Identification of a Novel De Novo Heterozygous Deletion in the SOX10 Gene in Waardenburg Syndrome Type II Using Next-Generation Sequencing.

PubMed

Li, Haonan; Jin, Peng; Hao, Qian; Zhu, Wei; Chen, Xia; Wang, Ping

2017-11-01

Waardenburg syndrome (WS) is a rare autosomal dominant disorder associated with pigmentation abnormalities and sensorineural hearing loss. In this study, we investigated the genetic cause of WSII in a patient and evaluated the reliability of the targeted next-generation exome sequencing method for the genetic diagnosis of WS. Clinical evaluations were conducted on the patient and targeted next-generation sequencing (NGS) was used to identify the candidate genes responsible for WSII. Multiplex ligation-dependent probe amplification (MLPA) and real-time quantitative polymerase chain reaction (qPCR) were performed to confirm the targeted NGS results. Targeted NGS detected the entire deletion of the coding sequence (CDS) of the SOX10 gene in the WSII patient. MLPA results indicated that all exons of the SOX10 heterozygous deletion were detected; no aberrant copy number in the PAX3 and microphthalmia-associated transcription factor (MITF) genes was found. Real-time qPCR results identified the mutation as a de novo heterozygous deletion. This is the first report of using a targeted NGS method for WS candidate gene sequencing; its accuracy was verified by using the MLPA and qPCR methods. Our research provides a valuable method for the genetic diagnosis of WS.

Deletion of exons 3-9 encompassing a mutational hot spot in the DMD gene presents an asymptomatic phenotype, indicating a target region for multiexon skipping therapy.

PubMed

Nakamura, Akinori; Fueki, Noboru; Shiba, Naoko; Motoki, Hirohiko; Miyazaki, Daigo; Nishizawa, Hitomi; Echigoya, Yusuke; Yokota, Toshifumi; Aoki, Yoshitsugu; Takeda, Shin'ichi

2016-07-01

Few cases of dystrophinopathy show an asymptomatic phenotype with mutations in the 5' (exons 3-7) hot spot in the Duchenne muscular dystrophy (DMD) gene. Our patient showed increased serum creatine kinase levels at 12 years of age. A muscle biopsy at 15 years of age led to a diagnosis of Becker muscular dystrophy. The patient showed a slight decrease in cardiac function at the age of 21 years and was administered a β-blocker, but there was no muscle involvement even at the age of 27 years. A deletion of exons 3-9 encompassing a mutational hot spot in the DMD gene was detected, and dystrophin protein expression was ∼15% that of control level. We propose that in-frame deletion of exons 3-9 may produce a functional protein, and that multiexon skipping therapy targeting these exons may be feasible for severe dystrophic patients with a mutation in the 5' hot spot of the DMD gene.

[Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System].

PubMed

Li, Lili; Wang, Zhan; Zhou, Yubai; Zhang, Fang; Shen, Sisi; Li, Zelin; Zeng, Yi

2015-09-01

For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.

Chassis organism from Corynebacterium glutamicum--a top-down approach to identify and delete irrelevant gene clusters.

PubMed

Unthan, Simon; Baumgart, Meike; Radek, Andreas; Herbst, Marius; Siebert, Daniel; Brühl, Natalie; Bartsch, Anna; Bott, Michael; Wiechert, Wolfgang; Marin, Kay; Hans, Stephan; Krämer, Reinhard; Seibold, Gerd; Frunzke, Julia; Kalinowski, Jörn; Rückert, Christian; Wendisch, Volker F; Noack, Stephan

2015-02-01

For synthetic biology applications, a robust structural basis is required, which can be constructed either from scratch or in a top-down approach starting from any existing organism. In this study, we initiated the top-down construction of a chassis organism from Corynebacterium glutamicum ATCC 13032, aiming for the relevant gene set to maintain its fast growth on defined medium. We evaluated each native gene for its essentiality considering expression levels, phylogenetic conservation, and knockout data. Based on this classification, we determined 41 gene clusters ranging from 3.7 to 49.7 kbp as target sites for deletion. 36 deletions were successful and 10 genome-reduced strains showed impaired growth rates, indicating that genes were hit, which are relevant to maintain biological fitness at wild-type level. In contrast, 26 deleted clusters were found to include exclusively irrelevant genes for growth on defined medium. A combinatory deletion of all irrelevant gene clusters would, in a prophage-free strain, decrease the size of the native genome by about 722 kbp (22%) to 2561 kbp. Finally, five combinatory deletions of irrelevant gene clusters were investigated. The study introduces the novel concept of relevant genes and demonstrates general strategies to construct a chassis suitable for biotechnological application. © 2014 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-Non-Commercial-NoDerivs Licence, which permits use and distribution in any medium, provided the original work is properly cited, the use is non- commercial and no modifications or adaptations are made.

A high-throughput method for the detection of homoeologous gene deletions in hexaploid wheat

PubMed Central

2010-01-01

Background Mutational inactivation of plant genes is an essential tool in gene function studies. Plants with inactivated or deleted genes may also be exploited for crop improvement if such mutations/deletions produce a desirable agronomical and/or quality phenotype. However, the use of mutational gene inactivation/deletion has been impeded in polyploid plant species by genetic redundancy, as polyploids contain multiple copies of the same genes (homoeologous genes) encoded by each of the ancestral genomes. Similar to many other crop plants, bread wheat (Triticum aestivum L.) is polyploid; specifically allohexaploid possessing three progenitor genomes designated as 'A', 'B', and 'D'. Recently modified TILLING protocols have been developed specifically for mutation detection in wheat. Whilst extremely powerful in detecting single nucleotide changes and small deletions, these methods are not suitable for detecting whole gene deletions. Therefore, high-throughput methods for screening of candidate homoeologous gene deletions are needed for application to wheat populations generated by the use of certain mutagenic agents (e.g. heavy ion irradiation) that frequently generate whole-gene deletions. Results To facilitate the screening for specific homoeologous gene deletions in hexaploid wheat, we have developed a TaqMan qPCR-based method that allows high-throughput detection of deletions in homoeologous copies of any gene of interest, provided that sufficient polymorphism (as little as a single nucleotide difference) amongst homoeologues exists for specific probe design. We used this method to identify deletions of individual TaPFT1 homoeologues, a wheat orthologue of the disease susceptibility and flowering regulatory gene PFT1 in Arabidopsis. This method was applied to wheat nullisomic-tetrasomic lines as well as other chromosomal deletion lines to locate the TaPFT1 gene to the long arm of chromosome 5. By screening of individual DNA samples from 4500 M2 mutant wheat

In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology

PubMed Central

Fazli, Mustafa; Harrison, Joe J.; Gambino, Michela; Givskov, Michael

2015-01-01

Burkholderia cenocepacia is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. Understanding the genetic basis of virulence in this emerging pathogen is important for the development of novel treatment regimes. Generation of deletion mutations in genes predicted to encode virulence determinants is fundamental to investigating the mechanisms of pathogenesis. However, there is a lack of appropriate selectable and counterselectable markers for use in B. cenocepacia, making its genetic manipulation problematic. Here we describe a Gateway-compatible allelic exchange system based on the counterselectable pheS gene and the I-SceI homing endonuclease. This system provides efficiency in cloning homology regions of target genes and allows the generation of precise and unmarked gene deletions in B. cenocepacia. As a proof of concept, we demonstrate its utility by deleting the Bcam1349 gene, encoding a cyclic di-GMP (c-di-GMP)-responsive regulator protein important for biofilm formation. PMID:25795676

Homozygous hereditary C3 deficiency due to a partial gene deletion.

PubMed Central

Botto, M; Fong, K Y; So, A K; Barlow, R; Routier, R; Morley, B J; Walport, M J

1992-01-01

The molecular mechanism of C3 deficiency in an Afrikaans patient with recurrent pyogenic infections was studied. Restriction enzyme analysis showed a gene deletion of 800 base pairs (bp) mapping to the alpha chain of C3. Amplification of genomic DNA, using the PCR, demonstrated that the deletion included exons 22 and 23 of the C3 gene. Truncated mRNA was shown in an Epstein-Barr virus-transformed B-cell line by PCR amplification of first-strand cDNA. A consequence of this deletion was that the RNA transcribed 3' to the deletion was out of frame, resulting in formation of a stop codon 19 bp downstream from the deletion. The molecular basis of the deletion was compatible with homologous recombination between two Alu sequences located in introns 21 and 23. An unrelated nonconsanguineous relative and two of a sample of 174 Afrikaans-speaking individuals were heterozygous carriers of the same gene deletion. The wide prevalence of this null allele in this population is probably due to the effects of a small founder population. The presence of this deletion in the C3 gene is not compatible with production of any functional C3, supporting the idea that study of such patients offers a valid model for understanding physiological activities of C3 in vivo in humans. Images PMID:1350678

A Convenient Cas9-based Conditional Knockout Strategy for Simultaneously Targeting Multiple Genes in Mouse.

PubMed

Chen, Jiang; Du, Yinan; He, Xueyan; Huang, Xingxu; Shi, Yun S

2017-03-31

The most powerful way to probe protein function is to characterize the consequence of its deletion. Compared to conventional gene knockout (KO), conditional knockout (cKO) provides an advanced gene targeting strategy with which gene deletion can be performed in a spatially and temporally restricted manner. However, for most species that are amphiploid, the widely used Cre-flox conditional KO (cKO) system would need targeting loci in both alleles to be loxP flanked, which in practice, requires time and labor consuming breeding. This is considerably significant when one is dealing with multiple genes. CRISPR/Cas9 genome modulation system is advantaged in its capability in targeting multiple sites simultaneously. Here we propose a strategy that could achieve conditional KO of multiple genes in mouse with Cre recombinase dependent Cas9 expression. By transgenic construction of loxP-stop-loxP (LSL) controlled Cas9 (LSL-Cas9) together with sgRNAs targeting EGFP, we showed that the fluorescence molecule could be eliminated in a Cre-dependent manner. We further verified the efficacy of this novel strategy to target multiple sites by deleting c-Maf and MafB simultaneously in macrophages specifically. Compared to the traditional Cre-flox cKO strategy, this sgRNAs-LSL-Cas9 cKO system is simpler and faster, and would make conditional manipulation of multiple genes feasible.

Acute morphine effects on respiratory activity in mice with target deletion of the tachykinin 1 gene (Tac1-/-).

PubMed

Shvarev, Yuri; Berner, Jonas; Bilkei-Gorzo, Andras; Lagercrantz, Hugo; Wickström, Ronny

2010-01-01

Search for physiological mechanisms which could antagonize the opioid-induced respiratory depression is of important clinical value. In this study, we investigated the acute effects of morphine on respiratory activity in genetically modified newborn (P2) mice with target deletion of the (Tac1 -/-) gene lacking substance P (SP) and neurokinin A (NKA). In vivo, as shown with whole-body flow barometric plethysmography technique, morphine induced significantly attenuated minute ventilation during intermittent hypoxia in control animals. In contrast, knockout mice revealed significant increase in minute ventilation. In vitro, in brainstem preparation, knockout mice demonstrated greater changes in burst frequency during intermittent anoxia challenge. The data suggest that hereditary deficiency in tachykinins, SP and NKA results in more robust hypoxic response in newborn Tac1-/- mice during respiratory depression induced by morphine.

Deletions spanning the neurofibromatosis I gene: Identification and phenotype of five patients

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

Kayes, L.M.; Burke, W.; Bennett, R.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder characterized by marked variation in clinical severity. To investigate the contribution to variability by genes either contiguous to or contained within the NF1 gene, the authors screened six NF1 patients with mild facial dysmorphology, mental retardation, and/or learning disabilities, for DNA rearrangement of the NF1 region. Five of the six patients had NF1 gene deletions on the basis of quantitative densitometry, locus hemizygosity, and analysis of somatic cell hybrid lines. Analysis of hybrid lines carrying each of the patient's chromosomes 17, with 15 regional DNA markers, demonstrated that each of themore » five patients carried a deletion >700 kb in size. Minimally, each of the deletions involved the entire 350-kb NF1 gene; the three genes - EVI2A, EVI2B, and OMG-that are contained within an NF1 intron; and considerable flanking DNA. For four of the patients, the deletions mapped to the same interval; the deletion in the fifth patient was larger, extending farther in both directions. The remaining NF1 allele presumably produced functional neurofibromin; no gene rearrangements were detected, and RNA-PCR demonstrated that it was transcribed. These data provide compelling evidence that the NF1 disorder results from haploid insufficiency of neurofibromin. Of the three documented de novo deletion cases, two involved the paternal NF1 allele and one the maternal allele. The parental origin of the single remaining expresses NF1 allele had no dramatic effect on patient phenotype. The deletion patients exhibited a variable number of physical anomalies that were not correlated with the extent of their deletion. All five patients with deletions were remarkable for exhibiting a large number of neurfibromas for their age, suggesting that deletion of an unknown gene in the NF1 region may affect tumor initiation or development. 69 refs., 5 figs., 1 tab.« less

In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology.

PubMed

Fazli, Mustafa; Harrison, Joe J; Gambino, Michela; Givskov, Michael; Tolker-Nielsen, Tim

2015-06-01

Burkholderia cenocepacia is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. Understanding the genetic basis of virulence in this emerging pathogen is important for the development of novel treatment regimes. Generation of deletion mutations in genes predicted to encode virulence determinants is fundamental to investigating the mechanisms of pathogenesis. However, there is a lack of appropriate selectable and counterselectable markers for use in B. cenocepacia, making its genetic manipulation problematic. Here we describe a Gateway-compatible allelic exchange system based on the counterselectable pheS gene and the I-SceI homing endonuclease. This system provides efficiency in cloning homology regions of target genes and allows the generation of precise and unmarked gene deletions in B. cenocepacia. As a proof of concept, we demonstrate its utility by deleting the Bcam1349 gene, encoding a cyclic di-GMP (c-di-GMP)-responsive regulator protein important for biofilm formation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Deletion of Tsc2 in Nociceptors Reduces Target Innervation, Ion Channel Expression, and Sensitivity to Heat

PubMed Central

Carlin, Dan; Golden, Judith P.; Monk, Kelly R.

2018-01-01

Abstract The mechanistic target of rapamycin complex 1 (mTORC1) is known to regulate cellular growth pathways, and its genetic activation is sufficient to enhance regenerative axon growth following injury to the central or peripheral nervous systems. However, excess mTORC1 activation may promote innervation defects, and mTORC1 activity mediates injury-induced hypersensitivity, reducing enthusiasm for the pathway as a therapeutic target. While mTORC1 activity is required for full expression of some pain modalities, the effects of pathway activation on nociceptor phenotypes and sensory behaviors are currently unknown. To address this, we genetically activated mTORC1 in mouse peripheral sensory neurons by conditional deletion of its negative regulator Tuberous Sclerosis Complex 2 (Tsc2). Consistent with the well-known role of mTORC1 in regulating cell size, soma size and axon diameter of C-nociceptors were increased in Tsc2-deleted mice. Glabrous skin and spinal cord innervation by C-fiber neurons were also disrupted. Transcriptional profiling of nociceptors enriched by fluorescence-associated cell sorting (FACS) revealed downregulation of multiple classes of ion channels as well as reduced expression of markers for peptidergic nociceptors in Tsc2-deleted mice. In addition to these changes in innervation and gene expression, Tsc2-deleted mice exhibited reduced noxious heat sensitivity and decreased injury-induced cold hypersensitivity, but normal baseline sensitivity to cold and mechanical stimuli. Together, these data show that excess mTORC1 activity in sensory neurons produces changes in gene expression, neuron morphology and sensory behavior. PMID:29766046

A comprehensive analysis of replicative lifespan in 4,698 single-gene deletion strains uncovers conserved mechanisms of aging

PubMed Central

McCormick, Mark A.; Delaney, Joe R.; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; Shemorry, Anna; Sim, Sylvia; Chou, Annie Chia-Zong; Ahmed, Umema; Carr, Daniel; Murakami, Christopher J.; Schleit, Jennifer; Sutphin, George L.; Wasko, Brian M.; Bennett, Christopher F.; Wang, Adrienne M.; Olsen, Brady; Beyer, Richard P.; Bammler, Theodor K.; Prunkard, Donna; Johnson, Simon C.; Pennypacker, Juniper K.; An, Elroy; Anies, Arieanna; Castanza, Anthony S.; Choi, Eunice; Dang, Nick; Enerio, Shiena; Fletcher, Marissa; Fox, Lindsay; Goswami, Sarani; Higgins, Sean A.; Holmberg, Molly A.; Hu, Di; Hui, Jessica; Jelic, Monika; Jeong, Ki-Soo; Johnston, Elijah; Kerr, Emily O.; Kim, Jin; Kim, Diana; Kirkland, Katie; Klum, Shannon; Kotireddy, Soumya; Liao, Eric; Lim, Michael; Lin, Michael S.; Lo, Winston C.; Lockshon, Dan; Miller, Hillary A.; Moller, Richard M.; Muller, Brian; Oakes, Jonathan; Pak, Diana N.; Peng, Zhao Jun; Pham, Kim M.; Pollard, Tom G.; Pradeep, Prarthana; Pruett, Dillon; Rai, Dilreet; Robison, Brett; Rodriguez, Ariana A.; Ros, Bopharoth; Sage, Michael; Singh, Manpreet K.; Smith, Erica D.; Snead, Katie; Solanky, Amrita; Spector, Benjamin L.; Steffen, Kristan K.; Tchao, Bie Nga; Ting, Marc K.; Wende, Helen Vander; Wang, Dennis; Welton, K. Linnea; Westman, Eric A.; Brem, Rachel B.; Liu, Xin-guang; Suh, Yousin; Zhou, Zhongjun; Kaeberlein, Matt; Kennedy, Brian K.

2015-01-01

SUMMARY Many genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is non-additive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging. PMID:26456335

Salivary IgG subclasses in individuals with and without homozygous IGHG gene deletions.

PubMed Central

Engström, P E; Norhagen, G; Osipova, L; Helal, A; Wiebe, V; Brusco, A; Carbonara, A O; Lefranc, G; Lefranc, M P

1996-01-01

In this study, the levels of salivary IgG1, IgG2, IgG3 and IgG4 from individuals with and without homozygous immunoglobulin heavy chain constant gene deletions were quantified by enzyme-linked immunosorbent assay (ELISA). To analyse the restriction of salivary IgG subclasses, we used unstimulated whole saliva and sera collected at the same time from individuals with homozygous gene deletions, two with G1 deletion, one with G4 deletion, six with both G2 and G4 deletions and from eight individuals without IGHG gene deletions and expressing all four IgG subclasses. The median values of salivary IgG from individuals with homozygous G1, or G4, or both G2 and G4 deletions, and from individuals expressing all four subclasses were 24.2 mg/l and 23.4 mg/l, respectively. The median values of serum IgG were 13.7 g/l and 15.9 g/l, respectively. Our results show that the salivary and serum IgG levels were both within the normal range in individuals with homozygous gene deletions of either G1, or G4, or both G2 and G4. PMID:8943711

Mucopolysaccharidosis type IVA: Common double deletion in the N-Acetylgalactosamine-6-sulfatase gene (GALNS)

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

Hori, Toshinori; Tomatsu, Shunji; Fukuda, Seiji

1995-04-10

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency in N-acetylgalactosamine-6-sulfatase (GALNS). We found two separate deletions of nearly 8.0 and 6.0 kb in the GALNS gene, including some exons. There are Alu repetitive elements near the breakpoints of the 8.0-kb deletion, and this deletion resulted from an Alu-Alu recombination. The other 6.0-kb deletion involved illegitimate recombinational events between incomplete short direct repeats of 8 bp at deletion breakpoints. The same rearrangement has been observed in a heteroallelic state in four unrelated patients. This is the first documentation of a common double deletion a gene thatmore » is not a member of a gene cluster. 39 refs., 5 figs.« less

A large homozygous deletion in the SAMHD1 gene causes atypical Aicardi–Goutiéres syndrome associated with mtDNA deletions

PubMed Central

Leshinsky-Silver, Esther; Malinger, Gustavo; Ben-Sira, Liat; Kidron, Dvora; Cohen, Sarit; Inbar, Shani; Bezaleli, Tali; Levine, Arie; Vinkler, Chana; Lev, Dorit; Lerman-Sagie, Tally

2011-01-01

Aicardi–Goutiéres syndrome (AGS) is a genetic neurodegenerative disorder with clinical symptoms mimicking a congenital viral infection. Five causative genes have been described: three prime repair exonuclease1 (TREX1), ribonucleases H2A, B and C, and most recently SAM domain and HD domain 1 (SAMHD1). We performed a detailed clinical and molecular characterization of a family with autosomal recessive neurodegenerative disorder showing white matter destruction and calcifications, presenting in utero and associated with multiple mtDNA deletions. A muscle biopsy was normal and did not show any evidence of respiratory chain dysfunction. Southern blot analysis of tissue from a living child and affected fetuses demonstrated multiple mtDNA deletions. Molecular analysis of genes involved in mtDNA synthesis and maintenance (POLGα, POLGβ, Twinkle, ANT1, TK2, SUCLA1 and DGOUK) revealed normal sequences. Sequencing of TREX1 and ribonucleases H2A, B and C failed to reveal any mutations. Whole-genome homozygosity mapping revealed a candidate region containing the SAMHD1 gene. Sequencing of the gene in the affected child and two affected fetuses revealed a large deletion (9 kb), spanning the promoter, exon1 and intron 1. The parents were found to be heterozygous for this deletion. The identification of a homozygous large deletion in the SAMHD1 gene causing atypical AGS with multiple mtDNA deletions may add information regarding the involvement of mitochondria in self-activation of innate immunity by cell intrinsic components. PMID:21102625

Analysis of a new homozygous deletion in the tumor suppressor region at 3p12.3 reveals two novel intronic noncoding RNA genes.

PubMed

Angeloni, Debora; ter Elst, Arja; Wei, Ming Hui; van der Veen, Anneke Y; Braga, Eleonora A; Klimov, Eugene A; Timmer, Tineke; Korobeinikova, Luba; Lerman, Michael I; Buys, Charles H C M

2006-07-01

Homozygous deletions or loss of heterozygosity (LOH) at human chromosome band 3p12 are consistent features of lung and other malignancies, suggesting the presence of a tumor suppressor gene(s) (TSG) at this location. Only one gene has been cloned thus far from the overlapping region deleted in lung and breast cancer cell lines U2020, NCI H2198, and HCC38. It is DUTT1 (Deleted in U Twenty Twenty), also known as ROBO1, FLJ21882, and SAX3, according to HUGO. DUTT1, the human ortholog of the fly gene ROBO, has homology with NCAM proteins. Extensive analyses of DUTT1 in lung cancer have not revealed any mutations, suggesting that another gene(s) at this location could be of importance in lung cancer initiation and progression. Here, we report the discovery of a new, small, homozygous deletion in the small cell lung cancer (SCLC) cell line GLC20, nested in the overlapping, critical region. The deletion was delineated using several polymorphic markers and three overlapping P1 phage clones. Fiber-FISH experiments revealed the deletion was approximately 130 kb. Comparative genomic sequence analysis uncovered short sequence elements highly conserved among mammalian genomes and the chicken genome. The discovery of two EST clusters within the deleted region led to the isolation of two noncoding RNA (ncRNA) genes. These were subsequently found differentially expressed in various tumors when compared to their normal tissues. The ncRNA and other highly conserved sequence elements in the deleted region may represent miRNA targets of importance in cancer initiation or progression. Published 2006 Wiley-Liss, Inc.

Systematic Phenotyping of a Large-Scale Candida glabrata Deletion Collection Reveals Novel Antifungal Tolerance Genes

PubMed Central

Hiller, Ekkehard; Istel, Fabian; Tscherner, Michael; Brunke, Sascha; Ames, Lauren; Firon, Arnaud; Green, Brian; Cabral, Vitor; Marcet-Houben, Marina; Jacobsen, Ilse D.; Quintin, Jessica; Seider, Katja; Frohner, Ingrid; Glaser, Walter; Jungwirth, Helmut; Bachellier-Bassi, Sophie; Chauvel, Murielle; Zeidler, Ute; Ferrandon, Dominique; Gabaldón, Toni; Hube, Bernhard; d'Enfert, Christophe; Rupp, Steffen; Cormack, Brendan; Haynes, Ken; Kuchler, Karl

2014-01-01

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes. PMID:24945925

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92

PubMed Central

Jin, Hyun Yong; Oda, Hiroyo; Chen, Pengda; Kang, Seung Goo; Valentine, Elizabeth; Liao, Lujian; Zhang, Yaoyang; Gonzalez-Martin, Alicia; Shepherd, Jovan; Head, Steven R.; Kim, Pyeung-Hyeun; Fu, Guo; Liu, Wen-Hsien; Han, Jiahuai

2017-01-01

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes. PMID:28241004

A resource of vectors and ES cells for targeted deletion of microRNAs in mice

PubMed Central

Prosser, Haydn M.; Koike-Yusa, Hiroko; Cooper, James D.; Law, Frances C.; Bradley, Allan

2011-01-01

The 21-23 nucleotide single-stranded RNAs classified as microRNAs (miRNA) perform fundamental roles in a wide range of cellular and developmental processes. miRNAs regulate protein expression through sequence-specific base pairing with target messenger RNAs (mRNA) reducing both their stability and the process of protein translation1, 2. At least 30% of protein coding genes appear to be conserved targets for miRNAs1. In contrast to the protein coding genes3, 4, no public resource of miRNA mouse mutant alleles exists. We have generated a library of highly germ-line transmissible C57BL/6N mouse mutant embryonic stem (ES) cells with targeted deletions for the majority of miRNA genes currently annotated within the miRBase registry5. These alleles have been designed to be highly adaptable research tools that can be efficiently altered to create reporter, conditional and other allelic variants. This ES cell resource can be searched electronically and is available from ES cell repositories for distribution to the scientific community6. PMID:21822254

A Comprehensive Analysis of Replicative Lifespan in 4,698 Single-Gene Deletion Strains Uncovers Conserved Mechanisms of Aging.

PubMed

McCormick, Mark A; Delaney, Joe R; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; Shemorry, Anna; Sim, Sylvia; Chou, Annie Chia-Zong; Ahmed, Umema; Carr, Daniel; Murakami, Christopher J; Schleit, Jennifer; Sutphin, George L; Wasko, Brian M; Bennett, Christopher F; Wang, Adrienne M; Olsen, Brady; Beyer, Richard P; Bammler, Theodor K; Prunkard, Donna; Johnson, Simon C; Pennypacker, Juniper K; An, Elroy; Anies, Arieanna; Castanza, Anthony S; Choi, Eunice; Dang, Nick; Enerio, Shiena; Fletcher, Marissa; Fox, Lindsay; Goswami, Sarani; Higgins, Sean A; Holmberg, Molly A; Hu, Di; Hui, Jessica; Jelic, Monika; Jeong, Ki-Soo; Johnston, Elijah; Kerr, Emily O; Kim, Jin; Kim, Diana; Kirkland, Katie; Klum, Shannon; Kotireddy, Soumya; Liao, Eric; Lim, Michael; Lin, Michael S; Lo, Winston C; Lockshon, Dan; Miller, Hillary A; Moller, Richard M; Muller, Brian; Oakes, Jonathan; Pak, Diana N; Peng, Zhao Jun; Pham, Kim M; Pollard, Tom G; Pradeep, Prarthana; Pruett, Dillon; Rai, Dilreet; Robison, Brett; Rodriguez, Ariana A; Ros, Bopharoth; Sage, Michael; Singh, Manpreet K; Smith, Erica D; Snead, Katie; Solanky, Amrita; Spector, Benjamin L; Steffen, Kristan K; Tchao, Bie Nga; Ting, Marc K; Vander Wende, Helen; Wang, Dennis; Welton, K Linnea; Westman, Eric A; Brem, Rachel B; Liu, Xin-Guang; Suh, Yousin; Zhou, Zhongjun; Kaeberlein, Matt; Kennedy, Brian K

2015-11-03

Many genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is nonadditive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging. Copyright © 2015 Elsevier Inc. All rights reserved.

Dopaminergic Neuron-Specific Deletion of p53 Gene Attenuates Methamphetamine Neurotoxicity.

PubMed

Lu, Tao; Kim, Paul P; Greig, Nigel H; Luo, Yu

2017-08-01

p53 plays an essential role in the regulation of cell death in dopaminergic (DA) neurons and its activation has been implicated in the neurotoxic effects of methamphetamine (MA). However, how p53 mediates MA neurotoxicity remains largely unknown. In this study, we examined the effect of DA-specific p53 gene deletion in DAT-p53KO mice. Whereas in vivo MA binge exposure reduced locomotor activity in wild-type (WT) mice, this was significantly attenuated in DAT-p53KO mice and associated with significant differences in the levels of the p53 target genes BAX and p21 between WT and DAT-p53KO. Notably, DA-specific deletion of p53 provided protection of substantia nigra pars reticulata (SNpr) tyrosine hydroxylase (TH) positive fibers following binge MA, with DAT-p53KO mice having less decline of TH protein levels in striatum versus WT mice. Whereas DAT-p53KO mice demonstrated a consistently higher density of TH fibers in striatum compared to WT mice at 10 days after MA exposure, DA neuron counts within the substantia nigra pars compacta (SNpc) were similar. Finally, supportive of these results, administration of a p53-specific inhibitor (PFT-α) provided a similarly protective effect on MA binge-induced behavioral deficits. Neither DA specific p53 deletion nor p53 pharmacological inhibition affected hyperthermia induced by MA binge. These findings demonstrate a specific contribution of p53 activation in behavioral deficits and DA neuronal terminal loss by MA binge exposure.

Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae.

PubMed

Park, Yang-Nim; Masison, Daniel; Eisenberg, Evan; Greene, Lois E

2011-09-01

The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast. Published in 2011 by John Wiley & Sons, Ltd.

Antibiotic Combinations That Enable One-Step, Targeted Mutagenesis of Chromosomal Genes.

PubMed

Lee, Wonsik; Do, Truc; Zhang, Ge; Kahne, Daniel; Meredith, Timothy C; Walker, Suzanne

2018-06-08

Targeted modification of bacterial chromosomes is necessary to understand new drug targets, investigate virulence factors, elucidate cell physiology, and validate results of -omics-based approaches. For some bacteria, reverse genetics remains a major bottleneck to progress in research. Here, we describe a compound-centric strategy that combines new negative selection markers with known positive selection markers to achieve simple, efficient one-step genome engineering of bacterial chromosomes. The method was inspired by the observation that certain nonessential metabolic pathways contain essential late steps, suggesting that antibiotics targeting a late step can be used to select for the absence of genes that control flux into the pathway. Guided by this hypothesis, we have identified antibiotic/counterselectable markers to accelerate reverse engineering of two increasingly antibiotic-resistant pathogens, Staphylococcus aureus and Acinetobacter baumannii. For S. aureus, we used wall teichoic acid biosynthesis inhibitors to select for the absence of tarO and for A. baumannii, we used colistin to select for the absence of lpxC. We have obtained desired gene deletions, gene fusions, and promoter swaps in a single plating step with perfect efficiency. Our method can also be adapted to generate markerless deletions of genes using FLP recombinase. The tools described here will accelerate research on two important pathogens, and the concept we outline can be readily adapted to any organism for which a suitable target pathway can be identified.

HMG-CoA lyase (HL) gene: Cloning and characterization of the 5{prime} end of the mouse gene, gene targeting in ES cells, and demonstration of large deletions in three HL-deficient patients

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

Wang, S.; Robert, M.F.; Mitchell, G.A.

1994-09-01

3-hydroxy-3-methylglutaryl CoA lyase (HL) is a mitochondrial matrix enzyme which catalyzes the last step of leucine catabolism and of ketogenesis. Autosomal recessive HL deficiency in humans results in episodes of hypoglycemia and coma. We are interested in the pathophysiology of HL deficiency as a model for both amino acid and fatty acid inborn errors. We have cloned the human and mouse HL genes. In order to analyze the 5{prime} nontranslated region of mouse HL gene, we cloned and sequenced a 1.8 kb fragment containing the 5{prime} extremity including exon 1 and about 1.6 kb of 5{prime} nontranslated sequence. The regionmore » surrounding exon 1 is CpG-rich (66.4%). Using the criteria of West, the Observed/Expected ratio for CpG dinucleotides is 0.7 ({ge}0.6 is consistent with a CpG island). We are carrying out primer extension and RNase protection experiments to determine the transcription initiation site. We constructed a gene targeting vector by introducing the neomycin resistance gene into exon 2 of a 7.5 kb genomic subclone of the mouse HL gene. Targeting was performed by electroporating 10 mg linearized vector into 10{sup 7} ES cells and selecting for 12 days with G418. 5/228 colonies (2.2%) had homologous recombination as shown by PCR screening and Southern analysis. We are microinjecting the 5 targeted clones into blastocysts to create an HL-deficient mouse. To date we have obtained two chimeras with contributions of 95% and 55% from 129, by coat color estimates. Three of 27 (11%) of the HL-deficient patients studied were suggested by genomic Southern analysis to be homozygous for large intragenic deletions. We confirmed this and defined the boundaries using exonic PCR.« less

Lysis Delay and Burst Shrinkage of Coliphage T7 by Deletion of Terminator Tφ Reversed by Deletion of Early Genes

PubMed Central

Nguyen, Huong Minh

2014-01-01

ABSTRACT Bacteriophage T7 terminator Tφ is a class I intrinsic terminator coding for an RNA hairpin structure immediately followed by oligo(U), which has been extensively studied in terms of its transcription termination mechanism, but little is known about its physiological or regulatory functions. In this study, using a T7 mutant phage, where a 31-bp segment of Tφ was deleted from the genome, we discovered that deletion of Tφ from T7 reduces the phage burst size but delays lysis timing, both of which are disadvantageous for the phage. The burst downsizing could directly result from Tφ deletion-caused upregulation of gene 17.5, coding for holin, among other Tφ downstream genes, because infection of gp17.5-overproducing Escherichia coli by wild-type T7 phage showed similar burst downsizing. However, the lysis delay was not associated with cellular levels of holin or lysozyme or with rates of phage adsorption. Instead, when allowed to evolve spontaneously in five independent adaptation experiments, the Tφ-lacking mutant phage, after 27 or 29 passages, recovered both burst size and lysis time reproducibly by deleting early genes 0.5, 0.6, and 0.7 of class I, among other mutations. Deletion of genes 0.5 to 0.7 from the Tφ-lacking mutant phage decreased expression of several Tφ downstream genes to levels similar to that of the wild-type phage. Accordingly, phage T7 lysis timing is associated with cellular levels of Tφ downstream gene products. This suggests the involvement of unknown factor(s) besides the known lysis proteins, lysozyme and holin, and that Tφ plays a role of optimizing burst size and lysis time during T7 infection. IMPORTANCE E. coli PMID:24335287

[Gene deletion and functional analysis of the heptyl glycosyltransferase (waaF) gene in Vibrio parahemolyticus O-antigen cluster].

PubMed

Zhao, Feng; Meng, Songsong; Zhou, Deqing

2016-02-04

To construct heptyl glycosyltransferase gene II (waaF) gene deletion mutant of Vibrio parahaemolyticus, and explore the function of the waaF gene in Vibrio parahaemolyticus. The waaF gene deletion mutant was constructed by chitin-based transformation technology using clinical isolates, and then the growth rate, morphology and serotypes were identified. The different sources (O3, O5 and O10) waaF gene complementations were constructed through E. coli S17λpir strains conjugative transferring with Vibrio parahaemolyticus, and the function of the waaF gene was further verified by serotypes. The waaF gene deletion mutant strain was successfully constructed and it grew normally. The growth rate and morphology of mutant were similar with the wild type strains (WT), but the mutant could not occurred agglutination reaction with O antisera. The O3 and O5 sources waaF gene complementations occurred agglutination reaction with O antisera, but the O10 sources waaF gene complementations was not. The waaF gene was related with O-antigen synthesis and it was the key gene of O-antigen synthesis pathway in Vibrio parahaemolyticus. The function of different sources waaF gene were not the same.

Prevalence of the Prefoldin Subunit 5 Gene Deletion in Canine Mammary Tumors

PubMed Central

Bornemann-Kolatzki, Kirsten; Neumann, Stephan; Escobar, Hugo Murua; Nolte, Ingo; Hammer, Susanne Conradine; Hewicker-Trautwein, Marion; Junginger, Johannes; Kaup, Franz-Josef; Brenig, Bertram; Schütz, Ekkehard

2015-01-01

Background A somatic deletion at the proximal end of canine chromosome 27 (CFA27) was recently reported in 50% of malignant mammary tumors. This region harbours the tumor suppressor gene prefoldin subunit 5 (PFDN5) and the deletion correlated with a higher Ki-67 score. PFDN5 has been described to repress c-MYC and is, therefore, a candidate tumor-suppressor and cancer-driver gene in canine mammary cancer. Aim of this study was to confirm the recurrent deletion in a larger number of tumors. Methods Droplet digital PCR for PFDN5 was performed in DNA from 102 malignant, 40 benign mammary tumors/dysplasias, 11 non-neoplastic mammary tissues and each corresponding genomic DNA from leukocytes. The copy number of PFDN5 was normalized to a reference amplicon on canine chromosome 32 (CFA32). Z-scores were calculated, based on Gaussian distributed normalized PFDN5 copy numbers of the leukocyte DNA. Z-scores ≤ -3.0 in tissue were considered as being indicative of the PFDN5 deletion and called as such. The Ki-67 proliferation index was assessed in a subset of 79 tissue samples by immunohistochemistry. Results The deletion was confirmed in 24% of all malignant tumors, detected in only 7.5% of the benign tumors and was not present in any normal mammary tissue sample. The subgroup of solid carcinomas (n = 9) showed the highest frequency of the deletion (67%) and those malignomas without microscopical high fraction of benign tissue (n = 71) had a 32% frequency (p<0.01 vs. benign samples). The Ki-67 score was found to be significantly higher (p<0.05) in the PFDN5-deleted group compared to malignant tumors without the deletion. Conclusions A somatic deletion of the PFDN5 gene is recurrently present in canine mammary cancer, supporting a potential role in carcinogenesis. The association of this deletion with higher Ki-67 indicates an increased proliferation rate and thus a link to tumor aggressiveness can be hypothesized. The confirmation of earlier results warrants further studies

CuZnSOD gene deletion targeted to skeletal muscle leads to loss of contractile force but does not cause muscle atrophy in adult mice

PubMed Central

Zhang, Yiqiang; Davis, Carol; Sakellariou, George K.; Shi, Yun; Kayani, Anna C.; Pulliam, Daniel; Bhattacharya, Arunabh; Richardson, Arlan; Jackson, Malcolm J.; McArdle, Anne; Brooks, Susan V.; Van Remmen, Holly

2013-01-01

We have previously shown that deletion of CuZnSOD in mice (Sod1−/− mice) leads to accelerated loss of muscle mass and contractile force during aging. To dissect the relative roles of skeletal muscle and motor neurons in this process, we used a Cre-Lox targeted approach to establish a skeletal muscle-specific Sod1-knockout (mKO) mouse to determine whether muscle-specific CuZnSOD deletion is sufficient to cause muscle atrophy. Surprisingly, mKO mice maintain muscle masses at or above those of wild-type control mice up to 18 mo of age. In contrast, maximum isometric specific force measured in gastrocnemius muscle is significantly reduced in the mKO mice. We found no detectable increases in global measures of oxidative stress or ROS production, no reduction in mitochondrial ATP production, and no induction of adaptive stress responses in muscle from mKO mice. However, Akt-mTOR signaling is elevated and the number of muscle fibers with centrally located nuclei is increased in skeletal muscle from mKO mice, which suggests elevated regenerative pathways. Our data demonstrate that lack of CuZnSOD restricted to skeletal muscle does not lead to muscle atrophy but does cause muscle weakness in adult mice and suggest loss of CuZnSOD may potentiate muscle regenerative pathways.—Zhang, Y., Davis, C., Sakellariou, G.K., Shi, Y., Kayani, A.C., Pulliam, D., Bhattacharya, A., Richardson, A., Jackson, M.J., McArdle, A., Brooks, S.V., Van Remmen, H. CuZnSOD gene deletion targeted to skeletal muscle leads to loss of contractile force but does not cause muscle atrophy in adult mice. PMID:23729587

Prevalence of pfhrp2 and pfhrp3 gene deletions in Puerto Lempira, Honduras.

PubMed

Abdallah, Joseph F; Okoth, Sheila Akinyi; Fontecha, Gustavo A; Torres, Rosa Elena Mejia; Banegas, Engels I; Matute, María Luisa; Bucheli, Sandra Tamara Mancero; Goldman, Ira F; de Oliveira, Alexandre Macedo; Barnwell, John W; Udhayakumar, Venkatachalam

2015-01-21

Recent studies have demonstrated the deletion of the histidine-rich protein 2 (PfHRP2) gene (pfhrp2) in field isolates of Plasmodium falciparum, which could result in false negative test results when PfHRP2-based rapid diagnostic tests (RDTs) are used for malaria diagnosis. Although primary diagnosis of malaria in Honduras is determined based on microscopy, RDTs may be useful in remote areas. In this study, it was investigated whether there are deletions of the pfhrp2, pfhrp3 and their respective flanking genes in 68 P. falciparum parasite isolates collected from the city of Puerto Lempira, Honduras. In addition, further investigation considered the possible correlation between parasite population structure and the distribution of these gene deletions by genotyping seven neutral microsatellites. Sixty-eight samples used in this study, which were obtained from a previous chloroquine efficacy study, were utilized in the analysis. All samples were genotyped for pfhrp2, pfhrp3 and flanking genes by PCR. The samples were then genotyped for seven neutral microsatellites in order to determine the parasite population structure in Puerto Lempira at the time of sample collection. It was found that all samples were positive for pfhrp2 and its flanking genes on chromosome 8. However, only 50% of the samples were positive for pfhrp3 and its neighboring genes while the rest were either pfhrp3-negative only or had deleted a combination of pfhrp3 and its neighbouring genes on chromosome 13. Population structure analysis predicted that there are at least two distinct parasite population clusters in this sample population. It was also determined that a greater proportion of parasites with pfhrp3-(and flanking gene) deletions belonged to one cluster compared to the other. The findings indicate that the P. falciparum parasite population in the municipality of Puerto Lempira maintains the pfhrp2 gene and that PfHRP2-based RDTs could be considered for use in this region; however

Targeted exome sequencing of Korean triple-negative breast cancer reveals homozygous deletions associated with poor prognosis of adjuvant chemotherapy-treated patients

PubMed Central

Jeong, Hae Min; Kim, Ryong Nam; Kwon, Mi Jeong; Oh, Ensel; Han, Jinil; Lee, Se Kyung; Choi, Jong-Sun; Park, Sara; Nam, Seok Jin; Gong, Gyung Yup; Nam, Jin Wu; Choi, Doo Ho; Lee, Hannah; Nam, Byung-Ho; Choi, Yoon-La; Shin, Young Kee

2017-01-01

Triple-negative breast cancer is characterized by the absence of estrogen and progesterone receptors and human epidermal growth factor receptor 2, and is associated with a poorer outcome than other subtypes of breast cancer. Moreover, there are no accurate prognostic genes or effective therapeutic targets, thereby necessitating continued intensive investigation. This study analyzed the genetic mutation landscape in 70 patients with triple-negative breast cancer by targeted exome sequencing of tumor and matched normal samples. Sequencing showed that more than 50% of these patients had deleterious mutations and homozygous deletions of DNA repair genes, such as ATM, BRCA1, BRCA2, WRN, and CHEK2. These findings suggested that a large number of patients with triple-negative breast cancer have impaired DNA repair function and that therefore a poly ADP-ribose polymerase inhibitor may be an effective drug in the treatment of this disease. Notably, homozygous deletion of three genes, EPHA5, MITF, and ACSL3, was significantly associated with an increased risk of recurrence or distant metastasis in adjuvant chemotherapy-treated patients. PMID:28977883

A Catalog of Genes Homozygously Deleted in Human Lung Cancer and the Candidacy of PTPRD as a Tumor Suppressor Gene

PubMed Central

Kohno, Takashi; Otsuka, Ayaka; Girard, Luc; Sato, Masanori; Iwakawa, Reika; Ogiwara, Hideaki; Sanchez-Cespedes, Montse; Minna, John D.; Yokota, Jun

2010-01-01

A total of 176 genes homozygously deleted in human lung cancer were identified by DNA array-based whole genome scanning of 52 lung cancer cell lines and subsequent genomic PCR in 74 cell lines, including the 52 cell lines scanned. One or more exons of these genes were homozygously deleted in one (1%) to 20 (27%) cell lines. These genes included known tumor suppressor genes, e.g., CDKN2A/p16, RB1, and SMAD4, and candidate tumor suppressor genes whose hemizygous or homozygous deletions were reported in several types of human cancers, such as FHIT, KEAP1, and LRP1B/LRP-DIP. CDKN2A/p16 and p14ARF located in 9p21 were most frequently deleted (20/74, 27%). The PTPRD gene was most frequently deleted (8/74, 11%) among genes mapping to regions other than 9p21. Somatic mutations, including a nonsense mutation, of the PTPRD gene were detected in 8/74 (11%) of cell lines and 4/95 (4%) of surgical specimens of lung cancer. Reduced PTPRD expression was observed in the majority (>80%) of cell lines and surgical specimens of lung cancer. Therefore, PTPRD is a candidate tumor suppressor gene in lung cancer. Microarray-based expression profiling of 19 lung cancer cell lines also indicated that some of the 176 genes, such as KANK and ADAMTS1, are preferentially inactivated by epigenetic alterations. Genetic/epigenetic as well as functional studies of these 176 genes will increase our understanding of molecular mechanisms behind lung carcinogenesis. PMID:20073072

Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants.

PubMed

Luo, Ming; Gilbert, Brian; Ayliffe, Michael

2016-07-01

Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described.

Lysis delay and burst shrinkage of coliphage T7 by deletion of terminator Tφ reversed by deletion of early genes.

PubMed

Nguyen, Huong Minh; Kang, Changwon

2014-02-01

Bacteriophage T7 terminator Tϕ is a class I intrinsic terminator coding for an RNA hairpin structure immediately followed by oligo(U), which has been extensively studied in terms of its transcription termination mechanism, but little is known about its physiological or regulatory functions. In this study, using a T7 mutant phage, where a 31-bp segment of Tϕ was deleted from the genome, we discovered that deletion of Tϕ from T7 reduces the phage burst size but delays lysis timing, both of which are disadvantageous for the phage. The burst downsizing could directly result from Tϕ deletion-caused upregulation of gene 17.5, coding for holin, among other Tϕ downstream genes, because infection of gp17.5-overproducing Escherichia coli by wild-type T7 phage showed similar burst downsizing. However, the lysis delay was not associated with cellular levels of holin or lysozyme or with rates of phage adsorption. Instead, when allowed to evolve spontaneously in five independent adaptation experiments, the Tϕ-lacking mutant phage, after 27 or 29 passages, recovered both burst size and lysis time reproducibly by deleting early genes 0.5, 0.6, and 0.7 of class I, among other mutations. Deletion of genes 0.5 to 0.7 from the Tϕ-lacking mutant phage decreased expression of several Tϕ downstream genes to levels similar to that of the wild-type phage. Accordingly, phage T7 lysis timing is associated with cellular levels of Tϕ downstream gene products. This suggests the involvement of unknown factor(s) besides the known lysis proteins, lysozyme and holin, and that Tϕ plays a role of optimizing burst size and lysis time during T7 infection. IMPORTANCE Bacteriophages are bacterium-infecting viruses. After producing numerous progenies inside bacteria, phages lyse bacteria using their lysis protein(s) to get out and start a new infection cycle. Normally, lysis is tightly controlled to ensure phage progenies are maximally produced and released at an optimal time. Here, we have

Mcm2 deficiency results in short deletions allowing high resolution identification of genes contributing to lymphoblastic lymphoma

PubMed Central

Rusiniak, Michael E.; Kunnev, Dimiter; Freeland, Amy; Cady, Gillian K.; Pruitt, Steven C.

2011-01-01

Mini-chromosome maintenance (Mcm) proteins are part of the replication licensing complex that is loaded onto chromatin during the G1-phase of the cell cycle and required for initiation of DNA replication in the subsequent S-phase. Mcm proteins are typically loaded in excess of the number of locations that are utilized during S-phase. Nonetheless, partial depletion of Mcm proteins leads to cancers and stem cell deficiencies. Mcm2 deficient mice, on a 129Sv genetic background, display a high rate of thymic lymphoblastic lymphoma. Here array comparative genomic hybridization (aCGH) is utilized to characterize the genetic damage accruing in these tumors. The predominant events are deletions averaging less than 0.5 Mb, considerably shorter than observed in prior studies using alternative mouse lymphoma models or human tumors. Such deletions facilitate identification of specific genes and pathways responsible for the tumors. Mutations in many genes that have been implicated in human lymphomas are recapitulated in this mouse model. These features, and the fact that the mutation underlying the accelerated genetic damage does not target a specific gene or pathway a priori, are valuable features of this mouse model for identification of tumor suppressor genes. Genes affected in all tumors include Pten, Tcfe2a, Mbd3 and Setd1b. Notch1 and additional genes are affected in subsets of tumors. The high frequency of relatively short deletions is consistent with elevated recombination between nearby stalled replication forks in Mcm2 deficient mice. PMID:22158038

High-density array-CGH with targeted NGS unmask multiple noncontiguous minute deletions on chromosome 3p21 in mesothelioma.

PubMed

Yoshikawa, Yoshie; Emi, Mitsuru; Hashimoto-Tamaoki, Tomoko; Ohmuraya, Masaki; Sato, Ayuko; Tsujimura, Tohru; Hasegawa, Seiki; Nakano, Takashi; Nasu, Masaki; Pastorino, Sandra; Szymiczek, Agata; Bononi, Angela; Tanji, Mika; Pagano, Ian; Gaudino, Giovanni; Napolitano, Andrea; Goparaju, Chandra; Pass, Harvey I; Yang, Haining; Carbone, Michele

2016-11-22

We used a custom-made comparative genomic hybridization array (aCGH; average probe interval 254 bp) to screen 33 malignant mesothelioma (MM) biopsies for somatic copy number loss throughout the 3p21 region (10.7 Mb) that harbors 251 genes, including BRCA1 (breast cancer 1)-associated protein 1 (BAP1), the most commonly mutated gene in MM. We identified frequent minute biallelic deletions (<3 kb) in 46 of 251 genes: four were cancer-associated genes: SETD2 (SET domain-containing protein 2) (7 of 33), BAP1 (8 of 33), PBRM1 (polybromo 1) (3 of 33), and SMARCC1 (switch/sucrose nonfermentable- SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily c, member 1) (2 of 33). These four genes were further investigated by targeted next-generation sequencing (tNGS), which revealed sequence-level mutations causing biallelic inactivation. Combined high-density aCGH and tNGS revealed biallelic gene inactivation in SETD2 (9 of 33, 27%), BAP1 (16 of 33, 48%), PBRM1 (5 of 33, 15%), and SMARCC1 (2 of 33, 6%). The incidence of genetic alterations detected is much higher than reported in the literature because minute deletions are not detected by NGS or commercial aCGH. Many of these minute deletions were not contiguous, but rather alternated with segments showing oscillating copy number changes along the 3p21 region. In summary, we found that in MM: (i) multiple minute simultaneous biallelic deletions are frequent in chromosome 3p21, where they occur as distinct events involving multiple genes; (ii) in addition to BAP1, mutations of SETD2, PBRM1, and SMARCC1 are frequent in MM; and (iii) our results suggest that high-density aCGH combined with tNGS provides a more precise estimate of the frequency and types of genes inactivated in human cancer than approaches based exclusively on NGS strategy.

Opposing roles for DNA structure-specific proteins Rad1, Msh2, Msh3, and Sgs1 in yeast gene targeting.

PubMed

Langston, Lance D; Symington, Lorraine S

2005-06-15

Targeted gene replacement (TGR) in yeast and mammalian cells is initiated by the two free ends of the linear targeting molecule, which invade their respective homologous sequences in the chromosome, leading to replacement of the targeted locus with a selectable gene from the targeting DNA. To study the postinvasion steps in recombination, we examined the effects of DNA structure-specific proteins on TGR frequency and heteroduplex DNA formation. In strains deleted of RAD1, MSH2, or MSH3, we find that the frequency of TGR is reduced and the mechanism of TGR is altered while the reverse is true for deletion of SGS1, suggesting that Rad1 and Msh2:Msh3 facilitate TGR while Sgs1 opposes it. The altered mechanism of TGR in the absence of Msh2:Msh3 and Rad1 reveals a separate role for these proteins in suppressing an alternate gene replacement pathway in which incorporation of both homology regions from a single strand of targeting DNA into heteroduplex with the targeted locus creates a mismatch between the selectable gene on the targeting DNA and the targeted gene in the chromosome.

Problem-Solving Test: Conditional Gene Targeting Using the Cre/loxP Recombination System

ERIC Educational Resources Information Center

Szeberényi, József

2013-01-01

Terms to be familiar with before you start to solve the test: gene targeting, knock-out mutation, bacteriophage, complementary base-pairing, homologous recombination, deletion, transgenic organisms, promoter, polyadenylation element, transgene, DNA replication, RNA polymerase, Shine-Dalgarno sequence, restriction endonuclease, polymerase chain…

[Gene Diagnosis and Analysis of Clinical Hematological Phenotype of Thailand Deleted α-Thalassemia 1].

PubMed

Lin, Na; Huang, Hai-Long; Wang, Yan; Zheng, Lin; Fang, Xiang-Qun; Cai, Mei-Ying; Wang, Lin-Shuo; Liu, He-Kun; Xu, Liang-Pu; Lin, Yuan

2016-08-01

To investigate the hematologic characteristics and gene diagnosis of patients with Thailand deleted α-thalassemia 1, so as to provide the information for clinical genetic counseling. The clinical data of 32 patients with Thailand delated α-thalassemia 1 were analyzed retrospectively; the hematologic characteristics and gene diagnosis of Thailand deleted type were investigated by using routine hematologic examination, genetic detection of common thalassemia and Thailand deleted α-thalassemia 1. Among 32 cases, the Thailand deleted α-thalassemia 1 heterozygote was found in 29 cases, the Thailand deleted α-thalassemia 1 and α(3.7) gene deletion double heterozygote were found in 1 case, the Thailand deleted α-thalassemia 1 with β-thalassemia (1 case with codons 41-42 mutation heterozygous, 1 case with CD17 mutation heterozygous) was found in 2 cases by detection. The MCV and MCH levels were decreased in all cases of Thailand deleted thalassemia 1, there were significant differences in RBC, MCV, MCH (P<0.05) between normal control and Thailand deletion α-thalassemia 1 group; there were also significant differences in MCHC (P<0.05) between Southeast asia thalassemia and Thailand deleted α-thalassemia 1 group. There are no significant differences in hematological parameters except MCHC between Southeast asia thalassemia and Thailand deleted α-thalassemia 1 group. moreover the Thailand deleted α-thalassemia 1 in a certain proportion exists in area with high incidence of thalassemia, therefor the clinicians should pay more attention to the screen and diagnosis of Thailand delated α-thalassemia and can exactly diagnose the Thailand delected α-thalassemia 1 on the basis of comprehensive analysis of conventional and Thailand delected α-thalassemia 1 detection results, clinical presentation, hematologic parameters and ultrasonic examination, so as to avoid the birth of child with severe and intermidiate type α-thalassemia caused by Thailand deleted �

Rapid deletion plasmid construction methods for protoplast and Agrobacterium based fungal transformation systems

USDA-ARS?s Scientific Manuscript database

Increasing availability of genomic data and sophistication of analytical methodology in fungi has elevated the need for functional genomics tools in these organisms. Gene deletion is a critical tool for functional analysis. The targeted deletion of genes requires both a suitable method for the trans...

Deletion of the "OPHN1" Gene Detected by aCGH

ERIC Educational Resources Information Center

Madrigal, I.; Rodriguez-Revenga, L.; Badenas, C.; Sanchez, A.; Mila, M.

2008-01-01

Background: The oligophrenin 1 gene ("OPHN1") is an Rho-GTPase-activating protein involved in the regulation of the G-protein cycle required for dendritic spine morphogenesis. Mutations in this gene are implicated in X-linked mental retardation (XLMR). Methods: We report a deletion spanning exons 21 and 22 of the "OPHN1" gene identified by a…

Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9

PubMed Central

Satheka, Achim Cchitvsanzwhoh; Togo, Jacques; An, Yao; Humphrey, Mabwi; Ban, Luying; Ji, Yan; Jin, Honghong; Feng, Xuechao; Zheng, Yaowu

2015-01-01

ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. However, the most mutations reported up to date are small insertions and deletions. In this report, CRISPR/Cas9 system was used to make large DNA fragment deletions and insertions, including entire Dip2a gene deletion, about 65kb in size, and β-galactosidase (lacZ) reporter gene insertion of larger than 5kb in mouse. About 11.8% (11/93) are positive for 65kb deletion from transfected and diluted ES clones. High targeting efficiencies in ES cells were also achieved with G418 selection, 46.2% (12/26) and 73.1% (19/26) for left and right arms respectively. Targeted large fragment deletion efficiency is about 21.4% of live pups or 6.0% of injected embryos. Targeted insertion of lacZ reporter with NEO cassette showed 27.1% (13/48) of targeting rate by ES cell transfection and 11.1% (2/18) by direct zygote injection. The procedures have bypassed in vitro transcription by directly co-injection of zygotes or co-transfection of embryonic stem cells with circular plasmid DNA. The methods are technically easy, time saving, and cost effective in generating mouse models and will certainly facilitate gene function studies. PMID:25803037

Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus.

PubMed

Yang, Ying-Jie; Wang, Ye; Li, Zhi-Feng; Gong, Ya; Zhang, Peng; Hu, Wen-Chao; Sheng, Duo-Hong; Li, Yue-Zhong

2017-08-16

The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA-sgRNA-tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency. By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content.

Opposite effects of GSTM1--and GSTT1: gene deletion variants on bone mineral density.

PubMed

Mlakar, Simona Jurkovic; Osredkar, Josko; Prezelj, Janez; Marc, Janja

2011-01-01

Oxidative stress is associated with osteoporosis. The glutathione S-transferases form the major detoxifying group of enzymes responsible for eliminating products of oxidative stress. We have therefore proposed GSTM1 and GSTT1 genes as candidates for studying the genetics of osteoporosis. The aim of the present study was to examine possible association of GSTM1 and GSTT1 gene deletion polymorphisms, alone or in combination, with bone mineral density at femoral neck (BMD_fn), lumbar spine (BMD_ls) and total hip (BMD_th) in Slovenian elderly women and men.GSTM1 and GSTT1 gene deletion polymorphisms in 712 elderly people were analyzed using the triplex PCR method for the presence of GSTM1 and GSTT1 gene segments. BMD_fn, BMD_ls and BMD_th were measured by the dual-energy X-ray absorptiometry (DEXA) method. Results were analyzed using univariate statistic model adjusted for sex, body mass index (BMI) and age. Our results showed the significant differences in BMD_th, BMD_ls and BMD_fn values (p=0.031, 0.017 and 0.023, respectively) in subgroups of GSTT1 gene deletion polymorphism. For GSTM1 gene deletion polymorphism borderline significant association was found with BMD_ls (p=0.100). Furthermore, subjects with homozygous deletion of GSTT1 gene showed higher BMD values on all measured skeletal sites and, in contrast, subjects with homozygous deletion of GSTM1 gene showed lower BMD values. Moreover, a gene-gene interaction study showed significant association of GSTM1-null and GSTT1-null polymorphisms with BMD_ls values (p=0.044). Carriers with a combination of the presence of GSTT1 gene and the homozygous absence of GSTM1 gene fragment were associated with the lower BMD values at all skeletal sites. The significant association of combination of GSTT1 gene presence and homozygous absence of GSTM1 gene with BMD was demonstrated, suggesting that it could be used, if validated in other studies, as genetic marker for low BMD.

[Deletion of two genes from the genome of fission yeast Schizosaccharomyces pombe. Genetic manipulation and phenotype study].

PubMed

Petrescu, Elena; Voicu, Pia-Maneula; Poiţelea, M; Stoica, B; Stănescu, Raluca; Rusu, M

2005-01-01

The aim of this study was to delete two genes from the genome of the fission yeast S. pombe in order to search for their functions in the cell. These genes are SPAC869.02c (MRI) and SPBC21C3.19 (MR2) and previous studies reported their significant induction after gamma irradiation. We carried out the deletions of the two genes and we replaced them with the selection marker ura4. Among the phenotype characteristics we tested the viability, the sexual behaviour and the radiosensitivity to ultraviolet and gamma irradiation. Our results indicate that MR1-deleted strain is sensitive to both UV and gamma irradiation, while the survival of the irradiated MR2-deleted strain doesn't appear to be influenced by the deletion. This suggests an involvement of MR1 gene in the adaptive response triggered by these types of genotoxic aggression. The comparison of MR1-d and MR2-d with the double deleted strains containing the deletion of MR1 or MR2 combined with the deletion of sty1 or rad3 genes led to a surprising result: the double mutants MR1-d sty1-d and MR1-d rad3-d were more resistant to both UV and gamma irradiation than the simple deleted strains sty1-d and rad3-d, respectively. This suggests a possible contribution of MR1 gene to the lethal process taking place in irradiated cells.

Seven gene deletions in seven days: Fast generation of Escherichia coli strains tolerant to acetate and osmotic stress

PubMed Central

Jensen, Sheila I.; Lennen, Rebecca M.; Herrgård, Markus J.; Nielsen, Alex T.

2015-01-01

Generation of multiple genomic alterations is currently a time consuming process. Here, a method was established that enables highly efficient and simultaneous deletion of multiple genes in Escherichia coli. A temperature sensitive plasmid containing arabinose inducible lambda Red recombineering genes and a rhamnose inducible flippase recombinase was constructed to facilitate fast marker-free deletions. To further speed up the procedure, we integrated the arabinose inducible lambda Red recombineering genes and the rhamnose inducible FLP into the genome of E. coli K-12 MG1655. This system enables growth at 37 °C, thereby facilitating removal of integrated antibiotic cassettes and deletion of additional genes in the same day. Phosphorothioated primers were demonstrated to enable simultaneous deletions during one round of electroporation. Utilizing these methods, we constructed strains in which four to seven genes were deleted in E. coli W and E. coli K-12. The growth rate of an E. coli K-12 quintuple deletion strain was significantly improved in the presence of high concentrations of acetate and NaCl. In conclusion, we have generated a method that enables efficient and simultaneous deletion of multiple genes in several E. coli variants. The method enables deletion of up to seven genes in as little as seven days. PMID:26643270

A novel large deletion mutation of FERMT1 gene in a Chinese patient with Kindler syndrome.

PubMed

Gao, Ying; Bai, Jin-li; Liu, Xiao-yan; Qu, Yu-jin; Cao, Yan-yan; Wang, Jian-cai; Jin, Yu-wei; Wang, Hong; Song, Fang

2015-11-01

Kindler syndrome (KS; OMIM 173650) is a rare autosomal recessive skin disorder, which results in symptoms including blistering, epidermal atrophy, increased risk of cancer, and poor wound healing. The majority of mutations of the disease-determining gene (FERMT1 gene) are single nucleotide substitutions, including missense mutations, nonsense mutations, etc. Large deletion mutations are seldom reported. To determine the mutation in the FERMT1 gene associated with a 7-year-old Chinese patient who presented clinical manifestation of KS, we performed direct sequencing of all the exons of FERMT1 gene. For the exons 2-6 without amplicons, we analyzed the copy numbers using quantitative real-time polymerase chain reaction (qRT-PCR) with specific primers. The deletion breakpoints were sublocalized and the range of deletion was confirmed by PCR and direct sequencing. In this study, we identified a new 17-kb deletion mutation spanning the introns 1-6 of FERMT1 gene in a Chinese patient with severe KS phenotypes. Her parents were carriers of the same mutation. Our study reported a newly identified large deletion mutation of FERMT1 gene involved in KS, which further enriched the mutation spectrum of the FERMT1 gene.

RNA-guided genome editing for target gene mutations in wheat.

PubMed

Upadhyay, Santosh Kumar; Kumar, Jitesh; Alok, Anshu; Tuli, Rakesh

2013-12-09

The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas-mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3' end of the target site abolished the cleavage activity completely. The mismatches at the 5' end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3' end. This approach provides a powerful method for genome engineering in plants.

DIA1R is an X-linked gene related to Deleted In Autism-1.

PubMed

Aziz, Azhari; Harrop, Sean P; Bishop, Naomi E

2011-01-17

Autism spectrum disorders (ASDS) are frequently occurring disorders diagnosed by deficits in three core functional areas: social skills, communication, and behaviours and/or interests. Mental retardation frequently accompanies the most severe forms of ASDs, while overall ASDs are more commonly diagnosed in males. Most ASDs have a genetic origin and one gene recently implicated in the etiology of autism is the Deleted-In-Autism-1 (DIA1) gene. Using a bioinformatics-based approach, we have identified a human gene closely related to DIA1, we term DIA1R (DIA1-Related). While DIA1 is autosomal (chromosome 3, position 3q24), DIA1R localizes to the X chromosome at position Xp11.3 and is known to escape X-inactivation. The gene products are of similar size, with DIA1 encoding 430, and DIA1R 433, residues. At the amino acid level, DIA1 and DIA1R are 62% similar overall (28% identical), and both encode signal peptides for targeting to the secretory pathway. Both genes are ubiquitously expressed, including in fetal and adult brain tissue. Examination of published literature revealed point mutations in DIA1R are associated with X-linked mental retardation (XLMR) and DIA1R deletion is associated with syndromes with ASD-like traits and/or XLMR. Together, these results support a model where the DIA1 and DIA1R gene products regulate molecular traffic through the cellular secretory pathway or affect the function of secreted factors, and functional deficits cause disorders with ASD-like symptoms and/or mental retardation.

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.

Targeted disruption of the 3p12 gene, Dutt1/Robo1, predisposes mice to lung adenocarcinomas and lymphomas with methylation of the gene promoter.

PubMed

Xian, Jian; Aitchison, Alan; Bobrow, Linda; Corbett, Gerard; Pannell, Richard; Rabbitts, Terence; Rabbitts, Pamela

2004-09-15

The DUTT1 gene is located on human chromosome 3, band p12, within a region of nested homozygous deletions in breast and lung tumors. It is therefore a candidate tumor suppressor gene in humans and is the homologue (ROBO1) of the Drosophila axonal guidance receptor gene, Roundabout. We have shown previously that mice with a targeted homozygous deletion within the Dutt1/Robo1 gene generally die at birth due to incomplete lung development: survivors die within the first year of life with epithelial bronchial hyperplasia as a common feature. Because Dutt1/Robo1 heterozygous mice develop normally, we have determined their tumor susceptibility. Mice with a targeted deletion within one Dutt1/Robo1 allele spontaneously develop lymphomas and carcinomas in their second year of life with a 3-fold increase in incidence compared with controls: invasive lung adenocarcinomas are by far the predominant carcinoma. In addition to the mutant allele, loss of heterozygosity analysis indicates that these tumors retain the structurally normal allele but with substantial methylation of the gene's promoter. Substantial reduction of Dutt1/Robo1 protein expression in tumors is observed by Western blotting and immunohistochemistry. This suggests that Dutt1/Robo1 is a classic tumor suppressor gene requiring inactivation of both alleles to elicit tumorigenesis in these mice.

The frequency of previously undetectable deletions involving 3' Exons of the PMS2 gene.

PubMed

Vaughn, Cecily P; Baker, Christine L; Samowitz, Wade S; Swensen, Jeffrey J

2013-01-01

Lynch syndrome is characterized by mutations in one of four mismatch repair genes, MLH1, MSH2, MSH6, or PMS2. Clinical mutation analysis of these genes includes sequencing of exonic regions and deletion/duplication analysis. However, detection of deletions and duplications in PMS2 has previously been confined to Exons 1-11 due to gene conversion between PMS2 and the pseudogene PMS2CL in the remaining 3' exons (Exons 12-15). We have recently described an MLPA-based method that permits detection of deletions of PMS2 Exons 12-15; however, the frequency of such deletions has not yet been determined. To address this question, we tested for 3' deletions in 58 samples that were reported to be negative for PMS2 mutations using previously available methods. All samples were from individuals whose tumors exhibited loss of PMS2 immunohistochemical staining without concomitant loss of MLH1 immunostaining. We identified seven samples in this cohort with deletions in the 3' region of PMS2, including three previously reported samples with deletions of Exons 13-15 (two samples) and Exons 14-15. Also detected were deletions of Exons 12-15, Exon 13, and Exon 14 (two samples). Breakpoint analysis of the intragenic deletions suggests they occurred through Alu-mediated recombination. Our results indicate that ∼12% of samples suspected of harboring a PMS2 mutation based on immunohistochemical staining, for which mutations have not yet been identified, would benefit from testing using the new methodology. Copyright © 2012 Wiley Periodicals, Inc.

Improved pestalotiollide B production by deleting competing polyketide synthase genes in Pestalotiopsis microspora.

PubMed

Chen, Longfei; Li, Yingying; Zhang, Qian; Wang, Dan; Akhberdi, Oren; Wei, Dongsheng; Pan, Jiao; Zhu, Xudong

2017-02-01

Pestalotiollide B, an analog of dibenzodioxocinones which are inhibitors of cholesterol ester transfer proteins, is produced by Pestalotiopsis microspora NK17. To increase the production of pestalotiollide B, we attempted to eliminate competing polyketide products by deleting the genes responsible for their biosynthesis. We successfully deleted 41 out of 48 putative polyketide synthases (PKSs) in the genome of NK17. Nine of the 41 PKS deleted strains had significant increased production of pestalotiollide B (P < 0.05). For instance, deletion of pks35, led to an increase of pestalotiollide B by 887%. We inferred that these nine PKSs possibly lead to branch pathways that compete for precursors with pestalotiollide B, or that convert the product. Deletion of some other PKS genes such as pks8 led to a significant decrease of pestalotiollide B, suggesting they are responsible for its biosynthesis. Our data demonstrated that improvement of pestalotiollide B production can be achieved by eliminating competing polyketides.

The Immature Fiber Mutant Phenotype of Cotton (Gossypium hirsutum) Is Linked to a 22-bp Frame-Shift Deletion in a Mitochondria Targeted Pentatricopeptide Repeat Gene.

PubMed

Thyssen, Gregory N; Fang, David D; Zeng, Linghe; Song, Xianliang; Delhom, Christopher D; Condon, Tracy L; Li, Ping; Kim, Hee Jin

2016-06-01

Cotton seed trichomes are the most important source of natural fibers globally. The major fiber thickness properties influence the price of the raw material, and the quality of the finished product. The recessive immature fiber (im) gene reduces the degree of fiber cell wall thickening by a process that was previously shown to involve mitochondrial function in allotetraploid Gossypium hirsutum Here, we present the fine genetic mapping of the im locus, gene expression analysis of annotated proteins near the locus, and association analysis of the linked markers. Mapping-by-sequencing identified a 22-bp deletion in a pentatricopeptide repeat (PPR) gene that is completely linked to the immature fiber phenotype in 2837 F2 plants, and is absent from all 163 cultivated varieties tested, although other closely linked marker polymorphisms are prevalent in the diversity panel. This frame-shift mutation results in a transcript with two long open reading frames: one containing the N-terminal transit peptide that targets mitochondria, the other containing only the RNA-binding PPR domains, suggesting that a functional PPR protein cannot be targeted to mitochondria in the im mutant. Taken together, these results suggest that PPR gene Gh_A03G0489 is involved in the cotton fiber wall thickening process, and is a promising candidate gene at the im locus. Our findings expand our understanding of the molecular mechanisms that modulate cotton fiber fineness and maturity, and may facilitate the development of cotton varieties with superior fiber attributes. Copyright © 2016 Thyssen et al.

Editor’s Highlight: A Genome-wide Screening of Target Genes Against Silver Nanoparticles in Fission Yeast

PubMed Central

Lee, Sook-Jeong; Lee, Minho; Nam, Miyoung; Lee, Sol; Choi, Jian; Lee, Hye-Jin; Kim, Dong-Uk; Hoe, Kwang-Lae

2018-01-01

Abstract To identify target genes against silver nanoparticles (AgNPs), we screened a genome-wide gene deletion library of 4843 fission yeast heterozygous mutants covering 96% of all protein encoding genes. A total of 33 targets were identified by a microarray and subsequent individual confirmation. The target pattern of AgNPs was more similar to those of AgNO3 and H2O2, followed by Cd and As. The toxic effect of AgNPs on fission yeast was attributed to the intracellular uptake of AgNPs, followed by the subsequent release of Ag+, leading to the generation of reactive oxygen species (ROS). Next, we focused on the top 10 sensitive targets for further studies. As described previously, 7 nonessential targets were associated with detoxification of ROS, because their heterozygous mutants showed elevated ROS levels. Three novel essential targets were related to folate metabolism or cellular component organization, resulting in cell cycle arrest and no induction in the transcriptional level of antioxidant enzymes such as Sod1 and Gpx1 when 1 of the 2 copies was deleted. Intriguingly, met9 played a key role in combating AgNP-induced ROS generation via NADPH production and was also conserved in a human cell line. PMID:29294138

Relatively low proportion of dystrophin gene deletions in Israeili Duchenne and Becker muscular dystrophy patients

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

Shomrat, R.; Gluck, E.; Legum, C.

1994-02-15

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the X-linked dystrophin gene. The most common mutations in western populations are deletions that are spread non-randomly throughout the gene. Molecular analysis of the dystrophin gene structure by hybridization of the full length cDNA to Southern blots and by PCR in 62 unrelated Israeli male DMD/BMD patients showed deletions in 23 (37%). This proportion is significantly lower than that found in European and North American populations (55-65%). Seventy-eight percent of the deletions were confined to exons 44-52, half of these exons 44-45, and themore » remaining 22% to exons 1 and 19. There was no correlation between the size of the deletion and the severity of the disease. All the deletions causing frameshift resulted in the DMD phenotypes. 43 refs., 1 fig., 1 tab.« less

Assessment of the Toxicity of CuO Nanoparticles by Using Saccharomyces cerevisiae Mutants with Multiple Genes Deleted

PubMed Central

Bao, Shaopan; Lu, Qicong; Dai, Heping; Zhang, Chao

2015-01-01

To develop applicable and susceptible models to evaluate the toxicity of nanoparticles, the antimicrobial effects of CuO nanoparticles (CuO-NPs) on various Saccharomyces cerevisiae (S. cerevisiae) strains (wild type, single-gene-deleted mutants, and multiple-gene-deleted mutants) were determined and compared. Further experiments were also conducted to analyze the mechanisms associated with toxicity using copper salt, bulk CuO (bCuO), carbon-shelled copper nanoparticles (C/Cu-NPs), and carbon nanoparticles (C-NPs) for comparisons. The results indicated that the growth inhibition rates of CuO-NPs for the wild-type and the single-gene-deleted strains were comparable, while for the multiple-gene deletion mutant, significantly higher toxicity was observed (P < 0.05). When the toxicity of the CuO-NPs to yeast cells was compared with the toxicities of copper salt and bCuO, we concluded that the toxicity of CuO-NPs should be attributed to soluble copper rather than to the nanoparticles. The striking difference in adverse effects of C-NPs and C/Cu-NPs with equivalent surface areas also proved this. A toxicity assay revealed that the multiple-gene-deleted mutant was significantly more sensitive to CuO-NPs than the wild type. Specifically, compared with the wild-type strain, copper was readily taken up by mutant strains when cell permeability genes were knocked out, and the mutants with deletions of genes regulated under oxidative stress (OS) were likely producing more reactive oxygen species (ROS). Hence, as mechanism-based gene inactivation could increase the susceptibility of yeast, the multiple-gene-deleted mutants should be improved model organisms to investigate the toxicity of nanoparticles. PMID:26386067

Screening for microsatellite instability target genes in colorectal cancers

PubMed Central

Vilkki, S; Launonen, V; Karhu, A; Sistonen, P; Vastrik, I; Aaltonen, L

2002-01-01

Background: Defects in the DNA repair system lead to genetic instability because replication errors are not corrected. This type of genetic instability is a key event in the malignant progression of HNPCC and a subset of sporadic colon cancers and mutation rates are particularly high at short repetitive sequences. Somatic deletions of coding mononucleotide repeats have been detected, for example, in the TGFßRII and BAX genes, and recently many novel target genes for microsatellite instability (MSI) have been proposed. Novel target genes are likely to be discovered in the future. More data should be created on background mutation rates in MSI tumours to evaluate mutation rates observed in the candidate target genes. Methods: Mutation rates in 14 neutral intronic repeats were evaluated in MSI tumours. Bioinformatic searches combined with keywords related to cancer and tumour suppressor or CRC related gene homology were used to find new candidate MSI target genes. By comparison of mutation frequencies observed in intronic mononucleotide repeats versus exonic coding repeats of potential MSI target genes, the significance of the exonic mutations was estimated. Results: As expected, the length of an intronic mononucleotide repeat correlated positively with the number of slippages for both G/C and A/T repeats (p=0.0020 and p=0.0012, respectively). BRCA1, CtBP1, and Rb1 associated CtIP and other candidates were found in a bioinformatic search combined with keywords related to cancer. Sequencing showed a significantly increased mutation rate in the exonic A9 repeat of CtIP (25/109=22.9%) as compared with similar intronic repeats (p≤0.001). Conclusions: We propose a new candidate MSI target gene CtIP to be evaluated in further studies. PMID:12414815

Studies on the Nucleotide Sequence, Transcription and Deletion Analysis of the BmNPV Protein Kinase Gene.

PubMed

Zhang, Chuan-Xi; Hu, Cui; Wu, Xiang-Fu

1998-01-01

The coding region of BmvPK-1 gene of Bombyx mori NPV (Strain ZJ8) is 828 nt long and encodes a 276 aa polypeptide with predicted molecular mass of 32 kD. Dot blot analysis showed its mRNA to be gene is first detectable at 18 h p.i. and reaching the highest transcriptional level at 48 h p.i. The result suggested that BmvPK-1 gene is a late or very late gene. The most conserved 365 bp of the BmvPK-1 gene was deleted in a transfer vector (pUCPK-lac), and a report gene (lacZ) was inserted in the deleted position. Cotransfection of BmN cells with pUCPK-lac DNA and BmNPV DNA resulted in the recombinant virus which expressed detectable product of lacZ gene. But the virus with the deleted BmvPK-1 gene could not be isolated from the wild BmNPV by plaque purification method. The result showed that the BmvPK-1 gene deleted virus can multiply only with the help of the product of this gene from the wild type virus, and the gene is necessary for the virus to finish its life cycle in the cultured cells.

First report of a deletion encompassing an entire exon in the homogentisate 1,2-dioxygenase gene causing alkaptonuria.

PubMed

Zouheir Habbal, Mohammad; Bou-Assi, Tarek; Zhu, Jun; Owen, Renius; Chehab, Farid F

2014-01-01

Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5-16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband's phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin.

First Report of a Deletion Encompassing an Entire Exon in the Homogentisate 1,2-Dioxygenase Gene Causing Alkaptonuria

PubMed Central

Habbal, Mohammad Zouheir; Bou-Assi, Tarek; Zhu, Jun; Owen, Renius; Chehab, Farid F.

2014-01-01

Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5–16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband’s phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin. PMID:25233259

Targeted deletion of miR-132/-212 impairs memory and alters the hippocampal transcriptome.

PubMed

Hansen, Katelin F; Sakamoto, Kensuke; Aten, Sydney; Snider, Kaitlin H; Loeser, Jacob; Hesse, Andrea M; Page, Chloe E; Pelz, Carl; Arthur, J Simon C; Impey, Soren; Obrietan, Karl

2016-02-01

miR-132 and miR-212 are structurally related microRNAs that have been found to exert powerful modulatory effects within the central nervous system (CNS). Notably, these microRNAs are tandomly processed from the same noncoding transcript, and share a common seed sequence: thus it has been difficult to assess the distinct contribution of each microRNA to gene expression within the CNS. Here, we employed a combination of conditional knockout and transgenic mouse models to examine the contribution of the miR-132/-212 gene locus to learning and memory, and then to assess the distinct effects that each microRNA has on hippocampal gene expression. Using a conditional deletion approach, we show that miR-132/-212 double-knockout mice exhibit significant cognitive deficits in spatial memory, recognition memory, and in tests of novel object recognition. Next, we utilized transgenic miR-132 and miR-212 overexpression mouse lines and the miR-132/-212 double-knockout line to explore the distinct effects of these two miRNAs on the transcriptional profile of the hippocampus. Illumina sequencing revealed that miR-132/-212 deletion increased the expression of 1138 genes; Venn analysis showed that 96 of these genes were also downregulated in mice overexpressing miR-132. Of the 58 genes that were decreased in animals overexpressing miR-212, only four of them were also increased in the knockout line. Functional gene ontology analysis of downregulated genes revealed significant enrichment of genes related to synaptic transmission, neuronal proliferation, and morphogenesis, processes known for their roles in learning, and memory formation. These data, coupled with previous studies, firmly establish a role for the miR-132/-212 gene locus as a key regulator of cognitive capacity. Further, although miR-132 and miR-212 share a seed sequence, these data indicate that these miRNAs do not exhibit strongly overlapping mRNA targeting profiles, thus indicating that these two genes may function in

Targeted deletion of miR-132/-212 impairs memory and alters the hippocampal transcriptome

PubMed Central

Hansen, Katelin F.; Sakamoto, Kensuke; Aten, Sydney; Snider, Kaitlin H.; Loeser, Jacob; Hesse, Andrea M.; Page, Chloe E.; Pelz, Carl; Arthur, J. Simon C.; Impey, Soren

2016-01-01

miR-132 and miR-212 are structurally related microRNAs that have been found to exert powerful modulatory effects within the central nervous system (CNS). Notably, these microRNAs are tandomly processed from the same noncoding transcript, and share a common seed sequence: thus it has been difficult to assess the distinct contribution of each microRNA to gene expression within the CNS. Here, we employed a combination of conditional knockout and transgenic mouse models to examine the contribution of the miR-132/-212 gene locus to learning and memory, and then to assess the distinct effects that each microRNA has on hippocampal gene expression. Using a conditional deletion approach, we show that miR-132/-212 double-knockout mice exhibit significant cognitive deficits in spatial memory, recognition memory, and in tests of novel object recognition. Next, we utilized transgenic miR-132 and miR-212 overexpression mouse lines and the miR-132/-212 double-knockout line to explore the distinct effects of these two miRNAs on the transcriptional profile of the hippocampus. Illumina sequencing revealed that miR-132/-212 deletion increased the expression of 1138 genes; Venn analysis showed that 96 of these genes were also downregulated in mice overexpressing miR-132. Of the 58 genes that were decreased in animals overexpressing miR-212, only four of them were also increased in the knockout line. Functional gene ontology analysis of downregulated genes revealed significant enrichment of genes related to synaptic transmission, neuronal proliferation, and morphogenesis, processes known for their roles in learning, and memory formation. These data, coupled with previous studies, firmly establish a role for the miR-132/-212 gene locus as a key regulator of cognitive capacity. Further, although miR-132 and miR-212 share a seed sequence, these data indicate that these miRNAs do not exhibit strongly overlapping mRNA targeting profiles, thus indicating that these two genes may function in

Identification of apoptosis-related PLZF target genes

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

Bernardo, Maria Victoria; Yelo, Estefania; Gimeno, Lourdes

2007-07-27

The PLZF gene encodes a BTB/POZ-zinc finger-type transcription factor, involved in physiological development, proliferation, differentiation, and apoptosis. In this paper, we investigate proliferation, survival, and gene expression regulation in stable clones from the human haematopoietic K562, DG75, and Jurkat cell lines with inducible expression of PLZF. In Jurkat cells, but not in K562 and DG75 cells, PLZF induced growth suppression and apoptosis in a cell density-dependent manner. Deletion of the BTB/POZ domain of PLZF abrogated growth suppression and apoptosis. PLZF was expressed with a nuclear speckled pattern distinctively in the full-length PLZF-expressing Jurkat clones, suggesting that the nuclear speckled localizationmore » is required for PLZF-induced apoptosis. By microarray analysis, we identified that the apoptosis-inducer TP53INP1, ID1, and ID3 genes were upregulated, and the apoptosis-inhibitor TERT gene was downregulated. The identification of apoptosis-related PLZF target genes may have biological and clinical relevance in cancer typified by altered PLZF expression.« less

Deletion of the transforming growth factor β receptor type II gene in articular chondrocytes leads to a progressive osteoarthritis-like phenotype in mice.

PubMed

Shen, Jie; Li, Jia; Wang, Baoli; Jin, Hongting; Wang, Meina; Zhang, Yejia; Yang, Yunzhi; Im, Hee-Jeong; O'Keefe, Regis; Chen, Di

2013-12-01

While transforming growth factor β (TGFβ) signaling plays a critical role in chondrocyte metabolism, the TGFβ signaling pathways and target genes involved in cartilage homeostasis and the development of osteoarthritis (OA) remain unclear. Using an in vitro cell culture method and an in vivo mouse genetic approach, we undertook this study to investigate TGFβ signaling in chondrocytes and to determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling. TGFβ receptor type II (TGFβRII)-conditional knockout (KO) (TGFβRII(Col2ER)) mice were generated by breeding TGFβRII(flox/flox) mice with Col2-CreER-transgenic mice. Histologic, histomorphometric, and gene expression analyses were performed. In vitro TGFβ signaling studies were performed using chondrogenic rat chondrosarcoma cells. To determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling, TGFβRII/matrix metalloproteinase 13 (MMP-13)- and TGFβRII/ADAMTS-5-double-KO mice were generated and analyzed. Inhibition of TGFβ signaling (deletion of the Tgfbr2 gene in chondrocytes) resulted in up-regulation of Runx2, Mmp13, and Adamts5 expression in articular cartilage tissue and progressive OA development in TGFβRII(Col2ER) mice. Deletion of the Mmp13 or Adamts5 gene significantly ameliorated the OA-like phenotype induced by the loss of TGFβ signaling. Treatment of TGFβRII(Col2ER) mice with an MMP-13 inhibitor also slowed OA progression. Mmp13 and Adamts5 are critical downstream target genes involved in the TGFβ signaling pathway during the development of OA. Copyright © 2013 by the American College of Rheumatology.

Whole Wiskott‑Aldrich syndrome protein gene deletion identified by high throughput sequencing.

PubMed

He, Xiangling; Zou, Runying; Zhang, Bing; You, Yalan; Yang, Yang; Tian, Xin

2017-11-01

Wiskott‑Aldrich syndrome (WAS) is a rare X‑linked recessive immunodeficiency disorder, characterized by thrombocytopenia, small platelets, eczema and recurrent infections associated with increased risk of autoimmunity and malignancy disorders. Mutations in the WAS protein (WASP) gene are responsible for WAS. To date, WASP mutations, including missense/nonsense, splicing, small deletions, small insertions, gross deletions, and gross insertions have been identified in patients with WAS. In addition, WASP‑interacting proteins are suspected in patients with clinical features of WAS, in whom the WASP gene sequence and mRNA levels are normal. The present study aimed to investigate the application of next generation sequencing in definitive diagnosis and clinical therapy for WAS. A 5 month‑old child with WAS who displayed symptoms of thrombocytopenia was examined. Whole exome sequence analysis of genomic DNA showed that the coverage and depth of WASP were extremely low. Quantitative polymerase chain reaction indicated total WASP gene deletion in the proband. In conclusion, high throughput sequencing is useful for the verification of WAS on the genetic profile, and has implications for family planning guidance and establishment of clinical programs.

Contiguous gene deletion of chromosome 2p16.3-p21 as a cause of Lynch syndrome.

PubMed

Salo-Mullen, Erin E; Lynn, Patricio B; Wang, Lu; Walsh, Michael; Gopalan, Anuradha; Shia, Jinru; Tran, Christina; Man, Fung Ying; McBride, Sean; Schattner, Mark; Zhang, Liying; Weiser, Martin R; Stadler, Zsofia K

2018-01-01

Lynch syndrome is an autosomal dominant condition caused by pathogenic mutations in the DNA mismatch repair (MMR) genes. Although commonly associated with clinical features such as intellectual disability and congenital anomalies, contiguous gene deletions may also result in cancer predisposition syndromes. We report on a 52-year-old male with Lynch syndrome caused by deletion of chromosome 2p16.3-p21. The patient had intellectual disability and presented with a prostatic adenocarcinoma with an incidentally identified synchronous sigmoid adenocarcinoma that exhibited deficient MMR with an absence of MSH2 and MSH6 protein expression. Family history was unrevealing. Physical exam revealed short stature, brachycephaly with a narrow forehead and short philtrum, brachydactyly of the hands, palmar transverse crease, broad and small feet with hyperpigmentation of the soles. The patient underwent total colectomy with ileorectal anastomosis for a pT3N1 sigmoid adenocarcinoma. Germline genetic testing of the MSH2, MSH6, and EPCAM genes revealed full gene deletions. SNP-array based DNA copy number analysis identified a deletion of 4.8 Mb at 2p16.3-p21. In addition to the three Lynch syndrome associated genes, the deleted chromosomal section encompassed genes including NRXN1, CRIPT, CALM2, FBXO11, LHCGR, MCFD2, TTC7A, EPAS1, PRKCE, and 15 others. Contiguous gene deletions have been described in other inherited cancer predisposition syndromes, such as Familial Adenomatous Polyposis. Our report and review of the literature suggests that contiguous gene deletion within the 2p16-p21 chromosomal region is a rare cause of Lynch syndrome, but presents with distinct phenotypic features, highlighting the need for recognition and awareness of this syndromic entity.

Deletion and reduced expression of the Fanconi anemia FANCA gene in sporadic acute myeloid leukemia.

PubMed

Tischkowitz, M D; Morgan, N V; Grimwade, D; Eddy, C; Ball, S; Vorechovsky, I; Langabeer, S; Stöger, R; Hodgson, S V; Mathew, C G

2004-03-01

Fanconi anemia (FA) is an autosomal recessive chromosomal instability disorder caused by mutations in one of seven known genes (FANCA,C,D2,E,F,G and BRCA2). Mutations in the FANCA gene are the most prevalent, accounting for two-thirds of FA cases. Affected individuals have greatly increased risks of acute myeloid leukemia (AML). This raises the question as to whether inherited or acquired mutations in FA genes might be involved in the development of sporadic AML. Quantitative fluorescent PCR was used to screen archival DNA from sporadic AML cases for FANCA deletions, which account for 40% of FANCA mutations in FA homozygotes. Four heterozygous deletions were found in 101 samples screened, which is 35-fold higher than the expected population frequency for germline FANCA deletions (P<0.0001). Sequencing FANCA in the AML samples with FANCA deletions did not detect mutations in the second allele and there was no evidence of epigenetic silencing by hypermethylation. However, real-time quantitative PCR analysis in these samples showed reduced expression of FANCA compared to nondeleted AML samples and to controls. These findings suggest that gene deletions and reduced expression of FANCA may be involved in the promotion of genetic instability in a subset of cases of sporadic AML.

Early-onset obesity and paternal 2pter deletion encompassing the ACP1, TMEM18, and MYT1L genes.

PubMed

Doco-Fenzy, Martine; Leroy, Camille; Schneider, Anouck; Petit, Florence; Delrue, Marie-Ange; Andrieux, Joris; Perrin-Sabourin, Laurence; Landais, Emilie; Aboura, Azzedine; Puechberty, Jacques; Girard, Manon; Tournaire, Magali; Sanchez, Elodie; Rooryck, Caroline; Ameil, Agnès; Goossens, Michel; Jonveaux, Philippe; Lefort, Geneviève; Taine, Laurence; Cailley, Dorothée; Gaillard, Dominique; Leheup, Bruno; Sarda, Pierre; Geneviève, David

2014-04-01

Obesity is a common but highly, clinically, and genetically heterogeneous disease. Deletion of the terminal region of the short arm of chromosome 2 is rare and has been reported in about 13 patients in the literature often associated with a Prader-Willi-like phenotype. We report on five unrelated patients with 2p25 deletion of paternal origin presenting with early-onset obesity, hyperphagia, intellectual deficiency, and behavioural difficulties. Among these patients, three had de novo pure 2pter deletions, one presented with a paternal derivative der(2)t(2;15)(p25.3;q26) with deletion in the 2pter region and the last patient presented with an interstitial 2p25 deletion. The size of the deletions was characterized by SNP array or array-CGH and was confirmed by fluorescence in situ hybridization (FISH) studies. Four patients shared a 2p25.3 deletion with a minimal critical region estimated at 1.97 Mb and encompassing seven genes, namely SH3HYL1, ACP1, TMEMI8, SNTG2, TPO, PXDN, and MYT1L genes. The fifth patient had a smaller interstitial deletion encompassing the TPO, PXDN, and MYT1L genes. Paternal origin of the deletion was determined by genotyping using microsatellite markers. Analysis of the genes encompassed in the deleted region led us to speculate that the ACP1, TMEM18, and/or MYT1L genes might be involved in early-onset obesity. In addition, intellectual deficiency and behavioural troubles can be explained by the heterozygous loss of the SNTG2 and MYT1L genes. Finally, we discuss the parent-of-origin of the deletion.

Deletion of the SHOX gene in patients with short stature of unknown cause.

PubMed

Morizio, E; Stuppia, L; Gatta, V; Fantasia, D; Guanciali Franchi, P; Rinaldi, M M; Scarano, G; Concolino, D; Giannotti, A; Verrotti, A; Chiarelli, F; Calabrese, G; Palka, G

2003-06-15

A fluorescence in situ hybridization (FISH) study was performed in 56 patients with short stature of unknown cause in order to establish the role of deletion of the SHOX gene in this population. FISH analysis was carried out on metaphase spreads and interphase lymphocytes from blood smears using a probe specific for the SHOX gene. Deletion of SHOX was found in four patients (7.1%). No skeletal abnormalities were detected in these patients either at the physical examination or at X-rays of the upper and lower limbs. Present results indicate that SHOX plays an important role also in short stature of unknown cause, and FISH analysis appears as an easy, appropriate, and inexpensive method for the detection of SHOX deletion. Copyright 2003 Wiley-Liss, Inc.

Opposite Effects of GSTM1 – and GSTT1 – Gene Deletion Variants on Bone Mineral Density

PubMed Central

Mlakar, Simona Jurkovic; Osredkar, Josko; Prezelj, Janez; Marc, Janja

2011-01-01

Oxidative stress is associated with osteoporosis. The glutathione S-transferases form the major detoxifying group of enzymes responsible for eliminating products of oxidative stress. We have therefore proposed GSTM1 and GSTT1 genes as candidates for studying the genetics of osteoporosis. The aim of the present study was to examine possible association of GSTM1 and GSTT1 gene deletion polymorphisms, alone or in combination, with bone mineral density at femoral neck (BMD_fn), lumbar spine (BMD_ls) and total hip (BMD_th) in Slovenian elderly women and men. GSTM1 and GSTT1 gene deletion polymorphisms in 712 elderly people were analyzed using the triplex PCR method for the presence of GSTM1 and GSTT1 gene segments. BMD_fn, BMD_ls and BMD_th were measured by the dual-energy X-ray absorptiometry (DEXA) method. Results were analyzed using univariate statistic model adjusted for sex, body mass index (BMI) and age. Our results showed the significant differences in BMD_th, BMD_ls and BMD_fn values (p = 0.031, 0.017 and 0.023, respectively) in subgroups of GSTT1 gene deletion polymorphism. For GSTM1 gene deletion polymorphism borderline significant association was found with BMD_ls (p = 0.100). Furthermore, subjects with homozygous deletion of GSTT1 gene showed higher BMD values on all measured skeletal sites and, in contrast, subjects with homozygous deletion of GSTM1 gene showed lower BMD values. Moreover, a gene-gene interaction study showed significant association of GSTM1-null and GSTT1-null polymorphisms with BMD_ls values (p = 0.044). Carriers with a combination of the presence of GSTT1 gene and the homozygous absence of GSTM1 gene fragment were associated with the lower BMD values at all skeletal sites. The significant association of combination of GSTT1 gene presence and homozygous absence of GSTM1 gene with BMD was demonstrated, suggesting that it could be used, if validated in other studies, as genetic marker for low BMD. PMID:22048269

Targeted deletion and lipidomic analysis identify epithelial cell COX-2 as a major driver of chemically induced skin cancer.

PubMed

Jiao, Jing; Ishikawa, Tomo-O; Dumlao, Darren S; Norris, Paul C; Magyar, Clara E; Mikulec, Carol; Catapang, Art; Dennis, Edward A; Fischer, Susan M; Herschman, Harvey R

2014-11-01

Pharmacologic and global gene deletion studies demonstrate that cyclooxygenase-2 (PTGS2/COX-2) plays a critical role in DMBA/TPA-induced skin tumor induction. Although many cell types in the tumor microenvironment express COX-2, the cell types in which COX-2 expression is required for tumor promotion are not clearly established. Here, cell type-specific Cox-2 gene deletion reveals a vital role for skin epithelial cell COX-2 expression in DMBA/TPA tumor induction. In contrast, myeloid Cox-2 gene deletion has no effect on DMBA/TPA tumorigenesis. The infrequent, small tumors that develop on mice with an epithelial cell-specific Cox-2 gene deletion have decreased proliferation and increased cell differentiation properties. Blood vessel density is reduced in tumors with an epithelial cell-specific Cox-2 gene deletion, compared with littermate control tumors, suggesting a reciprocal relationship in tumor progression between COX-2-expressing tumor epithelial cells and microenvironment endothelial cells. Lipidomics analysis of skin and tumors from DMBA/TPA-treated mice suggests that the prostaglandins PGE2 and PGF2α are likely candidates for the epithelial cell COX-2-dependent eicosanoids that mediate tumor progression. This study both illustrates the value of cell type-specific gene deletions in understanding the cellular roles of signal-generating pathways in complex microenvironments and emphasizes the benefit of a systems-based lipidomic analysis approach to identify candidate lipid mediators of biologic responses. Cox-2 gene deletion demonstrates that intrinsic COX-2 expression in initiated keratinocytes is a principal driver of skin carcinogenesis; lipidomic analysis identifies likely prostanoid effectors. ©2014 American Association for Cancer Research.

Deletion of alpha-synuclein decreases impulsivity in mice.

PubMed

Peña-Oliver, Y; Buchman, V L; Dalley, J W; Robbins, T W; Schumann, G; Ripley, T L; King, S L; Stephens, D N

2012-03-01

The presynaptic protein alpha-synuclein, associated with Parkinson's Disease (PD), plays a role in dopaminergic neurotransmission and is implicated in impulse control disorders (ICDs) such as drug addiction. In this study we investigated a potential causal relationship between alpha-synuclein and impulsivity, by evaluating differences in motor impulsivity in the 5-choice serial reaction time task (5-CSRTT) in strains of mice that differ in the expression of the alpha-synuclein gene. C57BL/6JOlaHsd mice differ from their C57BL/6J ancestors in possessing a chromosomal deletion resulting in the loss of two genes, snca, encoding alpha-synuclein, and mmrn1, encoding multimerin-1. C57BL/6J mice displayed higher impulsivity (more premature responding) than C57BL/6JOlaHsd mice when the pre-stimulus waiting interval was increased in the 5-CSRTT. In order to ensure that the reduced impulsivity was indeed related to snca, and not adjacent gene deletion, wild type (WT) and mice with targeted deletion of alpha-synuclein (KO) were tested in the 5-CSRTT. Similarly, WT mice were more impulsive than mice with targeted deletion of alpha-synuclein. Interrogation of our ongoing analysis of impulsivity in BXD recombinant inbred mouse lines revealed an association of impulsive responding with levels of alpha-synuclein expression in hippocampus. Expression of beta- and gamma-synuclein, members of the synuclein family that may substitute for alpha-synuclein following its deletion, revealed no differential compensations among the mouse strains. These findings suggest that alpha-synuclein may contribute to impulsivity and potentially, to ICDs which arise in some PD patients treated with dopaminergic medication. © 2011 The Authors. Genes, Brain and Behavior © 2011 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

An atypical case of fragile X syndrome caused by a deletion that includes the FMR1 gene.

PubMed Central

Quan, F; Zonana, J; Gunter, K; Peterson, K L; Magenis, R E; Popovich, B W

1995-01-01

Fragile X syndrome is the most common form of inherited mental retardation and results from the transcriptional inactivation of the FMR1 gene. In the vast majority of cases, this is caused by the expansion of an unstable CGG repeat in the first exon of the FMR1 gene. We describe here a phenotypically atypical case of fragile X syndrome, caused by a deletion that includes the entire FMR1 gene and > or = 9.0 Mb of flanking DNA. The proband, RK, was a 6-year-old mentally retarded male with obesity and anal atresia. A diagnosis of fragile X syndrome was established by the failure of RK's DNA to hybridize to a 558-bp PstI-XhoI fragment (pfxa3) specific for the 5'-end of the FMR1 gene. The analysis of flanking markers in the interval from Xq26.3-q28 indicated a deletion extending from between 160-500 kb distal and 9.0 Mb proximal to the FMR1 gene. High-resolution chromosome banding confirmed a deletion with breakpoints in Xq26.3 and Xq27.3. This deletion was maternally transmitted and arose as a new mutation on the grandpaternal X chromosome. The maternal transmission of the deletion was confirmed by FISH using a 34-kb cosmid (c31.4) containing most of the FMR1 gene. These results indicated that RK carried a deletion of the FMR1 region with the most proximal breakpoint described to date. This patient's unusual clinical presentation may indicate the presence of genes located in the deleted interval proximal to the FMR1 locus that are able to modify the fragile X syndrome phenotype. Images Figure 5 Figure 2 Figure 4 Figure 6 Figure 7 PMID:7726157

Gene Deletions in Mycobacterium bovis BCG Stimulate Increased CD8+ T Cell Responses

PubMed Central

Panas, Michael W.; Sixsmith, Jaimie D.; White, KeriAnn; Korioth-Schmitz, Birgit; Shields, Shana T.; Moy, Brian T.; Lee, Sunhee; Schmitz, Joern E.; Jacobs, William R.; Porcelli, Steven A.; Haynes, Barton F.; Letvin, Norman L.

2014-01-01

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8+ T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8+ T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8+ T cell responses in vivo. This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8+ T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors. PMID:25287928

Gene deletions in Mycobacterium bovis BCG stimulate increased CD8+ T cell responses.

PubMed

Panas, Michael W; Sixsmith, Jaimie D; White, KeriAnn; Korioth-Schmitz, Birgit; Shields, Shana T; Moy, Brian T; Lee, Sunhee; Schmitz, Joern E; Jacobs, William R; Porcelli, Steven A; Haynes, Barton F; Letvin, Norman L; Gillard, Geoffrey O

2014-12-01

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8(+) T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8(+) T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8(+) T cell responses in vivo. This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8(+) T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The drug target genes show higher evolutionary conservation than non-target genes.

PubMed

Lv, Wenhua; Xu, Yongdeng; Guo, Yiying; Yu, Ziqi; Feng, Guanglong; Liu, Panpan; Luan, Meiwei; Zhu, Hongjie; Liu, Guiyou; Zhang, Mingming; Lv, Hongchao; Duan, Lian; Shang, Zhenwei; Li, Jin; Jiang, Yongshuai; Zhang, Ruijie

2016-01-26

Although evidence indicates that drug target genes share some common evolutionary features, there have been few studies analyzing evolutionary features of drug targets from an overall level. Therefore, we conducted an analysis which aimed to investigate the evolutionary characteristics of drug target genes. We compared the evolutionary conservation between human drug target genes and non-target genes by combining both the evolutionary features and network topological properties in human protein-protein interaction network. The evolution rate, conservation score and the percentage of orthologous genes of 21 species were included in our study. Meanwhile, four topological features including the average shortest path length, betweenness centrality, clustering coefficient and degree were considered for comparison analysis. Then we got four results as following: compared with non-drug target genes, 1) drug target genes had lower evolutionary rates; 2) drug target genes had higher conservation scores; 3) drug target genes had higher percentages of orthologous genes and 4) drug target genes had a tighter network structure including higher degrees, betweenness centrality, clustering coefficients and lower average shortest path lengths. These results demonstrate that drug target genes are more evolutionarily conserved than non-drug target genes. We hope that our study will provide valuable information for other researchers who are interested in evolutionary conservation of drug targets.

Targeted Deletion and Lipidomic Analysis Identify Epithelial Cell COX-2 as a Major Driver of Chemically-induced Skin Cancer

PubMed Central

Jiao, Jing; Ishikawa, Tomo-o; Dumlao, Darren S.; Norris, Paul C.; Magyar, Clara E.; Mikulec, Carol; Catapang, Art; Dennis, Edward A.; Fischer, Susan M.; Herschman, Harvey R.

2014-01-01

Pharmacologic and global gene deletion studies demonstrate that cyclooxygenase-2 (PTGS2/COX2) plays a critical role in DMBA/TPA-induced skin tumor induction. While many cell types in the tumor microenvironment express COX-2, the cell types in which COX-2 expression is required for tumor promotion are not clearly established. Here, cell-type specific Cox-2 gene deletion reveals a vital role for skin epithelial cell COX-2 expression in DMBA/TPA tumor induction. In contrast, myeloid Cox-2 gene deletion has no effect on DMBA/TPA tumorigenesis. The infrequent, small tumors that develop on mice with an epithelial cell-specific Cox-2 gene deletion have decreased proliferation and increased cell differentiation properties. Blood vessel density is reduced in tumors with an epithelial cell-specific Cox-2 gene deletion, compared to littermate control tumors, suggesting a reciprocal relationship in tumor progression between COX-2 expressing tumor epithelial cells and microenvironment endothelial cells. Lipidomics analysis of skin and tumors from DMBA/TPA-treated mice suggests that the prostaglandins PGE2 and PGF2α are likely candidates for the epithelial cell COX-2-dependent eicosanoids that mediate tumor progression. This study both illustrates the value of cell-type specific gene deletions in understanding the cellular roles of signal-generating pathways in complex microenvironments and emphasizes the benefit of a systems-based lipidomic analysis approach to identify candidate lipid mediators of biological responses. PMID:25063587

Targeted Gene Knock Out Using Nuclease-Assisted Vector Integration: Hemi- and Homozygous Deletion of JAG1.

PubMed

Gapinske, Michael; Tague, Nathan; Winter, Jackson; Underhill, Gregory H; Perez-Pinera, Pablo

2018-01-01

Gene editing technologies are revolutionizing fields such as biomedicine and biotechnology by providing a simple means to manipulate the genetic makeup of essentially any organism. Gene editing tools function by introducing double-stranded breaks at targeted sites within the genome, which the host cells repair preferentially by Non-Homologous End Joining. While the technologies to introduce double-stranded breaks have been extensively optimized, this progress has not been matched by the development of methods to integrate heterologous DNA at the target sites or techniques to detect and isolate cells that harbor the desired modification. We present here a technique for rapid introduction of vectors at target sites in the genome that enables efficient isolation of successfully edited cells.

Monitoring of Dual CRISPR/Cas9-Mediated Steroidogenic Acute Regulatory Protein Gene Deletion and Cholesterol Accumulation Using High-Resolution Fluorescence In Situ Hybridization in a Single Cell

PubMed Central

Lee, Jinwoo; Jefcoate, Colin

2017-01-01

Recent advances in fluorescence microscopy, coupled with CRISPR/Cas9 gene editing technology, provide opportunities for understanding gene regulation at the single-cell level. The application of direct imaging shown here provides an in situ side-by-side comparison of CRISPR/Cas9-edited cells and adjacent unedited cells. We apply this methodology to the steroidogenic acute regulatory protein (StAR) gene in Y-1 adrenal cells and MA-10 testis cells. StAR is a gatekeeper protein that controls the access of cholesterol from the cytoplasm to the inner mitochondria. The loss of this mitochondrial cholesterol transfer mediator rapidly increases lipid droplets in cells, as seen in StAR−/− mice. Here, we describe a dual CRISPR/Cas9 strategy marked by GFP/mCherry expression that deletes StAR activity within 12 h. We used single-molecule fluorescence in situ hybridization (sm-FISH) imaging to directly monitor the time course of gene editing in single cells. We achieved StAR gene deletion at high efficiency dual gRNA targeting to the proximal promoter and exon 2. Seventy percent of transfected cells showed a slow DNA deletion as measured by PCR, and loss of Br-cAMP stimulated transcription. This DNA deletion was seen by sm-FISH in both loci of individual cells relative to non-target Cyp11a1 and StAR exon 7. sm-FISH also distinguishes two effects on stimulated StAR expression without this deletion. Br-cAMP stimulation of primary and spliced StAR RNA at the gene loci were removed within 4 h in this dual CRISPR/Cas9 strategy before any effect on cytoplasmic mRNA and protein occurred. StAR mRNA disappeared between 12 and 24 h in parallel with this deletion, while cholesterol ester droplets increased fourfold. These alternative changes match distinct StAR expression processes. This dual gRNA and sm-FISH approach to CRISPR/Cas9 editing facilitates rapid testing of editing strategies and immediate assessment of single-cell adaptation responses without the perturbation of

Is NF-1 gene deletion the molecular mechanism of neurofibromatosis type 1 with destinctive facies?

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

Leppig, K.A.; Stephens, K.G.; Viskochill, D.

We have studied a patient with neurofibromatosis type 1 and unusual facial features using fluorescence in situ hybridization (FISH) and found that the patient had a deletion that minimially encompasses exon 2-11 of the NF-1 gene. The patient was one of two individuals initially described by Kaplan and Rosenblatt who suggested that another condition aside from neurofibromatosis type 1 may account for the unusual facial features observed in these patients with neurofibromatosis type 1. FISH studies were performed using a P1 clone probe, P1-9, which contains exons 2-11 of the NF-1 gene on chromosomes prepared from the patients. In allmore » 20 metaphase cells analyzed, one of the chromosome 17 homologues was deleted for the P1-9 probe. Therefore, this patient had neurofibromatosis type 1 and unusual facial features as the result of a deletion which minimally includes exons 2-11 of the NF-1 gene. The extent of the deletion is being mapped by FISH and somatic cell hybrid analysis. The patient studied was a 7-year-old male with mild developmental delays, normal growth parameters, and physical findings consistent with neurofibromatosis type 1, including multiple cafe au lait spots, several curaneous neurofibroma, and speckling of the irises. In addition, his unusual facial features consisted of telecanthus, antimongoloid slant of the palpebral fissures, a broad base of the nose, low set and mildly posteriorly rotated ears, thick helices, high arched palate, short and pointed chin, and low posterior hairline. We propose that deletions of the NF-1 gene and/or contiguous genes are the etiology of neurofibromatosis type 1 and unusual facial features. This particular facial appearance was inherited from the patient`s mother and has been described in other individuals with neurofibromatosis type 1. We are using FISH to rapidly screen patients with this phenotype for large deletions involving the NF-1 gene and flanking DNA sequences.« less

Alport syndrome, mental retardation, midface hypoplasia, and elliptocytosis: a new X linked contiguous gene deletion syndrome?

PubMed Central

Jonsson, J J; Renieri, A; Gallagher, P G; Kashtan, C E; Cherniske, E M; Bruttini, M; Piccini, M; Vitelli, F; Ballabio, A; Pober, B R

1998-01-01

We describe a family with four members, a mother, two sons, and a daughter, who show clinical features consistent with X linked Alport syndrome. The two males presented with additional features including mental retardation, dysmorphic facies with marked midface hypoplasia, and elliptocytosis. The elliptocytosis was not associated with any detectable abnormalities in red cell membrane proteins; red cell membrane stability and rigidity was normal on ektacytometry. Molecular characterisation suggests a submicroscopic X chromosome deletion encompassing the entire COL4A5 gene. We propose that the additional abnormalities found in the affected males of this family are attributable to deletion or disruption of X linked recessive genes adjacent to the COL4A5 gene and that this constellation of findings may represent a new X linked contiguous gene deletion syndrome. Images PMID:9598718

Testicular expressed genes are missing in familial X-Linked Kallmann syndrome due to two large different deletions in daughter's X chromosomes.

PubMed

Hershkovitz, Eli; Loewenthal, Neta; Peretz, Asaf; Parvari, Ruti

2008-01-01

X-linked Kallmann syndrome (KS) is caused mainly by point mutations, in the KAL1 gene. Large deletions >1 Mb are rare events in the human population and commonly result in contiguous gene syndromes. A search for the mutation causing KS carried out on two pairs of first-degree cousins of 2 sisters. Two different apparently independent deletions were found. The deleted sequences encompass the KAL1 gene and four known additional genes exclusively expressed in testis. Two of these genes belong to the FAM9 gene family, which shares some homology with the SCYP3 gene, previously implicated in azoospermia. One of the events causing the deletion may have been mediated by an L1 transposition, the other by a non-homologous end joining. Such non-homologous recombinations have not yet been reported in the KAL genomic region and thus this area may be more prone to deletions than previously expected. This is the first report on genetic characterization of KS with a deletion of solely testis-expressed genes. The absence of these genes may have unfavorable implications for the patients regarding future fertility. (c) 2008 S. Karger AG, Basel

Screening for single nucleotide variants, small indels and exon deletions with a next-generation sequencing based gene panel approach for Usher syndrome

PubMed Central

Krawitz, Peter M; Schiska, Daniela; Krüger, Ulrike; Appelt, Sandra; Heinrich, Verena; Parkhomchuk, Dmitri; Timmermann, Bernd; Millan, Jose M; Robinson, Peter N; Mundlos, Stefan; Hecht, Jochen; Gross, Manfred

2014-01-01

Usher syndrome is an autosomal recessive disorder characterized both by deafness and blindness. For the three clinical subtypes of Usher syndrome causal mutations in altogether 12 genes and a modifier gene have been identified. Due to the genetic heterogeneity of Usher syndrome, the molecular analysis is predestined for a comprehensive and parallelized analysis of all known genes by next-generation sequencing (NGS) approaches. We describe here the targeted enrichment and deep sequencing for exons of Usher genes and compare the costs and workload of this approach compared to Sanger sequencing. We also present a bioinformatics analysis pipeline that allows us to detect single-nucleotide variants, short insertions and deletions, as well as copy number variations of one or more exons on the same sequence data. Additionally, we present a flexible in silico gene panel for the analysis of sequence variants, in which newly identified genes can easily be included. We applied this approach to a cohort of 44 Usher patients and detected biallelic pathogenic mutations in 35 individuals and monoallelic mutations in eight individuals of our cohort. Thirty-nine of the sequence variants, including two heterozygous deletions comprising several exons of USH2A, have not been reported so far. Our NGS-based approach allowed us to assess single-nucleotide variants, small indels, and whole exon deletions in a single test. The described diagnostic approach is fast and cost-effective with a high molecular diagnostic yield. PMID:25333064

Screening for single nucleotide variants, small indels and exon deletions with a next-generation sequencing based gene panel approach for Usher syndrome.

PubMed

Krawitz, Peter M; Schiska, Daniela; Krüger, Ulrike; Appelt, Sandra; Heinrich, Verena; Parkhomchuk, Dmitri; Timmermann, Bernd; Millan, Jose M; Robinson, Peter N; Mundlos, Stefan; Hecht, Jochen; Gross, Manfred

2014-09-01

Usher syndrome is an autosomal recessive disorder characterized both by deafness and blindness. For the three clinical subtypes of Usher syndrome causal mutations in altogether 12 genes and a modifier gene have been identified. Due to the genetic heterogeneity of Usher syndrome, the molecular analysis is predestined for a comprehensive and parallelized analysis of all known genes by next-generation sequencing (NGS) approaches. We describe here the targeted enrichment and deep sequencing for exons of Usher genes and compare the costs and workload of this approach compared to Sanger sequencing. We also present a bioinformatics analysis pipeline that allows us to detect single-nucleotide variants, short insertions and deletions, as well as copy number variations of one or more exons on the same sequence data. Additionally, we present a flexible in silico gene panel for the analysis of sequence variants, in which newly identified genes can easily be included. We applied this approach to a cohort of 44 Usher patients and detected biallelic pathogenic mutations in 35 individuals and monoallelic mutations in eight individuals of our cohort. Thirty-nine of the sequence variants, including two heterozygous deletions comprising several exons of USH2A, have not been reported so far. Our NGS-based approach allowed us to assess single-nucleotide variants, small indels, and whole exon deletions in a single test. The described diagnostic approach is fast and cost-effective with a high molecular diagnostic yield.

Norrie disease gene: characterization of deletions and possible function.

PubMed

Chen, Z Y; Battinelli, E M; Hendriks, R W; Powell, J F; Middleton-Price, H; Sims, K B; Breakefield, X O; Craig, I W

1993-05-01

Positional cloning experiments have resulted recently in the isolation of a candidate gene for Norrie disease (pseudoglioma; NDP), a severe X-linked neurodevelopmental disorder. Here we report the isolation and analysis of human genomic DNA clones encompassing the NDP gene. The gene spans 28 kb and consists of 3 exons, the first of which is entirely contained within the 5' untranslated region. Detailed analysis of genomic deletions in Norrie patients shows that they are heterogeneous, both in size and in position. By PCR analysis, we found that expression of the NDP gene was not confined to the eye or to the brain. An extensive DNA and protein sequence comparison between the human NDP gene and related genes from the database revealed homology with cysteine-rich protein-binding domains of immediate--early genes implicated in the regulation of cell proliferation. We propose that NDP is a molecule related in function to these genes and may be involved in a pathway that regulates neural cell differentiation and proliferation.

[Changes of biological behavioral of E. coli K1 after ppk1 gene deletion].

PubMed

Peng, Liang; Pan, Jiayun; Luo, Su; Yang, Zhenghui; Huang, Mufang; Cao, Hong

2014-06-01

To study the changes in biological behaviors of meningitis E. coli K1 strain E44 after deletion of polyphosphate kinase 1 (ppk1) gene and explore the role of ppk1 in the pathogenesis of E. coli K1-induced meningitis. The wild-type strain E. coli K1 and ppk1 deletion mutant were exposed to heat at 56 degrees celsius; for 6 min, and their survival rates were determined. The adhesion and invasion of the bacteria to human brain microvascular endothelial cells (HBMECs) were observed using electron microscopy and quantitative tests. HBMECs were co-incubated with wild-type strain or ppk1 deletion mutant, and the cytoskeleton rearrangement was observed under laser scanning confocal microscope. The survival rate of the ppk1 deletion mutant was significantly lower than that of the wild-type strain after heat exposure. The ppk1 deletion mutant also showed lowered cell adhesion and invasion abilities and weakened ability to induce cytoskeleton rearrangement in HBMECs. ppk1 gene is important for E.coli K1 for heat resistance, cell adhesion and invasion, and for inducing cytoskeletal rearrangement in HBMECs.

Analysis of the Functions of Recombination-Related Genes in the Generation of Large Chromosomal Deletions by Loop-Out Recombination in Aspergillus oryzae

PubMed Central

Ogawa, Masahiro; Koyama, Yasuji

2012-01-01

Loop-out-type recombination is a type of intrachromosomal recombination followed by the excision of a chromosomal region. The detailed mechanism underlying this recombination and the genes involved in loop-out recombination remain unknown. In the present study, we investigated the functions of ku70, ligD, rad52, rad54, and rdh54 in the construction of large chromosomal deletions via loop-out recombination and the effect of the position of the targeted chromosomal region on the efficiency of loop-out recombination in Aspergillus oryzae. The efficiency of generation of large chromosomal deletions in the near-telomeric region of chromosome 3, including the aflatoxin gene cluster, was compared with that in the near-centromeric region of chromosome 8, including the tannase gene. In the Δku70 and Δku70-rdh54 strains, only precise loop-out recombination occurred in the near-telomeric region. In contrast, in the ΔligD, Δku70-rad52, and Δku70-rad54 strains, unintended chromosomal deletions by illegitimate loop-out recombination occurred in the near-telomeric region. In addition, large chromosomal deletions via loop-out recombination were efficiently achieved in the near-telomeric region, but barely achieved in the near-centromeric region, in the Δku70 strain. Induction of DNA double-strand breaks by I-SceI endonuclease facilitated large chromosomal deletions in the near-centromeric region. These results indicate that ligD, rad52, and rad54 play a role in the generation of large chromosomal deletions via precise loop-out-type recombination in the near-telomeric region and that loop-out recombination between distant sites is restricted in the near-centromeric region by chromosomal structure. PMID:22286092

R3-R4 deletion in the PRNP gene is associated with Creutzfeldt-Jakob disease (CJD)

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

Cervenakova, L.; Brown, P.; Nagle, J.

1994-09-01

There are conflicting reports on the association of deletions in the PRNP gene on chromosome 20 with CJD, a rapidly progressive fatal spongiform encephalopathy. We accumulated data suggesting that a deletion of R3-R4 type (parts of the third and fourth repeats are deleted from the area of four repeating 24 bp sequences in the 5{prime} region of the gene) is causing CJD. Screening of 129 unaffected control individuals demonstrated presence of a deletion of R2 type in four (1.55% of the studied chromosomes), but none of them had the R3-R4 type. Of 181 screened patients with spongiform encephalopathies, two hadmore » a deletion of R3-R4 type with no other mutations in the coding sequence. Both patients had a classical rapidly progressive dementing disease and diffuse spongiform degeneration, and both cases were apparently sporadic. The same R3-R4 type of deletion was detected in three additional neuropathologically confirmed spongiform encephalopathy patients, of which two had other known pathogenic mutations in the PRNP gene: at codon 178 on the methionine allele exhibiting the phenotype of fatal familial insomnia, and codon 200 causing CJD with severe dementia; the third was a patient with iatrogenic CJD who developed the disease after treatment with growth hormone extracted from cadaveric human pituitary glands. In all cases the deletion coincided with a variant sequence at position 129 coding for methionine.« less

A recessive contiguous gene deletion causing infantile hyperinsulinism, enteropathy and deafness identifies the Usher type 1C gene.

PubMed

Bitner-Glindzicz, M; Lindley, K J; Rutland, P; Blaydon, D; Smith, V V; Milla, P J; Hussain, K; Furth-Lavi, J; Cosgrove, K E; Shepherd, R M; Barnes, P D; O'Brien, R E; Farndon, P A; Sowden, J; Liu, X Z; Scanlan, M J; Malcolm, S; Dunne, M J; Aynsley-Green, A; Glaser, B

2000-09-01

Usher syndrome type 1 describes the association of profound, congenital sensorineural deafness, vestibular hypofunction and childhood onset retinitis pigmentosa. It is an autosomal recessive condition and is subdivided on the basis of linkage analysis into types 1A through 1E. Usher type 1C maps to the region containing the genes ABCC8 and KCNJ11 (encoding components of ATP-sensitive K + (KATP) channels), which may be mutated in patients with hyperinsulinism. We identified three individuals from two consanguineous families with severe hyperinsulinism, profound congenital sensorineural deafness, enteropathy and renal tubular dysfunction. The molecular basis of the disorder is a homozygous 122-kb deletion of 11p14-15, which includes part of ABCC8 and overlaps with the locus for Usher syndrome type 1C and DFNB18. The centromeric boundary of this deletion includes part of a gene shown to be mutated in families with type 1C Usher syndrome, and is hence assigned the name USH1C. The pattern of expression of the USH1C protein is consistent with the clinical features exhibited by individuals with the contiguous gene deletion and with isolated Usher type 1C.

Deletion of Specific Immune-Modulatory Genes from Modified Vaccinia Virus Ankara-Based HIV Vaccines Engenders Improved Immunogenicity in Rhesus Macaques

PubMed Central

O'Mara, Leigh A.; Gangadhara, Sailaja; McQuoid, Monica; Zhang, Xiugen; Zheng, Rui; Gill, Kiran; Verma, Meena; Yu, Tianwei; Johnson, Brent; Li, Bing; Derdeyn, Cynthia A.; Ibegbu, Chris; Altman, John D.; Hunter, Eric; Feinberg, Mark B.

2012-01-01

Modified vaccinia virus Ankara (MVA) is a safe, attenuated orthopoxvirus that is being developed as a vaccine vector but has demonstrated limited immunogenicity in several early-phase clinical trials. Our objective was to rationally improve the immunogenicity of MVA-based HIV/AIDS vaccines via the targeted deletion of specific poxvirus immune-modulatory genes. Vaccines expressing codon-optimized HIV subtype C consensus Env and Gag antigens were generated from MVA vector backbones that (i) harbor simultaneous deletions of four viral immune-modulatory genes, encoding an interleukin-18 (IL-18) binding protein, an IL-1β receptor, a dominant negative Toll/IL-1 signaling adapter, and CC-chemokine binding protein (MVAΔ4-HIV); (ii) harbor a deletion of an additional (fifth) viral gene, encoding uracil-DNA glycosylase (MVAΔ5-HIV); or (iii) represent the parental MVA backbone as a control (MVA-HIV). We performed head-to-head comparisons of the cellular and humoral immune responses that were elicited by these vectors during homologous prime-boost immunization regimens utilizing either high-dose (2 × 108 PFU) or low-dose (1 × 107 PFU) intramuscular immunization of rhesus macaques. At all time points, a majority of the HIV-specific T cell responses, elicited by all vectors, were directed against Env, rather than Gag, determinants, as previously observed with other vector systems. Both modified vectors elicited up to 6-fold-higher frequencies of HIV-specific CD8 and CD4 T cell responses and up to 25-fold-higher titers of Env (gp120)-specific binding (nonneutralizing) antibody responses that were relatively transient in nature. While the correlates of protection against HIV infection remain incompletely defined, our results indicate that the rational deletion of specific genes from MVA vectors can positively alter their cellular and humoral immunogenicity profiles in nonhuman primates. PMID:22973033

MicroRNA-182 drives metastasis of primary sarcomas by targeting multiple genes

PubMed Central

Sachdeva, Mohit; Mito, Jeffrey K.; Lee, Chang-Lung; Zhang, Minsi; Li, Zhizhong; Dodd, Rebecca D.; Cason, David; Luo, Lixia; Ma, Yan; Van Mater, David; Gladdy, Rebecca; Lev, Dina C.; Cardona, Diana M.; Kirsch, David G.

2014-01-01

Metastasis causes most cancer deaths, but is incompletely understood. MicroRNAs can regulate metastasis, but it is not known whether a single miRNA can regulate metastasis in primary cancer models in vivo. We compared the expression of miRNAs in metastatic and nonmetastatic primary mouse sarcomas and found that microRNA-182 (miR-182) was markedly overexpressed in some tumors that metastasized to the lungs. By utilizing genetically engineered mice with either deletion of or overexpression of miR-182 in primary sarcomas, we discovered that deletion of miR-182 substantially decreased, while overexpression of miR-182 considerably increased, the rate of lung metastasis after amputation of the tumor-bearing limb. Additionally, deletion of miR-182 decreased circulating tumor cells (CTCs), while overexpression of miR-182 increased CTCs, suggesting that miR-182 regulates intravasation of cancer cells into the circulation. We identified 4 miR-182 targets that inhibit either the migration of tumor cells or the degradation of the extracellular matrix. Notably, restoration of any of these targets in isolation did not alter the metastatic potential of sarcoma cells injected orthotopically, but the simultaneous restoration of all 4 targets together substantially decreased the number of metastases. These results demonstrate that a single miRNA can regulate metastasis of primary tumors in vivo by coordinated regulation of multiple genes. PMID:25180607

PRIMED: PRIMEr Database for Deleting and Tagging All Fission and Budding Yeast Genes Developed Using the Open-Source Genome Retrieval Script (GRS)

PubMed Central

Cummings, Michael T.; Joh, Richard I.; Motamedi, Mo

2015-01-01

The fission (Schizosaccharomyces pombe) and budding (Saccharomyces cerevisiae) yeasts have served as excellent models for many seminal discoveries in eukaryotic biology. In these organisms, genes are deleted or tagged easily by transforming cells with PCR-generated DNA inserts, flanked by short (50-100bp) regions of gene homology. These PCR reactions use especially designed long primers, which, in addition to the priming sites, carry homology for gene targeting. Primer design follows a fixed method but is tedious and time-consuming especially when done for a large number of genes. To automate this process, we developed the Python-based Genome Retrieval Script (GRS), an easily customizable open-source script for genome analysis. Using GRS, we created PRIMED, the complete PRIMEr D atabase for deleting and C-terminal tagging genes in the main S. pombe and five of the most commonly used S. cerevisiae strains. Because of the importance of noncoding RNAs (ncRNAs) in many biological processes, we also included the deletion primer set for these features in each genome. PRIMED are accurate and comprehensive and are provided as downloadable Excel files, removing the need for future primer design, especially for large-scale functional analyses. Furthermore, the open-source GRS can be used broadly to retrieve genome information from custom or other annotated genomes, thus providing a suitable platform for building other genomic tools by the yeast or other research communities. PMID:25643023

Total alpha-globin gene cluster deletion has high frequency in Filipinos

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

Hunt, J.A.; Haruyama, A.Z.; Chu, B.M.

1994-09-01

Most {alpha}-thalassemias [Thal] are due to large deletions. In Southeast Asians, the (--{sup SEA}) double {alpha}-globin gene deletion is common, 3 (--{sup Tot}) total {alpha}-globin cluster deletions are known: Filipino (--{sup Fil}), Thai (--{sup Thai}), and Chinese (--{sup Chin}). In a Hawaii Thal project, provisional diagnosis of {alpha}-Thal-1 heterozygotes was based on microcytosis, normal isoelectric focusing, and no iron deficiency. One in 10 unselected Filipinos was an {alpha}-Thal-1 heterozygote, 2/3 of these had a (--{sup Tot}) deletion: a {var_sigma}-cDNA probe consistently showed fainter intensity of the constant 5.5 kb {var_sigma}{sub 2} BamHI band, with no heterzygosity for {var_sigma}-globin region polymorphisms;more » {alpha}-cDNA or {var_sigma}-cDNA probes showed no BamHI or BglII bands diagnostic of the (--{sup SEA}) deletion; bands for the (-{alpha}) {alpha}-Thal-2 single {alpha}-globin deletions were only seen in Hb H cases. A reliable monoclonal anti-{var_sigma}-peptide antibody test for the (--{sup SEA}) deletion was always negative in (--{sup Tot}) samples. Southern digests with the Lo probe, a gift from D. Higgs of Oxford Univ., confirmed that 49 of 50 (--{sup Tot}) chromosomes in Filipinos were (--{sup Fil}). Of 20 {alpha}-Thal-1 hydrops born to Filipinos, 11 were (--{sup Fil}/--{sup SEA}) compound heterozygotes; 9 were (--{sup SEA}/--{sup SEA}) homozygotes, but none was a (--{sup Fil}/--{sup Fil}).« less

Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy

PubMed Central

Bornert, Olivier; Kühl, Tobias; Bremer, Jeroen; van den Akker, Peter C; Pasmooij, Anna MG; Nyström, Alexander

2016-01-01

Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB)—a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The therapeutic potential of this approach is critically dependent on gene, protein, and disease intrinsic factors. Naturally occurring exon skipping in COL7A1, translating collagen VII, suggests that skipping of exons containing disease-causing mutations may be feasible for the treatment of DEB. However, despite a primarily in-frame arrangement of exons in the COL7A1 gene, no general conclusion of the aptitude of exon skipping for DEB can be drawn, since regulation of collagen VII functionality is complex involving folding, intra- and intermolecular interactions. To directly address this, we deleted two conceptually important exons located at both ends of COL7A1, exon 13, containing recurrent mutations, and exon 105, predicted to impact folding. The resulting recombinantly expressed proteins showed conserved functionality in biochemical and in vitro assays. Injected into DEB mice, the proteins promoted skin stability. By demonstrating functionality of internally deleted collagen VII variants, our study provides support of targeted exon deletion or skipping as a potential therapy to treat a large number of individuals with DEB. PMID:27157667

Genomic anatomy of the Tyrp1 (brown) deletion complex

PubMed Central

Smyth, Ian M.; Wilming, Laurens; Lee, Angela W.; Taylor, Martin S.; Gautier, Phillipe; Barlow, Karen; Wallis, Justine; Martin, Sancha; Glithero, Rebecca; Phillimore, Ben; Pelan, Sarah; Andrew, Rob; Holt, Karen; Taylor, Ruth; McLaren, Stuart; Burton, John; Bailey, Jonathon; Sims, Sarah; Squares, Jan; Plumb, Bob; Joy, Ann; Gibson, Richard; Gilbert, James; Hart, Elizabeth; Laird, Gavin; Loveland, Jane; Mudge, Jonathan; Steward, Charlie; Swarbreck, David; Harrow, Jennifer; North, Philip; Leaves, Nicholas; Greystrong, John; Coppola, Maria; Manjunath, Shilpa; Campbell, Mark; Smith, Mark; Strachan, Gregory; Tofts, Calli; Boal, Esther; Cobley, Victoria; Hunter, Giselle; Kimberley, Christopher; Thomas, Daniel; Cave-Berry, Lee; Weston, Paul; Botcherby, Marc R. M.; White, Sharon; Edgar, Ruth; Cross, Sally H.; Irvani, Marjan; Hummerich, Holger; Simpson, Eleanor H.; Johnson, Dabney; Hunsicker, Patricia R.; Little, Peter F. R.; Hubbard, Tim; Campbell, R. Duncan; Rogers, Jane; Jackson, Ian J.

2006-01-01

Chromosome deletions in the mouse have proven invaluable in the dissection of gene function. The brown deletion complex comprises >28 independent genome rearrangements, which have been used to identify several functional loci on chromosome 4 required for normal embryonic and postnatal development. We have constructed a 172-bacterial artificial chromosome contig that spans this 22-megabase (Mb) interval and have produced a contiguous, finished, and manually annotated sequence from these clones. The deletion complex is strikingly gene-poor, containing only 52 protein-coding genes (of which only 39 are supported by human homologues) and has several further notable genomic features, including several segments of >1 Mb, apparently devoid of a coding sequence. We have used sequence polymorphisms to finely map the deletion breakpoints and identify strong candidate genes for the known phenotypes that map to this region, including three lethal loci (l4Rn1, l4Rn2, and l4Rn3) and the fitness mutant brown-associated fitness (baf). We have also characterized misexpression of the basonuclin homologue, Bnc2, associated with the inversion-mediated coat color mutant white-based brown (Bw). This study provides a molecular insight into the basis of several characterized mouse mutants, which will allow further dissection of this region by targeted or chemical mutagenesis. PMID:16505357

Recurrent Deletions of Puroindoline Genes at the Grain Hardness Locus in Four Independent Lineages of Polyploid Wheat1[W][OA

PubMed Central

Li, Wanlong; Huang, Li; Gill, Bikram S.

2008-01-01

Polyploidy is known to induce numerous genetic and epigenetic changes but little is known about their physiological bases. In wheat, grain texture is mainly determined by the Hardness (Ha) locus consisting of genes Puroindoline a (Pina) and b (Pinb). These genes are conserved in diploid progenitors but were deleted from the A and B genomes of tetraploid Triticum turgidum (AB). We now report the recurrent deletions of Pina-Pinb in other lineages of polyploid wheat. We analyzed the Ha haplotype structure in 90 diploid and 300 polyploid accessions of Triticum and Aegilops spp. Pin genes were conserved in all diploid species and deletion haplotypes were detected in all polyploid Triticum and most of the polyploid Aegilops spp. Two Pina-Pinb deletion haplotypes were found in hexaploid wheat (Triticum aestivum; ABD). Pina and Pinb were eliminated from the G genome, but maintained in the A genome of tetraploid Triticum timopheevii (AG). Subsequently, Pina and Pinb were deleted from the A genome but retained in the Am genome of hexaploid Triticum zhukovskyi (AmAG). Comparison of deletion breakpoints demonstrated that the Pina-Pinb deletion occurred independently and recurrently in the four polyploid wheat species. The implications of Pina-Pinb deletions for polyploid-driven evolution of gene and genome and its possible physiological significance are discussed. PMID:18024553

Deletion of ETS-1, a gene in the Jacobsen syndrome critical region, causes ventricular septal defects and abnormal ventricular morphology in mice

PubMed Central

Ye, Maoqing; Coldren, Chris; Liang, Xingqun; Mattina, Teresa; Goldmuntz, Elizabeth; Benson, D. Woodrow; Ivy, Dunbar; Perryman, M.B.; Garrett-Sinha, Lee Ann; Grossfeld, Paul

2010-01-01

Congenital heart defects comprise the most common form of major birth defects, affecting 0.7% of all newborn infants. Jacobsen syndrome (11q-) is a rare chromosomal disorder caused by deletions in distal 11q. We have previously determined that a wide spectrum of the most common congenital heart defects occur in 11q-, including an unprecedented high frequency of hypoplastic left heart syndrome (HLHS). We identified an ∼7 Mb ‘cardiac critical region’ in distal 11q that contains a putative causative gene(s) for congenital heart disease. In this study, we utilized chromosomal microarray mapping to characterize three patients with 11q- and congenital heart defects that carry interstitial deletions overlapping the 7 Mb cardiac critical region. We propose that this 1.2 Mb region of overlap harbors a gene(s) that causes at least a subset of the congenital heart defects that occur in 11q-. We demonstrate that one gene in this region, ETS-1 (a member of the ETS family of transcription factors), is expressed in the endocardium and neural crest during early mouse heart development. Gene-targeted deletion of ETS-1 in mice in a C57/B6 background causes, with high penetrance, large membranous ventricular septal defects and a bifid cardiac apex, and less frequently a non-apex-forming left ventricle (one of the hallmarks of HLHS). Our results implicate an important role for the ETS-1 transcription factor in mammalian heart development and should provide important insights into some of the most common forms of congenital heart disease. PMID:19942620

SHOX gene and conserved noncoding element deletions/duplications in Colombian patients with idiopathic short stature.

PubMed

Sandoval, Gloria Tatiana Vinasco; Jaimes, Giovanna Carola; Barrios, Mauricio Coll; Cespedes, Camila; Velasco, Harvy Mauricio

2014-03-01

SHOX gene mutations or haploinsufficiency cause a wide range of phenotypes such as Leri Weill dyschondrosteosis (LWD), Turner syndrome, and disproportionate short stature (DSS). However, this gene has also been found to be mutated in cases of idiopathic short stature (ISS) with a 3-15% frequency. In this study, the multiplex ligation-dependent probe amplification (MLPA) technique was employed to determine the frequency of SHOX gene mutations and their conserved noncoding elements (CNE) in Colombian patients with ISS. Patients were referred from different centers around the county. From a sample of 62 patients, 8.1% deletions and insertions in the intragenic regions and in the CNE were found. This result is similar to others published in other countries. Moreover, an isolated case of CNE 9 duplication and a new intron 6b deletion in another patient, associated with ISS, are described. This is one of the first studies of a Latin American population in which deletions/duplications of the SHOX gene and its CNE are examined in patients with ISS.

SHOX gene and conserved noncoding element deletions/duplications in Colombian patients with idiopathic short stature

PubMed Central

Sandoval, Gloria Tatiana Vinasco; Jaimes, Giovanna Carola; Barrios, Mauricio Coll; Cespedes, Camila; Velasco, Harvy Mauricio

2014-01-01

SHOX gene mutations or haploinsufficiency cause a wide range of phenotypes such as Leri Weill dyschondrosteosis (LWD), Turner syndrome, and disproportionate short stature (DSS). However, this gene has also been found to be mutated in cases of idiopathic short stature (ISS) with a 3–15% frequency. In this study, the multiplex ligation-dependent probe amplification (MLPA) technique was employed to determine the frequency of SHOX gene mutations and their conserved noncoding elements (CNE) in Colombian patients with ISS. Patients were referred from different centers around the county. From a sample of 62 patients, 8.1% deletions and insertions in the intragenic regions and in the CNE were found. This result is similar to others published in other countries. Moreover, an isolated case of CNE 9 duplication and a new intron 6b deletion in another patient, associated with ISS, are described. This is one of the first studies of a Latin American population in which deletions/duplications of the SHOX gene and its CNE are examined in patients with ISS. PMID:24689071

Cyp1b1 deletion and retinol deficiency coordinately suppress mouse liver lipogenic genes and hepcidin expression during post-natal development

PubMed Central

Maguire, Meghan; Larsen, Michele Campaigne; Foong, Yee Hoon; Tanumihardjo, Sherry; Jefcoate, Colin R.

2018-01-01

Cyp1b1 deletion and gestational vitamin A deficiency (GVAD) redirect adult liver gene expression. A matched sufficient pre- and post-natal diet, which has high carbohydrate and normal iron content (LF12), increased inflammatory gene expression markers in adult livers that were suppressed by GVAD and Cyp1b1 deletion. At birth on the LF12 diet, Cyp1b1 deletion and GVAD each suppress liver expression of the iron suppressor, hepcidin (Hepc), while increasing stellate cell activation markers and suppressing post-natal increases in lipogenesis. Hepc was less suppressed in Cyp1b1−/− pups with a standard breeder diet, but was restored by iron supplementation of the LF12 diet. Conclusions The LF12 diet delivered low post-natal iron and attenuated Hepc. Hepc decreases in Cyp1b1−/− and GVAD mice resulted in stellate activation and lipogenesis suppression. Endothelial BMP6, a Hepc stimulant, is a potential coordinator and Cyp1b1 target. These neonatal changes in Cyp1b1−/− mice link to diminished adult obesity and liver inflammation. PMID:28583802

Deletion of ribosomal protein genes is a common vulnerability in human cancer, especially in concert with TP53 mutations.

PubMed

Ajore, Ram; Raiser, David; McConkey, Marie; Jöud, Magnus; Boidol, Bernd; Mar, Brenton; Saksena, Gordon; Weinstock, David M; Armstrong, Scott; Ellis, Steven R; Ebert, Benjamin L; Nilsson, Björn

2017-04-01

Heterozygous inactivating mutations in ribosomal protein genes (RPGs) are associated with hematopoietic and developmental abnormalities, activation of p53, and altered risk of cancer in humans and model organisms. Here we performed a large-scale analysis of cancer genome data to examine the frequency and selective pressure of RPG lesions across human cancers. We found that hemizygous RPG deletions are common, occurring in about 43% of 10,744 cancer specimens and cell lines. Consistent with p53-dependent negative selection, such lesions are underrepresented in TP53 -intact tumors ( P  ≪ 10 -10 ), and shRNA-mediated knockdown of RPGs activated p53 in TP53 -wild-type cells. In contrast, we did not see negative selection of RPG deletions in TP53 -mutant tumors. RPGs are conserved with respect to homozygous deletions, and shRNA screening data from 174 cell lines demonstrate that further suppression of hemizygously deleted RPGs inhibits cell growth. Our results establish RPG haploinsufficiency as a strikingly common vulnerability of human cancers that associates with TP53 mutations and could be targetable therapeutically. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Reduction of Aspergillus niger Virulence in Apple Fruits by Deletion of the Catalase Gene cpeB.

PubMed

Zhang, Meng-Ke; Tang, Jun; Huang, Zhong-Qin; Hu, Kang-Di; Li, Yan-Hong; Han, Zhuo; Chen, Xiao-Yan; Hu, Lan-Ying; Yao, Gai-Fang; Zhang, Hua

2018-05-30

Aspergillus niger, a common saprophytic fungus, causes rot in many fruits. We studied the role of a putative catalase-peroxidase-encoding gene, cpeB, in oxidative stress and virulence in fruit. The cpeB gene was deleted in A. niger by homologous recombination, and the Δ cpeB mutant showed decreased CAT activity compared with that of the wild type. The cpeB gene deletion caused increased sensitivity to H 2 O 2 stress, and spore germination was significantly reduced; in addition, the reactive-oxygen-species (ROS) metabolites superoxide anions (·O 2 - ), hydrogen peroxide (H 2 O 2 ), and malondialdehyde (MDA) accumulated in the Δ cpeB mutant during H 2 O 2 stress. Furthermore, ROS metabolism in A. niger infected apples was determined, and our results showed that the Δ cpeB mutant induced an attenuated response in apple fruit during the fruit-pathogen interaction; the cpeB gene deletion significantly reduced the development of lesions, suggesting that the cpeB gene in A. niger is essential for full virulence in apples.

Alu-mediated large deletion of the CDSN gene as a cause of peeling skin disease.

PubMed

Wada, T; Matsuda, Y; Muraoka, M; Toma, T; Takehara, K; Fujimoto, M; Yachie, A

2014-10-01

Peeling skin disease (PSD) is an autosomal recessive skin disorder caused by mutations in CDSN and is characterized by superficial peeling of the upper epidermis. Corneodesmosin (CDSN) is a major component of corneodesmosomes that plays an important role in maintaining epidermis integrity. Herein, we report a patient with PSD caused by a novel homozygous large deletion in the 6p21.3 region encompassing the CDSN gene, which abrogates CDSN expression. Several genes including C6orf15, PSORS1C1, PSORS1C2, CCHCR1, and TCF19 were also deleted, however, the patient showed only clinical features typical of PSD. The deletion size was 59.1 kb. Analysis of the sequence surrounding the breakpoint showed that both telomeric and centromeric breakpoints existed within Alu-S sequences that were oriented in opposite directions. These results suggest an Alu-mediated recombination event as the mechanism underlying the deletion in our patient. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Deletions at the SOX10 gene locus cause Waardenburg syndrome types 2 and 4.

PubMed

Bondurand, Nadege; Dastot-Le Moal, Florence; Stanchina, Laure; Collot, Nathalie; Baral, Viviane; Marlin, Sandrine; Attie-Bitach, Tania; Giurgea, Irina; Skopinski, Laurent; Reardon, William; Toutain, Annick; Sarda, Pierre; Echaieb, Anis; Lackmy-Port-Lis, Marilyn; Touraine, Renaud; Amiel, Jeanne; Goossens, Michel; Pingault, Veronique

2007-12-01

Waardenburg syndrome (WS) is an auditory-pigmentary disorder that exhibits varying combinations of sensorineural hearing loss and abnormal pigmentation of the hair and skin. Depending on additional symptoms, WS is classified into four subtypes, WS1-WS4. Absence of additional features characterizes WS2. The association of facial dysmorphic features defines WS1 and WS3, whereas the association with Hirschsprung disease (aganglionic megacolon) characterizes WS4, also called "Waardenburg-Hirschsprung disease." Mutations within the genes MITF and SNAI2 have been identified in WS2, whereas mutations of EDN3, EDNRB, and SOX10 have been observed in patients with WS4. However, not all cases are explained at the molecular level, which raises the possibility that other genes are involved or that some mutations within the known genes are not detected by commonly used genotyping methods. We used a combination of semiquantitative fluorescent multiplex polymerase chain reaction and fluorescent in situ hybridization to search for SOX10 heterozygous deletions. We describe the first characterization of SOX10 deletions in patients presenting with WS4. We also found SOX10 deletions in WS2 cases, making SOX10 a new gene of WS2. Interestingly, neurological phenotypes reminiscent of that observed in WS4 (PCWH syndrome [peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, WS, and Hirschsprung disease]) were observed in some WS2-affected patients with SOX10 deletions. This study further characterizes the molecular complexity and the close relationship that links the different subtypes of WS.

A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1

PubMed Central

de Berardinis, Véronique; Vallenet, David; Castelli, Vanina; Besnard, Marielle; Pinet, Agnès; Cruaud, Corinne; Samair, Sumitta; Lechaplais, Christophe; Gyapay, Gabor; Richez, Céline; Durot, Maxime; Kreimeyer, Annett; Le Fèvre, François; Schächter, Vincent; Pezo, Valérie; Döring, Volker; Scarpelli, Claude; Médigue, Claudine; Cohen, Georges N; Marlière, Philippe; Salanoubat, Marcel; Weissenbach, Jean

2008-01-01

We have constructed a collection of single-gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2-3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches. PMID:18319726

Identification of Genes Related to Fungicide Resistance in Fusarium fujikuroi

PubMed Central

Choi, Younghae; Jung, Boknam; Li, Taiying

2017-01-01

We identified two genes related to fungicide resistance in Fusarium fujikuroi through random mutagenesis. Targeted gene deletions showed that survival factor 1 deletion resulted in higher sensitivity to fungicides, while deletion of the gene encoding F-box/WD-repeat protein increased resistance, suggesting that the genes affect fungicide resistance in different ways. PMID:28781543

Detection of large deletion in human BRCA1 gene in human breast carcinoma MCF-7 cells by using DNA-Silver Nanoclusters

NASA Astrophysics Data System (ADS)

Borghei, Yasaman-Sadat; Hosseini, Morteza; Ganjali, Mohammad Reza

2018-01-01

Here we describe a label-free detection strategy for large deletion mutation in breast cancer (BC) related gene BRCA1 based on a DNA-silver nanocluster (NC) fluorescence upon recognition-induced hybridization. The specific hybridization of DNA templated silver NCs fluorescent probe to target DNAs can act as effective templates for enhancement of AgNCs fluorescence, which can be used to distinguish the deletion of BRCA1 due to different fluorescence intensities. Under the optimal conditions, the fluorescence intensity of the DNA-AgNCs at emission peaks around 440 nm (upon excitation at 350 nm) increased with the increasing deletion type within a dynamic range from 1.0 × 10-10 to 2.4 × 10-6 M with a detection limit (LOD) of 6.4 × 10-11 M. In this sensing system, the normal type shows no significant fluorescence; on the other hand, the deletion type emits higher fluorescence than normal type. Using this nanobiosensor, we successfully determined mutation using the non-amplified genomic DNAs that were isolated from the BC cell line.

Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan

PubMed Central

Lopez-Moya, Federico; Kowbel, David; Nueda, Ma José; Palma-Guerrero, Javier; Glass, N. Louise; Lopez-Llorca, Luis Vicente

2016-01-01

Chitosan is a natural polymer with antimicrobial activity. Chitosan causes plasma membrane permeabilization and induction of intracellular reactive oxygen species (ROS) in Neurospora crassa. We have determined the transcriptional profile of N. crassa to chitosan and identified the main gene targets involved in the cellular response to this compound. Global network analyses showed membrane, transport and oxidoreductase activity as key nodes affected by chitosan. Activation of oxidative metabolism indicates the importance of ROS and cell energy together with plasma membrane homeostasis in N. crassa response to chitosan. Deletion strain analysis of chitosan susceptibility pointed, NCU03639 encoding a class 3 lipase, involved in plasma membrane repair by lipid replacement and NCU04537 a MFS monosaccharide transporter related with assimilation of simple sugars, as main gene targets of chitosan. NCU10521, a glutathione S-transferase-4 involved in the generation of reducing power for scavenging intracellular ROS is also a determinant chitosan gene target. Ca2+ increased tolerance to chitosan in N. crassa. Growth of NCU10610 (fig 1 domain) and SYT1 (a synaptotagmin) deletion strains was significantly increased by Ca2+ in presence of chitosan. Both genes play a determinant role in N. crassa membrane homeostasis. Our results are of paramount importance for developing chitosan as antifungal. PMID:26694141

Encephalopathy and bilateral cataract in a boy with an interstitial deletion of Xp22 comprising the CDKL5 and NHS genes.

PubMed

Van Esch, Hilde; Jansen, Anna; Bauters, Marijke; Froyen, Guy; Fryns, Jean-Pierre

2007-02-15

We describe a male patient with a deletion at Xp22, detected by high resolution X-array CGH. The clinical phenotype present in this infant boy, consists of severe encephalopathy, congenital cataracts and tetralogy of Fallot and can be attributed to the deletion of the genes within the interval. Among these deleted genes are the gene for Nance-Horan syndrome and the cyclin-dependent kinase-like 5 gene (CDKL5), responsible for the early seizure variant of Rett syndrome. This is the first description of a male patient with a deletion of these genes, showing the involvement of CDKL5 in severe epileptic encephalopathy in males. Moreover it illustrates the added value of high resolution array-CGH in molecular diagnosis of mental retardation-multiple congenital anomaly cases. (c) 2007 Wiley-Liss, Inc.

Deletion of a Stay-Green Gene Associates with Adaptive Selection in Brassica napus.

PubMed

Qian, Lunwen; Voss-Fels, Kai; Cui, Yixin; Jan, Habib U; Samans, Birgit; Obermeier, Christian; Qian, Wei; Snowdon, Rod J

2016-12-05

Chlorophyll levels provide important information about plant growth and physiological plasticity in response to changing environments. The stay-green gene NON-YELLOWING 1 (NYE1) is believed to regulate chlorophyll degradation during senescence, concomitantly affecting the disassembly of the light-harvesting complex and hence indirectly influencing photosynthesis. We identified Brassica napus accessions carrying an NYE1 deletion associated with increased chlorophyll content, and with upregulated expression of light-harvesting complex and photosynthetic reaction center (PSI and PSII) genes. Comparative analysis of the seed oil content of accessions with related genetic backgrounds revealed that the B. napus NYE1 gene deletion (bnnye1) affected oil accumulation, and linkage disequilibrium signatures suggested that the locus has been subject to artificial selection by breeding in oilseed B. napus forms. Comparative analysis of haplotype diversity groups (haplogroups) between three different ecotypes of the allopolyploid B. napus and its A-subgenome diploid progenitor, Brassica rapa, indicated that introgression of the bnnye1 deletion from Asian B. rapa into winter-type B. napus may have simultaneously improved its adaptation to cooler environments experienced by autumn-sown rapeseed. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Screening for large genomic rearrangements in the FANCA gene reveals extensive deletion in a Finnish breast cancer family.

PubMed

Solyom, Szilvia; Winqvist, Robert; Nikkilä, Jenni; Rapakko, Katrin; Hirvikoski, Pasi; Kokkonen, Hannaleena; Pylkäs, Katri

2011-03-28

A portion of familial breast cancer cases are caused by mutations in the same genes that are inactivated in the downstream part of Fanconi anemia (FA) signaling pathway. Here we have assessed the FANCA gene for breast cancer susceptibility by examining blood DNA for aberrations from 100 Northern Finnish breast cancer families using the MLPA method. We identified a novel heterozygous deletion, removing the promoter and 12 exons of the gene in one family. This allele was absent from 124 controls. We conclude that FANCA deletions might contribute to breast cancer susceptibility, potentially in combination with other germline mutations. To our knowledge, this is the first study reporting a large deletion in an upstream FA gene in familial breast cancer. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

High frequency of TTTY2-like gene-related deletions in patients with idiopathic oligozoospermia and azoospermia.

PubMed

Yapijakis, C; Serefoglou, Z; Papadimitriou, K; Makrinou, E

2015-06-01

Genes located on Y chromosome and expressed in testis are likely to be involved in spermatogenesis. TTTY2 is a Y-linked multicopy gene family of unknown function that includes TTTY2L2A and TTTY2L12A at Yq11 and Yp11 loci respectively. Using PCR amplification, we screened for TTTY2L2A- and TTTY2L12A-associated deletions, in 94 Greek men with fertility problems. Patients were divided into three groups as following: group A (n = 28) included men with idiopathic moderate oligozoospermia, group B (n = 34) with idiopathic severe oligozoospermia and azoospermia, and group C (n = 32) with oligo- and azoospermia of various known etiologies. No deletions were detected in group C patients and 50 fertile controls. However, two patients from group A had deletions in TTTY2L2A (7.1%) and six in TTTY2L12A (21.4%), whereas from group B, four patients had deletions in TTTY2L2A (11.8%) and 10 in TTTY2L12A (29.4%). In addition, five patients from both groups A and B (8%) appeared to have deletions in both studied TTTY2 genes, although these are located very far apart. These results indicate that the TTTY2 gene family may play a significant role in spermatogenesis and suggest a possible mechanism of nonhomologous recombinational events that may cause genomic instability and ultimately lead to male infertility. © 2014 Blackwell Verlag GmbH.

Investigation of tumor suppressor genes apart from VHL on 3p by deletion mapping in sporadic clear cell renal cell carcinoma (cRCC).

PubMed

Singh, Rashmi Bhat; Amare Kadam, Pratibha S

2013-10-01

To investigate the most recurrent deletion loci on 3p12-p26 by deletion mapping studies by PCR-LOH and BAC array-FISH in sporadic conventional renal cell carcinoma (cRCC) and further, to evaluate the their clinicopathologic significance in cRCC. Comparative allelotyping studies in cRCC and major epithelial carcinomas (MEC) such as lung, breast, and bladder tumors were also carried out to investigate the specificity of the targeted loci in cRCC. A total of 40 c-RCC patients were enrolled in this study, categorized in to 2 groups: group I comprises of patients of stages I and II and group II includes patients at stages III and IV. Loss of heterozygosity (LOH) studies were performed by PCR using 15 microsatellite markers of region 3p12-p26 on paired normal-tumor tissues. The recurrent LOH loci found in 27 cRCC tumors were further validated by BAC array-FISH using 23 serially mapped BAC clones. Simultaneously, the allelic deletion status of fragile histidine triad (FHIT) gene was studied by FISH in cRCC and major epithelial carcinoma (MEC) tumors. The numerical aberrations of chromosome 3 were also studied using the centromere enumeration probe (CEP) probe for chromosome 3 to validate the observed allelic losses by BAC array-FISH in cRCC as well as MECs. Our study revealed 3 affected regions of LOH on 3p in cRCC: 3p12.2-p14.1, 3p14.2-p21.1, and 3p24.2-p26.1 in both group I (stages I and II) and group II (stage III and IV). Comparative allelotyping studies revealed that except for LOH loci D3S2406 (20%), D3S1766 (14%), and D3S1560 (20%), remaining affected loci revealed retention of heterozygosity (ROH) in breast carcinomas. Lung and bladder tumors revealed ROH at all affected LOH loci. FISH with FHIT gene probe revealed deletions in cRCC (88%), breast (30%), and lung tumors (10%). FHIT gene deletions frequency was almost equal in both groups I and II (>70%), whereas a locus 3p13 (D3S2454) revealed the highest LOH in group II (83%) patients in comparison to group I (16

Weak D caused by a founder deletion in the RHD gene.

PubMed

Fichou, Yann; Chen, Jian-Min; Le Maréchal, Cédric; Jamet, Déborah; Dupont, Isabelle; Chuteau, Claude; Durousseau, Cécile; Loirat, Marie-Jeanne; Bailly, Pascal; Férec, Claude

2012-11-01

The RhD blood group system exemplifies a genotype-phenotype correlation by virtue of its highly polymorphic and immunogenic nature. Weak D phenotypes are generally thought to result from missense mutations leading to quantitative change of the D antigen in the red blood cell membrane or intracellularly. Different sets of polymerase chain reaction primers were designed to map and clone a deletion involving RHD Exon 10, which was found in approximately 3% of approximately 2000 RHD hemizygous subjects with D phenotype ambiguity. D antigen density was measured by flow cytometry. Transcript analysis was carried out by 3'-rapid amplification of complementary DNA ends. Haplotype analysis was performed by microsatellite genotyping. A 5405-bp deletion that removed nearly two-thirds of Intron 9 and almost all of Exon 10 of the RHD gene was characterized. It is predicted to result in the replacement of the last eight amino acids of the wild-type RhD protein by another four amino acids. The mean RhD antigen density from two deletion carriers was determined to be only 30. A consensus haplotype could be deduced from the deletion carriers based on the microsatellite genotyping data. The currently reported deletion was derived from a common founder. This deletion appears to represent not only the first large deletion associated with weak D but also the weakest of weak D alleles so far reported. This highly unusual genotype-phenotype relationship may be attributable to the additive effect of three distinct mechanisms that affect mRNA formation, mRNA stability, and RhD/ankyrin-R interaction, respectively. © 2012 American Association of Blood Banks.

Image-aided Suicide Gene Therapy Utilizing Multifunctional hTERT-targeting Adenovirus for Clinical Translation in Hepatocellular Carcinoma.

PubMed

Kim, Yun-Hee; Kim, Kyung Tae; Lee, Sang-Jin; Hong, Seung-Hee; Moon, Ju Young; Yoon, Eun Kyung; Kim, Sukyoung; Kim, Eun Ok; Kang, Se Hun; Kim, Seok Ki; Choi, Sun Il; Goh, Sung Ho; Kim, Daehong; Lee, Seong-Wook; Ju, Mi Ha; Jeong, Jin Sook; Kim, In-Hoo

2016-01-01

Trans-splicing ribozyme enables to sense and reprogram target RNA into therapeutic transgene and thereby becomes a good sensing device for detection of cancer cells, judging from transgene expression. Previously we proposed PEPCK-Rz-HSVtk (PRT), hTERT targeting trans-splicing ribozyme (Rz) driven by liver-specific promoter phosphoenolpyruvate carboxykinase (PEPCK) with downstream suicide gene, herpes simplex virus thymidine kinase (HSVtk) for hepatocellular carcinoma (HCC) gene therapy. Here, we describe success of a re-engineered adenoviral vector harboring PRT in obtaining greater antitumor activity with less off-target effect for clinical application as a theranostics. We introduced liver-selective apolipoprotein E (ApoE) enhancer to the distal region of PRT unit to augment activity and liver selectivity of PEPCK promoter, and achieved better transduction into liver cancer cells by replacement of serotype 35 fiber knob on additional E4orf1-4 deletion of E1&E3-deleted serotype 5 back bone. We demonstrated that our refined adenovirus harboring PEPCK/ApoE-Rz-HSVtk (Ad-PRT-E) achieved great anti-tumor efficacy and improved ability to specifically target HCC without damaging normal hepatocytes. We also showed noninvasive imaging modalities were successfully employed to monitor both how well a therapeutic gene (HSVtk) was expressed inside tumor and how effectively a gene therapy took an action in terms of tumor growth. Collectively, this study suggests that the advanced therapeutic adenoviruses Ad-PRT-E and its image-aided evaluation system may lead to the powerful strategy for successful clinical translation and the development of clinical protocols for HCC therapy.

A Novel Whole Gene Deletion of BCKDHB by Alu-Mediated Non-allelic Recombination in a Chinese Patient With Maple Syrup Urine Disease.

PubMed

Liu, Gang; Ma, Dingyuan; Hu, Ping; Wang, Wen; Luo, Chunyu; Wang, Yan; Sun, Yun; Zhang, Jingjing; Jiang, Tao; Xu, Zhengfeng

2018-01-01

Maple syrup urine disease (MSUD) is an autosomal recessive inherited metabolic disorder caused by mutations in the BCKDHA, BCKDHB, DBT , and DLD genes. Among the wide range of disease-causing mutations in BCKDHB , only one large deletion has been associated with MSUD. Compound heterozygous mutations in BCKDHB were identified in a Chinese patient with typical MSUD using next-generation sequencing, quantitative PCR, and array comparative genomic hybridization. One allele presented a missense mutation (c.391G > A), while the other allele had a large deletion; both were inherited from the patient's unaffected parents. The deletion breakpoints were characterized using long-range PCR and sequencing. A novel 383,556 bp deletion (chr6: g.80811266_81194921del) was determined, which encompassed the entire BCKDHB gene. The junction site of the deletion was localized within a homologous sequence in two AluYa5 elements. Hence, Alu-mediated non-allelic homologous recombination is speculated as the mutational event underlying the large deletion. In summary, this study reports a recombination mechanism in the BCKDHB gene causing a whole gene deletion in a newborn with MSUD.

PAX3 gene deletion detected by microarray analysis in a girl with hearing loss.

PubMed

Drozniewska, Malgorzata; Haus, Olga

2014-01-01

Deletions of the PAX3 gene have been rarely reported in the literature. Mutations of this gene are a common cause of Waardenburg syndrome type 1 and 3. We report a 16 year old female presenting hearing loss and normal intellectual development, without major features of Waardenburg syndrome type 1, and without family history of the syndrome. Her phenotype, however, overlaps with features of craniofacial-deafness-hand syndrome. Microarray analysis showed ~862 kb de novo deletion at 2q36.1 including PAX3. The above findings suggest that the rearrangement found in our patient appeared de novo and with high probability is a cause of her phenotype.

A Population of Deletion Mutants and an Integrated Mapping and Exome-seq Pipeline for Gene Discovery in Maize

PubMed Central

Jia, Shangang; Li, Aixia; Morton, Kyla; Avoles-Kianian, Penny; Kianian, Shahryar F.; Zhang, Chi; Holding, David

2016-01-01

To better understand maize endosperm filling and maturation, we used γ-irradiation of the B73 maize reference line to generate mutants with opaque endosperm and reduced kernel fill phenotypes, and created a population of 1788 lines including 39 Mo17 × F2s showing stable, segregating, and viable kernel phenotypes. For molecular characterization of the mutants, we developed a novel functional genomics platform that combined bulked segregant RNA and exome sequencing (BSREx-seq) to map causative mutations and identify candidate genes within mapping intervals. To exemplify the utility of the mutants and provide proof-of-concept for the bioinformatics platform, we present detailed characterization of line 937, an opaque mutant harboring a 6203 bp in-frame deletion covering six exons within the Opaque-1 gene. In addition, we describe mutant line 146 which contains a 4.8 kb intragene deletion within the Sugary-1 gene and line 916 in which an 8.6 kb deletion knocks out a Cyclin A2 gene. The publically available algorithm developed in this work improves the identification of causative deletions and its corresponding gaps within mapping peaks. This study demonstrates the utility of γ-irradiation for forward genetics in large nondense genomes such as maize since deletions often affect single genes. Furthermore, we show how this classical mutagenesis method becomes applicable for functional genomics when combined with state-of-the-art genomics tools. PMID:27261000

Xp22.3 genomic deletions involving the CDKL5 gene in girls with early onset epileptic encephalopathy.

PubMed

Mei, Davide; Marini, Carla; Novara, Francesca; Bernardina, Bernardo D; Granata, Tiziana; Fontana, Elena; Parrini, Elena; Ferrari, Anna R; Murgia, Alessandra; Zuffardi, Orsetta; Guerrini, Renzo

2010-04-01

Mutations of the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause an X-linked encephalopathy with early onset intractable epilepsy, including infantile spasms and other seizure types, and a Rett syndrome (RTT)-like phenotype. Very limited information is available on the frequency and phenotypic spectrum associated with CDKL5 deletions/duplications. We investigated the role of CDKL5 deletions/duplications in causing early onset intractable epilepsy of unknown etiology in girls. We studied 49 girls with early onset intractable epilepsy, with or without infantile spasms, and developmental impairment, for whom no etiologic factors were obvious after clinical examination, brain magnetic resonance imaging (MRI) and expanded screening for inborn errors of metabolism. We performed CDKL5 gene mutation analysis in all and multiplex ligation dependent probe amplification assay (MLPA) in those who were mutation negative. Custom Array-comparative genomic hybridization (CGH), breakpoint polymerase chain reaction (PCR) analysis, and X-inactivation studies were performed in patients in whom MLPA uncovered a genomic alteration. We found CDKL5 mutations in 8.2% (4 of 49) of patients and genomic deletions in 8.2% (4 of 49). Overall, abnormalities of the CDKL5 gene accounted for 16.3% (8 of 49) of patients. CDKL5 gene deletions are an under-ascertained cause of early onset intractable epilepsy in girls. Genetic testing of CDKL5, including both mutation and deletion/duplication analysis, should be considered in this clinical subgroup.

A recessive contiguous gene deletion of chromosome 2p16 associated with cystinuria and a mitochondrial disease.

PubMed

Parvari, R; Brodyansky, I; Elpeleg, O; Moses, S; Landau, D; Hershkovitz, E

2001-10-01

Deletions ranging from 100 Kb to 1 Mb--too small to be detected under the microscope--may still involve dozens of genes, thus causing microdeletion syndromes. The vast majority of these syndromes are caused by haploinsufficiency of one or several genes and are transmitted as dominant traits. We identified seven patients originating from an extended family and presenting with a unique syndrome, inherited in a recessive mode, consisting of cystinuria, neonatal seizures, hypotonia, severe somatic and developmental delay, facial dysmorphism, and lactic acidemia. Reduced activity of all the respiratory chain enzymatic complexes that are encoded in the mitochondria was found in muscle biopsy specimens of the patients examined. The molecular basis of this disorder is a homozygous deletion of 179,311 bp on chromosome 2p16, which includes the type I cystinuria gene (SLC3A1), the protein phosphatase 2Cbeta gene (PP2Cbeta), an unidentified gene (KIAA0436), and several expressed sequence tags. The extent of the deletion suggests that this unique syndrome is related to the complete absence of these genes' products, one of which may be essential for the synthesis of mitochondrial encoded proteins.

Deletion of a Single-Copy Trna Affects Microtubule Function in Saccharomyces Cerevisiae

PubMed Central

Reijo, R. A.; Cho, D. S.; Huffaker, T. C.

1993-01-01

rts1-1 was identified as an extragenic suppressor of tub2-104, a cold-sensitive allele of the sole gene encoding β-tubulin in the yeast, Saccharomyces cerevisiae. In addition, rts1-1 cells are heat sensitive and resistant to the microtubule-destabilizing drug, benomyl. The rts1-1 mutation is a deletion of approximately 5 kb of genomic DNA on chromosome X that includes one open reading frame and three tRNA genes. Dissection of this region shows that heat sensitivity is due to deletion of the open reading frame (HIT1). Suppression and benomyl resistance are caused by deletion of the gene encoding a tRNA(AGG)(Arg) (HSX1). Northern analysis of rts1-1 cells indicates that HSX1 is the only gene encoding this tRNA. Deletion of HSX1 does not suppress the tub2-104 mutation by misreading at the AGG codons in TUB2. It also does not suppress by interfering with the protein arginylation that targets certain proteins for degradation. These results leave open the prospect that this tRNA(AGG)(Arg) plays a novel role in the cell. PMID:8307335

Gene Deletion in Barley Mediated by LTR-retrotransposon BARE

PubMed Central

Shang, Yi; Yang, Fei; Schulman, Alan H.; Zhu, Jinghuan; Jia, Yong; Wang, Junmei; Zhang, Xiao-Qi; Jia, Qiaojun; Hua, Wei; Yang, Jianming; Li, Chengdao

2017-01-01

A poly-row branched spike (prbs) barley mutant was obtained from soaking a two-rowed barley inflorescence in a solution of maize genomic DNA. Positional cloning and sequencing demonstrated that the prbs mutant resulted from a 28 kb deletion including the inflorescence architecture gene HvRA2. Sequence annotation revealed that the HvRA2 gene is flanked by two LTR (long terminal repeat) retrotransposons (BARE) sharing 89% sequence identity. A recombination between the integrase (IN) gene regions of the two BARE copies resulted in the formation of an intact BARE and loss of HvRA2. No maize DNA was detected in the recombination region although the flanking sequences of HvRA2 gene showed over 73% of sequence identity with repetitive sequences on 10 maize chromosomes. It is still unknown whether the interaction of retrotransposons between barley and maize has resulted in the recombination observed in the present study. PMID:28252053

A large deletion of PROP1 gene in patients with combined pituitary hormone deficiency from two unrelated Chinese pedigrees.

PubMed

Zhang, Huiwen; Wang, Yi; Han, Lianshu; Gu, Xuefan; Shi, Dingping

2010-01-01

Familial combined pituitary hormone deficiency (CPHD) appears to have a genetic cause, PROP1 gene mutations being the most common one. We investigated whether PROP1 plays a role in two Chinese familial cases of CPHD. PROP1 gene and adjacent sequences from genomic samples from two unrelated families were amplified to investigate molecular variations and define the extension of a potential deletion. A quantitative real-time polymerase chain reaction was conducted to analyze the copy number of PROP1 gene in the probands' mothers. The relationship of the two distantly located families was further analyzed using microsatellite markers. A segment of about 53.2 kilobases (kb) comprehending PROP1 and another gene encoding a hypothetical protein Q6ZTH3 was deleted in both pedigrees. The mother of one of the probands was hemizygous for this large deletion, which confirmed the assumption that the affected children inherited the deletion allele from their consanguineous parents. The difference of three microsatellites surrounding the absent segment indicated that the two pedigrees were genetically unrelated. We report the largest genomic deletion including PROP1 gene associated with CPHD. Q6ZTH3 is unlikely to exert an indispensable function during embryogenesis or organogenesis. The 7.7-kb segment upstream of the transcription of PROP1 probably harbors a fragile site that favors the occurrence of breakpoints. Copyright (c) 2010 S. Karger AG, Basel.

A new deletion refines the boundaries of the murine Prader–Willi syndrome imprinting center

PubMed Central

DuBose, Amanda J.; Smith, Emily Y.; Yang, Thomas P.; Johnstone, Karen A.; Resnick, James L.

2011-01-01

The human chromosomal 15q11–15q13 region is subject to both maternal and paternal genomic imprinting. Absence of paternal gene expression from this region results in Prader–Willi syndrome (PWS), while absence of maternal gene expression leads to Angelman syndrome. Transcription of paternally expressed genes in the region depends upon an imprinting center termed the PWS-IC. Imprinting defects in PWS can be caused by microdeletions and the smallest commonly deleted region indicates that the PWS-IC lies within a region of 4.3 kb. The function and location of the PWS-IC is evolutionarily conserved, but delineation of the PWS-IC in mouse has proven difficult. The first targeted mutation of the PWS-IC, a deletion of 35 kb spanning Snrpn exon 1, exhibited a complete PWS-IC deletion phenotype. Pups inheriting this mutation paternally showed a complete loss of paternal gene expression and died neonatally. A reported deletion of 4.8 kb showed only a reduction in paternal gene expression and incomplete penetrance of neonatal lethality, suggesting that some PWS-IC function had been retained. Here, we report that a 6 kb deletion spanning Snrpn exon 1 exhibits a complete PWS-IC deletion phenotype. Pups inheriting this mutation paternally lack detectable expression of all PWS genes and paternal silencing of Ube3a, exhibit maternal DNA methylation imprints at Ndn and Mkrn3 and suffer failure to thrive leading to a fully penetrant neonatal lethality. PMID:21659337

A new deletion refines the boundaries of the murine Prader-Willi syndrome imprinting center.

PubMed

Dubose, Amanda J; Smith, Emily Y; Yang, Thomas P; Johnstone, Karen A; Resnick, James L

2011-09-01

The human chromosomal 15q11-15q13 region is subject to both maternal and paternal genomic imprinting. Absence of paternal gene expression from this region results in Prader-Willi syndrome (PWS), while absence of maternal gene expression leads to Angelman syndrome. Transcription of paternally expressed genes in the region depends upon an imprinting center termed the PWS-IC. Imprinting defects in PWS can be caused by microdeletions and the smallest commonly deleted region indicates that the PWS-IC lies within a region of 4.3 kb. The function and location of the PWS-IC is evolutionarily conserved, but delineation of the PWS-IC in mouse has proven difficult. The first targeted mutation of the PWS-IC, a deletion of 35 kb spanning Snrpn exon 1, exhibited a complete PWS-IC deletion phenotype. Pups inheriting this mutation paternally showed a complete loss of paternal gene expression and died neonatally. A reported deletion of 4.8 kb showed only a reduction in paternal gene expression and incomplete penetrance of neonatal lethality, suggesting that some PWS-IC function had been retained. Here, we report that a 6 kb deletion spanning Snrpn exon 1 exhibits a complete PWS-IC deletion phenotype. Pups inheriting this mutation paternally lack detectable expression of all PWS genes and paternal silencing of Ube3a, exhibit maternal DNA methylation imprints at Ndn and Mkrn3 and suffer failure to thrive leading to a fully penetrant neonatal lethality.

The Role of the Regulator Fur in Gene Regulation and Virulence of Riemerella anatipestifer Assessed Using an Unmarked Gene Deletion System

PubMed Central

Guo, Yunqing; Hu, Di; Guo, Jie; Li, Xiaowen; Guo, Jinyue; Wang, Xiliang; Xiao, Yuncai; Jin, Hui; Liu, Mei; Li, Zili; Bi, Dingren; Zhou, Zutao

2017-01-01

Riemerella anatipestifer, an avian pathogen, has resulted in enormous economic losses to the duck industry globally. Notwithstanding, little is known regarding the physiological, pathogenic and virulence mechanisms of Riemerella anatipestifer (RA) infection. However, the role of Ferric uptake regulator (Fur) in the virulence of R. anatipestifer has not, to date, been demonstrated. Using a genetic approach, unmarked gene deletion system, we evaluated the function of fur gene in the virulence of R. anatipestifer. For this purpose, we constructed a suicide vector containing pheS as a counter selectable marker for unmarked deletion of fur gene to investigate its role in the virulence. After successful transformation of the newly constructed vector, a mutant strain was characterized for genes regulated by iron and Fur using RNA-sequencing and a comparison was made between wild type and mutant strains in both iron restricted and enriched conditions. RNA-seq analysis of the mutant strain in a restricted iron environment showed the downregulation and upregulation of genes which were involved in either important metabolic pathways, transport processes, growth or cell membrane synthesis. Electrophoretic mobility shift assay was performed to identify the putative sequences recognized by Fur. The putative Fur-box sequence was 5′-GATAATGATAATCATTATC-3′. Lastly, the median lethal dose and histopathological investigations of animal tissues also illustrated mild pathological lesions produced by the mutant strain as compared to the wild type RA strain, hence showing declined virulence. Conclusively, an unmarked gene deletion system was successfully developed for RA and the role of the fur gene in virulence was explored comprehensively. PMID:28971067

Rapid deletion production in fungi via Agrobacterium mediated transformation of OSCAR deletion contructs.

USDA-ARS?s Scientific Manuscript database

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene’s function in the living organism. In this protocol we describe the OSCAR method of precise and rapid deletion plasmid construction. OSCAR relies on t...

A Novel Whole Gene Deletion of BCKDHB by Alu-Mediated Non-allelic Recombination in a Chinese Patient With Maple Syrup Urine Disease

PubMed Central

Liu, Gang; Ma, Dingyuan; Hu, Ping; Wang, Wen; Luo, Chunyu; Wang, Yan; Sun, Yun; Zhang, Jingjing; Jiang, Tao; Xu, Zhengfeng

2018-01-01

Maple syrup urine disease (MSUD) is an autosomal recessive inherited metabolic disorder caused by mutations in the BCKDHA, BCKDHB, DBT, and DLD genes. Among the wide range of disease-causing mutations in BCKDHB, only one large deletion has been associated with MSUD. Compound heterozygous mutations in BCKDHB were identified in a Chinese patient with typical MSUD using next-generation sequencing, quantitative PCR, and array comparative genomic hybridization. One allele presented a missense mutation (c.391G > A), while the other allele had a large deletion; both were inherited from the patient’s unaffected parents. The deletion breakpoints were characterized using long-range PCR and sequencing. A novel 383,556 bp deletion (chr6: g.80811266_81194921del) was determined, which encompassed the entire BCKDHB gene. The junction site of the deletion was localized within a homologous sequence in two AluYa5 elements. Hence, Alu-mediated non-allelic homologous recombination is speculated as the mutational event underlying the large deletion. In summary, this study reports a recombination mechanism in the BCKDHB gene causing a whole gene deletion in a newborn with MSUD. PMID:29740478

SOX2 anophthalmia syndrome: 12 new cases demonstrating broader phenotype and high frequency of large gene deletions.

PubMed

Bakrania, P; Robinson, D O; Bunyan, D J; Salt, A; Martin, A; Crolla, J A; Wyatt, A; Fielder, A; Ainsworth, J; Moore, A; Read, S; Uddin, J; Laws, D; Pascuel-Salcedo, D; Ayuso, C; Allen, L; Collin, J R O; Ragge, N K

2007-11-01

Developmental eye anomalies, which include anophthalmia (absent eye) or microphthalmia (small eye) are an important cause of severe visual impairment in infants and young children. Heterozygous mutations in SOX2, a SOX1B-HMG box transcription factor, have been found in up to 10% of individuals with severe microphthalmia or anophthalmia and such mutations could also be associated with a range of non-ocular abnormalities. We performed mutation analysis on a new cohort of 120 patients with congenital eye abnormalities, mainly anophthalmia, microphthalmia and coloboma. Multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridisation (FISH) were used to detect whole gene deletion. We identified four novel intragenic SOX2 mutations (one single base deletion, one single base duplication and two point mutations generating premature translational termination codons) and two further cases with the previously reported c.70del20 mutation. Of 52 patients with severe microphthalmia or anophthalmia analysed by MLPA, 5 were found to be deleted for the whole SOX2 gene and 1 had a partial deletion. In two of these, FISH studies identified sub-microscopic deletions involving a minimum of 328 Kb and 550 Kb. The SOX2 phenotypes include a patient with anophthalmia, oesophageal abnormalities and horseshoe kidney, and a patient with a retinal dystrophy implicating SOX2 in retinal development. Our results provide further evidence that SOX2 haploinsufficiency is a common cause of severe developmental ocular malformations and that background genetic variation determines the varying phenotypes. Given the high incidence of whole gene deletion we recommend that all patients with severe microphthalmia or anophthalmia, including unilateral cases be screened by MLPA and FISH for SOX2 deletions.

Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan

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

Lopez-Moya, Federico; Kowbel, David; Nueda, Ma Jose

Chitosan is a natural polymer with antimicrobial activity. Chitosan causes plasma membrane permeabilization and induction of intracellular reactive oxygen species (ROS) in Neurospora crassa. In this paper, we have determined the transcriptional profile of N. crassa to chitosan and identified the main gene targets involved in the cellular response to this compound. Global network analyses showed membrane, transport and oxidoreductase activity as key nodes affected by chitosan. Activation of oxidative metabolism indicates the importance of ROS and cell energy together with plasma membrane homeostasis in N. crassa response to chitosan. Deletion strain analysis of chitosan susceptibility pointed NCU03639 encoding amore » class 3 lipase, involved in plasma membrane repair by lipid replacement, and NCU04537 a MFS monosaccharide transporter related to assimilation of simple sugars, as main gene targets of chitosan. NCU10521, a glutathione S-transferase-4 involved in the generation of reducing power for scavenging intracellular ROS is also a determinant chitosan gene target. Ca 2+ increased tolerance to chitosan in N. crassa. Growth of NCU10610 (fig 1 domain) and SYT1 (a synaptotagmin) deletion strains was significantly increased by Ca 2+ in the presence of chitosan. Both genes play a determinant role in N. crassa membrane homeostasis. Finally, our results are of paramount importance for developing chitosan as an antifungal.« less

Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan

DOE PAGES

Lopez-Moya, Federico; Kowbel, David; Nueda, Ma Jose; ...

2015-12-01

Chitosan is a natural polymer with antimicrobial activity. Chitosan causes plasma membrane permeabilization and induction of intracellular reactive oxygen species (ROS) in Neurospora crassa. In this paper, we have determined the transcriptional profile of N. crassa to chitosan and identified the main gene targets involved in the cellular response to this compound. Global network analyses showed membrane, transport and oxidoreductase activity as key nodes affected by chitosan. Activation of oxidative metabolism indicates the importance of ROS and cell energy together with plasma membrane homeostasis in N. crassa response to chitosan. Deletion strain analysis of chitosan susceptibility pointed NCU03639 encoding amore » class 3 lipase, involved in plasma membrane repair by lipid replacement, and NCU04537 a MFS monosaccharide transporter related to assimilation of simple sugars, as main gene targets of chitosan. NCU10521, a glutathione S-transferase-4 involved in the generation of reducing power for scavenging intracellular ROS is also a determinant chitosan gene target. Ca 2+ increased tolerance to chitosan in N. crassa. Growth of NCU10610 (fig 1 domain) and SYT1 (a synaptotagmin) deletion strains was significantly increased by Ca 2+ in the presence of chitosan. Both genes play a determinant role in N. crassa membrane homeostasis. Finally, our results are of paramount importance for developing chitosan as an antifungal.« less

Genome-Wide Analysis of Syntenic Gene Deletion in the Grasses

PubMed Central

Schnable, James C.; Freeling, Michael; Lyons, Eric

2012-01-01

The grasses, Poaceae, are one of the largest and most successful angiosperm families. Like many radiations of flowering plants, the divergence of the major grass lineages was preceded by a whole-genome duplication (WGD), although these events are not rare for flowering plants. By combining identification of syntenic gene blocks with measures of gene pair divergence and different frequencies of ancient gene loss, we have separated the two subgenomes present in modern grasses. Reciprocal loss of duplicated genes or genomic regions has been hypothesized to reproductively isolate populations and, thus, speciation. However, in contrast to previous studies in yeast and teleost fishes, we found very little evidence of reciprocal loss of homeologous genes between the grasses, suggesting that post-WGD gene loss may not be the cause of the grass radiation. The sets of homeologous and orthologous genes and predicted locations of deleted genes identified in this study, as well as links to the CoGe comparative genomics web platform for analyzing pan-grass syntenic regions, are provided along with this paper as a resource for the grass genetics community. PMID:22275519

Tackling the issue of environmental survival of live Salmonella Typhimurium vaccines: deletion of the lon gene.

PubMed

Leyman, Bregje; Boyen, Filip; Van Parys, Alexander; Verbrugghe, Elin; Haesebrouck, Freddy; Pasmans, Frank

2012-12-01

Vaccination is an important measure to control Salmonella contamination in the meat production chain. A previous study showed that both the ΔrfaJ and ΔrfaL strains are suitable markers and allow serological differentiation of infected and vaccinated animals. The aim of this study was to verify whether deletion of the lon gene in a Salmonella Typhimurium ΔrfaJ marker strain resulted in decreased environmental survival. Our results indicate that deletion of the lon gene in the ΔrfaJ strain did not affect invasiveness in IPEC-J2 cells and resulted in an increased susceptibility to UV, disinfectants (such as hydrogen peroxide and tosylchloramide sodium) and citric acid. Immunization of pigs with inactivated ΔrfaJ or ΔlonΔrfaJ vaccines allowed differentiation of infected and vaccinated pigs. Furthermore, deletion of the lon gene did not reduce the protection conferred by live wild type or ΔrfaJ vaccines against subsequent challenge with a virulent Salmonella Typhimurium strain in BALB/c mice. Based on our results in mice, we conclude that deletion of lon in ΔrfaJ contributes to environmental safety of the ΔrfaJ DIVA strain. Copyright © 2012 Elsevier Ltd. All rights reserved.

TBR1 is the candidate gene for intellectual disability in patients with a 2q24.2 interstitial deletion.

PubMed

Palumbo, Orazio; Fichera, Marco; Palumbo, Pietro; Rizzo, Renata; Mazzolla, Elisabetta; Cocuzza, Donatella Maria; Carella, Massimo; Mattina, Teresa

2014-03-01

Interstitial deletion of 2q24.2 is a rarely described cytogenetic aberration in patients with intellectual disability (ID). Previously reported genotype-phenotype correlation identified a minimum deleted region of 2.65 Mb including 15 genes. Recently, a patient with a de novo 2q24.2 microdeletion of 0.4 Mb encompassing only three genes was described. However, the precise relationship between most deleted genes and the clinical features remains unclear. Here we describe a 12-year-old male patient diagnosed with growth retardation and ID. He also showed microcephaly, right palpebral ptosis, scapular winging, and pectus excavatum. Single nucleotide polymorphisms (SNP) array analysis showed a de novo interstitial deletion of 0.122 Mb at 2q24.2 region harboring only TBR1 (T-box, brain, 1; OMIM: 604616), which encodes a T-box family transcription factor expressed in post-mitotic projection neurons and functionally significant in embryologic corticogenesis. This is the first case of a deletion at 2q24.2 involving only TBR1. This finding narrows the smallest region of overlap (SRO) for deletions in this region and strengthens the previously suggested hypothesis that this gene is a strong candidate for the ID phenotype. The identification of TBR1 as candidate for ID encourages further molecular studies to identify novel mutations to understand the pathogenic effects of its haploinsufficiency. Finally, this report provides a review on 10 2q24.2 microdeletion patients. © 2014 Wiley Periodicals, Inc.

Nonalcoholic fatty liver in patients with Laron syndrome and GH gene deletion - preliminary report.

PubMed

Laron, Zvi; Ginsberg, Shira; Webb, Muriel

2008-10-01

There is little information on the relationship between growth hormone/insulin-like growth factor-I (GH/IGF-I) deficiency or IGF-I treatment on nonalcoholic fatty liver disease (NAFLD) a disorder linked to obesity and insulin resistance. To find out whether the markedly obese patients with Laron syndrome (LS) and GH gene deletion have fatty livers. We studied 11 untreated adult patients with LS (5M, 6F), five girls with LS treated by IGF-I and five adult patients with GH gene deletion (3M, 3F), four previously treated by hGH in childhood. Fatty liver was quantitatively evaluated by ultrasonography using a phase array US system (HITACHI 6500, Japan). Body adiposity was determined by DEXA, and insulin resistance was estimated by HOMA-IR using the fasting serum glucose and insulin values. Six out of 11 adult patients with LS, two out of the five IGF-I treated girls with LS and three out of five adult hGH gene deletion patients were found to have NAFLD (nonalcoholic fatty liver disease). NAFLD is a frequent complication in untreated and treated congenital IGF-I deficiency. No correlation between NAFLD and age, sex, degree of obesity, blood lipids, or degree of insulin resistance was observed.

Identification of a basic helix-loop-helix-type transcription regulator gene in Aspergillus oryzae by systematically deleting large chromosomal segments.

PubMed

Jin, Feng Jie; Takahashi, Tadashi; Machida, Masayuki; Koyama, Yasuji

2009-09-01

We previously developed two methods (loop-out and replacement-type recombination) for generating large-scale chromosomal deletions that can be applied to more effective chromosomal engineering in Aspergillus oryzae. In this study, the replacement-type method is used to systematically delete large chromosomal DNA segments to identify essential and nonessential regions in chromosome 7 (2.93 Mb), which is the smallest A. oryzae chromosome and contains a large number of nonsyntenic blocks. We constructed 12 mutants harboring deletions that spanned 16- to 150-kb segments of chromosome 7 and scored phenotypic changes in the resulting mutants. Among the deletion mutants, strains designated Delta5 and Delta7 displayed clear phenotypic changes involving growth and conidiation. In particular, the Delta5 mutant exhibited vigorous growth and conidiation, potentially beneficial characteristics for certain industrial applications. Further deletion analysis allowed identification of the AO090011000215 gene as the gene responsible for the Delta5 mutant phenotype. The AO090011000215 gene was predicted to encode a helix-loop-helix binding protein belonging to the bHLH family of transcription factors. These results illustrate the potential of the approach for identifying novel functional genes.

Smaller and larger deletions of the Williams Beuren syndrome region implicate genes involved in mild facial phenotype, epilepsy and autistic traits.

PubMed

Fusco, Carmela; Micale, Lucia; Augello, Bartolomeo; Teresa Pellico, Maria; Menghini, Deny; Alfieri, Paolo; Cristina Digilio, Maria; Mandriani, Barbara; Carella, Massimo; Palumbo, Orazio; Vicari, Stefano; Merla, Giuseppe

2014-01-01

Williams Beuren syndrome (WBS) is a multisystemic disorder caused by a hemizygous deletion of 1.5 Mb on chromosome 7q11.23 spanning 28 genes. A few patients with larger and smaller WBS deletion have been reported. They show clinical features that vary between isolated SVAS to the full spectrum of WBS phenotype, associated with epilepsy or autism spectrum behavior. Here we describe four patients with atypical WBS 7q11.23 deletions. Two carry ~3.5 Mb larger deletion towards the telomere that includes Huntingtin-interacting protein 1 (HIP1) and tyrosine 3-monooxygenase/tryptophan 5-monooxigenase activation protein gamma (YWHAG) genes. Other two carry a shorter deletion of ~1.2 Mb at centromeric side that excludes the distal WBS genes BAZ1B and FZD9. Along with previously reported cases, genotype-phenotype correlation in the patients described here further suggests that haploinsufficiency of HIP1 and YWHAG might cause the severe neurological and neuropsychological deficits including epilepsy and autistic traits, and that the preservation of BAZ1B and FZD9 genes may be related to mild facial features and moderate neuropsychological deficits. This report highlights the importance to characterize additional patients with 7q11.23 atypical deletions comparing neuropsychological and clinical features between these individuals to shed light on the pathogenic role of genes within and flanking the WBS region.

A Recessive Contiguous Gene Deletion of Chromosome 2p16 Associated with Cystinuria and a Mitochondrial Disease

PubMed Central

Parvari, Ruti; Brodyansky, Irena; Elpeleg, Orly; Moses, Shimon; Landau, Daniel; Hershkovitz, Eli

2001-01-01

Deletions ranging from 100 Kb to 1 Mb—too small to be detected under the microscope—may still involve dozens of genes, thus causing microdeletion syndromes. The vast majority of these syndromes are caused by haploinsufficiency of one or several genes and are transmitted as dominant traits. We identified seven patients originating from an extended family and presenting with a unique syndrome, inherited in a recessive mode, consisting of cystinuria, neonatal seizures, hypotonia, severe somatic and developmental delay, facial dysmorphism, and lactic acidemia. Reduced activity of all the respiratory chain enzymatic complexes that are encoded in the mitochondria was found in muscle biopsy specimens of the patients examined. The molecular basis of this disorder is a homozygous deletion of 179,311 bp on chromosome 2p16, which includes the type I cystinuria gene (SLC3A1), the protein phosphatase 2Cβ gene (PP2Cβ), an unidentified gene (KIAA0436), and several expressed sequence tags. The extent of the deletion suggests that this unique syndrome is related to the complete absence of these genes’ products, one of which may be essential for the synthesis of mitochondrial encoded proteins. PMID:11524703

Deletion of 8.5 Mb, including the FMR1 gene, in a male with the fragile X syndrome phenotype and overgrowth.

PubMed

Parvari, R; Mumm, S; Galil, A; Manor, E; Bar-David, Y; Carmi, R

1999-04-02

A four-year-old boy with severe psychomotor retardation, facial appearance consistent with the fragile X syndrome, hypotonia, and overgrowth was found to have a deletion including the fragile X gene (FMR1). The breakpoints of the deletion were established between CDR1 and sWXD2905 (approximately 200 kb apart) at Xq27.1 (centromeric) and between DXS8318 (612-1078L) and DXS7847 (576-291L) (approximately 250 kb apart) at Xq28, about 500 kb telomeric to the FMR1 gene. The total length of the deletion is approximately 8.5 Mb. The propositus's mother, who was found to be a carrier of the deletion, showed very mild mental impairment. Except for mental retardation, which is a common finding in all cases reported with similar deletions of chromosome Xq, this patient had generalized overgrowth, exceeding the 97th centile for height and weight. Obesity and increased growth parameters have been reported in other patients with deletions either overlapping or within a distance of 0.5 Mb from the deletion in the present patient. Thus, it is suggested that a deletion of the 8-Mb fragment centromeric to the FMR1 gene might have an effect on growth.

Heterozygous deletion at the SOX10 gene locus in two patients from a Chinese family with Waardenburg syndrome type II.

PubMed

Wenzhi, He; Ruijin, Wen; Jieliang, Li; Xiaoyan, Ma; Haibo, Liu; Xiaoman, Wang; Jiajia, Xian; Shaoying, Li; Shuanglin, Li; Qing, Li

2015-10-01

Waardenburg syndrome (WS) is a rare disease characterized by sensorineural deafness and pigment disturbance. To date, almost 100 mutations have been reported, but few reports on cases with SOX10 gene deletion. The inheritance pattern of SOX10 gene deletion is still unclear. Our objective was to identify the genetic causes of Waardenburg syndrome type II in a two-generation Chinese family. Clinical evaluations were conducted in both of the patients. Microarray analysis and multiplex ligation-dependent probe amplification (MLPA) were performed to identify disease-related copy number variants (CNVs). DNA sequencing of the SOX10, MITF and SNAI2 genes was performed to identify the pathogenic mutation responsible for WS2. A 280kb heterozygous deletion at the 22q13.1 chromosome region (including SOX10) was detected in both of the patients. No mutation was found in the patients, unaffected family members and 30 unrelated healthy controls. This report is the first to describe SOX10 heterozygous deletions in Chinese WS2 patients. Our result conform the thesis that heterozygous deletions at SOX10 is an important pathogenicity for WS, and present as autosomal dominant inheritance. Nevertheless, heterozygous deletion of the SOX10 gene would be worth investigating to understand their functions and contributions to neurologic phenotypes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Single-gene deletions that restore mating competence to diploid yeast.

PubMed

Schmidlin, Tom; Kaeberlein, Matt; Kudlow, Brian A; MacKay, Vivian; Lockshon, Daniel; Kennedy, Brian K

2008-03-01

Using the Saccharomyces cerevisiae MATa/MATalpha ORF deletion collection, homozygous deletion strains were identified that undergo mating with MATa or MATalpha haploids. Seven homozygous deletions were identified that confer enhanced mating. Three of these, lacking CTF8, CTF18, and DCC1, mate at a low frequency with either MATa or MATalpha haploids. The products of these genes form a complex involved in sister chromatid cohesion. Each of these strains also exhibits increased chromosome loss rates, and mating likely occurs due to loss of one copy of chromosome III, which bears the MAT locus. Three other homozygous diploid deletion strains, ylr193cDelta/ylr193cDelta, yor305wDelta/yor305wDelta, and ypr170cDelta/ypr170cDelta, mate at very low frequencies with haploids of either or both mating types. However, an ist3Delta/ist3Delta strain mates only with MATa haploids. It is shown that IST3, previously linked to splicing, is required for efficient processing of the MATa1 message, particularly the first intron. As a result, the ist3Delta/ist3Delta strain expresses unbalanced ratios of Matalpha to Mata proteins and therefore mates with MATa haploids. Accordingly, mating in this diploid can be repressed by introduction of a MATa1 cDNA. In summary, this study underscores and elaborates upon predicted pathways by which mutations restore mating function to yeast diploids and identifies new mutants warranting further study.

Inflammatory peeling skin syndrome caused by homozygous genomic deletion in the PSORS1 region encompassing the CDSN gene.

PubMed

Ishida-Yamamoto, Akemi; Furio, Laetitia; Igawa, Satomi; Honma, Masaru; Tron, Elodie; Malan, Valerie; Murakami, Masamoto; Hovnanian, Alain

2014-01-01

Peeling skin syndrome (PSS) type B is a rare recessive genodermatosis characterized by lifelong widespread, reddish peeling of the skin with pruritus. The disease is caused by small-scale mutations in the Corneodesmosin gene (CDSN) leading to premature termination codons. We report for the first time a Japanese case resulting from complete deletion of CDSN. Corneodesmosin was undetectable in the epidermis, and CDSN was unamplifiable by PCR. QMPSF analysis demonstrated deletion of CDSN exons inherited from each parent. Deletion mapping using microsatellite haplotyping, CGH array and PCR analysis established that the genomic deletion spanned 49-72 kb between HCG22 and TCF19, removing CDSN as well as five other genes within the psoriasis susceptibility region 1 (PSORS1) on 6p21.33. This observation widens the spectrum of molecular defects underlying PSS type B and shows that loss of these five genes from the PSORS1 region does not result in an additional cutaneous phenotype. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Target genes discovery through copy number alteration analysis in human hepatocellular carcinoma.

PubMed

Gu, De-Leung; Chen, Yen-Hsieh; Shih, Jou-Ho; Lin, Chi-Hung; Jou, Yuh-Shan; Chen, Chian-Feng

2013-12-21

High-throughput short-read sequencing of exomes and whole cancer genomes in multiple human hepatocellular carcinoma (HCC) cohorts confirmed previously identified frequently mutated somatic genes, such as TP53, CTNNB1 and AXIN1, and identified several novel genes with moderate mutation frequencies, including ARID1A, ARID2, MLL, MLL2, MLL3, MLL4, IRF2, ATM, CDKN2A, FGF19, PIK3CA, RPS6KA3, JAK1, KEAP1, NFE2L2, C16orf62, LEPR, RAC2, and IL6ST. Functional classification of these mutated genes suggested that alterations in pathways participating in chromatin remodeling, Wnt/β-catenin signaling, JAK/STAT signaling, and oxidative stress play critical roles in HCC tumorigenesis. Nevertheless, because there are few druggable genes used in HCC therapy, the identification of new therapeutic targets through integrated genomic approaches remains an important task. Because a large amount of HCC genomic data genotyped by high density single nucleotide polymorphism arrays is deposited in the public domain, copy number alteration (CNA) analyses of these arrays is a cost-effective way to reveal target genes through profiling of recurrent and overlapping amplicons, homozygous deletions and potentially unbalanced chromosomal translocations accumulated during HCC progression. Moreover, integration of CNAs with other high-throughput genomic data, such as aberrantly coding transcriptomes and non-coding gene expression in human HCC tissues and rodent HCC models, provides lines of evidence that can be used to facilitate the identification of novel HCC target genes with the potential of improving the survival of HCC patients.

Neurofibromatosis-1 gene deletions and mutations in de novo adult acute myeloid leukemia.

PubMed

Boudry-Labis, Elise; Roche-Lestienne, Catherine; Nibourel, Olivier; Boissel, Nicolas; Terre, Christine; Perot, Christine; Eclache, Virginie; Gachard, Nathalie; Tigaud, Isabelle; Plessis, Ghislaine; Cuccuini, Wendy; Geffroy, Sandrine; Villenet, Céline; Figeac, Martin; Leprêtre, Frederic; Renneville, Aline; Cheok, Meyling; Soulier, Jean; Dombret, Hervé; Preudhomme, Claude

2013-04-01

Germline heterozygous alterations of the tumor-suppressor gene neurofibromatosis-1 (NF1) lead to neurofibromatosis type 1, a genetic disorder characterized by a higher risk to develop juvenile myelomonocytic leukemia and/or acute myeloid leukemia (AML). More recently, somatic 17q11 deletions encompassing NF1 have been described in many adult myeloid malignancies. In this context, we aimed to define NF1 involvement in AML. We screened a total of 488 previously untreated de novo AML patients for the NF1 deletion using either array comparative genomic hybridization (aCGH) or real-time quantitative PCR/fluorescence in situ hybridization approaches. We also applied massively parallel sequencing for in depth mutation analysis of NF1 in 20 patients including five NF1-deleted patients. We defined a small ∼0.3 Mb minimal deleted region involving NF1 by aCGH and an overall frequency of NF1 deletion of 3.5% (17/485). NF1 deletion is significantly associated with unfavorable cytogenetics and with monosomal karyotype notably. We discovered six NF1 variants of unknown significance in 7/20 patients of which only one out of four disappeared in corresponding complete remission sample. In addition, only one out of five NF1-deleted patients has an acquired coding mutation in the remaining allele. In conclusion, direct NF1 inactivation is infrequent in de novo AML and may be a secondary event probably involved in leukemic progression. Copyright © 2013 Wiley Periodicals, Inc.

A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality

PubMed Central

van der Geize, R.; de Jong, W.; Hessels, G. I.; Grommen, A. W. F.; Jacobs, A. A. C.; Dijkhuizen, L.

2008-01-01

A novel method to efficiently generate unmarked in-frame gene deletions in Rhodococcus equi was developed, exploiting the cytotoxic effect of 5-fluorocytosine (5-FC) by the action of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) enzymes. The opportunistic, intracellular pathogen R. equi is resistant to high concentrations of 5-FC. Introduction of Escherichia coli genes encoding CD and UPRT conferred conditional lethality to R. equi cells incubated with 5-FC. To exemplify the use of the codA::upp cassette as counter-selectable marker, an unmarked in-frame gene deletion mutant of R. equi was constructed. The supA and supB genes, part of a putative cholesterol catabolic gene cluster, were efficiently deleted from the R. equi wild-type genome. Phenotypic analysis of the generated ΔsupAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol. Macrophage survival assays revealed that the ΔsupAB mutant is able to survive and proliferate in macrophages comparable to wild type. Thus, cholesterol metabolism does not appear to be essential for macrophage survival of R. equi. The CD-UPRT based 5-FC counter-selection may become a useful asset in the generation of unmarked in-frame gene deletions in other actinobacteria as well, as actinobacteria generally appear to be 5-FC resistant and 5-FU sensitive. PMID:18984616

[Clinical features of patients with Becker muscular dystrophy and deletions of the rod domain of dystrophin gene].

PubMed

Wang, Yanyun; Zhu, Yuling; Yang, Juan; Li, Yaqin; Sun, Jiangwen; Zhan, Yixin; Zhang, Cheng

2018-02-10

OBJECTIVE To explore the clinical features of patients carrying deletions of the rod domain of the dystrophin gene. METHODS Clinical data of 12 Chinese patients with Becker muscular dystrophy (BMD) and such deletions was reviewed. RESULTS Most patients complained of muscle weakness of lower limbs. Two patients had muscle cramps, one had increased creatine kinase (CK) level, and one had dilated cardiomyopathy. CONCLUSION Compared with DMD, the clinical features of BMD are much more variable, particularly for those carrying deletions of the rod domain of the dystrophin gene. Muscular weakness may not be the sole complaint of BMD. The diagnosis of BMD cannot be excluded by moderately elevated CK. For male patients with dilated cardiomyopathy, the possibility of BMD should be considered.

Chronic lymphocytic leukemia cells with allelic deletions at 13q14 commonly have one intact RB1 gene: Evidence for a role of an adjacent locus

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

Leu, Y.; Grander, D.; Linder, S.

The authors have previously shown that 30% of patients with B-cell chronic lymphocytic leukemia (B-CLL) have hemizygous deletions of the retinoblastoma (RB1) gene at 13q14. RB1 gene deletions may thus participate in malignant transformation of B-CLL, but is it also possible that a neighboring gene on 13q is the relevant one. To answer this question the remaining RB1 allele of eight clones with hemizygous deletions was studied by reverse transcription-polymerase chain reaction (RT-PCR), single-strand conformation polymorphism (SSCP) analysis, and immunofluorescense techniques. Cells from 10 patients without RB1 gene deletions were also studied by these methods. Lack of RB1 mRNA andmore » RB protein expression was seen in leukemia cells from one of the patients. All other cases were found to be normal with regard to immunofluorescense, RT-PCR, and SSCP analysis, indicating at least one functional RB1 allele and supporting the importance of another gene in the 13q14 deletions. The authors then performed extended Southern blot analysis of the 13q region, using probes for 10 different loci. In 14 of 31 CLL clones (45%), deletions of a region telomeric to the RB1 gene (D13S25) were observed. In 4 of the cases the deletions were homozygous. Hemizygous deletions of the RB1 gene were observed in 11 of these patients and in one of the patients without D13S25 deletions. These data thus indicate that a gene(s) telomeric to RB1 is involved in the malignant transformation of CLL clones and that deletions of this region are a common event in this disease. 20 refs., 3 figs., 3 tabs.« less

Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

PubMed

Chen, Ding-Ping; Tseng, Ching-Ping; Lin, Chi-Jui; Wang, Wei-Ting; Sun, Chien-Feng

2015-01-01

In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene. © 2015 by the Association of Clinical Scientists, Inc.

Human homologue sequences to the Drosophila dishevelled segment-polarity gene are deleted in the DiGeorge syndrome

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

Pizzuti, A.; Ratti, A.; Penso, D.

DiGeorge syndrome (DGS) is a developmental defect of some of the neural crest derivatives. Most DGS patients show haploinsufficiency due to interstitial deletions of the proximal long arm of chromosome 22. Deletions of 22q11 have also been reported in patients with the velo-cardio-facial syndrome and familial conotruncal heart defects. It has been suggested that the wide phenotype spectrum associated with 22q11 monosomy is a consequence of contiguous-gene deletions. We report the isolation of human cDNAs homologous to the Drosophila dishevelled (dsh) segment-polarity gene. Sequences homologous to the 3{prime} UTR of these transcripts (DVL-22) were positioned within the DGS critical regionmore » and were found to be deleted in DGS patients. Human DVL mRNAs are expressed in several fetal and adult tissues, including the thymus and, at high levels, the heart. Two transcripts, 3.2 and 5 kb, were detected, in Northern blot analysis, with different expression patterns in the surveyed tissues when different cDNAs were used. The isolated cDNAs exhibit high amino acid homology with the mouse and Xenopus Dvl-1 gene, the only other vertebrate dsh homologues so far isolated. The pivotal role of dsh in fly development suggests an analogous key function in vertebrate embryogenesis of its homologue genes. Since DGS may be due to perturbation of differentiation mechanisms at decisive embryological stages, a Dsh-like gene in the small-region overlap (SRO) might be a candidate for the pathogenesis of this disorder. 52 refs., 3 figs.« less

Radiation-induced total-deletion mutations in the human hprt gene: a biophysical model based on random walk interphase chromatin geometry

NASA Technical Reports Server (NTRS)

Wu, H.; Sachs, R. K.; Yang, T. C.

1998-01-01

PURPOSE: To develop a biophysical model that explains the sizes of radiation-induced hprt deletions. METHODS: Key assumptions: (1) Deletions are produced by two DSB that are closer than an interaction distance at the time of DSB induction; (2) Interphase chromatin is modelled by a biphasic random walk distribution; and (3) Misrejoining of DSB from two separate tracks dominates at low-LET and misrejoining of DSB from a single track dominates at high-LET. RESULTS: The size spectra for radiation-induced total deletions of the hprt gene are calculated. Comparing with the results of Yamada and coworkers for gamma-irradiated human fibroblasts the study finds that an interaction distance of 0.75 microm will fit both the absolute frequency and the size spectrum of the total deletions. It is also shown that high-LET radiations produce, relatively, more total deletions of sizes below 0.5 Mb. The model predicts an essential gene to be located between 2 and 3 Mb from the hprt locus towards the centromere. Using the same assumptions and parameters as for evaluating mutation frequencies, a frequency of intra-arm chromosome deletions is calculated that is in agreement with experimental data. CONCLUSIONS: Radiation-induced total-deletion mutations of the human hprt gene and intrachange chromosome aberrations share a common mechanism for their induction.

Limb Girdle Muscular Dystrophy Type 2E Due to a Novel Large Deletion in SGCB Gene.

PubMed

Ghafouri-Fard, Soudeh; Hashemi-Gorji, Feyzollah; Fardaei, Majid; Miryounesi, Mohammad

2017-01-01

Autosomal recessive limb-girdle muscular dystrophies (LGMD type 2) are a group of clinically and genetically heterogeneous diseases with the main characteristics of weakness and wasting of the pelvic and shoulder girdle muscles. Among them are sarcoglycanopathies caused by mutations in at least four genes named SGCA, SGCB, SGCG and SGCD. Here we report a consanguineous Iranian family with two children affected with LGMD type 2E. Mutation analysis revealed a novel homozygous exon 2 deletion of SGCB gene in the patients with the parents being heterozygous for this deletion. This result presents a novel underlying genetic mechanism for LGMD type 2E.

Identification of a novel deletion in SURF1 gene: Heterogeneity in Leigh syndrome with COX deficiency.

PubMed

Ribeiro, Carolina; do Carmo Macário, Maria; Viegas, Ana Teresa; Pratas, João; Santos, Maria João; Simões, Marta; Mendes, Cândida; Bacalhau, Mafalda; Garcia, Paula; Diogo, Luísa; Grazina, Manuela

2016-11-01

Leigh syndrome (LS) is a rare, progressive neurodegenerative mitochondrial disorder of infancy. It is a genetically heterogeneous disease. The mutations in SURF1 gene are the most frequently known cause. Here two cases of LS likely caused by SURF1 gene variants are reported: a 39-year-old male patient with a novel homozygous deletion (c.-11_13del), and a case of a 6-year-old boy with the same deletion and a nonsense mutation (c.868dupT), both in heterozygosity. Blue native PAGE showed absence of assembled complex IV. This is the first report of a variant that may abolish the SURF1 gene initiation codon in two LS patients. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

A new gene deletion in the alpha-like globin gene cluster as the molecular basis for the rare alpha-thalassemia-1(--/alpha alpha) in blacks: HbH disease in sickle cell trait.

PubMed

Steinberg, M H; Coleman, M B; Adams, J G; Hartmann, R C; Saba, H; Anagnou, N P

1986-02-01

A novel deletion of at least 26 kilobase of DNA, including both alpha-globin genes, the psi alpha- and psi zeta-globin genes, but sparing the functional zeta-gene was found in a 10-year-old black boy with HbH disease and sickle cell trait. This particular deletion has not previously been described in blacks. Its existence makes it likely that the absence of Hb Barts hydrops fetalis in blacks is due to the rarity of the chromosome lacking two alpha-globin genes rather than a result of early embryonic death due to the failure to synthesize embryonic hemoglobins because of deletion of functional zeta-globin genes.

Characterization of contiguous gene deletions in COL4A6 and COL4A5 in Alport syndrome-diffuse leiomyomatosis.

PubMed

Nozu, Kandai; Minamikawa, Shogo; Yamada, Shiro; Oka, Masafumi; Yanagita, Motoko; Morisada, Naoya; Fujinaga, Shuichiro; Nagano, China; Gotoh, Yoshimitsu; Takahashi, Eihiko; Morishita, Takahiro; Yamamura, Tomohiko; Ninchoji, Takeshi; Kaito, Hiroshi; Morioka, Ichiro; Nakanishi, Koichi; Vorechovsky, Igor; Iijima, Kazumoto

2017-07-01

Alport syndrome-diffuse leiomyomatosis (AS-DL, OMIM: 308940) is a rare variant of the X-linked Alport syndrome that shows overgrowth of visceral smooth muscles in the gastrointestinal, respiratory and female reproductive tracts in addition to renal symptoms. AS-DL results from deletions that encompass the 5' ends of the COL4A5 and COL4A6 genes, but deletion breakpoints between COL4A5 and COL4A6 have been determined in only four cases. Here, we characterize deletion breakpoints in five AS-DL patients and show a contiguous COL4A6/COL4A5 deletion in each case. We also demonstrate that eight out of nine deletion alleles involved sequences homologous between COL4A5 and COL4A6. Most breakpoints took place in recognizable transposed elements, including long and short interspersed repeats, DNA transposons and long-terminal repeat retrotransposons. Because deletions involved the bidirectional promoter region in each case, we suggest that the occurrence of leiomyomatosis in AS-DL requires inactivation of both genes. Altogether, our study highlights the importance of homologous recombination involving multiple transposed elements for the development of this continuous gene syndrome and other atypical loss-of-function phenotypes.

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease.

PubMed

Wolfs, Jason M; Hamilton, Thomas A; Lant, Jeremy T; Laforet, Marcon; Zhang, Jenny; Salemi, Louisa M; Gloor, Gregory B; Schild-Poulter, Caroline; Edgell, David R

2016-12-27

The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

Lack of association between deletion polymorphism of BIM gene and in vitro drug sensitivity in B-cell precursor acute lymphoblastic leukemia.

PubMed

Huang, Meixian; Miyake, Kunio; Kagami, Keiko; Abe, Masako; Shinohara, Tamao; Watanabe, Atsushi; Somazu, Shinpei; Oshiro, Hiroko; Goi, Kumiko; Goto, Hiroaki; Minegishi, Masayoshi; Iwamoto, Shotaro; Kiyokawa, Nobutaka; Sugita, Kanji; Inukai, Takeshi

2017-09-01

A deletion polymorphism in the BIM gene was identified as an intrinsic mechanism for resistance to tyrosine kinase inhibitor in chronic myeloid leukemia patients in East Asia. BIM is also involved in the responses to glucocorticoid and chemotherapy in acute lymphoblastic leukemia (ALL), suggesting a possible association between deletion polymorphism of BIM and the chemosensitivity of ALL. Thus, we analyzed 72 B-cell precursor (BCP)-ALL cell lines established from Japanese patients. Indeed, higher BIM gene expression was associated with good in vitro sensitivities to glucocorticoid and chemotherapeutic agents used in induction therapy. We also analyzed the methylation status of the BIM gene promoter by next generation sequencing of genome bisulfite PCR products, since genetic polymorphism could be insignificant when epigenetically inactivated. Hypermethylation of the BIM gene promoter was associated with lower BIM gene expression and poorer sensitivity to vincristine. Of note, however, the prevalence of a deletion polymorphism was not associated with the BIM gene expression level or drug sensitivities in BCP-ALL cell lines, in which the BIM gene was unmethylated. These observations suggest that an association of a deletion polymorphism of BIM and the response to induction therapy in BCP-ALL may be clinically minimal. Copyright © 2017. Published by Elsevier Ltd.

HisB as novel selection marker for gene targeting approaches in Aspergillus niger.

PubMed

Fiedler, Markus R M; Gensheimer, Tarek; Kubisch, Christin; Meyer, Vera

2017-03-08

For Aspergillus niger, a broad set of auxotrophic and dominant resistance markers is available. However, only few offer targeted modification of a gene of interest into or at a genomic locus of choice, which hampers functional genomics studies. We thus aimed to extend the available set by generating a histidine auxotrophic strain with a characterized hisB locus for targeted gene integration and deletion in A. niger. A histidine-auxotrophic strain was established via disruption of the A. niger hisB gene by using the counterselectable pyrG marker. After curing, a hisB - , pyrG - strain was obtained, which served as recipient strain for further studies. We show here that both hisB orthologs from A. nidulans and A. niger can be used to reestablish histidine prototrophy in this recipient strain. Whereas the hisB gene from A. nidulans was suitable for efficient gene targeting at different loci in A. niger, the hisB gene from A. niger allowed efficient integration of a Tet-on driven luciferase reporter construct at the endogenous non-functional hisB locus. Subsequent analysis of the luciferase activity revealed that the hisB locus is tight under non-inducing conditions and allows even higher luciferase expression levels compared to the pyrG integration locus. Taken together, we provide here an alternative selection marker for A. niger, hisB, which allows efficient homologous integration rates as well as high expression levels which compare favorably to the well-established pyrG selection marker.

A VNTR element associated with steroid sulfatase gene deletions stimulates recombination in cultured cells

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

Gong, Y.; Li, X.M.; Shapiro, L.J.

1994-09-01

Steroid sulfatase deficiency is a common genetic disorder, with a prevalence of approximately one in every 3500 males world wide. About 90% of these patients have complete gene deletions, which appear to result from recombination between members of a low-copy repeat family (CRI-232 is the prototype) that flank the gene. RU1 and RU2 are two VNTR elements found within each of these family members. RU1 consists of 30 bp repeating units and its length shows minimal variation among individuals. The RU2 element consists of repeating sequences which are highly asymmetric, with about 90% purines and no C`s on one strand,more » and range from 0.6 kb to over 23 kb among different individuals. We conducted a study to determine if the RU1 or RU2 elements can promote recombination in an in vivo test system. We inserted these elements adjacent to the neo gene in each of two pSV2neo derivatives, one of which has a deletion in the 5{prime} portion of the neo gene and the other having a deletion in the 3{prime} portion. These plasmids were combined and used to transfect EJ cells. Survival of cells in G418 indicates restoration of a functional neo gene by recombination between two deletion constructs. Thus counting G418 resistant colonies gives a quantitative measure of the enhancement of recombination by the inserted VNTR elements. The results showed no effect on recombination by the inserted RU1 element (compared to the insertion of a nonspecific sequence), while the RU2 element stimulated recombination by 3.5-fold (P<0.01). A separate set of constructs placed RU1 or RU2 within the intron of an exon trapping vector. Following tranfection of cells, recombination events were monitored by a PCR assay that detected the approximation of primer binding sites (as a result of recombination). These studies showed that, as in the first set of experiments, the highly variable RU2 element is capable of stimulating somatic recombination in mammalian cells.« less

A plasmid collection for PCR-based gene targeting in the filamentous ascomycete Ashbya gossypii.

PubMed

Kaufmann, Andreas

2009-08-01

PCR-based gene targeting with heterologous markers is an efficient method to delete genes, generate gene fusions, and modulate gene expression. For the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, several plasmid collections are available covering a wide range of tags and markers. For several reasons, many of these cassettes cannot be used in the filamentous ascomycete Ashbya gossypii. This article describes the construction of 93 heterologous modules for C- and N-terminal tagging and promoter replacements in A. gossypii. The performance of 12 different fluorescent tags was evaluated by monitoring their brightness, detectability, and photostability when fused to the myosin light-chain protein Mlc2. Furthermore, the thiamine-repressible S. cerevisiae THI13 promoter was established to regulate gene expression in A. gossypii. This collection will help accelerate analysis of gene function in A. gossypii and in other ascomycetes where S. cerevisiae promoter elements are functional.

Let-7b regulates the expression of the growth hormone receptor gene in deletion-type dwarf chickens.

PubMed

Lin, Shumao; Li, Hongmei; Mu, Heping; Luo, Wen; Li, Ying; Jia, Xinzheng; Wang, Sibing; Jia, Xiaolu; Nie, Qinghua; Li, Yugu; Zhang, Xiquan

2012-07-10

A deletion mutation in the growth hormone receptor (GHR) gene results in the inhibition of skeletal muscle growth and fat deposition in dwarf chickens. We used microarray techniques to determine microRNA (miRNA) and mRNA expression profiles of GHR in the skeletal muscles of 14-day-old embryos as well as 7-week-old deletion-type dwarf and normal-type chickens. Our aim was to elucidate the miRNA regulation of GHR expression with respect to growth inhibition and fat deposition. At the same developmental stages, different expression profiles in skeletal muscles of dwarf and normal chickens occurred for four miRNAs (miR-1623, miR-181b, let-7b, and miR-128). At different developmental stages, there was a significant difference in the expression profiles of a greater number of miRNAs. Eleven miRNAs were up-regulated and 18 down-regulated in the 7-week-old dwarf chickens when compared with profiles in 14-day-old embryos. In 7-week-old normal chickens, seven miRNAs were up-regulated and nine down-regulated compared with those in 14-day-old embryos. In skeletal muscles, 22 genes were up-regulated and 33 down-regulated in 14-day-old embryos compared with 7-week-old dwarf chickens. Sixty-five mRNAs were up-regulated and 108 down-regulated in 14-day-old embryos as compared with 7-week-old normal chickens. Thirty-four differentially expressed miRNAs were grouped into 18 categories based on overlapping seed and target sequences. Only let-7b was found to be complementary to its target in the 3' untranslated region of GHR, and was able to inhibit its expression. Kyoto Encyclopedia of Genes and Genomes pathway analysis and quantitative polymerase chain reactions indicated there were three main signaling pathways regulating skeletal muscle growth and fat deposition of chickens. These were influenced by let-7b-regulated GHR. Suppression of the cytokine signaling 3 (SOCS3) gene was found to be involved in the signaling pathway of adipocytokines. There is a critical miRNA, let-7b

Schizophrenia and chromosomal deletions

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

Lindsay, E.A.; Baldini, A.; Morris, M. A.

Recent genetic linkage analysis studies have suggested the presence of a schizophrenia locus on the chromosomal region 22q11-q13. Schizophrenia has also been frequently observed in patients affected with velo-cardio-facial syndrome (VCFS), a disorder frequently associated with deletions within 22q11.1. It has been hypothesized that psychosis in VCFS may be due to deletion of the catechol-o-methyl transferase gene. Prompted by these observations, we screened for 22q11 deletions in a population of 100 schizophrenics selected from the Maryland Epidemiological Sample. Our results show that there are schizophrenic patients carrying a deletion of 22q11.1 and a mild VCFS phenotype that might remain unrecognized.more » These findings should encourage a search for a schizophrenia-susceptibility gene within the deleted region and alert those in clinical practice to the possible presence of a mild VCFS phenotype associated with schizophrenia. 9 refs.« less

fabH deletion increases DHA production in Escherichia coli expressing Pfa genes.

PubMed

Giner-Robles, Laura; Lázaro, Beatriz; de la Cruz, Fernando; Moncalián, Gabriel

2018-06-08

Some marine bacteria, such as Moritella marina, produce the nutraceutical docosahexaenoic acid (DHA) thanks to a specific enzymatic complex called Pfa synthase. Escherichia coli heterologously expressing the pfa gene cluster from M. marina also produces DHA. The aim of this study was to find genetic or metabolic conditions to increase DHA production in E. coli. First, we analysed the effect of the antibiotic cerulenin, showing that DHA production increased twofold. Then, we tested a series of single gene knockout mutations affecting fatty acid biosynthesis, in order to optimize the synthesis of DHA. The most effective mutant, fabH, showed a threefold increase compared to wild type strain. The combination of cerulenin inhibition and fabH deletion rendered a 6.5-fold improvement compared to control strain. Both strategies seem to have the same mechanism of action, in which fatty acid synthesis via the canonical pathway (fab pathway) is affected in its first catalytic step, which allows the substrates to be used by the heterologous pathway to synthesize DHA. DHA-producing E. coli strain that carries a fabH gene deletion boosts DHA production by tuning down the competing canonical biosynthesis pathway. Our approach can be used for optimization of DHA production in different organisms.

Large deletions encompassing the TCOF1 and CAMK2A genes are responsible for Treacher Collins syndrome with intellectual disability

PubMed Central

Vincent, Marie; Collet, Corinne; Verloes, Alain; Lambert, Laetitia; Herlin, Christian; Blanchet, Catherine; Sanchez, Elodie; Drunat, Séverine; Vigneron, Jacqueline; Laplanche, Jean-Louis; Puechberty, Jacques; Sarda, Pierre; Geneviève, David

2014-01-01

Mandibulofacial dysostosis is part of a clinically and genetically heterogeneous group of disorders of craniofacial development, which lead to malar and mandibular hypoplasia. Treacher Collins syndrome is the major cause of mandibulofacial dysostosis and is due to mutations in the TCOF1 gene. Usually patients with Treacher Collins syndrome do not present with intellectual disability. Recently, the EFTUD2 gene was identified in patients with mandibulofacial dysostosis associated with microcephaly, intellectual disability and esophageal atresia. We report on two patients presenting with mandibulofacial dysostosis characteristic of Treacher Collins syndrome, but associated with unexpected intellectual disability, due to a large deletion encompassing several genes including the TCOF1 gene. We discuss the involvement of the other deleted genes such as CAMK2A or SLC6A7 in the cognitive development delay of the patients reported, and we propose the systematic investigation for 5q32 deletion when intellectual disability is associated with Treacher Collins syndrome. PMID:23695276

Large deletions encompassing the TCOF1 and CAMK2A genes are responsible for Treacher Collins syndrome with intellectual disability.

PubMed

Vincent, Marie; Collet, Corinne; Verloes, Alain; Lambert, Laetitia; Herlin, Christian; Blanchet, Catherine; Sanchez, Elodie; Drunat, Séverine; Vigneron, Jacqueline; Laplanche, Jean-Louis; Puechberty, Jacques; Sarda, Pierre; Geneviève, David

2014-01-01

Mandibulofacial dysostosis is part of a clinically and genetically heterogeneous group of disorders of craniofacial development, which lead to malar and mandibular hypoplasia. Treacher Collins syndrome is the major cause of mandibulofacial dysostosis and is due to mutations in the TCOF1 gene. Usually patients with Treacher Collins syndrome do not present with intellectual disability. Recently, the EFTUD2 gene was identified in patients with mandibulofacial dysostosis associated with microcephaly, intellectual disability and esophageal atresia. We report on two patients presenting with mandibulofacial dysostosis characteristic of Treacher Collins syndrome, but associated with unexpected intellectual disability, due to a large deletion encompassing several genes including the TCOF1 gene. We discuss the involvement of the other deleted genes such as CAMK2A or SLC6A7 in the cognitive development delay of the patients reported, and we propose the systematic investigation for 5q32 deletion when intellectual disability is associated with Treacher Collins syndrome.

A novel heterozygous germline deletion in MSH2 gene in a five generation Chinese family with Lynch syndrome

PubMed Central

Liang, Shengran; Ling, Chao; You, Yan; Xu, Lai; Zhong, Min-Er; Xiao, Yi; Qiu, Hui-Zhong; Lu, Jun-Yang; Banerjee, Santasree

2017-01-01

Lynch syndrome (LS) is one of the most common familial forms of colorectal cancer predisposing syndrome with an autosomal dominant mode of inheritance. LS is caused by the germline mutations in DNA mismatch repair (MMR) genes including MSH2, MLH1, MSH6 and PMS2. Clinically, LS is characterized by high incidence of early-onset colorectal cancer as well as endometrial, small intestinal and urinary tract cancers, usually occur in the third to fourth decade of the life. Here we describe a five generation Chinese family with LS clinically diagnosed according to the Amsterdam II criteria. Immuno-histochemical staining of MSH2 and MSH6 shows only foci nuclear positive on the surface of the tumor with strong expression of MLH1 and PMS2 with diffuse immunoreactivity. In order to dig into the molecular basis of this LS pedigree, we collected the proband's blood sample, extracted the genomic DNA and applied the genetic screening. As a result, we identified a novel heterozygous deletion in MSH2 gene by targeted next generation sequencing, which is also proved to be co-segregated among other affected family members by following validation. To our knowledge, this novel heterozygous deletion (c.1676_1679 delTAAA) in MSH2 gene causes frameshift mutation (p.Asn560Lysfs*29) and leads to the formation of a truncated MSH2 protein which is confirmed to be a deleterious mutation according to the variant interpretation guidelines of American College of Medical Genetics and Genomics (ACMG). Identification of novel DNA mismatch repair (MMR) gene mutations can definitely benefit to the clinical diagnosis and management. PMID:28903413

The gene for replication factor C subunit 2 (RFC2) is within the 7q11.23 Williams syndrome deletion

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

Peoples, R.; Perez-Jurado, L.; Francke, U.

1996-06-01

Williams syndrome (WS) is a developmental disorder with multiple system manifestations, including supraval var aortic stenosis (SVAS), peripheral pulmonic stenosis, connective tissue abnormalities, short stature, characteristic personality profile and cognitive deficits, and variable hypercalcemia in infancy. It is caused by heterozygosity for a chromosomal deletion of part of band 7q11.23 including the elastin locus (ELN). Since disruption of the ELN gene causes autosomal dominant SVAS, it is assumed that ELN haploinsufficiency is responsible for the cardiovascular features of WS. The deletion that extends from the ELN locus in both directions is {ge}200 kb in size, although estimates of {ge}2 Mbmore » are suggested by high-resolution chromosome banding and physical mapping studies. We have searched for additional dosage-sensitive genes within the deletion that may be responsible for the noncardiovascular features. We report here that the gene for replication factor C subunit 2 (RFC2) maps within the WS deletion region and was found to be deleted in all of 18 WS patients studied. The protein product of RFC2 is part of a multimeric complex involved in DNA elongation during replication. 14 refs., 3 figs.« less

Molecular basis and consequences of a deletion in the amelogenin gene, analyzed by capture PCR

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

Lagerstroem-Fermer, M.; Pettersson, U.; Landegren, U.

1993-07-01

A mutation that disrupts the gene for one of the major proteins in tooth enamel has been investigated. The mutation is located in the amelogenin gene and causes X-linked amelogenesis imperfecta, characterized by defective mineralization of tooth enamel. The authors have isolated the breakpoints of a 5-kb deletion in the amelogenin gene on the basis of nucleotide sequence information located upstream of the lesion, using a technique termed capture PCR. The deletion removes five of the seven exons, spanning from the second intron to the last exon. Only the first two codons for the mature protein remain, consistent with themore » relatively severe phenotype of affected individuals in the present family. The mutation appears to have arisen as an illegitimate recombination event since of 11 nucleotide positions immediately surrounding the two breakpoints, 9 are identical. 17 refs., 3 figs., 1 tab.« less

[Markerless DNA deletion based on Red recombination and in vivo I-Sec I endonuclease cleavage in Escherichia coli chromosome].

PubMed

Zhu, Meiqin; Yu, Jian; Zhou, Changlin; Fang, Hongqing

2016-01-01

Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.

Genes commonly deleted in childhood B-cell precursor acute lymphoblastic leukemia: association with cytogenetics and clinical features

PubMed Central

Schwab, Claire J.; Chilton, Lucy; Morrison, Heather; Jones, Lisa; Al-Shehhi, Halima; Erhorn, Amy; Russell, Lisa J.; Moorman, Anthony V.; Harrison, Christine J.

2013-01-01

In childhood B-cell precursor acute lymphoblastic leukemia, cytogenetics is important in diagnosis and as an indicator of response to therapy, thus playing a key role in risk stratification of patients for treatment. Little is known of the relationship between different cytogenetic subtypes in B-cell precursor acute lymphoblastic leukemia and the recently reported copy number abnormalities affecting significant leukemia associated genes. In a consecutive series of 1427 childhood B-cell precursor acute lymphoblastic leukemia patients, we have determined the incidence and type of copy number abnormalities using multiplex ligation-dependent probe amplification. We have shown strong links between certain deletions and cytogenetic subtypes, including the novel association between RB1 deletions and intrachromosomal amplification of chromosome 21. In this study, we characterized the different copy number abnormalities and show heterogeneity of PAX5 and IKZF1 deletions and the recurrent nature of RB1 deletions. Whole gene losses are often indicative of larger deletions, visible by conventional cytogenetics. An increased number of copy number abnormalities is associated with NCI high risk, specifically deletions of IKZF1 and CDKN2A/B, which occur more frequently among these patients. IKZF1 deletions and rearrangements of CRLF2 among patients with undefined karyotypes may point to the poor risk BCR-ABL1-like group. In conclusion, this study has demonstrated in a large representative cohort of children with B-cell precursor acute lymphoblastic leukemia that the pattern of copy number abnormalities is highly variable according to the primary genetic abnormality. PMID:23508010

Unexpected effects of gene deletion on mercury interactions with the methylation-deficient mutant hgcAB

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

Lin, Hui; Hurt, Jr., Richard Ashley; Johs, Alexander

2014-01-01

The hgcA and hgcB gene pair is essential for mercury (Hg) methylation by certain anaerobic bacteria,1 but little is known about how deletion of hgcAB affects cell surface interactions and intracellular uptake of Hg. Here, we compare hgcAB mutants with the wild-type (WT) strains of both Geobacter sulfurreducens PCA and Desulfovibrio desulfuricans ND132 and observe differences in Hg redox transformations, adsorption, and uptake in laboratory incubation studies. In both strains, deletion of hgcAB increased the reduction of Hg(II) but decreased the oxidation of Hg(0) under anaerobic conditions. The measured cellular thiol content in hgcAB mutants was lower than the WT,more » accounting for decreased adsorption and uptake of Hg. Despite the lack of methylation activity, Hg uptake by the hgcAB continued, albeit at a slower rate than the WT. These findings demonstrate that deletion of the hgcAB gene not only eliminates Hg methylation but also alters cell physiology, resulting in changes to Hg redox reactions, sorption, and uptake by cells.« less

Homozygous deletion of six genes including corneodesmosin on chromosome 6p21.3 is associated with generalized peeling skin disease.

PubMed

Teye, Kwesi; Hamada, Takahiro; Krol, Rafal P; Numata, Sanae; Ishii, Norito; Matsuda, Mitsuhiro; Ohata, Chika; Furumura, Minao; Hashimoto, Takashi

2014-07-01

Peeling skin syndrome (PSS) is a rare autosomal recessive form of ichthyosis showing skin exfoliation. PSS is divided into acral and generalized PSS, and the latter is further classified into non-inflammatory type (PSS type A) and inflammatory type (PSS type B). PSS type B is now called peeling skin disease (PSD). Different loss-of-function mutations in the corneodesmosin (CDSN) gene have been reported to cause PSD. The aim of this study was to determine genetic basis of disease in a 14-year-old Japanese patient with PSD. Immunohistochemical study showed lack of corneodesmosin (CDSN) in the skin, and standard PCR for genomic DNA failed to amplify CDSN product, suggesting CDSN defect. Multiplex ligation-dependent probe amplification and genomic quantitative real-time PCR analyses detected large homozygous deletion of 59,184bp extending from 40.6kb upstream to 13.2kb downstream of CDSN, which included 6 genes (TCF19, CCHCR1, PSORS1C2, PSORS1C1, CDSN and C6orf15). The continuous gene lost did not result in additional clinical features. Inverted repeats with 85% similarity flanking the deletion breakpoint were considered to mediate the deletion by non-homologous end joining or fork stalling and template switching/microhomology-mediated break-induced replication. Parents were clinically unaffected and were heterozygote carriers of the same deletion, which was absent in 284 ethnically matched control alleles. We also developed simple PCR method, which is useful for detection of this deletion. Although 5 other genes were also deleted, homozygous deletion of CDSN was considered to be responsible for this PSD. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia.

PubMed

Rachid Viana, Giselle Maria; Akinyi Okoth, Sheila; Silva-Flannery, Luciana; Lima Barbosa, Danielle Regina; Macedo de Oliveira, Alexandre; Goldman, Ira F; Morton, Lindsay C; Huber, Curtis; Anez, Arletta; Dantas Machado, Ricardo Luiz; Aranha Camargo, Luís Marcelo; Costa Negreiros do Valle, Suiane; Marins Póvoa, Marinete; Udhayakumar, Venkatachalam; Barnwell, John W

2017-01-01

More than 80% of available malaria rapid diagnostic tests (RDTs) are based on the detection of histidine-rich protein-2 (PfHRP2) for diagnosis of Plasmodium falciparum malaria. Recent studies have shown the genes that code for this protein and its paralog, histidine-rich protein-3 (PfHRP3), are absent in parasites from the Peruvian Amazon Basin. Lack of PfHRP2 protein through deletion of the pfhrp2 gene leads to false-negative RDT results for P. falciparum. We have evaluated the extent of pfhrp2 and pfhrp3 gene deletions in a convenience sample of 198 isolates from six sites in three states across the Brazilian Amazon Basin (Acre, Rondonia and Para) and 25 isolates from two sites in Bolivia collected at different times between 2010 and 2012. Pfhrp2 and pfhrp3 gene and their flanking genes on chromosomes 7 and 13, respectively, were amplified from 198 blood specimens collected in Brazil. In Brazil, the isolates collected in Acre state, located in the western part of the Brazilian Amazon, had the highest percentage of deletions for pfhrp2 25 (31.2%) of 79, while among those collected in Rondonia, the prevalence of pfhrp2 gene deletion was only 3.3% (2 out of 60 patients). In isolates from Para state, all parasites were pfhrp2-positive. In contrast, we detected high proportions of isolates from all 3 states that were pfhrp3-negative ranging from 18.3% (11 out of 60 samples) to 50.9% (30 out of 59 samples). In Bolivia, only one of 25 samples (4%) tested had deleted pfhrp2 gene, while 68% (17 out of 25 samples) were pfhrp3-negative. Among the isolates tested, P. falciparum pfhrp2 gene deletions were present mainly in those from Acre State in the Brazilian Amazon. These results indicate it is important to reconsider the use of PfHRP2-based RDTs in the western region of the Brazilian Amazon and to implement appropriate surveillance systems to monitor pfhrp2 gene deletions in this and other parts of the Amazon region.

Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia

PubMed Central

Rachid Viana, Giselle Maria; Akinyi Okoth, Sheila; Silva-Flannery, Luciana; Lima Barbosa, Danielle Regina; Macedo de Oliveira, Alexandre; Goldman, Ira F.; Morton, Lindsay C.; Huber, Curtis; Anez, Arletta; Dantas Machado, Ricardo Luiz; Aranha Camargo, Luís Marcelo; Costa Negreiros do Valle, Suiane; Marins Póvoa, Marinete; Barnwell, John W.

2017-01-01

More than 80% of available malaria rapid diagnostic tests (RDTs) are based on the detection of histidine-rich protein-2 (PfHRP2) for diagnosis of Plasmodium falciparum malaria. Recent studies have shown the genes that code for this protein and its paralog, histidine-rich protein-3 (PfHRP3), are absent in parasites from the Peruvian Amazon Basin. Lack of PfHRP2 protein through deletion of the pfhrp2 gene leads to false-negative RDT results for P. falciparum. We have evaluated the extent of pfhrp2 and pfhrp3 gene deletions in a convenience sample of 198 isolates from six sites in three states across the Brazilian Amazon Basin (Acre, Rondonia and Para) and 25 isolates from two sites in Bolivia collected at different times between 2010 and 2012. Pfhrp2 and pfhrp3 gene and their flanking genes on chromosomes 7 and 13, respectively, were amplified from 198 blood specimens collected in Brazil. In Brazil, the isolates collected in Acre state, located in the western part of the Brazilian Amazon, had the highest percentage of deletions for pfhrp2 25 (31.2%) of 79, while among those collected in Rondonia, the prevalence of pfhrp2 gene deletion was only 3.3% (2 out of 60 patients). In isolates from Para state, all parasites were pfhrp2-positive. In contrast, we detected high proportions of isolates from all 3 states that were pfhrp3-negative ranging from 18.3% (11 out of 60 samples) to 50.9% (30 out of 59 samples). In Bolivia, only one of 25 samples (4%) tested had deleted pfhrp2 gene, while 68% (17 out of 25 samples) were pfhrp3-negative. Among the isolates tested, P. falciparum pfhrp2 gene deletions were present mainly in those from Acre State in the Brazilian Amazon. These results indicate it is important to reconsider the use of PfHRP2-based RDTs in the western region of the Brazilian Amazon and to implement appropriate surveillance systems to monitor pfhrp2 gene deletions in this and other parts of the Amazon region. PMID:28301474

[Detection of bcr/abl fusion gene and its derivative chromosome 9 deletions in CML by using home-made bcr/abl extra-signal probe].

PubMed

Lai, Yue-Yun; Feng, Lin; Wang, Zheng; Lü, Shan; Dang, Hui; Shi, Yan; He, Qi; Huang, Xiao-Jun

2010-02-01

This study was aimed to verify the efficacy of home-made LSI bcr/abl ES probe for detection of bcr/abl fusion gene and derivative chromosome 9 deletions in chronic myeloid leukemia (CML). Fluorescence in situ hybridization (FISH) was carried out with dual color bcr/abl extra signal (ES) probe in 97 cases of CML based on morphology and cytogenetic karyotype and 129 cases of non-hematological malignancies/non-myeloproliferative diseases with normal cytogenetic karyotype. For the patients with signals of 1R1G1F indicating der(9) deletions, FISH were done using ASS DNA probe. The results showed that 91 cases with standard t(9;22) and 6 cases with variant translocation of t(9;22) were detected by conventional G banding technique. All of the 97 patients displayed bcr/abl fusion gene by ES-FISH, including 16 cases with signal patterns of 1R1G1F showing der(9) deletions. Among the 16 cases with der(9) deletions, 13 cases were detected to have deletions of ASS gene. Meanwhile, none of the 129 cases of negative control showed bcr/abl fusion gene by ES-FISH. It is concluded that home-made LSI bcr/abl ES probe is effective to identify the bcr/abl fusion gene and der(9) deletions in CML, and the ES-FISH results are consistent with conventional cytogenetic karyotype.

Identification of single gene deletions at 15q13.3: further evidence that CHRNA7 causes the 15q13.3 microdeletion syndrome phenotype.

PubMed

Hoppman-Chaney, N; Wain, K; Seger, P R; Superneau, D W; Hodge, J C

2013-04-01

The 15q13.3 microdeletion syndrome (OMIM #612001) is characterized by a wide range of phenotypic features, including intellectual disability, seizures, autism, and psychiatric conditions. This deletion is inherited in approximately 75% of cases and has been found in mildly affected and normal parents, consistent with variable expressivity and incomplete penetrance. The common deletion is approximately 2 Mb and contains several genes; however, the gene(s) responsible for the resulting clinical features have not been clearly defined. Recently, four probands were reported with small deletions including only the CHRNA7 gene. These patients showed a wide range of phenotypic features similar to those associated with the larger 15q13.3 microdeletion. To further correlate genotype and phenotype, we queried our database of >15,000 patients tested in the Mayo Clinic Cytogenetics Laboratory from 2008 to 2011 and identified 19 individuals (10 probands and 9 family members) with isolated heterozygous CHRNA7 gene deletions. All but two infants displayed multiple features consistent with 15q13.3 microdeletion syndrome. We also identified the first de novo deletion confined to CHRNA7 as well as the second known case with homozygous deletion of CHRNA7 only. These results provide further evidence implicating CHRNA7 as the gene responsible for the clinical findings associated with 15q13.3 microdeletion. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

A novel whole exon deletion in WWOX gene causes early epilepsy, intellectual disability and optic atrophy.

PubMed

Ben-Salem, Salma; Al-Shamsi, Aisha M; John, Anne; Ali, Bassam R; Al-Gazali, Lihadh

2015-05-01

Recent studies have implicated the WW domain-containing oxidoreductase encoding gene (WWOX) in a severe form of autosomal recessive neurological disorder. This condition showed an overlapping spectrum of clinical features including spinocerebellar ataxia associated with generalized seizures and delayed psychomotor development to growth retardation, spasticity, and microcephaly. We evaluated a child from a consanguineous Emirati family that presented at birth with growth retardation, microcephaly, epileptic seizures, and later developed spasticity and delayed psychomotor development. Screening for deletions and duplications using whole-chromosomal microarray analysis identified a novel homozygous microdeletion encompassing exon 5 of the WWOX gene. Analysis of parental DNA indicated that this deletion was inherited from both parents and lies within a large region of homozygosity. Sanger sequencing of the cDNA showed that the deletion resulted in exon 5 skipping leading to a frame-shift and creating a premature stop codon at amino acid position 212. Quantification of mRNA revealed striking low level of WWOX expression in the child and moderate level of expression in the mother compared to a healthy control. To the best of our knowledge, this is the first homozygous germline structural variation in WWOX gene resulting in truncated transcripts that were presumably subject to NMD pathway. Our findings extend the clinical and genetic spectrum of WWOX mutations and support a crucial role of this gene in neurological development.

High frequency of ribosomal protein gene deletions in Italian Diamond-Blackfan anemia patients detected by multiplex ligation-dependent probe amplification assay

PubMed Central

Quarello, Paola; Garelli, Emanuela; Brusco, Alfredo; Carando, Adriana; Mancini, Cecilia; Pappi, Patrizia; Vinti, Luciana; Svahn, Johanna; Dianzani, Irma; Ramenghi, Ugo

2012-01-01

Diamond-Blackfan anemia is an autosomal dominant disease due to mutations in nine ribosomal protein encoding genes. Because most mutations are loss of function and detected by direct sequencing of coding exons, we reasoned that part of the approximately 50% mutation negative patients may have carried a copy number variant of ribosomal protein genes. As a proof of concept, we designed a multiplex ligation-dependent probe amplification assay targeted to screen the six genes that are most frequently mutated in Diamond-Blackfan anemia patients: RPS17, RPS19, RPS26, RPL5, RPL11, and RPL35A. Using this assay we showed that deletions represent approximately 20% of all mutations. The combination of sequencing and multiplex ligation-dependent probe amplification analysis of these six genes allows the genetic characterization of approximately 65% of patients, showing that Diamond-Blackfan anemia is indisputably a ribosomopathy. PMID:22689679

RCSD1-ABL1 Translocation Associated with IKZF1 Gene Deletion in B-Cell Acute Lymphoblastic Leukemia.

PubMed

Kamran, Shawana; Raca, Gordana; Nazir, Kamran

2015-01-01

The RCSD1 gene has recently been identified as a novel gene fusion partner of the ABL1 gene in cases of B-cell Acute Lymphoblastic Leukemia (B-ALL). The RCSD1 gene is located at 1q23 and ABL1 is located at 9q34, so that the RCSD1-ABL1 fusion typically arises through a rare reciprocal translocation t(1;9)(q23;q34). Only a small number of RCSD1-ABL1 positive cases of B-ALL have been described in the literature, and the full spectrum of clinical, morphological, immunophenotypic, and molecular features associated with this genetic abnormality has not been defined. We describe extensive genetic characterization of a case of B-ALL with RCSD1-ABL1 fusion, by using conventional cytogenetic analysis, Fluorescence In Situ Hybridization (FISH) studies, and Chromosomal Microarray Analysis (CMA). The use of CMA resulted in detection of an approximately 70 kb deletion at 7p12.2, which caused a disruption of the IKZF1 gene. Deletions and mutations of IKZF1 are recurring abnormalities in B-ALL and are associated with a poor prognosis. Our findings highlight the association of the deletion of IKZF1 gene with the t(1;9)(q24;q34) and illustrate the importance of comprehensive cytogenetic and molecular evaluation for accurate prediction of prognosis in patients with B-cell ALL.

RCSD1-ABL1 Translocation Associated with IKZF1 Gene Deletion in B-Cell Acute Lymphoblastic Leukemia

PubMed Central

Kamran, Shawana; Nazir, Kamran

2015-01-01

The RCSD1 gene has recently been identified as a novel gene fusion partner of the ABL1 gene in cases of B-cell Acute Lymphoblastic Leukemia (B-ALL). The RCSD1 gene is located at 1q23 and ABL1 is located at 9q34, so that the RCSD1-ABL1 fusion typically arises through a rare reciprocal translocation t(1;9)(q23;q34). Only a small number of RCSD1-ABL1 positive cases of B-ALL have been described in the literature, and the full spectrum of clinical, morphological, immunophenotypic, and molecular features associated with this genetic abnormality has not been defined. We describe extensive genetic characterization of a case of B-ALL with RCSD1-ABL1 fusion, by using conventional cytogenetic analysis, Fluorescence In Situ Hybridization (FISH) studies, and Chromosomal Microarray Analysis (CMA). The use of CMA resulted in detection of an approximately 70 kb deletion at 7p12.2, which caused a disruption of the IKZF1 gene. Deletions and mutations of IKZF1 are recurring abnormalities in B-ALL and are associated with a poor prognosis. Our findings highlight the association of the deletion of IKZF1 gene with the t(1;9)(q24;q34) and illustrate the importance of comprehensive cytogenetic and molecular evaluation for accurate prediction of prognosis in patients with B-cell ALL. PMID:26600955

Novel deletion of the E3A ubiquitin protein ligase gene detected by multiplex ligation-dependent probe amplification in a patient with Angelman syndrome

PubMed Central

Calì, Francesco; Ragalmuto, Alda; Chiavetta, Valeria; Calabrese, Giuseppe; Fichera, Marco; Vinci, Mirella; Ruggeri, Giuseppa; Schinocca, Pietro; Sturnio, Maurizio; Romano, Salvatore; Elia, Maurizio

2010-01-01

Angelman syndrome (AS) is a severe neurobehavioural disorder caused by failure of expression of the maternal copy of the imprinted domain located on 15q11-q13. There are different mechanisms leading to AS: maternal microdeletion, uniparental disomy, defects in a putative imprinting centre, mutations of the E3 ubiquitin protein ligase (UBE3A) gene. However, some of suspected cases of AS are still scored negative to all the latter mutations. Recently, it has been shown that a proportion of negative cases bear large deletions overlapping one or more exons of the UBE3A gene. These deletions are difficult to detect by conventional gene-scanning methods due to the masking effect by the non-deleted allele. In this study, we have used for the first time multiplex ligation-dependent probe amplification (MLPA) and comparative multiplex dosage analysis (CMDA) to search for large deletions affecting the UBE3A gene. Using this approach, we identified a novel causative deletion involving exon 8 in an affected sibling. Based on our results, we propose the use of MLPA as a fast, accurate and inexpensive test to detect large deletions in the UBE3A gene in a small but significant percentage of AS patients. PMID:21072004

Enhanced freeze tolerance of baker's yeast by overexpressed trehalose-6-phosphate synthase gene (TPS1) and deleted trehalase genes in frozen dough.

PubMed

Tan, Haigang; Dong, Jian; Wang, Guanglu; Xu, Haiyan; Zhang, Cuiying; Xiao, Dongguang

2014-08-01

Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker's yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301(TPS1) overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301(TPS1) were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301(TPS1) was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker's yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker's yeast.

An atypical deletion of the Williams–Beuren syndrome interval implicates genes associated with defective visuospatial processing and autism

PubMed Central

Edelmann, Lisa; Prosnitz, Aaron; Pardo, Sherly; Bhatt, Jahnavi; Cohen, Ninette; Lauriat, Tara; Ouchanov, Leonid; González, Patricia J; Manghi, Elina R; Bondy, Pamela; Esquivel, Marcela; Monge, Silvia; Delgado, Marietha F; Splendore, Alessandra; Francke, Uta; Burton, Barbara K; McInnes, L Alison

2007-01-01

Background During a genetic study of autism, a female child who met diagnostic criteria for autism spectrum disorder, but also exhibited the cognitive–behavioural profile (CBP) associated with Williams–Beuren syndrome (WBS) was examined. The WBS CBP includes impaired visuospatial ability, an overly friendly personality, excessive non‐social anxiety and language delay. Methods Using array‐based comparative genomic hybridisation (aCGH), a deletion corresponding to BAC RP11‐89A20 in the distal end of the WBS deletion interval was detected. Hemizygosity was confirmed using fluorescence in situ hybridisation and fine mapping was performed by measuring the copy number of genomic DNA using quantitative polymerase chain reaction. Results The proximal breakpoint was mapped to intron 1 of GTF2IRD1 and the distal breakpoint lies 2.4–3.1 Mb towards the telomere. The subject was completely hemizygous for GTF2I, commonly deleted in carriers of the classic ∼1.5 Mb WBS deletion, and GTF2IRD2, deleted in carriers of the rare ∼1.84 Mb WBS deletion. Conclusion Hemizygosity of the GTF2 family of transcription factors is sufficient to produce many aspects of the WBS CBP, and particularly implicate the GTF2 transcription factors in the visuospatial construction deficit. Symptoms of autism in this case may be due to deletion of additional genes outside the typical WBS interval or remote effects on gene expression at other loci. PMID:16971481

Prevalence of pfhrp2 and/or pfhrp3 Gene Deletion in Plasmodium falciparum Population in Eight Highly Endemic States in India

PubMed Central

Bharti, Praveen Kumar; Chandel, Himanshu Singh; Ahmad, Amreen; Krishna, Sri; Udhayakumar, Venkatachalam; Singh, Neeru

2016-01-01

Background Plasmodium falciparum encoded histidine rich protein (HRP2) based malaria rapid diagnostic tests (RDTs) are used in India. Deletion of pfhrp2 and pfhrp3 genes contributes to false negative test results, and large numbers of such deletions have been reported from South America, highlighting the importance of surveillance to detect such deletions. Methods This is the first prospective field study carried out at 16 sites located in eight endemic states of India to assess the performance of PfHRP2 based RDT kits used in the national malaria control programme. In this study, microscopically confirmed P. falciparum but RDT negative samples were assessed for presence of pfhrp2, pfhrp3, and their flanking genes using PCR. Results Among 1521 microscopically positive P. falciparum samples screened, 50 were negative by HRP2 based RDT test. Molecular testing was carried out using these 50 RDT negative samples by assuming that 1471 RDT positive samples carried pfhrp2 gene. It was found that 2.4% (36/1521) and 1.8% (27/1521) of samples were negative for pfhrp2 and pfhrp3 genes, respectively. However, the frequency of pfhrp2 deletions varied between the sites ranging from 0–25% (2.4, 95% CI; 1.6–3.3). The frequency of both pfhrp2 and pfhrp3 gene deletion varied from 0–8% (1.6, 95% CI; 1.0–2.4). Conclusion This study provides evidence for low level presence of pfhrp2 and pfhrp3 deleted P. falciparum parasites in different endemic regions of India, and periodic surveillance is warranted for reliable use of PfHRP2 based RDTs. PMID:27518538

Deletion Mapping of zwf, the Gene for a Constitutive Enzyme, Glucose 6-Phosphate Dehydrogenase in ESCHERICHIA COLI

PubMed Central

Fraenkel, D. G.; Banerjee, Santimoy

1972-01-01

Genes for three enzymes of intermediary sugar metabolism in E. coli, zwf (glucose 6-phosphate dehydrogenase, constitutive), edd (gluconate 6-phosphate dehydrase, inducible), and eda (2-keto-3-deoxygluconate 6-phosphate aldolase, differently inducible) are closely linked on the E. coli genetic map, the overall gene order being man... old... eda. edd. zwf... cheB... uvrC... his. One class of apparent revertants of an eda mutant strain contains a secondary mutation in edd, and some of these mutations are deletions extending into zwf. We have used a series of spontaneous edd-zwf deletions to map a series of point mutants in zwf and thus report the first fine structure map of a gene for a constitutive enzyme (zwf). PMID:4560065

Human retinoblastoma susceptibility gene: genomic organization and analysis of heterozygous intragenic deletion mutants.

PubMed Central

Bookstein, R; Lee, E Y; To, H; Young, L J; Sery, T W; Hayes, R C; Friedmann, T; Lee, W H

1988-01-01

A gene in chromosome region 13q14 has been identified as the human retinoblastoma susceptibility (RB) gene on the basis of altered gene expression found in virtually all retinoblastomas. In order to further characterize the RB gene and its structural alterations, we examined genomic clones of the RB gene isolated from both a normal human genomic library and a library made from DNA of the retinoblastoma cell line Y79. First, a restriction and exon map of the RB gene was constructed by aligning overlapping genomic clones, yielding three contiguous regions ("contigs") of 150 kilobases total length separated by two gaps. At least 20 exons were identified in genomic clones, and these were provisionally numbered. Second, two overlapping genomic clones that demonstrated a DNA deletion of exons 2 through 6 from one RB allele were isolated from the Y79 library. To confirm and extend this result, a unique sequence probe from intron 1 was used to detect similar and possibly identical heterozygous deletions in genomic DNA from three retinoblastoma cell lines, thereby explaining the origins of their shortened RB mRNA transcripts. The same probe detected genomic rearrangements in fibroblasts from two hereditary retinoblastoma patients, indicating that intron 1 includes a frequent site for mutations conferring predisposition to retinoblastoma. Third, this probe also detected a polymorphic site for BamHI with allele frequencies near 0.5/0.5. Identification of commonly mutated regions will contribute significantly to genetic diagnosis in retinoblastoma patients and families. Images PMID:2895471

Identification of the first intragenic deletion of the PITX2 gene causing an Axenfeld-Rieger Syndrome: case report.

PubMed

de la Houssaye, Guillaume; Bieche, Ivan; Roche, Olivier; Vieira, Véronique; Laurendeau, Ingrid; Arbogast, Laurence; Zeghidi, Hatem; Rapp, Philippe; Halimi, Philippe; Vidaud, Michel; Dufier, Jean-Louis; Menasche, Maurice; Abitbol, Marc

2006-11-29

Axenfeld-Rieger syndrome (ARS) is characterized by bilateral congenital abnormalities of the anterior segment of the eye associated with abnormalities of the teeth, midface, and umbilicus. Most cases of ARS are caused by mutations in the genes encoding PITX2 or FOXC1. Here we describe a family affected by a severe form of ARS. Two members of this family (father and daughter) presented with typical ARS and developed severe glaucoma. The ocular phenotype was much more severe in the daughter than in the father. Magnetic resonance imaging (MRI) detected an aggressive form of meningioma in the father. There was no mutation in the PITX2 gene, determined by exon screening. We identified an intragenic deletion by quantitative genomic PCR analysis and characterized this deletion in detail. Our findings implicate the first intragenic deletion of the PITX2 gene in the pathogenesis of a severe form of ARS in an affected family. This study stresses the importance of a systematic search for intragenic deletions in families affected by ARS and in sporadic cases for which no mutations in the exons or introns of PITX2 have been found. The molecular genetics of some ARS pedigrees should be re-examined with enzymes that can amplify medium and large genomic fragments.

Identification of the first intragenic deletion of the PITX2 gene causing an Axenfeld-Rieger Syndrome: case report

PubMed Central

de la Houssaye, Guillaume; Bieche, Ivan; Roche, Olivier; Vieira, Véronique; Laurendeau, Ingrid; Arbogast, Laurence; Zeghidi, Hatem; Rapp, Philippe; Halimi, Philippe; Vidaud, Michel; Dufier, Jean-Louis; Menasche, Maurice; Abitbol, Marc

2006-01-01

Background Axenfeld-Rieger syndrome (ARS) is characterized by bilateral congenital abnormalities of the anterior segment of the eye associated with abnormalities of the teeth, midface, and umbilicus. Most cases of ARS are caused by mutations in the genes encoding PITX2 or FOXC1. Here we describe a family affected by a severe form of ARS. Case presentation Two members of this family (father and daughter) presented with typical ARS and developed severe glaucoma. The ocular phenotype was much more severe in the daughter than in the father. Magnetic resonance imaging (MRI) detected an aggressive form of meningioma in the father. There was no mutation in the PITX2 gene, determined by exon screening. We identified an intragenic deletion by quantitative genomic PCR analysis and characterized this deletion in detail. Conclusion Our findings implicate the first intragenic deletion of the PITX2 gene in the pathogenesis of a severe form of ARS in an affected family. This study stresses the importance of a systematic search for intragenic deletions in families affected by ARS and in sporadic cases for which no mutations in the exons or introns of PITX2 have been found. The molecular genetics of some ARS pedigrees should be re-examined with enzymes that can amplify medium and large genomic fragments. PMID:17134502

Novel contiguous gene deletion in peruvian girl with Trichothiodystrophy type 4 and glutaric aciduria type 3.

PubMed

La Serna-Infantes, Jorge; Pastor, Miguel Chávez; Trubnykova, Milana; Velásquez, Félix Chavesta; Sotomayor, Flor Vásquez; Barriga, Hugo Abarca

2018-07-01

Trichothiodystrophy type 4 is a rare autosomal recessive and ectodermal disorder, characterized by dry, brittle, sparse and sulfur-deficient hair and other features like intellectual disability, ichthyotic skin and short stature, caused by a homozygous mutation in MPLKIP gene. Glutaric aciduria type 3 is caused by a homozygous mutation in SUGCT gene with no distinctive phenotype. Both genes are localized on chromosome 7 (7p14). We report an 8-year-old female with short stature, microcephaly, development delay, intellectual disability and hair characterized for dark, short, coarse, sparse and brittle associated to classical trichorrhexis microscopy pattern. Chromosome microarray analysis showed a 125 kb homozygous pathogenic deletion, which includes genes MPLKIP and SUGCT, not described before. This is the first case described in Peru of a novel contiguous gene deletion of Trichothiodystrophy type 4 and Glutaric aciduria type 3 performed by chromosome microarray analysis, highlighting the contribution and importance of molecular technologies on diagnosis of rare genetic conditions. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Quantitative PCR analysis reveals a high incidence of large intragenic deletions in the FANCA gene in Spanish Fanconi anemia patients.

PubMed

Callén, E; Tischkowitz, M D; Creus, A; Marcos, R; Bueren, J A; Casado, J A; Mathew, C G; Surrallés, J

2004-01-01

Fanconi anaemia is an autosomal recessive disease characterized by chromosome fragility, multiple congenital abnormalities, progressive bone marrow failure and a high predisposition to develop malignancies. Most of the Fanconi anaemia patients belong to complementation group FA-A due to mutations in the FANCA gene. This gene contains 43 exons along a 4.3-kb coding sequence with a very heterogeneous mutational spectrum that makes the mutation screening of FANCA a difficult task. In addition, as the FANCA gene is rich in Alu sequences, it was reported that Alu-mediated recombination led to large intragenic deletions that cannot be detected in heterozygous state by conventional PCR, SSCP analysis, or DNA sequencing. To overcome this problem, a method based on quantitative fluorescent multiplex PCR was proposed to detect intragenic deletions in FANCA involving the most frequently deleted exons (exons 5, 11, 17, 21 and 31). Here we apply the proposed method to detect intragenic deletions in 25 Spanish FA-A patients previously assigned to complementation group FA-A by FANCA cDNA retroviral transduction. A total of eight heterozygous deletions involving from one to more than 26 exons were detected. Thus, one third of the patients carried a large intragenic deletion that would have not been detected by conventional methods. These results are in agreement with previously published data and indicate that large intragenic deletions are one of the most frequent mutations leading to Fanconi anaemia. Consequently, this technology should be applied in future studies on FANCA to improve the mutation detection rate. Copyright 2003 S. Karger AG, Basel

Identification of a Novel Deletion in AVP-NPII Gene in a Patient with Central Diabetes Insipidus.

PubMed

Deniz, Ferhat; Acar, Ceren; Saglar, Emel; Erdem, Beril; Karaduman, Tugce; Yonem, Arif; Cagiltay, Eylem; Ay, Seyit Ahmet; Mergen, Hatice

2015-01-01

Central Diabetes Insipidus (CDI) is caused by a deficiency of antidiuretic hormone and characterized by polyuria, polydipsia and inability to concentrate urine. Our objective was to present the results of the molecular analyses of AVP-neurophysin II (AVP-NPII) gene in a large familial neurohypophyseal (central) DI pedigree. A male patient and his family members were analyzed and the prospective clinical data were collected. The proband applied to hospital for eligibility to be a recruit in Armed Forces. The patient had severe polyuria (20 L/day), polydipsia (20.5 L/day), fatique, and deep thirstiness. CDI was confirmed with the water deprivation-desmopressin test according to an increase in urine osmolality from 162 mOsm/kg to 432 mOsm/kg after desmopressin acetate injection. To evaluate the coding regions of AVP-NPII gene, polymerase chain reactions were performed and amplified regions were submitted to direct sequence analysis. We detected a heterozygous three base pair deletion at codon 69-70 (207_209delGGC) in exon 2, which lead to a deletion of the amino acid alanine. A three-dimensional protein structure prediction was shown for the deleted AVP-NPII and compared with the wild type. The three base pair deletion may yield an abnormal AVP precursor in neurophysin moiety, but further functional analyses are needed to understand the function of the deleted protein. © 2015 by the Association of Clinical Scientists, Inc.

Seventeen {alpha}-hydroxylase deficiency with one base pair deletion of the cytochrome P450c17 (CYP17) gene

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

Oshiro, Chikara; Takasu, Nobuyuki; Wakugami, Tamio

1995-08-01

Mutation of the cytochrome P450c17 (CYP17) gene causes 17{alpha}-hydroxylase deficiency (170HD). Recently, several researchers have elucidated the molecular basis of 170HD by gene analysis. We experienced a case of 170HD and intended to reveal the abnormality of the CYP17 gene in this Japanese female with 170HD. Leukocytes were obtained from the patient, her mother and sister, and normal control subjects. We amplified the CYP17 gene using polymerase chain reaction and performed the sequence analysis using the dideoxy terminator method and restriction enzyme analysis. We found that the patient had one base-pair deletion at the position of amino acid 438. Anmore » indentical result was obtained with restriction enzyme analysis. This G deletion altered the reading frame and resulted in a premature stop codon at position 443; the ligand of heme iron (Cys: cystine 442) was absent. This small mutation may account for the patient`s clinical manifestations of 170HD. This is the first case of 170HD with only one base pair deletion of the CYP17 gene. 18 refs., 3 figs.« less

Deletion of the TNFAIP3/A20 gene detected by FICTION analysis in classical Hodgkin lymphoma

PubMed Central

2012-01-01

Background The TNFAIP3 gene, which encodes a ubiquitin-modifying enzyme (A20) involved in the negative regulation of NF-κB signaling, is frequently inactivated by gene deletions/mutations in a variety of B-cell malignancies. However, the detection of this in primary Hodgkin lymphoma (HL) specimens is hampered by the scarcity of Hodgkin Reed-Sternberg (HR-S) cells even after enrichment by micro-dissection. Methods We used anti-CD30 immunofluorescence with fluorescence in-situ hybridization (FISH) to evaluate the relative number of TNFAIP3/CEP6 double-positive signals in CD30-positive cells. Results From a total of 47 primary classical Hodgkin lymphoma (cHL) specimens, 44 were evaluable. We found that the relative numbers of TNFAIP3/CD30 cells were distributed among three groups, corresponding to those having homozygous (11%), heterozygous (32%), and no (57%) deletions in TNFAIP3. This shows that TNFAIP3 deletions could be sensitively detected using our chosen methods. Conclusions Comparing the results with mutation analysis, TNFAIP3 inactivation was shown to have escaped detection in many samples with homozygous deletions. This suggests that TNFAIP3 inactivation in primary cHL specimens might be more frequent than previously reported. PMID:23039325

Novel intra-genic large deletions of CTNNB1 gene identified in WT desmoid-type fibromatosis.

PubMed

Colombo, Chiara; Urbini, Milena; Astolfi, Annalisa; Collini, Paola; Indio, Valentina; Belfiore, Antonino; Paielli, Nicholas; Perrone, Federica; Tarantino, Giuseppe; Palassini, Elena; Fiore, Marco; Pession, Andrea; Stacchiotti, Silvia; Pantaleo, Maria Abbondanza; Gronchi, Alessandro

2018-06-14

A wait and see approach for desmoid tumors (DT) has become part of the routine treatment strategy. However, predictive factors to select the risk of progressive disease are still lacking. A translational project was run in order to identify genomic signatures in patients enrolled within an Italian prospective observational study. Among 12 DT patients (ten CTNNB1-mutated and two WT) enrolled from our Institution only two patients (17%) showed a progressive disease. Tumor biopsies were collected for whole exome sequencing. Overall, DT exhibited low somatic sequence mutation rate and no additional recurrent mutation was found. In the two WT cases, two novel alterations were detected: a complex deletion of APC and a pathogenic mutation of LAMTOR2. Focusing on WT DT subtype, deep sequencing of CTNNB1, APC and LAMTOR2 was conducted on a retrospective series of 11 WT DT using a targeted approach. No other mutation of LAMTOR2 was detected, while APC was mutated in two cases. Low-frequency (mean reads of 16%) CTNNB1 mutations were discovered in five samples (45%) and two novel intra-genic deletions in CTNNB1 were detected in two cases. Both deletions and low frequency mutations of CTNNB1 were highly expressed. In conclusion, a minority of DT is WT for either CTNNB1, APC or any other gene involved in the WNT pathway. In this subgroup novel and hard to be detected molecular alterations in APC and CTNNB1 were discovered, contributing to explain a portion of the allegedly WT DT cases. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Deletion and Gene Expression Analyses Define the Paxilline Biosynthetic Gene Cluster in Penicillium paxilli

PubMed Central

Scott, Barry; Young, Carolyn A.; Saikia, Sanjay; McMillan, Lisa K.; Monahan, Brendon J.; Koulman, Albert; Astin, Jonathan; Eaton, Carla J.; Bryant, Andrea; Wrenn, Ruth E.; Finch, Sarah C.; Tapper, Brian A.; Parker, Emily J.; Jameson, Geoffrey B.

2013-01-01

The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis. PMID:23949005

High frequency of EBV association and 30-bp deletion in the LMP-1 gene in CD56 lymphomas of the upper aerodigestive tract.

PubMed

Tai, Yan-Chin; Kim, Lian-Hua; Peh, Suat-Cheng

2004-03-01

Natural killer (NK)/T-cell lymphomas are frequently associated with Epstein-Barr virus (EBV), and usually lack TCR gene rearrangement. Studies from Asia have reported frequent deletion in the LMP-1 gene in EBV-associated nasopharyngeal carcinoma (NPC). The present study aims to investigate LMP-1 and TCRgamma gene status in upper aerodigestive tract lymphomas. A total of 43 cases were classified into T-, B-, and NK/T-cell tumors based on the phenotype expressions of CD3(+)/CD20(-)/CD56(-), CD3(-)/CD20(+)/CD56(-), and CD3(+)/CD20(-)/CD56(+), respectively. The presence of EBV in the tumor was confirmed by EBV early RNA-in situ hybridization. LMP-1 gene deletion and TCR gamma gene rearrangement were analyzed by polymerase chain reaction on paraffin-embedded tissues. There were 20 NK/T-, eight T-, and 15 B-cell phenotype lymphomas in the present series, and EBV was detected in 19 (95%), two (25%), and three (20%) cases in the respective groups. All EBV+ cases carried 30-bp deletion in the LMP-1 gene, and two of the NK/T-cell cases were infected by both the wild type and deleted strains. Five (25%) of the NK/T-cell phenotype lymphomas showed rearranged TCR gamma gene. The present study revealed a high frequency of EBV association, and a high frequency of 30-bp deletion in the LMP-1 gene in the virus in the present series of lymphoma. The NK/T-phenotype lymphomas are comprised of both NK-cell and cytotoxic T-lymphocyte-derived tumors.

A large deletion of the AVPR2 gene causing severe nephrogenic diabetes insipidus in a Turkish family.

PubMed

Saglar, Emel; Deniz, Ferhat; Erdem, Beril; Karaduman, Tugce; Yönem, Arif; Cagiltay, Eylem; Mergen, Hatice

2014-05-01

X-linked nephrogenic diabetes insipidus (NDI) is a rare hereditary disease caused by mutations in arginine vasopressin type 2 receptor (AVPR2) and characterized by the production of large amounts of urine and an inability to concentrate urine in response to the antidiuretic hormone vasopressin. We have identified a novel 388 bp deletion starting in intron 1 and ending in exon 2 in the AVPR2 gene in a patient with NDI and in his family. We have revealed that this mutation is a de novo mutation for the mother of the proband patient. Prospective clinical data were collected for all family members. The water deprivation test confirmed the diagnosis of diabetes insipidus. The patient has severe symptoms like deep polyuria nocturia, polydipsia, and fatigue. He was given arginine vasopressin treatment while he was a child. However, he could not get well due to his nephrogenic type of illness. Both of his nephews have the same complains in addition to failure to grow. We have sequenced all exons and intron-exon boundaries of the AVPR2 gene of all family members. The analyses of bioinformatics and comparative genomics of the deletion were done via considering the DNA level damage. AVPR2 gene mutation results in the absence of the three transmembrane domains, two extracellular domains, and one cytoplasmic domain. Three-dimensional protein structure prediction was shown. We concluded that X-linked NDI and severity of illness in this family is caused by a novel 388 bp deletion in the AVPR2 gene that is predicted to truncate the receptor protein, and also this deletion may lead to dysfunctioning in protein activity and inefficient or inadequate binding abilities.

Single-cell RNA sequencing reveals an altered gene expression pattern as a result of CRISPR/cas9-mediated deletion of Gene 33/Mig6 and chronic exposure to hexavalent chromium in human lung epithelial cells.

PubMed

Park, Soyoung; Zhang, Xiaowen; Li, Cen; Yin, Changhong; Li, Jiangwei; Fallon, John T; Huang, Weihua; Xu, Dazhong

2017-09-01

Gene 33 (Mig6, ERRFI1) is an adaptor protein with multiple cellular functions. We recently reported that depletion of this protein promotes lung epithelial cell transformation induced by hexavalent chromium [Cr(VI)]. However, the early molecular events that mediate this process are not clear. In the present study, we used single-cell RNA sequencing to compare gene expression profiles between BEAS-2B lung epithelial cells chronically exposed to a sublethal dose of Cr(VI) with or without CRISPR/cas9-mediated deletion of Gene 33. Our data reveal 83 differentially expressed genes. The most notable changes are genes associated with cell adhesion, oxidative stresses, protein ubiquitination, epithelial-mesenchymal transition/metastasis, and WNT signaling. Up-regulation of some neuro-specific genes is also evident, particularly ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a deubiquitinase and potential biomarker for lung cancer. Gene 33 deletion and/or Cr(VI) exposure did not cause discernable changes in cell morphology. However, Gene 33 deletion led to a modest but significant reduction of cells in the G2/M phase of the cell cycle regardless of Cr(VI) exposure. Gene 33 deletion also significantly reduced cell proliferation. Interestingly, Cr(VI) exposure eliminated the difference in cell proliferation between the two genotypes. Gene 33 deletion also significantly elevated cell migration. Our data indicate that combined Gene 33 deletion and chronic Cr(VI) exposure produces a gene expression pattern and a phenotype resemble those of the transformed lung epithelial cells. Given the known association of UCHL1 with lung cancer, we propose that UCHL1 is an important player in the early stage of lung epithelial cell transformation and tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

A large deletion in the succinate dehydrogenase B gene (SDHB) in a Japanese patient with abdominal paraganglioma and concomitant metastasis.

PubMed

Kodama, Hitomi; Iihara, Masatoshi; Nissato, Sumiko; Isobe, Kazumasa; Kawakami, Yasushi; Okamoto, Takahiro; Takekoshi, Kazuhiro

2010-01-01

Recently, mutations in nuclear genes encoding two mitochondrial complex II subunit proteins, Succinate dehydrogenase D (SDHD) and SDHB, have been found to be associated with the development of familial pheochromocytomas and paragangliomas (hereditary pheochromocytoma/paraganglioma syndrome: HPPS). Growing evidence suggests that the mutation of SDHB is highly associated with abdominal paraganglioma and the following distant metastasis (malignant paraganglioma). In the present study, we used multiplex ligation dependent probe amplification (MLPA) analysis to identify a large heterozygous SDHB gene deletion encompassing sequences corresponding to the promoter region, in addition to exon 1 and exon 2 malignant paraganglioma patient in whom previously characterized SDHB mutations were undetectable. This is the first Japanese case report of malignant paraganglioma, with a large SDHB deletions. Our present findings strongly support the notion that large deletions in the SDHB gene should be considered in patients lacking characterized SDHB mutations.

Genetically Validated Drug Targets in Leishmania: Current Knowledge and Future Prospects.

PubMed

Jones, Nathaniel G; Catta-Preta, Carolina M C; Lima, Ana Paula C A; Mottram, Jeremy C

2018-04-13

There has been a very limited number of high-throughput screening campaigns carried out with Leishmania drug targets. In part, this is due to the small number of suitable target genes that have been shown by genetic or chemical methods to be essential for the parasite. In this perspective, we discuss the state of genetic target validation in the field of Leishmania research and review the 200 Leishmania genes and 36 Trypanosoma cruzi genes for which gene deletion attempts have been made since the first published case in 1990. We define a quality score for the different genetic deletion techniques that can be used to identify potential drug targets. We also discuss how the advances in genome-scale gene disruption techniques have been used to assist target-based and phenotypic-based drug development in other parasitic protozoa and why Leishmania has lacked a similar approach so far. The prospects for this scale of work are considered in the context of the application of CRISPR/Cas9 gene editing as a useful tool in Leishmania.

TARGET Researchers Identify Mutations in SIX1/2 and microRNA Processing Genes in Favorable Histology Wilms Tumor | Office of Cancer Genomics

Cancer.gov

TARGET researchers molecularly characterized favorable histology Wilms tumor (FHWT), a pediatric renal cancer. Comprehensive genome and transcript analyses revealed single-nucleotide substitution/deletion mutations in microRNA processing genes (15% of FHWT patients) and Sine Oculis Homeobox Homolog 1/2 (SIX1/2) genes (7% of FHWT patients). SIX1/2 genes play a critical role in renal development and were not previously associated with FHWT, thus presenting a novel role for SIX1/2 pathway aberrations in this disease.

Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants

USDA-ARS?s Scientific Manuscript database

Direct cellobiose production from cellulose by a genetically modified fungus—Neurospora crassa, was explored in this study. A library of N. crassa sextuple beta-glucosidase (bgl) gene deletion strains was constructed. Various concentrations of cellobiose were detected in the culture broth of the N. ...

Diverse fission yeast genes required for responding to oxidative and metal stress: Comparative analysis of glutathione-related and other defense gene deletions.

PubMed

Pluskal, Tomáš; Sajiki, Kenichi; Becker, Joanne; Takeda, Kojiro; Yanagida, Mitsuhiro

2016-06-01

Living organisms have evolved multiple sophisticated mechanisms to deal with reactive oxygen species. We constructed a collection of twelve single-gene deletion strains of the fission yeast Schizosaccharomyces pombe designed for the study of oxidative and heavy metal stress responses. This collection contains deletions of biosynthetic enzymes of glutathione (Δgcs1 and Δgsa1), phytochelatin (Δpcs2), ubiquinone (Δabc1) and ergothioneine (Δegt1), as well as catalase (Δctt1), thioredoxins (Δtrx1 and Δtrx2), Cu/Zn- and Mn- superoxide dismutases (SODs; Δsod1 and Δsod2), sulfiredoxin (Δsrx1) and sulfide-quinone oxidoreductase (Δhmt2). First, we employed metabolomic analysis to examine the mutants of the glutathione biosynthetic pathway. We found that ophthalmic acid was produced by the same enzymes as glutathione in S. pombe. The identical genetic background of the strains allowed us to assess the severity of the individual gene knockouts by treating the deletion strains with oxidative agents. Among other results, we found that glutathione deletion strains were not particularly sensitive to peroxide or superoxide, but highly sensitive to cadmium stress. Our results show the astonishing diversity in cellular adaptation mechanisms to various types of oxidative and metal stress and provide a useful tool for further research into stress responses. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Whole-Genome Sequences of Variants of Bacillus anthracis Sterne and Their Toxin Gene Deletion Mutants

PubMed Central

Staab, A.; Plaut, R. D.; Pratt, C.; Lovett, S. P.; Wiley, M. R.; Biggs, T. D.; Bernhards, R. C.; Beck, L. C.; Palacios, G. F.; Stibitz, S.; Jones, K. L.; Goodwin, B. G.; Smith, M. A.

2017-01-01

ABSTRACT Here, we report the draft genome sequences of three laboratory variants of Bacillus anthracis Sterne and their double (Δlef Δcya) and triple (Δpag Δlef Δcya) toxin gene deletion derivatives. PMID:29122874

New traits in crops produced by genome editing techniques based on deletions.

PubMed

van de Wiel, C C M; Schaart, J G; Lotz, L A P; Smulders, M J M

2017-01-01

One of the most promising New Plant Breeding Techniques is genome editing (also called gene editing) with the help of a programmable site-directed nuclease (SDN). In this review, we focus on SDN-1, which is the generation of small deletions or insertions (indels) at a precisely defined location in the genome with zinc finger nucleases (ZFN), TALENs, or CRISPR-Cas9. The programmable nuclease is used to induce a double-strand break in the DNA, while the repair is left to the plant cell itself, and mistakes are introduced, while the cell is repairing the double-strand break using the relatively error-prone NHEJ pathway. From a biological point of view, it could be considered as a form of targeted mutagenesis. We first discuss improvements and new technical variants for SDN-1, in particular employing CRISPR-Cas, and subsequently explore the effectiveness of targeted deletions that eliminate the function of a gene, as an approach to generate novel traits useful for improving agricultural sustainability, including disease resistances. We compare them with examples of deletions that resulted in novel functionality as known from crop domestication and classical mutation breeding (both using radiation and chemical mutagens). Finally, we touch upon regulatory and access and benefit sharing issues regarding the plants produced.

Systematic deletion of homeobox genes in Podospora anserina uncovers their roles in shaping the fruiting body.

PubMed

Coppin, Evelyne; Berteaux-Lecellier, Véronique; Bidard, Frédérique; Brun, Sylvain; Ruprich-Robert, Gwenaël; Espagne, Eric; Aït-Benkhali, Jinane; Goarin, Anne; Nesseir, Audrey; Planamente, Sara; Debuchy, Robert; Silar, Philippe

2012-01-01

Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures.

Systematic Deletion of Homeobox Genes in Podospora anserina Uncovers Their Roles in Shaping the Fruiting Body

PubMed Central

Coppin, Evelyne; Berteaux-Lecellier, Véronique; Bidard, Frédérique; Brun, Sylvain; Ruprich-Robert, Gwenaël; Espagne, Eric; Aït-Benkhali, Jinane; Goarin, Anne; Nesseir, Audrey; Planamente, Sara; Debuchy, Robert; Silar, Philippe

2012-01-01

Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures. PMID:22662159

Let-7b regulates the expression of the growth hormone receptor gene in deletion-type dwarf chickens

PubMed Central

2012-01-01

Background A deletion mutation in the growth hormone receptor (GHR) gene results in the inhibition of skeletal muscle growth and fat deposition in dwarf chickens. We used microarray techniques to determine microRNA (miRNA) and mRNA expression profiles of GHR in the skeletal muscles of 14-day-old embryos as well as 7-week-old deletion-type dwarf and normal-type chickens. Our aim was to elucidate the miRNA regulation of GHR expression with respect to growth inhibition and fat deposition. Results At the same developmental stages, different expression profiles in skeletal muscles of dwarf and normal chickens occurred for four miRNAs (miR-1623, miR-181b, let-7b, and miR-128). At different developmental stages, there was a significant difference in the expression profiles of a greater number of miRNAs. Eleven miRNAs were up-regulated and 18 down-regulated in the 7-week-old dwarf chickens when compared with profiles in 14-day-old embryos. In 7-week-old normal chickens, seven miRNAs were up-regulated and nine down-regulated compared with those in 14-day-old embryos. In skeletal muscles, 22 genes were up-regulated and 33 down-regulated in 14-day-old embryos compared with 7-week-old dwarf chickens. Sixty-five mRNAs were up-regulated and 108 down-regulated in 14-day-old embryos as compared with 7-week-old normal chickens. Thirty-four differentially expressed miRNAs were grouped into 18 categories based on overlapping seed and target sequences. Only let-7b was found to be complementary to its target in the 3′ untranslated region of GHR, and was able to inhibit its expression. Kyoto Encyclopedia of Genes and Genomes pathway analysis and quantitative polymerase chain reactions indicated there were three main signaling pathways regulating skeletal muscle growth and fat deposition of chickens. These were influenced by let-7b-regulated GHR. Suppression of the cytokine signaling 3 (SOCS3) gene was found to be involved in the signaling pathway of adipocytokines. Conclusions There

The First Report of a 290-bp Deletion in β-Globin Gene in the South of Iran

PubMed Central

Hamid, Mohammad; Nejad, Ladan Dawoody; Shariati, Gholamreza; Galehdari, Hamid; Saberi, Alihossein; Mohammadi-Anaei, Marziye

2017-01-01

Background: β-thalassemia is one of the most widespread diseases in the world, including Iran. In this study, we reported, for the first time, a 290-bp β-globin gene deletion in the south of Iran. Methods: Four individuals from three unrelated families with Arabic ethnic background were studied in Khuzestan Province. Red blood cell indices and hemoglobin analysis were carried out according to the standard methods. Genomic DNA was obtained from peripheral blood cells by salting out procedures. β-globin gene amplification, multiplex ligation-dependent probe amplification (MLPA), and DNA sequencing were performed. Results: The PCR followed by sequencing and MLPA test of the β-globin gene confirmed the presence of a 290-bp deletion in the heterozygous form, along with -88C>A mutation. All the individuals had elevated hemoglobin A2 and normal fetal hemoglobin levels. Conclusions: This mutation causes β0-thalassemia and can be highly useful for prenatal diagnosis in compound heterozygous condition with different β-globin gene mutations. PMID:26948378

Novel large deletion in AVPR2 gene causing copy number variation in a patient with X-linked nephrogenic diabetes insipidus.

PubMed

Cho, Sun Young; Law, Chun Yiu; Ng, Kwok Leung; Lam, Ching Wan

2016-04-01

The diagnosis of cranial and nephrogenic diabetes insipidus (DI) can be clinically challenging. The application of molecular genetic analysis can help in resolving diagnostic difficulties. A 3 month-old boy presented with recurrent polyuria was admitted to Intensive Care Unit and was treated as DI. The patient also had a strong family history of polyuria affecting his maternal uncles. Molecular genetic analysis using Single Nucleotide Polymorphism (SNP) array detected a large deletion located at Xq28 region and the breakpoint was identified using PCR and Sanger sequencing. An 11,535 bp novel deletion affecting the entire APVR2 gene and the last intron and exon of the ARHGAP4 gene was confirmed. This large deletion is likely due to the 7-bp microhomology sequence at the junctions of both 5' and 3' breakpoints. No disease-causing mutation was identified for AQP2. We report a novel deletion in a Chinese patient with congenital nephrogenic DI. We suggested that patients with suspected congenital DI should undergo genetic analysis of AVPR2 and AQP2 genes. A definitive diagnosis can benefit patient by treatment of hydrochlorothiazide and amiloride and avoiding unnecessary investigations. Copyright © 2016 Elsevier B.V. All rights reserved.

Network-based analysis of oligodendrogliomas predicts novel cancer gene candidates within the region of the 1p/19q co-deletion.

PubMed

Gladitz, Josef; Klink, Barbara; Seifert, Michael

2018-06-11

Oligodendrogliomas are primary human brain tumors with a characteristic 1p/19q co-deletion of important prognostic relevance, but little is known about the pathology of this chromosomal mutation. We developed a network-based approach to identify novel cancer gene candidates in the region of the 1p/19q co-deletion. Gene regulatory networks were learned from gene expression and copy number data of 178 oligodendrogliomas and further used to quantify putative impacts of differentially expressed genes of the 1p/19q region on cancer-relevant pathways. We predicted 8 genes with strong impact on signaling pathways and 14 genes with strong impact on metabolic pathways widespread across the region of the 1p/19 co-deletion. Many of these candidates (e.g. ELTD1, SDHB, SEPW1, SLC17A7, SZRD1, THAP3, ZBTB17) are likely to push, whereas others (e.g. CAP1, HBXIP, KLK6, PARK7, PTAFR) might counteract oligodendroglioma development. For example, ELTD1, a functionally validated glioblastoma oncogene located on 1p, was overexpressed. Further, the known glioblastoma tumor suppressor SLC17A7 located on 19q was underexpressed. Moreover, known epigenetic alterations triggered by mutated SDHB in paragangliomas suggest that underexpressed SDHB in oligodendrogliomas may support and possibly enhance the epigenetic reprogramming induced by the IDH-mutation. We further analyzed rarely observed deletions and duplications of chromosomal arms within oligodendroglioma subcohorts identifying putative oncogenes and tumor suppressors that possibly influence the development of oligodendroglioma subgroups. Our in-depth computational study contributes to a better understanding of the pathology of the 1p/19q co-deletion and other chromosomal arm mutations. This might open opportunities for functional validations and new therapeutic strategies.

The evolution of small insertions and deletions in the coding genes of Drosophila melanogaster.

PubMed

Chong, Zechen; Zhai, Weiwei; Li, Chunyan; Gao, Min; Gong, Qiang; Ruan, Jue; Li, Juan; Jiang, Lan; Lv, Xuemei; Hungate, Eric; Wu, Chung-I

2013-12-01

Studies of protein evolution have focused on amino acid substitutions with much less systematic analysis on insertion and deletions (indels) in protein coding genes. We hence surveyed 7,500 genes between Drosophila melanogaster and D. simulans, using D. yakuba as an outgroup for this purpose. The evolutionary rate of coding indels is indeed low, at only 3% of that of nonsynonymous substitutions. As coding indels follow a geometric distribution in size and tend to fall in low-complexity regions of proteins, it is unclear whether selection or mutation underlies this low rate. To resolve the issue, we collected genomic sequences from an isogenic African line of D. melanogaster (ZS30) at a high coverage of 70× and analyzed indel polymorphism between ZS30 and the reference genome. In comparing polymorphism and divergence, we found that the divergence to polymorphism ratio (i.e., fixation index) for smaller indels (size ≤ 10 bp) is very similar to that for synonymous changes, suggesting that most of the within-species polymorphism and between-species divergence for indels are selectively neutral. Interestingly, deletions of larger sizes (size ≥ 11 bp and ≤ 30 bp) have a much higher fixation index than synonymous mutations and 44.4% of fixed middle-sized deletions are estimated to be adaptive. To our surprise, this pattern is not found for insertions. Protein indel evolution appear to be in a dynamic flux of neutrally driven expansion (insertions) together with adaptive-driven contraction (deletions), and these observations provide important insights for understanding the fitness of new mutations as well as the evolutionary driving forces for genomic evolution in Drosophila species.

A single-base deletion in soybean flavonol synthase gene is associated with magenta flower color.

PubMed

Takahashi, Ryoji; Githiri, Stephen M; Hatayama, Kouta; Dubouzet, Emilyn G; Shimada, Norimoto; Aoki, Toshio; Ayabe, Shin-ichi; Iwashina, Tsukasa; Toda, Kyoko; Matsumura, Hisakazu

2007-01-01

The Wm locus of soybean [Glycine max (L.) Merr.] controls flower color. Dominant Wm and recessive wm allele of the locus produce purple and magenta flower, respectively. A putative full-length cDNA of flavonol synthase (FLS), gmfls1 was isolated by 5' RACE and end-to-end PCR from a cultivar Harosoy with purple flower (WmWm). Sequence analysis revealed that gmfls1 consisted of 1,208 nucleotides encoding 334 amino acids. It had 59-72% homology with FLS proteins of other plant species. Conserved dioxygenase domains A and B were found in the deduced polypeptide. Sequence comparison between Harosoy and Harosoy-wm (magenta flower mutant of Harosoy; wmwm) revealed that they differed by a single G deletion in the coding region of Harosoy-wm. The deletion changed the subsequent reading frame resulting in a truncated polypeptide consisting of 37 amino acids that lacked the dioxygenase domains A and B. Extracts of E. coli cells expressing gmfls1 of Harosoy catalyzed the formation of quercetin from dihydroquercetin, whereas cell extracts expressing gmfls1 of Harosoy-wm had no FLS activity. Genomic Southern analysis suggested the existence of three to four copies of the FLS gene in the soybean genome. CAPS analysis was performed to detect the single-base deletion. Harosoy and Clark (WmWm) exhibited longer fragments, while Harosoy-wm had shorter fragments due to the single-base deletion. The CAPS marker co-segregated with genotypes at Wm locus in a F(2) population segregating for the locus. Linkage mapping using SSR markers revealed that the Wm and gmfls1 were mapped at similar position in the molecular linkage group F. The above results strongly suggest that gmfls1 represents the Wm gene and that the single-base deletion may be responsible for magenta flower color.

[Application of single nucleotide polymorphism-microarray and target gene sequencing in the study of genetic etiology of children with unexplained intellectual disability or developmental delay].

PubMed

Gao, Z J; Jiang, Q; Cheng, D Z; Yan, X X; Chen, Q; Xu, K M

2016-10-02

Objective: To evaluate the application of single nucleotide polymorphism (SNP)-microarray and target gene sequencing technology in the clinical molecular genetic diagnosis of unexplained intellectual disability(ID) or developmental delay (DD). Method: Patients with ID or DD were recruited in the Department of Neurology, Affiliated Children's Hospital of Capital Institute of Pediatrics between September 2015 and February 2016. The intellectual assessment of the patients was performed using 0-6-year-old pediatric examination table of neuropsychological development or Wechsler intelligence scale (>6 years). Patients with a DQ less than 49 or IQ less than 51 were included in this study. The patients were scanned by SNP-array for detection of genomic copy number variations (CNV), and the revealed genomic imbalance was confirmed by quantitative real time-PCR. Candidate gene mutation screening was carried out by target gene sequencing technology.Causal mutations or likely pathogenic variants were verified by polymerase chain reaction and direct sequencing. Result: There were 15 children with ID or DD enrolled, 9 males and 6 females. The age of these patients was 7 months-16 years and 9 months. SNP-array revealed that two of the 15 patients had genomic CNV. Both CNV were de novo micro deletions, one involved 11q24.1q25 and the other micro deletion located on 21q22.2q22.3. Both micro deletions were proved to have a clinical significance due to their association with ID, brain DD, unusual faces etc. by querying Decipher database. Thirteen patients with negative findings in SNP-array were consequently examined with target gene sequencing technology, genotype-phenotype correlation analysis and genetic analysis. Five patients were diagnosed with monogenic disorder, two were diagnosed with suspected genetic disorder and six were still negative. Conclusion: Sequential use of SNP-array and target gene sequencing technology can significantly increase the molecular genetic etiologic

Targeted Deletion of the Gene Encoding the La Autoantigen (Sjögren's Syndrome Antigen B) in B Cells or the Frontal Brain Causes Extensive Tissue Loss

PubMed Central

Gaidamakov, Sergei; Maximova, Olga A.; Chon, Hyongi; Blewett, Nathan H.; Wang, Hongsheng; Crawford, Amanda K.; Day, Amanda; Tulchin, Natalie; Crouch, Robert J.; Morse, Herbert C.; Blitzer, Robert D.

2014-01-01

La antigen (Sjögren's syndrome antigen B) is a phosphoprotein associated with nascent precursor tRNAs and other RNAs, and it is targeted by autoantibodies in patients with Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Increased levels of La are associated with leukemias and other cancers, and various viruses usurp La to promote their replication. Yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) genetically depleted of La grow and proliferate, whereas deletion from mice causes early embryonic lethality, raising the question of whether La is required by mammalian cells generally or only to surpass a developmental stage. We developed a conditional La allele and used it in mice that express Cre recombinase in either B cell progenitors or the forebrain. B cell Mb1Cre La-deleted mice produce no B cells. Consistent with αCamKII Cre, which induces deletion in hippocampal CA1 cells in the third postnatal week and later throughout the neocortex, brains develop normally in La-deleted mice until ∼5 weeks and then lose a large amount of forebrain cells and mass, with evidence of altered pre-tRNA processing. The data indicate that La is required not only in proliferating cells but also in nondividing postmitotic cells. Thus, La is essential in different cell types and required for normal development of various tissue types. PMID:24190965

Zinc-finger Nuclease-induced Gene Repair With Oligodeoxynucleotides: Wanted and Unwanted Target Locus Modifications

PubMed Central

Radecke, Sarah; Radecke, Frank; Cathomen, Toni; Schwarz, Klaus

2010-01-01

Correcting a mutated gene directly at its endogenous locus represents an alternative to gene therapy protocols based on viral vectors with their risk of insertional mutagenesis. When solely a single-stranded oligodeoxynucleotide (ssODN) is used as a repair matrix, the efficiency of the targeted gene correction is low. However, as shown with the homing endonuclease I-SceI, ssODN-mediated gene correction can be enhanced by concomitantly inducing a DNA double-strand break (DSB) close to the mutation. Because I-SceI is hardly adjustable to cut at any desired position in the human genome, here, customizable zinc-finger nucleases (ZFNs) were used to stimulate ssODN-mediated repair of a mutated single-copy reporter locus stably integrated into human embryonic kidney-293 cells. The ZFNs induced faithful gene repair at a frequency of 0.16%. Six times more often, ZFN-induced DSBs were found to be modified by unfaithful addition of ssODN between the termini and about 60 times more often by nonhomologous end joining-related deletions and insertions. Additionally, ZFN off-target activity based on binding mismatch sites at the locus of interest was detected in in vitro cleavage assays and also in chromosomal DNA isolated from treated cells. Therefore, the specificity of ZFN-induced ssODN-mediated gene repair needs to be improved, especially regarding clinical applications. PMID:20068556

Novel drug targets in cell wall biosynthesis exploited by gene disruption in Pseudomonas aeruginosa.

PubMed

Elamin, Ayssar A; Steinicke, Susanne; Oehlmann, Wulf; Braun, Yvonne; Wanas, Hanaa; Shuralev, Eduard A; Huck, Carmen; Maringer, Marko; Rohde, Manfred; Singh, Mahavir

2017-01-01

For clinicians, Pseudomonas aeruginosa is a nightmare pathogen that is one of the top three causes of opportunistic human infections. Therapy of P. aeruginosa infections is complicated due to its natural high intrinsic resistance to antibiotics. Active efflux and decreased uptake of drugs due to cell wall/membrane permeability appear to be important issues in the acquired antibiotic tolerance mechanisms. Bacterial cell wall biosynthesis enzymes have been shown to be essential for pathogenicity of Gram-negative bacteria. However, the role of these targets in virulence has not been identified in P. aeruginosa. Here, we report knockout (k.o) mutants of six cell wall biosynthesis targets (murA, PA4450; murD, PA4414; murF, PA4416; ppiB, PA1793; rmlA, PA5163; waaA, PA4988) in P. aeruginosa PAO1, and characterized these in order to find out whether these genes and their products contribute to pathogenicity and virulence of P. aeruginosa. Except waaA k.o, deletion of cell wall biosynthesis targets significantly reduced growth rate in minimal medium compared to the parent strain. The k.o mutants showed exciting changes in cell morphology and colonial architectures. Remarkably, ΔmurF cells became grossly enlarged. Moreover, the mutants were also attenuated in vivo in a mouse infection model except ΔmurF and ΔwaaA and proved to be more sensitive to macrophage-mediated killing than the wild-type strain. Interestingly, the deletion of the murA gene resulted in loss of virulence activity in mice, and the virulence was restored in a plant model by unknown mechanism. This study demonstrates that cell wall targets contribute significantly to intracellular survival, in vivo growth, and pathogenesis of P. aeruginosa. In conclusion, these findings establish a link between cell wall targets and virulence of P. aeruginosa and thus may lead to development of novel drugs for the treatment of P. aeruginosa infection.

Nance-Horan syndrome: a contiguous gene syndrome involving deletion of the amelogenin gene? A case report and molecular analysis.

PubMed

Franco, E; Hodgson, S; Lench, N; Roberts, G J

1995-03-01

A case of Nance-Horan syndrome in a male is presented, with some features of the condition in his carrier mother and her mother. It is proposed that Nance-Horan syndrome might be a contiguous gene syndrome mapping to chromosome Xp21.2-p22.3. The proband had congenital cataract microphthalmia and dental abnormalities including screwdriver shaped incisors and evidence of enamel pitting hypoplasia. The region Xp21.2-p22.3 also contains the tooth enamel protein gene, amelogenin (AMGX). Using molecular genetic techniques, we have shown that there is no evidence that the AMGX gene is deleted in this case of the Nance-Horan syndrome.

Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant.

PubMed

Mizutani, Osamu; Arazoe, Takayuki; Toshida, Kenji; Hayashi, Risa; Ohsato, Shuichi; Sakuma, Tetsushi; Yamamoto, Takashi; Kuwata, Shigeru; Yamada, Osamu

2017-03-01

Transcription activator-like effector nucleases (TALENs), which can generate DNA double-strand breaks at specific sites in the desired genome locus, have been used in many organisms as a tool for genome editing. In Aspergilli, including Aspergillus oryzae, however, the use of TALENs has not been validated. In this study, we performed genome editing of A. oryzae wild-type strain via error of nonhomologous end-joining (NHEJ) repair by transient expression of high-efficiency Platinum-Fungal TALENs (PtFg TALENs). Targeted mutations were observed as various mutation patterns. In particular, approximately half of the PtFg TALEN-mediated deletion mutants had deletions larger than 1 kb in the TALEN-targeting region. We also conducted PtFg TALEN-based genome editing in A. oryzae ligD disruptant (ΔligD) lacking the ligD gene involved in the final step of the NHEJ repair and found that mutations were still obtained as well as wild-type. In this case, the ratio of the large deletions reduced compared to PtFg TALEN-based genome editing in the wild-type. In conclusion, we demonstrate that PtFg TALENs are sufficiently functional to cause genome editing via error of NHEJ in A. oryzae. In addition, we reveal that genome editing using TALENs in A. oryzae tends to cause large deletions at the target region, which were partly suppressed by deletion of ligD. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Generation of megabase-scale deletions, inversions and duplications involving the Contactin-6 gene in mice by CRISPR/Cas9 technology.

PubMed

Korablev, Alexei N; Serova, Irina A; Serov, Oleg L

2017-12-28

Copy Number Variation (CNV) of the human CNTN6 gene (encoding the contactin-6 protein), caused by deletions or duplications, is responsible for severe neurodevelopmental impairments, often in combination with facial dysmorphias. Conversely, deleterious point mutations of this gene do not show any clinical phenotypes. The aim of this study is to generate mice carrying large deletions, duplications and inversions involving the Cntn6 gene as a new experimental model to study CNV of the human CNTN6 locus. To generate large chromosomal rearrangements on mouse chromosome 6, we applied CRISPR/Cas9 technology in zygotes. Two guide RNAs (gRNAs) (flanking a DNA fragment of 1137 Mb) together with Cas9 mRNA and single-stranded DNA oligonucleotides (ssODN) were microinjected into the cytoplasm of 599 zygotes of F1 (C57BL x CBA) mice, and 256 of them were transplanted into oviducts of CD-1 females. As a result, we observed the birth of 41 viable F0 offspring. Genotyping of these mice was performed by PCR analysis and sequencing of PCR products. Among the 41 F0 offspring, we identified seven mice with deletions, two animals carrying duplications of the gene and four carrying inversions. Interestingly, two F0 offspring had both deletions and duplications. It is important to note that while three of seven deletion carriers showed expected sequences at the new joint sites, in another three, we identified an absence of 1-10 nucleotides at the CRISPR/Cas9 cut sites, and in one animal, 103 bp were missing, presumably due to error-prone non-homologous end joining. In addition, we detected the absence of 5 and 13 nucleotides at these sites in two F0 duplication carriers. Similar sequence changes at CRISPR/Cas9 cut sites were observed at the right and left boundaries of inversions. Thus, megabase-scale deletions, duplications and inversions were identified in 11 F0 offspring among 41 analyzed, i.e., approximately 25% efficiency. All genetically modified F0 offspring were viable and

Increased frequency of DNA deletions in pink-eyed unstable mice carrying a mutation in the Werner syndrome gene homologue.

PubMed

Lebel, Michel

2002-01-01

Werner syndrome (WS) is a rare autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases, including cancers. Accumulating evidence indicates that the WS gene product is involved in resolving aberrant DNA structures that may arise during the process of DNA replication and/or transcription. To estimate the frequency of DNA deletions directly in the skin of mouse embryos, mice with a deletion of part of the murine WRN helicase domain were created. These mutant mice were then crossed to the pink-eyed unstable animals, which have a 70 kb internal duplication at the pink-eyed dilution (p) gene. This report indicates that the frequency of deletion of the duplicated sequence at the p locus is elevated in mice with a mutation in the WRN allele when compared with wild-type mice. In addition, the inhibitor of topoisomerase I camptothecin also increases the frequency of deletion at the p locus. This frequency is even more elevated in WRN mutant mice treated with camptothecin. In contrast, while the inhibition of poly(ADP-ribose) polymerase (PARP) activity by 3-aminobenzamide increases the frequency of DNA deletion, mutant WRN mice are not significantly more sensitive to the inhibition of PARP activity than wild-type animals.

Constitutional von Hippel-Lindau (VHL) gene deletions detected in VHL families by fluorescence in situ hybridization.

PubMed

Pack, S D; Zbar, B; Pak, E; Ault, D O; Humphrey, J S; Pham, T; Hurley, K; Weil, R J; Park, W S; Kuzmin, I; Stolle, C; Glenn, G; Liotta, L A; Lerman, M I; Klausner, R D; Linehan, W M; Zhuang, Z

1999-11-01

von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited cancer syndrome predisposing to a variety of tumor types that include retinal hemangioblastomas, hemangioblastomas of the central nervous system, renal cell carcinomas, pancreatic cysts and tumors, pheochromocytomas, endolymphatic sac tumors, and epididymal cystadenomas [W. M. Linehan et al., J. Am. Med. Assoc., 273: 564-570, 1995; E. A. Maher and W. G. Kaelin, Jr., Medicine (Baltimore), 76: 381-391, 1997; W. M. Linehan and R. D. Klausner, In: B. Vogelstein and K. Kinzler (eds.), The Genetic Basis of Human Cancer, pp. 455-473, McGraw-Hill, 1998]. The VHL gene was localized to chromosome 3p25-26 and cloned [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. Germline mutations in the VHL gene have been detected in the majority of VHL kindreds. The reported frequency of detection of VHL germline mutations has varied from 39 to 80% (J. M. Whaley et al., Am. J. Hum. Genet., 55: 1092-1102, 1994; Clinical Research Group for Japan, Hum. Mol. Genet., 4: 2233-2237, 1995; F. Chen et al., Hum. Mutat., 5: 66-75, 1995; E. R. Maher et al., J. Med. Genet., 33: 328-332, 1996; B. Zbar, Cancer Surv., 25: 219-232, 1995). Recently a quantitative Southern blotting procedure was found to improve this frequency (C. Stolle et al., Hum. Mutat., 12: 417-423, 1998). In the present study, we report the use of fluorescence in situ hybridization (FISH) as a method to detect and characterize VHL germline deletions. We reexamined a group of VHL patients shown previously by single-strand conformation and sequencing analysis not to harbor point mutations in the VHL locus. We found constitutional deletions in 29 of 30 VHL patients in this group using cosmid and P1 probes that cover the VHL locus. We then tested six phenotypically normal offspring from four of these VHL families: two were found to carry the deletion and the other four were deletion-free. In addition, germline mosaicism of the VHL gene was identified in

Novel deletions involving the USH2A gene in patients with Usher syndrome and retinitis pigmentosa.

PubMed

García-García, Gema; Aller, Elena; Jaijo, Teresa; Aparisi, Maria J; Larrieu, Lise; Faugère, Valérie; Blanco-Kelly, Fiona; Ayuso, Carmen; Roux, Anne-Francoise; Millán, José M

2014-01-01

The aim of the present work was to identify and characterize large rearrangements involving the USH2A gene in patients with Usher syndrome and nonsyndromic retinitis pigmentosa. The multiplex ligation-dependent probe amplification (MLPA) technique combined with a customized array-based comparative genomic hybridization (aCGH) analysis was applied to 40 unrelated patients previously screened for point mutations in the USH2A gene in which none or only one pathologic mutation was identified. We detected six large deletions involving USH2A in six out of the 40 cases studied. Three of the patients were homozygous for the deletion, and the remaining three were compound heterozygous with a previously identified USH2A point mutation. In five of these cases, the patients displayed Usher type 2, and the remaining case displayed nonsyndromic retinitis pigmentosa. The exact breakpoint junctions of the deletions found in USH2A in four of these cases were characterized. Our study highlights the need to develop improved efficient strategies of mutation screening based upon next generation sequencing (NGS) that reduce cost, time, and complexity and allow simultaneous identification of all types of disease-causing mutations in diagnostic procedures.

Confirmation that a deletion in the POMC gene is associated with body weight of Labrador Retriever dogs.

PubMed

Mankowska, M; Krzeminska, P; Graczyk, M; Switonski, M

2017-06-01

A 14-bp deletion present in the proopiomelanocortin (POMC) gene of Labrador and Flat Coat Retrievers (FCR), but absent in POMC of other breeds, disrupts the β-MSH and β-endorphin coding sequences. This deletion was recently reported as strongly associated with increased body weight and obesity. We searched for this mutation in a cohort of 272 dogs, representing four breeds with a known predisposition to obesity (Labrador and Golden Retrievers, Beagle, and Cocker Spaniel) and, as expected, we found it only in Labradors. Further, we confirmed the association between the deletion variant and body weight of Labradors but not with a 5-point body condition score (BCS). We suspect that the deletion variant in our cohort may act as a recessive allele, unlike the previous study, which suggested its additive effect. Copyright © 2017 Elsevier Ltd. All rights reserved.

A novel deletion in the thyrotropin Beta-subunit gene identified by array comparative genomic hybridization analysis causes central congenital hypothyroidism in a boy originating from Turkey.

PubMed

Hermanns, Pia; Couch, Robert; Leonard, Norma; Klotz, Cherise; Pohlenz, Joachim

2014-01-01

Isolated central congenital hypothyroidism (ICCH) is rare but important. Most ICCH patients are diagnosed later, which results in severe growth failure and intellectual disability. We describe a boy with ICCH due to a large homozygous TSHβ gene deletion. A 51-day-old male Turkish infant, whose parents were first cousins, was admitted for evaluation of prolonged jaundice. His clinical appearance was compatible with hypothyroidism. Venous thyrotropin (TSH) was undetectably low, with a subsequent low free T4 and a low free T3, suggestive of central hypothyroidism. Using different PCR protocols, we could not amplify both coding exons of the boy's TSHβ gene, which suggested a deletion. An array comparative genomic hybridization (aCGH) using specific probes around the TSHβ gene locus showed him to be homozygous for a 6-kb deletion spanning all exons and parts of the 5' untranslated region of the gene. Infants who are clinically suspected of having hypothyroidism should be evaluated thoroughly, even if their TSH-based screening result is normal. In cases with ICCH and undetectably low TSH serum concentrations, a TSHβ gene deletion should be considered; aCGH should be performed when gene deletions are suspected. In such cases, PCR-based sequencing techniques give negative results.

Two novel types of contiguous gene deletion of the AVPR2 and ARHGAP4 genes in unrelated Japanese kindreds with nephrogenic diabetes insipidus.

PubMed

Demura, Masashi; Takeda, Yoshiyu; Yoneda, Takashi; Furukawa, Kenji; Usukura, Mikiya; Itoh, Yuji; Mabuchi, Hiroshi

2002-01-01

Study of two families containing individuals with nephrogenic diabetes insipidus (NDI) indicated different types of 21.3 kb and 26.3 kb deletions involving the AVPR2 and ARHGAP4 (RhoGAP C1) genes. In the case of the 21.3 kb deletion, the deletion consensus motif (5'-TGAAGG-3') and polypurine runs, known as the arrest site of polymerase alpha, were detected in the vicinity of the deletion junction. Inverted repeats (7/8 matches), believed to potentiate DNA loop formation, flank the deletion breakpoint. We propose this deletion to be the result of slipped mispairing during DNA replication. In the case of the 26.3 kb deletion, the 12,945 bp inverted region with the 10,003 bp internal deletion was accompanied with the 2,509 bp deletion in the 5'-side and the 13,785 bp deletion in the 3'-side. We defined three deletion junctions in this rearrangement (DJ1, DJ2, and DJ3) from the 5'-side. The surrounding sequence of DJ1 (5'-CCC-3') closely resembled that of DJ3 (5'-AGGG-3') (DJ1; 5'-cCCCgaggg-3', DJ3; 5'-ccccAGGG-3'), and DJ1 was located in the 5'-side of DJ3 without any overlapping in sequence. The immunoglobulin class switch (ICS) motif (5'-TGGGG-3') was found around the complementary sequence of DJ3. There was a 10-base palindrome (5'-aGACAtgtct-3') in the alignment of the DJ2 (5'-GACA-3') region. From these findings, we propose a novel mutation process with the rearrangement probably resulting from stem-loop induced non-homologous recombination in an ICS-like fashion. Both patients, despite lacking ARHGAP4, had no morphological, clinical, or laboratory abnormalities except for those usually found in patients with NDI. Copyright 2001 Wiley-Liss, Inc.

The effects of upaB deletion and the double/triple deletion of upaB, aatA, and aatB genes on pathogenicity of avian pathogenic Escherichia coli.

PubMed

Zhu-Ge, Xiang-Kai; Pan, Zi-Hao; Tang, Fang; Mao, Xiang; Hu, Lin; Wang, Shao-Hui; Xu, Bin; Lu, Cheng-Ping; Fan, Hong-Jie; Dai, Jian-Jun

2015-12-01

Autotransporters (ATs) are associated with pathogenesis of Avian Pathogenic Escherichia coli (APEC). The molecular characterization of APEC ATs can provide insights about their relevance to APEC pathogenesis. Here, we characterized a conventional autotransporter UpaB in APEC DE205B genome. The upaB existed in 41.9 % of 236 APEC isolates and was predominantly associated with ECOR B2 and D. Our studies showed that UpaB mediates the DE205B adhesion in DF-1 cells, and enhances autoaggregation and biofilm formation of fimbria-negative E. coli AAEC189 (MG1655Δfim) in vitro. Deletion of upaB of DE205B attenuates the virulence in duck model and early colonization in the duck lungs during APEC systemic infection. Furthermore, double and triple deletion of upaB, aatA, and aatB genes cumulatively attenuated DE205B adhesion in DF-1 cells, accompanying with decreased 50 % lethal dose (LD50) in duck model and the early colonization in the duck lungs. However, DE205BΔupaB/ΔaatA/ΔaatB might "compensate" the influence of gene deletion by upregulating the expression of fimbrial adhesin genes yqiL, yadN, and vacuolating autotransporter vat during early colonization of APEC. Finally, we demonstrated that vaccination with recombinant UpaB, AatA, and AatB proteins conferred protection against colisepticemia caused by DE205B infection in duck model.

Application of oligonucleotide array CGH to the simultaneous detection of a deletion in the nuclear TK2 gene and mtDNA depletion.

PubMed

Zhang, Shulin; Li, Fang-Yuan; Bass, Harold N; Pursley, Amber; Schmitt, Eric S; Brown, Blaire L; Brundage, Ellen K; Mardach, Rebecca; Wong, Lee-Jun

2010-01-01

Thymidine kinase 2 (TK2), encoded by the TK2 gene on chromosome 16q22, is one of the deoxyribonucleoside kinases responsible for the maintenance of mitochondrial deoxyribonucleotide pools. Defects in TK2 mainly cause a myopathic form of the mitochondrial DNA depletion syndrome (MDDS). Currently, only point mutations and small insertions and deletions have been reported in TK2 gene; gross rearrangements of TK2 gene and possible hepatic involvement in patients with TK2 mutations have not been described. We report a non-consanguineous Jordanian family with three deceased siblings due to mtDNA depletion. Sequence analysis of the father detected a heterozygous c.761T>A (p.I254N) mutation in his TK2 gene; however, point mutations in the mother were not detected. Subsequent gene dosage analysis using oligonucleotide array CGH identified an intragenic approximately 5.8-kb deletion encompassing the 5'UTR to intron 2 of her TK2 gene. Sequence analysis confirmed that the deletion spans c.1-495 to c.283-2899 of the TK2 gene (nucleotide 65,136,256-65,142,086 of chromosome 16). Analysis of liver and muscle specimens from one of the deceased infants in this family revealed compound heterozygosity for the paternal point mutation and maternal intragenic deletion. In addition, a significant reduction of the mtDNA content in liver and muscle was detected (10% and 20% of age- and tissue-matched controls, respectively). Prenatal diagnosis was performed in the third pregnancy. The fetus was found to carry both the point mutation and the deletion. This child died 6months after birth due to myopathy. A serum specimen demonstrated elevated liver transaminases in two of the infants from whom results were available. This report expands the mutation spectrum associated with TK2 deficiency. While the myopathic form of MDDS appears to be the main phenotype of TK2 mutations, liver dysfunction may also be a part of the mitochondrial depletion syndrome caused by TK2 gene defects.

Large FVIII gene deletion confers very high risk of inhibitor development in three related severe haemophiliacs.

PubMed

Salviato, R; Belvini, D; Are, A; Radossi, P; Tagariello, G

2002-01-01

Haemophilia A displays a broad heterogeneity of genetic defects and of clinical severity. Inhibitor development is the main complication of replacement therapy in severe cases and most patients with inhibitors have gross gene rearrangement or point mutations, which hamper the production of normal circulating factor VIII (FVIII). We have investigated three related severe haemophilia A patients, all of whom have high titre inhibitors. By using long-range polymerase chain reaction (PCR) for FVIII gene inversion, we observed an unusual pattern in these patients. We therefore decided to screen the whole FVIII gene by conformation-sensitive gel electrophoresis. A large FVIII gene deletion spanning exon 2 to exon 25 was identified and we were able to obtain a 18.5 kb PCR product, which is specific for this mutation and useful for determining the carrier state in this family. All three haemophiliacs carrying this very large gene deletion show similar clinical history and very high-titre inhibitors, supporting the observation that inhibitor development seems to be an inherited characteristic. On the basis of our observations we think that this subgroup of patients at very high risk of inhibitor development should be identified by mutation analysis whenever possible, before the beginning of replacement therapy.

Pattern of deletions of the dystrophin gene in Mexican Duchenne/Becker muscular dystrophy patients: the use of new designed primers for the analysis of the major deletion "hot spot" region.

PubMed

Coral-Vazquez, R; Arenas, D; Cisneros, B; Peñaloza, L; Salamanca, F; Kofman, S; Mercado, R; Montañez, C

1997-06-13

We have analyzed 59 unrelated Mexican Duchenne/Becker muscular dystrophy patients (DMD/BMD) using PCR analysis of the 2 prone deletion regions in the DMD gene. Thirty one (52%) of the patients had a deletion of one or several of the exons. Most of the alterations (87%) were clustered in exons 44-52, this being the highest percentage reported until now. In order to improve the molecular diagnosis in the Mexican population, we designed a new multiplex assay to PCR amplify exons 44-52. This assay allowed for the identification of a greater number of deletions in this region compared with the 9 and 5-plex assays previously described and to determine most of the deletion end boundaries. This is a reliable alternative for the initial screening of the DMD patients in the Mexican population.

Identification of a novel AluSx-mediated deletion of exon 3 in the SBDS gene in a patient with Shwachman-Diamond syndrome.

PubMed

Costa, Elísio; Duque, Frederico; Oliveira, Jorge; Garcia, Paula; Gonçalves, Isabel; Diogo, Luísa; Santos, Rosário

2007-01-01

Shwachman-Diamond syndrome (SDS) is caused by mutations in the SBDS gene, most of which are the result of gene conversion events involving its highly homologous pseudogene SBDSP. Here we describe the molecular characterization of the first documented gross deletion in the SBDS gene, in a 4-year-old Portuguese girl with SDS. The clinical diagnosis was based on the presence of hematological symptoms (severe anemia and cyclic neutropenia), pancreatic exocrine insufficiency and skeletal abnormalities. Routine molecular screening revealed heterozygosity for the common splicing mutation c.258+2T>C, and a further step-wise approach led to the detection of a large deletion encompassing exon 3, the endpoints of which were subsequently delineated at the gDNA level. This novel mutation (c.258+374_459+250del), predictably giving rise to an internally deleted polypeptide (p.Ile87_Gln153del), appears to have arisen from an excision event mediated by AluSx elements which are present in introns 2 and 3. Our case illustrates the importance of including gross deletion screening in the SDS diagnostic setting, especially in cases where only one deleterious mutation is detected by routine screening methods. In particular, deletional rearrangements involving exon 3 should be considered, since Alu sequences are known to be an important cause of recurrent mutations.

Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells

PubMed Central

Bressan, Raul Bardini; Dewari, Pooran Singh; Kalantzaki, Maria; Gangoso, Ester; Matjusaitis, Mantas; Garcia-Diaz, Claudia; Blin, Carla; Grant, Vivien; Bulstrode, Harry; Gogolok, Sabine; Skarnes, William C.

2017-01-01

Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable – experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci (Rosa26 and AAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry); and (4) engineering of glioma mutations (TP53 deletion; H3F3A point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis. PMID:28096221

About miRNAs, miRNA seeds, target genes and target pathways.

PubMed

Kehl, Tim; Backes, Christina; Kern, Fabian; Fehlmann, Tobias; Ludwig, Nicole; Meese, Eckart; Lenhof, Hans-Peter; Keller, Andreas

2017-12-05

miRNAs are typically repressing gene expression by binding to the 3' UTR, leading to degradation of the mRNA. This process is dominated by the eight-base seed region of the miRNA. Further, miRNAs are known not only to target genes but also to target significant parts of pathways. A logical line of thoughts is: miRNAs with similar (seed) sequence target similar sets of genes and thus similar sets of pathways. By calculating similarity scores for all 3.25 million pairs of 2,550 human miRNAs, we found that this pattern frequently holds, while we also observed exceptions. Respective results were obtained for both, predicted target genes as well as experimentally validated targets. We note that miRNAs target gene set similarity follows a bimodal distribution, pointing at a set of 282 miRNAs that seems to target genes with very high specificity. Further, we discuss miRNAs with different (seed) sequences that nonetheless regulate similar gene sets or pathways. Most intriguingly, we found miRNA pairs that regulate different gene sets but similar pathways such as miR-6886-5p and miR-3529-5p. These are jointly targeting different parts of the MAPK signaling cascade. The main goal of this study is to provide a general overview on the results, to highlight a selection of relevant results on miRNAs, miRNA seeds, target genes and target pathways and to raise awareness for artifacts in respective comparisons. The full set of information that allows to infer detailed results on each miRNA has been included in miRPathDB, the miRNA target pathway database (https://mpd.bioinf.uni-sb.de).

A Deletion of More than 800 kb Is the Most Recurrent Mutation in Chilean Patients with SHOX Gene Defects.

PubMed

Poggi, Helena; Vera, Alejandra; Avalos, Carolina; Lagos, Marcela; Mellado, Cecilia; Aracena, Mariana; Aravena, Teresa; Garcia, Hernan; Godoy, Claudia; Cattani, Andreina; Reyes, Loreto; Lacourt, Patricia; Rumie, Hana; Mericq, Veronica; Arriaza, Marta; Martinez-Aguayo, Alejandro

2015-01-01

Deletions in the SHOX gene are the most frequent genetic cause of Leri-Weill syndrome and Langer mesomelic dysplasia, which are also present in idiopathic short stature. To describe the molecular and clinical findings observed in 23 of 45 non-consanguineous Chilean patients with different phenotypes related to SHOX deficiency. Multiplex ligation-dependent probe amplification was used to detect the deletions; the SHOX coding region and deletion-flanking areas were sequenced to identify point mutations and single-nucleotide polymorphisms (SNPs). The main genetic defects identified in 21 patients consisted of deletions; one of them, a large deletion of >800 kb, was found in 8 patients. Also, a smaller deletion of >350 kb was observed in 4 patients. Although we could not precisely determine the deletion breakpoint, we were able to identify a common haplotype in 7 of the 8 patients with the larger deletion based on 22 informative SNPs. These results suggest that the large deletion-bearing allele has a common ancestor and was either introduced by European immigrants or had originated in our Amerindian population. This study allowed us to identify one recurrent deletion in Chilean patients; also, it contributed to expanding our knowledge about the genetic background of our population. © 2015 S. Karger AG, Basel.

Large Deletions of TSPAN12 Cause Familial Exudative Vitreoretinopathy (FEVR).

PubMed

Seo, Soo Hyun; Kim, Man Jin; Park, Sung Wook; Kim, Jeong Hun; Yu, Young Suk; Song, Ji Yun; Cho, Sung Im; Ahn, Joo Hyun; Oh, Yeon Hee; Lee, Jee-Soo; Lee, Seungjun; Seong, Moon-Woo; Park, Sung Sup; Kim, Ji Yeon

2016-12-01

Familial exudative vitreoretinopathy (FEVR) is a rare, hereditary visual disorder. The gene TSPAN12 is associated with autosomal dominant inheritance of FEVR. The prevalence and impact of large deletions/duplications of TSPAN12 on FEVR patients is unknown. To glean better insight of TSPAN12 on FEVR pathology, herein, we describe three FEVR patients with TSPAN12 deletions. Thirty-three Korean FEVR patients, who previously screened negative for TSPAN12 mutations, mutations in other FEVR-associated genes such as NDP, FZD4, LRP5, and large deletions and duplications of NDP, FZD4, and LRP5, were selected for TSPAN12 large deletion and duplication analyses. Semiquantitative multiplex PCR for TSPAN12 gene dosage analyses were performed, followed by droplet digital PCR (ddPCR) for validation. Among the 33 patients, three patients were confirmed to carry large TSPAN12 deletions. Two of them had whole-gene deletions of TSPAN12, and the other patient possessed a deletion of TSPAN12 in exon 4. FEVR severity detected in these patients was not more severe than in a patient with TSPAN12 point mutation. Regarding previously reported proportions of FEVR-associated genes contributing to the disorder's autosomal dominant inheritance pattern in Korea, we determined that patients with TSPAN12 large deletions were more common than patients with single nucleotide variants in TSPAN12. Evaluating TSPAN12 large deletions and duplications should be considered in FEVR screening and diagnosis as well as in routine genetic workups for FEVR patients.

Cryptic intragenic deletion of the SHOX gene in a family with Léri-Weill dyschondrosteosis detected by Multiplex Ligation-Dependent Probe Amplification (MLPA).

PubMed

Funari, Mariana F A; Jorge, Alexander A L; Pinto, Emilia M; Arnhold, Ivo J P; Mendonca, Berenice B; Nishi, Mirian Y

2008-11-01

LWD is associated to SHOX haploinsufficiency, in most cases, due to gene deletion. Generally FISH and microsatellite analysis are used to identify SHOX deletion. MLPA is a new method of detecting gene copy variation, allowing simultaneous analysis of several regions. Here we describe the presence of a SHOX intragenic deletion in a family with LWD, analyzed through different methodologies. Genomic DNA of 11 subjects from one family were studied by microsatellite analysis, direct sequencing and MLPA. FISH was performed in two affected individuals. Microsatellite analysis showed that all affected members shared the same haplotype suggesting the involvement of SHOX. MLPA detected an intragenic deletion involving exons IV-VIa, which was not detected by FISH and microsatellite analysis. In conclusion, the MLPA technique was proved to be the best solution on detecting this small deletion, it has the advantage of being less laborious also allowing the analysis of several regions simultaneously.

Lethality of mice bearing a knockout of the Ngly1-gene is partially rescued by the additional deletion of the Engase gene

PubMed Central

Fujihira, Haruhiko; Masahara-Negishi, Yuki; Tamura, Masaru; Huang, Chengcheng; Harada, Yoichiro; Wakana, Shigeharu; Takakura, Daisuke; Kawasaki, Nana; Taniguchi, Naoyuki; Kondoh, Gen; Yamashita, Tadashi; Funakoshi, Yoko; Suzuki, Tadashi

2017-01-01

The cytoplasmic peptide:N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-β-N-acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1- or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder. PMID:28426790

Detection of large scale 3' deletions in the PMS2 gene amongst Colon-CFR participants: have we been missing anything?

PubMed

Clendenning, Mark; Walsh, Michael D; Gelpi, Judith Balmana; Thibodeau, Stephen N; Lindor, Noralane; Potter, John D; Newcomb, Polly; LeMarchand, Loic; Haile, Robert; Gallinger, Steve; Hopper, John L; Jenkins, Mark A; Rosty, Christophe; Young, Joanne P; Buchanan, Daniel D

2013-09-01

Current screening practices have been able to identify PMS2 mutations in 78 % of cases of colorectal cancer from the Colorectal Cancer Family Registry (Colon CFR) which showed solitary loss of the PMS2 protein. However the detection of large-scale deletions in the 3' end of the PMS2 gene has not been possible due to technical difficulties associated with pseudogene sequences. Here, we utilised a recently described MLPA/long-range PCR-based approach to screen the remaining 22 % (n = 16) of CRC-affected probands for mutations in the 3' end of the PMS2 gene. No deletions encompassing any or all of exons 12 through 15 were identified; therefore, our results suggest that 3' deletions in PMS2 are not a frequent occurrence in such families.

Design and Validation of a New MLPA-Based Assay for the Detection of RS1 Gene Deletions and Application in a Large Family with X-Linked Juvenile Retinoschisis.

PubMed

Nicoletti, Annalisa; Ziccardi, Lucia; Maltese, Paolo Enrico; Benedetti, Sabrina; Palumbo, Orazio; Rendina, Michelina; D'Agruma, Leonardo; Falsini, Benedetto; Wang, Xinjing; Bertelli, Matteo

2017-02-01

X-linked juvenile retinoschisis (XLRS) is a severe ocular disorder that can evolve to blindness. More than 200 different disease-causing mutations have been reported in the RS1 gene and approximately 10% of these are deletions. Since transmission is X-linked, males are always affected and females are usually carriers. The identification of female carriers is always important and poses a technical challenge. Therefore, we sought to develop a multiplex ligation dependent probe amplification (MLPA)-based method to identify deletions or duplications in this gene. We then used our assay to study a large XLRS family. We designed six probes specific for each RS1 exon and then optimized and validated our method using control samples with known gene deletions. In the XLRS family, RS1 gene copy number variation was assessed by "home-made" MLPA analysis and by single nucleotide polymorphism (SNP) array analysis using the CytoScan HD Array. Direct sequencing was used for deletion breakpoint mapping. Our assay detected all deletions in control samples. All affected males of the family were positive for a deletion of exon 2 of the RS1 gene (RS1:NM_000330:c.53-?_78+?del). Carrier females were also identified. Our method is easily replicated, reliable, and inexpensive and allows female carriers to be detected. This is the first report of deep characterization of a whole exon deletion in the RS1 gene.

Design and Validation of a New MLPA-Based Assay for the Detection of RS1 Gene Deletions and Application in a Large Family with X-Linked Juvenile Retinoschisis

PubMed Central

Nicoletti, Annalisa; Ziccardi, Lucia; Benedetti, Sabrina; Palumbo, Orazio; Rendina, Michelina; D'Agruma, Leonardo; Falsini, Benedetto; Wang, Xinjing; Bertelli, Matteo

2017-01-01

Aims: X-linked juvenile retinoschisis (XLRS) is a severe ocular disorder that can evolve to blindness. More than 200 different disease-causing mutations have been reported in the RS1 gene and approximately 10% of these are deletions. Since transmission is X-linked, males are always affected and females are usually carriers. The identification of female carriers is always important and poses a technical challenge. Therefore, we sought to develop a multiplex ligation dependent probe amplification (MLPA)-based method to identify deletions or duplications in this gene. We then used our assay to study a large XLRS family. Methods: We designed six probes specific for each RS1 exon and then optimized and validated our method using control samples with known gene deletions. In the XLRS family, RS1 gene copy number variation was assessed by “home-made” MLPA analysis and by single nucleotide polymorphism (SNP) array analysis using the CytoScan HD Array. Direct sequencing was used for deletion breakpoint mapping. Results: Our assay detected all deletions in control samples. All affected males of the family were positive for a deletion of exon 2 of the RS1 gene (RS1:NM_000330:c.53-?_78+?del). Carrier females were also identified. Conclusion: Our method is easily replicated, reliable, and inexpensive and allows female carriers to be detected. This is the first report of deep characterization of a whole exon deletion in the RS1 gene. PMID:27997221

Delimitation of the Earliness per se D1 (Eps-D1) flowering gene to a subtelomeric chromosomal deletion in bread wheat (Triticum aestivum).

PubMed

Zikhali, Meluleki; Wingen, Luzie U; Griffiths, Simon

2016-01-01

Earliness per se (Eps) genes account for the variation in flowering time when vernalization and photoperiod requirements are satisfied. Genomics and bioinformatics approaches were used to describe allelic variation for 40 Triticum aestivum genes predicted, by synteny with Brachypodium distachyon, to be in the 1DL Eps region. Re-sequencing 1DL genes revealed that varieties carrying early heading alleles at this locus, Spark and Cadenza, carry a subtelomeric deletion including several genes. The equivalent region in Rialto and Avalon is intact. A bimodal distribution in the segregating Spark X Rialto single seed descent (SSD) populations enabled the 1DL QTL to be defined as a discrete Mendelian factor, which we named Eps-D1. Near isogenic lines (NILs) and NIL derived key recombinants between markers flanking Eps-D1 suggest that the 1DL deletion contains the gene(s) underlying Eps-D1. The deletion spans the equivalent of the Triticum monoccocum Eps-A (m) 1 locus, and hence includes MODIFIER OF TRANSCRIPTION 1 (MOT1) and FTSH PROTEASE 4 (FTSH4), the candidates for Eps-A (m) 1. The deletion also contains T. aestivum EARLY FLOWERING 3-D1 (TaELF3-D1) a homologue of the Arabidopsis thaliana circadian clock gene EARLY FLOWERING 3. Eps-D1 is possibly a homologue of Eps-B1 on chromosome 1BL. NILs carrying the Eps-D1 deletion have significantly reduced total TaELF3 expression and altered TaGIGANTEA (TaGI) expression compared with wild type. Altered TaGI expression is consistent with an ELF3 mutant, hence we propose TaELF3-D1 as the more likely candidate for Eps-D1. This is the first direct fine mapping of Eps effect in bread wheat. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

A splice junction-targeted CRISPR approach (spJCRISPR) reveals human FOXO3B to be a protein-coding gene.

PubMed

Santo, Evan E; Paik, Jihye

2018-06-17

The rapid development of CRISPR technology is revolutionizing molecular approaches to the dissection of complex biological phenomena. Here we describe an alternative generally applicable implementation of the CRISPR-Cas9 system that allows for selective knockdown of extremely homologous genes. This strategy employs the lentiviral delivery of paired sgRNAs and nickase Cas9 (Cas9D10A) to achieve targeted deletion of splice junctions. This general strategy offers several advantages over standard single-guide exon-targeting CRISPR-Cas9 such as greatly reduced off-target effects, more restricted genomic editing, routine disruption of target gene mRNA expression and the ability to differentiate between closely related genes. Here we demonstrate the utility of this strategy by achieving selective knockdown of the highly homologous human genes FOXO3A and suspected pseudogene FOXO3B. We find the spJCRISPR strategy to efficiently and selectively disrupt FOXO3A and FOXO3B mRNA and protein expression; thus revealing that the human FOXO3B locus encodes a bona fide human gene. Unlike FOXO3A, we find the FOXO3B protein to be cytosolically localized in both the presence and absence of active Akt. The ability to selectively target and efficiently disrupt the expression of the closely-related FOXO3A and FOXO3B genes demonstrates the efficacy of the spJCRISPR approach. Copyright © 2018. Published by Elsevier B.V.

Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications.

PubMed Central

Den Dunnen, J T; Grootscholten, P M; Bakker, E; Blonden, L A; Ginjaar, H B; Wapenaar, M C; van Paassen, H M; van Broeckhoven, C; Pearson, P L; van Ommen, G J

1989-01-01

We have studied 34 Becker and 160 Duchenne muscular dystrophy (DMD) patients with the dystrophin cDNA, using conventional blots and FIGE analysis. One hundred twenty-eight mutations (65%) were found, 115 deletions and 13 duplications, of which 106 deletions and 11 duplications could be precisely mapped in relation to both the mRNA and the major and minor mutation hot spots. Junction fragments, ideal markers for carrier detection, were found in 23 (17%) of the 128 cases. We identified eight new cDNA RFLPs within the DMD gene. With the use of cDNA probes we have completed the long-range map of the DMD gene, by the identification of a 680-kb SfiI fragment containing the gene's 3' end. The size of the DMD gene is now determined to be about 2.3 million basepairs. The combination of cDNA hybridizations with long-range analysis of deletion and duplication patients yields a global picture of the exon spacing within the dystrophin gene. The gene shows a large variability of intron size, ranging from only a few kilobases to 160-180 kb for the P20 intron. Images Figure 1 Figure 4 PMID:2573997

Prevalence of glutathione S-transferase gene deletions and their effect on sickle cell patients.

PubMed

Sanjay, Pandey; Mani, Mishra Rahasy; Sweta, Pandey; Vineet, Shah; Kumar, Ahuja Rajesh; Renu, Saxena

2012-01-01

Glutathione S-transferase gene deletions are known detoxification agents and cause oxidative damage. Due to the different pathophysiology of anemia in thalassemia and sickle cell disease, there are significant differences in the pathophysiology of iron overload and iron-related complications in these disorders. The aim of this study was to estimate the frequency of the GSTM1 and GSTT1 genotypes in sickle cell disease patients and their effect on iron status. Forty sickle cell anemia and sixty sickle ß-thalassemia patients and 100 controls were evaluated to determine the frequency of GST gene deletions. Complete blood counts were performed by an automated cell analyzer. Hemoglobin F, hemoglobin A, hemoglobin A2 and hemoglobin S were measured and diagnosis of patients was achieved by high performance liquid chromatography with DNA extraction by the phenol-chloroform method. The GST null genotype was determined using multiplex polymerase chain reaction and serum ferritin was measured using an ELISA kit. Statistical analysis was by EpiInfo and GraphPad statistics software. An increased frequency of the GSTT1 null genotype (p-value = 0.05) was seen in the patients. The mean serum ferritin level was higher in patients with the GST genotypes than in controls; this was statistically significant for all genotypes except GSTM1, however the higher levels of serum ferritin were due to blood transfusions in patients. GST deletions do not play a direct role in iron overload of sickle cell patients.

Two novel partial deletions of LDL-receptor gene in Italian patients with familial hypercholesterolemia (FH Siracusa and FH Reggio Emilia).

PubMed

Garuti, R; Lelli, N; Barozzini, M; Tiozzo, R; Ghisellini, M; Simone, M L; Li Volti, S; Garozzo, R; Mollica, F; Vergoni, W; Bertolini, S; Calandra, S

1996-03-01

In the present study we report two novel partial deletions of the LDL-R gene. The first (FH Siracusa), found in an FH-heterozygote, consists of a 20 kb deletion spanning from the 5' flanking region to the intron 2 of the LDL-receptor gene. The elimination of the promoter and the first two exons prevents the transcription of the deleted allele, as shown by Northern blot analysis of LDL-R mRNA isolated from the proband's fibroblasts. The second deletion (FH Reggio Emilia), which eliminates 11 nucleotides of exon 10, was also found in an FH heterozygote. The characterization of this deletion was made possible by a combination of techniques such as single strand conformation polymorphism (SSCP) analysis, direct sequence of exon 10 and cloning of the normal and deleted exon 10 from the proband's DNA. The 11 nt deletion occurs in a region of exon 10 which contains three triplets (CTG) and two four-nucleotides (CTGG) direct repeats. This structural feature might render this region more susceptible to a slipped mispairing during DNA duplication. Since this deletion causes a shift of the BamHI site at the 5' end of exon 10, a method has been devised for its rapid screening which is based on the PCR amplification of exon 10 followed by BamHI digestion. FH Reggio Emilia deletion produces a shift in the reading frame downstream from Lys458, leading to a sequence of 51 novel amino acids before the occurrence of a premature stop codon (truncated receptor). However, since RT-PCR failed to demonstrate the presence of the mutant LDL-R mRNA in proband fibroblasts, it is likely that the amount of truncated receptor produced in these cells is negligible.

Kcne3 deletion initiates extracardiac arrhythmogenesis in mice

PubMed Central

Hu, Zhaoyang; Crump, Shawn M.; Anand, Marie; Kant, Ritu; Levi, Roberto; Abbott, Geoffrey W.

2014-01-01

Mutations in the human KCNE3 potassium channel ancillary subunit gene are associated with life-threatening ventricular arrhythmias. Most genes underlying inherited cardiac arrhythmias, including KCNE3, are not exclusively expressed in the heart, suggesting potentially complex disease etiologies. Here we investigated mechanisms of KCNE3-linked arrhythmogenesis in Kcne3−/− mice using real-time qPCR, echo- and electrocardiography, ventricular myocyte patch-clamp, coronary artery ligation/reperfusion, blood analysis, cardiac synaptosome exocytosis, microarray and pathway analysis, and multitissue histology. Kcne3 transcript was undetectable in adult mouse atria, ventricles, and adrenal glands, but Kcne3−/− mice exhibited 2.3-fold elevated serum aldosterone (P=0.003) and differentially expressed gene networks consistent with an adrenal-targeted autoimmune response. Furthermore, 8/8 Kcne3−/− mice vs. 0/8 Kcne3+/+ mice exhibited an activated-lymphocyte adrenal infiltration (P=0.0002). Kcne3 deletion also caused aldosterone-dependent ventricular repolarization delay (19.6% mean QTc prolongation in females; P<0.05) and aldosterone-dependent predisposition to postischemia arrhythmogenesis. Thus, 5/11 Kcne3−/− mice vs. 0/10 Kcne3+/+ mice exhibited sustained ventricular tachycardia during reperfusion (P<0.05). Kcne3 deletion is therefore arrhythmogenic by a novel mechanism in which secondary hyperaldosteronism, associated with an adrenal-specific lymphocyte infiltration, impairs ventricular repolarization. The findings highlight the importance of considering extracardiac pathogenesis when investigating arrhythmogenic mechanisms, even in inherited, monogenic channelopathies.—Hu, Z., Crump, S. M., Anand, M., Kant, R., Levi, R., Abbott, G. W. Kcne3 deletion initiates extracardiac arrhythmogenesis in mice. PMID:24225147

Sorting genomes by reciprocal translocations, insertions, and deletions.

PubMed

Qi, Xingqin; Li, Guojun; Li, Shuguang; Xu, Ying

2010-01-01

The problem of sorting by reciprocal translocations (abbreviated as SBT) arises from the field of comparative genomics, which is to find a shortest sequence of reciprocal translocations that transforms one genome Pi into another genome Gamma, with the restriction that Pi and Gamma contain the same genes. SBT has been proved to be polynomial-time solvable, and several polynomial algorithms have been developed. In this paper, we show how to extend Bergeron's SBT algorithm to include insertions and deletions, allowing to compare genomes containing different genes. In particular, if the gene set of Pi is a subset (or superset, respectively) of the gene set of Gamma, we present an approximation algorithm for transforming Pi into Gamma by reciprocal translocations and deletions (insertions, respectively), providing a sorting sequence with length at most OPT + 2, where OPT is the minimum number of translocations and deletions (insertions, respectively) needed to transform Pi into Gamma; if Pi and Gamma have different genes but not containing each other, we give a heuristic to transform Pi into Gamma by a shortest sequence of reciprocal translocations, insertions, and deletions, with bounds for the length of the sorting sequence it outputs. At a conceptual level, there is some similarity between our algorithm and the algorithm developed by El Mabrouk which is used to sort two chromosomes with different gene contents by reversals, insertions, and deletions.

Kidney epithelium specific deletion of kelch-like ECH-associated protein 1 (Keap1) causes hydronephrosis in mice.

PubMed

Noel, Sanjeev; Arend, Lois J; Bandapalle, Samatha; Reddy, Sekhar P; Rabb, Hamid

2016-08-02

Transcription factor Nrf2 protects from experimental acute kidney injury (AKI) and is promising to limit progression in human chronic kidney disease (CKD) by upregulating multiple antioxidant genes. We recently demonstrated that deletion of Keap1, the endogenous inhibitor of Nrf2, in T lymphocytes significantly protects from AKI. In this study, we investigated the effect of Keap1 deletion on Nrf2 mediated antioxidant response in the renal tubular epithelial cells. We deleted Keap1 exon 2 and 3 in the renal tubular epithelial cells by crossing Ksp-Cre mice with Keap1 floxed (Keap1 (f/f)) mice. Deletion of Keap1 gene in the kidney epithelial cells of Ksp-Keap1 (-/-) mice and its effect on Nrf2 target gene expression was performed using PCR and real-time PCR respectively. Histological evaluation was performed on H&E stained sections. Complete blood count, serum and urine analysis were performed to assess systemic effects of defective kidney development. Student's T test was used to determine statistical difference between the groups. Ksp-Cre resulted in the deletion of Keap1 exon 2 and 3 and subsequent upregulation of Nrf2 target genes, Nqo1, Gclm and Gclc in the kidney epithelial cells of Ksp-Keap1 (-/-) mice at baseline. Renal epithelial cell specific deletion of Keap1 in Ksp-Keap1 (-/-) mice caused marked renal pelvic expansion and significant compression of medullary parenchyma consistent with hydronephrosis in both (3 month-old) males and females. Kidneys from 6 month-old Ksp-Keap1 (-/-) mice showed progressive hydronephrosis. Hematological, biochemical and urinary analysis showed significantly higher red blood cell count (p = 0.04), hemoglobin (p = 0.01), hematocrit (p = 0.02), mean cell volume (p = 0.02) and mean cell hemoglobin concentration (p = 0.003) in Ksp-Keap1 (-/-) mice in comparison to Keap1 (f/f) mice. These unexpected findings demonstrate that Keap1 deletion in renal tubular epithelial cells results in an abnormal kidney

Deletion of ultraconserved elements yields viable mice

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

Ahituv, Nadav; Zhu, Yiwen; Visel, Axel

2007-07-15

Ultraconserved elements have been suggested to retainextended perfect sequence identity between the human, mouse, and ratgenomes due to essential functional properties. To investigate thenecessities of these elements in vivo, we removed four non-codingultraconserved elements (ranging in length from 222 to 731 base pairs)from the mouse genome. To maximize the likelihood of observing aphenotype, we chose to delete elements that function as enhancers in amouse transgenic assay and that are near genes that exhibit markedphenotypes both when completely inactivated in the mouse as well as whentheir expression is altered due to other genomic modifications.Remarkably, all four resulting lines of mice lackingmore » these ultraconservedelements were viable and fertile, and failed to reveal any criticalabnormalities when assayed for a variety of phenotypes including growth,longevity, pathology and metabolism. In addition more targeted screens,informed by the abnormalities observed in mice where genes in proximityto the investigated elements had been altered, also failed to revealnotable abnormalities. These results, while not inclusive of all thepossible phenotypic impact of the deleted sequences, indicate thatextreme sequence constraint does not necessarily reflect crucialfunctions required for viability.« less

A new case of interstitial 6q16.2 deletion in a patient with Prader-Willi-like phenotype and investigation of SIM1 gene deletion in 87 patients with syndromic obesity.

PubMed

Varela, Monica C; Simões-Sato, Alex Y; Kim, Chong A; Bertola, Débora R; De Castro, Claudia I E; Koiffmann, Celia P

2006-01-01

The association of obesity, phenotypic abnormalities and mental retardation characterizes syndromic obesity. Its most common form is the Prader-Willi syndrome (PWS-- neonatal hypotonia, poor sucking, delayed psychomotor development, hyperphagia, severe obesity, short stature, small hands and feet, hypogonadism, mild to moderate mental retardation and behavioral disorders). A PWS-like phenotype has been described in patients with chromosome abnormalities involving the chromosome region 6q16.2 that includes the SIM1 gene. Herein we report cytogenetic and gene studies including a screening for the SIM1 gene deletion, performed on 87 patients with PWS-like phenotype, and describe the fifth case of syndromic obesity with an interstitial deletion of the chromosome segment 6q16-q21 and suggest that mutational analysis and further studies of the parental origin of chromosome alterations of 6q16.2 in patients with and without PWS-like phenotype are needed to evaluate possible imprinting effects of SIM1 gene and establish the contribution that alterations in this gene makes to the etiology of syndromic and non-syndromic obesity.

Heterozygous Hfe gene deletion leads to impaired glucose homeostasis, but not liver injury in mice fed a high-calorie diet.

PubMed

Britton, Laurence; Jaskowski, Lesley; Bridle, Kim; Santrampurwala, Nishreen; Reiling, Janske; Musgrave, Nick; Subramaniam, V Nathan; Crawford, Darrell

2016-06-01

Heterozygous mutations of the Hfe gene have been proposed as cofactors in the development and progression of nonalcoholic fatty liver disease (NAFLD). Homozygous Hfe deletion previously has been shown to lead to dysregulated hepatic lipid metabolism and accentuated liver injury in a dietary mouse model of NAFLD We sought to establish whether heterozygous deletion of Hfe is sufficient to promote liver injury when mice are exposed to a high-calorie diet (HCD). Eight-week-old wild-type and Hfe(+/-) mice received 8 weeks of a control diet or HCD Liver histology and pathways of lipid and iron metabolism were analyzed. Liver histology demonstrated that mice fed a HCD had increased NAFLD activity score (NAS), steatosis, and hepatocyte ballooning. However, liver injury was unaffected by Hfe genotype. Hepatic iron concentration (HIC) was increased in Hfe(+/-) mice of both dietary groups. HCD resulted in a hepcidin-independent reduction in HIC Hfe(+/-) mice demonstrated raised fasting serum glucose concentrations and HOMA-IR score, despite unaltered serum adiponectin concentrations. Downstream regulators of hepatic de novo lipogenesis (pAKT, SREBP-1, Fas, Scd1) and fatty acid oxidation (AdipoR2, Pparα, Cpt1) were largely unaffected by genotype. In summary, heterozygous Hfe gene deletion is associated with impaired iron and glucose metabolism. However, unlike homozygous Hfe deletion, heterozygous gene deletion did not affect lipid metabolism pathways or liver injury in this model. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Analysis of Pax6 contiguous gene deletions in the mouse, Mus musculus, identifies regions distinct from Pax6 responsible for extreme small-eye and belly-spotting phenotypes.

PubMed

Favor, Jack; Bradley, Alan; Conte, Nathalie; Janik, Dirk; Pretsch, Walter; Reitmeir, Peter; Rosemann, Michael; Schmahl, Wolfgang; Wienberg, Johannes; Zaus, Irmgard

2009-08-01

In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca(+2)-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.

Familial 46,XY sex reversal without campomelic dysplasia caused by a deletion upstream of the SOX9 gene

PubMed Central

Layman, Lawrence C.; Ullmann, Reinhard; Shen, Yiping; Ha, Kyungsoo; Rehman, Khurram; Looney, Stephen; McDonough, Paul G.; Kim, Hyung-Goo; Carr, Bruce R.

2014-01-01

Background 46,XY sex reversal is a rare disorder and familial cases are even more rare. The purpose of the present study was to determine the molecular basis for a family with three affected siblings who had 46,XY sex reversal. Methods DNA was extracted from three females with 46,XY sex reversal, two normal sisters, and both unaffected parents. All protein coding exons of the SRY and NR5A1 genes were subjected to PCR-based DNA sequencing. In addition, array comparative genomic hybridization was performed on DNA from all seven family members. A deletion was confirmed using quantitative polymerase chain reaction. Expression of SOX9 gene was quantified using reverse transcriptase polymerase chain reaction. Results A 349kb heterozygous deletion located 353kb upstream of the SOX9 gene on the long arm of chromosome 17 was discovered in the father and three affected siblings, but not in the mother. The expression of SOX9 was significantly decreased in the affected siblings. Two of three affected sisters had gonadoblastomas. Conclusion This is the first report of 46,XY sex reversal in three siblings who have a paternally inherited deletion upstream of SOX9 associated with reduced SOX9 mRNA expression. PMID:24907458

Antiviral resistance due to deletion in the neuraminidase gene and defective interfering-like viral polymerase basic 2 RNA of influenza A virus subtype H3N2.

PubMed

Trebbien, Ramona; Christiansen, Claus Bohn; Fischer, Thea Kølsen

2018-05-01

Antiviral treatment of influenza virus infections can lead to drug resistance of virus. This study investigates a selection of mutations in the full genome of H3N2 influenza A virus isolated from a patient in treatment with oseltamivir. Respiratory samples from a patient were collected before, during, and after antiviral treatment. Whole genome sequencing of the influenza virus by next generation sequencing, and low-frequency-variant analysis was performed. Neuraminidase-inhibition tests were performed with oseltamivir and zanamivir, and viruses were propagated in sial-transferase gene transfected Madin-Darby Canine Kidney cells. A deletion at amino acid position 245-248 in the neuraminidase gene occurred after initiation of treatment with oseltamivir. The deleted virus had highly reduced inhibition against oseltamivir but was sensitive to zanamivir. Nine days after discontinuation of oseltamivir treatment the deleted H3N2 virus was still present in the patient. After three passages of the deleted virus in cell culture, the deletion was retained. Several variant mutations appeared in the other genes of the H3N2 virus, where most striking were two major out-of-frame deletions in the polymerase basic 2 (PB2) gene, indicating defective interfering-like viral RNA. The viruses harboring the 245-248 deletion in the neuraminidase gene were still present after discontinuation of oseltamivir treatment and passages in cell cultures, indicating a potential risk for transmission of the deleted virus. Full genome deep sequencing was useful to reveal variant mutations that might be selected due to antiviral treatment, and defective interfering-like viral PB2 RNA in the respiratory samples was detected. Copyright © 2018 Elsevier B.V. All rights reserved.

An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology

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

Geller, A.I.; Keyomarsi, K.; Bryan, J.

1990-11-01

The authors have previously described a defective herpes simplex virus (HSV-1) vector system that permits that introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli {beta}-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; tsmore » mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. They now report an efficient packaging system for HSV-1 VECTORS USING A DELETION MUTANT, d30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses {beta}-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system.« less

Interrelationship between TP53 gene deletion, protein expression and chromosome 17 aneusomy in gastric adenocarcinoma

PubMed Central

2009-01-01

Background This study evaluates the existence of numerical alterations of chromosome 17 and TP53 gene deletion in gastric adenocarcinoma. The p53 protein expression was also evaluated, as well as, possible associations with clinicopathological characteristics. Methods Dual-color fluorescence in situ hybridization and immunostaining were performed in twenty gastric cancer samples of individuals from Northern Brazil. Results Deletion of TP53 was found in all samples. TP53 was inactivated mainly by single allelic deletion, varying to 7–39% of cells/case. Aneusomy of chromosome 17 was observed in 85% of cases. Chromosome 17 monosomy and gain were both observed in about half of cases. Cells with gain of chromosome 17 frequently presented TP53 deletion. The frequency of cells with two chr17 and one TP53 signals observed was higher in diffuse than in intestinal-type GC. Immunoreactivity of p53 was found only in intestinal-type samples. The frequency of cells with two chr17 and two TP53 signals found was higher in samples with positive p53 expression than in negative cases in intestinal-type GC. Conclusion We suggest that TP53 deletion and chromosome 17 aneusomy is a common event in GC and other TP53 alterations, as mutation, may be implicated in the distinct carcinogenesis process of diffuse and intestinal types. PMID:19619279

Associations between neurodevelopmental genes, neuroanatomy, and ultra high risk symptoms of psychosis in 22q11.2 deletion syndrome.

PubMed

Thompson, Carlie A; Karelis, Jason; Middleton, Frank A; Gentile, Karen; Coman, Ioana L; Radoeva, Petya D; Mehta, Rashi; Fremont, Wanda P; Antshel, Kevin M; Faraone, Stephen V; Kates, Wendy R

2017-04-01

22q11.2 deletion syndrome is a neurogenetic disorder resulting in the deletion of over 40 genes. Up to 40% of individuals with 22q11.2DS develop schizophrenia, though little is known about the underlying mechanisms. We hypothesized that allelic variation in functional polymorphisms in seven genes unique to the deleted region would affect lobar brain volumes, which would predict risk for psychosis in youth with 22q11.2DS. Participants included 56 individuals (30 males) with 22q11.2DS. Anatomic MR images were collected and processed using Freesurfer. Participants were genotyped for 10 SNPs in the COMT, DGCR8, GNB1L, PIK4CA, PRODH, RTN4R, and ZDHHC8 genes. All subjects were assessed for ultra high risk symptoms of psychosis. Allelic variation of the rs701428 SNP of RTN4R was significantly associated with volumetric differences in gray matter of the lingual gyrus and cuneus of the occipital lobe. Moreover, occipital gray matter volumes were robustly associated with ultra high risk symptoms of psychosis in the presence of the G allele of rs701428. Our results suggest that RTN4R, a relatively under-studied gene at the 22q11 locus, constitutes a susceptibility gene for psychosis in individuals with this syndrome through its alteration of the architecture of the brain. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Treacher Collins syndrome with a de Novo 5-bp deletion in the TCOF1 gene.

PubMed

Su, Pen-Hua; Chen, Jia-Yu; Chen, Suh-Jen; Yu, Ju-Shan

2006-06-01

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development with features including malar hypoplasia, micrognathia, microtia, downward slanting palpebral fissures, lower eyelid coloboma, conductive hearing loss, and cleft palate. TCS is caused by mutations in the TCOF1 gene, which encodes the nuclear phosphoprotein treacle. Here, we describe a 1-day-old male infant with classical TCS presentation. A 5-bp deletion in exon 22 of the TCOF1 gene (3469del ACTCT) was found to cause a premature stop codon. This is the first report of TCOF1 gene mutation in the Taiwanese population.

Screening for mutations in exon 4 of the LDL receptor gene: identification of a new deletion mutation.

PubMed Central

Theart, L; Kotze, M J; Langenhoven, E; Loubser, O; Peeters, A V; Lintott, C J; Scott, R S

1995-01-01

DNA from 14 unrelated New Zealand familial hypercholesterolaemia (FH) heterozygotes, originating from the United Kingdom, was screened for mutations in exon 4 of the low density lipoprotein receptor (LDLR) gene. One patient was heterozygous for mutation D206E, which was initially identified in South Africa. The chromosomal background of this mutant allele was compatible with that described previously in Afrikaner and English patients, suggesting that this mutation originated in the United Kingdom. The 2 bp deletion in codon 206 and mutations D154N and D200G, previously reported in English FH patients, were not detected in this sample. In one of the patients, however, a new deletion of 7 bp was identified after nucleotide 581 (or 582) in exon 4 of the LDLR gene. Images PMID:7616546

Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.

PubMed Central

Yang, Y; Nunes, F A; Berencsi, K; Furth, E E; Gönczöl, E; Wilson, J M

1994-01-01

An important limitation that has emerged in the use of adenoviruses for gene therapy has been loss of recombinant gene expression that occurs concurrent with the development of pathology in the organ expressing the transgene. We have used liver-directed approaches to gene therapy in mice to study mechanisms that underlie the problems with transient expression and pathology that have characterized in vivo applications of first-generation recombinant adenoviruses (i.e., those deleted of E1a and E1b). Our data are consistent with the following hypothesis. Cells harboring the recombinant viral genome express the transgene as desired; however, low-level expression of viral genes also occurs. A virus-specific cellular immune response is stimulated that leads to destruction of the genetically modified hepatocytes, massive hepatitis, and repopulation of the liver with nontransgene-containing hepatocytes. These findings suggest approaches for improving recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral genes. Images PMID:8183921

Novel deletions involving the USH2A gene in patients with Usher syndrome and retinitis pigmentosa

PubMed Central

García-García, Gema; Jaijo, Teresa; Aparisi, Maria J.; Larrieu, Lise; Faugère, Valérie; Blanco-Kelly, Fiona; Ayuso, Carmen; Roux, Anne-Francoise; Millán, José M.

2014-01-01

Purpose The aim of the present work was to identify and characterize large rearrangements involving the USH2A gene in patients with Usher syndrome and nonsyndromic retinitis pigmentosa. Methods The multiplex ligation-dependent probe amplification (MLPA) technique combined with a customized array-based comparative genomic hybridization (aCGH) analysis was applied to 40 unrelated patients previously screened for point mutations in the USH2A gene in which none or only one pathologic mutation was identified. Results We detected six large deletions involving USH2A in six out of the 40 cases studied. Three of the patients were homozygous for the deletion, and the remaining three were compound heterozygous with a previously identified USH2A point mutation. In five of these cases, the patients displayed Usher type 2, and the remaining case displayed nonsyndromic retinitis pigmentosa. The exact breakpoint junctions of the deletions found in USH2A in four of these cases were characterized. Conclusions Our study highlights the need to develop improved efficient strategies of mutation screening based upon next generation sequencing (NGS) that reduce cost, time, and complexity and allow simultaneous identification of all types of disease-causing mutations in diagnostic procedures. PMID:25352746

Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites from Peru

PubMed Central

Akinyi, Sheila; Hayden, Tonya; Gamboa, Dionicia; Torres, Katherine; Bendezu, Jorge; Abdallah, Joseph F.; Griffing, Sean M.; Quezada, Wilmer Marquiño; Arrospide, Nancy; De Oliveira, Alexandre Macedo; Lucas, Carmen; Magill, Alan J.; Bacon, David J.; Barnwell, John W.; Udhayakumar, Venkatachalam

2013-01-01

The majority of malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), encoded by the pfhrp2 gene. Recently, P. falciparum isolates from Peru were found to lack pfhrp2 leading to false-negative RDT results. We hypothesized that pfhrp2-deleted parasites in Peru derived from a single genetic event. We evaluated the parasite population structure and pfhrp2 haplotype of samples collected between 1998 and 2005 using seven neutral and seven chromosome 8 microsatellite markers, respectively. Five distinct pfhrp2 haplotypes, corresponding to five neutral microsatellite-based clonal lineages, were detected in 1998-2001; pfhrp2 deletions occurred within four haplotypes. In 2003-2005, outcrossing among the parasite lineages resulted in eight population clusters that inherited the five pfhrp2 haplotypes seen previously and a new haplotype; pfhrp2 deletions occurred within four of these haplotypes. These findings indicate that the genetic origin of pfhrp2 deletion in Peru was not a single event, but likely occurred multiple times. PMID:24077522

Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides.

PubMed Central

Falcone, D L; Tabita, F R

1991-01-01

A Rhodobacter sphaeroides ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strain was constructed that was complemented by plasmids containing either the form I or form II CO2 fixation gene cluster. This strain was also complemented by genes encoding foreign RubisCO enzymes expressed from a Rhodospirillum rubrum RubisCO promoter. In R. sphaeroides, the R. rubrum promoter was regulated, resulting in variable levels of disparate RubisCO molecules under different growth conditions. Photosynthetic growth of the R. sphaeroides deletion strain complemented with cyanobacterial RubisCO revealed physiological properties reflective of the unique cellular environment of the cyanobacterial enzyme. The R. sphaeroides RubisCO deletion strain and R. rubrum promoter system may be used to assess the properties of mutagenized proteins in vivo, as well as provide a potential means to select for altered RubisCO molecules after random mutagenesis of entire genes or gene regions encoding RubisCO enzymes. Images PMID:1900508

Deletion and aberrant CpG island methylation of Caspase 8 gene in medulloblastoma.

PubMed

Gonzalez-Gomez, Pilar; Bello, M Josefa; Inda, M Mar; Alonso, M Eva; Arjona, Dolores; Amiñoso, Cinthia; Lopez-Marin, Isabel; de Campos, Jose M; Sarasa, Jose L; Castresana, Javier S; Rey, Juan A

2004-09-01

Aberrant methylation of promoter CpG islands in human genes is an alternative genetic inactivation mechanism that contributes to the development of human tumors. Nevertheless, few studies have analyzed methylation in medulloblastomas. We determined the frequency of aberrant CpG island methylation for Caspase 8 (CASP8) in a group of 24 medulloblastomas arising in 8 adult and 16 pediatric patients. Complete methylation of CASP8 was found in 15 tumors (62%) and one case displayed hemimethylation. Three samples amplified neither of the two primer sets for methylated or unmethylated alleles, suggesting that genomic deletion occurred in the 5' flanking region of CASP8. Our findings suggest that methylation commonly contributes to CASP8 silencing in medulloblastomas and that homozygous deletion or severe sequence changes involving the promoter region may be another mechanism leading to CASP8 inactivation in this neoplasm.

Contiguous deletion of the NDP, MAOA, MAOB, and EFHC2 genes in a patient with Norrie disease, severe psychomotor retardation and myoclonic epilepsy.

PubMed

Rodriguez-Revenga, L; Madrigal, I; Alkhalidi, L S; Armengol, L; González, E; Badenas, C; Estivill, X; Milà, M

2007-05-01

Norrie disease (ND) is an X-linked disorder, inherited as a recessive trait that, therefore, mostly affects males. The gene responsible for ND, called NDP, maps to the short arm of chromosome X (Xp11.4-p11.3). We report here an atypical case of ND, consisting of a patient harboring a large submicroscopic deletion affecting not only the NDP gene but also the MAOA, MAOB, and EFHC2 genes. Microarray comparative genomic hybridization (CGH) analysis showed that 11 consecutive bacterial artificial chromosome (BAC) clones, mapping around the NDP gene, were deleted. These clones span a region of about 1 Mb on Xp11.3. The deletion was ascertained by fluorescent in situ hybridization (FISH) analysis with different BAC clones located within the region. Clinical features of the proband include bilateral retinal detachment, microcephaly, severe psychomotor retardation without verbal language skills acquired, and epilepsy. The identification and molecular characterization of this case reinforces the idea of a new contiguous gene syndrome that would explain the complex phenotype shared by atypical ND patients.

Speckle-type POZ (pox virus and zinc finger protein) protein gene deletion in ovarian cancer: Fluorescence in situ hybridization analysis of a tissue microarray.

PubMed

Hu, Xiaoyu; Yang, Zhu; Zeng, Manman; Liu, Y I; Yang, Xiaotao; Li, Yanan; Li, X U; Yu, Qiubo

2016-07-01

The aim of the present study was to investigate the status of speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) gene located on chromosome 17q21 in ovarian cancer (OC). The present study evaluated a tissue microarray, which contained 90 samples of ovarian cancer and 10 samples of normal ovarian tissue, using fluorescence in situ hybridization (FISH). FISH is a method where a SPOP-specific DNA red fluorescence probe was used for the experimental group and a centromere-specific DNA green fluorescence probe for chromosome 17 was used for the control group. The present study demonstrated that a deletion of the SPOP gene was observed in 52.27% (46/88) of the ovarian cancer tissues, but was not identified in normal ovarian tissues. Simultaneously, monosomy 17 was frequently identified in the ovarian cancer tissues, but not in the normal ovarian tissues. Furthermore, the present data revealed that the ovarian cancer histological subtype and grade were significantly associated with a deletion of the SPOP gene, which was assessed by the appearance of monosomy 17 in the ovarian cancer samples; the deletion of the SPOP gene was observed in a large proportion of serous epithelial ovarian cancer (41/61; 67.21%), particularly in grade 3 (31/37; 83.78%). In conclusion, deletion of the SPOP gene on chromosome 17 in ovarian cancer samples, which results from monosomy 17, indicates that the SPOP gene may serve as a tumor suppressor gene in ovarian cancer.

Functional Genomics Using the Saccharomyces cerevisiae Yeast Deletion Collections.

PubMed

Nislow, Corey; Wong, Lai Hong; Lee, Amy Huei-Yi; Giaever, Guri

2016-09-01

Constructed by a consortium of 16 laboratories, the Saccharomyces genome-wide deletion collections have, for the past decade, provided a powerful, rapid, and inexpensive approach for functional profiling of the yeast genome. Loss-of-function deletion mutants were systematically created using a polymerase chain reaction (PCR)-based gene deletion strategy to generate a start-to-stop codon replacement of each open reading frame by homologous recombination. Each strain carries two molecular barcodes that serve as unique strain identifiers, enabling their growth to be analyzed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays or through the use of next-generation sequencing technologies. Functional profiling of the deletion collections, using either strain-by-strain or parallel assays, provides an unbiased approach to systematically survey the yeast genome. The Saccharomyces yeast deletion collections have proved immensely powerful in contributing to the understanding of gene function, including functional relationships between genes and genetic pathways in response to diverse genetic and environmental perturbations. © 2016 Cold Spring Harbor Laboratory Press.

Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis.

PubMed

Wright, Robin; Parrish, Mark L; Cadera, Emily; Larson, Lynnelle; Matson, Clinton K; Garrett-Engele, Philip; Armour, Chris; Lum, Pek Yee; Shoemaker, Daniel D

2003-07-30

Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation. Copyright 2003 John Wiley & Sons, Ltd.

Pleiotropy in microdeletion syndromes: Neurologic and spermatogenic abnormalities in mice homozygous for the p{sup 6H} deletion are likely due to dysfunction of a single gene

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

Rinchik, E.M.; Carpenter, D.A.; Handel, M.A.

1995-07-03

Variability and complexity of phenotypes observed in microdeletion syndromes can be due to deletion of a single gene whose product participates in several aspects of development or can be due to the deletion of a number of tightly linked genes, each adding its own effect to the syndrome. The p{sup 6H} deletion in mouse chromosome 7 presents a good model with which to address this question of multigene vs. single-gene pleiotropy. Mice homozygous for the p{sup 6H} deletion are diluted in pigmentation, are smaller than their littermates, and manifest a nervous jerky-gait phenotype. Male homozygotes are sterile and exhibit profoundmore » abnormalities in spermiogenesis. By using N-ethyl-N-nitrosourea (EtNU) mutagenesis and a breeding protocol designed to recover recessive mutations expressed hemizygously opposite a large p-locus deletion, we have generated three noncomplementing mutations that map to the p{sup 6H} deletion. Each of these EtNU-induced mutations has adverse effects on the size, nervous behavior, and progression of spermiogenesis that characterize p{sup 6H} deletion homozygotes. Because etNU is thought to induce primarily intragenic (point) mutations in mouse stem-cell spermatogonia, we propose that the trio of phenotypes (runtiness, nervous jerky gait, and male sterility) expressed in p{sup 6H} deletion homozygotes is the result of deletion of a single highly pleiotropic gene. We also predict that a homologous single locus, quite possibly tightly linked and distal to the D15S12 (P) locus in human chromosome 15q11-q13, may be associated with similar developmental abnormalities in humans. 29 refs., 3 figs., 1 tab.« less

Deletion analysis of Streptococcus pneumoniae late competence genes distinguishes virulence determinants that are dependent or independent of competence induction.

PubMed

Zhu, Luchang; Lin, Jingjun; Kuang, Zhizhou; Vidal, Jorge E; Lau, Gee W

2015-07-01

The competence regulon of Streptococcus pneumoniae (pneumococcus) is crucial for genetic transformation. During competence development, the alternative sigma factor ComX is activated, which in turn, initiates transcription of 80 'late' competence genes. Interestingly, only 16 late genes are essential for genetic transformation. We hypothesized that these late genes that are dispensable for competence are beneficial to pneumococcal fitness during infection. These late genes were systematically deleted, and the resulting mutants were examined for their fitness during mouse models of bacteremia and acute pneumonia. Among these, 14 late genes were important for fitness in mice. Significantly, deletion of some late genes attenuated pneumococcal fitness to the same level in both wild-type and ComX-null genetic backgrounds, suggesting that the constitutive baseline expression of these genes was important for bacterial fitness. In contrast, some mutants were attenuated only in the wild-type genetic background but not in the ComX-null background, suggesting that specific expression of these genes during competence state contributed to pneumococcal fitness. Increased virulence during competence state was partially caused by the induction of allolytic enzymes that enhanced pneumolysin release. These results distinguish the role of basal expression versus competence induction in virulence functions encoded by ComX-regulated late competence genes. © 2015 John Wiley & Sons Ltd.

Insertion/Deletion Within the KDM6A Gene Is Significantly Associated With Litter Size in Goat

PubMed Central

Cui, Yang; Yan, Hailong; Wang, Ke; Xu, Han; Zhang, Xuelian; Zhu, Haijing; Liu, Jinwang; Qu, Lei; Lan, Xianyong; Pan, Chuanying

2018-01-01

A previous whole-genome association analysis identified lysine demethylase 6A (KDM6A), which encodes a type of histone demethylase, as a candidate gene associated to goat fecundity. KDM6A gene knockout mouse disrupts gametophyte development, suggesting that it has a critical role in reproduction. In this study, goat KDM6A mRNA expression profiles were determined, insertion/deletion (indel) variants in the gene identified, indel variants effect on KDM6A gene expression assessed, and their association with first-born litter size analyzed in 2326 healthy female Shaanbei white cashmere goats. KDM6A mRNA was expressed in all tissues tested (heart, liver, spleen, lung, kidney, muscle, brain, skin and testis); the expression levels in testes at different developmental stages [1-week-old (wk), 2, 3 wk, 1-month-old (mo), 1.5 and 2 mo] indicated a potential association with the mitosis-to-meiosis transition, implying that KDM6A may have an essential role in goat fertility. Meanwhile, two novel intronic indels of 16 bp and 5 bp were identified. Statistical analysis revealed that only the 16 bp indel was associated with first-born litter size (P < 0.01), and the average first-born litter size of individuals with an insertion/insertion genotype higher than that of those with the deletion/deletion genotype (P < 0.05). There was also a significant difference in genotype distributions of the 16 bp indel between mothers of single-lamb and multi-lamb litters in the studied goat population (P = 0.001). Consistently, the 16 bp indel also had a significant effect on KDM6A gene expression. Additionally, there was no significant linkage disequilibrium (LD) between these two indel loci, consistent with the association analysis results. Together, these findings suggest that the 16 bp indel in KDM6A may be useful for marker-assisted selection (MAS) of goats. PMID:29616081

UGT2B17 gene deletion associated with an increase in bone mineral density similar to the effect of hormone replacement in postmenopausal women.

PubMed

Giroux, S; Bussières, J; Bureau, A; Rousseau, F

2012-03-01

UGT2B17 is one of the most important enzymes for androgen metabolism. In addition, the UGT2B17 gene is one of the most commonly deleted regions of the human genome. The deletion was previously found associated with higher femoral bone density in men and women, and we replicated this association in a sample of postmenopausal who never used hormone therapy. Deletion of the UGT2B17 gene was previously shown to be associated with a higher hip bone mineral density (BMD). Using a PCR assay, we tried to replicate the association among a large group of 2,379 women. We examined the effect of the deletion on femoral neck BMD and lumbar spine BMD according to the menopausal status and hormone replacement therapy (HRT). We used a high-throughput PCR assay to identify the gene and the deletion in a population of well-characterized women. Two additional polymorphisms, UGT2B28 deletion and UGT2B15 rs1902023 G > T were also investigated. Only UGT2B17 deletion was associated with LS and FN BMD. Furthermore, the association was seen only among postmenopausal women who had never used hormone replacement as in the first reported association. We confirmed the association between UGT2B17 deletion and a higher LS and FN BMD. In addition, we show that the association is observed among postmenopausal women who never used HRT consistent with the enzymatic function of UGT2B17. The analysis shows that those having one or two UGT2B17 alleles benefit from HRT, which is not the case for null carriers.

Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3.

PubMed

Zhang, Wei; He, Yulian; Gao, Weixia; Feng, Jun; Cao, Mingfeng; Yang, Chao; Song, Cunjiang; Wang, Shufang

2015-02-01

Here, we attempted to elevate poly-gamma-glutamic acid (γ-PGA) production by modifying genes involved in glutamate metabolism in Bacillus amyloliquefaciens LL3. Products of rocR, rocG and gudB facilitate the conversion from glutamate to 2-oxoglutarate in Bacillus subtillis. The gene odhA is responsible for the synthesis of a component of the 2-oxoglutarate dehydrogenase complex that catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl coenzyme A. In-frame deletions of these four genes were performed. In shake flask experiments the gudB/rocG double mutant presented enhanced production of γ-PGA, a 38 % increase compared with wild type. When fermented in a 5-L fermenter with pH control, the γ-PGA yield of the rocR mutant was increased to 5.83 g/L from 4.55 g/L for shake flask experiments. The gudB/rocG double mutant produced 5.68 g/L γ-PGA compared with that of 4.03 g/L for the wild type, a 40 % increase. Those results indicated the possibility of improving γ-PGA production by modifying glutamate metabolism, and identified potential genetic targets to improve γ-PGA production.

Rapid Diagnostic Tests for Malaria Diagnosis in the Peruvian Amazon: Impact of pfhrp2 Gene Deletions and Cross-Reactions

PubMed Central

Maltha, Jessica; Gamboa, Dionicia; Bendezu, Jorge; Sanchez, Luis; Cnops, Lieselotte; Gillet, Philippe; Jacobs, Jan

2012-01-01

Background In the Peruvian Amazon, Plasmodium falciparum and Plasmodium vivax malaria are endemic in rural areas, where microscopy is not available. Malaria rapid diagnostic tests (RDTs) provide quick and accurate diagnosis. However, pfhrp2 gene deletions may limit the use of histidine-rich protein-2 (PfHRP2) detecting RDTs. Further, cross-reactions of P. falciparum with P. vivax-specific test lines and vice versa may impair diagnostic specificity. Methods Thirteen RDT products were evaluated on 179 prospectively collected malaria positive samples. Species diagnosis was performed by microscopy and confirmed by PCR. Pfhrp2 gene deletions were assessed by PCR. Results Sensitivity for P. falciparum diagnosis was lower for PfHRP2 compared to P. falciparum-specific Plasmodium lactate dehydrogenase (Pf-pLDH)- detecting RDTs (71.6% vs. 98.7%, p<0.001). Most (19/21) false negative PfHRP2 results were associated with pfhrp2 gene deletions (25.7% of 74 P. falciparum samples). Diagnostic sensitivity for P. vivax (101 samples) was excellent, except for two products. In 10/12 P. vivax-detecting RDT products, cross-reactions with the PfHRP2 or Pf-pLDH line occurred at a median frequency of 2.5% (range 0%–10.9%) of P. vivax samples assessed. In two RDT products, two and one P. falciparum samples respectively cross-reacted with the Pv-pLDH line. Two Pf-pLDH/pan-pLDH-detecting RDTs showed excellent sensitivity with few (1.0%) cross-reactions but showed faint Pf-pLDH lines in 24.7% and 38.9% of P. falciparum samples. Conclusion PfHRP2-detecting RDTs are not suitable in the Peruvian Amazon due to pfhrp2 gene deletions. Two Pf-pLDH-detecting RDTs performed excellently and are promising RDTs for this region although faint test lines are of concern. PMID:22952633

Rapid diagnostic tests for malaria diagnosis in the Peruvian Amazon: impact of pfhrp2 gene deletions and cross-reactions.

PubMed

Maltha, Jessica; Gamboa, Dionicia; Bendezu, Jorge; Sanchez, Luis; Cnops, Lieselotte; Gillet, Philippe; Jacobs, Jan

2012-01-01

In the Peruvian Amazon, Plasmodium falciparum and Plasmodium vivax malaria are endemic in rural areas, where microscopy is not available. Malaria rapid diagnostic tests (RDTs) provide quick and accurate diagnosis. However, pfhrp2 gene deletions may limit the use of histidine-rich protein-2 (PfHRP2) detecting RDTs. Further, cross-reactions of P. falciparum with P. vivax-specific test lines and vice versa may impair diagnostic specificity. Thirteen RDT products were evaluated on 179 prospectively collected malaria positive samples. Species diagnosis was performed by microscopy and confirmed by PCR. Pfhrp2 gene deletions were assessed by PCR. Sensitivity for P. falciparum diagnosis was lower for PfHRP2 compared to P. falciparum-specific Plasmodium lactate dehydrogenase (Pf-pLDH)-detecting RDTs (71.6% vs. 98.7%, p<0.001). Most (19/21) false negative PfHRP2 results were associated with pfhrp2 gene deletions (25.7% of 74 P. falciparum samples). Diagnostic sensitivity for P. vivax (101 samples) was excellent, except for two products. In 10/12 P. vivax-detecting RDT products, cross-reactions with the PfHRP2 or Pf-pLDH line occurred at a median frequency of 2.5% (range 0%-10.9%) of P. vivax samples assessed. In two RDT products, two and one P. falciparum samples respectively cross-reacted with the Pv-pLDH line. Two Pf-pLDH/pan-pLDH-detecting RDTs showed excellent sensitivity with few (1.0%) cross-reactions but showed faint Pf-pLDH lines in 24.7% and 38.9% of P. falciparum samples. PfHRP2-detecting RDTs are not suitable in the Peruvian Amazon due to pfhrp2 gene deletions. Two Pf-pLDH-detecting RDTs performed excellently and are promising RDTs for this region although faint test lines are of concern.

Nature of frequent deletions in CEBPA.

PubMed

Fuchs, Ota; Kostecka, Arnost; Provaznikova, Dana; Krasna, Blazena; Brezinova, Jana; Filkukova, Jitka; Kotlin, Roman; Kouba, Michal; Kobylka, Petr; Neuwirtova, Radana; Jonasova, Anna; Caniga, Miroslav; Schwarz, Jiri; Markova, Jana; Maaloufova, Jacqueline; Sponerova, Dana; Novakova, Ludmila; Cermak, Jaroslav

2009-01-01

C/EBPalpha (CCAAT/enhancer binding protein alpha) belongs to the family of leucine zipper transcription factors and is necessary for transcriptional control of granulocyte, adipocyte and hepatocyte differentiation, glucose metabolism and lung development. C/EBPalpha is encoded by an intronless gene. CEBPA mutations cause a myeloid differentiation block and were detected in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), multiple myeloma and non-Hodgkin's lymphoma (NHL) patients. In this study we identified in 41 individuals from 824 screened individuals (290 AML patients, 382 MDS patients, 56 NHL patients and 96 healthy individuals) a single class of 23 deletions in CEBPA gene which involved a direct repeat of at least 2 bp. These mutations are characterised by the loss of one of two same repeats at the ends of deleted sequence. Three most frequent repeats included in these deletions in CEBPA gene are CGCGAG (493-498_865-870), GCCAAGCAGC (508-517_907-916) and GG (486-487_885-886), all according to GenBank accession no. NM_004364.2. A mechanism for deletion formation between two repetitive sequences can be recombination events in the repair process. Double-stranded cut in DNA can initiate these recombination events of adjacent DNA sequences.

Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo

PubMed Central

Abdel-Wahab, Omar; Gao, Jie; Adli, Mazhar; Dey, Anwesha; Trimarchi, Thomas; Chung, Young Rock; Kuscu, Cem; Hricik, Todd; Ndiaye-Lobry, Delphine; LaFave, Lindsay M.; Koche, Richard; Shih, Alan H.; Guryanova, Olga A.; Kim, Eunhee; Li, Sheng; Pandey, Suveg; Shin, Joseph Y.; Telis, Leon; Liu, Jinfeng; Bhatt, Parva K.; Monette, Sebastien; Zhao, Xinyang; Mason, Christopher E.; Park, Christopher Y.; Bernstein, Bradley E.

2013-01-01

Somatic Addition of Sex Combs Like 1 (ASXL1) mutations occur in 10–30% of patients with myeloid malignancies, most commonly in myelodysplastic syndromes (MDSs), and are associated with adverse outcome. Germline ASXL1 mutations occur in patients with Bohring-Opitz syndrome. Here, we show that constitutive loss of Asxl1 results in developmental abnormalities, including anophthalmia, microcephaly, cleft palates, and mandibular malformations. In contrast, hematopoietic-specific deletion of Asxl1 results in progressive, multilineage cytopenias and dysplasia in the context of increased numbers of hematopoietic stem/progenitor cells, characteristic features of human MDS. Serial transplantation of Asxl1-null hematopoietic cells results in a lethal myeloid disorder at a shorter latency than primary Asxl1 knockout (KO) mice. Asxl1 deletion reduces hematopoietic stem cell self-renewal, which is restored by concomitant deletion of Tet2, a gene commonly co-mutated with ASXL1 in MDS patients. Moreover, compound Asxl1/Tet2 deletion results in an MDS phenotype with hastened death compared with single-gene KO mice. Asxl1 loss results in a global reduction of H3K27 trimethylation and dysregulated expression of known regulators of hematopoiesis. RNA-Seq/ChIP-Seq analyses of Asxl1 in hematopoietic cells identify a subset of differentially expressed genes as direct targets of Asxl1. These findings underscore the importance of Asxl1 in Polycomb group function, development, and hematopoiesis. PMID:24218140

Targeted deletion of the 9p21 noncoding coronary artery disease risk interval in mice

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

Visel, Axel; Zhu, Yiwen; May, Dalit

2010-01-01

Sequence polymorphisms in a 58kb interval on chromosome 9p21 confer a markedly increased risk for coronary artery disease (CAD), the leading cause of death worldwide 1,2. The variants have a substantial impact on the epidemiology of CAD and other life?threatening vascular conditions since nearly a quarter of Caucasians are homozygous for risk alleles. However, the risk interval is devoid of protein?coding genes and the mechanism linking the region to CAD risk has remained enigmatic. Here we show that deletion of the orthologous 70kb noncoding interval on mouse chromosome 4 affects cardiac expression of neighboring genes, as well as proliferation propertiesmore » of vascular cells. Chr4delta70kb/delta70kb mice are viable, but show increased mortality both during development and as adults. Cardiac expression of two genes near the noncoding interval, Cdkn2a and Cdkn2b, is severely reduced in chr4delta70kb/delta70kb mice, indicating that distant-acting gene regulatory functions are located in the noncoding CAD risk interval. Allelespecific expression of Cdkn2b transcripts in heterozygous mice revealed that the deletion affects expression through a cis-acting mechanism. Primary cultures of chr4delta70kb/delta70kb aortic smooth muscle cells exhibited excessive proliferation and diminished senescence, a cellular phenotype consistent with accelerated CAD pathogenesis. Taken together, our results provide direct evidence that the CAD risk interval plays a pivotal role in regulation of cardiac Cdkn2a/b expression and suggest that this region affects CAD progression by altering the dynamics of vascular cell proliferation.« less

High incidence of large deletions in the PMS2 gene in Spanish Lynch syndrome families.

PubMed

Brea-Fernández, A J; Cameselle-Teijeiro, J M; Alenda, C; Fernández-Rozadilla, C; Cubiella, J; Clofent, J; Reñé, J M; Anido, U; Milá, M; Balaguer, F; Castells, A; Castellvi-Bel, S; Jover, R; Carracedo, A; Ruiz-Ponte, C

2014-06-01

Lynch syndrome (LS) is caused by germline mutations in one of the four mismatch repair (MMR) genes. Defects in this pathway lead to microsatellite instability (MSI) in DNA tumors, which constitutes the molecular hallmark of this disease. Selection of patients for genetic testing in LS is usually based on fulfillment of diagnostic clinical criteria (i.e. Amsterdam criteria or the revised Bethesda guidelines). However, following these criteria PMS2 mutations have probably been underestimated as their penetrances appear to be lower than those of the other MMR genes. The use of universal MMR study-based strategies, using MSI testing and immunohistochemical (IHC) staining, is being one proposed alternative. Besides, germline mutation detection in PMS2 is complicated by the presence of highly homologous pseudogenes. Nevertheless, specific amplification of PMS2 by long-range polymerase chain reaction (PCR) and the improvement of the analysis of large deletions/duplications by multiplex ligation-dependent probe amplification (MLPA) overcome this difficulty. By using both approaches, we analyzed 19 PMS2-suspected carriers who have been selected by clinical or universal strategies and found five large deletions and one frameshift mutation in PMS2 in six patients (31%). Owing to the high incidence of large deletions found in our cohort, we recommend MLPA analysis as the first-line method for searching germline mutations in PMS2. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Analysis of BIM (BCL-2 like 11 gene) deletion polymorphism in Chinese non-small cell lung cancer patients

PubMed Central

Zhong, Jia; Li, Zheng-Xiang; Zhao, Jun; Duan, Jian-Chun; Bai, Hua; An, Tong-Tong; Yang, Xiao-Dan; Wang, Jie

2014-01-01

Background Drug resistance significantly weakens the efficacy of cancer treatment, and the BIM (also known as the BCL2L11 gene) deletion polymorphism has been identified as a potential biomarker for drug resistance. In this retrospective study, we included a total of 290 patients with advanced non-small cell lung cancer (NSCLC) who received treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and chemotherapy. Methods The BIM deletion polymorphism of each patient was detected by polymerase chain reaction. EGFR mutations were detected by denaturing high-performance liquid chromatography methods and the amplification refractory mutation system. Results The BIM deletion polymorphism was detected in 45/290 (15.5%) Chinese NSCLC patients. No associations were observed between the BIM deletion and clinic-pathologic characteristics of patients. The BIM deletion polymorphism was predictive of shorter progression-free survival in Chinese patients with EGFR-mutant adenocarcinoma and who were treated with EGFR-TKIs (7.30 vs. 9.53 months, P = 0.034). Additionally, we found that the BIM deletion polymorphism was an effective predictor of short progression-free survival in individuals with EGFR-mutant NSCLC and treated with chemotherapy containing pemetrexed (3.32 vs. 5.30, P = 0.012) or second-/beyond-line chemotherapy containing taxanes (1.53 vs. 2.61 months, P = 0.025). The BIM deletion was not correlated with overall survival. Conclusion The BIM deletion polymorphism occurs in 15.5% of Chinese NSCLC patients, and is a biomarker for resistance to TKIs and chemotherapy. However, BIM deletion was not a decisive factor in overall survival. PMID:26767045

Analysis of BIM (BCL-2 like 11 gene) deletion polymorphism in Chinese non-small cell lung cancer patients.

PubMed

Zhong, Jia; Li, Zheng-Xiang; Zhao, Jun; Duan, Jian-Chun; Bai, Hua; An, Tong-Tong; Yang, Xiao-Dan; Wang, Jie

2014-11-01

Drug resistance significantly weakens the efficacy of cancer treatment, and the BIM (also known as the BCL2L11 gene) deletion polymorphism has been identified as a potential biomarker for drug resistance. In this retrospective study, we included a total of 290 patients with advanced non-small cell lung cancer (NSCLC) who received treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and chemotherapy. The BIM deletion polymorphism of each patient was detected by polymerase chain reaction. EGFR mutations were detected by denaturing high-performance liquid chromatography methods and the amplification refractory mutation system. The BIM deletion polymorphism was detected in 45/290 (15.5%) Chinese NSCLC patients. No associations were observed between the BIM deletion and clinic-pathologic characteristics of patients. The BIM deletion polymorphism was predictive of shorter progression-free survival in Chinese patients with EGFR-mutant adenocarcinoma and who were treated with EGFR-TKIs (7.30 vs. 9.53 months, P = 0.034). Additionally, we found that the BIM deletion polymorphism was an effective predictor of short progression-free survival in individuals with EGFR-mutant NSCLC and treated with chemotherapy containing pemetrexed (3.32 vs. 5.30, P = 0.012) or second-/beyond-line chemotherapy containing taxanes (1.53 vs. 2.61 months, P = 0.025). The BIM deletion was not correlated with overall survival. The BIM deletion polymorphism occurs in 15.5% of Chinese NSCLC patients, and is a biomarker for resistance to TKIs and chemotherapy. However, BIM deletion was not a decisive factor in overall survival.

Molecular characterization of the breakpoints of a 12-kb deletion in the NF1 gene in a family showing germ-line mosaicism.

PubMed Central

Lázaro, C; Gaona, A; Lynch, M; Kruyer, H; Ravella, A; Estivill, X

1995-01-01

Neurofibromatosis type 1 (NF1) is caused by deletions, insertions, translocations, and point mutations in the NF1 gene, which spans 350 kb on the long arm of human chromosome 17. Although several point mutations have been described, large molecular abnormalities have rarely been characterized in detail. We describe here the molecular breakpoints of a 12-kb deletion of the NF1 gene, which is responsible for the NF1 phenotype in a kindred with two children affected because of germline mosaicism in the unaffected father, who has the mutation in 10% of his spermatozoa. The mutation spans introns 31-39, removing 12,021 nt and inserting 30 bp, of which 19 bp are a direct repetition of a sequence located in intron 31, just 4 bp before the 5' breakpoint. The 5' and 3' breakpoints contain the sequence TATTTTA, which could be involved in the generation of the deletion. The most plausible explanation for the mechanism involved in the generation of this 12-kb deletion is homologous/nonhomologous recombination. Since sperm of the father does not contain the corresponding insertion of the 12-kb deleted sequence, this deletion could have occurred within the NF1 chromosome through loop formation. RNA from lymphocytes of one of the NF1 patients showed similar levels of the mutated and normal transcripts, suggesting that the NF1-mRNA from mutations causing frame shifts of the reading frame or stop codons in this gene is not degraded during its processing. The mutation was not detected in fresh lymphocytes from the unaffected father by PCR analysis, supporting the case for true germ-line mosaicism. Images Figure 1 Figure 3 PMID:7485153

A novel contiguous deletion involving NDP, MAOB and EFHC2 gene in a patient with familial Norrie disease: bilateral blindness and leucocoria without other deficits.

PubMed

Jia, Bei; Huang, Liping; Chen, Yaoyu; Liu, Siping; Chen, Cuihua; Xiong, Ke; Song, Lanlin; Zhou, Yulai; Yang, Xinping; Zhong, Mei

2017-12-01

Contiguous microdeletions of the Norrie disease pseudoglioma (NDP) region on chromosome Xp11.3 have been widely confirmed as contributing to the typical clinical features of Norrie disease (ND). However, the precise relation between genotype and phenotype could vary. The contiguous deletion of NDP and its neighbouring genes, MAOA/B and EFHC2, reportedly leads to syndromic clinical features such as microcephaly, intellectual disability, and epilepsy. Herewe report a novel contiguous microdeletion of the NDP region containing the MAOB and EFHC2 genes,which causes eye defects but no cognitive disability.We detected a deletion of 494.6 kb atXp11.3 in both the proband and carrier mother. This deletionwas then used as the molecular marker in prenatal diagnosis for two subsequent pregnancies. The deletion was absent in one of the foetuses, who remain without any abnormalities at 2 years of age. The proband shows the typical ocular clinical features of ND including bilateral retinal detachment, microphthalmia, atrophic irides, corneal opacification, and cataracts, but no symptoms of microcephaly, intellectual disability, and epilepsy. This familial study demonstrates that a deficiency in one of two MAO genes may not lead to psychomotor delay, and deletion of EFHC2 may not cause epilepsy. Our observations provide new information on the genotype-phenotype relations of MAOA/B and EFHC2 genes involved in the contiguous deletions of ND.

Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

PubMed

Osato, Naoki

2018-01-19

Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional

The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting.

PubMed

Lu, Zhi Hong; Kaliberov, Sergey; Zhang, Jingzhu; Muz, Barbara; Azab, Abdel K; Sohn, Rebecca E; Kaliberova, Lyudmila; Du, Yingqiu; Curiel, David T; Arbeit, Jeffrey M

2014-08-01

Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

Targeted gene insertion for molecular medicine.

PubMed

Voigt, Katrin; Izsvák, Zsuzsanna; Ivics, Zoltán

2008-11-01

Genomic insertion of a functional gene together with suitable transcriptional regulatory elements is often required for long-term therapeutical benefit in gene therapy for several genetic diseases. A variety of integrating vectors for gene delivery exist. Some of them exhibit random genomic integration, whereas others have integration preferences based on attributes of the targeted site, such as primary DNA sequence and physical structure of the DNA, or through tethering to certain DNA sequences by host-encoded cellular factors. Uncontrolled genomic insertion bears the risk of the transgene being silenced due to chromosomal position effects, and can lead to genotoxic effects due to mutagenesis of cellular genes. None of the vector systems currently used in either preclinical experiments or clinical trials displays sufficient preferences for target DNA sequences that would ensure appropriate and reliable expression of the transgene and simultaneously prevent hazardous side effects. We review in this paper the advantages and disadvantages of both viral and non-viral gene delivery technologies, discuss mechanisms of target site selection of integrating genetic elements (viruses and transposons), and suggest distinct molecular strategies for targeted gene delivery.

Establishment of a Cre recombinase based mutagenesis protocol for markerless gene deletion in Streptococcus suis.

PubMed

Koczula, A; Willenborg, J; Bertram, R; Takamatsu, D; Valentin-Weigand, P; Goethe, R

2014-12-01

The lack of knowledge about pathogenicity mechanisms of Streptococcus (S.) suis is, at least partially, attributed to limited methods for its genetic manipulation. Here, we established a Cre-lox based recombination system for markerless gene deletions in S. suis serotype 2 with high selective pressure and without undesired side effects. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel growth hormone receptor gene deletion mutation in a patient with primary growth hormone insensitivity syndrome (Laron syndrome).

PubMed

Yamamoto, Hiroyasu; Kouhara, Haruhiko; Iida, Keiji; Chihara, Kazuo; Kasayama, Soji

2008-04-01

Growth hormone (GH) insensitivity syndrome (Laron syndrome) is known to be caused by genetic disorders of the GH-IGF-1 axis. Although many mutations in the GH receptor have been identified, there have been only a few reports of deletions of the GH receptor gene. A Japanese adult female patient with Laron syndrome was subjected to chromosome analysis with basic G-banding and also with a high accuracy technique. Each exon of the GH receptor gene was amplified by means of PCR. Since this patient was diagnosed with osteoporosis, the effects of alendronate on bone mineral density (BMD) were also examined. The chromosome analysis with the high accuracy technique demonstrated a large deletion of the short arm in one allele of chromosome 5 from p11 to p13.1 [46, XX, del (5) (p11-p13.1)]. PCR amplification of exons of the GH receptor gene showed that only exons 2 and 3 were amplified. Low-dose IGF-1 administration (30microg/kg body weight) failed to increase her BMD, whereas alendronate administration resulted in an increase associated with a decrease in urinary deoxypyridinoline (DPD) and serum osteocalcin concentrations. The GH receptor gene of the patient was shown to lack exons 4-10. To the best of our knowledge, this is the third case report of Laron syndrome with large GH receptor deletion. Alendronate was effective for the enhancement of BMD.

Prevalence and Spectrum of Large Deletions or Duplications in the Major Long QT Syndrome-Susceptibility Genes and Implications for Long QT Syndrome Genetic Testing

PubMed Central

Tester, David J.; Benton, Amber J.; Train, Laura; Deal, Barbara; Baudhuin, Linnea M.; Ackerman, Michael J.

2010-01-01

Long QT Syndrome (LQTS) is a cardiac channelopathy associated with syncope, seizures, and sudden death. Approximately 75% of LQTS is due to mutations in genes encoding for three cardiac ion channel alpha-subunits (LQT1-3). However, traditional mutational analyses have limited detection capabilities for atypical mutations such as large gene rearrangements. Here, we set out to determine the prevalence and spectrum of large deletions/duplications in the major LQTS-susceptibility genes among unrelated patients who were mutation-negative following point mutation analysis of LQT1-12-susceptibility genes. Forty-two unrelated clinically strong LQTS patients were analyzed using multiplex ligation-dependent probe amplification (MLPA), a quantitative fluorescent technique for detecting multiple exon deletions and duplications. The SALSA-MLPA LQTS Kit from MRC-Holland was used to analyze the three major LQTS-associated genes: KCNQ1, KCNH2, and SCN5A and the two minor genes: KCNE1 and KCNE2. Overall, 2 gene rearrangements were found in 2/42 (4.8%, CI, 1.7–11%) unrelated patients. A deletion of KCNQ1 exon 3 was identified in a 10 year-old Caucasian boy with a QTc of 660 milliseconds (ms), a personal history of exercise-induced syncope, and a family history of syncope. A deletion of KCNQ1 exon 7 was identified in a 17 year-old Caucasian girl with a QTc of 480 ms, a personal history of exercise-induced syncope, and a family history of sudden cardiac death. In conclusion, since nearly 5% of patients with genetically elusive LQTS had large genomic rearrangements involving the canonical LQTS-susceptibility genes, reflex genetic testing to investigate genomic rearrangements may be of clinical value. PMID:20920651

A Deletion in the Canine POMC Gene Is Associated with Weight and Appetite in Obesity-Prone Labrador Retriever Dogs.

PubMed

Raffan, Eleanor; Dennis, Rowena J; O'Donovan, Conor J; Becker, Julia M; Scott, Robert A; Smith, Stephen P; Withers, David J; Wood, Claire J; Conci, Elena; Clements, Dylan N; Summers, Kim M; German, Alexander J; Mellersh, Cathryn S; Arendt, Maja L; Iyemere, Valentine P; Withers, Elaine; Söder, Josefin; Wernersson, Sara; Andersson, Göran; Lindblad-Toh, Kerstin; Yeo, Giles S H; O'Rahilly, Stephen

2016-05-10

Sequencing of candidate genes for obesity in Labrador retriever dogs identified a 14 bp deletion in pro-opiomelanocortin (POMC) with an allele frequency of 12%. The deletion disrupts the β-MSH and β-endorphin coding sequences and is associated with body weight (per allele effect of 0.33 SD), adiposity, and greater food motivation. Among other dog breeds, the deletion was only found in the closely related flat-coat retriever (FCR), where it is similarly associated with body weight and food motivation. The mutation is significantly more common in Labrador retrievers selected to become assistance dogs than pets. In conclusion, the deletion in POMC is a significant modifier of weight and appetite in Labrador retrievers and FCRs and may influence other behavioral traits. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Detection of large scale 3′ deletions in the PMS2 gene amongst Colon-CFR participants – have we been missing anything?

PubMed Central

Clendenning, Mark; Walsh, Michael D; Gelpi, Judith Balmana; Thibodeau, Stephen N.; Lindor, Noralane; Potter, John D.; Newcomb, Polly; LeMarchand, Loic; Haile, Robert; Gallinger, Steve; Hopper, John L.; Jenkins, Mark A.; Rosty, Christophe; Young, Joanne P.; Buchanan, Daniel D.

2013-01-01

Current screening practices have been able to identify PMS2 mutations in 78% of cases of colorectal cancer from the Colorectal Cancer Family Registry (Colon CFR) which showed solitary loss of the PMS2 protein. However the detection of large-scale deletions in the 3′ end of the PMS2 gene has not been possible due to technical difficulties associated with pseudogene sequences. Here, we utilised a recently described MLPA/long-range PCR-based approach to screen the remaining 22% (n = 16) of CRC-affected probands for mutations in the 3′ end of the PMS2 gene. No deletions encompassing any or all of exons 12 through 15 were identified; therefore, our results suggest that 3′ deletions in PMS2 are not a frequent occurrence in such families. PMID:23288611

Deletion of the African Swine Fever Virus Gene DP148R Does Not Reduce Virus Replication in Culture but Reduces Virus Virulence in Pigs and Induces High Levels of Protection against Challenge

PubMed Central

Goatley, Lynnette C.; Jabbar, Tamara; Sanchez-Cordon, Pedro J.; Netherton, Christopher L.; Chapman, David A. G.; Dixon, Linda K.

2017-01-01

these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The virus with the gene deletion, BeninΔDP148R, caused mild clinical signs in pigs and induced high levels of protection against challenge with the parental virulent virus. Therefore, deletion of this gene can provide a target for the rational development of vaccines. PMID:28978700

Deletion of the African Swine Fever Virus Gene DP148R Does Not Reduce Virus Replication in Culture but Reduces Virus Virulence in Pigs and Induces High Levels of Protection against Challenge.

PubMed

Reis, Ana L; Goatley, Lynnette C; Jabbar, Tamara; Sanchez-Cordon, Pedro J; Netherton, Christopher L; Chapman, David A G; Dixon, Linda K

2017-12-15

148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The virus with the gene deletion, BeninΔDP148R, caused mild clinical signs in pigs and induced high levels of protection against challenge with the parental virulent virus. Therefore, deletion of this gene can provide a target for the rational development of vaccines. Copyright © 2017 Reis et al.

DiGeorge-like syndrome in a child with a 3p12.3 deletion involving MIR4273 gene born to a mother with gestational diabetes mellitus.

PubMed

Cirillo, Emilia; Giardino, Giuliana; Gallo, Vera; Galasso, Giovanni; Romano, Roberta; D'Assante, Roberta; Scalia, Giulia; Del Vecchio, Luigi; Nitsch, Lucio; Genesio, Rita; Pignata, Claudio

2017-04-24

Chromosome 22q11.2 deletion is the most common chromosomal alteration associated with DiGeorge syndrome (DGS), even though this is not the only underlying cause of DGS. In rare patients, mutations in a single gene, TBX1, have been described resulting in a DGS phenotype. Recently, it has been reported that at least part of the TBX1 mutant phenotype is due to excessive bone morphogenetic proteins (BMP) signaling. Evidence suggests that miRNA may modulate the expression of critical T-box transcriptional regulators during midface development and Bmp-signaling. We report on a 7-year-old Caucasian male born to a mother affected with gestational diabetes (GDM) who had a 371Kb-interstitial deletion of 3p12.3 identified by array CGH, involving the ZNF717, MIR1243, and 4273 genes. The child presented with a DiGeorge anomaly (DGA) associated with unilateral renal agenesis and language delay. The immunological evaluation revealed a severe reduction and impairment of T lymphocytes. FISH analysis and TBX1 sequencing were negative. Among the miRNA-4273 predicted target genes, we found BMP3, which is involved in several steps of embryogenesis including kidney and lung organogenesis and in insulin gene expression. Since, DGA is not commonly found in newborns of diabetic mothers, we hypothesize that the pathogenesis of DGA associated with GDM is multifactorial, involving both genetic and/or epigenetic cofactors. © 2017 Wiley Periodicals, Inc.

The prevalence of chromosomal deletions relating to developmental delay and/or intellectual disability in human euploid blastocysts.

PubMed

He, Wenyin; Sun, Xiaofang; Liu, Lian; Li, Man; Jin, Hua; Wang, Wei-Hua

2014-01-01

Chromosomal anomalies in human embryos produced by in vitro fertilization are very common, which include numerical (aneuploidy) and structural (deletion, duplication or others) anomalies. Our previous study indicated that chromosomal deletion(s) is the most common structural anomaly accounting for approximately 8% of euploid blastocysts. It is still unknown if these deletions in human euploid blastocysts have clinical significance. In this study, we analyzed 15 previously diagnosed euploid blastocysts that had chromosomal deletion(s) using Agilent oligonucleotide DNA microarray platform and localized the gene location in each deletion. Then, we used OMIM gene map and phenotype database to investigate if these deletions are related with some important genes that cause genetic diseases, especially developmental delay or intellectual disability. As results, we found that the detectable chromosomal deletion size with Agilent microarray is above 2.38 Mb, while the deletions observed in human blastocysts are between 11.6 to 103 Mb. With OMIM gene map and phenotype database information, we found that deletions can result in loss of 81-464 genes. Out of these genes, 34-149 genes are related with known genetic problems. Furthermore, we found that 5 out of 15 samples lost genes in the deleted region, which were related to developmental delay and/or intellectual disability. In conclusion, our data indicates that all human euploid blastocysts with chromosomal deletion(s) are abnormal and transfer of these embryos may cause birth defects and/or developmental and intellectual disabilities. Therefore, the embryos with chromosomal deletion revealed by DNA microarray should not be transferred to the patients, or further gene map and/or phenotype seeking is necessary before making a final decision.

Preferrential rearrangement in normal rabbits of the 3' VHa allotype gene that is deleted in Alicia mutants; somatic hypermutation/conversion may play a major role in generating the heterogeneity of rabbit heavy chain variable region sequences.

PubMed

Allegrucci, M; Young-Cooper, G O; Alexander, C B; Newman, B A; Mage, R G

1991-02-01

The rabbit is unique in having well-defined allotypes in the variable region of the heavy chain. Products of the VHa locus, (with alleles a1, a2, and a3), account for the majority of the serum immunoglobulins. A small percentage of the serum immunoglobulins are a-negative. In 1986, Kelus and Weiss described a mutation that depressed the expression of the Ig VH a2 genes in an a1/a2 rabbit. From this animal the Alicia rabbit strain was developed and the mutation was termed ali. We previously showed, using Southern analysis and the transverse alternating field electrophoresis technique, that the difference between the ali rabbit and normal is a relatively small deletion including some of the most 3' VH genes. The most JH proximal 3' VH1 genes in DNA from normal rabbits of a1, a2 and a3 haplotypes encode a1, a2 and a3 molecules respectively, and it has been suggested that these genes are responsible for allelic inheritance of VHa allotypes. The present study suggests that the 3' end of the VH locus probably plays a key role in regulation of VH gene expression in rabbits because VH gene(s) in this region are the target(s) of preferential VDJ rearrangements. This raises the possibility that mechanisms such as somatic gene conversion and hypermutation are at work to generate the antibody repertoire in this species. Our data support the view that the 3' VH1 gene may be the preferred target for rearrangement in normal rabbits, and for the normal chromosome in heterozygous ali animals. However, homozygous ali rabbits with a deletion that removed the a2-encoding VH1 on both chromosomes do survive, rearrange other VH genes and produce normal levels of immunoglobulins as well as a significant percentage of B cells which bear the a2 allotype. This challenges the view that one VH gene, VH1, is solely responsible for the inheritance pattern of VHa allotypes.

Characterization of novel RS1 exonic deletions in juvenile X-linked retinoschisis

PubMed Central

D’Souza, Leera; Cukras, Catherine; Antolik, Christian; Craig, Candice; He, Hong; Li, Shibo; Hejtmancik, James F.; Sieving, Paul A.; Wang, Xinjing

2013-01-01

Purpose X-linked juvenile retinoschisis (XLRS) is a vitreoretinal dystrophy characterized by schisis (splitting) of the inner layers of the neuroretina. Mutations within the retinoschisis (RS1) gene are responsible for this disease. The mutation spectrum consists of amino acid substitutions, splice site variations, small indels, and larger genomic deletions. Clinically, genomic deletions are rarely reported. Here, we characterize two novel full exonic deletions: one encompassing exon 1 and the other spanning exons 4–5 of the RS1 gene. We also report the clinical findings in these patients with XLRS with two different exonic deletions. Methods Unrelated XLRS men and boys and their mothers (if available) were enrolled for molecular genetics evaluation. The patients also underwent ophthalmologic examination and in some cases electroretinogram (ERG) recording. All the exons and the flanking intronic regions of the RS1 gene were analyzed with direct sequencing. Two patients with exonic deletions were further evaluated with array comparative genomic hybridization to define the scope of the genomic aberrations. After the deleted genomic region was identified, primer walking followed by direct sequencing was used to determine the exact breakpoints. Results Two novel exonic deletions of the RS1 gene were identified: one including exon 1 and the other spanning exons 4 and 5. The exon 1 deletion extends from the 5′ region of the RS1 gene (including the promoter) through intron 1 (c.(−35)-1723_c.51+2664del4472). The exon 4–5 deletion spans introns 3 to intron 5 (c.185–1020_c.522+1844del5764). Conclusions Here we report two novel exonic deletions within the RS1 gene locus. We have also described the clinical presentations and hypothesized the genomic mechanisms underlying these schisis phenotypes. PMID:24227916

Characterization of novel RS1 exonic deletions in juvenile X-linked retinoschisis.

PubMed

D'Souza, Leera; Cukras, Catherine; Antolik, Christian; Craig, Candice; Lee, Ji-Yun; He, Hong; Li, Shibo; Smaoui, Nizar; Hejtmancik, James F; Sieving, Paul A; Wang, Xinjing

2013-01-01

X-linked juvenile retinoschisis (XLRS) is a vitreoretinal dystrophy characterized by schisis (splitting) of the inner layers of the neuroretina. Mutations within the retinoschisis (RS1) gene are responsible for this disease. The mutation spectrum consists of amino acid substitutions, splice site variations, small indels, and larger genomic deletions. Clinically, genomic deletions are rarely reported. Here, we characterize two novel full exonic deletions: one encompassing exon 1 and the other spanning exons 4-5 of the RS1 gene. We also report the clinical findings in these patients with XLRS with two different exonic deletions. Unrelated XLRS men and boys and their mothers (if available) were enrolled for molecular genetics evaluation. The patients also underwent ophthalmologic examination and in some cases electroretinogram (ERG) recording. All the exons and the flanking intronic regions of the RS1 gene were analyzed with direct sequencing. Two patients with exonic deletions were further evaluated with array comparative genomic hybridization to define the scope of the genomic aberrations. After the deleted genomic region was identified, primer walking followed by direct sequencing was used to determine the exact breakpoints. Two novel exonic deletions of the RS1 gene were identified: one including exon 1 and the other spanning exons 4 and 5. The exon 1 deletion extends from the 5' region of the RS1 gene (including the promoter) through intron 1 (c.(-35)-1723_c.51+2664del4472). The exon 4-5 deletion spans introns 3 to intron 5 (c.185-1020_c.522+1844del5764). Here we report two novel exonic deletions within the RS1 gene locus. We have also described the clinical presentations and hypothesized the genomic mechanisms underlying these schisis phenotypes.

Recurrence and variability of germline EPCAM deletions in Lynch syndrome.

PubMed

Kuiper, Roland P; Vissers, Lisenka E L M; Venkatachalam, Ramprasath; Bodmer, Danielle; Hoenselaar, Eveline; Goossens, Monique; Haufe, Aline; Kamping, Eveline; Niessen, Renée C; Hogervorst, Frans B L; Gille, Johan J P; Redeker, Bert; Tops, Carli M J; van Gijn, Marielle E; van den Ouweland, Ans M W; Rahner, Nils; Steinke, Verena; Kahl, Philip; Holinski-Feder, Elke; Morak, Monika; Kloor, Matthias; Stemmler, Susanne; Betz, Beate; Hutter, Pierre; Bunyan, David J; Syngal, Sapna; Culver, Julie O; Graham, Tracy; Chan, Tsun L; Nagtegaal, Iris D; van Krieken, J Han J M; Schackert, Hans K; Hoogerbrugge, Nicoline; van Kessel, Ad Geurts; Ligtenberg, Marjolijn J L

2011-04-01

Recently, we identified 3' end deletions in the EPCAM gene as a novel cause of Lynch syndrome. These truncating EPCAM deletions cause allele-specific epigenetic silencing of the neighboring DNA mismatch repair gene MSH2 in tissues expressing EPCAM. Here we screened a cohort of unexplained Lynch-like families for the presence of EPCAM deletions. We identified 27 novel independent MSH2-deficient families from multiple geographical origins with varying deletions all encompassing the 3' end of EPCAM, but leaving the MSH2 gene intact. Within The Netherlands and Germany, EPCAM deletions appeared to represent at least 2.8% and 1.1% of the confirmed Lynch syndrome families, respectively. MSH2 promoter methylation was observed in epithelial tissues of all deletion carriers tested, thus confirming silencing of MSH2 as the causative defect. In a total of 45 families, 19 different deletions were found, all including the last two exons and the transcription termination signal of EPCAM. All deletions appeared to originate from Alu-repeat mediated recombination events. In 17 cases regions of microhomology around the breakpoints were found, suggesting nonallelic homologous recombination as the most likely mechanism. We conclude that 3' end EPCAM deletions are a recurrent cause of Lynch syndrome, which should be implemented in routine Lynch syndrome diagnostics. © 2011 Wiley-Liss, Inc.

Familial spinal neurofibromatosis due to a multiexonic NF1 gene deletion.

PubMed

Pizzuti, Antonio; Bottillo, Irene; Inzana, Francesca; Lanari, Valentina; Buttarelli, Francesca; Torrente, Isabella; Giallonardo, Anna Teresa; De Luca, Alessandro; Dallapiccola, Bruno

2011-08-01

We report the detailed clinical presentation and molecular features of a spinal neurofibromatosis familial case where a 40-year-old woman, presenting with multiple bilateral spinal neurofibromas and no other clinical feature of neurofibromatosis type 1 (NF1), inherited a paternal large multiexonic deletion (c.5944-?_7126+?del) which resulted in NF1 gene haploinsufficiency at the RNA level. In the clinically unaffected 73-year-old father, spinal cord MRI disclosed bilateral and symmetrical hypertrophy of spinal lumbosacral roots. Our study widens the phenotypic and mutational spectrum of NF1 and illustrates the difficulties of counseling patients with border-line or atypical presentation of this disorder.

Targeted deletion of RANKL in M cell inducer cells by the Col6a1-Cre driver.

PubMed

Nagashima, Kazuki; Sawa, Shinichiro; Nitta, Takeshi; Prados, Alejandro; Koliaraki, Vasiliki; Kollias, George; Nakashima, Tomoki; Takayanagi, Hiroshi

2017-11-04

The gut-associated lymphoid tissues (GALTs), including Peyer's patches (PPs), cryptopatches (CPs) and isolated lymphoid follicles (ILFs), establish a host-microbe symbiosis by the promotion of immune reactions against gut microbes. Microfold cell inducer (MCi) cells in GALTs are the recently identified mesenchymal cells that express the cytokine RANKL and initiate bacteria-specific immunoglobulin A (IgA) production via induction of microfold (M) cell differentiation. In the previous study, the Twist2-Cre driver was utilized for gene deletion in mesenchymal cells including MCi cells. In order to investigate MCi cells more extensively, it will be necessary to develop experimental tools in addition to the Twist2-Cre driver mice and characterize such drivers in specificity and efficiency. Here we show that M cell differentiation and IgA production are impaired in the targeted deletion of RANKL by the Col6a1-Cre driver. We compared Col6a1-Cre with Twist2-Cre in terms of the specificity for mesenchymal cells in GALTs. Col6a1-Cre CAG-CAT-EGFP mice exhibited EGFP expression in podoplanin + CD31 - cells including MCi cells, while Twist2-Cre mice were shown to target endothelial cells and podoplanin + CD31 - cells. Tnfsf11 fl/Δ Col6a1-Cre mice exhibited the absence of M cells and severe IgA reduction together with an alteration in gut microbial composition. Moreover, we analyzed germ free mice to test whether changes in the microbiota are the cause of M cell deficiency. M cell differentiation was normal in the CPs/ILFs of germ free mice, indicating that MCi cells induce M cells independently of microbial colonization. This study demonstrates that Col6a1-Cre driver mice are as useful as Twist2-Cre driver mice for functional analyses of GALT-resident mesenchymal cells, including MCi cells. Copyright © 2017 Elsevier Inc. All rights reserved.

[The prevalence of SHOX gene deletion in children with idiopathic short stature. A multicentric study].

PubMed

Dávid, Anna; Butz, Henriett; Halász, Zita; Török, Dóra; Nyirő, Gábor; Muzsnai, Ágota; Csákváry, Violetta; Luczay, Andrea; Sallai, Ágnes; Hosszú, Éva; Felszeghy, Enikő; Tar, Attila; Szántó, Zsuzsanna; Fekete, Gy László; Kun, Imre; Patócs, Attila; Bertalan, Rita

2017-08-01

The isolated haploinsufficiency of the SHOX gene is one of the most common cause of short stature determined by monogenic mutations. The heterozygous deviation of the gene can be detected in 2-15% of patients with idiopathic short stature (ISS), in 50-90% of patients with Leri-Weill dyschondrosteosis syndrome (LWS), and in almost 100% of patients with Turner syndrome. The aim of our study was to evaluate the frequency of SHOX gene haploinsufficiency in children with ISS, LWS and in patients having Turner syndrome phenotype (TF), but normal karyotype, and to identify the dysmorphic signs characteristic for SHOX gene deficiency. A total of 144 patients were included in the study. Multiplex Ligation-dependent Probe Amplification (MLPA) method was used to identify the SHOX gene haploinsufficiency. The relationships between clinical data (axiological parameters, skeletal disorders, dysmorphic signs) and genotype were analyzed by statistical methods. 11 (7.6%) of the 144 patients showed SHOX gene deficiency with female dominance (8/11, 81% female). The SHOX positive patients had a significantly higher BMI (in 5/11 vs. 20/133 cases, p<0.02) and presented more frequent dysmorphic signs (9/11vs 62/133, p = 0.02). Madelung deformity of the upper limbs was also significantly more frequent among the SHOX positive patients (4/11, i.e. 36%, vs. 14/133, i.e. 10%, p = 0.0066). There were no statistically significant differences between the mean age, mean height and auxological measurements (sitting height/height, arm span/height) between the two groups of patients. The occurrence of SHOX gene haploinsufficiency observed in our population corresponds to the literature data. In SHOX positive patients, in addition to short stature, the dysmorphic signs have a positive predictive value for SHOX gene alterations. However, the SHOX deletion detected in a patient with idiopathic short stature without dysmorphic signs suggest that SHOX deletion analysis can be recommended in patients with

A self-excising beta-recombinase/six cassette for repetitive gene deletion and homokaryon purification in Neurospora crassa

USDA-ARS?s Scientific Manuscript database

In a previous study we developed a cassette employing a bacterial beta-recombinase acting on six recognition sequences (beta-rec/six), which allowed repetitive site-specific gene deletion and marker recycling in Neurospora crassa. However, only one positive selection marker was used in the cassette...

Loss-of-function variants in NFIA provide further support that NFIA is a critical gene in 1p32-p31 deletion syndrome: A four patient series.

PubMed

Revah-Politi, Anya; Ganapathi, Mythily; Bier, Louise; Cho, Megan T; Goldstein, David B; Hemati, Parisa; Iglesias, Alejandro; Juusola, Jane; Pappas, John; Petrovski, Slavé; Wilson, Ashley L; Aggarwal, Vimla S; Anyane-Yeboa, Kwame

2017-12-01

The association between 1p32-p31 contiguous gene deletions and a distinct phenotype that includes anomalies of the corpus callosum, ventriculomegaly, developmental delay, seizures, and dysmorphic features has been long recognized and described. Recently, the observation of overlapping phenotypes in patients with chromosome translocations that disrupt NFIA (Nuclear factor I/A), a gene within this deleted region, and NFIA intragenic deletions has led to the hypothesis that NFIA is a critical gene within this region. The wide application and increasing accessibility of whole exome sequencing (WES) has helped identify new cases to support this hypothesis. Here, we describe four patients with loss-of-function variants in the NFIA gene identified through WES. The clinical presentation of these patients significantly overlaps with the phenotype described in previously reported cases of 1p32-p31 deletion syndrome, NFIA gene disruptions and intragenic NFIA deletions. Our cohort includes a mother and daughter as well as an unrelated individual who share the same nonsense variant (c.205C>T, p.Arg69Ter; NM_001145512.1). We also report a patient with a frameshift NFIA variant (c.159_160dupCC, p.Gln54ProfsTer49). We have compared published cases of 1p32-p31 microdeletion syndrome, translocations resulting in NFIA gene disruption, intragenic deletions, and loss-of-function mutations (including our four patients) to reveal that abnormalities of the corpus callosum, ventriculomegaly/hydrocephalus, macrocephaly, Chiari I malformation, dysmorphic features, developmental delay, hypotonia, and urinary tract defects are common findings. The consistent overlap in clinical presentation provides further evidence of the critical role of NFIA haploinsufficiency in the development of the 1p32-p31 microdeletion syndrome phenotype. © 2017 Wiley Periodicals, Inc.

Pathological mechanisms underlying single large‐scale mitochondrial DNA deletions

PubMed Central

Rocha, Mariana C.; Rosa, Hannah S.; Grady, John P.; Blakely, Emma L.; He, Langping; Romain, Nadine; Haller, Ronald G.; Newman, Jane; McFarland, Robert; Ng, Yi Shiau; Gorman, Grainne S.; Schaefer, Andrew M.; Tuppen, Helen A.; Taylor, Robert W.

2018-01-01

Objective Single, large‐scale deletions in mitochondrial DNA (mtDNA) are a common cause of mitochondrial disease. This study aimed to investigate the relationship between the genetic defect and molecular phenotype to improve understanding of pathogenic mechanisms associated with single, large‐scale mtDNA deletions in skeletal muscle. Methods We investigated 23 muscle biopsies taken from adult patients (6 males/17 females with a mean age of 43 years) with characterized single, large‐scale mtDNA deletions. Mitochondrial respiratory chain deficiency in skeletal muscle biopsies was quantified by immunoreactivity levels for complex I and complex IV proteins. Single muscle fibers with varying degrees of deficiency were selected from 6 patient biopsies for determination of mtDNA deletion level and copy number by quantitative polymerase chain reaction. Results We have defined 3 “classes” of single, large‐scale deletion with distinct patterns of mitochondrial deficiency, determined by the size and location of the deletion. Single fiber analyses showed that fibers with greater respiratory chain deficiency harbored higher levels of mtDNA deletion with an increase in total mtDNA copy number. For the first time, we have demonstrated that threshold levels for complex I and complex IV deficiency differ based on deletion class. Interpretation Combining genetic and immunofluorescent assays, we conclude that thresholds for complex I and complex IV deficiency are modulated by the deletion of complex‐specific protein‐encoding genes. Furthermore, removal of mt‐tRNA genes impacts specific complexes only at high deletion levels, when complex‐specific protein‐encoding genes remain. These novel findings provide valuable insight into the pathogenic mechanisms associated with these mutations. Ann Neurol 2018;83:115–130 PMID:29283441

Controllability analysis of the directed human protein interaction network identifies disease genes and drug targets

PubMed Central

Vinayagam, Arunachalam; Gibson, Travis E.; Lee, Ho-Joon; Yilmazel, Bahar; Roesel, Charles; Hu, Yanhui; Kwon, Young; Sharma, Amitabh; Liu, Yang-Yu; Perrimon, Norbert; Barabási, Albert-László

2016-01-01

The protein–protein interaction (PPI) network is crucial for cellular information processing and decision-making. With suitable inputs, PPI networks drive the cells to diverse functional outcomes such as cell proliferation or cell death. Here, we characterize the structural controllability of a large directed human PPI network comprising 6,339 proteins and 34,813 interactions. This network allows us to classify proteins as “indispensable,” “neutral,” or “dispensable,” which correlates to increasing, no effect, or decreasing the number of driver nodes in the network upon removal of that protein. We find that 21% of the proteins in the PPI network are indispensable. Interestingly, these indispensable proteins are the primary targets of disease-causing mutations, human viruses, and drugs, suggesting that altering a network’s control property is critical for the transition between healthy and disease states. Furthermore, analyzing copy number alterations data from 1,547 cancer patients reveals that 56 genes that are frequently amplified or deleted in nine different cancers are indispensable. Among the 56 genes, 46 of them have not been previously associated with cancer. This suggests that controllability analysis is very useful in identifying novel disease genes and potential drug targets. PMID:27091990

Prevalence and spectrum of large deletions or duplications in the major long QT syndrome-susceptibility genes and implications for long QT syndrome genetic testing.

PubMed

Tester, David J; Benton, Amber J; Train, Laura; Deal, Barbara; Baudhuin, Linnea M; Ackerman, Michael J

2010-10-15

Long QT syndrome (LQTS) is a cardiac channelopathy associated with syncope, seizures, and sudden death. Approximately 75% of LQTS is due to mutations in genes encoding for 3 cardiac ion channel α-subunits (LQT1 to LQT3). However, traditional mutational analyses have limited detection capabilities for atypical mutations such as large gene rearrangements. We set out to determine the prevalence and spectrum of large deletions/duplications in the major LQTS-susceptibility genes in unrelated patients who were mutation negative after point mutation analysis of LQT1- to LQT12-susceptibility genes. Forty-two unrelated, clinically strong LQTS patients were analyzed using multiplex ligation-dependent probe amplification, a quantitative fluorescent technique for detecting multiple exon deletions and duplications. The SALSA multiplex ligation-dependent probe amplification LQTS kit from MRC-Holland was used to analyze the 3 major LQTS-associated genes, KCNQ1, KCNH2, and SCN5A, and the 2 minor genes, KCNE1 and KCNE2. Overall, 2 gene rearrangements were found in 2 of 42 unrelated patients (4.8%, confidence interval 1.7 to 11). A deletion of KCNQ1 exon 3 was identified in a 10-year-old Caucasian boy with a corrected QT duration of 660 ms, a personal history of exercise-induced syncope, and a family history of syncope. A deletion of KCNQ1 exon 7 was identified in a 17-year-old Caucasian girl with a corrected QT duration of 480 ms, a personal history of exercise-induced syncope, and a family history of sudden cardiac death. In conclusion, because nearly 5% of patients with genetically elusive LQTS had large genomic rearrangements involving the canonical LQTS-susceptibility genes, reflex genetic testing to investigate genomic rearrangements may be of clinical value. Copyright © 2010 Elsevier Inc. All rights reserved.

Genome engineering using a synthetic gene circuit in Bacillus subtilis.

PubMed

Jeong, Da-Eun; Park, Seung-Hwan; Pan, Jae-Gu; Kim, Eui-Joong; Choi, Soo-Keun

2015-03-31

Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac-chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Haptoglobin genotyping of Vietnamese: global distribution of HP del, complete deletion allele of the HP gene.

PubMed

Soejima, Mikiko; Agusa, Tetsuro; Iwata, Hisato; Fujihara, Junko; Kunito, Takashi; Takeshita, Haruo; Lan, Vi Thi Mai; Minh, Tu Binh; Takahashi, Shin; Trang, Pham Thi Kim; Viet, Pham Hung; Tanabe, Shinsuke; Koda, Yoshiro

2015-01-01

The haptoglobin (HP) gene deletion allele (HP(del)) is responsible for anhaptoglobinemia and a genetic risk factor for anaphylaxis reaction after transfusion due to production of the anti-HP antibody. The distribution of this allele has been explored by several groups including ours. Here, we studied the frequency of HP(del) in addition to the distribution of common HP genotypes in 293 Vietnamese. The HP(del) was encountered with the frequency of 0.020. The present result suggested that this deletion allele is restricted to East and Southeast Asians. Thus, this allele seems to be a potential ancestry informative marker for these populations. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Increased production of biomass-degrading enzymes by double deletion of creA and creB genes involved in carbon catabolite repression in Aspergillus oryzae.

PubMed

Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2018-02-01

In a previous study, we reported that a double gene deletion mutant for CreA and CreB, which constitute the regulatory machinery involved in carbon catabolite repression, exhibited improved production of α-amylase compared with the wild-type strain and single creA or creB deletion mutants in Aspergillus oryzae. Because A. oryzae can also produce biomass-degrading enzymes, such as xylolytic and cellulolytic enzymes, we examined the production levels of those enzymes in deletion mutants in this study. Xylanase and β-glucosidase activities in the wild-type were hardly detected in submerged culture containing xylose as the carbon source, whereas those enzyme activities were significantly increased in the single creA deletion (ΔcreA) and double creA and creB deletion (ΔcreAΔcreB) mutants. In particular, the ΔcreAΔcreB mutant exhibited >100-fold higher xylanase and β-glucosidase activities than the wild-type. Moreover, in solid-state culture, the β-glucosidase activity of the double deletion mutant was >7-fold higher than in the wild-type. These results suggested that deletion of both creA and creB genes could also efficiently improve the production levels of biomass-degrading enzymes in A. oryzae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

CRISPR-mediated direct mutation of cancer genes in the mouse liver

PubMed Central

Xue, Wen; Chen, Sidi; Yin, Hao; Tammela, Tuomas; Papagiannakopoulos, Thales; Joshi, Nikhil S.; Cai, Wenxin; Yang, Gillian; Bronson, Roderick; Crowley, Denise G.; Zhang, Feng; Anderson, Daniel G.; Sharp, Phillip A.; Jacks, Tyler

2014-01-01

The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem (ES) cells1. Here we describe a new method of cancer model generation using the CRISPR/Cas system in vivo in wild-type mice. We have used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs)2–4 to the liver and directly target the tumor suppressor genes Pten5 and p536, alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology7, 8. Simultaneous targeting of Pten and p53 induced liver tumors that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumor tissue revealed insertion or deletion (indel) mutations of the tumor suppressor genes, including bi-allelic mutations of both Pten and p53 in tumors. Furthermore, co-injection of Cas9 plasmids harboring sgRNAs targeting the β-Catenin gene (Ctnnb1) and a single-stranded DNA (ssDNA) oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-Catenin. This study demonstrates the feasibility of direct mutation of tumor suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics. PMID:25119044

CRISPR-mediated direct mutation of cancer genes in the mouse liver.

PubMed

Xue, Wen; Chen, Sidi; Yin, Hao; Tammela, Tuomas; Papagiannakopoulos, Thales; Joshi, Nikhil S; Cai, Wenxin; Yang, Gillian; Bronson, Roderick; Crowley, Denise G; Zhang, Feng; Anderson, Daniel G; Sharp, Phillip A; Jacks, Tyler

2014-10-16

The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the β-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.

Angiotensin-converting enzyme (ACE) gene insertion/deletion polymorphism is not a risk factor for hypertension in SLE nephritis.

PubMed

Negi, Vir S; Devaraju, Panneer; Gulati, Reena

2015-09-01

SLE is a systemic autoimmune disease with high prevalence of hypertension. Around 40-75 % of SLE patients develop nephritis, a major cause of hypertension and mortality. Angiotensin-converting enzyme (ACE) maintains the blood pressure and blood volume homeostasis. An insertion/deletion (I/D) polymorphism in intron 16 of ACE gene was reported to influence the development of hypertension, nephritis, and cardiovascular diseases in different ethnic populations. Despite compelling evidence for the high prevalence of hypertension in individuals with SLE, underlying factors for its development are not well studied. With this background, we analyzed the influence of ACE insertion/deletion polymorphism on susceptibility to SLE, development of nephritis and hypertension, other clinical features and autoantibody phenotype in South Indian SLE patients. Three hundred patients with SLE and 460 age and sex similar ethnicity matched individuals were included as patients and healthy controls, respectively. The ACE gene insertion/deletion polymorphism was analyzed by PCR. Insertion (I) and deletion (D) alleles were observed to be equally distributed among patients (57 and 43 %) and controls (59 and 41 %), respectively. The mutant (D) allele did not confer significant risk for SLE (II vs. ID: p = 0.4, OR 1.15, 95 % CI 0.8-1.6; II vs. DD: p = 0.34, OR 1.22, 95 % CI 0.8-1.85). There was no association of the ACE genotype or the allele with development of lupus nephritis (II vs. ID: p = 0.19, OR 1.41, 95 % CI 0.84-2.36; II vs. DD: p = 0.41, OR 0.74, 95 % CI 0.38-1.41) or hypertension (II vs. ID: p = 0.85, OR 0.9, 95 % CI 0.43-1.8; II vs. DD: p = 0.66, OR 1.217, 95 % CI 0.5-2.8). The presence of mutant allele (D) was not found to influence any clinical features or autoantibody phenotype. The insertion/deletion polymorphism of the ACE gene is not a genetic risk factor for SLE and does not influence development of hypertension or lupus nephritis in South Indian

Severe visual impairment and retinal changes in a boy with a deletion of the gene for Nance-Horan syndrome.

PubMed

Mathys, R; Deconinck, H; Keymolen, K; Jansen, A; Van Esch, H

2007-01-01

We present the ophthalmologic findings in a boy with a deletion of Xp22 comprising the gene for Nance-Horan syndrome. Different mechanisms underlying the visual impairment in Nance-Horan syndrome are discussed.

Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector

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

Watanabe, Daisuke; Brockman, Mark A.; Ndung'u, Thumbi

2007-01-20

Herpes simplex virus (HSV) recombinants induce durable immune responses in rhesus macaques and mice and have induced partial protection in rhesus macaques against mucosal challenge with virulent simian immunodeficiency virus (SIV). In this study, we evaluated the properties of a new generation HSV vaccine vector, an HSV-1 multiple immediate-early (IE) gene deletion mutant virus, d106, which contains deletions in the ICP4, ICP27, ICP22, and ICP47 genes. Because several of the HSV IE genes have been implicated in immune evasion, inactivation of the genes encoding these proteins was expected to result in enhanced immunogenicity. The d106 virus expresses few HSV genemore » products and shows minimal cytopathic effect in cultured cells. When d106 was inoculated into mice, viral DNA accumulated at high levels in draining lymph nodes, consistent with an ability to transduce dendritic cells and activate their maturation and movement to lymph nodes. A d106 recombinant expressing Escherichia coli {beta}-galactosidase induced durable {beta}-gal-specific IgG and CD8{sup +} T cell responses in naive and HSV-immune mice. Finally, d106-based recombinants have been constructed that express simian immunodeficiency virus (SIV) gag, env, or a rev-tat-nef fusion protein for several days in cultured cells. Thus, d106 shows many of the properties desirable in a vaccine vector: limited expression of HSV gene products and cytopathogenicity, high level expression of transgenes, ability to induce durable immune responses, and an ability to transduce dendritic cells and induce their maturation and migration to lymph nodes.« less

Deletion of the thymidine kinase gene induces complete attenuation of the Georgia isolate of African swine fever virus.

PubMed

Sanford, B; Holinka, L G; O'Donnell, V; Krug, P W; Carlson, J; Alfano, M; Carrillo, C; Wu, Ping; Lowe, Andre; Risatti, G R; Gladue, D P; Borca, M V

2016-02-02

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. There are no vaccines to control Africa swine fever (ASF). Experimental vaccines have been developed using genetically modified live attenuated ASFVs obtained by specifically deleting virus genes involved in virulence, including the thymidine kinase (TK) gene. TK has been shown to be involved in the virulence of several viruses, including ASFV. Here we report the construction of a recombinant virus (ASFV-G/V-ΔTK) obtained by deleting the TK gene in a virulent strain of ASFV Georgia adapted to replicate in Vero cells (ASFV-G/VP30). ASFV-G/P-ΔTK demonstrated decreased replication both in primary swine macrophage cell cultures and in Vero cells compared with ASFV-G/VP30. In vivo, intramuscular administration of up to 10(6) TCID50 of ASFV-G/V-ΔTK does not result in ASF disease. However, these animals are not protected when challenged with the virulent parental Georgia strain. Published by Elsevier B.V.

Targeted disruption of the murine Facc gene: Towards the establishment of a mouse model for Fanconi anemia

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

Chen, M.; Auerbach, W.; Buchwald, M.

1994-09-01

Fanconi anemia (FA) is an autosomal recessive disease characterized by bone marrow failure, congenital malformations and predisposition to malignancies. The gene responsible for the defect in FA group C has been cloned and designated the Fanconi Anemia Complementation Group C gene (FACC). A murine cDNA for this gene (Facc) was also cloned. Here we report our progress in the establishment of a mouse model for FA. The mouse Facc cDNA was used as probe to screen a genomic library of mouse strain 129. More than twenty positive clones were isolated. Three of them were mapped and found to be overlappingmore » clones, encompassing the genomic region from exon 8 to the end of the 3{prime} UTR of the mouse cDNA. A targeting vector was constructed using the most 5{prime} mouse genomic sequence available. The end result of the homologous recombination is that exon 8 is deleted and the neo gene is inserted. The last exon, exon 14, is essential for the complementing function of the FACC gene product; the disruption in the middle of the murine Facc gene should render this locus biologically inactive. This targeting vector was linearized and electroporated into R1 embryonic stem (ES) cells which were derived from the 129 mouse. Of 102 clones screened, 19 positive cell lines were identified. Four targeted cell lines have been used to produce chimeric mice. 129-derived ES cells were aggregated ex vivo into the morulas derived from CD1 mice and then implanted into foster mothers. 22 chimeras have been obtained. Moderately and strongly chimeric mice have been bred to test for germline transmission. Progeny with the expected coat color derived from 2 chimeras are currently being examined to confirm transmission of the targeted allele.« less

First case of Hb Fontainebleau with sickle haemoglobin and other non-deletional α gene variants identified in neonates during newborn screening for sickle cell disorders.

PubMed

Upadhye, Dipti S; Jain, Dipty; Nair, Sona B; Nadkarni, Anita H; Ghosh, Kanjaksha; Colah, Roshan B

2012-07-01

To evaluate the significance of non-deletional α gene variants identified in neonates during newborn screening for sickle cell disorders. 1534 newborn babies were screened in the last 2 years for sickle cell disease using a targeted screening approach. Investigations included a complete blood count, high performance liquid chromatography analysis, cellulose acetate electrophoresis (pH 8.9), heat stability test, restriction digestion and Amplified Refractory Mutation System for confirmation of sickle haemoglobin (Hb S), α genotyping by multiplex PCR and DNA sequencing. Three non-deletional α gene variants, Hb Fontainebleau, Hb O Indonesia and Hb Koya Dora, were identified in heterozygous condition in newborns. This is the first report of Hb Fontainebleau in association with Hb S. The baby had anaemia at birth (Hb 11.4 g/dl) with no cyanosis, icterus or need for transfusion. She had occipital encephalocoele and was operated on day 24 to remove the mass. The baby diagnosed with Hb O Indonesia in combination with Hb S also had a low haemoglobin level of 12.7 g/dl. Newborn screening for sickle cell disorders also enabled us to identify three α globin chain variants. Two babies who inherited Hb Fontainebleau and Hb O Indonesia along with Hb S had reduced Hb levels at birth and need to be followed up.

Early-onset seizures due to mosaic exonic deletions of CDKL5 in a male and two females.

PubMed

Bartnik, Magdalena; Derwińska, Katarzyna; Gos, Monika; Obersztyn, Ewa; Kołodziejska, Katarzyna E; Erez, Ayelet; Szpecht-Potocka, Agnieszka; Fang, Ping; Terczyńska, Iwona; Mierzewska, Hanna; Lohr, Naomi J; Bellus, Gary A; Reimschisel, Tyler; Bocian, Ewa; Mazurczak, Tadeusz; Cheung, Sau Wai; Stankiewicz, Paweł

2011-05-01

Mutations in the CDKL5 gene have been associated with an X-linked dominant early infantile epileptic encephalopathy-2. The clinical presentation is usually of severe encephalopathy with refractory seizures and Rett syndrome (RTT)-like phenotype. We attempted to assess the role of mosaic intragenic copy number variation in CDKL5. We have used comparative genomic hybridization with a custom-designed clinical oligonucleotide array targeting exons of selected disease and candidate genes, including CDKL5. We have identified mosaic exonic deletions of CDKL5 in one male and two females with developmental delay and medically intractable seizures. These three mosaic changes represent 60% of all deletions detected in 12,000 patients analyzed by array comparative genomic hybridization and involving the exonic portion of CDKL5. We report the first case of an exonic deletion of CDKL5 in a male and emphasize the importance of underappreciated mosaic exonic copy number variation in patients with early-onset seizures and RTT-like features of both genders.

TARGETED DELETION OF INDUCIBLE HEAT SHOCK PROTEIN 70 ABROGATES THE LATE INFARCT-SPARING EFFECT OF MYOCARDIAL ISCHEMIC PRECONDITIONING

EPA Science Inventory

Abstract submitted for 82nd annual meeting of the American Association for Thoracic Surgery, May 4-8, 2002 in Washington D.C.

Targeted Deletion of Inducible Heat Shock Protein 70 Abrogates the Late Infarct-Sparing Effect of Myocardial Ischemic Preconditioning

Craig...

Metabolic Dysfunction Consistent with Premature Aging Results from Deletion of Pim Kinases

PubMed Central

Din, Shabana; Konstandin, Mathias H; Johnson, Bevan; Emathinger, Jacqueline; Völkers, Mirko; Toko, Haruhiro; Collins, Brett; Ormachea, Lucy; Samse, Kaitlen; Kubli, Dieter A; De La Torre, Andrea; Kraft, Andrew S; Gustafsson, Asa B; Kelly, Daniel P; Sussman, Mark A

2014-01-01

Rationale The senescent cardiac phenotype is accompanied by changes in mitochondrial function and biogenesis causing impairment in energy provision. The relationship between myocardial senescence and Pim kinases deserves attention since Pim-1 kinase is cardioprotective, in part, by preservation of mitochondrial integrity. Study of the pathological effects resulting from genetic deletion of all Pim kinase family members could provide important insight regarding cardiac mitochondrial biology and the aging phenotype. Objective Demonstrate myocardial senescence is promoted by loss of Pim leading to premature aging and aberrant mitochondrial function. Methods and Results Cardiac myocyte senescence was evident at three months of age in Pim Triple KnockOut (PTKO) mice, where all three isoforms of Pim kinase family members are genetically deleted. Cellular hypertrophic remodeling and fetal gene program activation was followed by heart failure at six months in PTKO mice. Metabolic dysfunction is an underlying cause of cardiac senescence and instigates a decline in cardiac function. Altered mitochondrial morphology is evident consequential to Pim deletion together with decreased ATP levels and increased phosphorylated AMPK, exposing an energy deficiency in PTKO mice. Expression of the genes encoding master regulators of mitochondrial biogenesis, PPARγ coactivator-1 (PGC-1) α and β were diminished in PTKO hearts, as were downstream targets included in mitochondrial energy transduction, including fatty acid oxidation. Reversal of the dysregulated metabolic phenotype was observed by overexpressing c-Myc, a downstream target of Pim kinases. Conclusion Pim kinases prevent premature cardiac aging and maintain a healthy pool of functional mitochondria leading to efficient cellular energetics. PMID:24916111

Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe

PubMed Central

Duhig, Trevor; Nam, Miyoung; Palmer, Georgia; Han, Sangjo; Jeffery, Linda; Baek, Seung-Tae; Lee, Hyemi; Shim, Young Sam; Lee, Minho; Kim, Lila; Heo, Kyung-Sun; Noh, Eun Joo; Lee, Ah-Reum; Jang, Young-Joo; Chung, Kyung-Sook; Choi, Shin-Jung; Park, Jo-Young; Park, Youngwoo; Kim, Hwan Mook; Park, Song-Kyu; Park, Hae-Joon; Kang, Eun-Jung; Kim, Hyong Bai; Kang, Hyun-Sam; Park, Hee-Moon; Kim, Kyunghoon; Song, Kiwon; Song, Kyung Bin; Nurse, Paul; Hoe, Kwang-Lae

2014-01-01

SUMMARY We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome. This resource provides a powerful tool for biotechnological and eukaryotic cell biology research. Comprehensive gene dispensability comparisons with budding yeast, the first time such studies have been possible between two eukaryotes, revealed that 83% of single copy orthologues in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than non-essential genes to be single copy, broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth. PMID:20473289

Unique and atypical deletions in Prader-Willi syndrome reveal distinct phenotypes.

PubMed

Kim, Soo-Jeong; Miller, Jennifer L; Kuipers, Paul J; German, Jennifer Ruth; Beaudet, Arthur L; Sahoo, Trilochan; Driscoll, Daniel J

2012-03-01

Prader-Willi syndrome (PWS) is a multisystem, contiguous gene disorder caused by an absence of paternally expressed genes within the 15q11.2-q13 region via one of the three main genetic mechanisms: deletion of the paternally inherited 15q11.2-q13 region, maternal uniparental disomy and imprinting defect. The deletion class is typically subdivided into Type 1 and Type 2 based on their proximal breakpoints (BP1-BP3 and BP2-BP3, respectively). Despite PWS being a well-characterized genetic disorder the role of the specific genes contributing to various aspects of the phenotype are not well understood. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is a recently developed technique that detects copy number changes and aberrant DNA methylation. In this study, we initially applied MS-MLPA to elucidate the deletion subtypes of 88 subjects. In our cohort, 32 had a Type 1 and 49 had a Type 2 deletion. The remaining seven subjects had unique or atypical deletions that were either smaller (n=5) or larger (n=2) than typically described and were further characterized by array-based comparative genome hybridization. In two subjects both the PWS region (15q11.2) and the newly described 15q13.3 microdeletion syndrome region were deleted. The subjects with a unique or an atypical deletion revealed distinct phenotypic features. In conclusion, unique or atypical deletions were found in ∼8% of the deletion subjects with PWS in our cohort. These novel deletions provide further insight into the potential role of several of the genes within the 15q11.2 and the 15q13.3 regions.

Deletion of the Sm1 encoding motif in the lsm gene results in distinct changes in the transcriptome and enhanced swarming activity of Haloferax cells.

PubMed

Maier, Lisa-Katharina; Benz, Juliane; Fischer, Susan; Alstetter, Martina; Jaschinski, Katharina; Hilker, Rolf; Becker, Anke; Allers, Thorsten; Soppa, Jörg; Marchfelder, Anita

2015-10-01

Members of the Sm protein family are important for the cellular RNA metabolism in all three domains of life. The family includes archaeal and eukaryotic Lsm proteins, eukaryotic Sm proteins and archaeal and bacterial Hfq proteins. While several studies concerning the bacterial and eukaryotic family members have been published, little is known about the archaeal Lsm proteins. Although structures for several archaeal Lsm proteins have been solved already more than ten years ago, we still do not know much about their biological function, however one can confidently propose that the archaeal Lsm proteins will also be involved in RNA metabolism. Therefore, we investigated this protein in the halophilic archaeon Haloferax volcanii. The Haloferax genome encodes a single Lsm protein, the lsm gene overlaps and is co-transcribed with the gene for the ribosomal L37.eR protein. Here, we show that the reading frame of the lsm gene contains a promoter which regulates expression of the overlapping rpl37R gene. This rpl37R specific promoter ensures high expression of the rpl37R gene in exponential growth phase. To investigate the biological function of the Lsm protein we generated a lsm deletion mutant that had the coding sequence for the Sm1 motif removed but still contained the internal promoter for the downstream rpl37R gene. The transcriptome of this deletion mutant was compared to the wild type transcriptome, revealing that several genes are down-regulated and many genes are up-regulated in the deletion strain. Northern blot analyses confirmed down-regulation of two genes. In addition, the deletion strain showed a gain of function in swarming, in congruence with the up-regulation of transcripts encoding proteins required for motility. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Deletion of Xpter encompassing the SHOX gene and PAR1 region in familial patients with Leri-Weill Dyschondrosteosis syndrome.

PubMed

Mutesa, L; Vanbellinghen, J F; Hellin, A C; Segers, K; Jamar, M; Pierquin, G; Bours, V

2009-01-01

Heterozygote deletions or mutations of pseudoautosomal 1 region (PAR1) encompassing the short stature homeobox-containing (SHOX) gene cause Leri-Weill Dyschondrosteosis (LWD), which is a dominantly inherited osteochondroplasia characterized by short stature with mesomelic shortening of the upper and lower limbs and Madelung deformity of the wrists. SHOX is expressed by both sex chromosomes in males and females and plays an important role in bone growth and development. Clinically, the LWD expression is variable and more severe in females than males due to sex differences in oestrogen levels. Here, we report two familial cases of LWD with a large Xp terminal deletion (approximately 943 kb) of distal PAR1 encompassing the SHOX gene. In addition, the proband had mental retardation which appeared to be from recessive inheritance in the family.

Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

PubMed Central

Eberhart, Lauren J.; Ohlert, Janet M.; Knutson, Carolann M.; Plunkett, Mary H.

2015-01-01

Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes. PMID:25888177

Submicroscopic deletions at the WAGR locus, revealed by nonradioactive in situ hybridization.

PubMed

Fantes, J A; Bickmore, W A; Fletcher, J M; Ballesta, F; Hanson, I M; van Heyningen, V

1992-12-01

Fluorescence in situ hybridization (FISH) with biotin-labeled probes mapping to 11p13 has been used for the molecular analysis of deletions of the WAGR (Wilms tumor, aniridia, genitourinary abnormalities, and mental retardation) locus. We have detected a submicroscopic 11p13 deletion in a child with inherited aniridia who subsequently presented with Wilms tumor in a horseshoe kidney, only revealed at surgery. The mother, who has aniridia, was also found to carry a deletion including both the aniridia candidate gene (AN2) and the Wilms tumor predisposition gene (WT1). This is therefore a rare case of an inherited WAGR deletion. Wilms tumor has so far only been associated with sporadic de novo aniridia cases. We have shown that a cosmid probe for a candidate aniridia gene, homologous to the mouse Pax-6 gene, is deleted in cell lines from aniridia patients with previously characterized deletions at 11p13, while another cosmid marker mapping between two aniridia-associated translocation breakpoints (and hence a second candidate marker) is present on both chromosomes. These results support the Pax-6 homologue as a strong candidate for the AN2 gene. FISH with cosmid probes has proved to be a fast and reliable technique for the molecular analysis of deletions. It can be used with limited amounts of material and has strong potential for clinical applications.

Co-consumption of sugars or ethanol and glucose in a Saccharomyces cerevisiae strain deleted in the HXK2 gene.

PubMed

Raamsdonk, L M; Diderich, J A; Kuiper, A; van Gaalen, M; Kruckeberg, A L; Berden, J A; Van Dam, K; Kruckberg, A L

2001-08-01

In previous studies it was shown that deletion of the HXK2 gene in Saccharomyces cerevisiae yields a strain that hardly produces ethanol and grows almost exclusively oxidatively in the presence of abundant glucose. This paper reports on physiological studies on the hxk2 deletion strain on mixtures of glucose/sucrose, glucose/galactose, glucose/maltose and glucose/ethanol in aerobic batch cultures. The hxk2 deletion strain co-consumed galactose and sucrose, together with glucose. In addition, co-consumption of glucose and ethanol was observed during the early exponential growth phase. In S.cerevisiae, co-consumption of ethanol and glucose (in the presence of abundant glucose) has never been reported before. The specific respiration rate of the hxk2 deletion strain growing on the glucose/ethanol mixture was 900 micromol.min(-1).(g protein)(-1), which is four to five times higher than that of the hxk2 deletion strain growing oxidatively on glucose, three times higher than its parent growing on ethanol (when respiration is fully derepressed) and is almost 10 times higher than its parent growing on glucose (when respiration is repressed). This indicates that the hxk2 deletion strain has a strongly enhanced oxidative capacity when grown on a mixture of glucose and ethanol. Copyright 2001 John Wiley & Sons, Ltd.

Evolving phage vectors for cell targeted gene delivery.

PubMed

Larocca, David; Burg, Michael A; Jensen-Pergakes, Kristen; Ravey, Edward Prenn; Gonzalez, Ana Maria; Baird, Andrew

2002-03-01

We adapted filamentous phage vectors for targeted gene delivery to mammalian cells by inserting a mammalian reporter gene expression cassette (GFP) into the vector backbone and fusing the pIII coat protein to a cell targeting ligand (i.e. FGF2, EGF). Like transfection with animal viral vectors, targeted phage gene delivery is concentration, time, and ligand dependent. Importantly, targeted phage particles are specific for the appropriate target cell surface receptor. Phage have distinct advantages over existing gene therapy vectors because they are simple, economical to produce at high titer, have no intrinsic tropism for mammalian cells, and are relatively simple to genetically modify and evolve. Initially transduction by targeted phage particles was low resulting in foreign gene expression in 1-2% of transfected cells. We increased transduction efficiency by modifying both the transfection protocol and vector design. For example, we stabilized the display of the targeting ligand to create multivalent phagemid-based vectors with transduction efficiencies of up to 45% in certain cell lines when combined with genotoxic treatment. Taken together, these studies establish that the efficiency of phage-mediated gene transfer can be significantly improved through genetic modification. We are currently evolving phage vectors with enhanced cell targeting, increased stability, reduced immunogenicity and other properties suitable for gene therapy.

Homozygous deletions at 3p12 in breast and lung cancer.

PubMed

Sundaresan, V; Chung, G; Heppell-Parton, A; Xiong, J; Grundy, C; Roberts, I; James, L; Cahn, A; Bench, A; Douglas, J; Minna, J; Sekido, Y; Lerman, M; Latif, F; Bergh, J; Li, H; Lowe, N; Ogilvie, D; Rabbitts, P

1998-10-01

We have constructed a physical map of the region homozygously deleted in the U2020 cell line at 3p12, including the location of putative CpG islands. Adjacent to one of these islands, we have identified and cloned a new gene (DUTT1) and used probes from this gene to detect two other homozygous deletions occurring in lung and breast carcinomas: the smallest deletion is within the gene itself and would result in a truncated protein. The DUTT1 gene is a member of the neural cell adhesion molecule family, although its widespread expression suggests it plays a less specialized role compared to other members of the family.

Targeting gene therapy to cancer: a review.

PubMed

Dachs, G U; Dougherty, G J; Stratford, I J; Chaplin, D J

1997-01-01

In recent years the idea of using gene therapy as a modality in the treatment of diseases other than genetically inherited, monogenic disorders has taken root. This is particularly obvious in the field of oncology where currently more than 100 clinical trials have been approved worldwide. This report will summarize some of the exciting progress that has recently been made with respect to both targeting the delivery of potentially therapeutic genes to tumor sites and regulating their expression within the tumor microenvironment. In order to specifically target malignant cells while at the same time sparing normal tissue, cancer gene therapy will need to combine highly selective gene delivery with highly specific gene expression, specific gene product activity, and, possibly, specific drug activation. Although the efficient delivery of DNA to tumor sites remains a formidable task, progress has been made in recent years using both viral (retrovirus, adenovirus, adeno-associated virus) and nonviral (liposomes, gene gun, injection) methods. In this report emphasis will be placed on targeted rather than high-efficiency delivery, although those would need to be combined in the future for effective therapy. To date delivery has been targeted to tumor-specific and tissue-specific antigens, such as epithelial growth factor receptor, c-kit receptor, and folate receptor, and these will be described in some detail. To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue. Targeted gene expression has been analyzed using tissue-specific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements. Another novel possibility that will be

A deletion affecting an LRR-RLK gene co-segregates with the fruit flat shape trait in peach.

PubMed

López-Girona, Elena; Zhang, Yu; Eduardo, Iban; Mora, José Ramón Hernández; Alexiou, Konstantinos G; Arús, Pere; Aranzana, María José

2017-07-27

In peach, the flat phenotype is caused by a partially dominant allele in heterozygosis (Ss), fruits from homozygous trees (SS) abort a few weeks after fruit setting. Previous research has identified a SSR marker (UDP98-412) highly associated with the trait, found suitable for marker assisted selection (MAS). Here we report a ∼10 Kb deletion affecting the gene PRUPE.6G281100, 400 Kb upstream of UDP98-412, co-segregating with the trait. This gene is a leucine-rich repeat receptor-like kinase (LRR-RLK) orthologous to the Brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) group. PCR markers suitable for MAS confirmed its strong association with the trait in a collection of 246 cultivars. They were used to evaluate the DNA from a round fruit derived from a somatic mutation of the flat variety 'UFO-4', revealing that the mutation affected the flat associated allele (S). Protein BLAST alignment identified significant hits with genes involved in different biological processes. Best protein hit occurred with AtRLP12, which may functionally complement CLAVATA2, a key regulator that controls the stem cell population size. RT-PCR analysis revealed the absence of transcription of the partially deleted allele. The data support PRUPE.6G281100 as a candidate gene for flat shape in peach.

A de novo whole gene deletion of XIAP detected by exome sequencing analysis in very early onset inflammatory bowel disease: a case report.

PubMed

Kelsen, Judith R; Dawany, Noor; Martinez, Alejandro; Martinez, Alejuandro; Grochowski, Christopher M; Maurer, Kelly; Rappaport, Eric; Piccoli, David A; Baldassano, Robert N; Mamula, Petar; Sullivan, Kathleen E; Devoto, Marcella

2015-11-18

Children with very early-onset inflammatory bowel disease (VEO-IBD), those diagnosed at less than 5 years of age, are a unique population. A subset of these patients present with a distinct phenotype and more severe disease than older children and adults. Host genetics is thought to play a more prominent role in this young population, and monogenic defects in genes related to primary immunodeficiencies are responsible for the disease in a small subset of patients with VEO-IBD. We report a child who presented at 3 weeks of life with very early-onset inflammatory bowel disease (VEO-IBD). He had a complicated disease course and remained unresponsive to medical and surgical therapy. The refractory nature of his disease, together with his young age of presentation, prompted utilization of whole exome sequencing (WES) to detect an underlying monogenic primary immunodeficiency and potentially target therapy to the identified defect. Copy number variation analysis (CNV) was performed using the eXome-Hidden Markov Model. Whole exome sequencing revealed 1,380 nonsense and missense variants in the patient. Plausible candidate variants were not detected following analysis of filtered variants, therefore, we performed CNV analysis of the WES data, which led us to identify a de novo whole gene deletion in XIAP. This is the first reported whole gene deletion in XIAP, the causal gene responsible for XLP2 (X-linked lymphoproliferative Disease 2). XLP2 is a syndrome resulting in VEO-IBD and can increase susceptibility to hemophagocytic lymphohistocytosis (HLH). This identification allowed the patient to be referred for bone marrow transplantation, potentially curative for his disease and critical to prevent the catastrophic sequela of HLH. This illustrates the unique etiology of VEO-IBD, and the subsequent effects on therapeutic options. This cohort requires careful and thorough evaluation for monogenic defects and primary immunodeficiencies.

Factor IX[sub Madrid 2]: A deletion/insertion in Facotr IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site

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

Solera, J.; Magallon, M.; Martin-Villar, J.

1992-02-01

DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5[prime] end of intron d and the two last coding nucleotides located at the 3[prime] end of exon IV in the normal factor IX gene; this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends ofmore » the deleted DNA fragment.« less

Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

PubMed Central

Tan, Siyuan; Guschin, Dmitry; Davalos, Albert; Lee, Ya-Li; Snowden, Andrew W.; Jouvenot, Yann; Zhang, H. Steven; Howes, Katherine; McNamara, Andrew R.; Lai, Albert; Ullman, Chris; Reynolds, Lindsey; Moore, Michael; Isalan, Mark; Berg, Lutz-Peter; Campos, Bradley; Qi, Hong; Spratt, S. Kaye; Case, Casey C.; Pabo, Carl O.; Campisi, Judith; Gregory, Philip D.

2003-01-01

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics. PMID:14514889

Construction of brewing-wine Aspergillus oryzae pyrG- mutant by pyrG gene deletion and its application in homology transformation.

PubMed

Du, Yu; Xie, Guizhen; Yang, Chunfa; Fang, Baishan; Chen, Hongwen

2014-06-01

pyrG(-) host cells are indispensable for pyrG(-) based transformation system. Isolations of pyrG(-) host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG(-) mutant by site-directed mutation of pyrG gene deletion which would be used as a host for further transformation. pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to construct pyrG(-) mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating that pyrG was completely excised. The ΔpyrG mutants were applied as pyrG(-) host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production of A. oryzae. The xdh disruption mutants were efficiently constructed by transforming a pMD-pyrG-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Δxdh mutant was three times as much as that of the ΔpyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG(-) host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding of A. oryzae. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Deletion of Plasmodium falciparum Histidine-Rich Protein 2 (pfhrp2) and Histidine-Rich Protein 3 (pfhrp3) Genes in Colombian Parasites.

PubMed