterogeneous East African region. The currently accepted “mountain refugia hypothesis” states that populations living in disjunct montane forests belong to a different lineage than those in savanna habitats surrounding these forests. We obtained microsatellite data, mitochondrial sequences, and morphometric data from worker honey bees collected from feral colonies in three montane forests and corresponding neighboring savanna regions in Kenya. Honey bee colonies from montane forests showed distinct worker morphology compared with colonies in savanna areas. Mitochondrial sequence data did not support the existence of the two currently accepted subspecies. Furthermore, analyses of the microsatellite data with a Bayesian clustering method did not support the existence of two source populations as it would be expected under the mountain refugia scenario. Our findings suggest that phenotypic plasticity rather than distinct ancestry is the leading cause behind the phenotypic divergence observed between montane forest and savanna honey bees. Our study thus corroborates the idea that high gene flow may select for increased plasticity. PMID:24223262

Phenotypic diversity identified by cardiac magnetic resonance in a large hypertrophic cardiomyopathy family with a single MYH7 mutation.

PubMed

Wang, Jie; Wan, Ke; Sun, Jiayu; Li, Weihao; Liu, Hong; Han, Yuchi; Chen, Yucheng

2018-01-17

Limited data is available on phenotypic variations with the same genotype in hypertrophic cardiomyopathy (HCM). The present study aims to explore the relationship between genotype and phenotype characterized by cardiovascular magnetic resonance (CMR) in a large Chinese family. A proband diagnosed with HCM from a multigenerational family underwent next-generation sequencing based on a custom sureSelect panel, including 117 candidate pathogenic genes associated with cardiomyopathies. All genetic results were confirmed by the Sanger sequencing method. All confirmed mutation carriers underwent CMR exam and myocardial tissue characterization using T1 mapping and late gadolinium enhancement (LGE) on a 3T scanner (Siemens Trio, Gemany). After clinical and genetic screening of 36 (including the proband) members of a large Chinese family, nineteen family members are determined to carry the single p.T1377M (c.4130C>T) mutation in the MYH7 gene. Of these 19 mutation carriers, eight are diagnosed with HCM, one was considered as borderline affected and ten are not clinically or phenotypically affected. Different HCM phenotypes are present in the nine affected individuals in this family. In addition, we have found different tissue characteristics assessed by T1 mapping and LGE in these individuals. We describe a family that demonstrates the diverse HCM phenotypes associated with a single MYH7 mutation.

Genomics of gene banks: A case study in rice.

PubMed

McCouch, Susan R; McNally, Kenneth L; Wang, Wen; Sackville Hamilton, Ruaraidh

2012-02-01

Only a small fraction of the naturally occurring genetic diversity available in the world's germplasm repositories has been explored to date, but this is expected to change with the advent of affordable, high-throughput genotyping and sequencing technology. It is now possible to examine genome-wide patterns of natural variation and link sequence polymorphisms with downstream phenotypic consequences. In this paper, we discuss how dramatic changes in the cost and efficiency of sequencing and genotyping are revolutionizing the way gene bank scientists approach the responsibilities of their job. Sequencing technology provides a set of tools that can be used to enhance the quality, efficiency, and cost-effectiveness of gene bank operations, the depth of scientific knowledge of gene bank holdings, and the level of public interest in natural variation. As a result, gene banks have the chance to take on new life. Previously seen as "warehouses" where seeds were diligently maintained, but evolutionarily frozen in time, gene banks could transform into vibrant research centers that actively investigate the genetic potential of their holdings. In this paper, we will discuss how genotyping and sequencing can be integrated into the activities of a modern gene bank to revolutionize the way scientists document the genetic identity of their accessions; track seed lots, varieties, and alleles; identify duplicates; and rationalize active collections, and how the availability of genomics data are likely to motivate innovative collaborations with the larger research and breeding communities to engage in systematic and rigorous phenotyping and multilocation evaluation of the genetic resources in gene banks around the world. The objective is to understand and eventually predict how variation at the DNA level helps determine the phenotypic potential of an individual or population. Leadership and vision are needed to coordinate the characterization of collections and to integrate genotypic and phenotypic information in ways that will illuminate the value of these resources. Genotyping of collections represents a powerful starting point that will enable gene banks to become more effective as stewards of crop biodiversity.

Whole genome sequencing of Saccharomyces cerevisiae: from genotype to phenotype for improved metabolic engineering applications.

PubMed

Otero, José Manuel; Vongsangnak, Wanwipa; Asadollahi, Mohammad A; Olivares-Hernandes, Roberto; Maury, Jérôme; Farinelli, Laurent; Barlocher, Loïc; Osterås, Magne; Schalk, Michel; Clark, Anthony; Nielsen, Jens

2010-12-22

The need for rapid and efficient microbial cell factory design and construction are possible through the enabling technology, metabolic engineering, which is now being facilitated by systems biology approaches. Metabolic engineering is often complimented by directed evolution, where selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering. In this work we performed whole genome high-throughput sequencing and annotation can be used to identify single nucleotide polymorphisms (SNPs) between Saccharomyces cerevisiae strains S288c and CEN.PK113-7D. The yeast strain S288c was the first eukaryote sequenced, serving as the reference genome for the Saccharomyces Genome Database, while CEN.PK113-7D is a preferred laboratory strain for industrial biotechnology research. A total of 13,787 high-quality SNPs were detected between both strains (reference strain: S288c). Considering only metabolic genes (782 of 5,596 annotated genes), a total of 219 metabolism specific SNPs are distributed across 158 metabolic genes, with 85 of the SNPs being nonsynonymous (e.g., encoding amino acid modifications). Amongst metabolic SNPs detected, there was pathway enrichment in the galactose uptake pathway (GAL1, GAL10) and ergosterol biosynthetic pathway (ERG8, ERG9). Physiological characterization confirmed a strong deficiency in galactose uptake and metabolism in S288c compared to CEN.PK113-7D, and similarly, ergosterol content in CEN.PK113-7D was significantly higher in both glucose and galactose supplemented cultivations compared to S288c. Furthermore, DNA microarray profiling of S288c and CEN.PK113-7D in both glucose and galactose batch cultures did not provide a clear hypothesis for major phenotypes observed, suggesting that genotype to phenotype correlations are manifested post-transcriptionally or post-translationally either through protein concentration and/or function. With an intensifying need for microbial cell factories that produce a wide array of target compounds, whole genome high-throughput sequencing and annotation for SNP detection can aid in better reducing and defining the metabolic landscape. This work demonstrates direct correlations between genotype and phenotype that provides clear and high-probability of success metabolic engineering targets. The genome sequence, annotation, and a SNP viewer of CEN.PK113-7D are deposited at http://www.sysbio.se/cenpk.

Whole genome sequencing of Saccharomyces cerevisiae: from genotype to phenotype for improved metabolic engineering applications

PubMed Central

2010-01-01

Background The need for rapid and efficient microbial cell factory design and construction are possible through the enabling technology, metabolic engineering, which is now being facilitated by systems biology approaches. Metabolic engineering is often complimented by directed evolution, where selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering. Results In this work we performed whole genome high-throughput sequencing and annotation can be used to identify single nucleotide polymorphisms (SNPs) between Saccharomyces cerevisiae strains S288c and CEN.PK113-7D. The yeast strain S288c was the first eukaryote sequenced, serving as the reference genome for the Saccharomyces Genome Database, while CEN.PK113-7D is a preferred laboratory strain for industrial biotechnology research. A total of 13,787 high-quality SNPs were detected between both strains (reference strain: S288c). Considering only metabolic genes (782 of 5,596 annotated genes), a total of 219 metabolism specific SNPs are distributed across 158 metabolic genes, with 85 of the SNPs being nonsynonymous (e.g., encoding amino acid modifications). Amongst metabolic SNPs detected, there was pathway enrichment in the galactose uptake pathway (GAL1, GAL10) and ergosterol biosynthetic pathway (ERG8, ERG9). Physiological characterization confirmed a strong deficiency in galactose uptake and metabolism in S288c compared to CEN.PK113-7D, and similarly, ergosterol content in CEN.PK113-7D was significantly higher in both glucose and galactose supplemented cultivations compared to S288c. Furthermore, DNA microarray profiling of S288c and CEN.PK113-7D in both glucose and galactose batch cultures did not provide a clear hypothesis for major phenotypes observed, suggesting that genotype to phenotype correlations are manifested post-transcriptionally or post-translationally either through protein concentration and/or function. Conclusions With an intensifying need for microbial cell factories that produce a wide array of target compounds, whole genome high-throughput sequencing and annotation for SNP detection can aid in better reducing and defining the metabolic landscape. This work demonstrates direct correlations between genotype and phenotype that provides clear and high-probability of success metabolic engineering targets. The genome sequence, annotation, and a SNP viewer of CEN.PK113-7D are deposited at http://www.sysbio.se/cenpk. PMID:21176163

The Autism Simplex Collection: an international, expertly phenotyped autism sample for genetic and phenotypic analyses.

PubMed

Buxbaum, Joseph D; Bolshakova, Nadia; Brownfeld, Jessica M; Anney, Richard Jl; Bender, Patrick; Bernier, Raphael; Cook, Edwin H; Coon, Hilary; Cuccaro, Michael; Freitag, Christine M; Hallmayer, Joachim; Geschwind, Daniel; Klauck, Sabine M; Nurnberger, John I; Oliveira, Guiomar; Pinto, Dalila; Poustka, Fritz; Scherer, Stephen W; Shih, Andy; Sutcliffe, James S; Szatmari, Peter; Vicente, Astrid M; Vieland, Veronica; Gallagher, Louise

2014-01-01

There is an urgent need for expanding and enhancing autism spectrum disorder (ASD) samples, in order to better understand causes of ASD. In a unique public-private partnership, 13 sites with extensive experience in both the assessment and diagnosis of ASD embarked on an ambitious, 2-year program to collect samples for genetic and phenotypic research and begin analyses on these samples. The program was called The Autism Simplex Collection (TASC). TASC sample collection began in 2008 and was completed in 2010, and included nine sites from North America and four sites from Western Europe, as well as a centralized Data Coordinating Center. Over 1,700 trios are part of this collection, with DNA from transformed cells now available through the National Institute of Mental Health (NIMH). Autism Diagnostic Interview-Revised (ADI-R) and Autism Diagnostic Observation Schedule-Generic (ADOS-G) measures are available for all probands, as are standardized IQ measures, Vineland Adaptive Behavioral Scales (VABS), the Social Responsiveness Scale (SRS), Peabody Picture Vocabulary Test (PPVT), and physical measures (height, weight, and head circumference). At almost every site, additional phenotypic measures were collected, including the Broad Autism Phenotype Questionnaire (BAPQ) and Repetitive Behavior Scale-Revised (RBS-R), as well as the non-word repetition scale, Communication Checklist (Children's or Adult), and Aberrant Behavior Checklist (ABC). Moreover, for nearly 1,000 trios, the Autism Genome Project Consortium (AGP) has carried out Illumina 1 M SNP genotyping and called copy number variation (CNV) in the samples, with data being made available through the National Institutes of Health (NIH). Whole exome sequencing (WES) has been carried out in over 500 probands, together with ancestry matched controls, and this data is also available through the NIH. Additional WES is being carried out by the Autism Sequencing Consortium (ASC), where the focus is on sequencing complete trios. ASC sequencing for the first 1,000 samples (all from whole-blood DNA) is complete and data will be released in 2014. Data is being made available through NIH databases (database of Genotypes and Phenotypes (dbGaP) and National Database for Autism Research (NDAR)) with DNA released in Dist 11.0. Primary funding for the collection, genotyping, sequencing and distribution of TASC samples was provided by Autism Speaks and the NIH, including the National Institute of Mental Health (NIMH) and the National Human Genetics Research Institute (NHGRI). TASC represents an important sample set that leverages expert sites. Similar approaches, leveraging expert sites and ongoing studies, represent an important path towards further enhancing available ASD samples.

Interval mapping for red/green skin color in Asian pears using a modified QTL-seq method

PubMed Central

Xue, Huabai; Shi, Ting; Wang, Fangfang; Zhou, Huangkai; Yang, Jian; Wang, Long; Wang, Suke; Su, Yanli; Zhang, Zhen; Qiao, Yushan; Li, Xiugen

2017-01-01

Pears with red skin are attractive to consumers and provide additional health benefits. Identification of the gene(s) responsible for skin coloration can benefit cultivar selection and breeding. The use of QTL-seq, a bulked segregant analysis method, can be problematic when heterozygous parents are involved. The present study modified the QTL-seq method by introducing a |Δ(SNP-index)| parameter to improve the accuracy of mapping the red skin trait in a group of highly heterozygous Asian pears. The analyses were based on mixed DNA pools composed of 28 red-skinned and 27 green-skinned pear lines derived from a cross between the ‘Mantianhong’ and ‘Hongxiangsu’ red-skinned cultivars. The ‘Dangshansuli’ cultivar genome was used as reference for sequence alignment. An average single-nucleotide polymorphism (SNP) index was calculated using a sliding window approach (200-kb windows, 20-kb increments). Nine scaffolds within the candidate QTL interval were in the fifth linkage group from 111.9 to 177.1 cM. There was a significant linkage between the insertions/deletions and simple sequence repeat markers designed from the candidate intervals and the red/green skin (R/G) locus, which was in a 582.5-kb candidate interval that contained 81 predicted protein-coding gene models and was composed of two subintervals at the bottom of the fifth chromosome. The ZFRI 130-16, In2130-12 and In2130-16 markers located near the R/G locus could potentially be used to identify the red skin trait in Asian pear populations. This study provides new insights into the genetics controlling the red skin phenotype in this fruit. PMID:29118994

Interval mapping for red/green skin color in Asian pears using a modified QTL-seq method.

PubMed

Xue, Huabai; Shi, Ting; Wang, Fangfang; Zhou, Huangkai; Yang, Jian; Wang, Long; Wang, Suke; Su, Yanli; Zhang, Zhen; Qiao, Yushan; Li, Xiugen

2017-01-01

Pears with red skin are attractive to consumers and provide additional health benefits. Identification of the gene(s) responsible for skin coloration can benefit cultivar selection and breeding. The use of QTL-seq, a bulked segregant analysis method, can be problematic when heterozygous parents are involved. The present study modified the QTL-seq method by introducing a |Δ(SNP-index)| parameter to improve the accuracy of mapping the red skin trait in a group of highly heterozygous Asian pears. The analyses were based on mixed DNA pools composed of 28 red-skinned and 27 green-skinned pear lines derived from a cross between the 'Mantianhong' and 'Hongxiangsu' red-skinned cultivars. The 'Dangshansuli' cultivar genome was used as reference for sequence alignment. An average single-nucleotide polymorphism (SNP) index was calculated using a sliding window approach (200-kb windows, 20-kb increments). Nine scaffolds within the candidate QTL interval were in the fifth linkage group from 111.9 to 177.1 cM. There was a significant linkage between the insertions/deletions and simple sequence repeat markers designed from the candidate intervals and the red/green skin (R/G) locus, which was in a 582.5-kb candidate interval that contained 81 predicted protein-coding gene models and was composed of two subintervals at the bottom of the fifth chromosome. The ZFRI 130-16, In2130-12 and In2130-16 markers located near the R/G locus could potentially be used to identify the red skin trait in Asian pear populations. This study provides new insights into the genetics controlling the red skin phenotype in this fruit.

Exome Sequencing in the Clinical Diagnosis of Sporadic or Familial Cerebellar Ataxia

PubMed Central

Fogel, Brent L.; Lee, Hane; Deignan, Joshua L.; Strom, Samuel P.; Kantarci, Sibel; Wang, Xizhe; Quintero-Rivera, Fabiola; Vilain, Eric; Grody, Wayne W.; Perlman, Susan; Geschwind, Daniel H.; Nelson, Stanley F.

2015-01-01

IMPORTANCE Cerebellar ataxias are a diverse collection of neurologic disorders with causes ranging from common acquired etiologies to rare genetic conditions. Numerous genetic disorders have been associated with chronic progressive ataxia and this consequently presents a diagnostic challenge for the clinician regarding how to approach and prioritize genetic testing in patients with such clinically heterogeneous phenotypes. Additionally, while the value of genetic testing in early-onset and/or familial cases seems clear, many patients with ataxia present sporadically with adult onset of symptoms and the contribution of genetic variation to the phenotype of these patients has not yet been established. OBJECTIVE To investigate the contribution of genetic disease in a population of patients with predominantly adult- and sporadic-onset cerebellar ataxia. DESIGN, SETTING, AND PARTICIPANTS We examined a consecutive series of 76 patients presenting to a tertiary referral center for evaluation of chronic progressive cerebellar ataxia. MAIN OUTCOMES AND MEASURES Next-generation exome sequencing coupled with comprehensive bioinformatic analysis, phenotypic analysis, and clinical correlation. RESULTS We identified clinically relevant genetic information in more than 60% of patients studied (n = 46), including diagnostic pathogenic gene variants in 21% (n = 16), a notable yield given the diverse genetics and clinical heterogeneity of the cerebellar ataxias. CONCLUSIONS AND RELEVANCE This study demonstrated that clinical exome sequencing in patients with adult-onset and sporadic presentations of ataxia is a high-yield test, providing a definitive diagnosis in more than one-fifth of patients and suggesting a potential diagnosis in more than one-third to guide additional phenotyping and diagnostic evaluation. Therefore, clinical exome sequencing is an appropriate consideration in the routine genetic evaluation of all patients presenting with chronic progressive cerebellar ataxia. PMID:25133958

Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains.

PubMed

van den Broek, M; Bolat, I; Nijkamp, J F; Ramos, E; Luttik, M A H; Koopman, F; Geertman, J M; de Ridder, D; Pronk, J T; Daran, J-M

2015-09-01

Lager brewing strains of Saccharomyces pastorianus are natural interspecific hybrids originating from the spontaneous hybridization of Saccharomyces cerevisiae and Saccharomyces eubayanus. Over the past 500 years, S. pastorianus has been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics of S. pastorianus genomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed that S. pastorianus strain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants in S. pastorianus CBS1483. Analysis of eight additional S. pastorianus strains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes. Copyright © 2015, van den Broek et al.

Solexa-Sequencing Based Transcriptome Study of Plaice Skin Phenotype in Rex Rabbits (Oryctolagus cuniculus)

PubMed Central

Pan, Lei; Liu, Yan; Wei, Qiang; Xiao, Chenwen; Ji, Quanan; Bao, Guolian; Wu, Xinsheng

2015-01-01

Background Fur is an important genetically-determined characteristic of domestic rabbits; rabbit furs are of great economic value. We used the Solexa sequencing technology to assess gene expression in skin tissues from full-sib Rex rabbits of different phenotypes in order to explore the molecular mechanisms associated with fur determination. Methodology/Principal Findings Transcriptome analysis included de novo assembly, gene function identification, and gene function classification and enrichment. We obtained 74,032,912 and 71,126,891 short reads of 100 nt, which were assembled into 377,618 unique sequences by Trinity strategy (N50=680 nt). Based on BLAST results with known proteins, 50,228 sequences were identified at a cut-off E-value ≥ 10-5. Using Blast to Gene Ontology (GO), Clusters of Orthologous Groups (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG), we obtained several genes with important protein functions. A total of 308 differentially expressed genes were obtained by transcriptome analysis of plaice and un-plaice phenotype animals; 209 additional differentially expressed genes were not found in any database. These genes included 49 that were only expressed in plaice skin rabbits. The novel genes may play important roles during skin growth and development. In addition, 99 known differentially expressed genes were assigned to PI3K-Akt signaling, focal adhesion, and ECM-receptor interactin, among others. Growth factors play a role in skin growth and development by regulating these signaling pathways. We confirmed the altered expression levels of seven target genes by qRT-PCR. And chosen a key gene for SNP to found the differentially between plaice and un-plaice phenotypes rabbit. Conclusions/Significance The rabbit transcriptome profiling data provide new insights in understanding the molecular mechanisms underlying rabbit skin growth and development. PMID:25955442

AGAPE (Automated Genome Analysis PipelinE) for Pan-Genome Analysis of Saccharomyces cerevisiae

PubMed Central

Song, Giltae; Dickins, Benjamin J. A.; Demeter, Janos; Engel, Stacia; Dunn, Barbara; Cherry, J. Michael

2015-01-01

The characterization and public release of genome sequences from thousands of organisms is expanding the scope for genetic variation studies. However, understanding the phenotypic consequences of genetic variation remains a challenge in eukaryotes due to the complexity of the genotype-phenotype map. One approach to this is the intensive study of model systems for which diverse sources of information can be accumulated and integrated. Saccharomyces cerevisiae is an extensively studied model organism, with well-known protein functions and thoroughly curated phenotype data. To develop and expand the available resources linking genomic variation with function in yeast, we aim to model the pan-genome of S. cerevisiae. To initiate the yeast pan-genome, we newly sequenced or re-sequenced the genomes of 25 strains that are commonly used in the yeast research community using advanced sequencing technology at high quality. We also developed a pipeline for automated pan-genome analysis, which integrates the steps of assembly, annotation, and variation calling. To assign strain-specific functional annotations, we identified genes that were not present in the reference genome. We classified these according to their presence or absence across strains and characterized each group of genes with known functional and phenotypic features. The functional roles of novel genes not found in the reference genome and associated with strains or groups of strains appear to be consistent with anticipated adaptations in specific lineages. As more S. cerevisiae strain genomes are released, our analysis can be used to collate genome data and relate it to lineage-specific patterns of genome evolution. Our new tool set will enhance our understanding of genomic and functional evolution in S. cerevisiae, and will be available to the yeast genetics and molecular biology community. PMID:25781462

Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains

PubMed Central

van den Broek, M.; Bolat, I.; Nijkamp, J. F.; Ramos, E.; Luttik, M. A. H.; Koopman, F.; Geertman, J. M.; de Ridder, D.; Pronk, J. T.

2015-01-01

Lager brewing strains of Saccharomyces pastorianus are natural interspecific hybrids originating from the spontaneous hybridization of Saccharomyces cerevisiae and Saccharomyces eubayanus. Over the past 500 years, S. pastorianus has been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics of S. pastorianus genomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed that S. pastorianus strain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants in S. pastorianus CBS1483. Analysis of eight additional S. pastorianus strains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes. PMID:26150454

Is This the Carbapenemase Test We've Been Waiting for? A Multicenter Evaluation of the Modified Carbapenem Inactivation Method.

PubMed

Butler-Wu, Susan M; Abbott, April N

2017-08-01

A plethora of phenotypic methods exist for the detection of carbapenemases; however, clinical laboratories have struggled for years with accurate, objective phenotypic detection of carbapenemase activity in Enterobacteriaceae In this issue of the Journal of Clinical Microbiology , V. M. Pierce et al. (J Clin Microbiol 55:2321-2333, 2017, https://doi.org/10.1128/JCM.00193-17) report on a multicenter evaluation of the modified carbapenem inactivation method (mCIM). The high sensitivity, specificity, reproducibility, and ease of interpretation associated with the mCIM for Enterobacteriaceae will likely lead to its adoption by clinical laboratories. Copyright © 2017 American Society for Microbiology.

A molecular deletion of distal chromosome 4p in two families with a satellited chromosome 4 lacking the Wolf-Hirschhorn syndrome phenotype.

PubMed

Estabrooks, L L; Lamb, A N; Kirkman, H N; Callanan, N P; Rao, K W

1992-11-01

We report two families with a satellited chromosome 4 short arm (4ps). Satellites and stalks normally occur on the short arms of acrocentric chromosomes; however, the literature cites several reports of satellited nonacrocentric chromosomes, which presumably result from a translocation with an acrocentric chromosome. This is the first report of 4ps chromosomes. Our families are remarkable in that both unaffected and affected individuals carry the 4ps chromosome. The phenotypes observed in affected individuals, although dissimilar, were sufficient to encourage a search for a deletion of chromosome 4p. By Southern blot analysis and fluorescence in situ hybridization, a deletion of material mapping approximately 150 kb from chromosome 4pter was discovered. This deletion is notable because it does not result in the Wolf-Hirschhorn syndrome and can result in an apparently normal phenotype. We speculate that homology between subterminal repeat sequences on 4p and sequences on the acrocentric short arms may explain the origin of the rearrangement and that position effect may play a role in the expression of the abnormal phenotype.

The modified iron avidity index: a promising phenotypic predictor in HFE-related haemochromatosis.

PubMed

Verhaegh, Pauline L M; Moris, Wenke; Koek, Ger H; van Deursen, Cees Th B M

2016-10-01

Phenotypes of the HFE-related haemochromatosis vary considerably, making it hard to predict the course of iron accumulation. The aim of this retrospective study was to determine if the Iron Avidity Index (IAI) is a good phenotypic predictor of the number of phlebotomies needed per year during maintenance treatment (NPDMT) in patients with homozygous p.C282Y hereditary haemochromatosis (HH). Patients with HH homozygous for p.C282Y, on maintenance treatment for at least 1 year were included. The IAI (ferritin level at diagnosis/age at diagnosis) was calculated. Ninety-five patients were included in the analysis. Linear regression analysis showed the confounding effect of sex on the relationship between IAI and NPDMT. A modified IAI, adjusted for sex, was calculated. As proton pump inhibitor (PPI) use was independently associated with NPDMT, the group was split in PPI- and non-PPI-users. A positive correlation between the modified IAI and the NPDMT was shown in both groups (PPI r = 0.367, P = 0.023; non-PPI r = 0.453, P < 0.001). An ROC was computed to measure the accuracy of the modified IAI to predict who needed 0-2 vs. ≥3 maintenance treatments per year. The AUROC in the PPI and non-PPI group were respectively 0.576 (0.368-0.784) and 0.752 (0.606-0.899). The modified IAI is a fairly good predictor in non-PPI-using homozygous C282Y HH patients, to differentiate who needs ≥3 maintenance phlebotomies per year. Therefore, this index might help to select patients that benefit from an alternative less frequent therapy, e.g. erythrocytapheresis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evidence for Interspecies Gene Transfer in the Evolution of 2,4-Dichlorophenoxyacetic Acid Degraders

PubMed Central

McGowan, Catherine; Fulthorpe, Roberta; Wright, Alice; Tiedje, J. M.

1998-01-01

Small-subunit ribosomal DNA (SSU rDNA) from 20 phenotypically distinct strains of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was partially sequenced, yielding 18 unique strains belonging to members of the alpha, beta, and gamma subgroups of the class Proteobacteria. To understand the origin of 2,4-D degradation in this diverse collection, the first gene in the 2,4-D pathway, tfdA, was sequenced. The sequences fell into three unique classes found in various members of the beta and gamma subgroups of Proteobacteria. None of the α-Proteobacteria yielded tfdA PCR products. A comparison of the dendrogram of the tfdA genes with that of the SSU rDNA genes demonstrated incongruency in phylogenies, and hence 2,4-D degradation must have originated from gene transfer between species. Only those strains with tfdA sequences highly similar to the tfdA sequence of strain JMP134 (tfdA class I) transferred all the 2,4-D genes and conferred the 2,4-D degradation phenotype to a Burkholderia cepacia recipient. PMID:9758850

Application of Genomic Technologies to the Breeding of Trees

PubMed Central

Badenes, Maria L.; Fernández i Martí, Angel; Ríos, Gabino; Rubio-Cabetas, María J.

2016-01-01

The recent introduction of next generation sequencing (NGS) technologies represents a major revolution in providing new tools for identifying the genes and/or genomic intervals controlling important traits for selection in breeding programs. In perennial fruit trees with long generation times and large sizes of adult plants, the impact of these techniques is even more important. High-throughput DNA sequencing technologies have provided complete annotated sequences in many important tree species. Most of the high-throughput genotyping platforms described are being used for studies of genetic diversity and population structure. Dissection of complex traits became possible through the availability of genome sequences along with phenotypic variation data, which allow to elucidate the causative genetic differences that give rise to observed phenotypic variation. Association mapping facilitates the association between genetic markers and phenotype in unstructured and complex populations, identifying molecular markers for assisted selection and breeding. Also, genomic data provide in silico identification and characterization of genes and gene families related to important traits, enabling new tools for molecular marker assisted selection in tree breeding. Deep sequencing of transcriptomes is also a powerful tool for the analysis of precise expression levels of each gene in a sample. It consists in quantifying short cDNA reads, obtained by NGS technologies, in order to compare the entire transcriptomes between genotypes and environmental conditions. The miRNAs are non-coding short RNAs involved in the regulation of different physiological processes, which can be identified by high-throughput sequencing of RNA libraries obtained by reverse transcription of purified short RNAs, and by in silico comparison with known miRNAs from other species. All together, NGS techniques and their applications have increased the resources for plant breeding in tree species, closing the former gap of genetic tools between trees and annual species. PMID:27895664

Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients.

PubMed

Hoshino, Tomonori; Fujiwara, Taku; Kilian, Mogens

2005-12-01

The aim of this study was to evaluate molecular and phenotypic methods for the identification of nonhemolytic streptococci. A collection of 148 strains consisting of 115 clinical isolates from cases of infective endocarditis, septicemia, and meningitis and 33 reference strains, including type strains of all relevant Streptococcus species, were examined. Identification was performed by phylogenetic analysis of nucleotide sequences of four housekeeping genes, ddl, gdh, rpoB, and sodA; by PCR analysis of the glucosyltransferase (gtf) gene; and by conventional phenotypic characterization and identification using two commercial kits, Rapid ID 32 STREP and STREPTOGRAM and the associated databases. A phylogenetic tree based on concatenated sequences of the four housekeeping genes allowed unequivocal differentiation of recognized species and was used as the reference. Analysis of single gene sequences revealed deviation clustering in eight strains (5.4%) due to homologous recombination with other species. This was particularly evident in S. sanguinis and in members of the anginosus group of streptococci. The rate of correct identification of the strains by both commercial identification kits was below 50% but varied significantly between species. The most significant problems were observed with S. mitis and S. oralis and 11 Streptococcus species described since 1991. Our data indicate that identification based on multilocus sequence analysis is optimal. As a more practical alternative we recommend identification based on sodA sequences with reference to a comprehensive set of sequences that is available for downloading from our server. An analysis of the species distribution of 107 nonhemolytic streptococci from bacteremic patients showed a predominance of S. oralis and S. anginosus with various underlying infections.

Application of Genomic Technologies to the Breeding of Trees.

PubMed

Badenes, Maria L; Fernández I Martí, Angel; Ríos, Gabino; Rubio-Cabetas, María J

2016-01-01

The recent introduction of next generation sequencing (NGS) technologies represents a major revolution in providing new tools for identifying the genes and/or genomic intervals controlling important traits for selection in breeding programs. In perennial fruit trees with long generation times and large sizes of adult plants, the impact of these techniques is even more important. High-throughput DNA sequencing technologies have provided complete annotated sequences in many important tree species. Most of the high-throughput genotyping platforms described are being used for studies of genetic diversity and population structure. Dissection of complex traits became possible through the availability of genome sequences along with phenotypic variation data, which allow to elucidate the causative genetic differences that give rise to observed phenotypic variation. Association mapping facilitates the association between genetic markers and phenotype in unstructured and complex populations, identifying molecular markers for assisted selection and breeding. Also, genomic data provide in silico identification and characterization of genes and gene families related to important traits, enabling new tools for molecular marker assisted selection in tree breeding. Deep sequencing of transcriptomes is also a powerful tool for the analysis of precise expression levels of each gene in a sample. It consists in quantifying short cDNA reads, obtained by NGS technologies, in order to compare the entire transcriptomes between genotypes and environmental conditions. The miRNAs are non-coding short RNAs involved in the regulation of different physiological processes, which can be identified by high-throughput sequencing of RNA libraries obtained by reverse transcription of purified short RNAs, and by in silico comparison with known miRNAs from other species. All together, NGS techniques and their applications have increased the resources for plant breeding in tree species, closing the former gap of genetic tools between trees and annual species.

Use of whole genome sequencing in surveillance of drug resistant tuberculosis.

PubMed

McNerney, Ruth; Zignol, Matteo; Clark, Taane G

2018-05-01

The threat of resistance to anti-tuberculosis drugs is of global concern. Current efforts to monitor resistance rely on phenotypic testing where cultured bacteria are exposed to critical concentrations of the drugs. Capacity for such testing is low in TB endemic countries. Drug resistance is caused by mutations in the Mycobacterium tuberculosis genome and whole genome sequencing to detect these mutations offers an alternative means of assessing resistance. Areas covered: The challenges of assessing TB drug resistance are discussed. Progress in elucidating the M. tuberculosis resistome and evidence of the accuracy of next generation sequencing for detecting resistance is reviewed. Expert Commentary: There are considerable advantages to using next generation sequencing for TB drug resistance surveillance. Accuracy is high for detecting resistance to the major first-line drugs but is currently lower for the second-line drugs due to our incomplete knowledge regarding resistance causing mutations. With the advances in sequencing technology and the opportunity to replace phenotypic drug susceptibility testing with safer and more cost effective methods it would appear that the question is when to implement. Current bottlenecks are sample extraction to allow whole genome sequencing directly from sputum and the lack of bioinformatics expertise in some TB endemic countries.

Influenza virus sequence feature variant type analysis: evidence of a role for NS1 in influenza virus host range restriction.

PubMed

Noronha, Jyothi M; Liu, Mengya; Squires, R Burke; Pickett, Brett E; Hale, Benjamin G; Air, Gillian M; Galloway, Summer E; Takimoto, Toru; Schmolke, Mirco; Hunt, Victoria; Klem, Edward; García-Sastre, Adolfo; McGee, Monnie; Scheuermann, Richard H

2012-05-01

Genetic drift of influenza virus genomic sequences occurs through the combined effects of sequence alterations introduced by a low-fidelity polymerase and the varying selective pressures experienced as the virus migrates through different host environments. While traditional phylogenetic analysis is useful in tracking the evolutionary heritage of these viruses, the specific genetic determinants that dictate important phenotypic characteristics are often difficult to discern within the complex genetic background arising through evolution. Here we describe a novel influenza virus sequence feature variant type (Flu-SFVT) approach, made available through the public Influenza Research Database resource (www.fludb.org), in which variant types (VTs) identified in defined influenza virus protein sequence features (SFs) are used for genotype-phenotype association studies. Since SFs have been defined for all influenza virus proteins based on known structural, functional, and immune epitope recognition properties, the Flu-SFVT approach allows the rapid identification of the molecular genetic determinants of important influenza virus characteristics and their connection to underlying biological functions. We demonstrate the use of the SFVT approach to obtain statistical evidence for effects of NS1 protein sequence variations in dictating influenza virus host range restriction.

Natural gene expression variation studies in yeast.

PubMed

Thompson, Dawn A; Cubillos, Francisco A

2017-01-01

The rise of sequence information across different yeast species and strains is driving an increasing number of studies in the emerging field of genomics to associate polymorphic variants, mRNA abundance and phenotypic differences between individuals. Here, we gathered evidence from recent studies covering several layers that define the genotype-phenotype gap, such as mRNA abundance, allele-specific expression and translation efficiency to demonstrate how genetic variants co-evolve and define an individual's genome. Moreover, we exposed several antecedents where inter- and intra-specific studies led to opposite conclusions, probably owing to genetic divergence. Future studies in this area will benefit from the access to a massive array of well-annotated genomes and new sequencing technologies, which will allow the fine breakdown of the complex layers that delineate the genotype-phenotype map. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

SLC52A2 mutations cause SCABD2 phenotype: A second report.

PubMed

Babanejad, Mojgan; Adeli, Omid Ali; Nikzat, Nooshin; Beheshtian, Maryam; Azarafra, Hakimeh; Sadeghnia, Farnaz; Mohseni, Marzieh; Najmabadi, Hossein; Kahrizi, Kimia

2018-01-01

Autosomal recessive cerebellar ataxias (ARCAs) are a large group of neurodegenerative disorders that manifest mainly in children and young adults. Most ARCAs are heterogeneous with respect to age at onset, severity of disease progression, and frequency of extracerebellar and systemic signs. The phenotype of a consanguineous Iranian family was characterized using clinical testing and pedigree analysis. Whole-exome sequencing was used to identify the disease-causing gene in this family. Using whole exome sequencing (WES), a novel missense mutation in SLC52A2 gene is reported in a consanguineous Iranian family with progressive severe hearing loss, optic atrophy and ataxia. This is the second report of the genotype-phenotype correlation between this syndrome named spinocerebellar ataxia with blindness and deafness type 2 (SCABD2) and SLC52A2 gene. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae.

PubMed Central

Colicelli, J; Nicolette, C; Birchmeier, C; Rodgers, L; Riggs, M; Wigler, M

1991-01-01

Saccharomyces cerevisiae strains expressing the activated RAS2Val19 gene or lacking both cAMP phosphodiesterase genes, PDE1 and PDE2, have impaired growth control and display an acute sensitivity to heat shock. We have isolated two classes of mammalian cDNAs from yeast expression libraries that suppress the heat shock-sensitive phenotype of RAS2Val19 strain. Members of the first class of cDNAs also suppress the heat shock-sensitive phenotype of pde1- pde2- strains and encode cAMP phosphodiesterases. Members of the second class fail to suppress the phenotype of pde1- pde2- strains and therefore are candidate cDNAs encoding proteins that interact with RAS proteins. We report the nucleotide sequence of three members of this class. Two of these cDNAs share considerable sequence similarity, but none are clearly similar to previously isolated genes. Images PMID:1849280

hnRNPA2B1 and hnRNPA1 mutations are rare in patients with "multisystem proteinopathy" and frontotemporal lobar degeneration phenotypes.

PubMed

Le Ber, Isabelle; Van Bortel, Inge; Nicolas, Gael; Bouya-Ahmed, Kawtar; Camuzat, Agnès; Wallon, David; De Septenville, Anne; Latouche, Morwena; Lattante, Serena; Kabashi, Edor; Jornea, Ludmila; Hannequin, Didier; Brice, Alexis

2014-04-01

hnRNPA2B1 and hnRNPA1 mutations have been recently identified by exome sequencing in three families presenting with multisystem proteinopathy (MSP), a rare complex phenotype associating frontotemporal lobar degeneration (FTLD), Paget disease of bone (PDB), inclusion body myopathy (IBM), and amyotrophic lateral sclerosis (ALS). No study has evaluated the exact frequency of these genes in cohorts of MSP or FTD patients so far. We sequenced both genes in 17 patients with MSP phenotypes, and in 60 patients with FTLD and FTLD-ALS to test whether mutations could be implicated in the pathogenesis of these disorders. No disease-causing mutation was identified. We conclude that hnRNPA2B1 and hnRNPA1 mutations are rare in MSP and FTLD spectrum of diseases, although further investigations in larger populations are needed. Copyright © 2014 Elsevier Inc. All rights reserved.

Pseudoexon activation increases phenotype severity in a Becker muscular dystrophy patient.

PubMed

Greer, Kane; Mizzi, Kayla; Rice, Emily; Kuster, Lukas; Barrero, Roberto A; Bellgard, Matthew I; Lynch, Bryan J; Foley, Aileen Reghan; O Rathallaigh, Eoin; Wilton, Steve D; Fletcher, Sue

2015-07-01

We report a dystrophinopathy patient with an in-frame deletion of DMD exons 45-47, and therefore a genetic diagnosis of Becker muscular dystrophy, who presented with a more severe than expected phenotype. Analysis of the patient DMD mRNA revealed an 82 bp pseudoexon, derived from intron 44, that disrupts the reading frame and is expected to yield a nonfunctional dystrophin. Since the sequence of the pseudoexon and canonical splice sites does not differ from the reference sequence, we concluded that the genomic rearrangement promoted recognition of the pseudoexon, causing a severe dystrophic phenotype. We characterized the deletion breakpoints and identified motifs that might influence selection of the pseudoexon. We concluded that the donor splice site was strengthened by juxtaposition of intron 47, and loss of intron 44 silencer elements, normally located downstream of the pseudoexon donor splice site, further enhanced pseudoexon selection and inclusion in the DMD transcript in this patient.

Exome sequencing and SNP analysis detect novel compound heterozygosity in fatty acid hydroxylase-associated neurodegeneration

PubMed Central

Pierson, Tyler Mark; Simeonov, Dimitre R; Sincan, Murat; Adams, David A; Markello, Thomas; Golas, Gretchen; Fuentes-Fajardo, Karin; Hansen, Nancy F; Cherukuri, Praveen F; Cruz, Pedro; Blackstone, Craig; Tifft, Cynthia; Boerkoel, Cornelius F; Gahl, William A

2012-01-01

Fatty acid hydroxylase-associated neurodegeneration due to fatty acid 2-hydroxylase deficiency presents with a wide range of phenotypes including spastic paraplegia, leukodystrophy, and/or brain iron deposition. All previously described families with this disorder were consanguineous, with homozygous mutations in the probands. We describe a 10-year-old male, from a non-consanguineous family, with progressive spastic paraplegia, dystonia, ataxia, and cognitive decline associated with a sural axonal neuropathy. The use of high-throughput sequencing techniques combined with SNP array analyses revealed a novel paternally derived missense mutation and an overlapping novel maternally derived ∼28-kb genomic deletion in FA2H. This patient provides further insight into the consistent features of this disorder and expands our understanding of its phenotypic presentation. The presence of a sural nerve axonal neuropathy had not been previously associated with this disorder and so may extend the phenotype. PMID:22146942

Exome sequence analysis suggests genetic burden contributes to phenotypic variability and complex neuropathy

PubMed Central

Gonzaga-Jauregui, Claudia; Harel, Tamar; Gambin, Tomasz; Kousi, Maria; Griffin, Laurie B.; Francescatto, Ludmila; Ozes, Burcak; Karaca, Ender; Jhangiani, Shalini; Bainbridge, Matthew N.; Lawson, Kim S.; Pehlivan, Davut; Okamoto, Yuji; Withers, Marjorie; Mancias, Pedro; Slavotinek, Anne; Reitnauer, Pamela J; Goksungur, Meryem T.; Shy, Michael; Crawford, Thomas O.; Koenig, Michel; Willer, Jason; Flores, Brittany N.; Pediaditrakis, Igor; Us, Onder; Wiszniewski, Wojciech; Parman, Yesim; Antonellis, Anthony; Muzny, Donna M.; Katsanis, Nicholas; Battaloglu, Esra; Boerwinkle, Eric; Gibbs, Richard A.; Lupski, James R.

2015-01-01

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous distal symmetric polyneuropathy. Whole-exome sequencing (WES) of 40 individuals from 37 unrelated families with CMT-like peripheral neuropathy refractory to molecular diagnosis identified apparent causal mutations in ~45% (17/37) of families. Three candidate disease genes are proposed, supported by a combination of genetic and in vivo studies. Aggregate analysis of mutation data revealed a significantly increased number of rare variants across 58 neuropathy associated genes in subjects versus controls; confirmed in a second ethnically discrete neuropathy cohort, suggesting mutation burden potentially contributes to phenotypic variability. Neuropathy genes shown to have highly penetrant Mendelizing variants (HMPVs) and implicated by burden in families were shown to interact genetically in a zebrafish assay exacerbating the phenotype established by the suppression of single genes. Our findings suggest that the combinatorial effect of rare variants contributes to disease burden and variable expressivity. PMID:26257172

Whole-genome resequencing reveals signatures of selection and timing of duck domestication.

PubMed

Zhang, Zebin; Jia, Yaxiong; Almeida, Pedro; Mank, Judith E; van Tuinen, Marcel; Wang, Qiong; Jiang, Zhihua; Chen, Yu; Zhan, Kai; Hou, Shuisheng; Zhou, Zhengkui; Li, Huifang; Yang, Fangxi; He, Yong; Ning, Zhonghua; Yang, Ning; Qu, Lujiang

2018-04-01

The genetic basis of animal domestication remains poorly understood, and systems with substantial phenotypic differences between wild and domestic populations are useful for elucidating the genetic basis of adaptation to new environments as well as the genetic basis of rapid phenotypic change. Here, we sequenced the whole genome of 78 individual ducks, from two wild and seven domesticated populations, with an average sequencing depth of 6.42X per individual. Our population and demographic analyses indicate a complex history of domestication, with early selection for separate meat and egg lineages. Genomic comparison of wild to domesticated populations suggests that genes that affect brain and neuronal development have undergone strong positive selection during domestication. Our FST analysis also indicates that the duck white plumage is the result of selection at the melanogenesis-associated transcription factor locus. Our results advance the understanding of animal domestication and selection for complex phenotypic traits.

Genetic Rearrangements Can Modify Chromatin Features at Epialleles

PubMed Central

Foerster, Andrea M.; Dinh, Huy Q.; Sedman, Laura; Wohlrab, Bonnie; Mittelsten Scheid, Ortrun

2011-01-01

Analogous to genetically distinct alleles, epialleles represent heritable states of different gene expression from sequence-identical genes. Alleles and epialleles both contribute to phenotypic heterogeneity. While alleles originate from mutation and recombination, the source of epialleles is less well understood. We analyze active and inactive epialleles that were found at a transgenic insert with a selectable marker gene in Arabidopsis. Both converse expression states are stably transmitted to progeny. The silent epiallele was previously shown to change its state upon loss-of-function of trans-acting regulators and drug treatments. We analyzed the composition of the epialleles, their chromatin features, their nuclear localization, transcripts, and homologous small RNA. After mutagenesis by T-DNA transformation of plants carrying the silent epiallele, we found new active alleles. These switches were associated with different, larger or smaller, and non-overlapping deletions or rearrangements in the 3′ regions of the epiallele. These cis-mutations caused different degrees of gene expression stability depending on the nature of the sequence alteration, the consequences for transcription and transcripts, and the resulting chromatin organization upstream. This illustrates a tight dependence of epigenetic regulation on local structures and indicates that sequence alterations can cause epigenetic changes at some distance in regions not directly affected by the mutation. Similar effects may also be involved in gene expression and chromatin changes in the vicinity of transposon insertions or excisions, recombination events, or DNA repair processes and could contribute to the origin of new epialleles. PMID:22028669

Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in Flavonoid Biosynthetic Pathway

PubMed Central

Sun, Wei; Meng, Xiangyu; Liang, Lingjie; Jiang, Wangshu; Huang, Yafei; He, Jing; Hu, Haiyan; Almqvist, Jonas; Gao, Xiang; Wang, Li

2015-01-01

Chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1) encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants. PMID:25742495

Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in flavonoid biosynthetic pathway.

PubMed

Sun, Wei; Meng, Xiangyu; Liang, Lingjie; Jiang, Wangshu; Huang, Yafei; He, Jing; Hu, Haiyan; Almqvist, Jonas; Gao, Xiang; Wang, Li

2015-01-01

Chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1) encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants.

Evolutionary diversification of type 2 porcine reproductive and respiratory syndrome virus.

PubMed

Brar, Manreetpal Singh; Shi, Mang; Murtaugh, Michael P; Leung, Frederick Chi-Ching

2015-07-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the leading swine pathogens causing tremendous economic loss to the global swine industry due to its virulence, pathogenesis, infectivity and transmissibility. Although formally recognized only two and half decades ago, molecular dating estimation indicates a more ancient evolutionary history, which involved divergence into two genotypes (type 1 and type 2) prior to the 'initial' outbreaks of the late 1980s. Type 2 PRRSV circulates primarily in North America and Asia. The relatively greater availability of sequence data for this genotype from widespread geographical territories has enabled a better understanding of the evolving genotype. However, there are a number of challenges in terms of the vastness of data available and what this indicates in the context of viral diversity. Accordingly, here we revisit the mechanisms by which PRRSV generates variability, describe a means of organizing type 2 diversity captured in voluminous ORF5 sequences in a phylogenetic framework and provide a holistic view of known global type 2 diversity in the same setting. The consequences of the expanding diversity for control measures such as vaccination are discussed, as well as the contribution of modified live vaccines to the circulation of field isolates. We end by highlighting some limitations of current molecular epidemiology studies in relation to inferring PRRSV diversity, and what steps can be taken to overcome these and additionally enable PRRSV sequence data to be informative about viral phenotypic traits such as virulence.

In vivo myosin step-size from zebrafish skeletal muscle

PubMed Central

Ajtai, Katalin; Sun, Xiaojing; Takubo, Naoko; Wang, Yihua

2016-01-01

Muscle myosins transduce ATP free energy into actin displacement to power contraction. In vivo, myosin side chains are modified post-translationally under native conditions, potentially impacting function. Single myosin detection provides the ‘bottom-up’ myosin characterization probing basic mechanisms without ambiguities inherent to ensemble observation. Macroscopic muscle physiological experimentation provides the definitive ‘top-down’ phenotype characterizations that are the concerns in translational medicine. In vivo single myosin detection in muscle from zebrafish embryo models for human muscle fulfils ambitions for both bottom-up and top-down experimentation. A photoactivatable green fluorescent protein (GFP)-tagged myosin light chain expressed in transgenic zebrafish skeletal muscle specifically modifies the myosin lever-arm. Strychnine induces the simultaneous contraction of the bilateral tail muscles in a live embryo, causing them to be isometric while active. Highly inclined thin illumination excites the GFP tag of single lever-arms and its super-resolution orientation is measured from an active isometric muscle over a time sequence covering many transduction cycles. Consecutive frame lever-arm angular displacement converts to step-size by its product with the estimated lever-arm length. About 17% of the active myosin steps that fall between 2 and 7 nm are implicated as powerstrokes because they are beyond displacements detected from either relaxed or ATP-depleted (rigor) muscle. PMID:27249818

Leber's hereditary optic neuropathy is associated with mitochondrial ND6 T14502C mutation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Fuxin; Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang 325003; Guan, Minqiang

2009-11-20

We report here the clinical, genetic, and molecular characterization of three Chinese families with Leber's hereditary optic neuropathy (LHON). There were variable severity and age of onset in visual impairment among these families. Strikingly, there were extremely low penetrances of visual impairment in these Chinese families. Sequence analysis of complete mitochondrial genomes in these pedigrees showed the homoplasmic T14502C (I58V) mutation, which localized at a highly conserved isoleucine at position 58 of ND6, and distinct sets of mtDNA polymorphisms belonging to haplogroups M10a, F1a1, and H2. The occurrence of T14502C mutation in these several genetically unrelated subjects affected by visualmore » impairment strongly indicates that this mutation is involved in the pathogenesis of visual impairment. Here, mtDNA variants I187T in the ND1, A122V in CO1, S99A in the A6, and V254I in CO3 exhibited an evolutionary conservation, indicating a potential modifying role in the development of visual impairment associated with T14502C mutation in those families. Furthermore, nuclear modifier gene(s) or environmental factor(s) may play a role in the phenotypic manifestation of the LHON-associated T14502C mutation in these Chinese families.« less

Harnessing the Versatility of Bacterial Collagen to Improve the Chondrogenic Potential of Porous Collagen Scaffolds

PubMed Central

Parmar, Paresh A.; St-Pierre, Jean-Philippe; Chow, Lesley W.; Puetzer, Jennifer L.; Stoichevska, Violet; Peng, Yong Y.; Werkmeister, Jerome A.; Ramshaw, John A. M.; Stevens, Molly M.

2017-01-01

Collagen I foams are used in the clinic as scaffolds to promote articular cartilage repair as they provide a bioactive environment for cells with chondrogenic potential. However, collagen I as a base material does not allow for precise control over bioactivity. Alternatively, recombinant bacterial collagens can be used as “blank slate” collagen molecules to offer a versatile platform for incorporation of selected bioactive sequences and fabrication into 3D scaffolds. Here, we show the potential of Streptococcal collagen-like 2 (Scl2) protein foams modified with peptides designed to specifically and noncovalently bind hyaluronic acid and chondroitin sulfate to improve chondrogenesis of human mesenchymal stem cells (hMSCs) compared to collagen I foams. Specific compositions of functionalized Scl2 foams lead to improved chondrogenesis compared to both nonfunctionalized Scl2 and collagen I foams, as indicated by gene expression, extracellular matrix accumulation, and compression moduli. hMSCs cultured in functionalized Scl2 foams exhibit decreased collagens I and X gene and protein expression, suggesting an advantage over collagen I foams in promoting a chondrocytic phenotype. These highly modular foams can be further modified to improve specific aspects chondrogenesis. As such, these scaffolds also have the potential to be tailored for other regenerative medicine applications. PMID:27219220

Food Allergy - Basic Mechanisms and Applications to Identifying Risks Associated with Plant Incorporated Pesticides and Other Genetically Modified Crops

EPA Science Inventory

Food allergy is a relatively new concern for toxicologists as a result of the incorporation of novel proteins into food crops in order to promote resistance to pests and other stresses, improve nutrition, or otherwise modify the phenotype. Food allergy can manifest as inflammatio...

Characterizing partial AZFc deletions of the Y chromosome with amplicon-specific sequence markers

PubMed Central

Navarro-Costa, Paulo; Pereira, Luísa; Alves, Cíntia; Gusmão, Leonor; Proença, Carmen; Marques-Vidal, Pedro; Rocha, Tiago; Correia, Sónia C; Jorge, Sónia; Neves, António; Soares, Ana P; Nunes, Joaquim; Calhaz-Jorge, Carlos; Amorim, António; Plancha, Carlos E; Gonçalves, João

2007-01-01

Background The AZFc region of the human Y chromosome is a highly recombinogenic locus containing multi-copy male fertility genes located in repeated DNA blocks (amplicons). These AZFc gene families exhibit slight sequence variations between copies which are considered to have functional relevance. Yet, partial AZFc deletions yield phenotypes ranging from normospermia to azoospermia, thwarting definite conclusions on their real impact on fertility. Results The amplicon content of partial AZFc deletion products was characterized with novel amplicon-specific sequence markers. Data indicate that partial AZFc deletions are a male infertility risk [odds ratio: 5.6 (95% CI: 1.6–30.1)] and although high diversity of partial deletion products and sequence conversion profiles were recorded, the AZFc marker profiles detected in fertile men were also observed in infertile men. Additionally, the assessment of rearrangement recurrence by Y-lineage analysis indicated that while partial AZFc deletions occurred in highly diverse samples, haplotype diversity was minimal in fertile men sharing identical marker profiles. Conclusion Although partial AZFc deletion products are highly heterogeneous in terms of amplicon content, this plasticity is not sufficient to account for the observed phenotypical variance. The lack of causative association between the deletion of specific gene copies and infertility suggests that AZFc gene content might be part of a multifactorial network, with Y-lineage evolution emerging as a possible phenotype modulator. PMID:17903263

Neural/Bayes network predictor for inheritable cardiac disease pathogenicity and phenotype.

PubMed

Burghardt, Thomas P; Ajtai, Katalin

2018-04-11

The cardiac muscle sarcomere contains multiple proteins contributing to contraction energy transduction and its regulation during a heartbeat. Inheritable heart disease mutants affect most of them but none more frequently than the ventricular myosin motor and cardiac myosin binding protein c (mybpc3). These co-localizing proteins have mybpc3 playing a regulatory role to the energy transducing motor. Residue substitution and functional domain assignment of each mutation in the protein sequence decides, under the direction of a sensible disease model, phenotype and pathogenicity. The unknown model mechanism is decided here using a method combing neural and Bayes networks. Missense single nucleotide polymorphisms (SNPs) are clues for the disease mechanism summarized in an extensive database collecting mutant sequence location and residue substitution as independent variables that imply the dependent disease phenotype and pathogenicity characteristics in 4 dimensional data points (4ddps). The SNP database contains entries with the majority having one or both dependent data entries unfulfilled. A neural network relating causes (mutant residue location and substitution) and effects (phenotype and pathogenicity) is trained, validated, and optimized using fulfilled 4ddps. It then predicts unfulfilled 4ddps providing the implicit disease model. A discrete Bayes network interprets fulfilled and predicted 4ddps with conditional probabilities for phenotype and pathogenicity given mutation location and residue substitution thus relating the neural network implicit model to explicit features of the motor and mybpc3 sequence and structural domains. Neural/Bayes network forecasting automates disease mechanism modeling by leveraging the world wide human missense SNP database that is in place and expanding. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Mutants of the Paf1 Complex Alter Phenotypic Expression of the Yeast Prion [PSI+

PubMed Central

Strawn, Lisa A.; Lin, Changyi A.; Tank, Elizabeth M.H.; Osman, Morwan M.; Simpson, Sarah A.

2009-01-01

The yeast [PSI+] prion is an epigenetic modifier of translation termination fidelity that causes nonsense suppression. The prion [PSI+] forms when the translation termination factor Sup35p adopts a self-propagating conformation. The presence of the [PSI+] prion modulates survivability in a variety of growth conditions. Nonsense suppression is essential for many [PSI+]-mediated phenotypes, but many do not appear to be due to read-through of a single stop codon, but instead are multigenic traits. We hypothesized that other global mechanisms act in concert with [PSI+] to influence [PSI+]-mediated phenotypes. We have identified one such global regulator, the Paf1 complex (Paf1C). Paf1C is conserved in eukaryotes and has been implicated in several aspects of transcriptional and posttranscriptional regulation. Mutations in Ctr9p and other Paf1C components reduced [PSI+]-mediated nonsense suppression. The CTR9 deletion also alters nonsense suppression afforded by other genetic mutations but not always to the same extent as the effects on [PSI+]-mediated read-through. Our data suggest that the Paf1 complex influences mRNA translatability but not solely through changes in transcript stability or abundance. Finally, we demonstrate that the CTR9 deletion alters several [PSI+]-dependent phenotypes. This provides one example of how [PSI+] and genetic modifiers can interact to uncover and regulate phenotypic variability. PMID:19225160

A GENOME MAY REDUCE YOUR CARBON FOOTPRINT

USDA-ARS?s Scientific Manuscript database

Cheap sequence information will place new emphasis on comparative approaches to mining the data for sequence alterations that lead to physiological contrasts between groups. The data will need to be accessible and coupled with phenotypic data to be made useful for breeders. Increased emphasis on b...

In Silico Analysis of Gene Expression Network Components Underlying Pigmentation Phenotypes in the Python Identified Evolutionarily Conserved Clusters of Transcription Factor Binding Sites

PubMed Central

2016-01-01

Color variation provides the opportunity to investigate the genetic basis of evolution and selection. Reptiles are less studied than mammals. Comparative genomics approaches allow for knowledge gained in one species to be leveraged for use in another species. We describe a comparative vertebrate analysis of conserved regulatory modules in pythons aimed at assessing bioinformatics evidence that transcription factors important in mammalian pigmentation phenotypes may also be important in python pigmentation phenotypes. We identified 23 python orthologs of mammalian genes associated with variation in coat color phenotypes for which we assessed the extent of pairwise protein sequence identity between pythons and mouse, dog, horse, cow, chicken, anole lizard, and garter snake. We next identified a set of melanocyte/pigment associated transcription factors (CREB, FOXD3, LEF-1, MITF, POU3F2, and USF-1) that exhibit relatively conserved sequence similarity within their DNA binding regions across species based on orthologous alignments across multiple species. Finally, we identified 27 evolutionarily conserved clusters of transcription factor binding sites within ~200-nucleotide intervals of the 1500-nucleotide upstream regions of AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR from Python bivittatus. Our results provide insight into pigment phenotypes in pythons. PMID:27698666

In Silico Analysis of Gene Expression Network Components Underlying Pigmentation Phenotypes in the Python Identified Evolutionarily Conserved Clusters of Transcription Factor Binding Sites.

PubMed

Irizarry, Kristopher J L; Bryden, Randall L

2016-01-01

Color variation provides the opportunity to investigate the genetic basis of evolution and selection. Reptiles are less studied than mammals. Comparative genomics approaches allow for knowledge gained in one species to be leveraged for use in another species. We describe a comparative vertebrate analysis of conserved regulatory modules in pythons aimed at assessing bioinformatics evidence that transcription factors important in mammalian pigmentation phenotypes may also be important in python pigmentation phenotypes. We identified 23 python orthologs of mammalian genes associated with variation in coat color phenotypes for which we assessed the extent of pairwise protein sequence identity between pythons and mouse, dog, horse, cow, chicken, anole lizard, and garter snake. We next identified a set of melanocyte/pigment associated transcription factors (CREB, FOXD3, LEF-1, MITF, POU3F2, and USF-1) that exhibit relatively conserved sequence similarity within their DNA binding regions across species based on orthologous alignments across multiple species. Finally, we identified 27 evolutionarily conserved clusters of transcription factor binding sites within ~200-nucleotide intervals of the 1500-nucleotide upstream regions of AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR from Python bivittatus . Our results provide insight into pigment phenotypes in pythons.

Empirical analysis of RNA robustness and evolution using high-throughput sequencing of ribozyme reactions.

PubMed

Hayden, Eric J

2016-08-15

RNA molecules provide a realistic but tractable model of a genotype to phenotype relationship. This relationship has been extensively investigated computationally using secondary structure prediction algorithms. Enzymatic RNA molecules, or ribozymes, offer access to genotypic and phenotypic information in the laboratory. Advancements in high-throughput sequencing technologies have enabled the analysis of sequences in the lab that now rivals what can be accomplished computationally. This has motivated a resurgence of in vitro selection experiments and opened new doors for the analysis of the distribution of RNA functions in genotype space. A body of computational experiments has investigated the persistence of specific RNA structures despite changes in the primary sequence, and how this mutational robustness can promote adaptations. This article summarizes recent approaches that were designed to investigate the role of mutational robustness during the evolution of RNA molecules in the laboratory, and presents theoretical motivations, experimental methods and approaches to data analysis. Copyright © 2016 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, Shea N.; McLoughlin, Kevin; Be, Nicholas A.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broadmore » panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Lastly, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.« less

Genetic architecture of retinal and macular degenerative diseases: the promise and challenges of next-generation sequencing

PubMed Central

2013-01-01

Inherited retinal degenerative diseases (RDDs) display wide variation in their mode of inheritance, underlying genetic defects, age of onset, and phenotypic severity. Molecular mechanisms have not been delineated for many retinal diseases, and treatment options are limited. In most instances, genotype-phenotype correlations have not been elucidated because of extensive clinical and genetic heterogeneity. Next-generation sequencing (NGS) methods, including exome, genome, transcriptome and epigenome sequencing, provide novel avenues towards achieving comprehensive understanding of the genetic architecture of RDDs. Whole-exome sequencing (WES) has already revealed several new RDD genes, whereas RNA-Seq and ChIP-Seq analyses are expected to uncover novel aspects of gene regulation and biological networks that are involved in retinal development, aging and disease. In this review, we focus on the genetic characterization of retinal and macular degeneration using NGS technology and discuss the basic framework for further investigations. We also examine the challenges of NGS application in clinical diagnosis and management. PMID:24112618

High prevalence of DUOX2 mutations in Japanese patients with permanent congenital hypothyroidism or transient hypothyroidism.

PubMed

Matsuo, Kumihiro; Tanahashi, Yusuke; Mukai, Tokuo; Suzuki, Shigeru; Tajima, Toshihiro; Azuma, Hiroshi; Fujieda, Kenji

2016-07-01

Dual oxidase 2 (DUOX2) mutations are a cause of dyshormonogenesis (DH) and have been identified in patients with permanent congenital hypothyroidism (PH) and with transient hypothyroidism (TH). We aimed to elucidate the prevalence and phenotypical variations of DUOX2 mutations. Forty-eight Japanese DH patients were enroled and analysed for sequence variants of DUOX2, DUOXA2, and TPO using polymerase chain reaction-amplified direct sequencing. Fourteen sequence variants of DUOX2, including 10 novel variants, were identified in 11 patients. DUOX2 variants were more prevalent (11/48, 22.9%) than TPO (3/48, 6.3%) (p=0.020). The prevalence of DUOX2 variants in TH was slightly, but not significantly, higher than in PH. Furthermore, one patient had digenic heterozygous sequence variants of both DUOX2 and TPO. Our results suggest that DUOX2 mutations might be the most common cause of both PH and TH, and that phenotypes of these mutations might be milder than those of other causes.

A Visual Reporter System for Virus-Induced Gene Silencing in Tomato Fruit Based on Anthocyanin Accumulation1[C][W

PubMed Central

Orzaez, Diego; Medina, Aurora; Torre, Sara; Fernández-Moreno, Josefina Patricia; Rambla, José Luis; Fernández-del-Carmen, Asun; Butelli, Eugenio; Martin, Cathie; Granell, Antonio

2009-01-01

Virus-induced gene silencing (VIGS) is a powerful tool for reverse genetics in tomato (Solanum lycopersicum). However, the irregular distribution of the effects of VIGS hampers the identification and quantification of nonvisual phenotypes. To overcome this limitation, a visually traceable VIGS system was developed for fruit, comprising two elements: (1) a transgenic tomato line (Del/Ros1) expressing Antirrhinum majus Delila and Rosea1 transcription factors under the control of the fruit-specific E8 promoter, showing a purple-fruited, anthocyanin-rich phenotype; and (2) a modified tobacco rattle virus VIGS vector incorporating partial Rosea1 and Delila sequences, which was shown to restore the red-fruited phenotype upon agroinjection in Del/Ros1 plants. Dissection of silenced areas for subsequent chemometric analysis successfully identified the relevant metabolites underlying gene function for three tomato genes, phytoene desaturase, TomloxC, and SlODO1, used for proof of concept. The C-6 aldehydes derived from lipid 13-hydroperoxidation were found to be the volatile compounds most severely affected by TomloxC silencing, whereas geranial and 6-methyl-5-hepten-2-one were identified as the volatiles most severely reduced by phytoene desaturase silencing in ripening fruit. In a third example, silencing of SlODO1, a tomato homolog of the ODORANT1 gene encoding a myb transcription factor, which regulates benzenoid metabolism in petunia (Petunia hybrida) flowers, resulted in a sharp accumulation of benzaldehyde in tomato fruit. Together, these results indicate that fruit VIGS, enhanced by anthocyanin monitoring, can be a powerful tool for reverse genetics in the study of the metabolic networks operating during fruit ripening. PMID:19429602

Phenotypic diversity in autosomal-dominant cone-rod dystrophy elucidated by adaptive optics retinal imaging

PubMed Central

Song, Hongxin; Rossi, Ethan A; Stone, Edwin; Latchney, Lisa; Williams, David; Dubra, Alfredo; Chung, Mina

2018-01-01

Purpose Several genes causing autosomal-dominant cone-rod dystrophy (AD-CRD) have been identified. However, the mechanisms by which genetic mutations lead to cellular loss in human disease remain poorly understood. Here we combine genotyping with high-resolution adaptive optics retinal imaging to elucidate the retinal phenotype at a cellular level in patients with AD-CRD harbouring a defect in the GUCA1A gene. Methods Nine affected members of a four-generation AD-CRD pedigree and three unaffected first-degree relatives underwent clinical examinations including visual acuity, fundus examination, Goldmann perimetry, spectral domain optical coherence tomography and electroretinography. Genome-wide scan followed by bidirectional sequencing was performed on all affected participants. High-resolution imaging using a custom adaptive optics scanning light ophthalmoscope (AOSLO) was performed for selected participants. Results Clinical evaluations showed a range of disease severity from normal fundus appearance in teenaged patients to pronounced macular atrophy in older patients. Molecular genetic testing showed a mutation in in GUCA1A segregating with disease. AOSLO imaging revealed that of the two teenage patients with mild disease, one had severe disruption of the photoreceptor mosaic while the other had a normal cone mosaic. Conclusions AOSLO imaging demonstrated variability in the pattern of cone and rod cell loss between two teenage cousins with early AD-CRD, who had similar clinical features and had the identical disease-causing mutation in GUCA1A. This finding suggests that a mutation in GUCA1A does not lead to the same degree of AD-CRD in all patients. Modifying factors may mitigate or augment disease severity, leading to different retinal cellular phenotypes. PMID:29074494

MicroRNA-31 is a positive modulator of endothelial-mesenchymal transition and associated secretory phenotype induced by TGF-β.

PubMed

Katsura, Akihiro; Suzuki, Hiroshi I; Ueno, Toshihide; Mihira, Hajime; Yamazaki, Tomoko; Yasuda, Takahiko; Watabe, Tetsuro; Mano, Hiroyuki; Yamada, Yoshitsugu; Miyazono, Kohei

2016-01-01

Transforming growth factor-β (TGF-β) plays central roles in endothelial-mesenchymal transition (EndMT) involved in development and pathogenesis. Although EndMT and epithelial-mesenchymal transition are similar processes, roles of microRNAs in EndMT are largely unknown. Here, we report that constitutively active microRNA-31 (miR-31) is a positive regulator of TGF-β-induced EndMT. Although the expression is not induced by TGF-β, miR-31 is required for induction of mesenchymal genes including α-SMA, actin reorganization and MRTF-A activation during EndMT. We identified VAV3, a regulator of actin remodeling and MRTF-A activity, as a miR-31 target. Global transcriptome analysis further showed that miR-31 positively regulates EndMT-associated unique secretory phenotype (EndMT-SP) characterized by induction of multiple inflammatory chemokines and cytokines including CCL17, CX3CL1, CXCL16, IL-6 and Angptl2. As a mechanism for this phenomenon, TGF-β and miR-31 suppress Stk40, a negative regulator of NF-κB pathway. Interestingly, TGF-β induces alternative polyadenylation (APA)-coupled miR-31-dependent Stk40 suppression without concomitant miR-31 induction, and APA-mediated exclusion of internal poly(A) sequence in Stk40 3'UTR enhances target efficiency of Stk40. Finally, miR-31 functions as a molecular hub to integrate TGF-β and TNF-α signaling to enhance EndMT. These data confirm that constitutively active microRNAs, as well as inducible microRNAs, serve as phenotypic modifiers interconnected with transcriptome dynamics during EndMT. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Recent Advances in the Molecular Genetics of Familial Hypertrophic Cardiomyopathy in South Asian Descendants

PubMed Central

Kraker, Jessica; Viswanathan, Shiv Kumar; Knöll, Ralph; Sadayappan, Sakthivel

2016-01-01

The South Asian population, numbered at 1.8 billion, is estimated to comprise around 20% of the global population and 1% of the American population, and has one of the highest rates of cardiovascular disease. While South Asians show increased classical risk factors for developing heart failure, the role of population-specific genetic risk factors has not yet been examined for this group. Hypertrophic cardiomyopathy (HCM) is one of the major cardiac genetic disorders among South Asians, leading to contractile dysfunction, heart failure, and sudden cardiac death. This disease displays autosomal dominant inheritance, and it is associated with a large number of variants in both sarcomeric and non-sarcomeric proteins. The South Asians, a population with large ethnic diversity, potentially carries region-specific polymorphisms. There is high variability in disease penetrance and phenotypic expression of variants associated with HCM. Thus, extensive studies are required to decipher pathogenicity and the physiological mechanisms of these variants, as well as the contribution of modifier genes and environmental factors to disease phenotypes. Conducting genotype-phenotype correlation studies will lead to improved understanding of HCM and, consequently, improved treatment options for this high-risk population. The objective of this review is to report the history of cardiovascular disease and HCM in South Asians, present previously published pathogenic variants, and introduce current efforts to study HCM using induced pluripotent stem cell-derived cardiomyocytes, next-generation sequencing, and gene editing technologies. The authors ultimately hope that this review will stimulate further research, drive novel discoveries, and contribute to the development of personalized medicine with the aim of expanding therapeutic strategies for HCM. PMID:27840609

Phenotypic diversity in autosomal-dominant cone-rod dystrophy elucidated by adaptive optics retinal imaging.

PubMed

Song, Hongxin; Rossi, Ethan A; Stone, Edwin; Latchney, Lisa; Williams, David; Dubra, Alfredo; Chung, Mina

2018-01-01

Several genes causing autosomal-dominant cone-rod dystrophy (AD-CRD) have been identified. However, the mechanisms by which genetic mutations lead to cellular loss in human disease remain poorly understood. Here we combine genotyping with high-resolution adaptive optics retinal imaging to elucidate the retinal phenotype at a cellular level in patients with AD-CRD harbouring a defect in the GUCA1A gene. Nine affected members of a four-generation AD-CRD pedigree and three unaffected first-degree relatives underwent clinical examinations including visual acuity, fundus examination, Goldmann perimetry, spectral domain optical coherence tomography and electroretinography. Genome-wide scan followed by bidirectional sequencing was performed on all affected participants. High-resolution imaging using a custom adaptive optics scanning light ophthalmoscope (AOSLO) was performed for selected participants. Clinical evaluations showed a range of disease severity from normal fundus appearance in teenaged patients to pronounced macular atrophy in older patients. Molecular genetic testing showed a mutation in in GUCA1A segregating with disease. AOSLO imaging revealed that of the two teenage patients with mild disease, one had severe disruption of the photoreceptor mosaic while the other had a normal cone mosaic. AOSLO imaging demonstrated variability in the pattern of cone and rod cell loss between two teenage cousins with early AD-CRD, who had similar clinical features and had the identical disease-causing mutation in GUCA1A . This finding suggests that a mutation in GUCA1A does not lead to the same degree of AD-CRD in all patients. Modifying factors may mitigate or augment disease severity, leading to different retinal cellular phenotypes. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Application of a modified selection index for honey bees (Hymenoptera: Apidae).

PubMed

van Engelsdorp, D; Otis, G W

2000-12-01

Nine different genetic families of honey bees (Apis mellifera L.) were compared using summed z-scores (phenotypic values) and a modified selection index (Imod). Imod values incorporated both the phenotypic scores of the different traits and the economic weightings of these traits, as determined by a survey of commercial Ontario beekeepers. Largely because of the high weight all beekeepers place on honey production, a distinct difference between line rankings based on phenotypic scores and Imod scores was apparent, thereby emphasizing the need to properly weight the traits being evaluated to select bee stocks most valuable for beekeepers. Furthermore, when beekeepers who made >10% of their income from queen and nucleus colony sales assigned relative values to the traits used in the Imod calculations, the results differed from those based on weightings assigned by honey producers. Our results underscore the difficulties the North American beekeeping industry must overcome to devise effective methods of evaluating colonies for breeding purposes.

Targeted next-generation sequencing reveals novel USH2A mutations associated with diverse disease phenotypes: implications for clinical and molecular diagnosis.

PubMed

Chen, Xue; Sheng, Xunlun; Liu, Xiaoxing; Li, Huiping; Liu, Yani; Rong, Weining; Ha, Shaoping; Liu, Wenzhou; Kang, Xiaoli; Zhao, Kanxing; Zhao, Chen

2014-01-01

USH2A mutations have been implicated in the disease etiology of several inherited diseases, including Usher syndrome type 2 (USH2), nonsyndromic retinitis pigmentosa (RP), and nonsyndromic deafness. The complex genetic and phenotypic spectrums relevant to USH2A defects make it difficult to manage patients with such mutations. In the present study, we aim to determine the genetic etiology and to characterize the correlated clinical phenotypes for three Chinese pedigrees with nonsyndromic RP, one with RP sine pigmento (RPSP), and one with USH2. Family histories and clinical details for all included patients were reviewed. Ophthalmic examinations included best corrected visual acuities, visual field measurements, funduscopy, and electroretinography. Targeted next-generation sequencing (NGS) was applied using two sequence capture arrays to reveal the disease causative mutations for each family. Genotype-phenotype correlations were also annotated. Seven USH2A mutations, including four missense substitutions (p.P2762A, p.G3320C, p.R3719H, and p.G4763R), two splice site variants (c.8223+1G>A and c.8559-2T>C), and a nonsense mutation (p.Y3745*), were identified as disease causative in the five investigated families, of which three reported to have consanguineous marriage. Among all seven mutations, six were novel, and one was recurrent. Two homozygous missense mutations (p.P2762A and p.G3320C) were found in one individual family suggesting a potential double hit effect. Significant phenotypic divergences were revealed among the five families. Three families of the five families were affected with early, moderated, or late onset RP, one with RPSP, and the other one with USH2. Our study expands the genotypic and phenotypic variability relevant to USH2A mutations, which would help with a clear insight into the complex genetic and phenotypic spectrums relevant to USH2A defects, and is complementary for a better management of patients with such mutations. We have also demonstrated that a targeted NGS approach is a valuable tool for the genetic diagnosis of USH2 and RP.

Targeted Next-Generation Sequencing Reveals Novel USH2A Mutations Associated with Diverse Disease Phenotypes: Implications for Clinical and Molecular Diagnosis

PubMed Central

Li, Huiping; Liu, Yani; Rong, Weining; Ha, Shaoping; Liu, Wenzhou; Kang, Xiaoli; Zhao, Kanxing; Zhao, Chen

2014-01-01

USH2A mutations have been implicated in the disease etiology of several inherited diseases, including Usher syndrome type 2 (USH2), nonsyndromic retinitis pigmentosa (RP), and nonsyndromic deafness. The complex genetic and phenotypic spectrums relevant to USH2A defects make it difficult to manage patients with such mutations. In the present study, we aim to determine the genetic etiology and to characterize the correlated clinical phenotypes for three Chinese pedigrees with nonsyndromic RP, one with RP sine pigmento (RPSP), and one with USH2. Family histories and clinical details for all included patients were reviewed. Ophthalmic examinations included best corrected visual acuities, visual field measurements, funduscopy, and electroretinography. Targeted next-generation sequencing (NGS) was applied using two sequence capture arrays to reveal the disease causative mutations for each family. Genotype-phenotype correlations were also annotated. Seven USH2A mutations, including four missense substitutions (p.P2762A, p.G3320C, p.R3719H, and p.G4763R), two splice site variants (c.8223+1G>A and c.8559-2T>C), and a nonsense mutation (p.Y3745*), were identified as disease causative in the five investigated families, of which three reported to have consanguineous marriage. Among all seven mutations, six were novel, and one was recurrent. Two homozygous missense mutations (p.P2762A and p.G3320C) were found in one individual family suggesting a potential double hit effect. Significant phenotypic divergences were revealed among the five families. Three families of the five families were affected with early, moderated, or late onset RP, one with RPSP, and the other one with USH2. Our study expands the genotypic and phenotypic variability relevant to USH2A mutations, which would help with a clear insight into the complex genetic and phenotypic spectrums relevant to USH2A defects, and is complementary for a better management of patients with such mutations. We have also demonstrated that a targeted NGS approach is a valuable tool for the genetic diagnosis of USH2 and RP. PMID:25133613

De Novo and Inherited Loss-of-Function Variants in TLK2: Clinical and Genotype-Phenotype Evaluation of a Distinct Neurodevelopmental Disorder.

PubMed

Reijnders, Margot R F; Miller, Kerry A; Alvi, Mohsan; Goos, Jacqueline A C; Lees, Melissa M; de Burca, Anna; Henderson, Alex; Kraus, Alison; Mikat, Barbara; de Vries, Bert B A; Isidor, Bertrand; Kerr, Bronwyn; Marcelis, Carlo; Schluth-Bolard, Caroline; Deshpande, Charu; Ruivenkamp, Claudia A L; Wieczorek, Dagmar; Baralle, Diana; Blair, Edward M; Engels, Hartmut; Lüdecke, Hermann-Josef; Eason, Jacqueline; Santen, Gijs W E; Clayton-Smith, Jill; Chandler, Kate; Tatton-Brown, Katrina; Payne, Katelyn; Helbig, Katherine; Radtke, Kelly; Nugent, Kimberly M; Cremer, Kirsten; Strom, Tim M; Bird, Lynne M; Sinnema, Margje; Bitner-Glindzicz, Maria; van Dooren, Marieke F; Alders, Marielle; Koopmans, Marije; Brick, Lauren; Kozenko, Mariya; Harline, Megan L; Klaassens, Merel; Steinraths, Michelle; Cooper, Nicola S; Edery, Patrick; Yap, Patrick; Terhal, Paulien A; van der Spek, Peter J; Lakeman, Phillis; Taylor, Rachel L; Littlejohn, Rebecca O; Pfundt, Rolph; Mercimek-Andrews, Saadet; Stegmann, Alexander P A; Kant, Sarina G; McLean, Scott; Joss, Shelagh; Swagemakers, Sigrid M A; Douzgou, Sofia; Wall, Steven A; Küry, Sébastien; Calpena, Eduardo; Koelling, Nils; McGowan, Simon J; Twigg, Stephen R F; Mathijssen, Irene M J; Nellaker, Christoffer; Brunner, Han G; Wilkie, Andrew O M

2018-06-07

Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

A probabilistic model to predict clinical phenotypic traits from genome sequencing.

PubMed

Chen, Yun-Ching; Douville, Christopher; Wang, Cheng; Niknafs, Noushin; Yeo, Grace; Beleva-Guthrie, Violeta; Carter, Hannah; Stenson, Peter D; Cooper, David N; Li, Biao; Mooney, Sean; Karchin, Rachel

2014-09-01

Genetic screening is becoming possible on an unprecedented scale. However, its utility remains controversial. Although most variant genotypes cannot be easily interpreted, many individuals nevertheless attempt to interpret their genetic information. Initiatives such as the Personal Genome Project (PGP) and Illumina's Understand Your Genome are sequencing thousands of adults, collecting phenotypic information and developing computational pipelines to identify the most important variant genotypes harbored by each individual. These pipelines consider database and allele frequency annotations and bioinformatics classifications. We propose that the next step will be to integrate these different sources of information to estimate the probability that a given individual has specific phenotypes of clinical interest. To this end, we have designed a Bayesian probabilistic model to predict the probability of dichotomous phenotypes. When applied to a cohort from PGP, predictions of Gilbert syndrome, Graves' disease, non-Hodgkin lymphoma, and various blood groups were accurate, as individuals manifesting the phenotype in question exhibited the highest, or among the highest, predicted probabilities. Thirty-eight PGP phenotypes (26%) were predicted with area-under-the-ROC curve (AUC)>0.7, and 23 (15.8%) of these were statistically significant, based on permutation tests. Moreover, in a Critical Assessment of Genome Interpretation (CAGI) blinded prediction experiment, the models were used to match 77 PGP genomes to phenotypic profiles, generating the most accurate prediction of 16 submissions, according to an independent assessor. Although the models are currently insufficiently accurate for diagnostic utility, we expect their performance to improve with growth of publicly available genomics data and model refinement by domain experts.

Delineation of C12orf65-related phenotypes: a genotype-phenotype relationship.

PubMed

Spiegel, Ronen; Mandel, Hanna; Saada, Ann; Lerer, Issy; Burger, Ayala; Shaag, Avraham; Shalev, Stavit A; Jabaly-Habib, Haneen; Goldsher, Dorit; Gomori, John M; Lossos, Alex; Elpeleg, Orly; Meiner, Vardiella

2014-08-01

C12orf65 participates in the process of mitochondrial translation and has been shown to be associated with a spectrum of phenotypes, including early onset optic atrophy, progressive encephalomyopathy, peripheral neuropathy, and spastic paraparesis.We used whole-genome homozygosity mapping as well as exome sequencing and targeted gene sequencing to identify novel C12orf65 disease-causing mutations in seven affected individuals originating from two consanguineous families. In four family members affected with childhood-onset optic atrophy accompanied by slowly progressive peripheral neuropathy and spastic paraparesis, we identified a homozygous frame shift mutation c.413_417 delAACAA, which predicts a truncated protein lacking the C-terminal portion. In the second family, we studied three affected individuals who presented with early onset optic atrophy, peripheral neuropathy, and spastic gait in addition to moderate intellectual disability. Muscle biopsy in two of the patients revealed decreased activities of the mitochondrial respiratory chain complexes I and IV. In these patients, we identified a homozygous splice mutation, g.21043 T>A (c.282+2 T>A) which leads to skipping of exon 2. Our study broadens the phenotypic spectrum of C12orf65 defects and highlights the triad of optic atrophy, axonal neuropathy and spastic paraparesis as its key clinical features. In addition, a clear genotype-phenotype correlation is anticipated in which deleterious mutations which disrupt the GGQ-containing domain in the first coding exon are expected to result in a more severe phenotype, whereas down-stream C-terminal mutations may result in a more favorable phenotype, typically lacking cognitive impairment.

Phenotypic heterogeneity and genetic modification of P102L inherited prion disease in an international series

PubMed Central

Webb, T. E. F.; Poulter, M.; Beck, J.; Uphill, J.; Adamson, G.; Campbell, T.; Linehan, J.; Powell, C.; Brandner, S.; Pal, S.; Siddique, D.; Wadsworth, J. D.; Joiner, S.; Alner, K.; Petersen, C.; Hampson, S.; Rhymes, C.; Treacy, C.; Storey, E.; Geschwind, M. D.; Nemeth, A. H.; Wroe, S.; Mead, S.

2008-01-01

The largest kindred with inherited prion disease P102L, historically Gerstmann-Sträussler-Scheinker syndrome, originates from central England, with émigrés now resident in various parts of the English-speaking world. We have collected data from 84 patients in the large UK kindred and numerous small unrelated pedigrees to investigate phenotypic heterogeneity and modifying factors. This collection represents by far the largest series of P102L patients so far reported. Microsatellite and genealogical analyses of eight separate European kindreds support multiple distinct mutational events at a cytosine-phosphate diester-guanidine dinucleotide mutation hot spot. All of the smaller P102L kindreds were linked to polymorphic human prion protein gene codon 129M and were not connected by genealogy or microsatellite haplotype background to the large kindred or each other. While many present with classical Gerstmann-Sträussler-Scheinker syndrome, a slowly progressive cerebellar ataxia with later onset cognitive impairment, there is remarkable heterogeneity. A subset of patients present with prominent cognitive and psychiatric features and some have met diagnostic criteria for sporadic Creutzfeldt-Jakob disease. We show that polymorphic human prion protein gene codon 129 modifies age at onset: the earliest eight clinical onsets were all MM homozygotes and overall age at onset was 7 years earlier for MM compared with MV heterozygotes (P = 0.02). Unexpectedly, apolipoprotein E4 carriers have a delayed age of onset by 10 years (P = 0.02). We found a preponderance of female patients compared with males (54 females versus 30 males, P = 0.01), which probably relates to ascertainment bias. However, these modifiers had no impact on a semi-quantitative pathological phenotype in 10 autopsied patients. These data allow an appreciation of the range of clinical phenotype, modern imaging and molecular investigation and should inform genetic counselling of at-risk individuals, with the identification of two genetic modifiers. PMID:18757886

CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus.

PubMed

Bhattacharya, Dipankan; Marfo, Chris A; Li, Davis; Lane, Maura; Khokha, Mustafa K

2015-12-15

Congenital malformations are the major cause of infant mortality in the US and Europe. Due to rapid advances in human genomics, we can now efficiently identify sequence variants that may cause disease in these patients. However, establishing disease causality remains a challenge. Additionally, in the case of congenital heart disease, many of the identified candidate genes are either novel to embryonic development or have no known function. Therefore, there is a pressing need to develop inexpensive and efficient technologies to screen these candidate genes for disease phenocopy in model systems and to perform functional studies to uncover their role in development. For this purpose, we sought to test F0 CRISPR based gene editing as a loss of function strategy for disease phenocopy in the frog model organism, Xenopus tropicalis. We demonstrate that the CRISPR/Cas9 system can efficiently modify both alleles in the F0 generation within a few hours post fertilization, recapitulating even early disease phenotypes that are highly similar to knockdowns from morpholino oligos (MOs) in nearly all cases tested. We find that injecting Cas9 protein is dramatically more efficacious and less toxic than cas9 mRNA. We conclude that CRISPR based F0 gene modification in X. tropicalis is efficient and cost effective and readily recapitulates disease and MO phenotypes. Copyright © 2015 Elsevier Inc. All rights reserved.

A novel variant in the SLC12A1 gene in two families with antenatal Bartter syndrome.

PubMed

Breinbjerg, Anders; Siggaard Rittig, Charlotte; Gregersen, Niels; Rittig, Søren; Hvarregaard Christensen, Jane

2017-01-01

Bartter syndrome is an autosomal-recessive inherited disease in which patients present with hypokalaemia and metabolic alkalosis. We present two apparently nonrelated cases with antenatal Bartter syndrome type I, due to a novel variant in the SLC12A1 gene encoding the bumetanide-sensitive sodium-(potassium)-chloride cotransporter 2 in the thick ascending limb of the loop of Henle. Blood samples were received from the two cases and 19 of their relatives, and deoxyribonucleic acid was extracted. The coding regions of the SLC12A1 gene were amplified using polymerase chain reaction, followed by bidirectional direct deoxyribonucleic acid sequencing. Each affected child in the two families was homozygous for a novel inherited variant in the SLC12A1gene, c.1614T>A. The variant predicts a change from a tyrosine codon to a stop codon (p.Tyr538Ter). The two cases presented antenatally and at six months of age, respectively. The two cases were homozygous for the same variant in the SLC12A1 gene, but presented clinically at different ages. This could eventually be explained by the presence of other gene variants or environmental factors modifying the phenotypes. The phenotypes of the patients were similar to other patients with antenatal Bartter syndrome. ©2016 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

Mutations in HAMP and HJV genes and their impact on expression of clinical hemochromatosis in a cohort of 100 Spanish patients homozygous for the C282Y mutation of HFE gene.

PubMed

Altès, Albert; Bach, Vanessa; Ruiz, Angels; Esteve, Anna; Felez, Jordi; Remacha, Angel F; Sardà, M Pilar; Baiget, Montserrat

2009-10-01

Most hereditary hemochromatosis (HH) patients are homozygous for the C282Y mutation of the HFE gene. Nevertheless, penetrance of the disease is very variable. In some patients, penetrance can be mediated by concomitant mutations in other iron master genes. We evaluated the clinical impact of hepcidin (HAMP) and hemojuvelin mutations in a cohort of 100 Spanish patients homozygous for the C282Y mutation of the HFE gene. HAMP and hemojuvelin mutations were evaluated in all patients by bidirectional direct cycle sequencing. Phenotype-genotype interactions were evaluated. A heterozygous mutation of the HAMP gene (G71D) was found in only one out of 100 cases. Following, we performed a study of several members of that family, and we observed several members had a digenic inheritance of the C282Y mutation of the HFE gene and the G71D mutation of the HAMP gene. This mutation in the HAMP gene did not modify the phenotype of the individuals who were homozygous for the C282Y mutation. One other patient presented a new polymorphism in the hemojuvelin gene, without consequences in iron load or clinical course of the disease. In conclusion, HAMP and hemojuvelin mutations are rare among Spanish HH patients, and their impact in this population is not significant.

Implication of LRRC4C and DPP6 in neurodevelopmental disorders

PubMed Central

Maussion, Gilles; Cruceanu, Cristiana; Rosenfeld, Jill A.; Bell, Scott C.; Jollant, Fabrice; Szatkiewicz, Jin; Collins, Ryan L.; Hanscom, Carrie; Kolobova, Ilaria; de Champfleur, Nicolas Menjot; Blumenthal, Ian; Chiang, Colby; Ota, Vanessa; Hultman, Christina; O’Dushlaine, Colm; McCarroll, Steve; Alda, Martin; Jacquemont, Sebastien; Ordulu, Zehra; Marshall, Christian R.; Carter, Melissa T.; Shaffer, Lisa G.; Sklar, Pamela; Girirajan, Santhosh; Morton, Cynthia C.; Gusella, James F.; Turecki, Gustavo; Stavropoulos, D. J.; Sullivan, Patrick F.; Scherer, Stephen W.; Talkowski, Michael E.; Ernst, Carl

2018-01-01

We performed whole-genome sequencing on an individual from a family with variable psychiatric phenotypes that had a sensory processing disorder, apraxia, and autism. The proband harbored a maternally inherited balanced translocation (46,XY,t(11;14)(p12;p12)mat) that disrupted LRRC4C, a member of the highly specialized netrin G family of axon guidance molecules. The proband also inherited a paternally derived chromosomal inversion that disrupted DPP6, a potassium channel interacting protein. Copy Number (CN) analysis in 14,077 cases with neurodevelopmental disorders and 8,960 control subjects revealed that 60% of cases with exonic deletions in LRRC4C had a second clinically recognizable syndrome associated with variable clinical phenotypes, including 16p11.2, 1q44, and 2q33.1 CN syndromes, suggesting LRRC4C deletion variants may be modifiers of neurodevelopmental disorders. In vitro, functional assessments modeling patient deletions in LRRC4C suggest a negative regulatory role of these exons found in the untranslated region of LRRC4C, which has a single, terminal coding exon. These data suggest that the proband’s autism may be due to the inheritance of disruptions in both DPP6 and LRRC4C, and may highlight the importance of the netrin G family and potassium channel interacting molecules in neurodevelopmental disorders. PMID:27759917

Recurrent Deletions and Reciprocal Duplications of 10q11.21q11.23 Including CHAT and SLC18A3 are Likely Mediated by Complex Low-Copy Repeats

PubMed Central

Stankiewicz, Paweł; Kulkarni, Shashikant; Dharmadhikari, Avinash V.; Sampath, Srirangan; Bhatt, Samarth S.; Shaikh, Tamim H.; Xia, Zhilian; Pursley, Amber N.; Cooper, M. Lance; Shinawi, Marwan; Paciorkowski, Alex R.; Grange, Dorothy K.; Noetzel, Michael J.; Saunders, Scott; Simons, Paul; Summar, Marshall; Lee, Brendan; Scaglia, Fernando; Fellmann, Florence; Martinet, Danielle; Beckmann, Jacques S.; Asamoah, Alexander; Platky, Kathryn; Sparks, Susan; Martin, Ann S.; Madan-Khetarpal, Suneeta; Hoover, Jacqueline; Medne, Livija; Bonnemann, Carsten G.; Moeschler, John B.; Vallee, Stephanie E.; Parikh, Sumit; Irwin, Polly; Dalzell, Victoria P.; Smith, Wendy E.; Banks, Valerie C.; Flannery, David B.; Lovell, Carolyn M.; Bellus, Gary A.; Golden-Grant, Kathryn; Gorski, Jerome L.; Kussmann, Jennifer L.; McGregor, Tracy L.; Hamid, Rizwan; Pfotenhauer, Jean; Ballif, Blake C.; Shaw, Chad A.; Kang, Sung-Hae L.; Bacino, Carlos A.; Patel, Ankita; Rosenfeld, Jill A.; Cheung, Sau Wai; Shaffer, Lisa G.

2013-01-01

We report 24 unrelated individuals with deletions and 17 additional cases with duplications at 10q11.21q21.1 identified by chromosomal microarray analysis. The rearrangements range in size from 0.3 to 12 Mb. Nineteen of the deletions and eight duplications are flanked by large, directly oriented segmental duplications of >98% sequence identity, suggesting that nonallelic homologous recombination (NAHR) caused these genomic rearrangements. Nine individuals with deletions and five with duplications have additional copy number changes. Detailed clinical evaluation of 20 patients with deletions revealed variable clinical features, with developmental delay (DD) and/or intellectual disability (ID) as the only features common to a majority of individuals. We suggest that some of the other features present in more than one patient with deletion, including hypotonia, sleep apnea, chronic constipation, gastroesophageal and vesicoureteral refluxes, epilepsy, ataxia, dysphagia, nystagmus, and ptosis may result from deletion of the CHAT gene, encoding choline acetyltransferase, and the SLC18A3 gene, mapping in the first intron of CHAT and encoding vesicular acetylcholine transporter. The phenotypic diversity and presence of the deletion in apparently normal carrier parents suggest that subjects carrying 10q11.21q11.23 deletions may exhibit variable phenotypic expressivity and incomplete penetrance influenced by additional genetic and nongenetic modifiers. PMID:21948486

Design and Management of Field Trials of Transgenic Cereals

NASA Astrophysics Data System (ADS)

Bedő, Zoltán; Rakszegi, Mariann; Láng, László

The development of gene transformation systems has allowed the introgression of alien genes into plant genomes, thus providing a mechanism for broadening the genetic resources available to plant breeders. The design and the management of field trials vary according to the purpose for which transgenic cereals are developed. Breeders study the phenotypic and genotypic stability of transgenic plants, monitor the increase in homozygosity of transgenic genotypes under field conditions, and develop backcross generations to transfer the introduced genes into secondary transgenic cereal genotypes. For practical purposes, they may also multiply seed of the transgenic lines to produce sufficient amounts of grain for the detailed analysis of trait(s) of interest, to determine the field performance of transgenic lines, and to compare them with the non-transformed parental genotypes. Prior to variety registration, the Distinctness, Uniformity and Stability (DUS) tests and Value for Cultivation and Use (VCU) experiments are carried out in field trials. Field testing includes specific requirements for transgenic cereals to assess potential environmental risks. The capacity of the pollen to survive, establish and disseminate in the field test environment, the potential for gene transfer, the effects of products expressed by the introduced sequences and phenotypic and genotypic instability that might cause deleterious effects must all be specifically monitored, as required by EU Directives 2003/701/EC (1) on the release of genetically modified higher plants in the environment.

Extensive molecular analysis suggested the strong genetic heterogeneity of idiopathic chronic pancreatitis.

PubMed

Sofia, Valentina Maria; Da Sacco, Letizia; Surace, Cecilia; Tomaiuolo, Anna Cristina; Genovese, Silvia; Grotta, Simona; Gnazzo, Maria; Petrocchi, Stefano; Ciocca, Laura; Alghisi, Federico; Montemitro, Enza; Martemucci, Luigi; Elce, Ausilia; Lucidi, Vincenzina; Castaldo, Giuseppe; Angioni, Adriano

2016-05-26

Genetic features of Chronic Pancreatitis (CP) have been extensively investigated mainly testing genes associated to the trypsinogen activation pathway. However, different molecular pathways involving other genes may be implicated in CP pathogenesis. 80 patients with Idiopathic CP were investigated using Next Generation Sequencing approach with a panel of 70 genes related to six different pancreatic pathways: premature activation of trypsinogen; modifier genes of Cystic Fibrosis phenotype; pancreatic secretion and ion homeostasis; Calcium signalling and zymogen granules exocytosis; autophagy; autoimmune pancreatitis related genes. We detected mutations in 34 out of 70 genes examined; 64/80 patients (80.0%) were positive for mutations in one or more genes, 16/80 patients (20.0%) had no mutations. Mutations in CFTR were detected in 32/80 patients (40.0%) and 22 of them exhibited at least one mutation in genes of other pancreatic pathways. Of the remaining 48 patients, 13/80 (16.3%) had mutations in genes involved in premature activation of trypsinogen and 19/80 (23.8%) had mutations only in genes of the other pathways: 38/64 patients positive for mutations showed variants in two or more genes (59.3%). Our data, although to be extended with functional analysis of novel mutations, suggest a high rate of genetic heterogeneity in chronic pancreatitis and that trans-heterozygosity may predispose to the idiopathic CP phenotype.

Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells.

PubMed

Wu, Yuxuan; Zhou, Hai; Fan, Xiaoying; Zhang, Ying; Zhang, Man; Wang, Yinghua; Xie, Zhenfei; Bai, Meizhu; Yin, Qi; Liang, Dan; Tang, Wei; Liao, Jiaoyang; Zhou, Chikai; Liu, Wujuan; Zhu, Ping; Guo, Hongshan; Pan, Hong; Wu, Chunlian; Shi, Huijuan; Wu, Ligang; Tang, Fuchou; Li, Jinsong

2015-01-01

Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc(-/-)) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.

Single nucleotide editing without DNA cleavage using CRISPR/Cas9-deaminase in the sea urchin embryo.

PubMed

Shevidi, Saba; Uchida, Alicia; Schudrowitz, Natalie; Wessel, Gary M; Yajima, Mamiko

2017-12-01

A single base pair mutation in the genome can result in many congenital disorders in humans. The recent gene editing approach using CRISPR/Cas9 has rapidly become a powerful tool to replicate or repair such mutations in the genome. These approaches rely on cleaving DNA, while presenting unexpected risks. In this study, we demonstrate a modified CRISPR/Cas9 system fused to cytosine deaminase (Cas9-DA), which induces a single nucleotide conversion in the genome. Cas9-DA was introduced into sea urchin eggs with sgRNAs targeted for SpAlx1, SpDsh, or SpPks, each of which is critical for skeletogenesis, embryonic axis formation, or pigment formation, respectively. We found that both Cas9 and Cas9-DA edit the genome, and cause predicted phenotypic changes at a similar efficiency. Cas9, however, resulted in significant deletions in the genome centered on the gRNA target sequence, whereas Cas9-DA resulted in single or double nucleotide editing of C to T conversions within the gRNA target sequence. These results suggest that the Cas9-DA approach may be useful for manipulating gene activity with decreased risks of genomic aberrations. Developmental Dynamics 246:1036-1046, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Breeding for blast-disease-resistant and high-yield Thai jasmine rice (Oryza sativa L. cv. KDML 105) mutants using low-energy ion beams

NASA Astrophysics Data System (ADS)

Mahadtanapuk, S.; Teraarusiri, W.; Phanchaisri, B.; Yu, L. D.; Anuntalabhochai, S.

2013-07-01

Low-energy ion beam was applied on mutation induction for plant breeding of blast-disease-resistant Thai jasmine rice (Oryza sativa L. cv. KDML 105). Seeds of the wild-type rice were bombarded in vacuum by nitrogen ion beam at energy of 60-80 keV to a beam fluence range of 2 × 1016-2 × 1017 ions/cm2. The ion-bombarded rice seeds were grown in soil for 2 weeks as transplanted rice in plastic pots at 1 seedling/pot. The seedlings were then screened for blast resistance by Pyricularia grisea inoculation with 106 spores/ml concentrations. The blast-resistant rice mutant was planted up to F6 generation with the consistent phenotypic variation. The high percentage of the blast-disease-resistant rice was analyzed with DNA fingerprint. The HAT-RAPD (high annealing temperature-random amplified polymorphic DNA) marker revealed the modified polymorphism fragment presenting in the mutant compared with wild type (KDML 105). The cDNA fingerprints were investigated and the polymorphism fragment was subcloned into pGEM-T easy vector and then sequenced. The sequence of this fragment was compared with those already contained in the database, and the fragment was found to be related to the Spotted leaf protein 11 (Spl11).

Whole exome sequencing confirms the clinical diagnosis of Marfan syndrome combined with X-linked hypophosphatemia.

PubMed

Sheng, Xunlun; Chen, Xue; Lei, Bo; Chen, Rui; Wang, Hui; Zhang, Fangxia; Rong, Weining; Ha, Ruoshui; Liu, Yani; Zhao, Feng; Yang, Peizeng; Zhao, Chen

2015-06-04

To determine the genetic lesions and to modify the clinical diagnosis for a Chinese family with significant intrafamilial phenotypic diversities and unusual presentations. Three affected patients and the asymptomatic father were included and received comprehensive systemic examinations. Whole exome sequencing (WES) was performed for mutation detection. Structural modeling test was applied to analyze the potential structural changes caused by the missense substitution. The proband showed a wide spectrum of systemic anomalies, including bilateral ectopia lentis, atrial septal defect, ventricular septal defect, widening of tibial metaphysis with medial bowing, and dolichostenomelia in digits, while her mother and elder brother only demonstrated similar skeletal changes. A recurrent mutation, PHEX p.R291*, was found in all patients, while a de novo mutation, FBN1 p.C792F, was only detected in the proband. The FBN1 substitution was also predicted to cause significant conformational change in fibrillin-1 protein, thus changing its physical and biological properties. Taken together, we finalized the diagnosis for this family as X-linked hypophosphatemia (XLH), and diagnosed this girl as Marfan syndrome combined with XLH, and congenital heart disease. Our study also emphasizes the importance of WES in assisting the clinical diagnosis for complicated cases when the original diagnoses are challenged.

The Role of the Gut Microbiome in Multiple Sclerosis Risk and Progression: Towards Characterization of the "MS Microbiome".

PubMed

Pröbstel, Anne-Katrin; Baranzini, Sergio E

2018-01-01

Multiple sclerosis (MS) is the prototypic complex disease, in which both genes and the environment contribute to its pathogenesis. To date, > 200 independent loci across the genome have been associated with MS risk. However, these only explain a fraction of the total phenotypic variance, suggesting the possible presence of additional genetic factors, and, most likely, also environmental factors. New DNA sequencing technologies have enabled the sequencing of all kinds of microorganisms, including those living in and around humans (i.e., microbiomes). The study of bacterial populations inhabiting the gut is of particular interest in autoimmune diseases owing to their key role in shaping immune responses. In this review, we address the potential crosstalk between B cells and the gut microbiota, a relevant scenario in light of recently approved anti-B-cell therapies for MS. In addition, we review recent efforts to characterize the gut microbiome in patients with MS and discuss potential challenges and future opportunities. Finally, we describe the international MS microbiome study, a multicenter effort to study a large population of patients with MS and their healthy household partners to define the core MS microbiome, how it is shaped by disease-modifying therapies, and to explore potential therapeutic interventions.

Molecular analysis in true hermaphrodites with different karyotypes and similar phenotypes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Torres, L.; Cervantes, A.; Kofman-Alfaro, S.

1996-05-17

True hermaphroditism is characterized by the development of ovarian and testicular tissue in the same individual. Muellerian and Wolffian structures are usually present, and external genitalia are often ambiguous. The most frequent karyotype in these patients is 46,XX or various forms of mosaicism, whereas 46,XY is very rarely found. The phenotype in all these subjects is similar. We studied 10 true hermaphrodites. Six of them had a 46,XX chromosomal complement: 3 had been reared as males and 3 as females. The other 4 patients were mosaics: 3 were 46,XX/46,XY and one had a 46,XX/47,XXY karyotype. One of the 46,XX/46,XY mosaicsmore » was reared as a female, whereas the other 3 mosaics were reared as males. The sex of assignment in the 10 patients depended only on labio-scrotal differentiation. Molecular studies in 46,XX subjects documented the absence of Y centromeric sequences in all cases, arguing against hidden mosaicism. One patient presented Yp sequences (ZFY+, SRY+), which contrast with South African black 46,XX true hermaphrodites in whom no Y sequences were found. Molecular analysis in the subjects with mosaicism demonstrated the presence of Y centromeric and Yp sequences confirming the presence of a Y chromosome. Gonadal development, endocrine function, and phenotype in the 10 patients did not correlate with the presence of a Y chromosome or Y-derived sequences in the genome, confirming that true hermaphroditism is a heterogeneous condition. Both Mexican and non-South African 46,XX true hermaphrodites may be SRY positive. 51 refs., 3 figs., 2 tabs.« less

Cloning, sequencing, disruption and phenotypic analysis of uvsC, an Aspergillus nidulans homologue of yeast RAD51.

PubMed

van Heemst, D; Swart, K; Holub, E F; van Dijk, R; Offenberg, H H; Goosen, T; van den Broek, H W; Heyting, C

1997-05-01

We have cloned the uvsC gene of Aspergillus nidulans by complementation of the A. nidulans uvsC114 mutant. The predicted protein UVSC shows 67.4% sequence identity to the Saccharomyces cerevisiae Rad51 protein and 27.4% sequence identity to the Escherichia coli RecA protein. Transcription of uvsC is induced by methyl-methane sulphonate (MMS), as is transcription of RAD51 of yeast. Similar levels of uvsC transcription were observed after MMS induction in a uvsC+ strain and the uvsC114 mutant. The coding sequence of the uvsC114 allele has a deletion of 6 bp, which results in deletion of two amino acids and replacement of one amino acid in the translation product. In order to gain more insight into the biological function of the uvsC gene, a uvsC null mutant was constructed, in which the entire uvsC coding sequence was replaced by a selectable marker gene. Meiotic and mitotic phenotypes of a uvsC+ strain, the uvsC114 mutant and the uvsC null mutant were compared. The uvsC null mutant was more sensitive to both UV and MMS than the uvsC114 mutant. The uvsC114 mutant arrested in meiotic prophase-I. The uvsC null mutant arrested at an earlier stage, before the onset of meiosis. One possible interpretation of these meiotic phenotypes is that the A. nidulans homologue of Rad51 of yeast has a role both in the specialized processes preceding meiosis and in meiotic prophase I.

Design of synthetic bacterial communities for predictable plant phenotypes

PubMed Central

Herrera Paredes, Sur; Gao, Tianxiang; Law, Theresa F.; Finkel, Omri M.; Mucyn, Tatiana; Teixeira, Paulo José Pereira Lima; Salas González, Isaí; Feltcher, Meghan E.; Powers, Matthew J.; Shank, Elizabeth A.; Jones, Corbin D.; Jojic, Vladimir; Dangl, Jeffery L.; Castrillo, Gabriel

2018-01-01

Specific members of complex microbiota can influence host phenotypes, depending on both the abiotic environment and the presence of other microorganisms. Therefore, it is challenging to define bacterial combinations that have predictable host phenotypic outputs. We demonstrate that plant–bacterium binary-association assays inform the design of small synthetic communities with predictable phenotypes in the host. Specifically, we constructed synthetic communities that modified phosphate accumulation in the shoot and induced phosphate starvation–responsive genes in a predictable fashion. We found that bacterial colonization of the plant is not a predictor of the plant phenotypes we analyzed. Finally, we demonstrated that characterizing a subset of all possible bacterial synthetic communities is sufficient to predict the outcome of untested bacterial consortia. Our results demonstrate that it is possible to infer causal relationships between microbiota membership and host phenotypes and to use these inferences to rationally design novel communities. PMID:29462153

Whole genome sequence analysis of unidentified genetically modified papaya for development of a specific detection method.

PubMed

Nakamura, Kosuke; Kondo, Kazunari; Akiyama, Hiroshi; Ishigaki, Takumi; Noguchi, Akio; Katsumata, Hiroshi; Takasaki, Kazuto; Futo, Satoshi; Sakata, Kozue; Fukuda, Nozomi; Mano, Junichi; Kitta, Kazumi; Tanaka, Hidenori; Akashi, Ryo; Nishimaki-Mogami, Tomoko

2016-08-15

Identification of transgenic sequences in an unknown genetically modified (GM) papaya (Carica papaya L.) by whole genome sequence analysis was demonstrated. Whole genome sequence data were generated for a GM-positive fresh papaya fruit commodity detected in monitoring using real-time polymerase chain reaction (PCR). The sequences obtained were mapped against an open database for papaya genome sequence. Transgenic construct- and event-specific sequences were identified as a GM papaya developed to resist infection from a Papaya ringspot virus. Based on the transgenic sequences, a specific real-time PCR detection method for GM papaya applicable to various food commodities was developed. Whole genome sequence analysis enabled identifying unknown transgenic construct- and event-specific sequences in GM papaya and development of a reliable method for detecting them in papaya food commodities. Copyright © 2016 Elsevier Ltd. All rights reserved.

Phenotypic spectrum associated with mutations of the mitochondrial polymerase gamma gene.

PubMed

Horvath, Rita; Hudson, Gavin; Ferrari, Gianfrancesco; Fütterer, Nancy; Ahola, Sofia; Lamantea, Eleonora; Prokisch, Holger; Lochmüller, Hanns; McFarland, Robert; Ramesh, V; Klopstock, Thomas; Freisinger, Peter; Salvi, Fabrizio; Mayr, Johannes A; Santer, Rene; Tesarova, Marketa; Zeman, Jiri; Udd, Bjarne; Taylor, Robert W; Turnbull, Douglass; Hanna, Michael; Fialho, Doreen; Suomalainen, Anu; Zeviani, Massimo; Chinnery, Patrick F

2006-07-01

Mutations in the gene coding for the catalytic subunit of the mitochondrial DNA (mtDNA) polymerase gamma (POLG1) have recently been described in patients with diverse clinical presentations, revealing a complex relationship between genotype and phenotype in patients and their families. POLG1 was sequenced in patients from different European diagnostic and research centres to define the phenotypic spectrum and advance understanding of the recurrence risks. Mutations were identified in 38 cases, with the majority being sporadic compound heterozygotes. Eighty-nine DNA sequence changes were identified, including 2 predicted to alter a splice site, 1 predicted to cause a premature stop codon and 13 predicted to cause novel amino acid substitutions. The majority of children had a mutation in the linker region, often 1399G-->A (A467T), and a mutation affecting the polymerase domain. Others had mutations throughout the gene, and 11 had 3 or more substitutions. The clinical presentation ranged from the neonatal period to late adult life, with an overlapping phenotypic spectrum from severe encephalopathy and liver failure to late-onset external ophthalmoplegia, ataxia, myopathy and isolated muscle pain or epilepsy. There was a strong gender bias in children, with evidence of an environmental interaction with sodium valproate. POLG1 mutations cause an overlapping clinical spectrum of disease with both dominant and recessive modes of inheritance. 1399G-->A (A467T) is common in children, but complete POLG1 sequencing is required to identify multiple mutations that can have complex implications for genetic counselling.

Novel (ovario) leukodystrophy related to AARS2 mutations

PubMed Central

Dallabona, Cristina; Diodato, Daria; Kevelam, Sietske H.; Haack, Tobias B.; Wong, Lee-Jun; Salomons, Gajja S.; Baruffini, Enrico; Melchionda, Laura; Mariotti, Caterina; Strom, Tim M.; Meitinger, Thomas; Prokisch, Holger; Chapman, Kim; Colley, Alison; Rocha, Helena; Őunap, Katrin; Schiffmann, Raphael; Salsano, Ettore; Savoiardo, Mario; Hamilton, Eline M.; Abbink, Truus E. M.; Wolf, Nicole I.; Ferrero, Ileana; Lamperti, Costanza; Zeviani, Massimo; Vanderver, Adeline

2014-01-01

Objectives: The study was focused on leukoencephalopathies of unknown cause in order to define a novel, homogeneous phenotype suggestive of a common genetic defect, based on clinical and MRI findings, and to identify the causal genetic defect shared by patients with this phenotype. Methods: Independent next-generation exome-sequencing studies were performed in 2 unrelated patients with a leukoencephalopathy. MRI findings in these patients were compared with available MRIs in a database of unclassified leukoencephalopathies; 11 patients with similar MRI abnormalities were selected. Clinical and MRI findings were investigated. Results: Next-generation sequencing revealed compound heterozygous mutations in AARS2 encoding mitochondrial alanyl-tRNA synthetase in both patients. Functional studies in yeast confirmed the pathogenicity of the mutations in one patient. Sanger sequencing revealed AARS2 mutations in 4 of the 11 selected patients. The 6 patients with AARS2 mutations had childhood- to adulthood-onset signs of neurologic deterioration consisting of ataxia, spasticity, and cognitive decline with features of frontal lobe dysfunction. MRIs showed a leukoencephalopathy with striking involvement of left-right connections, descending tracts, and cerebellar atrophy. All female patients had ovarian failure. None of the patients had signs of a cardiomyopathy. Conclusions: Mutations in AARS2 have been found in a severe form of infantile cardiomyopathy in 2 families. We present 6 patients with a new phenotype caused by AARS2 mutations, characterized by leukoencephalopathy and, in female patients, ovarian failure, indicating that the phenotypic spectrum associated with AARS2 variants is much wider than previously reported. PMID:24808023

Phenotypic integration in an extended phenotype: among-individual variation in nest-building traits of the alfalfa leafcutting bee (Megachile rotundata)

USDA-ARS?s Scientific Manuscript database

Structures such as nests and burrows are an essential component of many organisms’ life-cycle and requires a complex sequence of behaviors. Because behaviors can vary consistently among individuals and be correlated with one another, we hypothesized that these structures would 1) show evidence of am...

Comparative phenotypic analysis of Gossypium raimondii with Upland cotton

USDA-ARS?s Scientific Manuscript database

Gossypium raimondii Ulbr., a wild species with a diploid genome, has been sequenced due to its small genome size and sequence similarity with the polyploidy cultivated Gossypium species. Accessibility of the G. raimondii genome has made the species a reference used extensively in cotton genomic and...

Datasets for evolutionary comparative genomics

PubMed Central

Liberles, David A

2005-01-01

Many decisions about genome sequencing projects are directed by perceived gaps in the tree of life, or towards model organisms. With the goal of a better understanding of biology through the lens of evolution, however, there are additional genomes that are worth sequencing. One such rationale for whole-genome sequencing is discussed here, along with other important strategies for understanding the phenotypic divergence of species. PMID:16086856

Modified echo peak correction for radial acquisition regime (RADAR).

PubMed

Takizawa, Masahiro; Ito, Taeko; Itagaki, Hiroyuki; Takahashi, Tetsuhiko; Shimizu, Kanichirou; Harada, Junta

2009-01-01

Because radial sampling imposes many limitations on magnetic resonance (MR) imaging hardware, such as on the accuracy of the gradient magnetic field or the homogeneity of B(0), some correction of the echo signal is usually needed before image reconstruction. In our previous study, we developed an echo-peak-shift correction (EPSC) algorithm not easily affected by hardware performance. However, some artifacts remained in lung imaging, where tissue is almost absent, or in cardiac imaging, which is affected by blood flow. In this study, we modified the EPSC algorithm to improve the image quality of the radial aquisition regime (RADAR) and expand its application sequences. We assumed the artifacts were mainly caused by errors in the phase map for EPSC and used a phantom on a 1.5-tesla (T) MR scanner to investigate whether to modify the EPSC algorithm. To evaluate the effectiveness of EPSC, we compared results from T(1)- and T(2)-weighted images of a volunteer's lung region using the current and modified EPSC. We then applied the modified EPSC to RADAR spin echo (SE) and RADAR balanced steady-state acquisition with rewound gradient echo (BASG) sequence. The modified EPSC reduced phase discontinuity in the reference data used for EPSC and improved visualization of blood vessels in the lungs. Motion and blood flow caused no visible artifacts in the resulting images in either RADAR SE or RADAR BASG sequence. Use of the modified EPSC eliminated artifacts caused by signal loss in the reference data for EPSC. In addition, the modified EPSC was applied to RADAR SE and RADAR BASG sequences.

Transitions towards either slow-oxidative or fast-glycolytic phenotype can be induced in the murine WTt myogenic cell line.

PubMed

Peltzer, J; Carpentier, G; Martelly, I; Courty, J; Keller, A

2010-09-01

Contraction and energy metabolism are functions of skeletal muscles co-regulated by still largely unknown signals. To help elucidating these interconnecting pathways, we are developing new cellular models that will allow to control the switch from a neonatal to an adult slow-oxidative or fast-glycolytic phenotype of myofibers, during in vitro differentiation. Thus, our purpose was to direct the differentiation of the newly characterized WTt clone, from a mixed towards either fast or slow phenotype, by modifying amounts of two transcription factors respectively involved in control of glycolytic and oxidative energy metabolism, namely HIF-1alpha and PPARdelta. Our data support the idea that HIF-1alpha protein stabilization would favor expression of fast phenotypic markers, accompanied or not by a decreased expression of slow markers, depending on treatment conditions. Conversely, PPARdelta over-expression appears to enhance the slow-oxidative phenotype of WTt myotubes. Furthermore, we have observed that expression of PGC-1alpha, a coregulator of PPAR, is also modified in this cell line upon conditions that stabilize HIF-1alpha protein. This observation points to the existence of a regulatory link between pathways controlled by the two transcription factors HIF-1alpha and PPARdelta. Therefore, these cells should be useful to analyze the balance between oxidative and glycolytic energy production as a function of phenotypic transitions occurring during myogenic maturation. The newly characterized murine WTt clone will be a good tool to investigate molecular mechanisms implicating HIF-1alpha and PPARdelta in the coordinated metabolic and contractile regulations involved in myogenesis. (c) 2010 Wiley-Liss, Inc.

Complex small-molecule architectures regulate phenotypic plasticity in a nematode.

PubMed

Bose, Neelanjan; Ogawa, Akira; von Reuss, Stephan H; Yim, Joshua J; Ragsdale, Erik J; Sommer, Ralf J; Schroeder, Frank C

2012-12-07

Chemistry the worm's way: The nematode Pristionchus pacificus constructs elaborate small molecules from modified building blocks of primary metabolism, including an unusual xylopyranose-based nucleoside (see scheme). These compounds act as signaling molecules to control adult phenotypic plasticity and dauer development and provide examples of modular generation of structural diversity in metazoans. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

4p terminal deletion and 11p subtelomeric duplication detected by genomic microarray in a patient with Wolf-Hirschhorn syndrome and an atypical phenotype.

PubMed

Stevenson, David A; Carey, John C; Cowley, Brett C; Bayrak-Toydemir, Pinar; Mao, Rong; Brothman, Arthur R

2004-12-01

We report a de novo cryptic 11p duplication found by genomic microarray with a cytogenetically detected 4p deletion. Terminal 4p deletions cause Wolf-Hirschhorn syndrome, but the phenotype probably was modified by the paternally derived 11p duplication. This emphasizes the clinical utility of genomic microarray.

CYBRD1 as a modifier gene that modulates iron phenotype in HFE p.C282Y homozygous patients.

PubMed

Pelucchi, Sara; Mariani, Raffaella; Calza, Stefano; Fracanzani, Anna Ludovica; Modignani, Giulia Litta; Bertola, Francesca; Busti, Fabiana; Trombini, Paola; Fraquelli, Mirella; Forni, Gian Luca; Girelli, Domenico; Fargion, Silvia; Specchia, Claudia; Piperno, Alberto

2012-12-01

Most patients with hereditary hemochromatosis in the Caucasian population are homozygous for the p.C282Y mutation in the HFE gene. The penetrance and expression of hereditary hemochromatosis differ largely among cases of homozygous p.C282Y. Genetic factors might be involved in addition to environmental factors. In the present study, we analyzed 50 candidate genes involved in iron metabolism and evaluated the association between 214 single nucleotide polymorphisms in these genes and three phenotypic outcomes of iron overload (serum ferritin, iron removed and transferrin saturation) in a large group of 296 p.C282Y homozygous Italians. Polymorphisms were tested for genetic association with each single outcome using linear regression models adjusted for age, sex and alcohol consumption. We found a series of 17 genetic variants located in different genes with possible additive effects on the studied outcomes. In order to evaluate whether the selected polymorphisms could provide a predictive signature for adverse phenotype, we re-evaluated data by dividing patients in two extreme phenotype classes based on the three phenotypic outcomes. We found that only a small improvement in prediction could be achieved by adding genetic information to clinical data. Among the selected polymorphisms, a significant association was observed between rs3806562, located in the 5'UTR of CYBRD1, and transferrin saturation. This variant belongs to the same haplotype block that contains the CYBRD1 polymorphism rs884409, found to be associated with serum ferritin in another population of p.C282Y homozygotes, and able to modulate promoter activity. A luciferase assay indicated that rs3806562 does not have a significant functional role, suggesting that it is a genetic marker linked to the putative genetic modifier rs884409. While our results support the hypothesis that polymorphisms in genes regulating iron metabolism may modulate penetrance of HFE-hereditary hemochromatosis, with emphasis on CYBRD1, they strengthen the notion that none of these polymorphisms alone is a major modifier of the phenotype of hereditary hemochromatosis.

CYBRD1 as a modifier gene that modulates iron phenotype in HFE p.C282Y homozygous patients

PubMed Central

Pelucchi, Sara; Mariani, Raffaella; Calza, Stefano; Fracanzani, Anna Ludovica; Modignani, Giulia Litta; Bertola, Francesca; Busti, Fabiana; Trombini, Paola; Fraquelli, Mirella; Forni, Gian Luca; Girelli, Domenico; Fargion, Silvia; Specchia, Claudia; Piperno, Alberto

2012-01-01

Background Most patients with hereditary hemochromatosis in the Caucasian population are homozygous for the p.C282Y mutation in the HFE gene. The penetrance and expression of hereditary hemochromatosis differ largely among cases of homozygous p.C282Y. Genetic factors might be involved in addition to environmental factors. Design and Methods: In the present study, we analyzed 50 candidate genes involved in iron metabolism and evaluated the association between 214 single nucleotide polymorphisms in these genes and three phenotypic outcomes of iron overload (serum ferritin, iron removed and transferrin saturation) in a large group of 296 p.C282Y homozygous Italians. Polymorphisms were tested for genetic association with each single outcome using linear regression models adjusted for age, sex and alcohol consumption. Results We found a series of 17 genetic variants located in different genes with possible additive effects on the studied outcomes. In order to evaluate whether the selected polymorphisms could provide a predictive signature for adverse phenotype, we re-evaluated data by dividing patients in two extreme phenotype classes based on the three phenotypic outcomes. We found that only a small improvement in prediction could be achieved by adding genetic information to clinical data. Among the selected polymorphisms, a significant association was observed between rs3806562, located in the 5'UTR of CYBRD1, and transferrin saturation. This variant belongs to the same haplotype block that contains the CYBRD1 polymorphism rs884409, found to be associated with serum ferritin in another population of p.C282Y homozygotes, and able to modulate promoter activity. A luciferase assay indicated that rs3806562 does not have a significant functional role, suggesting that it is a genetic marker linked to the putative genetic modifier rs884409. Conclusions While our results support the hypothesis that polymorphisms in genes regulating iron metabolism may modulate penetrance of HFE-hereditary hemochromatosis, with emphasis on CYBRD1, they strengthen the notion that none of these polymorphisms alone is a major modifier of the phenotype of hereditary hemochromatosis. PMID:22773607

DNA polymerase having modified nucleotide binding site for DNA sequencing

DOEpatents

Tabor, Stanley; Richardson, Charles

1997-01-01

Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.

Combining high-throughput phenotyping and genome-wide association studies to reveal natural genetic variation in rice

PubMed Central

Yang, Wanneng; Guo, Zilong; Huang, Chenglong; Duan, Lingfeng; Chen, Guoxing; Jiang, Ni; Fang, Wei; Feng, Hui; Xie, Weibo; Lian, Xingming; Wang, Gongwei; Luo, Qingming; Zhang, Qifa; Liu, Qian; Xiong, Lizhong

2014-01-01

Even as the study of plant genomics rapidly develops through the use of high-throughput sequencing techniques, traditional plant phenotyping lags far behind. Here we develop a high-throughput rice phenotyping facility (HRPF) to monitor 13 traditional agronomic traits and 2 newly defined traits during the rice growth period. Using genome-wide association studies (GWAS) of the 15 traits, we identify 141 associated loci, 25 of which contain known genes such as the Green Revolution semi-dwarf gene, SD1. Based on a performance evaluation of the HRPF and GWAS results, we demonstrate that high-throughput phenotyping has the potential to replace traditional phenotyping techniques and can provide valuable gene identification information. The combination of the multifunctional phenotyping tools HRPF and GWAS provides deep insights into the genetic architecture of important traits. PMID:25295980

Are genes destiny? Have adenine, cytosine, guanine and thymine replaced Lachesis, Clotho and Atropos as the weavers of our fate?

PubMed

Eisenberg, Leon

2005-02-01

It is as futile to ask how much of the phenotype of an organism is due to nature and how much to its nurture as it is to determine how much of the area of a rectangle is due to its length and how much to its height. Phenotype and area are joint products. The spectacular success of genomics, unfortunately, threatens to re-awaken belief in genes as the principal determinants of human behavior. This paper develops the thesis that gene expression is modified by environmental inputs and that the impact of the environment on a given organism is modified by its genome. Genes set the boundaries of the possible; environments parse out the actual.

Fingerprints of Modified RNA Bases from Deep Sequencing Profiles.

PubMed

Kietrys, Anna M; Velema, Willem A; Kool, Eric T

2017-11-29

Posttranscriptional modifications of RNA bases are not only found in many noncoding RNAs but have also recently been identified in coding (messenger) RNAs as well. They require complex and laborious methods to locate, and many still lack methods for localized detection. Here we test the ability of next-generation sequencing (NGS) to detect and distinguish between ten modified bases in synthetic RNAs. We compare ultradeep sequencing patterns of modified bases, including miscoding, insertions and deletions (indels), and truncations, to unmodified bases in the same contexts. The data show widely varied responses to modification, ranging from no response, to high levels of mutations, insertions, deletions, and truncations. The patterns are distinct for several of the modifications, and suggest the future use of ultradeep sequencing as a fingerprinting strategy for locating and identifying modifications in cellular RNAs.

SU-E-I-62: Reduction of Susceptibility Artifacts by Increasing the Bandwidth (BW) and Echo Train Length (ETL)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mavroidis, P; Boci, N; Kostopoulos, S

2015-06-15

Purpose: The aim of this present study is to increase bandwidth (BW) and echo train length (ETL) in Proton Density Turbo Spin Echo (PD TSE) sequences with and without fat saturation (FS) as well as in Turbo Inversion Recovery Magnitude sequences (TIRM) in order to assess whether these sequences are capable of reducing susceptibility artifacts. Methods: We compared 1) TIRM coronal (COR) with the same sequence with increased both BW and ETL 2) Conventional PD TSE sagittal (SAG) with FS with an increased BW 3) Conventional PD TSE SAG without FS with an increased BW 4) Conventional PD TSE SAGmore » without FS with increased both BW and ETL. A quantitative analysis was performed to measure the extent of the susceptibility artifacts. Furthermore, a qualitative analysis was performed by two radiologists in order to evaluate the susceptibility artifacts, image distortion and fat suppression. The depiction of cartilage, menisci, muscles, tendons and bone marrow were also qualitatively analyzed. Results: The quantitative analysis found that the modified TIRM sequence is significantly superior to the conventional one regarding the extent of the susceptibility artifacts. In the qualitative analysis, the modified TIRM sequence was superior to the corresponding conventional one in eight characteristics out of ten that were analyzed. The modified PD TSE with FS was superior to the corresponding conventional one regarding the susceptibility artifacts, image distortion and depiction of bone marrow and cartilage while achieving effective fat saturation. The modified PD TSE sequence without FS with a high (H) BW was found to be superior corresponding to the conventional one in the case of cartilage. Conclusion: Consequently, TIRM sequence with an increased BW and ETL is proposed for producing images of high quality and modified PD TSE with H BW for smaller metals, especially when FS is used.« less

Copy number variation of individual cattle genomes using next-generation sequencing

USDA-ARS?s Scientific Manuscript database

Copy number variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next-generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one ...

Individualized cattle copy number and segmental duplication maps using next generation sequencing

USDA-ARS?s Scientific Manuscript database

Copy Number Variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one ...

Copy number variation of individual cattle genomes using next-generation sequencing

USDA-ARS?s Scientific Manuscript database

Copy Number Variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often difficult to track. Using a read depth approach based on next generation sequencing, we examined genome-wide copy number differences among five taurine (three Angu...

A high-resolution cattle CNV map by population-scale genome sequencing

USDA-ARS?s Scientific Manuscript database

Copy Number Variations (CNVs) are common genomic structural variations that have been linked to human diseases and phenotypic traits. Prior studies in cattle have produced low-resolution CNV maps. We constructed a draft, high-resolution map of cattle CNVs based on whole genome sequencing data from 7...

SNP-based genotyping in lentil: linking sequence information with phenotypes

USDA-ARS?s Scientific Manuscript database

Lentil (Lens culinaris) has been late to enter the world of high throughput molecular analysis due to a general lack of genomic resources. Using a 454 sequencing-based approach, SNPs have been identified in genes across the lentil genome. Several hundred have been turned into single SNP KASP assay...

Mining conifers’ mega-genome using rapid and efficient multiplexed high-throughput genotyping-by-sequencing (GBS) SNP discovery platform

USDA-ARS?s Scientific Manuscript database

Next-generation sequencing (NGS) technologies are revolutionizing both medical and biological research through generation of massive SNP data sets for identifying heritable genome variation underlying key traits, from rare human diseases to important agronomic phenotypes in crop species. We evaluate...

An Internet-Accessible DNA Sequence Database for Identifying Fusaria from Human and Animal Infections

USDA-ARS?s Scientific Manuscript database

Because less than one-third of clinically relevant fusaria can be accurately identified to species level using phenotypic data (i.e., morphological species recognition), we constructed a three-locus DNA sequence database to facilitate molecular identification of the 69 Fusarium species associated wi...

A Kinome RNAi Screen in Drosophila Identifies Novel Genes Interacting with Lgl, aPKC, and Crb Cell Polarity Genes in Epithelial Tissues.

PubMed

Parsons, Linda M; Grzeschik, Nicola A; Amaratunga, Kasun; Burke, Peter; Quinn, Leonie M; Richardson, Helena E

2017-08-07

In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila "cell polarity" eye and wing phenotypes. Initially, we identified kinase or phosphatase genes whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, Jun kinase (JNK), and Notch signaling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras, and lipid/Phospho-inositol-3-kinase (PI3K) signaling pathways. Additionally, we demonstrated that the "nutrient sensing" kinases Salt Inducible Kinase 2 and 3 ( SIK2 and 3 ) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, our screen has revealed novel cell polarity-interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development. Copyright © 2017 Parsons et al.

Sequencing and comparative genomic analysis of 1227 Felis catus cDNA sequences enriched for developmental, clinical and nutritional phenotypes

PubMed Central

2012-01-01

Background The feline genome is valuable to the veterinary and model organism genomics communities because the cat is an obligate carnivore and a model for endangered felids. The initial public release of the Felis catus genome assembly provided a framework for investigating the genomic basis of feline biology. However, the entire set of protein coding genes has not been elucidated. Results We identified and characterized 1227 protein coding feline sequences, of which 913 map to public sequences and 314 are novel. These sequences have been deposited into NCBI's genbank database and complement public genomic resources by providing additional protein coding sequences that fill in some of the gaps in the feline genome assembly. Through functional and comparative genomic analyses, we gained an understanding of the role of these sequences in feline development, nutrition and health. Specifically, we identified 104 orthologs of human genes associated with Mendelian disorders. We detected negative selection within sequences with gene ontology annotations associated with intracellular trafficking, cytoskeleton and muscle functions. We detected relatively less negative selection on protein sequences encoding extracellular networks, apoptotic pathways and mitochondrial gene ontology annotations. Additionally, we characterized feline cDNA sequences that have mouse orthologs associated with clinical, nutritional and developmental phenotypes. Together, this analysis provides an overview of the value of our cDNA sequences and enhances our understanding of how the feline genome is similar to, and different from other mammalian genomes. Conclusions The cDNA sequences reported here expand existing feline genomic resources by providing high-quality sequences annotated with comparative genomic information providing functional, clinical, nutritional and orthologous gene information. PMID:22257742

Retrospective genotype-phenotype analysis in a 305 patient cohort referred for testing of a targeted epilepsy panel.

PubMed

Hesse, Andrew N; Bevilacqua, Jennifer; Shankar, Kritika; Reddi, Honey V

2018-05-16

Epilepsy is a diverse neurological condition with extreme genetic and phenotypic heterogeneity. The introduction of next-generation sequencing into the clinical laboratory has made it possible to investigate hundreds of associated genes simultaneously for a patient, even in the absence of a clearly defined syndrome. This has resulted in the detection of rare and novel mutations at a rate well beyond our ability to characterize their effects. This retrospective study reviews genotype data in the context of available phenotypic information on 305 patients spanning the epileptic spectrum to identify established and novel patterns of correlation. Our epilepsy panel comprising 377 genes was used to sequence 305 patients referred for genetic testing. Qualifying variants were annotated with phenotypic data obtained from either the test requisition form or supporting clinical documentation. Observed phenotypes were compared with established phenotypes in OMIM, published literature and the ILAEs 2010 report on genetic testing to assess congruity with known gene aberrations. We identified a number of novel and recognized genetic variants consistent with established epileptic phenotypes. Forty-one pathogenic or predicted deleterious variants were detected in 39 patients with accompanying clinical documentation. Twenty-five of these variants across 15 genes were novel. Furthermore, evaluation of phenotype data for 194 patients with variants of unknown significance in genes with autosomal dominant and X-linked disease inheritance elucidated potentially disease-causing variants that were not currently characterized in the literature. Assessment of key genotype-phenotype correlations from our cohort provide insight into variant classification, as well as the importance of including ILAE recommended genes as part of minimum panel content for comprehensive epilepsy tests. Many of the reported VUSs are likely genuine pathogenic variants driving the observed phenotypes, but not enough evidence is available for assertive classifications. Similar studies will provide more utility via mounting independent genotype-phenotype data from unrelated patients. The possible outcome would be a better molecular diagnostic product, with fewer indeterminate reports containing only VUSs. Copyright © 2018. Published by Elsevier B.V.

The UK10K project identifies rare variants in health and disease.

PubMed

Walter, Klaudia; Min, Josine L; Huang, Jie; Crooks, Lucy; Memari, Yasin; McCarthy, Shane; Perry, John R B; Xu, ChangJiang; Futema, Marta; Lawson, Daniel; Iotchkova, Valentina; Schiffels, Stephan; Hendricks, Audrey E; Danecek, Petr; Li, Rui; Floyd, James; Wain, Louise V; Barroso, Inês; Humphries, Steve E; Hurles, Matthew E; Zeggini, Eleftheria; Barrett, Jeffrey C; Plagnol, Vincent; Richards, J Brent; Greenwood, Celia M T; Timpson, Nicholas J; Durbin, Richard; Soranzo, Nicole

2015-10-01

The contribution of rare and low-frequency variants to human traits is largely unexplored. Here we describe insights from sequencing whole genomes (low read depth, 7×) or exomes (high read depth, 80×) of nearly 10,000 individuals from population-based and disease collections. In extensively phenotyped cohorts we characterize over 24 million novel sequence variants, generate a highly accurate imputation reference panel and identify novel alleles associated with levels of triglycerides (APOB), adiponectin (ADIPOQ) and low-density lipoprotein cholesterol (LDLR and RGAG1) from single-marker and rare variant aggregation tests. We describe population structure and functional annotation of rare and low-frequency variants, use the data to estimate the benefits of sequencing for association studies, and summarize lessons from disease-specific collections. Finally, we make available an extensive resource, including individual-level genetic and phenotypic data and web-based tools to facilitate the exploration of association results.

Linkage Map of Escherichia coli K-12, Edition 10: The Traditional Map

PubMed Central

Berlyn, Mary K. B.

1998-01-01

This map is an update of the edition 9 map by Berlyn et al. (M. K. B. Berlyn, K. B. Low, and K. E. Rudd, p. 1715–1902, in F. C. Neidhardt et al., ed., Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed., vol. 2, 1996). It uses coordinates established by the completed sequence, expressed as 100 minutes for the entire circular map, and adds new genes discovered and established since 1996 and eliminates those shown to correspond to other known genes. The latter are included as synonyms. An alphabetical list of genes showing map location, synonyms, the protein or RNA product of the gene, phenotypes of mutants, and reference citations is provided. In addition to genes known to correspond to gene sequences, other genes, often older, that are described by phenotype and older mapping techniques and that have not been correlated with sequences are included. PMID:9729611

Diversity of halophilic archaea from six hypersaline environments in Turkey.

PubMed

Ozcan, Birgul; Ozcengiz, Gulay; Coleri, Arzu; Cokmus, Cumhur

2007-06-01

The diversity of archaeal strains from six hypersaline environments in Turkey was analyzed by comparing their phenotypic characteristics and 16S rDNA sequences. Thirty-three isolates were characterized in terms of their phenotypic properties including morphological and biochemical characteristics, susceptibility to different antibiotics, and total lipid and plasmid contents, and finally compared by 16S rDNA gene sequences. The results showed that all isolates belong to the family Halobacteriaceae. Phylogenetic analyses using approximately 1,388 bp comparisions of 16S rDNA sequences demonstrated that all isolates clustered closely to species belonging to 9 genera, namely Halorubrum (8 isolates), Natrinema (5 isolates), Haloarcula (4 isolates), Natronococcus (4 isolates), Natrialba (4 isolates), Haloferax (3 isolates), Haloterrigena (3 isolates), Halalkalicoccus (1 isolate), and Halomicrobium (1 isolate). The results revealed a high diversity among the isolated halophilic strains and indicated that some of these strains constitute new taxa of extremely halophilic archaea.

Properties of Cells Carrying the Herpes Simplex Virus Type 2 Thymidine Kinase Gene: Mechanisms of Reversion to a Thymidine Kinase-Negative Phenotype

PubMed Central

Bastow, K. F.; Darby, G.; Wildy, P.; Minson, A. C.

1980-01-01

We have isolated cells with a thymidine kinase-negative (tk−) phenotype from cells which carry the herpes simplex virus type 2 tk gene by selection in 5-bromodeoxyuridine or 9-(2-hydroxyethoxymethyl)guanine. Both selection routines generated revertants with a frequency of 10−3 to 10−4, and resistance to either compound conferred simultaneous resistance to the other. tk− revertants fell into three classes: (i) cells that arose by deletion of all virus sequences, (ii) cells that had lost the virus tk gene but retained a nonselected virus-specific function and arose by deletion of part of the virus-specific sequence, and (iii) cells that retained the potential to express all of the virus-specific functions of the parental cells and retained all of the virus-specific DNA sequences. Images PMID:16789205

Atrazine Resistance in Chenopodium album

PubMed Central

Bettini, Priscilla; McNally, Sheila; Sevignac, Mireille; Darmency, Henri; Gasquez, Jacques; Dron, Michel

1987-01-01

In Chenopodium album two different levels of atrazine resistance have been found according to following criteria: lethal dose and leaf fluorescence curve. The intermediate (I) phenotype is represented by a low level of resistance and a typical I fluorescence curve. It arose at high frequency, within one generation, after self-pollination of particular plants displaying a susceptible (S) phenotype. The resistance phenotype (Ri) has a high level of resistance and presents a typical resistant fluorescence curve. It appeared after self-pollination of chemically treated I plants. The I, Ri, and also R (resistant plants found in atrazine treated fields) phenotypes contain a serine to glycine mutation at amino acid position 264 in the chloroplast psbA gene product. The steady state level of the psbA gene transcript is not modified between S, I, Ri, and R phenotypes. Images Fig. 4 PMID:16665624

Whole-Genome Sequence Analysis of Antimicrobial Resistance Genes in Streptococcus uberis and Streptococcus dysgalactiae Isolates from Canadian Dairy Herds

PubMed Central

Vélez, Julián Reyes; Cameron, Marguerite; Rodríguez-Lecompte, Juan Carlos; Xia, Fangfang; Heider, Luke C.; Saab, Matthew; McClure, J. Trenton; Sánchez, Javier

2017-01-01

The objectives of this study are to determine the occurrence of antimicrobial resistance (AMR) genes using whole-genome sequence (WGS) of Streptococcus uberis (S. uberis) and Streptococcus dysgalactiae (S. dysgalactiae) isolates, recovered from dairy cows in the Canadian Maritime Provinces. A secondary objective included the exploration of the association between phenotypic AMR and the genomic characteristics (genome size, guanine–cytosine content, and occurrence of unique gene sequences). Initially, 91 isolates were sequenced, and of these isolates, 89 were assembled. Furthermore, 16 isolates were excluded due to larger than expected genomic sizes (>2.3 bp × 1,000 bp). In the final analysis, 73 were used with complete WGS and minimum inhibitory concentration records, which were part of the previous phenotypic AMR study, representing 18 dairy herds from the Maritime region of Canada (1). A total of 23 unique AMR gene sequences were found in the bacterial genomes, with a mean number of 8.1 (minimum: 5; maximum: 13) per genome. Overall, there were 10 AMR genes [ANT(6), TEM-127, TEM-163, TEM-89, TEM-95, Linb, Lnub, Ermb, Ermc, and TetS] present only in S. uberis genomes and 2 genes unique (EF-TU and TEM-71) to the S. dysgalactiae genomes; 11 AMR genes [APH(3′), TEM-1, TEM-136, TEM-157, TEM-47, TetM, bl2b, gyrA, parE, phoP, and rpoB] were found in both bacterial species. Two-way tabulations showed association between the phenotypic susceptibility to lincosamides and the presence of linB (P = 0.002) and lnuB (P < 0.001) genes and the between the presence of tetM (P = 0.015) and tetS (P = 0.064) genes and phenotypic resistance to tetracyclines only for the S. uberis isolates. The logistic model showed that the odds of resistance (to any of the phenotypically tested antimicrobials) was 4.35 times higher when there were >11 AMR genes present in the genome, compared with <7 AMR genes (P < 0.001). The odds of resistance was lower for S. dysgalactiae than S. uberis (P = 0.031). When the within-herd somatic cell count was >250,000 cells/mL, a trend toward higher odds of resistance compared with the baseline category of <150,000 cells/mL was observed. When the isolate corresponded to a post-mastitis sample, there were lower odds of resistance when compared with non-clinical isolates (P = 0.01). The results of this study showed the strength of associations between phenotypic AMR resistance of both mastitis pathogens and their genotypic resistome and other epidemiological characteristics. PMID:28589129

The Autism Simplex Collection: an international, expertly phenotyped autism sample for genetic and phenotypic analyses

PubMed Central

2014-01-01

Background There is an urgent need for expanding and enhancing autism spectrum disorder (ASD) samples, in order to better understand causes of ASD. Methods In a unique public-private partnership, 13 sites with extensive experience in both the assessment and diagnosis of ASD embarked on an ambitious, 2-year program to collect samples for genetic and phenotypic research and begin analyses on these samples. The program was called The Autism Simplex Collection (TASC). TASC sample collection began in 2008 and was completed in 2010, and included nine sites from North America and four sites from Western Europe, as well as a centralized Data Coordinating Center. Results Over 1,700 trios are part of this collection, with DNA from transformed cells now available through the National Institute of Mental Health (NIMH). Autism Diagnostic Interview-Revised (ADI-R) and Autism Diagnostic Observation Schedule-Generic (ADOS-G) measures are available for all probands, as are standardized IQ measures, Vineland Adaptive Behavioral Scales (VABS), the Social Responsiveness Scale (SRS), Peabody Picture Vocabulary Test (PPVT), and physical measures (height, weight, and head circumference). At almost every site, additional phenotypic measures were collected, including the Broad Autism Phenotype Questionnaire (BAPQ) and Repetitive Behavior Scale-Revised (RBS-R), as well as the non-word repetition scale, Communication Checklist (Children’s or Adult), and Aberrant Behavior Checklist (ABC). Moreover, for nearly 1,000 trios, the Autism Genome Project Consortium (AGP) has carried out Illumina 1 M SNP genotyping and called copy number variation (CNV) in the samples, with data being made available through the National Institutes of Health (NIH). Whole exome sequencing (WES) has been carried out in over 500 probands, together with ancestry matched controls, and this data is also available through the NIH. Additional WES is being carried out by the Autism Sequencing Consortium (ASC), where the focus is on sequencing complete trios. ASC sequencing for the first 1,000 samples (all from whole-blood DNA) is complete and data will be released in 2014. Data is being made available through NIH databases (database of Genotypes and Phenotypes (dbGaP) and National Database for Autism Research (NDAR)) with DNA released in Dist 11.0. Primary funding for the collection, genotyping, sequencing and distribution of TASC samples was provided by Autism Speaks and the NIH, including the National Institute of Mental Health (NIMH) and the National Human Genetics Research Institute (NHGRI). Conclusions TASC represents an important sample set that leverages expert sites. Similar approaches, leveraging expert sites and ongoing studies, represent an important path towards further enhancing available ASD samples. PMID:25392729

Draft Genome Sequence of Pedobacter sp. Strain V48, Isolated from a Coastal Sand Dune in the Netherlands

PubMed Central

Bitzer, Adam S.; Garbeva, Paolina

2014-01-01

Pedobacter sp. strain V48 participates in an interaction with Pseudomonas fluorescens which elicits interaction-induced phenotypes. We report the draft genome sequence of Pedobacter sp. V48, consisting of 6.46 Mbp. The sequence will contribute to improved understanding of the genus and facilitate genomic analysis of the model interspecies interaction with P. fluorescens. PMID:24578271

Cattle phenotypes can disguise their maternal ancestry.

PubMed

Srirattana, Kanokwan; McCosker, Kieren; Schatz, Tim; St John, Justin C

2017-06-26

Cattle are bred for, amongst other factors, specific traits, including parasite resistance and adaptation to climate. However, the influence and inheritance of mitochondrial DNA (mtDNA) are not usually considered in breeding programmes. In this study, we analysed the mtDNA profiles of cattle from Victoria (VIC), southern Australia, which is a temperate climate, and the Northern Territory (NT), the northern part of Australia, which has a tropical climate, to determine if the mtDNA profiles of these cattle are indicative of breed and phenotype, and whether these profiles are appropriate for their environments. A phylogenetic tree of the full mtDNA sequences of different breeds of cattle, which were obtained from the NCBI database, showed that the mtDNA profiles of cattle do not always reflect their phenotype as some cattle with Bos taurus phenotypes had Bos indicus mtDNA, whilst some cattle with Bos indicus phenotypes had Bos taurus mtDNA. Using D-loop sequencing, we were able to contrast the phenotypes and mtDNA profiles from different species of cattle from the 2 distinct cattle breeding regions of Australia. We found that 67 of the 121 cattle with Bos indicus phenotypes from NT (55.4%) had Bos taurus mtDNA. In VIC, 92 of the 225 cattle with Bos taurus phenotypes (40.9%) possessed Bos indicus mtDNA. When focusing on oocytes from cattle with the Bos taurus phenotype in VIC, their respective oocytes with Bos indicus mtDNA had significantly lower levels of mtDNA copy number compared with oocytes possessing Bos taurus mtDNA (P < 0.01). However, embryos derived from oocytes with Bos indicus mtDNA had the same ability to develop to the blastocyst stage and the levels of mtDNA copy number in their blastocysts were similar to blastocysts derived from oocytes harbouring Bos taurus mtDNA. Nevertheless, oocytes originating from the Bos indicus phenotype exhibited lower developmental potential due to low mtDNA copy number when compared with oocytes from cattle with a Bos taurus phenotype. The phenotype of cattle is not always related to their mtDNA profiles. MtDNA profiles should be considered for breeding programmes as they also influence phenotypic traits and reproductive capacity in terms of oocyte quality.

Analysis of CHRNA7 rare variants in autism spectrum disorder susceptibility.

PubMed

Bacchelli, Elena; Battaglia, Agatino; Cameli, Cinzia; Lomartire, Silvia; Tancredi, Raffaella; Thomson, Susanne; Sutcliffe, James S; Maestrini, Elena

2015-04-01

Chromosome 15q13.3 recurrent microdeletions are causally associated with a wide range of phenotypes, including autism spectrum disorder (ASD), seizures, intellectual disability, and other psychiatric conditions. Whether the reciprocal microduplication is pathogenic is less certain. CHRNA7, encoding for the alpha7 subunit of the neuronal nicotinic acetylcholine receptor, is considered the likely culprit gene in mediating neurological phenotypes in 15q13.3 deletion cases. To assess if CHRNA7 rare variants confer risk to ASD, we performed copy number variant analysis and Sanger sequencing of the CHRNA7 coding sequence in a sample of 135 ASD cases. Sequence variation in this gene remains largely unexplored, given the existence of a fusion gene, CHRFAM7A, which includes a nearly identical partial duplication of CHRNA7. Hence, attempts to sequence coding exons must distinguish between CHRNA7 and CHRFAM7A, making next-generation sequencing approaches unreliable for this purpose. A CHRNA7 microduplication was detected in a patient with autism and moderate cognitive impairment; while no rare damaging variants were identified in the coding region, we detected rare variants in the promoter region, previously described to functionally reduce transcription. This study represents the first sequence variant analysis of CHRNA7 in a sample of idiopathic autism. © 2015 Wiley Periodicals, Inc.

The Sequences of 1504 Mutants in the Model Rice Variety Kitaake Facilitate Rapid Functional Genomic Studies

PubMed Central

Pham, Nikki T.; Wei, Tong; Schackwitz, Wendy S.; Lipzen, Anna M.; Duong, Phat Q.; Jones, Kyle C.; Ruan, Deling; Bauer, Diane; Peng, Yi; Schmutz, Jeremy

2017-01-01

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa ssp japonica), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations. PMID:28576844

Scanning the Effects of Ethyl Methanesulfonate on the Whole Genome of Lotus japonicus Using Second-Generation Sequencing Analysis

PubMed Central

Mohd-Yusoff, Nur Fatihah; Ruperao, Pradeep; Tomoyoshi, Nurain Emylia; Edwards, David; Gresshoff, Peter M.; Biswas, Bandana; Batley, Jacqueline

2015-01-01

Genetic structure can be altered by chemical mutagenesis, which is a common method applied in molecular biology and genetics. Second-generation sequencing provides a platform to reveal base alterations occurring in the whole genome due to mutagenesis. A model legume, Lotus japonicus ecotype Miyakojima, was chemically mutated with alkylating ethyl methanesulfonate (EMS) for the scanning of DNA lesions throughout the genome. Using second-generation sequencing, two individually mutated third-generation progeny (M3, named AM and AS) were sequenced and analyzed to identify single nucleotide polymorphisms and reveal the effects of EMS on nucleotide sequences in these mutant genomes. Single-nucleotide polymorphisms were found in every 208 kb (AS) and 202 kb (AM) with a bias mutation of G/C-to-A/T changes at low percentage. Most mutations were intergenic. The mutation spectrum of the genomes was comparable in their individual chromosomes; however, each mutated genome has unique alterations, which are useful to identify causal mutations for their phenotypic changes. The data obtained demonstrate that whole genomic sequencing is applicable as a high-throughput tool to investigate genomic changes due to mutagenesis. The identification of these single-point mutations will facilitate the identification of phenotypically causative mutations in EMS-mutated germplasm. PMID:25660167

Progressive dopaminergic cell loss with unilateral-to-bilateral progression in a genetic model of Parkinson disease

PubMed Central

Rousseaux, Maxime W. C.; Marcogliese, Paul C.; Qu, Dianbo; Hewitt, Sarah J.; Seang, Sarah; Kim, Raymond H.; Slack, Ruth S.; Schlossmacher, Michael G.; Lagace, Diane C.; Mak, Tak W.; Park, David S.

2012-01-01

DJ-1 mutations cause autosomal recessive early-onset Parkinson disease (PD). We report a model of PD pathology: the DJ1-C57 mouse. A subset of DJ-1–nullizygous mice, when fully backcrossed to a C57BL/6J background, display dramatic early-onset unilateral loss of dopaminergic (DA) neurons in their substantia nigra pars compacta, progressing to bilateral degeneration of the nigrostriatal axis with aging. In addition, these mice exhibit age-dependent bilateral degeneration at the locus ceruleus nucleus and display mild motor behavior deficits at aged time points. These findings effectively recapitulate the early stages of PD. Therefore, the DJ1-C57 mouse provides a tool to study the preclinical aspects of neurodegeneration. Importantly, by exome sequencing, we identify candidate modifying genes that segregate with the phenotype, providing potentially critical clues into how certain genes may influence the penetrance of DJ-1–related degeneration in mice. PMID:23019375

A Cyfip2-Dependent Excitatory Interneuron Pathway Establishes the Innate Startle Threshold.

PubMed

Marsden, Kurt C; Jain, Roshan A; Wolman, Marc A; Echeverry, Fabio A; Nelson, Jessica C; Hayer, Katharina E; Miltenberg, Ben; Pereda, Alberto E; Granato, Michael

2018-04-17

Sensory experiences dynamically modify whether animals respond to a given stimulus, but it is unclear how innate behavioral thresholds are established. Here, we identify molecular and circuit-level mechanisms underlying the innate threshold of the zebrafish startle response. From a forward genetic screen, we isolated five mutant lines with reduced innate startle thresholds. Using whole-genome sequencing, we identify the causative mutation for one line to be in the fragile X mental retardation protein (FMRP)-interacting protein cyfip2. We show that cyfip2 acts independently of FMRP and that reactivation of cyfip2 restores the baseline threshold after phenotype onset. Finally, we show that cyfip2 regulates the innate startle threshold by reducing neural activity in a small group of excitatory hindbrain interneurons. Thus, we identify a selective set of genes critical to establishing an innate behavioral threshold and uncover a circuit-level role for cyfip2 in this process. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Bedside Back to Bench: Building Bridges between Basic and Clinical Genomic Research.

PubMed

Manolio, Teri A; Fowler, Douglas M; Starita, Lea M; Haendel, Melissa A; MacArthur, Daniel G; Biesecker, Leslie G; Worthey, Elizabeth; Chisholm, Rex L; Green, Eric D; Jacob, Howard J; McLeod, Howard L; Roden, Dan; Rodriguez, Laura Lyman; Williams, Marc S; Cooper, Gregory M; Cox, Nancy J; Herman, Gail E; Kingsmore, Stephen; Lo, Cecilia; Lutz, Cathleen; MacRae, Calum A; Nussbaum, Robert L; Ordovas, Jose M; Ramos, Erin M; Robinson, Peter N; Rubinstein, Wendy S; Seidman, Christine; Stranger, Barbara E; Wang, Haoyi; Westerfield, Monte; Bult, Carol

2017-03-23

Genome sequencing has revolutionized the diagnosis of genetic diseases. Close collaborations between basic scientists and clinical genomicists are now needed to link genetic variants with disease causation. To facilitate such collaborations, we recommend prioritizing clinically relevant genes for functional studies, developing reference variant-phenotype databases, adopting phenotype description standards, and promoting data sharing. Published by Elsevier Inc.

Bedside Back to Bench: Building Bridges between Basic and Clinical Genomic Research

PubMed Central

Manolio, Teri A.; Fowler, Douglas M.; Starita, Lea M.; Haendel, Melissa A.; MacArthur, Daniel G.; Biesecker, Leslie G.; Worthey, Elizabeth; Chisholm, Rex L.; Green, Eric D.; Jacob, Howard J.; McLeod, Howard L.; Roden, Dan; Rodriguez, Laura Lyman; Williams, Marc S.; Cooper, Gregory M.; Cox, Nancy J.; Herman, Gail E.; Kingsmore, Stephen; Lo, Cecilia; Lutz, Cathleen; MacRae, Calum A.; Nussbaum, Robert L.; Ordovas, Jose M.; Ramos, Erin M.; Robinson, Peter N.; Rubinstein, Wendy S.; Seidman, Christine; Stranger, Barbara E.; Wang, Haoyi; Westerfield, Monte; Bult, Carol

2017-01-01

Summary Genome sequencing has revolutionized the diagnosis of genetic diseases. Close collaborations between basic scientists and clinical genomicists are now needed to link genetic variants with disease causation. To facilitate such collaborations we recommend prioritizing clinically relevant genes for functional studies, developing reference variant-phenotype databases, adopting phenotype description standards, and promoting data sharing. PMID:28340351

A molecular deletion of distal chromosome 4p in two families with a satellited chromosome 4 lacking the Wolf-Hirschhorn syndrome phenotype.

PubMed Central

Estabrooks, L L; Lamb, A N; Kirkman, H N; Callanan, N P; Rao, K W

1992-01-01

We report two families with a satellited chromosome 4 short arm (4ps). Satellites and stalks normally occur on the short arms of acrocentric chromosomes; however, the literature cites several reports of satellited nonacrocentric chromosomes, which presumably result from a translocation with an acrocentric chromosome. This is the first report of 4ps chromosomes. Our families are remarkable in that both unaffected and affected individuals carry the 4ps chromosome. The phenotypes observed in affected individuals, although dissimilar, were sufficient to encourage a search for a deletion of chromosome 4p. By Southern blot analysis and fluorescence in situ hybridization, a deletion of material mapping approximately 150 kb from chromosome 4pter was discovered. This deletion is notable because it does not result in the Wolf-Hirschhorn syndrome and can result in an apparently normal phenotype. We speculate that homology between subterminal repeat sequences on 4p and sequences on the acrocentric short arms may explain the origin of the rearrangement and that position effect may play a role in the expression of the abnormal phenotype. Images Figure 2 Figure 3 PMID:1384329

DNA polymerase having modified nucleotide binding site for DNA sequencing

DOEpatents

Tabor, S.; Richardson, C.

1997-03-25

A modified gene encoding a modified DNA polymerase is disclosed. The modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase. 6 figs.

Whole exome sequencing in an Italian family with isolated maxillary canine agenesis and canine eruption anomalies.

PubMed

Barbato, Ersilia; Traversa, Alice; Guarnieri, Rosanna; Giovannetti, Agnese; Genovesi, Maria Luce; Magliozzi, Maria Rosa; Paolacci, Stefano; Ciolfi, Andrea; Pizzi, Simone; Di Giorgio, Roberto; Tartaglia, Marco; Pizzuti, Antonio; Caputo, Viviana

2018-07-01

The aim of this study was the clinical and molecular characterization of a family segregating a trait consisting of a phenotype specifically involving the maxillary canines, including agenesis, impaction and ectopic eruption, characterized by incomplete penetrance and variable expressivity. Clinical standardized assessment of 14 family members and a whole-exome sequencing (WES) of three affected subjects were performed. WES data analyses (sequence alignment, variant calling, annotation and prioritization) were carried out using an in-house implemented pipeline. Variant filtering retained coding and splice-site high quality private and rare variants. Variant prioritization was performed taking into account both the disruptive impact and the biological relevance of individual variants and genes. Sanger sequencing was performed to validate the variants of interest and to carry out segregation analysis. Prioritization of variants "by function" allowed the identification of multiple variants contributing to the trait, including two concomitant heterozygous variants in EDARADD (c.308C>T, p.Ser103Phe) and COL5A1 (c.1588G>A, p.Gly530Ser), specifically associated with a more severe phenotype (i.e. canine agenesis). Differently, heterozygous variants in genes encoding proteins with a role in the WNT pathway were shared by subjects showing a phenotype of impacted/ectopic erupted canines. This study characterized the genetic contribution underlying a complex trait consisting of isolated canine anomalies in a medium-sized family, highlighting the role of WNT and EDA cell signaling pathways in tooth development. Copyright © 2018 Elsevier Ltd. All rights reserved.

Phenotypic H-Antigen Typing by Mass Spectrometry Combined with Genetic Typing of H Antigens, O Antigens, and Toxins by Whole-Genome Sequencing Enhances Identification of Escherichia coli Isolates.

PubMed

Cheng, Keding; Chui, Huixia; Domish, Larissa; Sloan, Angela; Hernandez, Drexler; McCorrister, Stuart; Robinson, Alyssia; Walker, Matthew; Peterson, Lorea A M; Majcher, Miles; Ratnam, Sam; Haldane, David J M; Bekal, Sadjia; Wylie, John; Chui, Linda; Tyler, Shaun; Xu, Bianli; Reimer, Aleisha; Nadon, Celine; Knox, J David; Wang, Gehua

2016-08-01

Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinical Escherichia coli isolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprising wzx and wzy and that comprising wzm and wzt The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenic E. coli typing. Copyright © 2016 Cheng et al.

Genotypic and phenotypic diversity of Alicyclobacillus acidocaldarius isolates.

PubMed

Félix-Valenzuela, L; Guardiola-Avila, I; Burgara-Estrella, A; Ibarra-Zavala, M; Mata-Haro, V

2015-10-01

The fruit juice industry recognizes Alicyclobacillus as a major quality control target micro-organism. In this study, we analysed 19 bacterial isolates to identify Alicyclobacillus species by polymerase chain reaction (PCR) and sequencing analyses. Phenotypic and genomic diversity among isolates were investigated by API 50CHB system and ERIC-PCR (enterobacterial repetitive intergenic consensus-PCR) respectively. All bacterial isolates were identified as Alicyclobacillus acidocaldarius, and almost all showed identical DNA sequences according to their 16S rRNA (rDNA) gene partial sequences. Only few carbohydrates were fermented by A. acidocaldarius isolates, and there was little variability in the biochemical profile. Genotypic fingerprinting of the A. acidocaldarius isolates showed high diversity, and clusters by ERIC-PCR were distinct to those obtained from the 16S rRNA gene phylogenetic tree. There was no correlation between phenotypic and genotypic variability in the A. acidocaldarius isolates analysed in this study. Detection of Alicyclobacillus strains is imperative in fruit concentrates and juices due to the production of guaiacol. Identification of the genera originates rejection of the product by processing industry. However, not all the Alicyclobacillus species are deteriorative and hence the importance to differentiate among them. In this study, partial 16S ribosomal RNA sequence alignment allowed the differentiation of species. In addition, ERIC-PCR was introduced for the genotypic characterization of Alicyclobacillus, as an alternative for differentiation among isolates from the same species. © 2015 The Society for Applied Microbiology.

Basfia succiniciproducens gen. nov., sp. nov., a new member of the family Pasteurellaceae isolated from bovine rumen.

PubMed

Kuhnert, Peter; Scholten, Edzard; Haefner, Stefan; Mayor, Désirée; Frey, Joachim

2010-01-01

Gram-negative, coccoid, non-motile bacteria that are catalase-, urease- and indole-negative, facultatively anaerobic and oxidase-positive were isolated from the bovine rumen using an improved selective medium for members of the Pasteurellaceae. All strains produced significant amounts of succinic acid under anaerobic conditions with glucose as substrate. Phenotypic characterization and multilocus sequence analysis (MLSA) using 16S rRNA, rpoB, infB and recN genes were performed on seven independent isolates. All four genes showed high sequence similarity to their counterparts in the genome sequence of the patent strain MBEL55E, but less than 95 % 16S rRNA gene sequence similarity to any other species of the Pasteurellaceae. Genetically these strains form a very homogeneous group in individual as well as combined phylogenetic trees, clearly separated from other genera of the family from which they can also be separated based on phenotypic markers. Genome relatedness as deduced from the recN gene showed high interspecies similarities, but again low similarity to any of the established genera of the family. No toxicity towards bovine, human or fish cells was observed and no RTX toxin genes were detected in members of the new taxon. Based on phylogenetic clustering in the MLSA analysis, the low genetic similarity to other genera and the phenotypic distinction, we suggest to classify these bovine rumen isolates as Basfia succiniciproducens gen. nov., sp. nov. The type strain is JF4016(T) (=DSM 22022(T) =CCUG 57335(T)).

Demographic History of a Recent Invasion of House Mice on the Isolated Island of Gough

PubMed Central

Gray, Melissa M.; Wegmann, Daniel; Haasl, Ryan J.; White, Michael A.; Gabriel, Sofia I.; Searle, Jeremy B.; Cuthbert, Richard J.; Ryan, Peter G.; Payseur, Bret A.

2014-01-01

Island populations provide natural laboratories for studying key contributors to evolutionary change, including natural selection, population size, and the colonization of new environments. The demographic histories of island populations can be reconstructed from patterns of genetic diversity. House mice (Mus musculus) inhabit islands throughout the globe, making them an attractive system for studying island colonization from a genetic perspective. Gough Island, in the central South Atlantic Ocean, is one of the remotest islands in the world. House mice were introduced to Gough Island by sealers during the 19th century, and display unusual phenotypes, including exceptionally large body size and carnivorous feeding behavior. We describe genetic variation in Gough Island mice using mitochondrial sequences, nuclear sequences, and microsatellites. Phylogenetic analysis of mitochondrial sequences suggested that Gough Island mice belong to Mus musculus domesticus, with the maternal lineage possibly originating in England or France. Cluster analyses of microsatellites revealed genetic membership for Gough Island mice in multiple coastal populations in Western Europe, suggesting admixed ancestry. Gough Island mice showed substantial reductions in mitochondrial and nuclear sequence variation and weak reductions in microsatellite diversity compared with Western European populations, consistent with a population bottleneck. Approximate Bayesian Computation (ABC) estimated that mice recently colonized Gough Island (~100 years ago) and experienced a 98% reduction in population size followed by a rapid expansion. Our results indicate that the unusual phenotypes of Gough Island mice evolved rapidly, positioning these mice as useful models for understanding rapid phenotypic evolution. PMID:24617968

Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction

PubMed Central

Kirkby, Nicholas S.; Chan, Melissa V.; Finsterbusch, Michaela; Hogg, Nancy; Nourshargh, Sussan; Warner, Timothy D.

2015-01-01

Testing of platelet function is central to the cardiovascular phenotyping of genetically modified mice. Traditional platelet function tests have been developed primarily for testing human samples and the volumes required make them highly unsuitable for the testing of mouse platelets. This limits research in this area. To address this problem, we have developed a miniaturized whole blood aggregometry assay, based on a readily accessible 96-well plate format coupled with quantification of single platelet depletion by flow cytometric analysis. Using this approach, we observed a concentration-dependent loss of single platelets in blood exposed to arachidonic acid, collagen, U46619 or protease activated receptor 4 activating peptide. This loss was sensitive to well-established antiplatelet agents and genetic manipulation of platelet activation pathways. Observations were more deeply analyzed by flow cytometric imaging, confocal imaging, and measurement of platelet releasates. Phenotypic analysis of the reactivity of platelets taken from mice lacking intercellular adhesion molecule (ICAM)-1 identified a marked decrease in fibrinogen-dependent platelet-monocyte interactions, especially under inflammatory conditions. Such findings exemplify the value of screening platelet phenotypes of genetically modified mice and shed further light upon the roles and interactions of platelets in inflammation. PMID:26215112

Gtl2lacZ, an insertional mutation on mouse chromosome 12 with parental origin-dependent phenotype.

PubMed

Schuster-Gossler, K; Simon-Chazottes, D; Guenet, J L; Zachgo, J; Gossler, A

1996-01-01

We have produced a transgenic mouse line, Gtl2lacZ (Gene trap locus 2), that carries an insertional mutation with a dominant modified pattern of inheritance:heterozygous Gtl2lacZ mice that inherited the transgene from the father show a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype is strongly reduced in Gtl2lacZ mice that inherited the transgene from the mother. On a mixed genetic background this pattern of inheritance was reversible upon transmission of the transgene through the germ line of the opposite sex. On a predominantly 129/Sv genetic background, however, transgene passage through the female germ line modified the transgene effect, such that the penetrance of the mutation was drastically reduced and the phenotype was no longer obvious after subsequent male germ line transmission. Expression of the transgene, however, was neither affected by genetic background nor by parental legacy. Gtl2lacZ maps to mouse Chromosome 12 in a region that displays imprinting effects associated with maternal and paternal disomy. Our results suggest that the transgene insertion in Gtl2lacZ mice affects an endogenous gene(s) required for fetal and postnatal growth and that this gene(s) is predominantly paternally expressed.

Mapping QTL for popping expansion volume in popcorn with simple sequence repeat markers.

PubMed

Lu, H-J; Bernardo, R; Ohm, H W

2003-02-01

Popping expansion volume is the most important quality trait in popcorn ( Zea mays L.), but its genetics is not well understood. The objectives of this study were to map quantitative trait loci (QTLs) responsible for popping expansion volume in a popcorn x dent corn cross, and to compare the predicted efficiencies of phenotypic selection, marker-based selection, and marker-assisted selection for popping expansion volume. Of 259 simple sequence repeat (SSR) primer pairs screened, 83 pairs were polymorphic between the H123 (dent corn) and AG19 (popcorn) parental inbreds. Popping test data were obtained for 160 S(1) families developed from the [AG19(H123 x AG19)] BC(1) population. The heritability ( h(2)) for popping expansion volume on an S(1) family mean basis was 0.73. The presence of the gametophyte factor Ga1(s) in popcorn complicates the analysis of popcorn x dent corn crosses. But, from a practical perspective, the linkage between a favorable QTL allele and Ga1(s) in popcorn will lead to selection for the favorable QTL allele. Four QTLs, on chromosomes 1S, 3S, 5S and 5L, jointly explained 45% of the phenotypic variation. Marker-based selection for popping expansion volume would require less time and work than phenotypic selection. But due to the high h(2) of popping expansion volume, marker-based selection was predicted to be only 92% as efficient as phenotypic selection. Marker-assisted selection, which comprises index selection on phenotypic and marker scores, was predicted to be 106% as efficient as phenotypic selection. Overall, our results suggest that phenotypic selection will remain the preferred method for selection in popcorn x dent corn crosses.

Allelic heterogeneity of FGF5 mutations causes the long-hair phenotype in dogs.

PubMed

Dierks, C; Mömke, S; Philipp, U; Distl, O

2013-08-01

Hitherto, the only known mutant gene leading to the long-hair phenotype in mammals is the fibroblast growth factor 5 (FGF5). In many dog breeds, the previously discovered FGF5:p.Cys95Phe mutation appeared completely concordant with the long-hair phenotype, but for some breeds, the long-hair phenotype could not be resolved. First, we studied the role of the FGF5:p.Cys95Phe and FGF5:g.145_150dupACCAGC mutations in 268 dogs descending from 27 breeds and seven wolves. As these mutations did not explain all the long-hair phenotypes, all exons and their neighbouring regions of FGF5 were re-sequenced. We detected three novel mutations in the coding sequence and one novel non-coding splice-site mutation in FGF5 associated with the long-hair phenotype. The FGF5:p.Ala193Val polymorphism was perfectly consistent with long hair in Akitas and probably in Siberian huskies, too. Dogs of the long-hair breed Samoyed were either homozygous or compound heterozygous for the FGF5:p.Ala193Val or the FGF5:p.Cys95Phe polymorphisms respectively. The two newly detected polymorphisms FGF5:c.559_560dupGG and FGF5:g.8193T>A and the known mutation FGF5:p.Cys95Phe explained the long-hair phenotype of all Afghan hounds analysed. An FGF5:c.556_571del16 mutation was found in one longhaired Eurasier. All long-hair-associated mutations follow a recessive mode of inheritance, and allelic heterogeneity was a common finding in breeds other than Akita. © 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.

Whole genome sequencing of one complex pedigree illustrates challenges with genomic medicine.

PubMed

Fang, Han; Wu, Yiyang; Yang, Hui; Yoon, Margaret; Jiménez-Barrón, Laura T; Mittelman, David; Robison, Reid; Wang, Kai; Lyon, Gholson J

2017-02-23

Human Phenotype Ontology (HPO) has risen as a useful tool for precision medicine by providing a standardized vocabulary of phenotypic abnormalities to describe presentations of human pathologies; however, there have been relatively few reports combining whole genome sequencing (WGS) and HPO, especially in the context of structural variants. We illustrate an integrative analysis of WGS and HPO using an extended pedigree, which involves Prader-Willi Syndrome (PWS), hereditary hemochromatosis (HH), and dysautonomia-like symptoms. A comprehensive WGS pipeline was used to ensure reliable detection of genomic variants. Beyond variant filtering, we pursued phenotypic prioritization of candidate genes using Phenolyzer. Regarding PWS, WGS confirmed a 5.5 Mb de novo deletion of the parental allele at 15q11.2 to 15q13.1. Phenolyzer successfully returned the diagnosis of PWS, and pinpointed clinically relevant genes in the deletion. Further, Phenolyzer revealed how each of the genes is linked with the phenotypes represented by HPO terms. For HH, WGS identified a known disease variant (p.C282Y) in HFE of an affected female. Analysis of HPO terms alone fails to provide a correct diagnosis, but Phenolyzer successfully revealed the phenotype-genotype relationship using a disease-centric approach. Finally, Phenolyzer also revealed the complexity behind dysautonomia-like symptoms, and seven variants that might be associated with the phenotypes were identified by manual filtering based on a dominant inheritance model. The integration of WGS and HPO can inform comprehensive molecular diagnosis for patients, eliminate false positives and reveal novel insights into undiagnosed diseases. Due to extreme heterogeneity and insufficient knowledge of human diseases, it is also important that phenotypic and genomic data are standardized and shared simultaneously.

Role of reverse phenotyping in interpretation of next generation sequencing data and a review of INPP5E related disorders.

PubMed

de Goede, Christian; Yue, Wyatt W; Yan, Guanhua; Ariyaratnam, Shyamala; Chandler, Kate E; Downes, Laura; Khan, Nasaim; Mohan, Meyyammai; Lowe, Martin; Banka, Siddharth

2016-03-01

Next Generation Sequencing (NGS) is a useful tool in diagnosis of rare disorders but the interpretation of data can be challenging in clinical settings. We present results of extended studies on a family of multiple members with global developmental delay and learning disability, where another research group postulated the underlying cause to be a homozygous RABL6 missense variant. Using data from the Exome Variant Server, we show that missense RABL6 variants are unlikely to cause early onset rare developmental disorder. Protein structural analysis, cellular functional studies and reverse phenotyping proved that the condition in this family is due to a homozygous INPP5E mutation. An in-depth review of mutational and phenotypic spectrum associated with INPP5E demonstrated that mutations in this gene lead to a range of cilliopathy-phenotypes. We use this study as an example to demonstrate the importance of careful clinical evaluation of multiple family members, reverse phenotyping, considering the unknown phenotypic variability of rare diseases, utilizing publically available genomic databases and conducting appropriate bioinformatics and functional studies while interpreting results from NGS in uncertain cases. We emphasize that interpretation of NGS data is an iterative process and its dynamic nature should be explained to patients and families. Our study shows that developmental delay, intellectual disability, hypotonia and ocular motor apraxia are common in INPP5E-related disorders and considerable intra-familial phenotypic variability is possible. We have compiled the INPP5E mutational spectrum and provided novel insights into their molecular mechanisms. Copyright © 2015 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

Structure and function of neonatal social communication in a genetic mouse model of autism.

PubMed

Takahashi, T; Okabe, S; Broin, P Ó; Nishi, A; Ye, K; Beckert, M V; Izumi, T; Machida, A; Kang, G; Abe, S; Pena, J L; Golden, A; Kikusui, T; Hiroi, N

2016-09-01

A critical step toward understanding autism spectrum disorder (ASD) is to identify both genetic and environmental risk factors. A number of rare copy number variants (CNVs) have emerged as robust genetic risk factors for ASD, but not all CNV carriers exhibit ASD and the severity of ASD symptoms varies among CNV carriers. Although evidence exists that various environmental factors modulate symptomatic severity, the precise mechanisms by which these factors determine the ultimate severity of ASD are still poorly understood. Here, using a mouse heterozygous for Tbx1 (a gene encoded in 22q11.2 CNV), we demonstrate that a genetically triggered neonatal phenotype in vocalization generates a negative environmental loop in pup-mother social communication. Wild-type pups used individually diverse sequences of simple and complicated call types, but heterozygous pups used individually invariable call sequences with less complicated call types. When played back, representative wild-type call sequences elicited maternal approach, but heterozygous call sequences were ineffective. When the representative wild-type call sequences were randomized, they were ineffective in eliciting vigorous maternal approach behavior. These data demonstrate that an ASD risk gene alters the neonatal call sequence of its carriers and this pup phenotype in turn diminishes maternal care through atypical social communication. Thus, an ASD risk gene induces, through atypical neonatal call sequences, less than optimal maternal care as a negative neonatal environmental factor.

Structure and function of neonatal social communication in a genetic mouse model of autism

PubMed Central

Takahashi, Tomohisa; Okabe, Shota; Ó Broin, Pilib; Nishi, Akira; Ye, Kenny; Beckert, Michael V.; Izumi, Takeshi; Machida, Akihiro; Kang, Gina; Abe, Seiji; Pena, Jose L.; Golden, Aaron; Kikusui, Takefumi; Hiroi, Noboru

2015-01-01

A critical step toward understanding autism spectrum disorder (ASD) is to identify both genetic and environmental risk factors. A number of rare copy number variants (CNVs) have emerged as robust genetic risk factors for ASD, but not all CNV carriers exhibit ASD and the severity of ASD symptoms varies among CNV carriers. Although evidence exists that various environmental factors modulate symptomatic severity, the precise mechanisms by which these factors determine the ultimate severity of ASD are still poorly understood. Here, using a mouse heterozygous for Tbx1 (a gene encoded in 22q11.2 CNV), we demonstrate that a genetically-triggered neonatal phenotype in vocalization generates a negative environmental loop in pup-mother social communication. Wild-type pups used individually diverse sequences of simple and complicated call types, but heterozygous pups used individually invariable call sequences with less complicated call types. When played back, representative wild-type call sequences elicited maternal approach, but heterozygous call sequences were ineffective. When the representative wild-type call sequences were randomized, they were ineffective in eliciting vigorous maternal approach behavior. These data demonstrate that an ASD risk gene alters the neonatal call sequence of its carriers and this pup phenotype in turn diminishes maternal care through atypical social communication. Thus, an ASD risk gene induces, through atypical neonatal call sequences, less than optimal maternal care as a negative neonatal environmental factor. PMID:26666205

Phenotypic Characterization of pncA Mutants of Mycobacterium tuberculosis

PubMed Central

Morlock, Glenn P.; Crawford, Jack T.; Butler, W. Ray; Brim, Suzanne E.; Sikes, David; Mazurek, Gerald H.; Woodley, Charles L.; Cooksey, Robert C.

2000-01-01

We examined the correlation of mutations in the pyrazinamidase (PZase) gene (pncA) with the pyrazinamide (PZA) resistance phenotype with 60 Mycobacterium tuberculosis isolates. PZase activity was determined by the method of Wayne (L. G. Wayne, Am. Rev. Respir. Dis. 109:147–151, 1974), and the entire pncA nucleotide sequence, including the 74 bp upstream of the start codon, was determined. PZA susceptibility testing was performed by the method of proportions on modified Middlebrook and Cohn 7H10 medium. The PZA MICs were ≥100 μg/ml for 37 isolates, 34 of which had alterations in the pncA gene. These mutations included missense substitutions for 24 isolates, nonsense substitutions for 3 isolates, frameshifts by deletion for 4 isolates, a three-codon insertion for 1 isolate, and putative regulatory mutations for 2 isolates. Among 21 isolates for which PZA MICs were <100 μg/ml, 3 had the same mutation (Thr47→Ala) and 18 had the wild-type sequence. For the three Thr47→Ala mutants PZA MICs were 12.5 μg/ml by the method of proportions on 7H10 agar; two of these were resistant to 100 μg of PZA per ml and the third was resistant to 800 μg of PZA per ml by the BACTEC method. In all, 30 different pncA mutations were found among the 37 pncA mutants. No PZase activity was detected in 35 of 37 strains that were resistant to ≥100 μg of PZA per ml or in 34 of 37 pncA mutants. Reduced PZase activity was found in the three mutants with the Thr47→Ala mutation. This study demonstrates that mutations in the pncA gene may serve as a reliable indicator of resistance to ≥100 μg of PZA per ml. PMID:10952570

OVA: integrating molecular and physical phenotype data from multiple biomedical domain ontologies with variant filtering for enhanced variant prioritization.

PubMed

Antanaviciute, Agne; Watson, Christopher M; Harrison, Sally M; Lascelles, Carolina; Crinnion, Laura; Markham, Alexander F; Bonthron, David T; Carr, Ian M

2015-12-01

Exome sequencing has become a de facto standard method for Mendelian disease gene discovery in recent years, yet identifying disease-causing mutations among thousands of candidate variants remains a non-trivial task. Here we describe a new variant prioritization tool, OVA (ontology variant analysis), in which user-provided phenotypic information is exploited to infer deeper biological context. OVA combines a knowledge-based approach with a variant-filtering framework. It reduces the number of candidate variants by considering genotype and predicted effect on protein sequence, and scores the remainder on biological relevance to the query phenotype.We take advantage of several ontologies in order to bridge knowledge across multiple biomedical domains and facilitate computational analysis of annotations pertaining to genes, diseases, phenotypes, tissues and pathways. In this way, OVA combines information regarding molecular and physical phenotypes and integrates both human and model organism data to effectively prioritize variants. By assessing performance on both known and novel disease mutations, we show that OVA performs biologically meaningful candidate variant prioritization and can be more accurate than another recently published candidate variant prioritization tool. OVA is freely accessible at http://dna2.leeds.ac.uk:8080/OVA/index.jsp. Supplementary data are available at Bioinformatics online. umaan@leeds.ac.uk. © The Author 2015. Published by Oxford University Press.

Hypolocomotion, anxiety and serotonin syndrome-like behavior contribute to the complex phenotype of serotonin transporter knockout mice.

PubMed

Kalueff, A V; Fox, M A; Gallagher, P S; Murphy, D L

2007-06-01

Although mice with a targeted disruption of the serotonin transporter (SERT) have been studied extensively using various tests, their complex behavioral phenotype is not yet fully understood. Here we assess in detail the behavior of adult female SERT wild type (+/+), heterozygous (+/-) and knockout (-/-) mice on an isogenic C57BL/6J background subjected to a battery of behavioral paradigms. Overall, there were no differences in the ability to find food or a novel object, nest-building, self-grooming and its sequencing, and horizontal rod balancing, indicating unimpaired sensory functions, motor co-ordination and behavioral sequencing. In contrast, there were striking reductions in exploration and activity in novelty-based tests (novel object, sticky label and open field tests), accompanied by pronounced thigmotaxis, suggesting that combined hypolocomotion and anxiety (rather than purely anxiety) influence the SERT -/- behavioral phenotype. Social interaction behaviors were also markedly reduced. In addition, SERT -/- mice tended to move close to the ground, frequently displayed spontaneous Straub tail, tics, tremor and backward gait - a phenotype generally consistent with 'serotonin syndrome'-like behavior. In line with replicated evidence of much enhanced serotonin availability in SERT -/- mice, this serotonin syndrome-like state may represent a third factor contributing to their behavioral profile. An understanding of the emerging complexity of SERT -/- mouse behavior is crucial for a detailed dissection of their phenotype and for developing further neurobehavioral models using these mice.

The spectrum and clinical impact of epigenetic modifier mutations in myeloma

PubMed Central

Pawlyn, Charlotte; Kaiser, Martin F; Heuck, Christoph; Melchor, Lorenzo; Wardell, Christopher P; Murison, Alex; Chavan, Shweta; Johnson, David C; Begum, Dil; Dahir, Nasrin; Proszek, Paula; Cairns, David A; Boyle, Eileen M; Jones, John R; Cook, Gordon; Drayson, Mark T; Owen, Roger G; Gregory, Walter M; Jackson, Graham H; Barlogie, Bart; Davies, Faith E; Walker, Brian A; Morgan, Gareth J

2016-01-01

Purpose Epigenetic dysregulation is known to be an important contributor to myeloma pathogenesis but, unlike in other B cell malignancies, the full spectrum of somatic mutations in epigenetic modifiers has not been previously reported. We sought to address this using results from whole-exome sequencing in the context of a large prospective clinical trial of newly diagnosed patients and targeted sequencing in a cohort of previously treated patients for comparison. Experimental Design Whole-exome sequencing analysis of 463 presenting myeloma cases entered in the UK NCRI Myeloma XI study and targeted sequencing analysis of 156 previously treated cases from the University of Arkansas for Medical Sciences. We correlated the presence of mutations with clinical outcome from diagnosis and compared the mutations found at diagnosis with later stages of disease. Results In diagnostic myeloma patient samples we identify significant mutations in genes encoding the histone 1 linker protein, previously identified in other B-cell malignancies. Our data suggest an adverse prognostic impact from the presence of lesions in genes encoding DNA methylation modifiers and the histone demethylase KDM6A/UTX. The frequency of mutations in epigenetic modifiers appears to increase following treatment most notably in genes encoding histone methyltransferases and DNA methylation modifiers. Conclusions Numerous mutations identified raise the possibility of targeted treatment strategies for patients either at diagnosis or relapse supporting the use of sequencing-based diagnostics in myeloma to help guide therapy as more epigenetic targeted agents become available. PMID:27235425

CaMV-35S promoter sequence-specific DNA methylation in lettuce.

PubMed

Okumura, Azusa; Shimada, Asahi; Yamasaki, Satoshi; Horino, Takuya; Iwata, Yuji; Koizumi, Nozomu; Nishihara, Masahiro; Mishiba, Kei-ichiro

2016-01-01

We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery. We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.

The advantages of SMRT sequencing.

PubMed

Roberts, Richard J; Carneiro, Mauricio O; Schatz, Michael C

2013-07-03

Of the current next-generation sequencing technologies, SMRT sequencing is sometimes overlooked. However, attributes such as long reads, modified base detection and high accuracy make SMRT a useful technology and an ideal approach to the complete sequencing of small genomes.

Thorough Investigation of a Canine Autoinflammatory Disease (AID) Confirms One Main Risk Locus and Suggests a Modifier Locus for Amyloidosis

PubMed Central

Olsson, Mia; Tintle, Linda; Kierczak, Marcin; Perloski, Michele; Tonomura, Noriko; Lundquist, Andrew; Murén, Eva; Fels, Max; Tengvall, Katarina; Pielberg, Gerli; Dufaure de Citres, Caroline; Dorso, Laetitia; Abadie, Jérôme; Hanson, Jeanette; Thomas, Anne; Leegwater, Peter; Hedhammar, Åke; Lindblad-Toh, Kerstin; Meadows, Jennifer R. S.

2013-01-01

Autoinflammatory disease (AID) manifests from the dysregulation of the innate immune system and is characterised by systemic and persistent inflammation. Clinical heterogeneity leads to patients presenting with one or a spectrum of phenotypic signs, leading to difficult diagnoses in the absence of a clear genetic cause. We used separate genome-wide SNP analyses to investigate five signs of AID (recurrent fever, arthritis, breed specific secondary dermatitis, otitis and systemic reactive amyloidosis) in a canine comparative model, the pure bred Chinese Shar-Pei. Analysis of 255 DNA samples revealed a shared locus on chromosome 13 spanning two peaks of association. A three-marker haplotype based on the most significant SNP (p<2.6×10−8) from each analysis showed that one haplotypic pair (H13-11) was present in the majority of AID individuals, implicating this as a shared risk factor for all phenotypes. We also noted that a genetic signature (F ST) distinguishing the phenotypic extremes of the breed specific Chinese Shar-Pei thick and wrinkled skin, flanked the chromosome 13 AID locus; suggesting that breed development and differentiation has played a parallel role in the genetics of breed fitness. Intriguingly, a potential modifier locus for amyloidosis was revealed on chromosome 14, and an investigation of candidate genes from both this and the chromosome 13 regions revealed significant (p<0.05) renal differential expression in four genes previously implicated in kidney or immune health (AOAH, ELMO1, HAS2 and IL6). These results illustrate that phenotypic heterogeneity need not be a reflection of genetic heterogeneity, and that genetic modifiers of disease could be masked if syndromes were not first considered as individual clinical signs and then as a sum of their component parts. PMID:24130694

Ab initio genotype–phenotype association reveals intrinsic modularity in genetic networks

PubMed Central

Slonim, Noam; Elemento, Olivier; Tavazoie, Saeed

2006-01-01

Microbial species express an astonishing diversity of phenotypic traits, behaviors, and metabolic capacities. However, our molecular understanding of these phenotypes is based almost entirely on studies in a handful of model organisms that together represent only a small fraction of this phenotypic diversity. Furthermore, many microbial species are not amenable to traditional laboratory analysis because of their exotic lifestyles and/or lack of suitable molecular genetic techniques. As an adjunct to experimental analysis, we have developed a computational information-theoretic framework that produces high-confidence gene–phenotype predictions using cross-species distributions of genes and phenotypes across 202 fully sequenced archaea and eubacteria. In addition to identifying the genetic basis of complex traits, our approach reveals the organization of these genes into generic preferentially co-inherited modules, many of which correspond directly to known enzymatic pathways, molecular complexes, signaling pathways, and molecular machines. PMID:16732191

Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer.

PubMed

Yu, Honghao; Long, Weihu; Zhang, Xuezeng; Xu, Kaixiang; Guo, Jianxiong; Zhao, Heng; Li, Honghui; Qing, Yubo; Pan, Weirong; Jia, Baoyu; Zhao, Hong-Ye; Huang, Xingxu; Wei, Hong-Jiang

2018-02-27

Laron syndrome is an autosomal disease resulting from mutations in the growth hormone receptor (GHR) gene. The only therapeutic treatment for Laron syndrome is recombinant insulin-like growth factor I (IGF-I), which has been shown to have various side effects. The improved Laron syndrome models are important for better understanding the pathogenesis of the disease and developing corresponding therapeutics. Pigs have become attractive biomedical models for human condition due to similarities in anatomy, physiology, and metabolism relative to humans, which could serve as an appropriate model for Laron syndrome. To further improve the GHR knockout (GHRKO) efficiency and explore the feasibility of precise DNA deletion at targeted sites, the dual-sgRNAs/Cas9 system was designed to target GHR exon 3 in pig fetal fibroblasts (PFFs). The vectors encoding sgRNAs and Cas9 were co-transfected into PFFs by electroporation and GHRKO cell lines were established by single cell cloning culture. Two biallelic knockout cell lines were selected as the donor cell line for somatic cell nuclear transfer for the generation of GHRKO pigs. The genotype of colonies, cloned fetuses and piglets were identified by T7 endonuclease I (T7ENI) assay and sequencing. The GHR expression in the fibroblasts and piglets was analyzed by confocal microscopy, quantitative polymerase chain reaction (q-PCR), western blotting (WB) and immunohistochemical (IHC) staining. The phenotype of GHRKO pigs was recapitulated through level detection of IGF-I and glucose, and measurement of body weight and body size. GHRKO F1 generation were generated by crossing with wild-type pigs, and their genotype was detected by T7ENI assay and sequencing. GHRKO F2 generation was obtained via self-cross of GHRKO F1 pigs. Their genotypes of GHRKO F2 generation was also detected by Sanger sequencing. In total, 19 of 20 single-cell colonies exhibited biallelic modified GHR (95%), and the efficiency of DNA deletion mediated by dual-sgRNAs/Cas9 was as high as 90% in 40 GHR alleles of 20 single-cell colonies. Two types of GHR allelic single-cell colonies (GHR -47/-1 , GHR -47/-46 ) were selected as donor cells for the generation of GHRKO pigs. The reconstructed embryos were transferred into 15 recipient gilts, resulting in 15 GHRKO newborn piglets and 2 fetuses. The GHRKO pigs exhibited slow growth rates and small body sizes. From birth to 13 months old, the average body weight of wild-type pigs varied from 0.6 to 89.5 kg, but that of GHRKO pigs varied from only 0.9 to 37.0 kg. Biochemically, the knockout pigs exhibited decreased serum levels of IGF-I and glucose. Furthermore, the GHRKO pigs had normal reproduction ability, as eighteen GHRKO F1 piglets were obtained via mating a GHRKO pig with wild-type pigs and five GHRKO F2 piglets were obtained by self-cross of F1 generation, indicating that modified GHR alleles can pass to the next generation via germline transmission. The dual-sgRNAs/Cas9 is a reliable system for DNA deletion and that GHRKO pigs conform to typical phenotypes of those observed in Laron patients, suggesting that these pigs could serve as an appropriate model for Laron syndrome.

Targeted exome sequencing of suspected mitochondrial disorders

PubMed Central

Lieber, Daniel S.; Calvo, Sarah E.; Shanahan, Kristy; Slate, Nancy G.; Liu, Shangtao; Hershman, Steven G.; Gold, Nina B.; Chapman, Brad A.; Thorburn, David R.; Berry, Gerard T.; Schmahmann, Jeremy D.; Borowsky, Mark L.; Mueller, David M.; Sims, Katherine B.

2013-01-01

Objective: To evaluate the utility of targeted exome sequencing for the molecular diagnosis of mitochondrial disorders, which exhibit marked phenotypic and genetic heterogeneity. Methods: We considered a diverse set of 102 patients with suspected mitochondrial disorders based on clinical, biochemical, and/or molecular findings, and whose disease ranged from mild to severe, with varying age at onset. We sequenced the mitochondrial genome (mtDNA) and the exons of 1,598 nuclear-encoded genes implicated in mitochondrial biology, mitochondrial disease, or monogenic disorders with phenotypic overlap. We prioritized variants likely to underlie disease and established molecular diagnoses in accordance with current clinical genetic guidelines. Results: Targeted exome sequencing yielded molecular diagnoses in established disease loci in 22% of cases, including 17 of 18 (94%) with prior molecular diagnoses and 5 of 84 (6%) without. The 5 new diagnoses implicated 2 genes associated with canonical mitochondrial disorders (NDUFV1, POLG2), and 3 genes known to underlie other neurologic disorders (DPYD, KARS, WFS1), underscoring the phenotypic and biochemical overlap with other inborn errors. We prioritized variants in an additional 26 patients, including recessive, X-linked, and mtDNA variants that were enriched 2-fold over background and await further support of pathogenicity. In one case, we modeled patient mutations in yeast to provide evidence that recessive mutations in ATP5A1 can underlie combined respiratory chain deficiency. Conclusion: The results demonstrate that targeted exome sequencing is an effective alternative to the sequential testing of mtDNA and individual nuclear genes as part of the investigation of mitochondrial disease. Our study underscores the ongoing challenge of variant interpretation in the clinical setting. PMID:23596069

International Life Science Institute North America Cronobacter (Formerly Enterobacter sakazakii) isolate set.

PubMed

Ivy, Reid A; Farber, Jeffrey M; Pagotto, Franco; Wiedmann, Martin

2013-01-01

Foodborne pathogen isolate collections are important for the development of detection methods, for validation of intervention strategies, and to develop an understanding of pathogenesis and virulence. We have assembled a publicly available Cronobacter (formerly Enterobacter sakazakii) isolate set that consists of (i) 25 Cronobacter sakazakii isolates, (ii) two Cronobacter malonaticus isolates, (iii) one Cronobacter muytjensii isolate, which displays some atypical phenotypic characteristics, biochemical profiles, and colony color on selected differential media, and (iv) two nonclinical Enterobacter asburiae isolates, which show some phenotypic characteristics similar to those of Cronobacter spp. The set consists of human (n = 10), food (n = 11), and environmental (n = 9) isolates. Analysis of partial 16S rDNA sequence and seven-gene multilocus sequence typing data allowed for reliable identification of these isolates to species and identification of 14 isolates as sequence type 4, which had previously been shown to be the most common C. sakazakii sequence type associated with neonatal meningitis. Phenotypic characterization was carried out with API 20E and API 32E test strips and streaking on two selective chromogenic agars; isolates were also assessed for sorbitol fermentation and growth at 45°C. Although these strategies typically produced the same classification as sequence-based strategies, based on a panel of four biochemical tests, one C. sakazakii isolate yielded inconclusive data and one was classified as C. malonaticus. EcoRI automated ribotyping and pulsed-field gel electrophoresis (PFGE) with XbaI separated the set into 23 unique ribotypes and 30 unique PFGE types, respectively, indicating subtype diversity within the set. Subtype and source data for the collection are publicly available in the PathogenTracker database (www. pathogentracker. net), which allows for continuous updating of information on the set, including links to publications that include information on isolates from this collection.

A first linkage map and downy mildew resistance QTL discovery for sweet basil (Ocimum basilicum) facilitated by double digestion restriction site associated DNA sequencing (ddRADseq).

PubMed

Pyne, Robert; Honig, Josh; Vaiciunas, Jennifer; Koroch, Adolfina; Wyenandt, Christian; Bonos, Stacy; Simon, James

2017-01-01

Limited understanding of sweet basil (Ocimum basilicum L.) genetics and genome structure has reduced efficiency of breeding strategies. This is evidenced by the rapid, worldwide dissemination of basil downy mildew (Peronospora belbahrii) in the absence of resistant cultivars. In an effort to improve available genetic resources, expressed sequence tag simple sequence repeat (EST-SSR) and single nucleotide polymorphism (SNP) markers were developed and used to genotype the MRI x SB22 F2 mapping population, which segregates for response to downy mildew. SNP markers were generated from genomic sequences derived from double digestion restriction site associated DNA sequencing (ddRADseq). Disomic segregation was observed in both SNP and EST-SSR markers providing evidence of an O. basilicum allotetraploid genome structure and allowing for subsequent analysis of the mapping population as a diploid intercross. A dense linkage map was constructed using 42 EST-SSR and 1,847 SNP markers spanning 3,030.9 cM. Multiple quantitative trait loci (QTL) model (MQM) analysis identified three QTL that explained 37-55% of phenotypic variance associated with downy mildew response across three environments. A single major QTL, dm11.1 explained 21-28% of phenotypic variance and demonstrated dominant gene action. Two minor QTL dm9.1 and dm14.1 explained 5-16% and 4-18% of phenotypic variance, respectively. Evidence is provided for an additive effect between the two minor QTL and the major QTL dm11.1 increasing downy mildew susceptibility. Results indicate that ddRADseq-facilitated SNP and SSR marker genotyping is an effective approach for mapping the sweet basil genome.

A first linkage map and downy mildew resistance QTL discovery for sweet basil (Ocimum basilicum) facilitated by double digestion restriction site associated DNA sequencing (ddRADseq)

PubMed Central

Honig, Josh; Vaiciunas, Jennifer; Koroch, Adolfina; Wyenandt, Christian; Bonos, Stacy; Simon, James

2017-01-01

Limited understanding of sweet basil (Ocimum basilicum L.) genetics and genome structure has reduced efficiency of breeding strategies. This is evidenced by the rapid, worldwide dissemination of basil downy mildew (Peronospora belbahrii) in the absence of resistant cultivars. In an effort to improve available genetic resources, expressed sequence tag simple sequence repeat (EST-SSR) and single nucleotide polymorphism (SNP) markers were developed and used to genotype the MRI x SB22 F2 mapping population, which segregates for response to downy mildew. SNP markers were generated from genomic sequences derived from double digestion restriction site associated DNA sequencing (ddRADseq). Disomic segregation was observed in both SNP and EST-SSR markers providing evidence of an O. basilicum allotetraploid genome structure and allowing for subsequent analysis of the mapping population as a diploid intercross. A dense linkage map was constructed using 42 EST-SSR and 1,847 SNP markers spanning 3,030.9 cM. Multiple quantitative trait loci (QTL) model (MQM) analysis identified three QTL that explained 37–55% of phenotypic variance associated with downy mildew response across three environments. A single major QTL, dm11.1 explained 21–28% of phenotypic variance and demonstrated dominant gene action. Two minor QTL dm9.1 and dm14.1 explained 5–16% and 4–18% of phenotypic variance, respectively. Evidence is provided for an additive effect between the two minor QTL and the major QTL dm11.1 increasing downy mildew susceptibility. Results indicate that ddRADseq-facilitated SNP and SSR marker genotyping is an effective approach for mapping the sweet basil genome. PMID:28922359

The in vitro biocompatibility of d-(+) raffinose modified chitosan: Two-dimensional and three-dimensional systems for culturing of horse articular chondrocytes.

PubMed

De Angelis, Elena; Ravanetti, Francesca; Martelli, Paolo; Cacchioli, Antonio; Ivanovska, Ana; Corradi, Attilio; Nasi, Sonia; Bianchera, Annalisa; Passeri, Benedetta; Canelli, Elena; Bettini, Ruggero; Borghetti, Paolo

2017-12-01

The present study investigated the biocompatibility of chitosan films and scaffolds modified with d-(+)raffinose and their capability to support the growth and maintenance of the differentiation of articular chondrocytes in vitro. Primary equine articular chondrocytes were cultured on films and scaffolds of modified d-(+) raffinose chitosan. Their behavior was compared to that of chondrocytes grown in conventional bi- and three-dimensional culture systems, such as micromasses and alginate beads. Chitosan films maintained the phenotype of differentiated chondrocytes (typical round morphology) and sustained the synthesis of cartilaginous extracellular matrix (ECM), even at 4weeks of culture. Indeed, starting from 2weeks of culture, chondrocytes seeded on chitosan scaffolds were able to penetrate the surface pores and to colonize the internal matrix. Moreover they produced ECM expressing the genes of typical chondrocytes differentiation markers such as collagen II and aggrecan. In conclusion, chitosan modified with d-raffinose represents an ideal support for chondrocyte adhesion, proliferation and for the maintenance of cellular phenotypic and genotypic differentiation. This novel biomaterial could potentially be a reliable support for the re-differentiation of dedifferentiated chondrocytes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phenotypic models of evolution and development: geometry as destiny.

PubMed

François, Paul; Siggia, Eric D

2012-12-01

Quantitative models of development that consider all relevant genes typically are difficult to fit to embryonic data alone and have many redundant parameters. Computational evolution supplies models of phenotype with relatively few variables and parameters that allows the patterning dynamics to be reduced to a geometrical picture for how the state of a cell moves. The clock and wavefront model, that defines the phenotype of somitogenesis, can be represented as a sequence of two discrete dynamical transitions (bifurcations). The expression-time to space map for Hox genes and the posterior dominance rule are phenotypes that naturally follow from computational evolution without considering the genetics of Hox regulation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Phase 2 of the array automated assembly task for the low cost solar array project

NASA Technical Reports Server (NTRS)

Campbell, R. B.; Davis, J. R.; Ostroski, J. W.; Rai-Choudhury, P.; Rohatgi, A.; Seman, E. J.; Stapleton, R. E.

1979-01-01

The process sequence for the fabrication of dendritic web silicon into solar panels was modified to include aluminum back surface field formation. Plasma etching was found to be a feasible technique for pre-diffusion cleaning of the web. Several contacting systems were studied. The total plated Pd-Ni system was not compatible with the process sequence; however, the evaporated TiPd-electroplated Cu system was shown stable under life testing. Ultrasonic bonding parameters were determined for various interconnect and contact metals but the yield of the process was not sufficiently high to use for module fabrication at this time. Over 400 solar cells were fabricated according to the modified sequence. No sub-process incompatibility was seen. These cells were used to fabricate four demonstration modules. A cost analysis of the modified process sequence resulted in a selling price of $0.75/peak watt.

Secondary binding sites for heavily modified triplex forming oligonucleotides

PubMed Central

Cardew, Antonia S.; Brown, Tom; Fox, Keith R.

2012-01-01

In order to enhance DNA triple helix stability synthetic oligonucleotides have been developed that bear amino groups on the sugar or base. One of the most effective of these is bis-amino-U (B), which possesses 5-propargylamino and 2′-aminoethoxy modifications. Inclusion of this modified nucleotide not only greatly enhances triplex stability, but also increases the affinity for related sequences. We have used a restriction enzyme protection, selection and amplification assay (REPSA) to isolate sequences that are bound by the heavily modified 9-mer triplex-forming oligonucleotide B6CBT. The isolated sequences contain An tracts (n = 6), suggesting that the 5′-end of this TFO was responsible for successful triplex formation. DNase I footprinting with these sequences confirmed triple helix formation at these secondary targets and demonstrated no interaction with similar oligonucleotides containing T or 5-propargylamino-dU. PMID:22180535

Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica.

PubMed

Hoffmann, Maria; Pettengill, James B; Gonzalez-Escalona, Narjol; Miller, John; Ayers, Sherry L; Zhao, Shaohua; Allard, Marc W; McDermott, Patrick F; Brown, Eric W; Monday, Steven R

2017-01-01

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in plasmids, advances in plasmid sequencing, and phylogenetic analyses, and important insights about how MDR evolution occurs across diverse serotypes from different animal sources, particularly in agricultural settings where antimicrobial drug use practices vary.