Copy number variation and missense mutations of the agouti signaling protein (ASIP) gene in goat breeds with different coat colors.

PubMed

Fontanesi, L; Beretti, F; Riggio, V; Gómez González, E; Dall'Olio, S; Davoli, R; Russo, V; Portolano, B

2009-01-01

In goats, classical genetic studies reported a large number of alleles at the Agouti locus with effects on coat color and pattern distribution. From these early studies, the dominant A(Wt) (white/tan) allele was suggested to cause the white color of the Saanen breed. Here, we sequenced the coding region of the goat ASIP gene in 6 goat breeds (Girgentana, Maltese, Derivata di Siria, Murciano-Granadina, Camosciata delle Alpi, and Saanen), with different coat colors and patterns. Five single nucleotide polymorphisms (SNPs) were identified, 3 of which caused missense mutations in conserved positions of the cysteine-rich carboxy-terminal domain of the protein (p.Ala96Gly, p.Cys126Gly, and p.Val128Gly). Allele and genotype frequencies suggested that these mutations are not associated or not completely associated with coat color in the investigated goat breeds. Moreover, genotyping and sequencing results, deviation from Hardy-Weinberg equilibrium, as well as allele copy number evaluation from semiquantitative fluorescent multiplex PCR, indicated the presence of copy number variation (CNV) in all investigated breeds. To confirm the presence of CNV and evaluate its extension, we applied a bovine-goat cross-species array comparative genome hybridization (aCGH) experiment using a custom tiling array based on bovine chromosome 13. aCGH results obtained for 8 goat DNA samples confirmed the presence of CNV affecting a region of less that 100 kb including the ASIP and AHCY genes. In Girgentana and Saanen breeds, this CNV might cause the A(Wt) allele, as already suggested for a similar structural mutation in sheep affecting the ASIP and AHCY genes, providing evidence for a recurrent interspecies CNV. However, other mechanisms may also be involved in determining coat color in these 2 breeds. Copyright 2009 S. Karger AG, Basel.

An autosomal recessive mutation in SCL24A4 causing enamel hypoplasia in Samoyed and its relationship to breed-wide genetic diversity.

PubMed

Pedersen, Niels C; Shope, Bonnie; Liu, Hongwei

2017-01-01

Pure breeding of dogs has led to over 700 heritable disorders, of which almost 300 are Mendelian in nature. Seventy percent of the characterized mutations have an autosomal recessive mode of inheritance, indicative of positive selection during bouts of inbreeding primarily for new desired conformational traits. Samoyed suffer from several common complex genetic disorders, but up to this time only two X-linked and one autosomal dominant disorder have been identified. Previous studies based on pedigrees and SNP arrays have concluded that Samoyed breeders have done a good job in maintaining genetic diversity and avoiding excessive inbreeding. This may explain why autosomal recessive disorders have not occurred to the extent observed in many other breeds. However, an enamel hypoplasia analogous to a form of autosomal recessive amelogenesis imperfecta (ARAI) in humans has been recently characterized in Samoyed, although the causative mutation appears to have existed for three or more decades. The rise of such a mutation indicates that bouts of inbreeding for desired conformational traits are still occurring despite an old and well-defined breed standard. Therefore, the present study has two objectives: 1) measure genetic diversity in the breed using DNA and short tandem repeats (STR), and 2) identify the exact mutation responsible for enamel hypoplasia in the breed, possible explanations for its recent spread, and the effect of eliminating the mutation on existing genetic diversity. The recent discovery of an autosomal recessive amelogenesis imperfecta (ARAI) in Samoyed provides an opportunity to study the mutation as well as genetic factors that favored its occurrence and subsequent spread. The first step in the study was to use 33 short tandem repeat (STR) loci on 25/38 autosomes and seven STRs across the dog leukocyte antigen (DLA) class I and II regions on CFA12 to determine the DNA-based genetic profile of 182 individuals from North America, Europe and Australia

NCI-MATCH Trial Links Targeted Drugs to Mutations

Cancer.gov

Investigators for the nationwide trial, NCI-MATCH: Molecular Analysis for Therapy Choice, announced that the trial will seek to determine whether targeted therapies for people whose tumors have specific gene mutations will be effective regardless of their cancer type. NCI-MATCH will incorporate more than 20 different study drugs or drug combinations, each targeting a specific gene mutation, in order to match each patient in the trial with a therapy that targets a molecular abnormality in their tumor.

HER2 activating mutations are targets for colorectal cancer treatment.

PubMed

Kavuri, Shyam M; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M; Migliardi, Giorgia; Searleman, Adam C; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A; Bertotti, Andrea; Bose, Ron

2015-08-01

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs. HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer. ©2015 American Association for Cancer Research.

Novel mutations target distinct subgroups of medulloblastoma

PubMed Central

Robinson, Giles; Parker, Matthew; Kranenburg, Tanya A.; Lu, Charles; Chen, Xiang; Ding, Li; Phoenix, Timothy N.; Hedlund, Erin; Wei, Lei; Zhu, Xiaoyan; Chalhoub, Nader; Baker, Suzanne J.; Huether, Robert; Kriwacki, Richard; Curley, Natasha; Thiruvenkatam, Radhika; Wang, Jianmin; Wu, Gang; Rusch, Michael; Hong, Xin; Beckford, Jared; Gupta, Pankaj; Ma, Jing; Easton, John; Vadodaria, Bhavin; Onar-Thomas, Arzu; Lin, Tong; Li, Shaoyi; Pounds, Stanley; Paugh, Steven; Zhao, David; Kawauchi, Daisuke; Roussel, Martine F.; Finkelstein, David; Ellison, David W.; Lau, Ching C.; Bouffet, Eric; Hassall, Tim; Gururangan, Sridharan; Cohn, Richard; Fulton, Robert S.; Fulton, Lucinda L.; Dooling, David J.; Ochoa, Kerri; Gajjar, Amar; Mardis, Elaine R.; Wilson, Richard K.; Downing, James R.; Zhang, Jinghui; Gilbertson, Richard J.

2012-01-01

Summary Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups. To identify mutations that drive medulloblastoma we sequenced the entire genomes of 37 tumours and matched normal blood. One hundred and thirty-six genes harbouring somatic mutations in this discovery set were sequenced in an additional 56 medulloblastomas. Recurrent mutations were detected in 41 genes not yet implicated in medulloblastoma: several target distinct components of the epigenetic machinery in different disease subgroups, e.g., regulators of H3K27 and H3K4 trimethylation in subgroup-3 and 4 (e.g., KDM6A and ZMYM3), and CTNNB1-associated chromatin remodellers in WNT-subgroup tumours (e.g., SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours, identified genes that maintain this cell lineage (DDX3X) as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumourigenesis. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development. PMID:22722829

Frequency of canine nt230(del4) MDR1 mutation in prone pure breeds, their crosses and mongrels in Israel - insights from a worldwide comparative perspective.

PubMed

Dekel, Yaron; Machluf, Yossy; Stoler, Aviad; Aderet, Arava; Baumel, Daniel; Kellerman, Efrat; Plotsky, Yoram; Noked Partouche, Oshrat; Elhalal, Gal; Ben-Shlomo, Izhar; Bercovich, Dani

2017-11-13

Sensitivity to macrocyclic lactones, which are commonly used in veterinary clinics, was first found in Rough Collies, and was attributed in 2001 to a 4 bp deletion in the MDR1 gene. The list of affected breeds currently includes 13 breeds. Researchers from different countries and continents examined the allelic frequencies of the nt230(del4) MDR1 mutation, emphasizing the clinical importance of this test not only to mutation-prone dogs, but also to their crosses and mongrels, since treatment of a deletion carrier with these compounds may lead to its death. In this study, the allelic frequencies of nt230(del4) MDR1 mutation in affected breeds, their crosses, unrelated pure breeds and mongrels are reported for the state of Israel (n = 1416 dogs). The Israeli data were compared with reports from the US, Europe, UK, Australia and Japan. The allelic frequencies of nt230(del4) MDR1 mutation in Israel for Australian, Swiss and German Shepherds (31%, 17% and 2.4%, respectively) are similar to the corresponding frequencies worldwide, much higher for Border Collies (4.8%), twice lower for Rough Collies (28%, compared to 55% or more elsewhere), and ~1% for mongrels. The frequencies for crosses of Australian Shepherd and Border Collies in Israel are 4 and 1.6 times lower, respectively, compared to the frequencies for the respective pure breeds. This work, that for the first time presents the frequency of nt230(del4) MDR1 mutation in Israel, along with a worldwide survey, has implications for clinicians, owners and breeders of sheepdogs and their crosses and supports the need for extra care in treatment and in future breeding. Of note, the relative proportion of affected breeds, in the overall tested dogs, might be higher than their actual proportion in Israel due to directed samples collection by veterinarians for clinical purposes, as these are mainly limited to certain affected breeds or dogs that resemble them.

Targeted next-generation sequencing extends the phenotypic and mutational spectrums for EYS mutations

PubMed Central

Gu, Shun; Tian, Yuanyuan; Chen, Xue

2016-01-01

Purpose We aim to determine genetic lesions with a phenotypic correlation in four Chinese families with autosomal recessive retinitis pigmentosa (RP). Methods Medical histories were carefully reviewed. All patients received comprehensive ophthalmic evaluations. The next-generation sequencing (NGS) approach targeting a panel of 205 retinal disease–relevant genes and 15 candidate genes was selectively performed on probands from the four recruited families for mutation detection. Online predictive software and crystal structure modeling were also applied to test the potential pathogenic effects of identified mutations. Results Of the four families, two were diagnosed with RP sino pigmento (RPSP). Patients with RPSP claimed to have earlier RP age of onset but slower disease progression. Five mutations in the eyes shut homolog (EYS) gene, involving two novel (c.7228+1G>A and c.9248G>A) and three recurrent mutations (c.4957dupA, c.6416G>A and c.6557G>A), were found as RP causative in the four families. The missense variant c.5093T>C was determined to be a variant of unknown significance (VUS) due to the variant’s colocalization in the same allele with the reported pathogenic mutation c.6416G>A. The two novel variants were further confirmed absent in 100 unrelated healthy controls. Online predictive software indicated potential pathogenicity of the three missense mutations. Further, crystal structural modeling suggested generation of two abnormal hydrogen bonds by the missense mutation p.G2186E (c.6557G>A) and elongation of its neighboring β-sheet induced by p.G3083D (c.9248G>A), which could alter the tertiary structure of the eys protein and thus interrupt its physicochemical properties. Conclusions Taken together, with the targeted NGS approach, we reveal novel EYS mutations and prove the efficiency of targeted NGS in the genetic diagnoses of RP. We also first report the correlation between EYS mutations and RPSP. The genotypic-phenotypic relationship in all

Targeted next-generation sequencing extends the phenotypic and mutational spectrums for EYS mutations.

PubMed

Gu, Shun; Tian, Yuanyuan; Chen, Xue; Zhao, Chen

2016-01-01

We aim to determine genetic lesions with a phenotypic correlation in four Chinese families with autosomal recessive retinitis pigmentosa (RP). Medical histories were carefully reviewed. All patients received comprehensive ophthalmic evaluations. The next-generation sequencing (NGS) approach targeting a panel of 205 retinal disease-relevant genes and 15 candidate genes was selectively performed on probands from the four recruited families for mutation detection. Online predictive software and crystal structure modeling were also applied to test the potential pathogenic effects of identified mutations. Of the four families, two were diagnosed with RP sino pigmento (RPSP). Patients with RPSP claimed to have earlier RP age of onset but slower disease progression. Five mutations in the eyes shut homolog (EYS) gene, involving two novel (c.7228+1G>A and c.9248G>A) and three recurrent mutations (c.4957dupA, c.6416G>A and c.6557G>A), were found as RP causative in the four families. The missense variant c.5093T>C was determined to be a variant of unknown significance (VUS) due to the variant's colocalization in the same allele with the reported pathogenic mutation c.6416G>A. The two novel variants were further confirmed absent in 100 unrelated healthy controls. Online predictive software indicated potential pathogenicity of the three missense mutations. Further, crystal structural modeling suggested generation of two abnormal hydrogen bonds by the missense mutation p.G2186E (c.6557G>A) and elongation of its neighboring β-sheet induced by p.G3083D (c.9248G>A), which could alter the tertiary structure of the eys protein and thus interrupt its physicochemical properties. Taken together, with the targeted NGS approach, we reveal novel EYS mutations and prove the efficiency of targeted NGS in the genetic diagnoses of RP. We also first report the correlation between EYS mutations and RPSP. The genotypic-phenotypic relationship in all Chinese patients carrying mutations in the EYS

A KCNJ10 mutation previously identified in the Russell group of terriers also occurs in Smooth-Haired Fox Terriers with hereditary ataxia and in related breeds.

PubMed

Rohdin, Cecilia; Gilliam, Douglas; O'Leary, Caroline A; O'Brien, Dennis P; Coates, Joan R; Johnson, Gary S; Jäderlund, Karin Hultin

2015-05-23

Hereditary ataxias with similar phenotypes were reported in the Smooth-Haired Fox Terrier, the Jack Russell Terrier and the Parson Russell Terrier. However, segregation analyses showed differing inheritance modes in these breeds. Recently, molecular genetic studies on the Russell group of terriers found independent mutations in KCNJ10 and CAPN1, each associated with a specific clinical subtype of inherited ataxia. The aim of this study was to clarify whether or not Smooth-Haired Fox Terriers with hereditary ataxia and dogs of other related breeds harbor either of the same mutations. A sub goal was to update the results of KCNJ10 genotyping in Russell group terriers. Three Smooth-Haired Fox Terriers with hereditary ataxia and two Toy Fox Terriers with a similar phenotype were all homozygous for the KCNJ10 mutation. The same mutation was also found in a heterozygous state in clinically unaffected Tenterfield Terriers (n = 5) and, in agreement with previous studies, in Jack Russell Terriers, Parson Russell Terriers, and Russell Terriers. A KCNJ10 mutation, previously associated with an autosomal recessive spinocerebellar ataxia in Jack Russell Terriers, Parson Russell Terriers, and Russell Terriers segregates in at least three more breeds descended from British hunting terriers. Ataxic members of two of these breeds, the Smooth-Haired Fox Terrier and the Toy Fox Terrier, were homozygous for the mutation, strengthening the likelihood that this genetic defect is indeed the causative mutation for the disease known as "hereditary ataxia" in Fox Terriers and "spinocerebellar ataxia with myokymia, seizures or both" in the Russell group of terriers.

HER2 activating mutations are targets for colorectal cancer treatment

PubMed Central

Kavuri, Shyam M.; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M.; Migliardi, Giorgia; Searleman, Adam C.; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A.; Bertotti, Andrea; Bose, Ron

2015-01-01

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of colorectal cancer patients. Introduction of the HER2 mutations, S310F, L755S, V777L, V842I, and L866M, into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutations are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors, neratinib and afatinib. HER2 gene sequencing of 48 cetuximab resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) WT colorectal cancer patient-derived xenografts (PDX’s) identified 4 PDX’s with HER2 mutations. HER2 targeted therapies were tested on two PDX’s. Treatment with a single HER2 targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2 targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2 mutated PDX’s. PMID:26243863

Genetic mutations in human rectal cancers detected by targeted sequencing.

PubMed

Bai, Jun; Gao, Jinglong; Mao, Zhijun; Wang, Jianhua; Li, Jianhui; Li, Wensheng; Lei, Yu; Li, Shuaishuai; Wu, Zhuo; Tang, Chuanning; Jones, Lindsey; Ye, Hua; Lou, Feng; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; Huang, Xue F; Chen, Si-Yi; Zhang, Enke

2015-10-01

Colorectal cancer (CRC) is widespread with significant mortality. Both inherited and sporadic mutations in various signaling pathways influence the development and progression of the cancer. Identifying genetic mutations in CRC is important for optimal patient treatment and many approaches currently exist to uncover these mutations, including next-generation sequencing (NGS) and commercially available kits. In the present study, we used a semiconductor-based targeted DNA-sequencing approach to sequence and identify genetic mutations in 91 human rectal cancer samples. Analysis revealed frequent mutations in KRAS (58.2%), TP53 (28.6%), APC (16.5%), FBXW7 (9.9%) and PIK3CA (9.9%), and additional mutations in BRAF, CTNNB1, ERBB2 and SMAD4 were also detected at lesser frequencies. Thirty-eight samples (41.8%) also contained two or more mutations, with common combination mutations occurring between KRAS and TP53 (42.1%), and KRAS and APC (31.6%). DNA sequencing for individual cancers is of clinical importance for targeted drug therapy and the advantages of such targeted gene sequencing over other NGS platforms or commercially available kits in sensitivity, cost and time effectiveness may aid clinicians in treating CRC patients in the near future.

True-breeding targeted gene knock-out in barley using designer TALE-nuclease in haploid cells.

PubMed

Gurushidze, Maia; Hensel, Goetz; Hiekel, Stefan; Schedel, Sindy; Valkov, Vladimir; Kumlehn, Jochen

2014-01-01

Transcription activator-like effector nucleases (TALENs) are customizable fusion proteins able to cleave virtually any genomic DNA sequence of choice, and thereby to generate site-directed genetic modifications in a wide range of cells and organisms. In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A gfp-specific TALEN pair was expressed via Agrobacterium-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of gfp. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to gfp, and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered gfp alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species.

MutScan: fast detection and visualization of target mutations by scanning FASTQ data.

PubMed

Chen, Shifu; Huang, Tanxiao; Wen, Tiexiang; Li, Hong; Xu, Mingyan; Gu, Jia

2018-01-22

Some types of clinical genetic tests, such as cancer testing using circulating tumor DNA (ctDNA), require sensitive detection of known target mutations. However, conventional next-generation sequencing (NGS) data analysis pipelines typically involve different steps of filtering, which may cause miss-detection of key mutations with low frequencies. Variant validation is also indicated for key mutations detected by bioinformatics pipelines. Typically, this process can be executed using alignment visualization tools such as IGV or GenomeBrowse. However, these tools are too heavy and therefore unsuitable for validating mutations in ultra-deep sequencing data. We developed MutScan to address problems of sensitive detection and efficient validation for target mutations. MutScan involves highly optimized string-searching algorithms, which can scan input FASTQ files to grab all reads that support target mutations. The collected supporting reads for each target mutation will be piled up and visualized using web technologies such as HTML and JavaScript. Algorithms such as rolling hash and bloom filter are applied to accelerate scanning and make MutScan applicable to detect or visualize target mutations in a very fast way. MutScan is a tool for the detection and visualization of target mutations by only scanning FASTQ raw data directly. Compared to conventional pipelines, this offers a very high performance, executing about 20 times faster, and offering maximal sensitivity since it can grab mutations with even one single supporting read. MutScan visualizes detected mutations by generating interactive pile-ups using web technologies. These can serve to validate target mutations, thus avoiding false positives. Furthermore, MutScan can visualize all mutation records in a VCF file to HTML pages for cloud-friendly VCF validation. MutScan is an open source tool available at GitHub: https://github.com/OpenGene/MutScan.

Missense and nonsense mutations in melanocortin 1 receptor (MC1R) gene of different goat breeds: association with red and black coat colour phenotypes but with unexpected evidences

PubMed Central

2009-01-01

Background Agouti and Extension loci control the relative amount of eumelanin and pheomelanin production in melanocytes that, in turn, affects pigmentation of skin and hair. The Extension locus encodes the melanocortin 1 receptor (MC1R) whose permanent activation, caused by functional mutations, results in black coat colour, whereas other inactivating mutations cause red coat colour in different mammals. Results The whole coding region of the MC1R gene was sequenced in goats of six different breeds showing different coat colours (Girgentana, white cream with usually small red spots in the face; Maltese, white with black cheeks and ears; Derivata di Siria, solid red; Murciano-Granadina, solid black or solid brown; Camosciata delle Alpi, brown with black stripes; Saanen, white; F1 goats and the parental animals). Five single nucleotide polymorphisms (SNPs) were identified: one nonsense mutation (p.Q225X), three missense mutations (p.A81V, p.F250V, and p.C267W), and one silent mutation. The stop codon at position 225 should cause the production of a shorter MC1R protein whose functionality may be altered. These SNPs were investigated in a larger sample of animals belonging to the six breeds. The Girgentana breed was almost fixed for the p.225X allele. However, there was not complete association between the presence of red spots in the face and the presence of this allele in homozygous condition. The same allele was identified in the Derivata di Siria breed. However, its frequency was only 33%, despite the fact that these animals are completely red. The p.267W allele was present in all Murciano-Granadina black goats, whereas it was never identified in the brown ones. Moreover, the same substitution was present in almost all Maltese goats providing evidence of association between this mutation and black coat colour. Conclusion According to the results obtained in the investigated goat breeds, MC1R mutations may determine eumelanic and pheomelanic phenotypes. However

Missense and nonsense mutations in melanocortin 1 receptor (MC1R) gene of different goat breeds: association with red and black coat colour phenotypes but with unexpected evidences.

PubMed

Fontanesi, Luca; Beretti, Francesca; Riggio, Valentina; Dall'Olio, Stefania; González, Elena Gómez; Finocchiaro, Raffaella; Davoli, Roberta; Russo, Vincenzo; Portolano, Baldassare

2009-08-25

Agouti and Extension loci control the relative amount of eumelanin and pheomelanin production in melanocytes that, in turn, affects pigmentation of skin and hair. The Extension locus encodes the melanocortin 1 receptor (MC1R) whose permanent activation, caused by functional mutations, results in black coat colour, whereas other inactivating mutations cause red coat colour in different mammals. The whole coding region of the MC1R gene was sequenced in goats of six different breeds showing different coat colours (Girgentana, white cream with usually small red spots in the face; Maltese, white with black cheeks and ears; Derivata di Siria, solid red; Murciano-Granadina, solid black or solid brown; Camosciata delle Alpi, brown with black stripes; Saanen, white; F1 goats and the parental animals). Five single nucleotide polymorphisms (SNPs) were identified: one nonsense mutation (p.Q225X), three missense mutations (p.A81V, p.F250V, and p.C267W), and one silent mutation. The stop codon at position 225 should cause the production of a shorter MC1R protein whose functionality may be altered. These SNPs were investigated in a larger sample of animals belonging to the six breeds. The Girgentana breed was almost fixed for the p.225X allele. However, there was not complete association between the presence of red spots in the face and the presence of this allele in homozygous condition. The same allele was identified in the Derivata di Siria breed. However, its frequency was only 33%, despite the fact that these animals are completely red. The p.267W allele was present in all Murciano-Granadina black goats, whereas it was never identified in the brown ones. Moreover, the same substitution was present in almost all Maltese goats providing evidence of association between this mutation and black coat colour. According to the results obtained in the investigated goat breeds, MC1R mutations may determine eumelanic and pheomelanic phenotypes. However, they are probably not the only

A targeted mutational landscape of angioimmunoblastic T-cell lymphoma

PubMed Central

Odejide, Oreofe; Weigert, Oliver; Lane, Andrew A.; Toscano, Dan; Lunning, Matthew A.; Kopp, Nadja; Kim, Sunhee; van Bodegom, Diederik; Bolla, Sudha; Schatz, Jonathan H.; Teruya-Feldstein, Julie; Hochberg, Ephraim; Louissaint, Abner; Dorfman, David; Stevenson, Kristen; Rodig, Scott J.; Piccaluga, Pier Paolo; Jacobsen, Eric; Pileri, Stefano A.; Harris, Nancy L.; Ferrero, Simone; Inghirami, Giorgio; Horwitz, Steven M.

2014-01-01

The genetics of angioimmunoblastic T-cell lymphoma (AITL) are very poorly understood. We defined the mutational landscape of AITL across 219 genes in 85 cases from the United States and Europe. We identified ≥2 mutations in 34 genes, nearly all of which were not previously implicated in AITL. These included loss-of-function mutations in TP53 (n = 4), ETV6 (n = 3), CCND3 (n = 2), and EP300 (n = 5), as well as gain-of-function mutations in JAK2 (n = 2) and STAT3 (n = 4). TET2 was mutated in 65 (76%) AITLs, including 43 that harbored 2 or 3 TET2 mutations. DNMT3A mutations occurred in 28 (33%) AITLs; 100% of these also harbored TET2 mutations (P < .0001). Seventeen AITLs harbored IDH2 R172 substitutions, including 15 with TET2 mutations. In summary, AITL is characterized by high frequencies of overlapping mutations in epigenetic modifiers and targetable mutations in a subset of cases. PMID:24345752

Multi-breed and multi-trait co-association analysis of meat tenderness and other meat quality traits in three French beef cattle breeds.

PubMed

Ramayo-Caldas, Yuliaxis; Renand, Gilles; Ballester, Maria; Saintilan, Romain; Rocha, Dominique

2016-04-23

Studies to identify markers associated with beef tenderness have focused on Warner-Bratzler shear force (WBSF) but the interplay between the genes associated with WBSF has not been explored. We used the association weight matrix (AWM), a systems biology approach, to identify a set of interacting genes that are co-associated with tenderness and other meat quality traits, and shared across the Charolaise, Limousine and Blonde d'Aquitaine beef cattle breeds. Genome-wide association studies were performed using ~500K single nucleotide polymorphisms (SNPs) and 17 phenotypes measured on more than 1000 animals for each breed. First, this multi-trait approach was applied separately for each breed across 17 phenotypes and second, between- and across-breed comparisons at the AWM and functional levels were performed. Genetic heterogeneity was observed, and most of the variants that were associated with WBSF segregated within rather than across breeds. We identified 206 common candidate genes associated with WBSF across the three breeds. SNPs in these common genes explained between 28 and 30 % of the phenotypic variance for WBSF. A reduced number of common SNPs mapping to the 206 common genes were identified, suggesting that different mutations may target the same genes in a breed-specific manner. Therefore, it is likely that, depending on allele frequencies and linkage disequilibrium patterns, a SNP that is identified for one breed may not be informative for another unrelated breed. Well-known candidate genes affecting beef tenderness were identified. In addition, some of the 206 common genes are located within previously reported quantitative trait loci for WBSF in several cattle breeds. Moreover, the multi-breed co-association analysis detected new candidate genes, regulators and metabolic pathways that are likely involved in the determination of meat tenderness and other meat quality traits in beef cattle. Our results suggest that systems biology approaches that explore

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.

TILLING for plant breeding.

PubMed

Sharp, Peter; Dong, Chongmei

2014-01-01

TILLING is widely used in plant functional genomics. Mutagenesis and SNP detection is combined to allow for the isolation of mutations in genes of interest. It can also be used as a plant breeding tool, whereby variation in known or candidate genes of interest to breeding programs is generated. Here we describe a simple low-cost TILLING procedure.

Single molecule targeted sequencing for cancer gene mutation detection.

PubMed

Gao, Yan; Deng, Liwei; Yan, Qin; Gao, Yongqian; Wu, Zengding; Cai, Jinsen; Ji, Daorui; Li, Gailing; Wu, Ping; Jin, Huan; Zhao, Luyang; Liu, Song; Ge, Liangjin; Deem, Michael W; He, Jiankui

2016-05-19

With the rapid decline in cost of sequencing, it is now affordable to examine multiple genes in a single disease-targeted clinical test using next generation sequencing. Current targeted sequencing methods require a separate step of targeted capture enrichment during sample preparation before sequencing. Although there are fast sample preparation methods available in market, the library preparation process is still relatively complicated for physicians to use routinely. Here, we introduced an amplification-free Single Molecule Targeted Sequencing (SMTS) technology, which combined targeted capture and sequencing in one step. We demonstrated that this technology can detect low-frequency mutations using artificially synthesized DNA sample. SMTS has several potential advantages, including simple sample preparation thus no biases and errors are introduced by PCR reaction. SMTS has the potential to be an easy and quick sequencing technology for clinical diagnosis such as cancer gene mutation detection, infectious disease detection, inherited condition screening and noninvasive prenatal diagnosis.

Identification of T-cell Receptors Targeting KRAS-mutated Human Tumors

PubMed Central

Wang, Qiong J.; Yu, Zhiya; Griffith, Kayla; Hanada, Ken-ichi; Restifo, Nicholas P.; Yang, James C.

2015-01-01

KRAS is one of the most frequently mutated proto-oncogenes in human cancers. The dominant oncogenic mutations of KRAS are single amino acid substitutions at codon 12, in particular G12D and G12V present in 60–70% of pancreatic cancers and 20–30% of colorectal cancers. The consistency, frequency, and tumor specificity of these “neo-antigens” make them attractive therapeutic targets. Recent data associates T cells that target mutated antigens with clinical immunotherapy responses in patients with metastatic melanoma, lung cancer, or cholangiocarcinoma. Using HLA-peptide prediction algorithms, we noted that HLA-A*11:01 could potentially present mutated KRAS variants. By immunizing HLA-A*11:01 transgenic mice, we generated murine T cells and subsequently isolated T-cell receptors (TCRs) highly reactive to the mutated KRAS variants G12V and G12D. Peripheral blood lymphocytes (PBLs) transduced with these TCRs could recognize multiple HLA-A*11:01+ tumor lines bearing the appropriate KRAS mutations. In a xenograft model of large established tumor, adoptive transfer of these transduced PBLs reactive with an HLA-A*11:01, G12D-mutated pancreatic cell line could significantly reduce its growth in NSG mice (P = 0.002). The success of adoptive transfer of TCR-engineered T cells against melanoma and other cancers support clinical trials with these T cells that recognize mutated KRAS in patients with a variety of common cancer types. PMID:26701267

Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing.

PubMed

Li, Shan; Zheng, Yun-Chao; Cui, Hai-Rui; Fu, Hao-Wei; Shu, Qing-Yao; Huang, Jian-Zhong

Mutation breeding is based on the induction of genetic variations; hence knowledge of the frequency and type of induced mutations is of paramount importance for the design and implementation of a mutation breeding program. Although γ ray irradiation has been widely used since the 1960s in the breeding of about 200 economically important plant species, molecular elucidation of its genetic effects has so far been achieved largely by analysis of target genes or genomic regions. In the present study, the whole genomes of six γ-irradiated M 2 rice plants were sequenced; a total of 144-188 million high-quality (Q>20) reads were generated for each M 2 plant, resulting in genome coverage of >45 times for each plant. Single base substitution (SBS) and short insertion/deletion (Indel) mutations were detected at the average frequency of 7.5×10 -6 -9.8×10 -6 in the six M 2 rice plants (SBS being about 4 times more frequent than Indels). Structural and copy number variations, though less frequent than SBS and Indel, were also identified and validated. The mutations were scattered in all genomic regions across 12 rice chromosomes without apparent hotspots. The present study is the first genome-wide single-nucleotide resolution study on the feature and frequency of γ irradiation-induced mutations in a seed propagated crop; the findings are of practical importance for mutation breeding of rice and other crop species.

Selkirk Rex: Morphological and Genetic Characterization of a New Cat Breed

PubMed Central

2012-01-01

Rexoid, curly hair mutations have been selected to develop new domestic cat breeds. The Selkirk Rex is the most recently established curly-coated cat breed originating from a spontaneous mutation that was discovered in the United States in 1987. Unlike the earlier and well-established Cornish and Devon Rex breeds with curly-coat mutations, the Selkirk Rex mutation is suggested as autosomal dominant and has a different curl phenotype. This study provides a genetic analysis of the Selkirk Rex breed. An informal segregation analysis of genetically proven matings supported an autosomal, incomplete dominant expression of the curly trait in the Selkirk Rex. Homozygous curl cats can be distinguished from heterozygous cats by head and body type, as well as the presentation of the hair curl. Bayesian clustering of short tandem repeat (STR) genotypes from 31 cats that represent the future breeding stock supported the close relationship of the Selkirk Rex to the British Shorthair, Scottish Fold, Persian, and Exotic Shorthair, suggesting the Selkirk as part of the Persian breed family. The high heterozygosity of 0.630 and the low mean inbreeding coefficient of 0.057 suggest that Selkirk Rex has a diverse genetic foundation. A new locus for Selkirk autosomal dominant Rex, SADRE, is suggested for the curly trait. PMID:22837475

Targeted cancer exome sequencing reveals recurrent mutations in myeloproliferative neoplasms

PubMed Central

Tenedini, E; Bernardis, I; Artusi, V; Artuso, L; Roncaglia, E; Guglielmelli, P; Pieri, L; Bogani, C; Biamonte, F; Rotunno, G; Mannarelli, C; Bianchi, E; Pancrazzi, A; Fanelli, T; Malagoli Tagliazucchi, G; Ferrari, S; Manfredini, R; Vannucchi, A M; Tagliafico, E

2014-01-01

With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in vitro-expanded CD3+T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG and NRAS), we demonstrated a mutation frequency between 3 and 8%. We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest dynamic international prognostic scoring system (DIPSS)-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing a NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score. PMID:24150215

Haplotype diversity of the myostatin gene among beef cattle breeds

PubMed Central

Dunner, Susana; Miranda, M Eugenia; Amigues, Yves; Cañón, Javier; Georges, Michel; Hanset, Roger; Williams, John; Ménissier, François

2003-01-01

A total of 678 individuals from 28 European bovine breeds were both phenotyped and analysed at the myostatin locus by the Single Strand Conformation Polymorphism (SSCP) method. Seven new mutations were identified which contribute to the high polymorphism (1 SNP every 100 bp) present in this small gene; twenty haplotypes were described and a genotyping method was set up using the Oligonucleotide Ligation Assay (OLA) method. Some haplotypes appeared to be exclusive to a particular breed; this was the case for 5 in the Charolaise (involving mutation Q204X) and 7 in the Maine-Anjou (involving mutation E226X). The relationships between the different haplotypes were studied, thus allowing to test the earlier hypothesis on the origin of muscular hypertrophy in Europe: muscular hypertrophy (namely nt821(del11)) was mainly spread in different waves from northern Europe milk purpose populations in most breeds; however, other mutations (mostly disruptive) arose in a single breed, were highly selected and have since scarcely evolved to other populations. PMID:12605853

The cornerstone K-RAS mutation in pancreatic adenocarcinoma: From cell signaling network, target genes, biological processes to therapeutic targeting.

PubMed

Jonckheere, Nicolas; Vasseur, Romain; Van Seuningen, Isabelle

2017-03-01

RAS belongs to the super family of small G proteins and plays crucial roles in signal transduction from membrane receptors in the cell. Mutations of K-RAS oncogene lead to an accumulation of GTP-bound proteins that maintains an active conformation. In the pancreatic ductal adenocarcinoma (PDAC), one of the most deadly cancers in occidental countries, mutations of the K-RAS oncogene are nearly systematic (>90%). Moreover, K-RAS mutation is the earliest genetic alteration occurring during pancreatic carcinogenetic sequence. In this review, we discuss the central role of K-RAS mutations and their tremendous diversity of biological properties by the interconnected regulation of signaling pathways (MAPKs, NF-κB, PI3K, Ral…). In pancreatic ductal adenocarcinoma, transcriptome analysis and preclinical animal models showed that K-RAS mutation alters biological behavior of PDAC cells (promoting proliferation, migration and invasion, evading growth suppressors, regulating mucin pattern, and miRNA expression). K-RAS also impacts tumor microenvironment and PDAC metabolism reprogramming. Finally we discuss therapeutic targeting strategies of K-RAS that have been developed without significant clinical success so far. As K-RAS is considered as the undruggable target, targeting its multiple effectors and target genes should be considered as potential alternatives. Copyright © 2017 Elsevier B.V. All rights reserved.

Whole-genome resequencing reveals candidate mutations for pig prolificacy.

PubMed

Li, Wen-Ting; Zhang, Meng-Meng; Li, Qi-Gang; Tang, Hui; Zhang, Li-Fan; Wang, Ke-Jun; Zhu, Mu-Zhen; Lu, Yun-Feng; Bao, Hai-Gang; Zhang, Yuan-Ming; Li, Qiu-Yan; Wu, Ke-Liang; Wu, Chang-Xin

2017-12-20

Changes in pig fertility have occurred as a result of domestication, but are not understood at the level of genetic variation. To identify variations potentially responsible for prolificacy, we sequenced the genomes of the highly prolific Taihu pig breed and four control breeds. Genes involved in embryogenesis and morphogenesis were targeted in the Taihu pig, consistent with the morphological differences observed between the Taihu pig and others during pregnancy. Additionally, excessive functional non-coding mutations have been specifically fixed or nearly fixed in the Taihu pig. We focused attention on an oestrogen response element (ERE) within the first intron of the bone morphogenetic protein receptor type-1B gene ( BMPR1B ) that overlaps with a known quantitative trait locus (QTL) for pig fecundity. Using 242 pigs from 30 different breeds, we confirmed that the genotype of the ERE was nearly fixed in the Taihu pig. ERE function was assessed by luciferase assays, examination of histological sections, chromatin immunoprecipitation, quantitative polymerase chain reactions, and western blots. The results suggest that the ERE may control pig prolificacy via the cis-regulation of BMPR1B expression. This study provides new insight into changes in reproductive performance and highlights the role of non-coding mutations in generating phenotypic diversity between breeds. © 2017 The Author(s).

Somatic mutations in salivary duct carcinoma and potential therapeutic targets

PubMed Central

Smith, Joel A.; Clarke, Angus J.; Luk, Peter P.; Selinger, Christina I.; Mahon, Kate L.; Kraitsek, Spiridoula; Palme, Carsten; Boyer, Michael J.; Dinger, Marcel E.; Cowley, Mark J.; O’Toole, Sandra A.

2017-01-01

Background Salivary duct carcinomas (SDCa) are rare highly aggressive malignancies. Most patients die from distant metastatic disease within three years of diagnosis. There are limited therapeutic options for disseminated disease. Results 11 cases showed androgen receptor expression and 6 cases showed HER2 amplification. 6 Somatic mutations with additional available targeted therapies were identified: EGFR (p.G721A: Gefitinib), PDGFRA (p.H845Y: Imatinib and Crenolanib), PIK3CA (p.H1047R: Everolimus), ERBB2 (p.V842I: Lapatinib), HRAS (p.Q61R: Selumetinib) and KIT (p.T670I: Sorafenib). Furthermore, alterations in PTEN, PIK3CA and HRAS that alter response to androgen deprivation therapy and HER2 inhibition were also seen. Materials and Methods Somatic mutation analysis was performed on DNA extracted from 15 archival cases of SDCa using the targeted Illumina TruSeq Amplicon Cancer Panel. Potential targetable genetic alterations were identified using extensive literature and international somatic mutation database (COSMIC, KEGG) search. Immunohistochemistry for androgen receptor and immunohistochemistry and fluorescent in situ hybridization for HER2 were also performed. Conclusions SDCa show multiple somatic mutations, some that are amenable to pharmacologic manipulation and others that confer resistance to treatments currently under investigation. These findings emphasize the need to develop testing and treatment strategies for SDCa. PMID:29100278

Next-generation sequencing for targeted discovery of rare mutations in rice

USDA-ARS?s Scientific Manuscript database

Advances in DNA sequencing (i.e., next-generation sequencing, NGS) have greatly increased the power and efficiency of detecting rare mutations in large mutant populations. Targeting Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach for identifying gene mutations resulting fro...

Profiling of potential driver mutations in sarcomas by targeted next generation sequencing.

PubMed

Andersson, Carola; Fagman, Henrik; Hansson, Magnus; Enlund, Fredrik

2016-04-01

Comprehensive genetic profiling by massively parallel sequencing, commonly known as next generation sequencing (NGS), is becoming the foundation of personalized oncology. For sarcomas very few targeted treatments are currently in routine use. In clinical practice the preoperative diagnostic workup of soft tissue tumours largely relies on core needle biopsies. Although mostly sufficient for histopathological diagnosis, only very limited amounts of formalin fixated paraffin embedded tissue are often available for predictive mutation analysis. Targeted NGS may thus open up new possibilities for comprehensive characterization of scarce biopsies. We therefore set out to search for driver mutations by NGS in a cohort of 55 clinically and morphologically well characterized sarcomas using low input of DNA from formalin fixated paraffin embedded tissues. The aim was to investigate if there are any recurrent or targetable aberrations in cancer driver genes in addition to known chromosome translocations in different types of sarcomas. We employed a panel covering 207 mutation hotspots in 50 cancer-associated genes to analyse DNA from nine gastrointestinal stromal tumours, 14 synovial sarcomas, seven myxoid liposarcomas, 22 Ewing sarcomas and three Ewing-like small round cell tumours at a large sequencing depth to detect also mutations that are subclonal or occur at low allele frequencies. We found nine mutations in eight different potential driver genes, some of which are potentially actionable by currently existing targeted therapies. Even though no recurrent mutations in driver genes were found in the different sarcoma groups, we show that targeted NGS-based sequencing is clearly feasible in a diagnostic setting with very limited amounts of paraffin embedded tissue and may provide novel insights into mesenchymal cell signalling and potentially druggable targets. Interestingly, we also identify five non-synonymous sequence variants in 4 established cancer driver genes in DNA

New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis.

PubMed

Kuroyanagi, Miwa; Katayama, Takashi; Imai, Tadashi; Yamamoto, Yoshihisa; Chisada, Shin-ichi; Yoshiura, Yasutoshi; Ushijima, Tomokazu; Matsushita, Tomonao; Fujita, Masashi; Nozawa, Aoi; Suzuki, Yuzuru; Kikuchi, Kiyoshi; Okamoto, Hiroyuki

2013-11-13

In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported. In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING

A simple language to script and simulate breeding schemes: the breeding scheme language

USDA-ARS?s Scientific Manuscript database

It is difficult for plant breeders to determine an optimal breeding strategy given that the problem involves many factors, such as target trait genetic architecture and breeding resource availability. There are many possible breeding schemes for each breeding program. Although simulation study may b...

Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees.

PubMed

Peer, Reut; Rivlin, Gil; Golobovitch, Sara; Lapidot, Moshe; Gal-On, Amit; Vainstein, Alexander; Tzfira, Tzvi; Flaishman, Moshe A

2015-04-01

Targeting a gene in apple or fig with ZFN, introduced by transient or stable transformation, should allow genome editing with high precision to advance basic science and breeding programs. Genome editing is a powerful tool for precise gene manipulation in any organism; it has recently been shown to be of great value for annual plants. Classical breeding strategies using conventional cross-breeding and induced mutations have played an important role in the development of new cultivars in fruit trees. However, fruit-tree breeding is a lengthy process with many limitations. Efficient and widely applied methods for targeted modification of fruit-tree genomes are not yet available. In this study, transgenic apple and fig lines carrying a zinc-finger nuclease (ZFNs) under the control of a heat-shock promoter were developed. Editing of a mutated uidA gene, following expression of the ZFN genes by heat shock, was confirmed by GUS staining and PCR product sequencing. Finally, whole plants with a repaired uidA gene due to deletion of a stop codon were regenerated. The ZFN-mediated gene modifications were stable and passed onto regenerants from ZFN-treated tissue cultures. This is the first demonstration of efficient and precise genome editing, using ZFN at a specific genomic locus, in two different perennial fruit trees-apple and fig. We conclude that targeting a gene in apple or fig with a ZFN introduced by transient or stable transformation should allow knockout of a gene of interest. Using this technology for genome editing allows for marker gene-independent and antibiotic selection-free genome engineering with high precision in fruit trees to advance basic science as well as nontransgenic breeding programs.

Uveal melanoma hepatic metastases mutation spectrum analysis using targeted next-generation sequencing of 400 cancer genes.

PubMed

Luscan, A; Just, P A; Briand, A; Burin des Roziers, C; Goussard, P; Nitschké, P; Vidaud, M; Avril, M F; Terris, B; Pasmant, E

2015-04-01

Uveal melanoma (UM) is the most common malignant tumour of the eye. Diagnosis often occurs late in the course of disease, and prognosis is generally poor. Recently, recurrent somatic mutations were described, unravelling additional specific altered pathways in UM. Targeted next-generation sequencing (NGS) can now be applied to an accurate and fast identification of somatic mutations in cancer. The aim of the present study was to characterise the mutation pattern of five UM hepatic metastases with well-defined clinical and pathological features. We analysed the UM mutation spectrum using targeted NGS on 409 cancer genes. Four previous reported genes were found to be recurrently mutated. All tumours presented mutually exclusive GNA11 or GNAQ missense mutations. BAP1 loss-of-function mutations were found in three UMs. SF3B1 missense mutations were found in the two UMs with no BAP1 mutations. We then searched for additional mutation targets. We identified the Arg505Cys mutation in the tumour suppressor FBXW7. The same mutation was previously described in different cancer types, and FBXW7 was recently reported to be mutated in UM exomes. Further studies are required to confirm FBXW7 implication in UM tumorigenesis. Elucidating the molecular mechanisms underlying UM tumorigenesis holds the promise for novel and effective targeted UM therapies. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Development of Commercial Thermo-sensitive Genic Male Sterile Rice Accelerates Hybrid Rice Breeding Using the CRISPR/Cas9-mediated TMS5 Editing System

PubMed Central

Zhou, Hai; He, Ming; Li, Jing; Chen, Liang; Huang, Zhifeng; Zheng, Shaoyan; Zhu, Liya; Ni, Erdong; Jiang, Dagang; Zhao, Bingran; Zhuang, Chuxiong

2016-01-01

Hybrid rice breeding offers an important strategy to improve rice production, in which the cultivation of a male sterile line is the key to the success of cross-breeding. CRISPR/Cas9 systems have been widely used in target-site genome editing, whereas their application for crop genetic improvement has been rarely reported. Here, using the CRISPR/Cas9 system, we induced specific mutations in TMS5, which is the most widely applied thermo-sensitive genic male sterility (TGMS) gene in China, and developed new “transgene clean” TGMS lines. We designed 10 target sites in the coding region of TMS5 for targeted mutagenesis using the CRISPR/Cas9 system and assessed the potential rates of on- and off-target effects. Finally, we established the most efficient construct, the TMS5ab construct, for breeding potentially applicable “transgene clean” TGMS lines. We also discussed factors that affect the editing efficiency according to the characteristics of different target sequences. Notably, using the TMS5ab construct, we developed 11 new “transgene clean” TGMS lines with potential applications in hybrid breeding within only one year in both rice subspecies. The application of our system not only significantly accelerates the breeding of sterile lines but also facilitates the exploitation of heterosis. PMID:27874087

Development of Commercial Thermo-sensitive Genic Male Sterile Rice Accelerates Hybrid Rice Breeding Using the CRISPR/Cas9-mediated TMS5 Editing System.

PubMed

Zhou, Hai; He, Ming; Li, Jing; Chen, Liang; Huang, Zhifeng; Zheng, Shaoyan; Zhu, Liya; Ni, Erdong; Jiang, Dagang; Zhao, Bingran; Zhuang, Chuxiong

2016-11-22

Hybrid rice breeding offers an important strategy to improve rice production, in which the cultivation of a male sterile line is the key to the success of cross-breeding. CRISPR/Cas9 systems have been widely used in target-site genome editing, whereas their application for crop genetic improvement has been rarely reported. Here, using the CRISPR/Cas9 system, we induced specific mutations in TMS5, which is the most widely applied thermo-sensitive genic male sterility (TGMS) gene in China, and developed new "transgene clean" TGMS lines. We designed 10 target sites in the coding region of TMS5 for targeted mutagenesis using the CRISPR/Cas9 system and assessed the potential rates of on- and off-target effects. Finally, we established the most efficient construct, the TMS5ab construct, for breeding potentially applicable "transgene clean" TGMS lines. We also discussed factors that affect the editing efficiency according to the characteristics of different target sequences. Notably, using the TMS5ab construct, we developed 11 new "transgene clean" TGMS lines with potential applications in hybrid breeding within only one year in both rice subspecies. The application of our system not only significantly accelerates the breeding of sterile lines but also facilitates the exploitation of heterosis.

Mutational analysis of genes coding for cell surface proteins in colorectal cancer cell lines reveal novel altered pathways, druggable mutations and mutated epitopes for targeted therapy

PubMed Central

Correa, Bruna R.; Bettoni, Fabiana; Koyama, Fernanda C.; Navarro, Fabio C.P.; Perez, Rodrigo O.; Mariadason, John; Sieber, Oliver M.; Strausberg, Robert L.; Simpson, Andrew J.G.; Jardim, Denis L.F.; Reis, Luiz Fernando L.; Parmigiani, Raphael B.; Galante, Pedro A.F.; Camargo, Anamaria A.

2014-01-01

We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of “general” immune checkpoint drugs in this subset of patients. PMID:25193853

Generation and Inheritance of Targeted Mutations in Potato (Solanum tuberosum L.) Using the CRISPR/Cas System

PubMed Central

Butler, Nathaniel M.; Atkins, Paul A.; Voytas, Daniel F.; Douches, David S.

2015-01-01

Genome editing using sequence-specific nucleases (SSNs) offers an alternative approach to conventional genetic engineering and an opportunity to extend the benefits of genetic engineering in agriculture. Currently available SSN platforms, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)) have been used in a range of plant species for targeted mutagenesis via non-homologous end joining (NHEJ) are just beginning to be explored in crops such as potato (Solanum tuberosum Group Tuberosum L.). In this study, CRISPR/Cas reagents expressing one of two single-guide RNA (sgRNA) targeting the potato ACETOLACTATE SYNTHASE1 (StALS1) gene were tested for inducing targeted mutations in callus and stable events of diploid and tetraploid potato using Agrobacterium-mediated transformation with either a conventional T-DNA or a modified geminivirus T-DNA. The percentage of primary events with targeted mutations ranged from 3–60% per transformation and from 0–29% above an expected threshold based on the number of ALS alleles. Primary events with targeted mutation frequencies above the expected threshold were used for mutation cloning and inheritance studies using clonal propagation and crosses or selfing. Four of the nine primary events used for mutation cloning had more than one mutation type, and eight primary events contained targeted mutations that were maintained across clonal generations. Somatic mutations were most evident in the diploid background with three of the four primary events having more than two mutation types at a single ALS locus. Conversely, in the tetraploid background, four of the five candidates carried only one mutation type. Single targeted mutations were inherited through the germline of both diploid and tetraploid primary events with transmission percentages ranging from 87–100%. This

Generation and Inheritance of Targeted Mutations in Potato (Solanum tuberosum L.) Using the CRISPR/Cas System.

PubMed

Butler, Nathaniel M; Atkins, Paul A; Voytas, Daniel F; Douches, David S

2015-01-01

Genome editing using sequence-specific nucleases (SSNs) offers an alternative approach to conventional genetic engineering and an opportunity to extend the benefits of genetic engineering in agriculture. Currently available SSN platforms, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)) have been used in a range of plant species for targeted mutagenesis via non-homologous end joining (NHEJ) are just beginning to be explored in crops such as potato (Solanum tuberosum Group Tuberosum L.). In this study, CRISPR/Cas reagents expressing one of two single-guide RNA (sgRNA) targeting the potato ACETOLACTATE SYNTHASE1 (StALS1) gene were tested for inducing targeted mutations in callus and stable events of diploid and tetraploid potato using Agrobacterium-mediated transformation with either a conventional T-DNA or a modified geminivirus T-DNA. The percentage of primary events with targeted mutations ranged from 3-60% per transformation and from 0-29% above an expected threshold based on the number of ALS alleles. Primary events with targeted mutation frequencies above the expected threshold were used for mutation cloning and inheritance studies using clonal propagation and crosses or selfing. Four of the nine primary events used for mutation cloning had more than one mutation type, and eight primary events contained targeted mutations that were maintained across clonal generations. Somatic mutations were most evident in the diploid background with three of the four primary events having more than two mutation types at a single ALS locus. Conversely, in the tetraploid background, four of the five candidates carried only one mutation type. Single targeted mutations were inherited through the germline of both diploid and tetraploid primary events with transmission percentages ranging from 87-100%. This demonstration

Isocitrate dehydrogenase mutation as a therapeutic target in gliomas.

PubMed

Han, Catherine H; Batchelor, Tracy T

2017-06-01

Isocitrate dehydrogenases (IDH) are important enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG), producing NADPH in the process. More than 80% of low-grade gliomas and secondary glioblastoma (GBM) harbor an IDH mutation. IDH mutations involve the catalytic pocket of the enzyme and lead to a neomorphic ability to produce 2-hydroxyglutarate (2HG) while oxidizing NADPH to NADP+. 2HG is considered as an 'oncometabolite' which is thought to be responsible for many, if not all, biologic effects of IDH mutations. 2HG accumulation competitively inhibits α-KG-dependent dioxygenases, including histone lysine demethylases and DNA demethylases, resulting in a hypermethylation phenotype with alterations in cellular epigenetic status as well as a block in cellular differentiation. IDH mutations have been suggested as an important early event in tumorigenesis, however it remains unclear whether IDH mutation by itself causes cancer or if it requires other oncogenic events to initiate tumorigenesis. Significant efforts have been made to better understand the mechanisms of IDH mutations in tumor initiation and progression, and to develop targeted therapies for IDH-mutated tumors. This review provides an overview of the function of mutant IDH, and the current understanding of the role IDH mutations play in gliomagenesis. In addition, several potential therapeutic strategies for IDH-mutant gliomas, including mutant IDH inhibitors which have entered clinical evaluation in glioma patients, will be discussed.

E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

PubMed

Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B

2017-01-01

Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

Genome and transcriptome sequencing identifies breeding targets in the orphan crop tef (Eragrostis tef).

PubMed

Cannarozzi, Gina; Plaza-Wüthrich, Sonia; Esfeld, Korinna; Larti, Stéphanie; Wilson, Yi Song; Girma, Dejene; de Castro, Edouard; Chanyalew, Solomon; Blösch, Regula; Farinelli, Laurent; Lyons, Eric; Schneider, Michel; Falquet, Laurent; Kuhlemeier, Cris; Assefa, Kebebew; Tadele, Zerihun

2014-07-09

Tef (Eragrostis tef), an indigenous cereal critical to food security in the Horn of Africa, is rich in minerals and protein, resistant to many biotic and abiotic stresses and safe for diabetics as well as sufferers of immune reactions to wheat gluten. We present the genome of tef, the first species in the grass subfamily Chloridoideae and the first allotetraploid assembled de novo. We sequenced the tef genome for marker-assisted breeding, to shed light on the molecular mechanisms conferring tef's desirable nutritional and agronomic properties, and to make its genome publicly available as a community resource. The draft genome contains 672 Mbp representing 87% of the genome size estimated from flow cytometry. We also sequenced two transcriptomes, one from a normalized RNA library and another from unnormalized RNASeq data. The normalized RNA library revealed around 38000 transcripts that were then annotated by the SwissProt group. The CoGe comparative genomics platform was used to compare the tef genome to other genomes, notably sorghum. Scaffolds comprising approximately half of the genome size were ordered by syntenic alignment to sorghum producing tef pseudo-chromosomes, which were sorted into A and B genomes as well as compared to the genetic map of tef. The draft genome was used to identify novel SSR markers, investigate target genes for abiotic stress resistance studies, and understand the evolution of the prolamin family of proteins that are responsible for the immune response to gluten. It is highly plausible that breeding targets previously identified in other cereal crops will also be valuable breeding targets in tef. The draft genome and transcriptome will be of great use for identifying these targets for genetic improvement of this orphan crop that is vital for feeding 50 million people in the Horn of Africa.

Development of Novel Therapeutics Targeting Isocitrate Dehydrogenase Mutations in Cancer.

PubMed

Sharma, Horrick

2018-05-17

Isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that catalyze the conversion of isocitrate to α-ketoglutarate (αKG). IDH 1 and IDH2 regulate several cellular processes, including oxidative respiration, glutamine metabolism, lipogenesis, and cellular defense against oxidative damage. Mutations in IDH1 and IDH2 have recently been observed in multiple tumor types, including gliomas, acute myeloid leukemia, myelodysplastic syndromes, and chondrosarcoma. IDH1 and IDH2 mutations involve a gain in neomorphic activity that catalyze αKG conversion to (R)-2-hydroxyglutarate ((R)-2HG). IDH mutation-mediated accumulation of (R)-2HG result in epigenetic dysregulation, altered gene expression, and a block in cellular differentiation. Targeting mutant IDH by development of small molecule inhibitors is a rapidly emerging therapeutic approach as evidenced by the recent approval of the first selective mutant IDH2 inhibitor AG-221 (Enasidenib) for the treatment of IDH2-mutated AML. This review will focus on mutant isocitrate dehydrogenase as a therapeutic drug target and provides an update on selective and pan-mutant IDH 1/2 inhibitors in clinical trials and other mutant IDH inhibitors that are under development. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Breed relationships facilitate fine-mapping studies: A 7.8-kb deletion cosegregates with Collie eye anomaly across multiple dog breeds

PubMed Central

Parker, Heidi G.; Kukekova, Anna V.; Akey, Dayna T.; Goldstein, Orly; Kirkness, Ewen F.; Baysac, Kathleen C.; Mosher, Dana S.; Aguirre, Gustavo D.; Acland, Gregory M.; Ostrander, Elaine A.

2007-01-01

The features of modern dog breeds that increase the ease of mapping common diseases, such as reduced heterogeneity and extensive linkage disequilibrium, may also increase the difficulty associated with fine mapping and identifying causative mutations. One way to address this problem is by combining data from multiple breeds segregating the same trait after initial linkage has been determined. The multibreed approach increases the number of potentially informative recombination events and reduces the size of the critical haplotype by taking advantage of shortened linkage disequilibrium distances found across breeds. In order to identify breeds that likely share a trait inherited from the same ancestral source, we have used cluster analysis to divide 132 breeds of dog into five primary breed groups. We then use the multibreed approach to fine-map Collie eye anomaly (cea), a complex disorder of ocular development that was initially mapped to a 3.9-cM region on canine chromosome 37. Combined genotypes from affected individuals from four breeds of a single breed group significantly narrowed the candidate gene region to a 103-kb interval spanning only four genes. Sequence analysis revealed that all affected dogs share a homozygous deletion of 7.8 kb in the NHEJ1 gene. This intronic deletion spans a highly conserved binding domain to which several developmentally important proteins bind. This work both establishes that the primary cea mutation arose as a single disease allele in a common ancestor of herding breeds as well as highlights the value of comparative population analysis for refining regions of linkage. PMID:17916641

Cisgenesis strongly improves introgression breeding and induced translocation breeding of plants.

PubMed

Jacobsen, Evert; Schouten, Henk J

2007-05-01

There are two ways for genetic improvement in classical plant breeding: crossing and mutation. Plant varieties can also be improved through genetic modification; however, the present GMO regulations are based on risk assessments with the transgenes coming from non-crossable species. Nowadays, DNA sequence information of crop plants facilitates the isolation of cisgenes, which are genes from crop plants themselves or from crossable species. The increasing number of these isolated genes, and the development of transformation protocols that do not leave marker genes behind, provide an opportunity to improve plant breeding while remaining within the gene pool of the classical breeder. Compared with induced translocation and introgression breeding, cisgenesis is an improvement for gene transfer from crossable plants: it is a one-step gene transfer without linkage drag of other genes, whereas induced translocation and introgression breeding are multiple step gene transfer methods with linkage drag. The similarity of the genes used in cisgenesis compared with classical breeding is a compelling argument to treat cisgenic plants as classically bred plants. In the case of the classical breeding method induced translocation breeding, the insertion site of the genes is a priori unknown, as it is in cisgenesis. This provides another argument to treat cisgenic plants as classically bred plants, by exempting cisgenesis of plants from the GMO legislations.

HCK is a survival determinant transactivated by mutated MYD88, and a direct target of ibrutinib.

PubMed

Yang, Guang; Buhrlage, Sara J; Tan, Li; Liu, Xia; Chen, Jie; Xu, Lian; Tsakmaklis, Nicholas; Chen, Jiaji G; Patterson, Christopher J; Brown, Jennifer R; Castillo, Jorge J; Zhang, Wei; Zhang, Xiaofeng; Liu, Shuai; Cohen, Philip; Hunter, Zachary R; Gray, Nathanael; Treon, Steven P

2016-06-23

Activating mutations in MYD88 are present in ∼95% of patients with Waldenström macroglobulinemia (WM), as well as other B-cell malignancies including activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). In WM, mutated MYD88 triggers activation of Bruton tyrosine kinase (BTK). Ibrutinib, a pleiotropic kinase inhibitor that targets BTK, is highly active in patients with mutated MYD88. We observed that mutated MYD88 WM and ABC DLBCL cell lines, as well as primary WM cells show enhanced hematopoietic cell kinase (HCK) transcription and activation, and that HCK is activated by interleukin 6 (IL-6). Over-expression of mutated MYD88 triggers HCK and IL-6 transcription, whereas knockdown of HCK reduced survival and attenuated BTK, phosphoinositide 3-kinase/AKT, and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in mutated MYD88 WM and/or ABC DLBCL cells. Ibrutinib and the more potent HCK inhibitor A419259, blocked HCK activation and induced apoptosis in mutated MYD88 WM and ABC DLBCL cells. Docking and pull-down studies confirmed that HCK was a target of ibrutinib. Ibrutinib and A419259 also blocked adenosine triphosphate binding to HCK, whereas transduction of mutated MYD88 expressing WM cells with a mutated HCK gatekeeper greatly increased the half maximal effective concentration for ibrutinib and A419259. The findings support that HCK expression and activation is triggered by mutated MYD88, supports the growth and survival of mutated MYD88 WM and ABC DLBCL cells, and is a direct target of ibrutinib. HCK represents a novel target for therapeutic development in MYD88-mutated WM and ABC DLBCL, and possibly other diseases driven by mutated MYD88. © 2016 by The American Society of Hematology.

Mutation Breeding of β-carotene Producing Strain B. trispora by Low Energy Ion Implantation

NASA Astrophysics Data System (ADS)

Zhang, Ning; Yu, Long

2009-02-01

Ion beam bioengineering technology as a new mutation approach has been widely used in the biological breeding field. In this paper the application of low energy nitrogen ion implantation in the β-carotene producing strain, Blakeslea trispora(-) was investigated. The effects of different fermentation conditions on β-carotene production by a high yield strain were examined. Results showed that two β-carotene high yielding strains B.trispora(-) BH3-701 and BH3-728 were screened out and the averaged production of β-carotene was raised by 178.7% and 164.6% respectively after five passages in the shaking flasks. Compared with the original strain, the highest yield strain BH3-701 was potent in accumulating β-carotene, especially in the later stage, and greatly increased production efficiency.

Next generation sequencing gives an insight into the characteristics of highly selected breeds versus non-breed horses in the course of domestication.

PubMed

Metzger, Julia; Tonda, Raul; Beltran, Sergi; Agueda, Lídia; Gut, Marta; Distl, Ottmar

2014-07-04

Domestication has shaped the horse and lead to a group of many different types. Some have been under strong human selection while others developed in close relationship with nature. The aim of our study was to perform next generation sequencing of breed and non-breed horses to provide an insight into genetic influences on selective forces. Whole genome sequencing of five horses of four different populations revealed 10,193,421 single nucleotide polymorphisms (SNPs) and 1,361,948 insertion/deletion polymorphisms (indels). In comparison to horse variant databases and previous reports, we were able to identify 3,394,883 novel SNPs and 868,525 novel indels. We analyzed the distribution of individual variants and found significant enrichment of private mutations in coding regions of genes involved in primary metabolic processes, anatomical structures, morphogenesis and cellular components in non-breed horses and in contrast to that private mutations in genes affecting cell communication, lipid metabolic process, neurological system process, muscle contraction, ion transport, developmental processes of the nervous system and ectoderm in breed horses. Our next generation sequencing data constitute an important first step for the characterization of non-breed in comparison to breed horses and provide a large number of novel variants for future analyses. Functional annotations suggest specific variants that could play a role for the characterization of breed or non-breed horses.

Mutations in Lettuce Improvement.

USDA-ARS?s Scientific Manuscript database

Mutations can make profound impact on the evolution and improvement of a self-pollinated crop such as lettuce. Since it is nontransgenic, mutation breeding is more acceptable to consumers. Combined with genomic advances in new technologies like TILLING, mutagenesis is becoming an even more powerfu...

Inhibition of Axl improves the targeted therapy against ALK-mutated neuroblastoma

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

Xu, Fei; Li, Hongling; Sun, Yong, E-mail: sunfanqi2010@163.com

2014-11-28

Highlights: • First reported Axl is co-expressed with ALK in neuroblastoma tissues and cell lines. • Axl activation promotes cell growth and impairs the efficiency of ALK inhibitor. • Further found silence of Axl leads to increased sensitivity to ALK inhibitors. • Axl inhibitor promotes the efficiency of targeted therapy in vitro and in vivo. • Axl activation should be considered in the clinical application of ALK inhibitors. - Abstract: Neuroblastoma (NB) patients harboring mutated ALK can be expected to potentially benefit from targeted therapy based on ALK tyrosine kinase inhibitor (TKI), such as crizotinib and ceritinib. However, the effectmore » of the treatment varies with different individuals, although with the same genic changes. Axl receptor tyrosine kinase is expressed in a variety of human cancers, but little data are reported in NB, particularly in which carrying mutated ALK. In this study, we focus on the roles of Axl in ALK-mutated NB for investigating rational therapeutic strategy. We found that Axl is expressed in ALK-positive NB tissues and cell lines, and could be effectively activated by its ligand GAS6. Ligand-dependent Axl activation obviously rescued crizotinib-mediated suppression of cell proliferation in ALK-mutated NB cells. Genetic inhibition of Axl with specific small interfering RNA markedly increased the sensitivity of cells to ALK-TKIs. Furthermore, a small-molecule inhibitor of Axl significantly enhanced ALK-targeted therapy, as an increased frequency of apoptosis was observed in NB cells co-expressing ALK and Axl. Taken together, our results demonstrated that activation of Axl could lead to insensitivity to ALK inhibitors, and dual inhibition of ALK and Axl might be a potential therapeutic strategy against ALK-mutated NB.« less

[Research progress on mutation by spaceflight in medicinal plants breeding].

PubMed

Yan, Shuo; Gao, Wenyuan; Lu, Fuping; Zhao, Runhuai

2010-02-01

Space breeding in medicinal plants is special characteristics in China. Compared with other plants, in spite of a relatively small number, Medicinal plants have more obvious characteristics and advantages. Research on medicinal plants has also been carried into all aspects, such as biological traits, physiology and biochemistry, genomics, as well as differences in chemical composition, and chemical composition analysis is also involved. However, compared with other plants, especially crops and vegetables, biological research is an obvious deficiency, that is mainly reflected in the insufficient genetics and breeding researches, the stability of genetic traits from generation to generation were not followed up and in-depth study in breeding areas was not carried out. If medicinal plants resources from space with the genetic stability good quality were selected, it would address the problem of lack of resources and ease the pressure on wild resources of medicinal plants. It would at the same time play an important role in promoting the development of medicinal botany space breeding and the implementation of modernization of traditional Chinese medicine.

SETD2 is recurrently mutated in whole-exome sequenced canine osteosarcoma.

PubMed

Sakthikumar, Sharadha; Elvers, Ingegerd; Kim, Jaegil; Arendt, Maja L; Thomas, Rachael; Turner-Maier, Jason; Swofford, Ross; Johnson, Jeremy; Schumacher, Steven E; Alföldi, Jessica; Axelsson, Erik; Couto, Guillermo; Kisseberth, William; Pettersson, Mats E; Getz, Gad; Meadows, Jennifer R S; Modiano, Jaime F; Breen, Matthew; Kierczak, Marcin; Forsberg-Nilsson, Karin; Marinescu, Voichita D; Lindblad-Toh, Kerstin

2018-05-03

Osteosarcoma (OSA) is a debilitating bone cancer that affects humans, especially children and adolescents. A homologous form of OSA spontaneously occurs in dogs, and its differential incidence observed across breeds allows for the investigation of tumor mutations in the context of multiple genetic backgrounds. Using whole-exome sequencing and dogs from three susceptible breeds (22 golden retrievers, 21 Rottweilers, and 23 greyhounds), we found that OSA tumors show a high frequency of somatic copy number alterations (SCNA) affecting key oncogenes and tumor suppressor genes. The across-breed results are similar to what has been observed for human OSA, but the disease frequency and somatic mutation counts vary in the three breeds. For all breeds, three mutational signatures (one of which has not been previously reported), and eleven significantly mutated genes were identified. TP53 was the most frequently altered gene (83% of dogs have either mutations or SCNA in TP53), recapitulating observations in human OSA. The second most frequently mutated gene, histone methyltransferase SETD2, has known roles in multiple cancers, but has not previously been strongly implicated in OSA. This study points to the likely importance of histone modifications in OSA and highlights the strong genetic similarities between human and dog OSA, suggesting that canine OSA may serve as an excellent model for developing treatment strategies in both species. Copyright ©2018, American Association for Cancer Research.

Systems Pharmacology-Based Discovery of Natural Products for Precision Oncology Through Targeting Cancer Mutated Genes.

PubMed

Fang, J; Cai, C; Wang, Q; Lin, P; Zhao, Z; Cheng, F

2017-03-01

Massive cancer genomics data have facilitated the rapid revolution of a novel oncology drug discovery paradigm through targeting clinically relevant driver genes or mutations for the development of precision oncology. Natural products with polypharmacological profiles have been demonstrated as promising agents for the development of novel cancer therapies. In this study, we developed an integrated systems pharmacology framework that facilitated identifying potential natural products that target mutated genes across 15 cancer types or subtypes in the realm of precision medicine. High performance was achieved for our systems pharmacology framework. In case studies, we computationally identified novel anticancer indications for several US Food and Drug Administration-approved or clinically investigational natural products (e.g., resveratrol, quercetin, genistein, and fisetin) through targeting significantly mutated genes in multiple cancer types. In summary, this study provides a powerful tool for the development of molecularly targeted cancer therapies through targeting the clinically actionable alterations by exploiting the systems pharmacology of natural products. © 2017 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

[Medicinal plant DNA marker assisted breeding (Ⅱ) the assistant identification of SNPs assisted identification and breeding research of high yield Perilla frutescens new variety].

PubMed

Shen, Qi; Zhang, Dong; Sun, Wei; Zhang, Yu-Jun; Shang, Zhi-Wei; Chen, Shi-Lin

2017-05-01

Perilla frutescens is one of 60 kinds of food and medicine plants in the initial directory announced by health ministry of China. With the development of Perilla domain in recent , the breeding and application of good varieties has become the main bottleneck of its development. This study reported that applied to the system selection, add to marker-assisted method to breed perilla varieties. Through the whole genome sequencing and consistency matching, annotated the mutation locus according to genome data, and comparison analysis with Perilla common variants database, finally selected 30 non-synonymous mutation SNPs used as characteristic markers of Zhongyan Feishu No.1. those SNP marker were used as chosen standard of Perilla varieties. Finally breeding new perilla variety Zhongyan Feishu No.1, which possess to characters of the leaf and seed dual-used, high yield, high resistance, and could used to green fertilizer. The Zhongyan Feishu No.1 acquired the plant new varieties identification of Beijing city , the identification numbers is 2016054. Marker assisted identification guide new varieties breeding in plants, which can provide a new reference for breeding of medicinal plants. Copyright© by the Chinese Pharmaceutical Association.

Identify mutation in amyotrophic lateral sclerosis cases using HaloPlex target enrichment system.

PubMed

Liu, Zhi-Jun; Li, Hong-Fu; Tan, Guo-He; Tao, Qing-Qing; Ni, Wang; Cheng, Xue-Wen; Xiong, Zhi-Qi; Wu, Zhi-Ying

2014-12-01

To date, at least 18 causative genes have been identified in amyotrophic lateral sclerosis (ALS). Because of the clinical and genetic heterogeneity, molecular diagnosis for ALS faces great challenges. HaloPlex target enrichment system is a new targeted sequencing approach, which can detect already known mutations or candidate genes. We performed this approach to screen 18 causative genes of ALS, including SOD1, SETX, FUS, ANG, TARDBP, ALS2, FIG4, VAPB, OPTN, DAO, VCP, UBQLN2, SPG11, SIGMAR1, DCTN1, SQSTM1, PFN1, and CHMP2B in 8 ALS probands. Using this approach, we got an average of 9.5 synonymous or missense mutations per sample. After validation by Sanger sequencing, we identified 3 documented SOD1 mutations (p.F21C, p.G148D, and p.C147R) and 1 novel DCTN1 p.G59R mutation in 4 probands. The novel DCTN1 mutation appeared to segregate with the disease in the pedigree and was absent in 200 control subjects. The high throughput and efficiency of this approach indicated that it could be applied to diagnose ALS and other inherited diseases with multiple causative genes in clinical practice. Copyright © 2014 Elsevier Inc. All rights reserved.

USE OF NEUTRON IRRADIATIONS IN THE BROOKHAVEN MUTATIONS PROGRAM

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

Miksche, J.P.; Shapiro, S.

1963-01-01

Many plant species were irradiated with x rays, thermal and fast neutrons, andd gamma radiation during the past 10 yr of the cooperative mutations program and adjunct mutation breeding program. Four major concepts and/ or approaches related to the use of mutagenic agents in plant breeding that have evolved are discussed. It was concluded that outcrossing between treated and nontreated populations must be reckoned with, and consequently the two populations should be separated before a true measure of mutation induction can be ascertained; chromosome rearrangement studies are useful, with particular emphasis on inducing disease resistance; work concerned with tissue reorgandizationmore » and rearrangement as related to chimera production and basic understanding of tissue ontogeny, particularly with fruit crops andd horticultural crops is promising; and the effectiveness of responses of plant tissues to neutrons and other mutagenic agents is extremely variable and more basic work is needed before the full potentialities of mutation breeding as a tool in crop improvement can be appreciated. (auth)« less

Mutation testing for directing upfront targeted therapy and post-progression combination therapy strategies in lung adenocarcinoma

PubMed Central

Salgia, Ravi

2016-01-01

ABSTRACT Introduction: Advances in the biology of non-small-cell lung cancer, especially adenocarcinoma, reveal multiple molecular subtypes driving oncogenesis. Accordingly, individualized targeted therapeutics are based on mutational diagnostics. Areas covered: Advances in strategies and techniques for individualized treatment, particularly of adenocarcinoma, are described through literature review. Approved therapies are established for some molecular subsets, with new driver mutations emerging that represent increasing proportions of patients. Actionable mutations are de novo oncogenic drivers or acquired resistance mediators, and mutational profiling is important for directing therapy. Patients should be monitored for emerging actionable resistance mutations. Liquid biopsy and associated multiplex diagnostics will be important means to monitor patients during treatment. Expert commentary: Outcomes with targeted agents may be improved by integrating mutation screens during treatment to optimize subsequent therapy. In order for this to be translated into impactful patient benefit, appropriate platforms and strategies need to be optimized and then implemented universally. PMID:27139190

PALLAS BARLEY, A VARIETY PRODUCED BY IONIZING RADIATION: ITS SIGNIFICANCE FOR PLANT BREEDING AND EVOLUTION

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

Borg, G.; Froier, K.f Gustafsson, A.

1959-10-31

Results are reported from plant breeding programs in which mutations induced in barley by irradiation resulted in the development of a variety with characteristics superior to the parents. Good results were also reported in the development of superior varieties of oil rape, white mustard, and pea plants. The usefulness of radiation-induced mutations in plant breeding programs is discussed. (C.H.)

Frameshift mutational target gene analysis identifies similarities and differences in constitutional mismatch repair-deficiency and Lynch syndrome.

PubMed

Maletzki, Claudia; Huehns, Maja; Bauer, Ingrid; Ripperger, Tim; Mork, Maureen M; Vilar, Eduardo; Klöcking, Sabine; Zettl, Heike; Prall, Friedrich; Linnebacher, Michael

2017-07-01

Mismatch-repair deficient (MMR-D) malignancies include Lynch Syndrome (LS), which is secondary to germline mutations in one of the MMR genes, and the rare childhood-form of constitutional mismatch repair-deficiency (CMMR-D); caused by bi-allelic MMR gene mutations. A hallmark of LS-associated cancers is microsatellite instability (MSI), characterized by coding frameshift mutations (cFSM) in target genes. By contrast, tumors arising in CMMR-D patients are thought to display a somatic mutation pattern differing from LS. This study has the main goal to identify cFSM in MSI target genes relevant in CMMR-D and to compare the spectrum of common somatic mutations, including alterations in DNA polymerases POLE and D1 between LS and CMMR-D. CMMR-D-associated tumors harbored more somatic mutations compared to LS cases, especially in the TP53 gene and in POLE and POLD1, where novel mutations were additionally identified. Strikingly, MSI in classical mononucleotide markers BAT40 and CAT25 was frequent in CMMR-D cases. MSI-target gene analysis revealed mutations in CMMR-D-associated tumors, some of them known to be frequently hit in LS, such as RNaseT2, HT001, and TGFβR2. Our results imply a general role for these cFSM as potential new drivers of MMR-D tumorigenesis. © 2017 Wiley Periodicals, Inc.

HER2 Amplification and HER2 Mutation Are Distinct Molecular Targets in Lung Cancers.

PubMed

Li, Bob T; Ross, Dara S; Aisner, Dara L; Chaft, Jamie E; Hsu, Meier; Kako, Severine L; Kris, Mark G; Varella-Garcia, Marileila; Arcila, Maria E

2016-03-01

Human epidermal growth factor receptor 2 gene (HER2 [also known as ERBB2]) alterations have been identified as oncogenic drivers and potential therapeutic targets in lung cancers. The molecular associations of HER2 gene amplification, mutation, and HER2 protein overexpression in lung cancers have not been distinctly defined. To explore these associations, Memorial Sloan Kettering Cancer Center and the University of Colorado combined their data on HER2 alterations in lung cancers. Tumor specimens from 175 patients with lung adenocarcinomas and no prior targeted therapy were evaluated for the presence of HER2 amplification and mutation and HER2 protein overexpression. Amplification was assessed by fluorescence in situ hybridization (FISH) and defined as an HER2-to-chromosome enumeration probe 17 ratio of at least 2.0. Mutation was assessed by fragment analysis, mass spectrometry genotyping, and Sanger sequencing. Overexpression was assessed by immunohistochemical (IHC) staining. The frequencies of HER2 amplification and mutation and HER2 overexpression were calculated and their overlap examined. HER2 amplification was detected by FISH in 5 of 175 cases (3%). HER2 mutation was detected in 4 of 148 specimens (3%), including three identical 12-base pair insertions (p.A775_G776insYVMA) and a 9-base pair insertion, all in exon 20. None of the HER2-mutant cases was amplified. HER2 overexpression (2+ or 3+) on IHC staining was not detected in the 25 specimens available for testing, and negative IHC staining correlated with the negative results according to FISH. HER2 mutations are not associated with HER2 amplification, thus suggesting a distinct entity and therapeutic target. HER2-positive lung cancer may not be an adequate term, and patient cohorts for the study of HER2-targeted agents should be defined by the specific HER2 alteration present. Copyright © 2015 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Neurogenomics and the role of a large mutational target on rapid behavioral change.

PubMed

Stanley, Craig E; Kulathinal, Rob J

2016-11-08

Behavior, while complex and dynamic, is among the most diverse, derived, and rapidly evolving traits in animals. The highly labile nature of heritable behavioral change is observed in such evolutionary phenomena as the emergence of converged behaviors in domesticated animals, the rapid evolution of preferences, and the routine development of ethological isolation between diverging populations and species. In fact, it is believed that nervous system development and its potential to evolve a seemingly infinite array of behavioral innovations played a major role in the successful diversification of metazoans, including our own human lineage. However, unlike other rapidly evolving functional systems such as sperm-egg interactions and immune defense, the genetic basis of rapid behavioral change remains elusive. Here we propose that the rapid divergence and widespread novelty of innate and adaptive behavior is primarily a function of its genomic architecture. Specifically, we hypothesize that the broad diversity of behavioral phenotypes present at micro- and macroevolutionary scales is promoted by a disproportionately large mutational target of neurogenic genes. We present evidence that these large neuro-behavioral targets are significant and ubiquitous in animal genomes and suggest that behavior's novelty and rapid emergence are driven by a number of factors including more selection on a larger pool of variants, a greater role of phenotypic plasticity, and/or unique molecular features present in large genes. We briefly discuss the origins of these large neurogenic genes, as they relate to the remarkable diversity of metazoan behaviors, and highlight key consequences on both behavioral traits and neurogenic disease across, respectively, evolutionary and ontogenetic time scales. Current approaches to studying the genetic mechanisms underlying rapid phenotypic change primarily focus on identifying signatures of Darwinian selection in protein-coding regions. In contrast

[Detection of pathogenic mutations in Marfan syndrome by targeted next-generation semiconductor sequencing].

PubMed

Lu, Chaoxia; Wu, Wei; Xiao, Jifang; Meng, Yan; Zhang, Shuyang; Zhang, Xue

2013-06-01

To detect pathogenic mutations in Marfan syndrome (MFS) using an Ion Torrent Personal Genome Machine (PGM) and to validate the result of targeted next-generation semiconductor sequencing for the diagnosis of genetic disorders. Peripheral blood samples were collected from three MFS patients and a normal control with informed consent. Genomic DNA was isolated by standard method and then subjected to targeted sequencing using an Ion Ampliseq(TM) Inherited Disease Panel. Three multiplex PCR reactions were carried out to amplify the coding exons of 328 genes including FBN1, TGFBR1 and TGFBR2. DNA fragments from different samples were ligated with barcoded sequencing adaptors. Template preparation and emulsion PCR, and Ion Sphere Particles enrichment were carried out using an Ion One Touch system. The ion sphere particles were sequenced on a 318 chip using the PGM platform. Data from the PGM runs were processed using an Ion Torrent Suite 3.2 software to generate sequence reads. After sequence alignment and extraction of SNPs and indels, all the variants were filtered against dbSNP137. DNA sequences were visualized with an Integrated Genomics Viewer. The most likely disease-causing variants were analyzed by Sanger sequencing. The PGM sequencing has yielded an output of 855.80 Mb, with a > 100 × median sequencing depth and a coverage of > 98% for the targeted regions in all the four samples. After data analysis and database filtering, one known missense mutation (p.E1811K) and two novel premature termination mutations (p.E2264X and p.L871FfsX23) in the FBN1 gene were identified in the three MFS patients. All mutations were verified by conventional Sanger sequencing. Pathogenic FBN1 mutations have been identified in all patients with MFS, indicating that the targeted next-generation sequencing on the PGM sequencers can be applied for accurate and high-throughput testing of genetic disorders.

Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing

PubMed Central

Brownstein, Zippora; Abu-Rayyan, Amal; Karfunkel-Doron, Daphne; Sirigu, Serena; Davidov, Bella; Shohat, Mordechai; Frydman, Moshe; Houdusse, Anne; Kanaan, Moien; Avraham, Karen B

2014-01-01

Hereditary hearing loss is genetically heterogeneous, with a large number of genes and mutations contributing to this sensory, often monogenic, disease. This number, as well as large size, precludes comprehensive genetic diagnosis of all known deafness genes. A combination of targeted genomic capture and massively parallel sequencing (MPS), also referred to as next-generation sequencing, was applied to determine the deafness-causing genes in hearing-impaired individuals from Israeli Jewish and Palestinian Arab families. Among the mutations detected, we identified nine novel mutations in the genes encoding myosin VI, myosin VIIA and myosin XVA, doubling the number of myosin mutations in the Middle East. Myosin VI mutations were identified in this population for the first time. Modeling of the mutations provided predicted mechanisms for the damage they inflict in the molecular motors, leading to impaired function and thus deafness. The myosin mutations span all regions of these molecular motors, leading to a wide range of hearing phenotypes, reinforcing the key role of this family of proteins in auditory function. This study demonstrates that multiple mutations responsible for hearing loss can be identified in a relatively straightforward manner by targeted-gene MPS technology and concludes that this is the optimal genetic diagnostic approach for identification of mutations responsible for hearing loss. PMID:24105371

Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

DOE PAGES

Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric; ...

2015-03-30

Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

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

Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric

Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

Structure-Based Analysis Reveals Cancer Missense Mutations Target Protein Interaction Interfaces.

PubMed

Engin, H Billur; Kreisberg, Jason F; Carter, Hannah

2016-01-01

Recently it has been shown that cancer mutations selectively target protein-protein interactions. We hypothesized that mutations affecting distinct protein interactions involving established cancer genes could contribute to tumor heterogeneity, and that novel mechanistic insights might be gained into tumorigenesis by investigating protein interactions under positive selection in cancer. To identify protein interactions under positive selection in cancer, we mapped over 1.2 million nonsynonymous somatic cancer mutations onto 4,896 experimentally determined protein structures and analyzed their spatial distribution. In total, 20% of mutations on the surface of known cancer genes perturbed protein-protein interactions (PPIs), and this enrichment for PPI interfaces was observed for both tumor suppressors (Odds Ratio 1.28, P-value < 10(-4)) and oncogenes (Odds Ratio 1.17, P-value < 10(-3)). To study this further, we constructed a bipartite network representing structurally resolved PPIs from all available human complexes in the Protein Data Bank (2,864 proteins, 3,072 PPIs). Analysis of frequently mutated cancer genes within this network revealed that tumor-suppressors, but not oncogenes, are significantly enriched with functional mutations in homo-oligomerization regions (Odds Ratio 3.68, P-Value < 10(-8)). We present two important examples, TP53 and beta-2-microglobulin, for which the patterns of somatic mutations at interfaces provide insights into specifically perturbed biological circuits. In patients with TP53 mutations, patient survival correlated with the specific interactions that were perturbed. Moreover, we investigated mutations at the interface of protein-nucleotide interactions and observed an unexpected number of missense mutations but not silent mutations occurring within DNA and RNA binding sites. Finally, we provide a resource of 3,072 PPI interfaces ranked according to their mutation rates. Analysis of this list highlights 282 novel candidate cancer

Measuring genetic distances between breeds: use of some distances in various short term evolution models

PubMed Central

Laval, Guillaume; SanCristobal, Magali; Chevalet, Claude

2002-01-01

Many works demonstrate the benefits of using highly polymorphic markers such as microsatellites in order to measure the genetic diversity between closely related breeds. But it is sometimes difficult to decide which genetic distance should be used. In this paper we review the behaviour of the main distances encountered in the literature in various divergence models. In the first part, we consider that breeds are populations in which the assumption of equilibrium between drift and mutation is verified. In this case some interesting distances can be expressed as a function of divergence time, t, and therefore can be used to construct phylogenies. Distances based on allele size distribution (such as (δμ)2 and derived distances), taking a mutation model of microsatellites, the Stepwise Mutation Model, specifically into account, exhibit large variance and therefore should not be used to accurately infer phylogeny of closely related breeds. In the last section, we will consider that breeds are small populations and that the divergence times between them are too small to consider that the observed diversity is due to mutations: divergence is mainly due to genetic drift. Expectation and variance of distances were calculated as a function of the Wright-Malécot inbreeding coefficient, F. Computer simulations performed under this divergence model show that the Reynolds distance [57]is the best method for very closely related breeds. PMID:12270106

Differential Effects of HNF-1α Mutations Associated with Familial Young-Onset Diabetes on Target Gene Regulation

PubMed Central

Galán, Maria; García-Herrero, Carmen-Maria; Azriel, Sharona; Gargallo, Manuel; Durán, Maria; Gorgojo, Juan-Jose; Andía, Victor-Manuel; Navas, Maria-Angeles

2011-01-01

Hepatocyte nuclear factor 1-α (HNF-1α) is a homeodomain transcription factor expressed in a variety of tissues (including liver and pancreas) that regulates a wide range of genes. Heterozygous mutations in the gene encoding HNF-1α (HNF1A) cause familial young-onset diabetes, also known as maturity-onset diabetes of the young, type 3 (MODY3). The variability of the MODY3 clinical phenotype can be due to environmental and genetic factors as well as to the type and position of mutations. Thus, functional characterization of HNF1A mutations might provide insight into the molecular defects explaining the variability of the MODY3 phenotype. We have functionally characterized six HNF1A mutations identified in diabetic patients: two novel ones, p.Glu235Gly and c-57-64delCACGCGGT;c-55G>C; and four previously described, p.Val133Met, p.Thr196Ala, p.Arg271Trp and p.Pro379Arg. The effects of mutations on transcriptional activity have been measured by reporter assays on a subset of HNF-1α target promoters in Cos7 and Min6 cells. Target DNA binding affinities have been quantified by electrophoretic mobility shift assay using bacterially expressed glutathione-S-transferase (GST)-HNF-1α fusion proteins and nuclear extracts of transfected Cos7 cells. Our functional studies revealed that mutation c-57-64delCACGCGGT;c-55G>C reduces HNF1A promoter activity in Min6 cells and that missense mutations have variable effects. Mutation p.Arg271Trp impairs HNF-1α activity in all conditions tested, whereas mutations p.Val133Met, p.Glu235Gly and p.Pro379Arg exert differential effects depending on the target promoter. In contrast, substitution p.Thr196Ala does not appear to alter HNF-1α function. Our results suggest that HNF1A mutations may have differential effects on the regulation of specific target genes, which could contribute to the variability of the MODY3 clinical phenotype. PMID:21170474

Accuracies of genomically estimated breeding values from pure-breed and across-breed predictions in Australian beef cattle.

PubMed

Boerner, Vinzent; Johnston, David J; Tier, Bruce

2014-10-24

The major obstacles for the implementation of genomic selection in Australian beef cattle are the variety of breeds and in general, small numbers of genotyped and phenotyped individuals per breed. The Australian Beef Cooperative Research Center (Beef CRC) investigated these issues by deriving genomic prediction equations (PE) from a training set of animals that covers a range of breeds and crosses including Angus, Murray Grey, Shorthorn, Hereford, Brahman, Belmont Red, Santa Gertrudis and Tropical Composite. This paper presents accuracies of genomically estimated breeding values (GEBV) that were calculated from these PE in the commercial pure-breed beef cattle seed stock sector. PE derived by the Beef CRC from multi-breed and pure-breed training populations were applied to genotyped Angus, Limousin and Brahman sires and young animals, but with no pure-breed Limousin in the training population. The accuracy of the resulting GEBV was assessed by their genetic correlation to their phenotypic target trait in a bi-variate REML approach that models GEBV as trait observations. Accuracies of most GEBV for Angus and Brahman were between 0.1 and 0.4, with accuracies for abattoir carcass traits generally greater than for live animal body composition traits and reproduction traits. Estimated accuracies greater than 0.5 were only observed for Brahman abattoir carcass traits and for Angus carcass rib fat. Averaged across traits within breeds, accuracies of GEBV were highest when PE from the pooled across-breed training population were used. However, for the Angus and Brahman breeds the difference in accuracy from using pure-breed PE was small. For the Limousin breed no reasonable results could be achieved for any trait. Although accuracies were generally low compared to published accuracies estimated within breeds, they are in line with those derived in other multi-breed populations. Thus PE developed by the Beef CRC can contribute to the implementation of genomic selection in

TP53, PIK3CA, FBXW7 and KRAS Mutations in Esophageal Cancer Identified by Targeted Sequencing.

PubMed

Zheng, Huili; Wang, Yan; Tang, Chuanning; Jones, Lindsey; Ye, Hua; Zhang, Guangchun; Cao, Weihai; Li, Jingwen; Liu, Lifeng; Liu, Zhencong; Zhang, Chao; Lou, Feng; Liu, Zhiyuan; Li, Yangyang; Shi, Zhenfen; Zhang, Jingbo; Zhang, Dandan; Sun, Hong; Dong, Haichao; Dong, Zhishou; Guo, Baishuai; Yan, H E; Lu, Qingyu; Huang, Xue; Chen, Si-Yi

2016-01-01

Esophageal cancer (EC) is a common malignancy with significant morbidity and mortality. As individual cancers exhibit unique mutation patterns, identifying and characterizing gene mutations in EC that may serve as biomarkers might help predict patient outcome and guide treatment. Traditionally, personalized cancer DNA sequencing was impractical and expensive. Recent technological advancements have made targeted DNA sequencing more cost- and time-effective with reliable results. This technology may be useful for clinicians to direct patient treatment. The Ion PGM and AmpliSeq Cancer Panel was used to identify mutations at 737 hotspot loci of 45 cancer-related genes in 64 EC samples from Chinese patients. Frequent mutations were found in TP53 and less frequent mutations in PIK3CA, FBXW7 and KRAS. These results demonstrate that targeted sequencing can reliably identify mutations in individual tumors that make this technology a possibility for clinical use. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Detection of Somatic Mutations in Gastroenteropancreatic Neuroendocrine Tumors Using Targeted Deep Sequencing.

PubMed

Backman, Samuel; Norlén, Olov; Eriksson, Barbro; Skogseid, Britt; Stålberg, Peter; Crona, Joakim

2017-02-01

Mutations affecting the mechanistic target of rapamycin (MTOR) signalling pathway are frequent in human cancer and have been identified in up to 15% of pancreatic neuroendocrine tumours (NETs). Grade A evidence supports the efficacy of MTOR inhibition with everolimus in pancreatic NETs. Although a significant proportion of patients experience disease stabilization, only a minority will show objective tumour responses. It has been proposed that genomic mutations resulting in activation of MTOR signalling could be used to predict sensitivity to everolimus. Patients with NETs that underwent treatment with everolimus at our Institution were identified and those with available tumour tissue were selected for further analysis. Targeted next-generation sequencing (NGS) was used to re-sequence 22 genes that were selected on the basis of documented involvement in the MTOR signalling pathway or in the tumourigenesis of gastroenterpancreatic NETs. Radiological responses were documented using Response Evaluation Criteria in Solid Tumours. Six patients were identified, one had a partial response and four had stable disease. Sequencing of tumour tissue resulted in a median sequence depth of 667.1 (range=404-1301) with 1-fold coverage of 95.9-96.5% and 10-fold coverage of 87.6-92.2%. A total of 494 genetic variants were discovered, four of which were identified as pathogenic. All pathogenic variants were validated using Sanger sequencing and were found exclusively in menin 1 (MEN1) and death domain associated protein (DAXX) genes. No mutations in the MTOR pathway-related genes were observed. Targeted NGS is a feasible method with high diagnostic yield for genetic characterization of pancreatic NETs. A potential association between mutations in NETs and response to everolimus should be investigated by future studies. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Response of head and neck squamous cell carcinoma cells carrying PIK3CA mutations to selected targeted therapies.

PubMed

Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M

2015-06-01

The PIK3CA mutation is one of the most common mutations in head and neck squamous cell carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. (1) To determine the role of oncogene dependence on PIK3CA-one of the more common and targetable oncogenes in HNSCC, and (2) to evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. This was a cell culture-based, in vitro study performed at an academic research laboratory assessing the viability of PIK3CA-mutated head and neck cell lines when treated with targeted therapy. PIK3CA-mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R. Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred increased, rather than reduced, IC50 assay measurements when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared with the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared with control, whereas those expressing E545K showed slightly increased sensitivity of unclear significance. (1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared with control with dual PI3K/mTOR inhibition. (2) Oncogene addiction to PIK3CA hotspot mutations, if it occurs, is likely to evolve in vivo in the context of additional molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of

Response of Head and Neck Squamous Cell Carcinoma Cells Carrying PIK3CA Mutations to Select Targeted Therapies

PubMed Central

Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M

2015-01-01

Importance The PIK3CA mutation is one of the most common mutations in Head and Neck Squamous Cell Carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. Objectives 1) To determine the role of oncogene dependence on one of the more common and targetable oncogenes in HNSCC – PIK3CA; 2) To evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. Study Design In vitro study. Setting Academic research laboratory. Participants Cell culture based study assessing the viability of PIK3CA mutated head and neck cell lines when treated with targeted therapy. Exposures PIK3CA mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Main Outcome and Measures Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R Results Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred decreased, rather than increased, sensitivity as measured by IC50 when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared to the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared to control while those expressing E545K showed slightly increased sensitivity of unclear significance. Conclusions and Relevance 1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared to control with dual PI3K/mTOR inhibition. 2) Oncogene addiction to PIK3CA hot spot mutations, if it occurs, is likely to evolve in vivo molecular changes that remain to be identified. Additional study is required to develop new model systems and

Pituitary dwarfism in Saarloos and Czechoslovakian wolfdogs is associated with a mutation in LHX3.

PubMed

Voorbij, A M W Y; Leegwater, P A; Kooistra, H S

2014-01-01

Pituitary dwarfism in German Shepherd Dogs is associated with autosomal recessive inheritance and a mutation in LHX3, resulting in combined pituitary hormone deficiency. Congenital dwarfism also is encountered in breeds related to German Shepherd Dogs, such as Saarloos and Czechoslovakian wolfdogs. To investigate whether Saarloos and Czechoslovakian wolfdog dwarfs have the same LHX3 mutation as do Germans Shepherd Dog dwarfs. A specific aim was to determine the carrier frequency among Saarloos and Czechoslovakian wolfdogs used for breeding. Two client-owned Saarloos wolfdogs and 4 client-owned Czechoslovakian wolfdogs with pituitary dwarfism, 239 clinically healthy client-owned Saarloos wolfdogs, and 200 client-owned clinically healthy Czechoslovakian wolfdogs. Genomic DNA was amplified using polymerase chain reaction (PCR). In the Saarloos and Czechoslovakian wolfdog dwarfs, PCR products were analyzed by sequencing. DNA fragment length analysis was performed on the samples from the clinically healthy dogs. Saarloos and Czechoslovakian wolfdog dwarfs have the same 7 bp deletion in intron 5 of LHX3 as do German Shepherd Dog dwarfs. The frequency of carriers of this mutation among clinically healthy Saarloos and Czechoslovakian wolfdogs used for breeding was 31% and 21%, respectively. An LHX3 mutation is associated with pituitary dwarfism in Saarloos and Czechoslovakian wolfdogs. The rather high frequency of carriers of the mutated gene in the 2 breeds emphasizes the need for screening before breeding. If all breeding animals were genetically tested for the presence of the LHX3 mutation and a correct breeding policy would be implemented, this disease could be eradicated completely. Copyright © 2014 by the American College of Veterinary Internal Medicine.

Allelic heterogeneity of FGF5 mutations causes the long-hair phenotype in dogs.

PubMed

Dierks, C; Mömke, S; Philipp, U; Distl, O

2013-08-01

Hitherto, the only known mutant gene leading to the long-hair phenotype in mammals is the fibroblast growth factor 5 (FGF5). In many dog breeds, the previously discovered FGF5:p.Cys95Phe mutation appeared completely concordant with the long-hair phenotype, but for some breeds, the long-hair phenotype could not be resolved. First, we studied the role of the FGF5:p.Cys95Phe and FGF5:g.145_150dupACCAGC mutations in 268 dogs descending from 27 breeds and seven wolves. As these mutations did not explain all the long-hair phenotypes, all exons and their neighbouring regions of FGF5 were re-sequenced. We detected three novel mutations in the coding sequence and one novel non-coding splice-site mutation in FGF5 associated with the long-hair phenotype. The FGF5:p.Ala193Val polymorphism was perfectly consistent with long hair in Akitas and probably in Siberian huskies, too. Dogs of the long-hair breed Samoyed were either homozygous or compound heterozygous for the FGF5:p.Ala193Val or the FGF5:p.Cys95Phe polymorphisms respectively. The two newly detected polymorphisms FGF5:c.559_560dupGG and FGF5:g.8193T>A and the known mutation FGF5:p.Cys95Phe explained the long-hair phenotype of all Afghan hounds analysed. An FGF5:c.556_571del16 mutation was found in one longhaired Eurasier. All long-hair-associated mutations follow a recessive mode of inheritance, and allelic heterogeneity was a common finding in breeds other than Akita. © 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.

Recent advances of microbial breeding via heavy-ion mutagenesis at IMP.

PubMed

Hu, W; Li, W; Chen, J

2017-10-01

Nowadays, the value of heavy-ion mutagenesis has been accepted as a novel powerful mutagen technique to generate new microbial mutants due to its high linear energy transfer and high relative biological effectiveness. This paper briefly reviews recent progress in developing a more efficient mutagenesis technique for microbial breeding using heavy-ion mutagenesis, and also presents the outline of the beam line for microbial breeding in Heavy Ion Research Facility of Lanzhou. Then, new insights into microbial biotechnology via heavy-ion mutagenesis are also further explored. We hope that our concerns will give deep insight into microbial breeding biotechnology via heavy-ion mutagenesis. We also believe that heavy-ion mutagenesis breeding will greatly contribute to the progress of a comprehensive study industrial strain engineering for bioindustry in the future. There is currently a great interest in developing rapid and diverse microbial mutation tool for strain modification. Heavy-ion mutagenesis has been proved as a powerful technology for microbial breeding due to its broad spectrum of mutation phenotypes with high efficiency. In order to deeply understand heavy-ion mutagenesis technology, this paper briefly reviews recent progress in microbial breeding using heavy-ion mutagenesis at IMP, and also presents the outline of the beam line for microbial breeding in Heavy Ion Research Facility of Lanzhou (HIRFL) as well as new insights into microbial biotechnology via heavy-ion mutagenesis. Thus, this work can provide the guidelines to promote the development of novel microbial biotechnology cross-linking heavy-ion mutagenesis breeding that could make breeding process more efficiently in the future. © 2017 The Society for Applied Microbiology.

Generation of novel resistance genes using mutation and targeted gene editing.

PubMed

Gal-On, Amit; Fuchs, Marc; Gray, Stewart

2017-10-01

Classical breeding for virus resistance is a lengthy process and is restricted by the availability of resistance genes. Precise genome editing is a 'dream technology' to improve plants for virus resistance and these tools have opened new and very promising ways to generate virus resistant plants by disrupting host susceptibility genes, or by increasing the expression of viral resistance genes. However, precise targets must be identified and their roles understood to minimize potential negative effects on the plant. Nonetheless, the opportunities for genome editing are expanding, as are the technologies to generate effective and broad-spectrum resistance against plant viruses. Here we provide insights into recent progress related to gene targets and gene editing technologies. Published by Elsevier B.V.

Phenotypic Diagnosis of Lineage and Differentiation During Sake Yeast Breeding

PubMed Central

Ohnuki, Shinsuke; Okada, Hiroki; Friedrich, Anne; Kanno, Yoichiro; Goshima, Tetsuya; Hasuda, Hirokazu; Inahashi, Masaaki; Okazaki, Naoto; Tamura, Hiroyasu; Nakamura, Ryo; Hirata, Dai; Fukuda, Hisashi; Shimoi, Hitoshi; Kitamoto, Katsuhiko; Watanabe, Daisuke; Schacherer, Joseph; Akao, Takeshi; Ohya, Yoshikazu

2017-01-01

Sake yeast was developed exclusively in Japan. Its diversification during breeding remains largely uncharacterized. To evaluate the breeding processes of the sake lineage, we thoroughly investigated the phenotypes and differentiation of 27 sake yeast strains using high-dimensional, single-cell, morphological phenotyping. Although the genetic diversity of the sake yeast lineage is relatively low, its morphological diversity has expanded substantially compared to that of the Saccharomyces cerevisiae species as a whole. Evaluation of the different types of breeding processes showed that the generation of hybrids (crossbreeding) has more profound effects on cell morphology than the isolation of mutants (mutation breeding). Analysis of phenotypic robustness revealed that some sake yeast strains are more morphologically heterogeneous, possibly due to impairment of cellular network hubs. This study provides a new perspective for studying yeast breeding genetics and micro-organism breeding strategies. PMID:28642365

Targeted next generation sequencing of mucosal melanomas identifies frequent NF1 and RAS mutations.

PubMed

Cosgarea, Ioana; Ugurel, Selma; Sucker, Antje; Livingstone, Elisabeth; Zimmer, Lisa; Ziemer, Mirjana; Utikal, Jochen; Mohr, Peter; Pfeiffer, Christiane; Pföhler, Claudia; Hillen, Uwe; Horn, Susanne; Schadendorf, Dirk; Griewank, Klaus G; Roesch, Alexander

2017-06-20

Mucosal melanoma represents ~1% of all melanomas, frequently having a poor prognosis due to diagnosis at a late stage of disease. Mucosal melanoma differs from cutaneous melanoma not only in terms of poorer clinical outcome but also on the molecular level having e.g. less BRAF and more frequent KIT mutations than cutaneous melanomas. For the majority of mucosal melanomas oncogenic driver mutations remain unknown. In our study, 75 tumor tissues from patients diagnosed with mucosal melanoma were analyzed, applying a targeted next generation sequencing panel covering 29 known recurrently mutated genes in melanoma. NF1 and RAS mutations were identified as the most frequently mutated genes occurring in 18.3% and 16.9% of samples, respectively. Mutations in BRAF were identified in 8.4% and KIT in 7.0% of tumor samples. Our study identifies NF1 as the most frequently occurring driver mutation in mucosal melanoma. RAS alterations, consisting of NRAS and KRAS mutations, were the second most frequent mutation type. BRAF and KIT mutations were rare with frequencies below 10% each. Our data indicate that in mucosal melanomas RAS/NF1 alterations are frequent, implying a significant pathogenetic role for MAPK and potentially PI3K pathway activation in these tumors.

Targeted next generation sequencing of mucosal melanomas identifies frequent NF1 and RAS mutations

PubMed Central

Cosgarea, Ioana; Ugurel, Selma; Sucker, Antje; Livingstone, Elisabeth; Zimmer, Lisa; Ziemer, Mirjana; Utikal, Jochen; Mohr, Peter; Pfeiffer, Christiane; Pföhler, Claudia; Hillen, Uwe; Horn, Susanne; Schadendorf, Dirk

2017-01-01

Purpose Mucosal melanoma represents ~1% of all melanomas, frequently having a poor prognosis due to diagnosis at a late stage of disease. Mucosal melanoma differs from cutaneous melanoma not only in terms of poorer clinical outcome but also on the molecular level having e.g. less BRAF and more frequent KIT mutations than cutaneous melanomas. For the majority of mucosal melanomas oncogenic driver mutations remain unknown. Experimental Design and Results In our study, 75 tumor tissues from patients diagnosed with mucosal melanoma were analyzed, applying a targeted next generation sequencing panel covering 29 known recurrently mutated genes in melanoma. NF1 and RAS mutations were identified as the most frequently mutated genes occurring in 18.3% and 16.9% of samples, respectively. Mutations in BRAF were identified in 8.4% and KIT in 7.0% of tumor samples. Conclusions Our study identifies NF1 as the most frequently occurring driver mutation in mucosal melanoma. RAS alterations, consisting of NRAS and KRAS mutations, were the second most frequent mutation type. BRAF and KIT mutations were rare with frequencies below 10% each. Our data indicate that in mucosal melanomas RAS/NF1 alterations are frequent, implying a significant pathogenetic role for MAPK and potentially PI3K pathway activation in these tumors. PMID:28380455

Genome-Wide Analysis Reveals Selection for Important Traits in Domestic Horse Breeds

PubMed Central

Petersen, Jessica L.; Mickelson, James R.; Rendahl, Aaron K.; Valberg, Stephanie J.; Andersson, Lisa S.; Axelsson, Jeanette; Bailey, Ernie; Bannasch, Danika; Binns, Matthew M.; Borges, Alexandre S.; Brama, Pieter; da Câmara Machado, Artur; Capomaccio, Stefano; Cappelli, Katia; Cothran, E. Gus; Distl, Ottmar; Fox-Clipsham, Laura; Graves, Kathryn T.; Guérin, Gérard; Haase, Bianca; Hasegawa, Telhisa; Hemmann, Karin; Hill, Emmeline W.; Leeb, Tosso; Lindgren, Gabriella; Lohi, Hannes; Lopes, Maria Susana; McGivney, Beatrice A.; Mikko, Sofia; Orr, Nicholas; Penedo, M. Cecilia T.; Piercy, Richard J.; Raekallio, Marja; Rieder, Stefan; Røed, Knut H.; Swinburne, June; Tozaki, Teruaki; Vaudin, Mark; Wade, Claire M.; McCue, Molly E.

2013-01-01

Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an FST-based statistic calculated in 500-kb windows across the genome. A 5.5-Mb region of ECA18, in which the myostatin (MSTN) gene was centered, contained the highest signature of selection in both the Paint and Quarter Horse. Gene sequencing and histological analysis of gluteal muscle biopsies showed a promoter variant and intronic SNP of MSTN were each significantly associated with higher Type 2B and lower Type 1 muscle fiber proportions in the Quarter Horse, demonstrating a functional consequence of selection at this locus. Signatures of selection on ECA23 in all gaited breeds in the sample led to the identification of a shared, 186-kb haplotype including two doublesex related mab transcription factor genes (DMRT2 and 3). The recent identification of a DMRT3 mutation within this haplotype, which appears necessary for the ability to perform alternative gaits, provides further evidence for selection at this locus. Finally, putative loci for the determination of size were identified in the draft breeds and the Miniature horse on ECA11, as well as when signatures of selection surrounding candidate genes at other loci were examined. This work provides further evidence of the importance of MSTN in racing breeds, provides strong evidence for selection upon gait and size, and illustrates the potential for population-based techniques to find genomic regions driving important phenotypes in the modern horse. PMID:23349635

Genetic and phenotypic variations of inherited retinal diseases in dogs: the power of within- and across-breed studies

PubMed Central

Acland, Gregory M.

2014-01-01

Considerable clinical and molecular variations have been known in retinal blinding diseases in man and also in dogs. Different forms of retinal diseases occur in specific breed(s) caused by mutations segregating within each isolated breeding population. While molecular studies to find genes and mutations underlying retinal diseases in dogs have benefited largely from the phenotypic and genetic uniformity within a breed, within- and across-breed variations have often played a key role in elucidating the molecular basis. The increasing knowledge of phenotypic, allelic, and genetic heterogeneities in canine retinal degeneration has shown that the overall picture is rather more complicated than initially thought. Over the past 20 years, various approaches have been developed and tested to search for genes and mutations underlying genetic traits in dogs, depending on the availability of genetic tools and sample resources. Candidate gene, linkage analysis, and genome-wide association studies have so far identified 24 mutations in 18 genes underlying retinal diseases in at least 58 dog breeds. Many of these genes have been associated with retinal diseases in humans, thus providing opportunities to study the role in pathogenesis and in normal vision. Application in therapeutic interventions such as gene therapy has proven successful initially in a naturally occurring dog model followed by trials in human patients. Other genes whose human homologs have not been associated with retinal diseases are potential candidates to explain equivalent human diseases and contribute to the understanding of their function in vision. PMID:22065099

Genetic and phenotypic variations of inherited retinal diseases in dogs: the power of within- and across-breed studies.

PubMed

Miyadera, Keiko; Acland, Gregory M; Aguirre, Gustavo D

2012-02-01

Considerable clinical and molecular variations have been known in retinal blinding diseases in man and also in dogs. Different forms of retinal diseases occur in specific breed(s) caused by mutations segregating within each isolated breeding population. While molecular studies to find genes and mutations underlying retinal diseases in dogs have benefited largely from the phenotypic and genetic uniformity within a breed, within- and across-breed variations have often played a key role in elucidating the molecular basis. The increasing knowledge of phenotypic, allelic, and genetic heterogeneities in canine retinal degeneration has shown that the overall picture is rather more complicated than initially thought. Over the past 20 years, various approaches have been developed and tested to search for genes and mutations underlying genetic traits in dogs, depending on the availability of genetic tools and sample resources. Candidate gene, linkage analysis, and genome-wide association studies have so far identified 24 mutations in 18 genes underlying retinal diseases in at least 58 dog breeds. Many of these genes have been associated with retinal diseases in humans, thus providing opportunities to study the role in pathogenesis and in normal vision. Application in therapeutic interventions such as gene therapy has proven successful initially in a naturally occurring dog model followed by trials in human patients. Other genes whose human homologs have not been associated with retinal diseases are potential candidates to explain equivalent human diseases and contribute to the understanding of their function in vision.

Breeding for cuticle-associated traits in crop species: traits, targets, and strategies.

PubMed

Petit, Johann; Bres, Cécile; Mauxion, Jean-Philippe; Bakan, Bénédicte; Rothan, Christophe

2017-11-09

Improving crop productivity and quality while promoting sustainable agriculture have become major goals in plant breeding. The cuticle is a natural film covering the aerial organs of plants and consists of lipid polyesters covered and embedded with wax. The cuticle protects plants against water loss and pathogens and affects traits with strong impacts on crop quality such as, for horticultural crops, fruit brightness, cracking, russeting, netting, and shelf life. Here we provide an overview of the most important cuticle-associated traits that can be targeted for crop improvement. To date, most studies on cuticle-associated traits aimed at crop breeding have been done on fleshy fruits. Less information is available for staple crops such as rice, wheat or maize. Here we present new insights into cuticle formation and properties resulting from the study of genetic resources available for the various crop species. Our review also covers the current strategies and tools aimed at exploiting available natural and artificially induced genetic diversity and the technologies used to transfer the beneficial alleles affecting cuticle-associated traits to commercial varieties. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Degenerative myelopathy in the Collie breed: a retrospective immunohistochemical analysis of superoxide dismutase 1 in an affected Rough Collie, and a molecular epidemiological survey of the SOD1: c.118G>A mutation in Japan

PubMed Central

KOHYAMA, Moeko; KITAGAWA, Masato; KAMISHINA, Hiroaki; KOBATAKE, Yui; YABUKI, Akira; SAWA, Mariko; KAKITA, Shusaku; YAMATO, Osamu

2016-01-01

Canine degenerative myelopathy (DM) is an adult-onset, progressive neurodegenerative disease that occurs in multiple dog breeds. A DM-associated mutation of the canine superoxide dismutase 1 (SOD1) gene, designated as c.118G>A (p.E40K), has been implicated as one of pathogenetic determinants of the disease in many breeds, but it remains to be determined whether the c.118G>A mutation is responsible for development or progression of DM in Collies. Previously, a Rough Collie was diagnosed clinically and histopathologically as having DM in Japan, suggesting the possibility that the Collie breed may be predisposed to DM due to the high frequency of c.118G>A in Japan. In this study, accumulation and aggregate formation of SOD1 protein were retrospectively demonstrated in the spinal cord of the DM-affected dog by immunohistochemical analysis. Furthermore, a molecular epidemiological survey revealed a high carrier rate (27.6%) and mutant allele frequency (0.138) of c.118G>A in a population of Collies in Japan, suggesting that the Collie breed may be predisposed to DM associated with c.118G>A, and the prevention of DM in Collies in Japan should be addressed through epidemiological and genetic testing strategies. PMID:27941298

Erythrocytic Pyruvate Kinase Mutations Causing Hemolytic Anemia, Osteosclerosis, and Secondary Hemochromatosis in Dogs

PubMed Central

Gultekin, G. Inal; Raj, K.; Foureman, P.; Lehman, S.; Manhart, K.; Abdulmalik, O.; Giger, U.

2013-01-01

Background Erythrocytic pyruvate kinase (PK) deficiency, first documented in Basenjis, is the most common inherited erythroenzymopathy in dogs. Objectives To report 3 new breed-specific PK-LR gene mutations and a retrospective survey of PK mutations in a small and selected group of Beagles and West Highland White Terriers (WHWT). Animals Labrador Retrievers (2 siblings, 5 unrelated), Pugs (2 siblings, 1 unrelated), Beagles (39 anemic, 29 other), WHWTs (22 anemic, 226 nonanemic), Cairn Terrier (n = 1). Methods Exons of the PK-LR gene were sequenced from genomic DNA of young dogs (<2 years) with persistent highly regenerative hemolytic anemia. Results A nonsense mutation (c.799C>T) resulting in a premature stop codon was identified in anemic Labrador Retriever siblings that had osteosclerosis, high serum ferritin concentrations, and severe hepatic secondary hemochromatosis. Anemic Pug and Beagle revealed 2 different missense mutations (c.848T>C, c.994G>A, respectively) resulting in intolerable amino acid changes to protein structure and enzyme function. Breed-specific mutation tests were developed. Among the biased group of 248 WHWTs, 9% and 35% were homozygous (affected) and heterozygous, respectively, for the previously described mutation (mutant allele frequency 0.26). A PK-deficient Cairn Terrier had the same insertion mutation as the affected WHWTs. Of the selected group of 68 Beagles, 35% were PK-deficient and 3% were carriers (0.37). Conclusions and Clinical Importance Erythrocytic PK deficiency is caused by different mutations in different dog breeds and causes chronic severe hemolytic anemia, hemosiderosis, and secondary hemochromatosis because of chronic hemolysis and, an as yet unexplained osteosclerosis. The newly developed breed-specific mutation assays simplify the diagnosis of PK deficiency. PMID:22805166

Genome sequencing reveals a splice donor site mutation in the SNX14 gene associated with a novel cerebellar cortical degeneration in the Hungarian Vizsla dog breed.

PubMed

Fenn, Joe; Boursnell, Mike; Hitti, Rebekkah J; Jenkins, Christopher A; Terry, Rebecca L; Priestnall, Simon L; Kenny, Patrick J; Mellersh, Cathryn S; Forman, Oliver P

2016-08-26

Cerebellar cortical degeneration (CCD) is an increasingly recognised neurodegenerative disease process affecting many dog breeds. Typical presentation consists of a progressive cerebellar ataxia, with a variable age at onset and rate of progression between different breeds. Cerebellar histopathological findings typically consist of primary Purkinje neuronal degeneration and loss, with variable secondary depletion of the granular and molecular cell layers. Causative genes have been identified associated with CCD in several breeds, allowing screening for selective breeding to reduce the prevalence of these conditions. There have been no previous reports of CCD in Hungarian Vizslas. Two full-sibling Hungarian Vizsla puppies from a litter of nine presented with a history of progressive ataxia, starting around three months of age. Clinical signs included marked hypermetric and dysmetric ataxia, truncal sway, intention tremors and absent menace responses, with positional horizontal nystagmus in one dog. Routine diagnostic investigations were unremarkable, and magnetic resonance imaging performed in one dog revealed mild craniodorsal cerebellar sulci widening, supportive of cerebellar atrophy. Owners of both dogs elected for euthanasia shortly after the onset of signs. Histopathological examination revealed primary Purkinje neuron loss consistent with CCD. Whole genome sequencing was used to successfully identify a disease-associated splice donor site variant in the sorting nexin 14 gene (SNX14) as a strong causative candidate. An altered SNX14 splicing pattern for a CCD case was demonstrated by RNA analysis, and no SNX14 protein could be detected in CCD case cerebellum by western blotting. SNX14 is involved in maintaining normal neuronal excitability and synaptic transmission, and a mutation has recently been found to cause autosomal recessive cerebellar ataxia and intellectual disability syndrome in humans. Genetic screening of 133 unaffected Hungarian Vizslas revealed

Next-generation sequencing reveals the mutational landscape of clinically diagnosed Usher syndrome: copy number variations, phenocopies, a predominant target for translational read-through, and PEX26 mutated in Heimler syndrome.

PubMed

Neuhaus, Christine; Eisenberger, Tobias; Decker, Christian; Nagl, Sandra; Blank, Cornelia; Pfister, Markus; Kennerknecht, Ingo; Müller-Hofstede, Cornelie; Charbel Issa, Peter; Heller, Raoul; Beck, Bodo; Rüther, Klaus; Mitter, Diana; Rohrschneider, Klaus; Steinhauer, Ute; Korbmacher, Heike M; Huhle, Dagmar; Elsayed, Solaf M; Taha, Hesham M; Baig, Shahid M; Stöhr, Heidi; Preising, Markus; Markus, Susanne; Moeller, Fabian; Lorenz, Birgit; Nagel-Wolfrum, Kerstin; Khan, Arif O; Bolz, Hanno J

2017-09-01

Combined retinal degeneration and sensorineural hearing impairment is mostly due to autosomal recessive Usher syndrome (USH1: congenital deafness, early retinitis pigmentosa (RP); USH2: progressive hearing impairment, RP). Sanger sequencing and NGS of 112 genes (Usher syndrome, nonsyndromic deafness, overlapping conditions), MLPA, and array-CGH were conducted in 138 patients clinically diagnosed with Usher syndrome. A molecular diagnosis was achieved in 97% of both USH1 and USH2 patients, with biallelic mutations in 97% (USH1) and 90% (USH2), respectively. Quantitative readout reliably detected CNVs (confirmed by MLPA or array-CGH), qualifying targeted NGS as one tool for detecting point mutations and CNVs. CNVs accounted for 10% of identified USH2A alleles, often in trans to seemingly monoallelic point mutations. We demonstrate PTC124-induced read-through of the common p.Trp3955* nonsense mutation (13% of detected USH2A alleles), a potential therapy target. Usher gene mutations were found in most patients with atypical Usher syndrome, but the diagnosis was adjusted in case of double homozygosity for mutations in OTOA and NR2E3 , genes implicated in isolated deafness and RP. Two patients with additional enamel dysplasia had biallelic PEX26 mutations, for the first time linking this gene to Heimler syndrome. Targeted NGS not restricted to Usher genes proved beneficial in uncovering conditions mimicking Usher syndrome.

Combined mutation and copy-number variation detection by targeted next-generation sequencing in uveal melanoma.

PubMed

Smit, Kyra N; van Poppelen, Natasha M; Vaarwater, Jolanda; Verdijk, Robert; van Marion, Ronald; Kalirai, Helen; Coupland, Sarah E; Thornton, Sophie; Farquhar, Neil; Dubbink, Hendrikus-Jan; Paridaens, Dion; de Klein, Annelies; Kiliç, Emine

2018-05-01

Uveal melanoma is a highly aggressive cancer of the eye, in which nearly 50% of the patients die from metastasis. It is the most common type of primary eye cancer in adults. Chromosome and mutation status have been shown to correlate with the disease-free survival. Loss of chromosome 3 and inactivating mutations in BAP1, which is located on chromosome 3, are strongly associated with 'high-risk' tumors that metastasize early. Other genes often involved in uveal melanoma are SF3B1 and EIF1AX, which are found to be mutated in intermediate- and low-risk tumors, respectively. To obtain genetic information of all genes in one test, we developed a targeted sequencing method that can detect mutations in uveal melanoma genes and chromosomal anomalies in chromosome 1, 3, and 8. With as little as 10 ng DNA, we obtained enough coverage on all genes to detect mutations, such as substitutions, deletions, and insertions. These results were validated with Sanger sequencing in 28 samples. In >90% of the cases, the BAP1 mutation status corresponded to the BAP1 immunohistochemistry. The results obtained in the Ion Torrent single-nucleotide polymorphism assay were confirmed with several other techniques, such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, and Illumina SNP array. By validating our assay in 27 formalin-fixed paraffin-embedded and 43 fresh uveal melanomas, we show that mutations and chromosome status can reliably be obtained using targeted next-generation sequencing. Implementing this technique as a diagnostic pathology application for uveal melanoma will allow prediction of the patients' metastatic risk and potentially assess eligibility for new therapies.

Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy.

PubMed

Huang, Xiao-Yan; Zhuang, Hong; Wu, Ji-Hong; Li, Jian-Kang; Hu, Fang-Yuan; Zheng, Yu; Tellier, Laurent Christian Asker M; Zhang, Sheng-Hai; Gao, Feng-Juan; Zhang, Jian-Guo; Xu, Ge-Zhi

2017-01-01

Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [ LRP5 , c.4053 DelC (p.Ile1351IlefsX88); TSPAN12 , EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. We identified two novel heterozygous deletion mutations [ LRP5 , c.4053 DelC (p.Ile1351IlefsX88); TSPAN12 , EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype-phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling.

Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy

PubMed Central

Huang, Xiao-Yan; Zhuang, Hong; Wu, Ji-Hong; Li, Jian-Kang; Hu, Fang-Yuan; Zheng, Yu; Tellier, Laurent Christian Asker M.; Zhang, Sheng-Hai; Gao, Feng-Juan; Zhang, Jian-Guo

2017-01-01

Purpose Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. Methods To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). Results Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. Conclusions We identified two novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype–phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling. PMID:28867931

BREEDING AND GENETICS SYMPOSIUM:Breeding heat tolerant dairy cattle: the case for introgression of the "slick" prolactin receptor variant into dairy breeds.

PubMed

Davis, S R; Spelman, R J; Littlejohn, M D

2017-04-01

Increasing environmental temperatures are a threat to the sustainability of livestock production and, because of the high metabolic demands of lactation, to dairy production in particular. Summer heat waves in temperate climates reduce feed intake, milk production, and cow comfort. In extreme heat events, there is an increase in cow mortality. In tropical climates, dairy cattle are mostly (zebu) type or zebu crossbred with temperate dairy breeds. Crossbreeding is undertaken to combine the heat tolerance and tick resistance of zebu with the productivity of temperate dairy breeds. In the absence of improved heat tolerance, milk production and fertility of temperate cattle is severely impaired. We have recently identified a key role for the prolactin pathway in regulating heat tolerance. A de novo mutation in prolactin that impairs prolactin activity was discovered in hairy and heat intolerant, New Zealand dairy cattle. The phenotypes produced were remarkably similar to those seen in fescue toxicosis, a syndrome seen in grazing cattle in the U.S. where ingestion of ergovaline, a fungal toxin from infected pasture, inhibits prolactin secretion. Recognition of the role of prolactin in hairy cattle led us to identify a deletion in exon 10 of the long-form of the prolactin receptor in Senepol cattle that causes truncation of the protein and determines the slick coat and heat tolerance traits found in this , beef breed. The short form of the prolactin receptor is predicted to be unaffected by the deletion. Knowledge of this dominant mutation has provided the impetus to begin a crossbreeding program to investigate performance and heat tolerance of temperate dairy cattle carrying the slick, prolactin receptor variant. The perceived opportunity is to introgress this variant into temperate dairy cattle to enable performance and welfare improvement in hot climates. Heat tolerance of cattle with slick coats appears to be mostly associated with coat type although sweating ability

Efficient Generation of Somatic Cell Nuclear Transfer-Competent Porcine Cells with Mutated Alleles at Multiple Target Loci by Using CRISPR/Cas9 Combined with Targeted Toxin-Based Selection System.

PubMed

Sato, Masahiro; Miyoshi, Kazuchika; Nakamura, Shingo; Ohtsuka, Masato; Sakurai, Takayuki; Watanabe, Satoshi; Kawaguchi, Hiroaki; Tanimoto, Akihide

2017-12-04

The recent advancement in genome editing such a CRISPR/Cas9 system has enabled isolation of cells with knocked multiple alleles through a one-step transfection. Somatic cell nuclear transfer (SCNT) has been frequently employed as one of the efficient tools for the production of genetically modified (GM) animals. To use GM cells as SCNT donor, efficient isolation of transfectants with mutations at multiple target loci is often required. The methods for the isolation of such GM cells largely rely on the use of drug selection-based approach using selectable genes; however, it is often difficult to isolate cells with mutations at multiple target loci. In this study, we used a novel approach for the efficient isolation of porcine cells with at least two target loci mutations by one-step introduction of CRISPR/Cas9-related components. A single guide (sg) RNA targeted to GGTA1 gene, involved in the synthesis of cell-surface α-Gal epitope (known as xenogenic antigen), is always a prerequisite. When the transfected cells were reacted with toxin-labeled BS-I-B₄ isolectin for 2 h at 37 C to eliminate α-Gal epitope-expressing cells, the surviving clones lacked α-Gal epitope expression and were highly expected to exhibit induced mutations at another target loci. Analysis of these α-Gal epitope-negative surviving cells demonstrated a 100% occurrence of genome editing at target loci. SCNT using these cells as donors resulted in the production of cloned blastocysts with the genotype similar to that of the donor cells used. Thus, this novel system will be useful for SCNT-mediated acquisition of GM cloned piglets, in which multiple target loci may be mutated.

Genome-editing technologies and their potential application in horticultural crop breeding

PubMed Central

Xiong, Jin-Song; Ding, Jing; Li, Yi

2015-01-01

Plant breeding, one of the oldest agricultural activities, parallels human civilization. Many crops have been domesticated to satisfy human's food and aesthetical needs, including numerous specialty horticultural crops such as fruits, vegetables, ornamental flowers, shrubs, and trees. Crop varieties originated through selection during early human civilization. Other technologies, such as various forms of hybridization, mutation, and transgenics, have also been invented and applied to crop breeding over the past centuries. The progress made in these breeding technologies, especially the modern biotechnology-based breeding technologies, has had a great impact on crop breeding as well as on our lives. Here, we first review the developmental process and applications of these technologies in horticultural crop breeding. Then, we mainly describe the principles of the latest genome-editing technologies and discuss their potential applications in the genetic improvement of horticultural crops. The advantages and challenges of genome-editing technologies in horticultural crop breeding are also discussed. PMID:26504570

Targeted treatment of mutated EGFR-expressing non-small-cell lung cancer: focus on erlotinib with companion diagnostics

PubMed Central

Karachaliou, Niki; Rosell, Rafael

2014-01-01

Deeper understanding of the pathobiology of non-small-cell lung cancer (NSCLC) has led to the development of small molecules that target genetic mutations known to play critical roles in the progression to metastatic disease. The discovery of epidermal growth factor receptor (EGFR) mutations in 15%–20% of lung adenocarcinomas and the associated response to EGFR tyrosine kinase inhibitors have provided a successful avenue of attack in late-stage adenocarcinomas. Use of the EGFR tyrosine kinase inhibitors gefitinib, erlotinib, and afatinib is limited to patients who have adenocarcinomas with known activating EGFR mutations. However, the EGFR mutation testing landscape is varied and includes many screening and targeted methods, each with its own benefits and limitations. These tests can simplify the drug discovery process, make clinical trials more efficient and informative, and individualize cancer therapy. In practice, the choice of method should be determined by the nature of the sample to be tested, the testing laboratory’s expertise and access to equipment, and whether the detection of only known activating EGFR mutations, or of all possible mutations, is required. Development of companion diagnostic tests for this identification is advancing; nevertheless, the use of such tests merits greater attention. PMID:28210145

Screening Mutations of MYBPC3 in 114 Unrelated Patients with Hypertrophic Cardiomyopathy by Targeted Capture and Next-generation Sequencing.

PubMed

Liu, Xuxia; Jiang, Tengyong; Piao, Chunmei; Li, Xiaoyan; Guo, Jun; Zheng, Shuai; Zhang, Xiaoping; Cai, Tao; Du, Jie

2015-06-19

Hypertrophic cardiomyopathy (HCM) is a major cause of sudden cardiac death. Mutations in the MYBPC3 gene represent the cause of HCM in ~35% of patients with HCM. However, genetic testing in clinic setting has been limited due to the cost and relatively time-consuming by Sanger sequencing. Here, we developed a HCM Molecular Diagnostic Kit enabling ultra-low-cost targeted gene resequencing in a large cohort and investigated the mutation spectrum of MYBPC3. In a cohort of 114 patients with HCM, a total of 20 different mutations (8 novel and 12 known mutations) of MYBPC3 were identified from 25 patients (21.9%). We demonstrated that the power of targeted resequencing in a cohort of HCM patients, and found that MYBPC3 is a common HCM-causing gene in Chinese patients. Phenotype-genotype analyses showed that the patients with double mutations (n = 2) or premature termination codon mutations (n = 12) showed more severe manifestations, compared with patients with missense mutations (n = 11). Particularly, we identified a recurrent truncation mutation (p.Y842X) in four unrelated cases (4/25, 16%), who showed severe phenotypes, and suggest that the p.Y842X is a frequent mutation in Chinese HCM patients with severe phenotypes.

Development of a Targeted Next-Generation Sequencing Assay to Detect Diagnostically Relevant Mutations of JAK2, CALR, and MPL in Myeloproliferative Neoplasms.

PubMed

Frawley, Thomas; O'Brien, Cathal P; Conneally, Eibhlin; Vandenberghe, Elisabeth; Percy, Melanie; Langabeer, Stephen E; Haslam, Karl

2018-02-01

The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), consisting of polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are a heterogeneous group of neoplasms that harbor driver mutations in the JAK2, CALR, and MPL genes. The detection of mutations in these genes has been incorporated into the recent World Health Organization (WHO) diagnostic criteria for MPN. Given a pressing clinical need to screen for mutations in these genes in a routine diagnostic setting, a targeted next-generation sequencing (NGS) assay for the detection of MPN-associated mutations located in JAK2 exon 14, JAK2 exon 12, CALR exon 9, and MPL exon 10 was developed to provide a single platform alternative to reflexive, stepwise diagnostic algorithms. Polymerase chain reaction (PCR) primers were designed to target mutation hotspots in JAK2 exon 14, JAK2 exon 12, MPL exon 10, and CALR exon 9. Multiplexed PCR conditions were optimized by using qualitative PCR followed by NGS. Diagnostic genomic DNA from 35 MPN patients, known to harbor driver mutations in one of the target genes, was used to validate the assay. One hundred percent concordance was observed between the previously-identified mutations and those detected by NGS, with no false positives, nor any known mutations missed (specificity = 100%, CI = 0.96, sensitivity = 100%, CI = 0.89). Improved resolution of mutation sequences was also revealed by NGS analysis. Detection of diagnostically relevant driver mutations of MPN is enhanced by employing a targeted multiplex NGS approach. This assay presents a robust solution to classical MPN mutation screening, providing an alternative to time-consuming sequential analyses.

Advances in Maize Genomics and Their Value for Enhancing Genetic Gains from Breeding

PubMed Central

Xu, Yunbi; Skinner, Debra J.; Wu, Huixia; Palacios-Rojas, Natalia; Araus, Jose Luis; Yan, Jianbing; Gao, Shibin; Warburton, Marilyn L.; Crouch, Jonathan H.

2009-01-01

Maize is an important crop for food, feed, forage, and fuel across tropical and temperate areas of the world. Diversity studies at genetic, molecular, and functional levels have revealed that, tropical maize germplasm, landraces, and wild relatives harbor a significantly wider range of genetic variation. Among all types of markers, SNP markers are increasingly the marker-of-choice for all genomics applications in maize breeding. Genetic mapping has been developed through conventional linkage mapping and more recently through linkage disequilibrium-based association analyses. Maize genome sequencing, initially focused on gene-rich regions, now aims for the availability of complete genome sequence. Conventional insertion mutation-based cloning has been complemented recently by EST- and map-based cloning. Transgenics and nutritional genomics are rapidly advancing fields targeting important agronomic traits including pest resistance and grain quality. Substantial advances have been made in methodologies for genomics-assisted breeding, enhancing progress in yield as well as abiotic and biotic stress resistances. Various genomic databases and informatics tools have been developed, among which MaizeGDB is the most developed and widely used by the maize research community. In the future, more emphasis should be given to the development of tools and strategic germplasm resources for more effective molecular breeding of tropical maize products. PMID:19688107

Global impact of accelerated plant breeding: Evidence from a meta-analysis on rice breeding.

PubMed

Lenaerts, Bert; de Mey, Yann; Demont, Matty

2018-01-01

Rice breeders in Asia and elsewhere in the world have long overlooked trying to shorten the time it takes to develop new varieties. Plant breeders have proposed a technique called Rapid Generation Advance (RGA) as a way to accelerate the results of public rice breeding programs. However, little is known about RGA's potential impact. Here, we present the first results of a global impact study of RGA. More specifically, we calculated the multiplicator effects of RGA on the research benefits generated by conventional rice breeding programs and applied them to a meta-analysis of selected impact studies in the literature. These insights are a first crucial step in developing a targeted approach for disseminating RGA technology among rice breeders to accelerate the impact of their public rice breeding programs around the world. We show that the additional benefits due to time savings are considerable and offer some insights into the economics of breeding. Our results confirm that the adoption of accelerated breeding would lead to substantial advantages to rice breeding programs and the earlier variety release leads to significant economic benefits to society. This can be important to policy makers when reshaping their public breeding methods and optimising their return on research investments in breeding.

Identification of a novel LMF1 nonsense mutation responsible for severe hypertriglyceridemia by targeted next-generation sequencing.

PubMed

Cefalù, Angelo B; Spina, Rossella; Noto, Davide; Ingrassia, Valeria; Valenti, Vincenza; Giammanco, Antonina; Fayer, Francesca; Misiano, Gabriella; Cocorullo, Gianfranco; Scrimali, Chiara; Palesano, Ornella; Altieri, Grazia I; Ganci, Antonina; Barbagallo, Carlo M; Averna, Maurizio R

Severe hypertriglyceridemia (HTG) may result from mutations in genes affecting the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. The aim of this study was to develop a targeted next-generation sequencing panel for the molecular diagnosis of disorders characterized by severe HTG. We developed a targeted customized panel for next-generation sequencing Ion Torrent Personal Genome Machine to capture the coding exons and intron/exon boundaries of 18 genes affecting the main pathways of TG synthesis and metabolism. We sequenced 11 samples of patients with severe HTG (TG>885 mg/dL-10 mmol/L): 4 positive controls in whom pathogenic mutations had previously been identified by Sanger sequencing and 7 patients in whom the molecular defect was still unknown. The customized panel was accurate, and it allowed to confirm genetic variants previously identified in all positive controls with primary severe HTG. Only 1 patient of 7 with HTG was found to be carrier of a homozygous pathogenic mutation of the third novel mutation of LMF1 gene (c.1380C>G-p.Y460X). The clinical and molecular familial cascade screening allowed the identification of 2 additional affected siblings and 7 heterozygous carriers of the mutation. We showed that our targeted resequencing approach for genetic diagnosis of severe HTG appears to be accurate, less time consuming, and more economical compared with traditional Sanger resequencing. The identification of pathogenic mutations in candidate genes remains challenging and clinical resequencing should mainly intended for patients with strong clinical criteria for monogenic severe HTG. Copyright © 2017 National Lipid Association. Published by Elsevier Inc. All rights reserved.

Temporal Interplay between Efflux Pumps and Target Mutations in Development of Antibiotic Resistance in Escherichia coli

PubMed Central

Singh, Renu; Swick, Michelle C.; Ledesma, Kimberly R.; Yang, Zhen; Hu, Ming; Zechiedrich, Lynn

2012-01-01

The emergence of resistance presents a debilitating change in the management of infectious diseases. Currently, the temporal relationship and interplay between various mechanisms of drug resistance are not well understood. A thorough understanding of the resistance development process is needed to facilitate rational design of countermeasure strategies. Using an in vitro hollow-fiber infection model that simulates human drug treatment, we examined the appearance of efflux pump (acrAB) overexpression and target topoisomerase gene (gyrA and parC) mutations over time in the emergence of quinolone resistance in Escherichia coli. Drug-resistant isolates recovered early (24 h) had 2- to 8-fold elevation in the MIC due to acrAB overexpression, but no point mutations were noted. In contrast, high-level (≥64× MIC) resistant isolates with target site mutations (gyrA S83L with or without parC E84K) were selected more readily after 120 h, and regression of acrAB overexpression was observed at 240 h. Using a similar dosing selection pressure, the emergence of levofloxacin resistance was delayed in a strain with acrAB deleted compared to the isogenic parent. The role of efflux pumps in bacterial resistance development may have been underappreciated. Our data revealed the interplay between two mechanisms of quinolone resistance and provided a new mechanistic framework in the development of high-level resistance. Early low-level levofloxacin resistance conferred by acrAB overexpression preceded and facilitated high-level resistance development mediated by target site mutation(s). If this interpretation is correct, then these findings represent a paradigm shift in the way quinolone resistance is thought to develop. PMID:22232279

Genome-wide scan for selection signatures in six cattle breeds in South Africa.

PubMed

Makina, Sithembile O; Muchadeyi, Farai C; van Marle-Köster, Este; Taylor, Jerry F; Makgahlela, Mahlako L; Maiwashe, Azwihangwisi

2015-11-26

The detection of selection signatures in breeds of livestock species can contribute to the identification of regions of the genome that are, or have been, functionally important and, as a consequence, have been targeted by selection. This study used two approaches to detect signatures of selection within and between six cattle breeds in South Africa, including Afrikaner (n = 44), Nguni (n = 54), Drakensberger (n = 47), Bonsmara (n = 44), Angus (n = 31) and Holstein (n = 29). The first approach was based on the detection of genomic regions in which haplotypes have been driven towards complete fixation within breeds. The second approach identified regions of the genome that had very different allele frequencies between populations (F ST). Forty-seven candidate genomic regions were identified as harbouring putative signatures of selection using both methods. Twelve of these candidate selected regions were shared among the breeds and ten were validated by previous studies. Thirty-three of these regions were successfully annotated and candidate genes were identified. Among these genes the keratin genes (KRT222, KRT24, KRT25, KRT26, and KRT27) and one heat shock protein gene (HSPB9) on chromosome 19 between 42,896,570 and 42,897,840 bp were detected for the Nguni breed. These genes were previously associated with adaptation to tropical environments in Zebu cattle. In addition, a number of candidate genes associated with the nervous system (WNT5B, FMOD, PRELP, and ATP2B), immune response (CYM, CDC6, and CDK10), production (MTPN, IGFBP4, TGFB1, and AJAP1) and reproductive performance (ADIPOR2, OVOS2, and RBBP8) were also detected as being under selection. The results presented here provide a foundation for detecting mutations that underlie genetic variation of traits that have economic importance for cattle breeds in South Africa.

The polyploidy and its key role in plant breeding.

PubMed

Sattler, Mariana Cansian; Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo

2016-02-01

This article provides an up-to-date review concerning from basic issues of polyploidy to aspects regarding the relevance and role of both natural and artificial polyploids in plant breeding programs. Polyploidy is a major force in the evolution of both wild and cultivated plants. Polyploid organisms often exhibit increased vigor and, in some cases, outperform their diploid relatives in several aspects. This remarkable superiority of polyploids has been the target of many plant breeders in the last century, who have induced polyploidy and/or used natural polyploids in many ways to obtain increasingly improved plant cultivars. Some of the most important consequences of polyploidy for plant breeding are the increment in plant organs ("gigas" effect), buffering of deleterious mutations, increased heterozygosity, and heterosis (hybrid vigor). Regarding such features as tools, cultivars have been generated with higher yield levels, improving the product quality and increasing the tolerance to both biotic and abiotic stresses. In some cases, when the crossing between two species is not possible because of differences in ploidy level, polyploids can be used as a bridge for gene transferring between them. In addition, polyploidy often results in reduced fertility due to meiotic errors, allowing the production of seedless varieties. On the other hand, the genome doubling in a newly formed sterile hybrid allows the restoration of its fertility. Based on these aspects, the present review initially concerns the origin, frequency and classification of the polyploids, progressing to show the revolution promoted by the discovery of natural polyploids and polyploidization induction in the breeding program status of distinct crops.

The relative contribution of target-site mutations in complex acaricide resistant phenotypes as assessed by marker assisted backcrossing in Tetranychus urticae.

PubMed

Riga, Maria; Bajda, Sabina; Themistokleous, Christos; Papadaki, Stavrini; Palzewicz, Maria; Dermauw, Wannes; Vontas, John; Leeuwen, Thomas Van

2017-08-23

The mechanisms underlying insecticide and acaricide resistance in insects and mites are often complex, including additive effects of target-site insensitivity, increased metabolism and transport. The extent to which target-site resistance mutations contribute to the resistance phenotype is, however, not well studied. Here, we used marker-assisted backcrossing to create 30 congenic lines carrying nine mutations (alone, or in combination in a few cases) associated with resistance to avermectins, pyrethroids, mite growth inhibitors and mitochondrial complex III inhibitors (QoI) in a polyphagous arthropod pest, the spider mite Tetranychus urticae. Toxicity tests revealed that mutations in the voltage-gated sodium channel, chitin synthase 1 and cytochrome b confer high levels of resistance and, when fixed in a population, these mutations alone can result in field failure of acaricide treatment. In contrast, although we confirmed the implication of mutations in glutamate-gated chloride channels in abamectin and milbemectin insensitivity, these mutations do not lead to the high resistance levels that are often reported in abamectin resistant strains of T. urticae. Overall, this study functionally validates reported target-site resistance mutations in T. urticae, by uncoupling them from additional mechanisms, allowing to finally investigate the strength of the conferred phenotype in vivo.

SF3B1 mutations constitute a novel therapeutic target in breast cancer

PubMed Central

Maguire, Sarah L; Leonidou, Andri; Wai, Patty; Marchiò, Caterina; Ng, Charlotte KY; Sapino, Anna; Salomon, Anne-Vincent; Reis-Filho, Jorge S; Weigelt, Britta; Natrajan, Rachael C

2015-01-01

Mutations in genes encoding proteins involved in RNA splicing have been found to occur at relatively high frequencies in several tumour types including myelodysplastic syndromes, chronic lymphocytic leukaemia, uveal melanoma, and pancreatic cancer, and at lower frequencies in breast cancer. To investigate whether dysfunction in RNA splicing is implicated in the pathogenesis of breast cancer, we performed a re-analysis of published exome and whole genome sequencing data. This analysis revealed that mutations in spliceosomal component genes occurred in 5.6% of unselected breast cancers, including hotspot mutations in the SF3B1 gene, which were found in 1.8% of unselected breast cancers. SF3B1 mutations were significantly associated with ER-positive disease, AKT1 mutations, and distinct copy number alterations. Additional profiling of hotspot mutations in a panel of special histological subtypes of breast cancer showed that 16% and 6% of papillary and mucinous carcinomas of the breast harboured the SF3B1 K700E mutation. RNA sequencing identified differentially spliced events expressed in tumours with SF3B1 mutations including the protein coding genes TMEM14C, RPL31, DYNL11, UQCC, and ABCC5, and the long non-coding RNA CRNDE. Moreover, SF3B1 mutant cell lines were found to be sensitive to the SF3b complex inhibitor spliceostatin A and treatment resulted in perturbation of the splicing signature. Albeit rare, SF3B1 mutations result in alternative splicing events, and may constitute drivers and a novel therapeutic target in a subset of breast cancers. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. PMID:25424858

The callipyge mutation and other genes that affect muscle hypertrophy in sheep

PubMed Central

2005-01-01

Genetic strategies to improve the profitability of sheep operations have generally focused on traits for reproduction. However, natural mutations exist in sheep that affect muscle growth and development, and the exploitation of these mutations in breeding strategies has the potential to significantly improve lamb-meat quality. The best-documented mutation for muscle development in sheep is callipyge (CLPG), which causes a postnatal muscle hypertrophy that is localized to the pelvic limbs and loin. Enhanced skeletal muscle growth is also observed in animals with the Carwell (or rib-eye muscling) mutation, and a double-muscling phenotype has been documented for animals of the Texel sheep breed. However, the actual mutations responsible for these muscular hypertrophy phenotypes in sheep have yet to be identified, and further characterization of the genetic basis for these phenotypes will provide insight into the biological control of muscle growth and body composition. PMID:15601596

Spectrum of mutations in leiomyosarcomas identified by clinical targeted next-generation sequencing.

PubMed

Lee, Paul J; Yoo, Naomi S; Hagemann, Ian S; Pfeifer, John D; Cottrell, Catherine E; Abel, Haley J; Duncavage, Eric J

2017-02-01

Recurrent genomic mutations in uterine and non-uterine leiomyosarcomas have not been well established. Using a next generation sequencing (NGS) panel of common cancer-associated genes, 25 leiomyosarcomas arising from multiple sites were examined to explore genetic alterations, including single nucleotide variants (SNV), small insertions/deletions (indels), and copy number alterations (CNA). Sequencing showed 86 non-synonymous, coding region somatic variants within 151 gene targets in 21 cases, with a mean of 4.1 variants per case; 4 cases had no putative mutations in the panel of genes assayed. The most frequently altered genes were TP53 (36%), ATM and ATRX (16%), and EGFR and RB1 (12%). CNA were identified in 85% of cases, with the most frequent copy number losses observed in chromosomes 10 and 13 including PTEN and RB1; the most frequent gains were seen in chromosomes 7 and 17. Our data show that deletions in canonical cancer-related genes are common in leiomyosarcomas. Further, the spectrum of gene mutations observed shows that defects in DNA repair and chromosomal maintenance are central to the biology of leiomyosarcomas, and that activating mutations observed in other common cancer types are rare in leiomyosarcomas. Copyright © 2017 Elsevier Inc. All rights reserved.

Polymorphism of BMP4 gene in Indian goat breeds differing in prolificacy.

PubMed

Sharma, Rekha; Ahlawat, Sonika; Maitra, A; Roy, Manoranjan; Mandakmale, S; Tantia, M S

2013-12-10

Bone morphogenetic proteins (BMPs) are members of the TGF-β (transforming growth factor-beta) superfamily, of which BMP4 is the most important due to its crucial role in follicular growth and differentiation, cumulus expansion and ovulation. Reproduction is a crucial trait in goat breeding and based on the important role of BMP4 gene in reproduction it was considered as a possible candidate gene for the prolificacy of goats. The objective of the present study was to detect polymorphism in intronic, exonic and 3' un-translated regions of BMP4 gene in Indian goats. Nine different goat breeds (Barbari, Beetal, Black Bengal, Malabari, Jakhrana (Twinning>40%), Osmanabadi, Sangamneri (Twinning 20-30%), Sirohi and Ganjam (Twinning<10%)) differing in prolificacy and geographic distribution were employed for polymorphism scanning. Cattle sequence (AC_000167.1) was used to design primers for the amplification of a targeted region followed by direct DNA sequencing to identify the genetic variations. Single nucleotide polymorphisms (SNPs) were not detected in exon 3, the intronic region and the 3' flanking region. A SNP (G1534A) was identified in exon 2. It was a non-synonymous mutation resulting in an arginine to lysine change in a corresponding protein sequence. G to A transition at the 1534 locus revealed two genotypes GG and GA in the nine investigated goat breeds. The GG genotype was predominant with a genotype frequency of 0.98. The GA genotype was present in the Black Bengal as well as Jakhrana breed with a genotype frequency of 0.02. A microsatellite was identified in the 3' flanking region, only 20 nucleotides downstream from the termination site of the coding region, as a short sequence with more than nineteen continuous and repeated CA dinucleotides. Since the gene is highly evolutionarily conserved, identification of a non-synonymous SNP (G1534A) in the coding region gains further importance. To our knowledge, this is the first report of a mutation in the coding

CRISPR-mediated targeting of HER2 inhibits cell proliferation through a dominant negative mutation.

PubMed

Wang, Huajing; Sun, William

2017-01-28

With the discovery of the CRISPR/Cas9 technology, genome editing could be performed in a rapid, precise and effective manner. Its potential applications in functional interrogation of cancer-causing genes and cancer therapy have been extensively explored. In this study, we demonstrated the use of the CRISPR/Cas9 system to directly target the oncogene HER2. Directing Cas9 to exons of the HER2 gene inhibited cell growth in breast cancer cell lines that harbor amplification of the HER2 locus. The inhibitory effect was potentiated with the addition of PARP inhibitors. Unexpectedly, CRISPR-induced mutations did not significantly affect the level of HER2 protein expression. Instead, CRISPR targeting appeared to exert its effect through a dominant negative mutation. This HER2 mutant interfered with the MAPK/ERK axis of HER2 downstream signaling. Our work provides a novel mechanism underlying the anti-cancer effects of HER2-targeting by CRISPR/Cas9, which is distinct from the clinical drug Herceptin. In addition, it opens up the possibility that incomplete CRISPR targeting of certain oncogenes could still have therapeutic value by generation of dominant negative mutants. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Dengue vector management using insecticide treated materials and targeted interventions on productive breeding-sites in Guatemala.

PubMed

Rizzo, Nidia; Gramajo, Rodrigo; Escobar, Maria Cabrera; Arana, Byron; Kroeger, Axel; Manrique-Saide, Pablo; Petzold, Max

2012-10-30

In view of the epidemiological expansion of dengue worldwide and the availability of new tools and strategies particularly for controlling the primary dengue vector Aedes aegypti, an intervention study was set up to test the efficacy, cost and feasibility of a combined approach of insecticide treated materials (ITMs) alone and in combination with appropriate targeted interventions of the most productive vector breeding-sites. The study was conducted as a cluster randomized community trial using "reduction of the vector population" as the main outcome variable. The trial had two arms: 10 intervention clusters (neighborhoods) and 10 control clusters in the town of Poptun Guatemala. Activities included entomological assessments (characteristics of breeding-sites, pupal productivity, Stegomyia indices) at baseline, 6 weeks after the first intervention (coverage of window and exterior doorways made of PermaNet 2.0 netting, factory treated with deltamethrin at 55 mg/m2, and of 200 L drums with similar treated material) and 6 weeks after the second intervention (combination of treated materials and other suitable interventions targeting productive breeding-sites i.e larviciding with Temephos, elimination etc.). The second intervention took place 17 months after the first intervention. The insecticide residual activity and the insecticidal content were also studied at different intervals. Additionally, information about demographic characteristics, cost of the intervention, coverage of houses protected and satisfaction in the population with the interventions was collected. At baseline (during the dry season) a variety of productive container types for Aedes pupae were identified: various container types holding >20 L, 200 L drums, washbasins and buckets (producing 83.7% of all pupae). After covering 100% of windows and exterior doorways and a small number of drums (where the commercial cover could be fixed) in 970 study households, tropical rains occurred in the area and

Dengue vector management using insecticide treated materials and targeted interventions on productive breeding-sites in Guatemala

PubMed Central

2012-01-01

Background In view of the epidemiological expansion of dengue worldwide and the availability of new tools and strategies particularly for controlling the primary dengue vector Aedes aegypti, an intervention study was set up to test the efficacy, cost and feasibility of a combined approach of insecticide treated materials (ITMs) alone and in combination with appropriate targeted interventions of the most productive vector breeding-sites. Methods The study was conducted as a cluster randomized community trial using “reduction of the vector population” as the main outcome variable. The trial had two arms: 10 intervention clusters (neighborhoods) and 10 control clusters in the town of Poptun Guatemala. Activities included entomological assessments (characteristics of breeding-sites, pupal productivity, Stegomyia indices) at baseline, 6 weeks after the first intervention (coverage of window and exterior doorways made of PermaNet 2.0 netting, factory treated with deltamethrin at 55 mg/m2, and of 200 L drums with similar treated material) and 6 weeks after the second intervention (combination of treated materials and other suitable interventions targeting productive breeding-sites i.e larviciding with Temephos, elimination etc.). The second intervention took place 17 months after the first intervention. The insecticide residual activity and the insecticidal content were also studied at different intervals. Additionally, information about demographic characteristics, cost of the intervention, coverage of houses protected and satisfaction in the population with the interventions was collected. Results At baseline (during the dry season) a variety of productive container types for Aedes pupae were identified: various container types holding >20 L, 200 L drums, washbasins and buckets (producing 83.7% of all pupae). After covering 100% of windows and exterior doorways and a small number of drums (where the commercial cover could be fixed) in 970 study households, tropical

Sequence Characterization of the MC1R Gene in Yak (Poephagus grunniens) Breeds with Different Coat Colors

PubMed Central

Chen, Shi-Yi; Huang, Yi; Zhu, Qing; Fontanesi, Luca; Yao, Yong-Gang; Liu, Yi-Ping

2009-01-01

Melanocortin 1 receptor (MC1R) gene plays a key role in determining coat color in several species, including the cattle. However, up to now there is no report regarding the MC1R gene and the potential association of its mutations with coat colors in yak (Poephagus grunniens). In this study, we sequenced the encoding region of the MC1R gene in three yak breeds with completely white (Tianzhu breed) or black coat color (Jiulong and Maiwa breeds). The predicted coding region of the yak MC1R gene resulted of 954 bp, the same to that of the wild-type cattle sequence, with >99% identity. None of the mutation events reported in cattle was found. Comparing the yak obtained sequences, five nucleotide substitutions were detected, which defined three haplotypes (EY1, EY2, and EY3). Of the five mutations, two, characterizing the EY1 haplotype, were nonsynonymous substitutions (c.340C>A and c.871G>A) causing amino acid changes located in the first extracellular loop (p.Q114K) and in the seventh transmembrane region (p.A291T). In silico prediction might indicate a functional effect of the latter substitution. However, all three haplotypes were present in the three yak breeds with relatively consistent frequency distribution, despite of their distinguished coat colors, which suggested that there was no across-breed association between haplotypes or genotypes and black/white phenotypes, at least in the investigated breeds. Other genes may be involved in affecting coat color in the analyzed yaks. PMID:19584942

RANK ligand as a potential target for breast cancer prevention in BRCA1-mutation carriers.

PubMed

Nolan, Emma; Vaillant, François; Branstetter, Daniel; Pal, Bhupinder; Giner, Göknur; Whitehead, Lachlan; Lok, Sheau W; Mann, Gregory B; Rohrbach, Kathy; Huang, Li-Ya; Soriano, Rosalia; Smyth, Gordon K; Dougall, William C; Visvader, Jane E; Lindeman, Geoffrey J

2016-08-01

Individuals who have mutations in the breast-cancer-susceptibility gene BRCA1 (hereafter referred to as BRCA1-mutation carriers) frequently undergo prophylactic mastectomy to minimize their risk of breast cancer. The identification of an effective prevention therapy therefore remains a 'holy grail' for the field. Precancerous BRCA1(mut/+) tissue harbors an aberrant population of luminal progenitor cells, and deregulated progesterone signaling has been implicated in BRCA1-associated oncogenesis. Coupled with the findings that tumor necrosis factor superfamily member 11 (TNFSF11; also known as RANKL) is a key paracrine effector of progesterone signaling and that RANKL and its receptor TNFRSF11A (also known as RANK) contribute to mammary tumorigenesis, we investigated a role for this pathway in the pre-neoplastic phase of BRCA1-mutation carriers. We identified two subsets of luminal progenitors (RANK(+) and RANK(-)) in histologically normal tissue of BRCA1-mutation carriers and showed that RANK(+) cells are highly proliferative, have grossly aberrant DNA repair and bear a molecular signature similar to that of basal-like breast cancer. These data suggest that RANK(+) and not RANK(-) progenitors are a key target population in these women. Inhibition of RANKL signaling by treatment with denosumab in three-dimensional breast organoids derived from pre-neoplastic BRCA1(mut/+) tissue attenuated progesterone-induced proliferation. Notably, proliferation was markedly reduced in breast biopsies from BRCA1-mutation carriers who were treated with denosumab. Furthermore, inhibition of RANKL in a Brca1-deficient mouse model substantially curtailed mammary tumorigenesis. Taken together, these findings identify a targetable pathway in a putative cell-of-origin population in BRCA1-mutation carriers and implicate RANKL blockade as a promising strategy in the prevention of breast cancer.

A computational approach to animal breeding.

PubMed

Berger-Wolf, Tanya Y; Moore, Cristopher; Saia, Jared

2007-02-07

We propose a computational model of mating strategies for controlled animal breeding programs. A mating strategy in a controlled breeding program is a heuristic with some optimization criteria as a goal. Thus, it is appropriate to use the computational tools available for analysis of optimization heuristics. In this paper, we propose the first discrete model of the controlled animal breeding problem and analyse heuristics for two possible objectives: (1) breeding for maximum diversity and (2) breeding a target individual. These two goals are representative of conservation biology and agricultural livestock management, respectively. We evaluate several mating strategies and provide upper and lower bounds for the expected number of matings. While the population parameters may vary and can change the actual number of matings for a particular strategy, the order of magnitude of the number of expected matings and the relative competitiveness of the mating heuristics remains the same. Thus, our simple discrete model of the animal breeding problem provides a novel viable and robust approach to designing and comparing breeding strategies in captive populations.

BRD4-targeted therapy induces Myc-independent cytotoxicity in Gnaq/11-mutatant uveal melanoma cells.

PubMed

Ambrosini, Grazia; Sawle, Ashley D; Musi, Elgilda; Schwartz, Gary K

2015-10-20

Uveal melanoma (UM) is an aggressive intraocular malignancy with limited therapeutic options. Both primary and metastatic UM are characterized by oncogenic mutations in the G-protein alpha subunit q and 11. Furthermore, nearly 40% of UM has amplification of the chromosomal arm 8q and monosomy of chromosome 3, with consequent anomalies of MYC copy number. Chromatin regulators have become attractive targets for cancer therapy. In particular, the bromodomain and extra-terminal (BET) inhibitor JQ1 has shown selective inhibition of c-Myc expression with antiproliferative activity in hematopoietic and solid tumors. Here we provide evidence that JQ1 had cytotoxic activity in UM cell lines carrying Gnaq/11 mutations, while in cells without the mutations had little effects. Using microarray analysis, we identified a large subset of genes modulated by JQ1 involved in the regulation of cell cycle, apoptosis and DNA repair. Further analysis of selected genes determined that the concomitant silencing of Bcl-xL and Rad51 represented the minimal requirement to mimic the apoptotic effects of JQ1 in the mutant cells, independently of c-Myc. In addition, administration of JQ1 to mouse xenograft models of Gnaq-mutant UM resulted in significant inhibition of tumor growth.Collectively, our results define BRD4 targeting as a novel therapeutic intervention against UM with Gnaq/Gna11 mutations.

Targeted next generation sequencing of well-differentiated/dedifferentiated liposarcoma reveals novel gene amplifications and mutations.

PubMed

Somaiah, Neeta; Beird, Hannah C; Barbo, Andrea; Song, Juhee; Mills Shaw, Kenna R; Wang, Wei-Lien; Eterovic, Karina; Chen, Ken; Lazar, Alexander; Conley, Anthony P; Ravi, Vinod; Hwu, Patrick; Futreal, Andrew; Simon, George; Meric-Bernstam, Funda; Hong, David

2018-04-13

Well-differentiated/dedifferentiated liposarcoma is a common soft tissue sarcoma with approximately 1500 new cases per year. Surgery is the mainstay of treatment but recurrences are frequent and systemic options are limited. 'Tumor genotyping' is becoming more common in clinical practice as it offers the hope of personalized targeted therapy. We wanted to evaluate the results and the clinical utility of available next-generation sequencing panels in WD/DD liposarcoma. Patients who had their tumor sequenced by either FoundationOne ( n = 13) or the institutional T200/T200.1 panels ( n = 7) were included in this study. Significant copy number alterations were identified, but mutations were infrequent. Out of the 27 mutations detected in 7 samples, 8 ( CTNNB1, MECOM, ZNF536, EGFR, EML4, CSMD3, PBRM1, PPP1R3A ) were identified as deleterious (on Condel, PolyPhen and SIFT) and a truncating mutation was found in NF2 . Of these, EGFR and NF2 are potential driver mutations and have not been reported previously in liposarcoma. MDM2 and CDK4 amplification was universally present in all the tested samples and multiple other recurrent genes with high amplification or high deletion were detected. Many of these targets are potentially actionable. Eight patients went on to receive an MDM2 inhibitor with a median time to progression of 23 months (95% CI: 10-83 months).

Lethal mutagenesis: targeting the mutator phenotype in cancer.

PubMed

Fox, Edward J; Loeb, Lawrence A

2010-10-01

The evolution of cancer and RNA viruses share many similarities. Both exploit high levels of genotypic diversity to enable extensive phenotypic plasticity and thereby facilitate rapid adaptation. In order to accumulate large numbers of mutations, we have proposed that cancers express a mutator phenotype. Similar to cancer cells, many viral populations, by replicating their genomes with low fidelity, carry a substantial mutational load. As high levels of mutation are potentially deleterious, the viral mutation frequency is thresholded at a level below which viral populations equilibrate in a traditional mutation-selection balance, and above which the population is no longer viable, i.e., the population undergoes an error catastrophe. Because their mutation frequencies are fine-tuned just below this error threshold, viral populations are susceptible to further increases in mutational load and, recently this phenomenon has been exploited therapeutically by a concept that has been termed lethal mutagenesis. Here we review the application of lethal mutagenesis to the treatment of HIV and discuss how lethal mutagenesis may represent a novel therapeutic approach for the treatment of solid cancers. Copyright © 2010 Elsevier Ltd. All rights reserved.

Targeted mutation analysis of endometrial clear cell carcinoma.

PubMed

Hoang, Lien N; McConechy, Melissa K; Meng, Bo; McIntyre, John B; Ewanowich, Carol; Gilks, Cyril Blake; Huntsman, David G; Köbel, Martin; Lee, Cheng-Han

2015-04-01

Endometrial clear cell carcinomas (CCC) constitute fewer than 5% of all carcinomas of the endometrium. Currently, little is known regarding the genetic basis of endometrial CCC. We performed genomic and immunohistochemical analyses on 14 rigorously reviewed pure endometrial CCC. The genomic analysis consisted of sequencing the coding regions of 26 genes implicated previously in endometrial carcinoma. Twelve of 14 tumours displayed a prototypical CCC immunophenotype [napsin A+, hepatocyte nuclear factor-1β (HNF1β(+) ) and oestrogen receptor(-) ] and all showed intact mismatch repair protein expression. We detected mutations in 11 of 14 tumours, and there was a predominance of mutations involving genes that are mutated more frequently in endometrial serous carcinomas than in endometrioid carcinomas. Two tumours displayed a prototypical serous carcinoma mutation profile (concurrent TP53 and PPP2R1A mutations, without PTEN, CTNNB1 or ARID1A mutation). No mutations in PTEN, CTNNB1 or POLE were identified. The overall mutation profile of this cohort of endometrial CCC appears to be more serous-like than endometrioid-like, with a minor subset in the TP53-mutated CCC showing serous carcinoma profile. These findings provide new insights into the molecular features of morphologically prototypical endometrial CCC, and underscore the need for further investigations into the oncogenesis of endometrial CCC. © 2014 John Wiley & Sons Ltd.

Targeted disruption of sp7 and myostatin with CRISPR-Cas9 results in severe bone defects and more muscular cells in common carp

PubMed Central

Zhong, Zhaomin; Niu, Pengfei; Wang, Mingyong; Huang, Guodong; Xu, Shuhao; Sun, Yi; Xu, Xiaona; Hou, Yi; Sun, Xiaowen; Yan, Yilin; Wang, Han

2016-01-01

The common carp (Cyprinus carpio) as one of the most important aquaculture fishes produces over 3 million metric tones annually, approximately 10% the annual production of the all farmed freshwater fish worldwide. However, the tetraploidy genome and long generation-time of the common carp have made its breeding and genetic studies extremely difficult. Here, TALEN and CRISPR-Cas9, two versatile genome-editing tools, are employed to target common carp bone-related genes sp7, runx2, bmp2a, spp1, opg, and muscle suppressor gene mstn. TALEN were shown to induce mutations in the target coding sites of sp7, runx2, spp1 and mstn. With CRISPR-Cas9, the two common carp sp7 genes, sp7a and sp7b, were mutated individually, all resulting in severe bone defects; while mstnba mutated fish have grown significantly more muscle cells. We also employed CRISPR-Cas9 to generate double mutant fish of sp7a;mstnba with high efficiencies in a single step. These results demonstrate that both TALEN and CRISPR-Cas9 are highly efficient tools for modifying the common carp genome, and open avenues for facilitating common carp genetic studies and breeding. PMID:26976234

Assessment of canine BEST1 variations identifies new mutations and establishes an independent bestrophinopathy model (cmr3)

PubMed Central

Wickström, Kaisa; Slavik, Julianna; Lindauer, Sarah J.; Ahonen, Saija; Schelling, Claude; Lohi, Hannes; Guziewicz, Karina E.; Aguirre, Gustavo D.

2010-01-01

Purpose Mutations in bestrophin 1 (BEST1) are associated with a group of retinal disorders known as bestrophinopathies in man and canine multifocal retinopathies (cmr) in the dog. To date, the dog is the only large animal model suitable for the complex characterization and in-depth studies of Best-related disorders. In the first report of cmr, the disease was described in a group of mastiff-related breeds (cmr1) and the Coton de Tulear (cmr2). Additional breeds, e.g., the Lapponian herder (LH) and others, subsequently were recognized with similar phenotypes, but linked loci are unknown. Analysis of the BEST1 gene aimed to identify mutations in these additional populations and extend our understanding of genotype–phenotype associations. Methods Animals were subjected to routine eye exams, phenotypically characterized, and samples were collected for molecular studies. Known BEST1 mutations were assessed, and the canine BEST1 coding exons were amplified and sequenced in selected individuals that exhibited a cmr compatible phenotype but that did not carry known mutations. Resulting sequence changes were genotyped in several different breeds and evaluated in the context of the phenotype. Results Seven novel coding variants were identified in exon 10 of cBEST1. Two linked mutations were associated with cmr exclusive to the LH breed (cmr3). Two individuals of Jämthund and Norfolk terrier breeds were heterozygous for two conservative changes, but these were unlikely to have disease-causing potential. Another three substitutions were found in the Bernese mountain dog that were predicted to have a deleterious effect on protein function. Previously reported mutations were excluded from segregation in these populations, but cmr1 was confirmed in another mastiff-related breed, the Italian cane corso. Conclusions A third independent canine model for human bestrophinopathies has been established in the LH breed. While exhibiting a phenotype comparable to cmr1 and cmr2, the

Analysis of SOX10 mutations identified in Waardenburg-Hirschsprung patients: Differential effects on target gene regulation.

PubMed

Chan, Kwok Keung; Wong, Corinne Kung Yen; Lui, Vincent Chi Hang; Tam, Paul Kwong Hang; Sham, Mai Har

2003-10-15

SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis

A Bioinformatic Pipeline for Monitoring of the Mutational Stability of Viral Drug Targets with Deep-Sequencing Technology.

PubMed

Kravatsky, Yuri; Chechetkin, Vladimir; Fedoseeva, Daria; Gorbacheva, Maria; Kravatskaya, Galina; Kretova, Olga; Tchurikov, Nickolai

2017-11-23

The efficient development of antiviral drugs, including efficient antiviral small interfering RNAs (siRNAs), requires continuous monitoring of the strict correspondence between a drug and the related highly variable viral DNA/RNA target(s). Deep sequencing is able to provide an assessment of both the general target conservation and the frequency of particular mutations in the different target sites. The aim of this study was to develop a reliable bioinformatic pipeline for the analysis of millions of short, deep sequencing reads corresponding to selected highly variable viral sequences that are drug target(s). The suggested bioinformatic pipeline combines the available programs and the ad hoc scripts based on an original algorithm of the search for the conserved targets in the deep sequencing data. We also present the statistical criteria for the threshold of reliable mutation detection and for the assessment of variations between corresponding data sets. These criteria are robust against the possible sequencing errors in the reads. As an example, the bioinformatic pipeline is applied to the study of the conservation of RNA interference (RNAi) targets in human immunodeficiency virus 1 (HIV-1) subtype A. The developed pipeline is freely available to download at the website http://virmut.eimb.ru/. Brief comments and comparisons between VirMut and other pipelines are also presented.

B cell Variable genes have evolved their codon usage to focus the targeted patterns of somatic mutation on the complementarity determining regions

PubMed Central

Saini, Jasmine; Hershberg, Uri

2015-01-01

The exceptional ability of B cells to diversify through somatic mutation and improve affinity of the repertoire towards the antigens is the cornerstone of adaptive immunity. Somatic mutation is not evenly distributed and exhibits certain micro-sequence specificities. We show here that the combination of somatic mutation targeting and the codon usage in human B cell receptor (BCR) Variable (V) genes create expected patterns of mutation and post mutation changes that are focused on their complementarity determining regions (CDR). T cell V genes are also skewed in targeting mutations but to a lesser extent and are lacking the codon usage bias observed in BCRs. This suggests that the observed skew in T cell receptors is due to their amino acid usage, which is similar to that of BCRs. The mutation targeting and the codon bias allow B cell CDRs to diversify by specifically accumulating nonconservative changes. We counted the distribution of mutations to CDR in 4 different human datasets. In all four cases we found that the number of actual mutations in the CDR correlated significantly with the V gene mutation biases to the CDR predicted by our models. Finally, it appears that the mutation bias in V genes indeed relates to their long-term survival in actual human repertoires. We observed that resting repertoires of B cells overexpressed V genes that were especially biased towards focused mutation and change in the CDR. This bias in V gene usage was somewhat relaxed at the height of the immune response to a vaccine, presumably because of the need for a wider diversity in a primary response. However, older patients did not retain this flexibility and were biased towards using only highly skewed V genes at all stages of their response. PMID:25660968

B cell variable genes have evolved their codon usage to focus the targeted patterns of somatic mutation on the complementarity determining regions.

PubMed

Saini, Jasmine; Hershberg, Uri

2015-05-01

The exceptional ability of B cells to diversify through somatic mutation and improve affinity of the repertoire toward the antigens is the cornerstone of adaptive immunity. Somatic mutation is not evenly distributed and exhibits certain micro-sequence specificities. We show here that the combination of somatic mutation targeting and the codon usage in human B cell receptor (BCR) Variable (V) genes create expected patterns of mutation and post mutation changes that are focused on their complementarity determining regions (CDR). T cell V genes are also skewed in targeting mutations but to a lesser extent and are lacking the codon usage bias observed in BCRs. This suggests that the observed skew in T cell receptors is due to their amino acid usage, which is similar to that of BCRs. The mutation targeting and the codon bias allow B cell CDRs to diversify by specifically accumulating nonconservative changes. We counted the distribution of mutations to CDR in 4 different human datasets. In all four cases we found that the number of actual mutations in the CDR correlated significantly with the V gene mutation biases to the CDR predicted by our models. Finally, it appears that the mutation bias in V genes indeed relates to their long-term survival in actual human repertoires. We observed that resting repertoires of B cells overexpressed V genes that were especially biased toward focused mutation and change in the CDR. This bias in V gene usage was somewhat relaxed at the height of the immune response to a vaccine, presumably because of the need for a wider diversity in a primary response. However, older patients did not retain this flexibility and were biased toward using only highly skewed V genes at all stages of their response. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mutation Accumulation, Soft Selection and the Middle-Class Neighborhood

PubMed Central

Moorad, Jacob A.; Hall, David W.

2009-01-01

The “middle-class neighborhood” is a breeding design intended to allow new mutations to accumulate by lessening the effects of purifying selection through the elimination of among-line fitness variation. We show that this design effectively applies soft selection to the experimental population, potentially causing biased estimates of mutational effects if social effects contribute to fitness. PMID:19448272

Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes

PubMed Central

Fish, Margaret B.; Cho, Ken W. Y.

2016-01-01

CRISPR/Cas9 genome editing is revolutionizing genetic loss-of-function analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1 heterozygotes. Phenotypic analysis occurs in the F2 generation following F1 intercrosses. Thus, mutant analyses will require multi-generational studies. Second, when targeting essential genes, efficient mutagenesis of founders is often lethal, preventing the acquisition of mature animals. Reducing mutagenesis levels may improve founder survival, but results in lower, more variable rates of germline transmission. Therefore, an efficient approach to study lethal mutations would be useful. To overcome these shortfalls, we introduce ‘leapfrogging’, a method combining efficient CRISPR mutagenesis with transplantation of mutated primordial germ cells into a wild-type host. Tested using Xenopus tropicalis, we show that founders containing transplants transmit mutant alleles with high efficiency. F1 offspring from intercrosses between F0 animals that carry embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an entire generation of breeding. We anticipate that leapfrogging will be transferable to other species. PMID:27385011

Targeted next generation sequencing of well-differentiated/dedifferentiated liposarcoma reveals novel gene amplifications and mutations

PubMed Central

Somaiah, Neeta; Beird, Hannah C; Barbo, Andrea; Song, Juhee; Mills Shaw, Kenna R.; Wang, Wei-Lien; Eterovic, Karina; Chen, Ken; Lazar, Alexander; Conley, Anthony P.; Ravi, Vinod; Hwu, Patrick; Futreal, Andrew; Simon, George; Meric-Bernstam, Funda; Hong, David

2018-01-01

Well-differentiated/dedifferentiated liposarcoma is a common soft tissue sarcoma with approximately 1500 new cases per year. Surgery is the mainstay of treatment but recurrences are frequent and systemic options are limited. ‘Tumor genotyping’ is becoming more common in clinical practice as it offers the hope of personalized targeted therapy. We wanted to evaluate the results and the clinical utility of available next-generation sequencing panels in WD/DD liposarcoma. Patients who had their tumor sequenced by either FoundationOne (n = 13) or the institutional T200/T200.1 panels (n = 7) were included in this study. Significant copy number alterations were identified, but mutations were infrequent. Out of the 27 mutations detected in 7 samples, 8 (CTNNB1, MECOM, ZNF536, EGFR, EML4, CSMD3, PBRM1, PPP1R3A) were identified as deleterious (on Condel, PolyPhen and SIFT) and a truncating mutation was found in NF2. Of these, EGFR and NF2 are potential driver mutations and have not been reported previously in liposarcoma. MDM2 and CDK4 amplification was universally present in all the tested samples and multiple other recurrent genes with high amplification or high deletion were detected. Many of these targets are potentially actionable. Eight patients went on to receive an MDM2 inhibitor with a median time to progression of 23 months (95% CI: 10-83 months). PMID:29731991

Two recessive mutations in FGF5 are associated with the long-hair phenotype in donkeys.

PubMed

Legrand, Romain; Tiret, Laurent; Abitbol, Marie

2014-09-25

Seven donkey breeds are recognized by the French studbook. Individuals from the Pyrenean, Provence, Berry Black, Normand, Cotentin and Bourbonnais breeds are characterized by a short coat, while those from the Poitou breed (Baudet du Poitou) are characterized by a long-hair phenotype. We hypothesized that loss-of-function mutations in the FGF5 (fibroblast growth factor 5) gene, which are associated with a long-hair phenotype in several mammalian species, may account for the special coat feature of Poitou donkeys. To the best of our knowledge, mutations in FGF5 have never been described in Equidae. We sequenced the FGF5 gene from 35 long-haired Poitou donkeys, as well as from a panel of 67 short-haired donkeys from the six other French breeds and 131 short-haired ponies and horses. We identified a recessive c.433_434delAT frameshift deletion in FGF5, present in Poitou and three other donkey breeds and a recessive nonsense c.245G > A substitution, present in Poitou and four other donkey breeds. The frameshift deletion was associated with the long-hair phenotype in Poitou donkeys when present in two copies (n = 31) or combined with the nonsense mutation (n = 4). The frameshift deletion led to a stop codon at position 159 whereas the nonsense mutation led to a stop codon at position 82 in the FGF5 protein. In silico, the two truncated FGF5 proteins were predicted to lack the critical β strands involved in the interaction between FGF5 and its receptor, a mandatory step to inhibit hair growth. Our results highlight the allelic heterogeneity of the long-hair phenotype in donkeys and enlarge the panel of recessive FGF5 loss-of-function alleles described in mammals. Thanks to the DNA test developed in this study, breeders of non-Poitou breeds will have the opportunity to identify long-hair carriers in their breeding stocks.

Targeted next-generation sequencing reveals that a compound heterozygous mutation in phosphodiesterase 6a gene leads to retinitis pigmentosa in a Chinese family.

PubMed

Zhang, Shanshan; Li, Jie; Li, Shujin; Yang, Yeming; Yang, Mu; Yang, Zhenglin; Zhu, Xianjun; Zhang, Lin

2018-04-25

Retinitis pigmentosa (RP) is a genetically heterogeneous disease with over 70 causative genes identified to date. However, approximately 40% of RP cases remain genetically unsolved, suggesting that many novel disease-causing mutations are yet to be identified. The purpose of this study is to identify the causative mutations of a Chinese RP family. Targeted next-generation sequencing (NGS) for a total of 163 genes which involved in inherited retinal disorders were used to screen the possible causative mutations. Sanger sequencing was used to verify the mutations. As results, we identified two heterozygous mutations: a splicing site mutation c.1407 + 1G>C and a nonsense mutation c. 1957C>T (p.R653X) in phosphodiesterase 6A (PDE6A) gene in the RP patient. These two mutations are inherited from his father and mother, respectively. Furthermore, these mutations are unique in our in-house database and are rare in human genome databases, implicating that these two mutations are pathological. By using targeted NGS method, we identified a compound heterozygous mutation in PDE6A gene that is associated with RP in a Chinese family.

Genomic scan of selective sweeps in thin and fat tail sheep breeds for identifying of candidate regions associated with fat deposition

PubMed Central

2012-01-01

Background Identification of genomic regions that have been targets of selection for phenotypic traits is one of the most important and challenging areas of research in animal genetics. However, currently there are relatively few genomic regions identified that have been subject to positive selection. In this study, a genome-wide scan using ~50,000 Single Nucleotide Polymorphisms (SNPs) was performed in an attempt to identify genomic regions associated with fat deposition in fat-tail breeds. This trait and its modification are very important in those countries grazing these breeds. Results Two independent experiments using either Iranian or Ovine HapMap genotyping data contrasted thin and fat tail breeds. Population differentiation using FST in Iranian thin and fat tail breeds revealed seven genomic regions. Almost all of these regions overlapped with QTLs that had previously been identified as affecting fat and carcass yield traits in beef and dairy cattle. Study of selection sweep signatures using FST in thin and fat tail breeds sampled from the Ovine HapMap project confirmed three of these regions located on Chromosomes 5, 7 and X. We found increased homozygosity in these regions in favour of fat tail breeds on chromosome 5 and X and in favour of thin tail breeds on chromosome 7. Conclusions In this study, we were able to identify three novel regions associated with fat deposition in thin and fat tail sheep breeds. Two of these were associated with an increase of homozygosity in the fat tail breeds which would be consistent with selection for mutations affecting fat tail size several thousand years after domestication. PMID:22364287

Identification of the mutation causing progressive retinal atrophy in Old Danish Pointing Dog.

PubMed

Karlskov-Mortensen, P; Proschowsky, H F; Gao, F; Fredholm, M

2018-06-01

Progressive retinal atrophy (PRA) is a common cause of blindness in many dog breeds. It is most often inherited as a simple Mendelian trait, but great genetic heterogeneity has been demonstrated both within and between breeds. In many breeds the genetic cause of the disease is not known, and until now, the Old Danish Pointing Dog (ODP) has been one of those breeds. ODP is one of the oldest dog breeds in Europe. Seventy years ago the breed almost vanished, but today a population still exists, primarily in Denmark but with some dogs in Germany and Sweden. PRA has been diagnosed in ODP since the late 1990s. It resembles late onset PRA in other dog breeds, and it is inherited as an autosomal recessive trait. In the present study, we performed whole-genome sequencing and identified a single base insertion (c.3149_3150insC) in exon 1 of C17H2orf71. This is the same mutation previously found to cause PRA in Gordon Setters and Irish Setters, and it was later found in Tibetan Terrier, Standard Poodle and the Polski Owczarek Nizinny. The presence of the mutation in such a diverse range of breeds indicates an origin preceding creation of modern dog breeds. Hence, we screened 262 dogs from 44 different breeds plus four crossbred dogs, and can subsequently add Miniature Poodle and another polish sheepdog, the Polski Owczarek Podhalanski, to the list of affected breeds. © 2018 Stichting International Foundation for Animal Genetics.

Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy

NASA Astrophysics Data System (ADS)

Kühnemund, Malte; Wei, Qingshan; Darai, Evangelia; Wang, Yingjie; Hernández-Neuta, Iván; Yang, Zhao; Tseng, Derek; Ahlford, Annika; Mathot, Lucy; Sjöblom, Tobias; Ozcan, Aydogan; Nilsson, Mats

2017-01-01

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies.

Targeted rescue of a polycystic kidney disease mutation by lysosomal inhibition.

PubMed

Hofherr, Alexis; Wagner, Claudius J; Watnick, Terry; Köttgen, Michael

2016-04-01

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of end-stage renal disease. The molecular pathogenesis of ADPKD is not completely known, and there is no approved therapy. To date, there is limited knowledge concerning the molecular consequences of specific disease-causing mutations. Here we show that the ADPKD missense variant TRPP2(D511V) greatly reduces TRPP2 protein stability, and that TRPP2(D511V) function can be rescued in vivo by small molecules targeting the TRPP2 degradation pathway. Expression of the TRPP2(D511V) protein was significantly reduced compared to wild-type TRPP2. Inhibition of lysosomal degradation of TRPP2(D511V) by the US Food and Drug Administration (FDA)-approved drug chloroquine strongly increased TRPP2 protein levels in vitro. The validation of these results in vivo requires appropriate animal models. However, there are currently no mouse models harboring human PKD2 missense mutations, and screening for chemical rescue of patient mutations in rodent models is time-consuming and expensive. Therefore, we developed a Drosophila melanogaster model expressing the ortholog of TRPP2(D511V) to test chemical rescue of mutant TRPP2 in vivo. Notably, chloroquine was sufficient to improve the phenotype of flies expressing mutant TRPP2. Thus, this proof-of-concept study highlights the potential of directed therapeutic approaches for ADPKD, and provides a rapid-throughput experimental model to screen PKD2 patient mutations and small molecules in vivo. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing.

PubMed

King, Robert; Bird, Nicholas; Ramirez-Gonzalez, Ricardo; Coghill, Jane A; Patil, Archana; Hassani-Pak, Keywan; Uauy, Cristobal; Phillips, Andrew L

2015-01-01

Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.

Targeted mutation screening panels expose systematic population bias in detection of cystic fibrosis risk.

PubMed

Lim, Regine M; Silver, Ari J; Silver, Maxwell J; Borroto, Carlos; Spurrier, Brett; Petrossian, Tanya C; Larson, Jessica L; Silver, Lee M

2016-02-01

Carrier screening for mutations contributing to cystic fibrosis (CF) is typically accomplished with panels composed of variants that are clinically validated primarily in patients of European descent. This approach has created a static genetic and phenotypic profile for CF. An opportunity now exists to reevaluate the disease profile of CFTR at a global population level. CFTR allele and genotype frequencies were obtained from a nonpatient cohort with more than 60,000 unrelated personal genomes collected by the Exome Aggregation Consortium. Likely disease-contributing mutations were identified with the use of public database annotations and computational tools. We identified 131 previously described and likely pathogenic variants and another 210 untested variants with a high probability of causing protein damage. None of the current genetic screening panels or existing CFTR mutation databases covered a majority of deleterious variants in any geographical population outside of Europe. Both clinical annotation and mutation coverage by commercially available targeted screening panels for CF are strongly biased toward detection of reproductive risk in persons of European descent. South and East Asian populations are severely underrepresented, in part because of a definition of disease that preferences the phenotype associated with European-typical CFTR alleles.

Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy

PubMed Central

Kühnemund, Malte; Wei, Qingshan; Darai, Evangelia; Wang, Yingjie; Hernández-Neuta, Iván; Yang, Zhao; Tseng, Derek; Ahlford, Annika; Mathot, Lucy; Sjöblom, Tobias; Ozcan, Aydogan; Nilsson, Mats

2017-01-01

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies. PMID:28094784

Concordant and Discordant EGFR Mutations in Patients with Multifocal Adenocarcinomas: Implications for EGFR Targeted Therapy

PubMed Central

Chuang, Jody C.; Shrager, Joseph B.; Wakelee, Heather A.; Neal, Joel W.

2016-01-01

Purpose Adenocarcinoma remains the most common subtype of lung cancer in the United States. Most patients present with tumors that are invasive and often metastatic, but some patients develop multiple precursor in-situ or minimally invasive adenocarcinoma tumors which can be synchronous and metachronous. These precursor lesions harbor the same spectrum of genetic mutations found in pure-invasive adenocarcinomas, such as EGFR, KRAS and p53 mutations. It is less clear, however, if separate lesions in patients who present with multifocal disease share common underlying genetic driver mutations. Methods Here we review the relevant literature on molecular driver alterations in adenocarcinoma precursor lesions. We then report four cases with multifocal EGFR mutant adenocarcinomas in whom we performed molecular testing on 2 separate lesions. Findings In two of these patients, the mutations are concordant, and in two cases the mutations are discordant. A review of the literature demonstrates increasing evidence that lesions with discordant mutations may confer a more favorable prognosis, since they are unlikely to represent metastases. Implications Our findings suggest that the emergence of the dominant EGFR driver alteration is often independent between lesions in patients with multifocal adenocarcinomas, and thus the same targeted therapy may not be effective for all lesions. However, genetic testing of multiple lesions can help distinguish separate primary tumors from metastatic disease. PMID:27368115

A CNGB1 Frameshift Mutation in Papillon and Phalène Dogs with Progressive Retinal Atrophy

PubMed Central

Ahonen, Saija J.; Arumilli, Meharji; Lohi, Hannes

2013-01-01

Progressive retinal degenerations are the most common causes of complete blindness both in human and in dogs. Canine progressive retinal atrophy (PRA) or degeneration resembles human retinitis pigmentosa (RP) and is characterized by a progressive loss of rod photoreceptor cells followed by a loss of cone function. The primary clinical signs are detected as vision impairment in a dim light. Although several genes have been associated with PRAs, there are still PRAs of unknown genetic cause in many breeds, including Papillons and Phalènes. We have performed a genome wide association and linkage studies in cohort of 6 affected Papillons and Phalènes and 14 healthy control dogs to map a novel PRA locus on canine chromosome 2, with a 1.9 Mb shared homozygous region in the affected dogs. Parallel exome sequencing of a trio identified an indel mutation, including a 1-bp deletion, followed by a 6-bp insertion in the CNGB1 gene. This mutation causes a frameshift and premature stop codon leading to probable nonsense mediated decay (NMD) of the CNGB1 mRNA. The mutation segregated with the disease and was confirmed in a larger cohort of 145 Papillons and Phalènes (PFisher = 1.4×10−8) with a carrier frequency of 17.2 %. This breed specific mutation was not present in 334 healthy dogs from 10 other breeds or 121 PRA affected dogs from 44 other breeds. CNGB1 is important for the photoreceptor cell function its defects have been previously associated with retinal degeneration in both human and mouse. Our study indicates that a frameshift mutation in CNGB1 is a cause of PRA in Papillons and Phalènes and establishes the breed as a large functional animal model for further characterization of retinal CNGB1 biology and possible retinal gene therapy trials. This study enables also the development of a genetic test for breeding purposes. PMID:24015210

Rapid targeted mutational analysis of human tumours: a clinical platform to guide personalized cancer medicine

PubMed Central

Dias-Santagata, Dora; Akhavanfard, Sara; David, Serena S; Vernovsky, Kathy; Kuhlmann, Georgiana; Boisvert, Susan L; Stubbs, Hannah; McDermott, Ultan; Settleman, Jeffrey; Kwak, Eunice L; Clark, Jeffrey W; Isakoff, Steven J; Sequist, Lecia V; Engelman, Jeffrey A; Lynch, Thomas J; Haber, Daniel A; Louis, David N; Ellisen, Leif W; Borger, Darrell R; Iafrate, A John

2010-01-01

Targeted cancer therapy requires the rapid and accurate identification of genetic abnormalities predictive of therapeutic response. We sought to develop a high-throughput genotyping platform that would allow prospective patient selection to the best available therapies, and that could readily and inexpensively be adopted by most clinical laboratories. We developed a highly sensitive multiplexed clinical assay that performs very well with nucleic acid derived from formalin fixation and paraffin embedding (FFPE) tissue, and tests for 120 previously described mutations in 13 cancer genes. Genetic profiling of 250 primary tumours was consistent with the documented oncogene mutational spectrum and identified rare events in some cancer types. The assay is currently being used for clinical testing of tumour samples and contributing to cancer patient management. This work therefore establishes a platform for real-time targeted genotyping that can be widely adopted. We expect that efforts like this one will play an increasingly important role in cancer management. PMID:20432502

Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus.

PubMed

Merkle, Florian T; Neuhausser, Werner M; Santos, David; Valen, Eivind; Gagnon, James A; Maas, Kristi; Sandoe, Jackson; Schier, Alexander F; Eggan, Kevin

2015-05-12

The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Use of CRISPR/Cas Genome Editing Technology for Targeted Mutagenesis in Rice.

PubMed

Xu, Rongfang; Wei, Pengcheng; Yang, Jianbo

2017-01-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system is a newly emerging mutagenesis (gene-editing) tool in genetic engineering. Among the agriculturally important crops, several genes have been successfully mutated by the system, and some agronomic important traits have been rapidly generated, which indicates the potential applications in both scientific research and plant breeding. In this chapter, we describe a standard gene-editing procedure to effectively target rice genes and to make specific rice mutants using the CRISPR/Cas9 system mediated by Agrobacterium transformation.

Targeted exome sequencing reveals novel USH2A mutations in Chinese patients with simplex Usher syndrome.

PubMed

Shu, Hai-Rong; Bi, Huai; Pan, Yang-Chun; Xu, Hang-Yu; Song, Jian-Xin; Hu, Jie

2015-09-16

Usher syndrome (USH) is an autosomal recessive disorder characterized by hearing impairment and vision dysfunction due to retinitis pigmentosa. Phenotypic and genetic heterogeneities of this disease make it impractical to obtain a genetic diagnosis by conventional Sanger sequencing. In this study, we applied a next-generation sequencing approach to detect genetic abnormalities in patients with USH. Two unrelated Chinese families were recruited, consisting of two USH afflicted patients and four unaffected relatives. We selected 199 genes related to inherited retinal diseases as targets for deep exome sequencing. Through systematic data analysis using an established bioinformatics pipeline, all variants that passed filter criteria were validated by Sanger sequencing and co-segregation analysis. A homozygous frameshift mutation (c.4382delA, p.T1462Lfs*2) was revealed in exon20 of gene USH2A in the F1 family. Two compound heterozygous mutations, IVS47 + 1G > A and c.13156A > T (p.I4386F), located in intron 48 and exon 63 respectively, of USH2A, were identified as causative mutations for the F2 family. Of note, the missense mutation c.13156A > T has not been reported so far. In conclusion, targeted exome sequencing precisely and rapidly identified the genetic defects in two Chinese USH families and this technique can be applied as a routine examination for these disorders with significant clinical and genetic heterogeneity.

SLC3A1 and SLC7A9 mutations in autosomal recessive or dominant canine cystinuria: a new classification system.

PubMed

Brons, A-K; Henthorn, P S; Raj, K; Fitzgerald, C A; Liu, J; Sewell, A C; Giger, U

2013-01-01

Cystinuria, one of the first recognized inborn errors of metabolism, has been reported in many dog breeds. To determine urinary cystine concentrations, inheritance, and mutations in the SLC3A1 and SLC7A9 genes associated with cystinuria in 3 breeds. Mixed and purebred Labrador Retrievers (n = 6), Australian Cattle Dogs (6), Miniature Pinschers (4), and 1 mixed breed dog with cystine urolithiasis, relatives and control dogs. Urinary cystinuria and aminoaciduria was assessed and exons of the SLC3A1 and SLC7A9 genes were sequenced from genomic DNA. In each breed, male and female dogs, independent of neuter status, were found to form calculi. A frameshift mutation in SLC3A1 (c.350delG) resulting in a premature stop codon was identified in autosomal-recessive (AR) cystinuria in Labrador Retrievers and mixed breed dogs. A 6 bp deletion (c.1095_1100del) removing 2 threonines in SLC3A1 was found in autosomal-dominant (AD) cystinuria with a more severe phenotype in homozygous than in heterozygous Australian Cattle Dogs. A missense mutation in SLC7A9 (c.964G>A) was discovered in AD cystinuria in Miniature Pinschers with only heterozygous affected dogs observed to date. Breed-specific DNA tests were developed, but the prevalence of each mutation remains unknown. These studies describe the first AD inheritance and the first putative SLC7A9 mutation to cause cystinuria in dogs and expand our understanding of this phenotypically and genetically heterogeneous disease, leading to a new classification system for canine cystinuria and better therapeutic management and genetic control in these breeds. Copyright © 2013 by the American College of Veterinary Internal Medicine.

An equine chromosome 3 inversion is associated with the tobiano spotting pattern in German horse breeds.

PubMed

Haase, B; Jude, R; Brooks, S A; Leeb, T

2008-06-01

The tobiano white-spotting pattern is one of several known depigmentation phenotypes in horses and is desired by many horse breeders and owners. The tobiano spotting phenotype is inherited as an autosomal dominant trait. Horses that are heterozygous or homozygous for the tobiano allele (To) are phenotypically indistinguishable. A SNP associated with To had previously been identified in intron 13 of the equine KIT gene and was used for an indirect gene test. The test was useful in several horse breeds. However, genotyping this sequence variant in the Lewitzer horse breed revealed that 14% of horses with the tobiano pattern did not show the polymorphism in intron 13 and consequently the test was not useful to identify putative homozygotes for To within this breed. Speculations were raised that an independent mutation might cause the tobiano spotting pattern in this breed. Recently, the putative causative mutation for To was described as a large chromosomal inversion on equine chromosome 3. One of the inversion breakpoints is approximately 70 kb downstream of the KIT gene and probably disrupts a regulatory element of the KIT gene. We obtained genotypes for the intron 13 SNP and the chromosomal inversion for 204 tobiano spotted horses and 24 control animals of several breeds. The genotyping data confirmed that the chromosomal inversion was perfectly associated with the To allele in all investigated horses. Therefore, the new test is suitable to discriminate heterozygous To/+ and homozygous To/To horses in the investigated breeds.

Transforming activity and therapeutic targeting of C-terminal-binding protein 2 in Apc-mutated neoplasia.

PubMed

Sumner, E T; Chawla, A T; Cororaton, A D; Koblinski, J E; Kovi, R C; Love, I M; Szomju, B B; Korwar, S; Ellis, K C; Grossman, S R

2017-08-17

Overexpression of the transcriptional coregulators C-terminal binding proteins 1 and 2 (CtBP1 and 2) occurs in many human solid tumors and is associated with poor prognosis. CtBP modulates oncogenic gene expression programs and is an emerging drug target, but its oncogenic role is unclear. Consistent with this oncogenic potential, exogenous CtBP2 transformed primary mouse and human cells to anchorage independence similarly to mutant H-Ras. To investigate CtBP's contribution to in vivo tumorigenesis, Apc min/+ mice, which succumb to massive intestinal polyposis, were bred to Ctbp2 +/- mice. CtBP interacts with adenomatous polyposis coli (APC) protein, and is stabilized in both APC-mutated human colon cancers and Apc min/+ intestinal polyps. Ctbp2 heterozygosity increased the median survival of Apc min/+ mice from 21 to 48 weeks, and reduced polyp formation by 90%, with Ctbp2 +/- polyps exhibiting reduced levels of β-catenin and its oncogenic transcriptional target, cyclin D1. CtBP's potential as a therapeutic target was studied by treating Apc min/+ mice with the CtBP small-molecule inhibitors 4-methylthio-2-oxobutyric acid and 2-hydroxy-imino phenylpyruvic acid, both of which reduced polyposis by more than half compared with vehicle treatment. Phenocopying Ctbp2 deletion, both Ctbp inhibitors caused substantial decreases in the protein level of Ctbp2, as well its oncogenic partner β-catenin, and the effects of the inhibitors on CtBP and β-catenin levels could be modeled in an APC-mutated human colon cancer cell line. CtBP2 is thus a druggable transforming oncoprotein critical for the evolution of neoplasia driven by Apc mutation.

Polymorphism of the prion protein gene (PRNP) in two Chinese indigenous cattle breeds.

PubMed

Qin, L H; Zhao, Y M; Bao, Y H; Bai, W L; Chong, J; Zhang, G L; Zhang, J B; Zhao, Z H

2011-08-01

Prion protein (PRNP) gene has been located at position q17 of chromosome 13 in cattle. The polymorphisms of PRNP gene might be associated with BSE susceptibility. In the present work, we investigated the polymorphisms of PRNP gene, including SNP in exon 3, 23-bp indel in promoter region, 12-bp indel in intron 1 in 2 Chinese indigenous cattle breeds of northeast China. Eighty-six animals from Yanbian (34) and Chinese Red Steppes (52) were genotyped at PRNP locus by analyzing genomic DNA. A total of 4 single nucleotide polymorphism (SNP) sites were revealed in the PRNP gene exon 3 of the 2 cattle breeds investigated. Three of these SNPs were non-synonymous mutations that resulted in the amino acid exchanges (K119N, S154N, and M177V), and one is silent nucleotide substitutions (A234G). The two amino acid mutations of S154N and M177V were detected only in Yanbian with a very low frequency (0.0147), and they appears to be absent in Chinese Red Steppes. The average gene heterozygosity (He), effective allele numbers (Ne), Shannon's information index (I) and polymorphism information content (PIC) were 0.3088, 1.5013, 0.3814 and 0.2000 in Yanbian, respectively, being relatively higher than that of Chinese Red Steppes (0.2885, 1.4985, 0.3462 and 0.1873, respectively). In 23-bp indel and 12-bp indel loci, three different genotypes were identified in both Yanbian and Chinese Red Steppes breeds. Based 23- and 12-bp indels, four haplotypes was constructed in the 2 Chinese cattle breeds, of which the 23-bp (-)/12-bp (-) was main haplotypes accounting for more than 50% of the total in both Yanbian and Chinese Red Steppes breeds. These results might be useful in understanding the genetic characteristics of PRNP gene in Chinese indigenous cattle breeds.

Canine chondrodysplasia caused by a truncating mutation in collagen-binding integrin alpha subunit 10.

PubMed

Kyöstilä, Kaisa; Lappalainen, Anu K; Lohi, Hannes

2013-01-01

The skeletal dysplasias are disorders of the bone and cartilage tissues. Similarly to humans, several dog breeds have been reported to suffer from different types of genetic skeletal disorders. We have studied the molecular genetic background of an autosomal recessive chondrodysplasia that affects the Norwegian Elkhound and Karelian Bear Dog breeds. The affected dogs suffer from disproportionate short stature dwarfism of varying severity. Through a genome-wide approach, we mapped the chondrodysplasia locus to a 2-Mb region on canine chromosome 17 in nine affected and nine healthy Elkhounds (praw = 7.42×10(-6), pgenome-wide = 0.013). The associated locus contained a promising candidate gene, cartilage specific integrin alpha 10 (ITGA10), and mutation screening of its 30 exons revealed a nonsense mutation in exon 16 (c.2083C>T; p.Arg695*) that segregated fully with the disease in both breeds (p = 2.5×10(-23)). A 24% mutation carrier frequency was indicated in NEs and an 8% frequency in KBDs. The ITGA10 gene product, integrin receptor α10-subunit combines into a collagen-binding α10β1 integrin receptor, which is expressed in cartilage chondrocytes and mediates chondrocyte-matrix interactions during endochondral ossification. As a consequence of the nonsense mutation, the α10-protein was not detected in the affected cartilage tissue. The canine phenotype highlights the importance of the α10β1 integrin in bone growth, and the large animal model could be utilized to further delineate its specific functions. Finally, this study revealed a candidate gene for human chondrodysplasias and enabled the development of a genetic test for breeding purposes to eradicate the disease from the two dog breeds.

Frequency of MELAS main mutation in a phenotype-targeted young ischemic stroke patient population.

PubMed

Tatlisumak, Turgut; Putaala, Jukka; Innilä, Markus; Enzinger, Christian; Metso, Tiina M; Curtze, Sami; von Sarnowski, Bettina; Amaral-Silva, Alexandre; Jungehulsing, Gerhard Jan; Tanislav, Christian; Thijs, Vincent; Rolfs, Arndt; Norrving, Bo; Fazekas, Franz; Suomalainen, Anu; Kolodny, Edwin H

2016-02-01

Mitochondrial diseases, predominantly mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), may occasionally underlie or coincide with ischemic stroke (IS) in young and middle-aged individuals. We searched for undiagnosed patients with MELAS in a target subpopulation of unselected young IS patients enrolled in the Stroke in Young Fabry Patients study (sifap1). Among the 3291 IS patients aged 18-55 years recruited to the sifap1 study at 47 centers across 14 European countries, we identified potential MELAS patients with the following phenotypic features: (a) diagnosed cardiomyopathy or (b) presence of two of the three following findings: migraine, short stature (≤165 cm for males; ≤155 cm for females), and diabetes. Identified patients' blood samples underwent analysis of the common MELAS mutation, m.3243A>G in the MTTL1 gene of mitochondrial DNA. Clinical and cerebral MRI features of the mutation carriers were reviewed. We analyzed blood samples of 238 patients (177 with cardiomyopathy) leading to identification of four previously unrecognized MELAS main mutation carrier-patients. Their clinical and MRI characteristics were within the expectation for common IS patients except for severe hearing loss in one patient and hyperintensity of the pulvinar thalami on T1-weighted MRI in another one. Genetic testing for the m.3243A>G MELAS mutation in young patients with IS based on phenotypes suggestive of mitochondrial disease identifies previously unrecognized carriers of MELAS main mutation, but does not prove MELAS as the putative cause.

Mitochondrial-morphology-targeted breeding of industrial yeast strains for alcohol fermentation.

PubMed

Kitagaki, Hiroshi

2009-05-29

Since mitochondrial genes are repressed under high glucose and low O2, and these conditions correspond to the conditions in which yeast cells are exposed during alcohol fermentation, the existence and structure of yeast mitochondria during alcohol fermentation have not been elucidated. Yeast mitochondria can be observed throughout brewing of sake (Japanese rice wine) and fragment during brewing. Furthermore, it has been revealed that Fis1 [fission 1 (mitochondrial outer membrane) homologue (Saccharomyces cerevisiae)], which is a transmembrane protein with its C-terminal anchor embedded in the outer membrane of mitochondria, is required for fragmentation of yeast mitochondria during sake brewing. By utilizing this knowledge, a fis1 disruptant of a sake yeast strain has been generated that has a networked mitochondrial structure throughout sake brewing. It transpired that this strain produces a high content of malate, which imparts a crisp acidic taste, during sake brewing. This strategy is a useful and a completely novel strategy towards developing a new yeast strain which produces a high content of malate in sake, and mitochondrial morphology has now emerged as a promising target for the breeding of practical industrial strains.

Liquid Biopsies in the Screening of Oncogenic Mutations in NSCLC and its Application in Targeted Therapy.

PubMed

Tang, Jason H; Chia, David

2015-01-01

Non-small cell lung cancer (NSCLC) still dominates cancer-related deaths in America. Despite this, new discoveries and advancements in technology are helping with the detection and treatment of NSCLC. The discovery of circulating tumor DNA in blood and other biofluids is essential for the creation of a DNA biomarker. Limitations in technology and sequencing have stunted assay development, but with recent advancements in the next-generation sequencing, droplet digital PCR, and EFIRM, the detection of mutations in biofluids has become possible with reasonable sensitivity and specificity. These methods have been applied to the detection of mutations in NSCLC by measuring the levels of circulating tumor DNA. ALK fusion genes along with mutations in EGFR and KRAS have been shown to correlate to tumor size and metastasis. These methods allow for noninvasive, affordable, and efficient diagnoses of oncogenic mutations that overcome the issues of traditional biopsies. These issues include tumor heterogeneity and early detection of cancers with asymptomatic early stages. Early detection and treatment remain the best way to ensure survival. This review aims to describe these new technologies along with their application in mutation detection in NSCLC in order to proactively utilize targeted anticancer therapy.

Mutations in Lettuce Improvement

PubMed Central

Mou, Beiquan

2011-01-01

Lettuce is a major vegetable in western countries. Mutations generated genetic variations and played an important role in the domestication of the crop. Many traits derived from natural and induced mutations, such as dwarfing, early flowering, male sterility, and chlorophyll deficiency, are useful in physiological and genetic studies. Mutants were also used to develop new lettuce products including miniature and herbicide-tolerant cultivars. Mutant analysis was critical in lettuce genomic studies including identification and cloning of disease-resistance genes. Mutagenesis combined with genomic technology may provide powerful tools for the discovery of novel gene alleles. In addition to radiation and chemical mutagens, unconventional approaches such as tissue or protoplast culture, transposable elements, and space flights have been utilized to generate mutants in lettuce. Since mutation breeding is considered nontransgenic, it is more acceptable to consumers and will be explored more in the future for lettuce improvement. PMID:22287955

Target enrichment and high-throughput sequencing of 80 ribosomal protein genes to identify mutations associated with Diamond-Blackfan anaemia.

PubMed

Gerrard, Gareth; Valgañón, Mikel; Foong, Hui En; Kasperaviciute, Dalia; Iskander, Deena; Game, Laurence; Müller, Michael; Aitman, Timothy J; Roberts, Irene; de la Fuente, Josu; Foroni, Letizia; Karadimitris, Anastasios

2013-08-01

Diamond-Blackfan anaemia (DBA) is caused by inactivating mutations in ribosomal protein (RP) genes, with mutations in 13 of the 80 RP genes accounting for 50-60% of cases. The remaining 40-50% cases may harbour mutations in one of the remaining RP genes, but the very low frequencies render conventional genetic screening as challenging. We, therefore, applied custom enrichment technology combined with high-throughput sequencing to screen all 80 RP genes. Using this approach, we identified and validated inactivating mutations in 15/17 (88%) DBA patients. Target enrichment combined with high-throughput sequencing is a robust and improved methodology for the genetic diagnosis of DBA. © 2013 John Wiley & Sons Ltd.

Candidate genes for idiopathic epilepsy in four dog breeds.

PubMed

Ekenstedt, Kari J; Patterson, Edward E; Minor, Katie M; Mickelson, James R

2011-04-25

Idiopathic epilepsy (IE) is a naturally occurring and significant seizure disorder affecting all dog breeds. Because dog breeds are genetically isolated populations, it is possible that IE is attributable to common founders and is genetically homogenous within breeds. In humans, a number of mutations, the majority of which are genes encoding ion channels, neurotransmitters, or their regulatory subunits, have been discovered to cause rare, specific types of IE. It was hypothesized that there are simple genetic bases for IE in some purebred dog breeds, specifically in Vizslas, English Springer Spaniels (ESS), Greater Swiss Mountain Dogs (GSMD), and Beagles, and that the gene(s) responsible may, in some cases, be the same as those already discovered in humans. Candidate genes known to be involved in human epilepsy, along with selected additional genes in the same gene families that are involved in murine epilepsy or are expressed in neural tissue, were examined in populations of affected and unaffected dogs. Microsatellite markers in close proximity to each candidate gene were genotyped and subjected to two-point linkage in Vizslas, and association analysis in ESS, GSMD and Beagles. Most of these candidate genes were not significantly associated with IE in these four dog breeds, while a few genes remained inconclusive. Other genes not included in this study may still be causing monogenic IE in these breeds or, like many cases of human IE, the disease in dogs may be likewise polygenic.

[Retrospect and prospect of transgenic fish breeding in China].

PubMed

Wang, Yaping; He, Libo

2016-07-25

The first transgenic fish was generated in China about 30 years ago. Since then, considerable progress has been achieved for farmed fishes breeding with improvement of target traits of growth, disease resistance, stress tolerance, and nutrition qualities. Up to now, the technology of transgenic fish breeding is almost mature and the biosafety assessment is established. In this review, a successful example of the fast-growing transgenic common carp was presented and the foreground of transgenic fish breeding was also discussed and prospected.

Fine mapping of quantitative trait loci underlying sensory meat quality traits in three French beef cattle breeds.

PubMed

Allais, S; Levéziel, H; Hocquette, J F; Rousset, S; Denoyelle, C; Journaux, L; Renand, G

2014-10-01

Improving the traits that underlie meat quality is a major challenge in the beef industry. The objective of this paper was to detect QTL linked to sensory meat quality traits in 3 French beef cattle breeds. We genotyped 1,059, 1,219, and 947 young bulls and their sires belonging to the Charolais, Limousin, and Blonde d'Aquitaine breeds, respectively, using the Illumina BovineSNP50 BeadChip (Illumina Inc., San Diego, CA). After estimating relevant genetic parameters using VCE software, we performed a linkage disequilibrium and linkage analysis on 4 meat traits: intramuscular fat content, muscle lightness, shear force, and tenderness score. Heritability coefficients largely ranged between 0.10 and 0.24; however, they reached a maximum of 0.44 and 0.50 for intramuscular fat content and tenderness score, respectively, in the Charolais breed. The 2 meat texture traits, shear force and tenderness score, were strongly genetically correlated (-0.91 in the Charolais and Limousin breed and -0.86 in the Blonde d'Aquitaine breed), indicating that they are 2 different measures of approximately the same trait. The genetic correlation between tenderness and intramuscular fat content differed across breeds. Using a significance threshold of 5 × 10(-4) for QTL detection, we found more than 200 significant positions across the 29 autosomal chromosomes for the 4 traits in the Charolais and Blonde d'Aquitaine breeds; in contrast, there were only 78 significant positions in the Limousin breed. Few QTL were common across breeds. We detected QTL for intramuscular fat content located near the myostatin gene in the Charolais and Blonde d'Aquitaine breeds. No mutation in this gene has been reported for the Blonde d'Aquitaine breed; therefore, it suggests that an unknown mutation could be segregating in this breed. We confirmed that, in certain breeds, markers in the calpastatin and calpain 1 gene regions affect tenderness. We also found new QTL as several QTL on chromosome 3 that are

Toll-like receptor 3 as an immunotherapeutic target for KRAS mutated colorectal cancer

PubMed Central

Maitra, Radhashree; Augustine, Titto; Dayan, Yitzchak; Chandy, Carol; Coffey, Matthew; Goel, Sanjay

2017-01-01

New therapeutic interventions are essential for improved management of patients with metastatic colorectal cancer (mCRC). This is especially critical for those patients whose tumors harbor a mutation in the KRAS oncogene (40-45% of all patients). This patient cohort is excluded from receiving anti-EGFR monoclonal antibodies that have added a significant therapeutic benefit for KRAS wild type CRC patients. Reovirus, a double stranded (ds) RNA virus is in clinical development for patients with chemotherapy refractory KRAS mutated tumors. Toll Like Receptor (TLR) 3, a member of the toll like receptor family of the host innate immune system is the pattern recognition motif for dsRNA pathogens. Using TLR3 expressing commercial HEK-Blue™-hTLR3 cells we confirm that TLR3 is the host pattern recognition motif responsible for the detection of reovirus. Further, our investigation with KRAS mutated HCT116 cell line showed that effective expression of host TLR3 dampens the infection potential of reovirus by mounting a robust innate immune response. Down regulation of TLR3 expression with siRNA improves the anticancer activity of reovirus. In vivo experiments using human CRC cells derived xenografts in athymic mice further demonstrate the beneficial effects of TLR3 knock down by improving tumor response rates to reovirus. Strategies to mitigate the TLR3 response pathway can be utilized as a tool towards improved reovirus efficacy to specifically target the dissemination of KRAS mutated CRC. PMID:28422714

Characterization of Ribozymes Targeting a Congenital Night Blindness Mutation in Rhodopsin Mutation.

PubMed

Conley, Shannon M; Whalen, Patrick; Lewin, Alfred S; Naash, Muna I

2016-01-01

The G90D mutation in the rhodopsin gene leads to autosomal dominant congenital stationary night blindness (CSNB) in patients. This occurs because the G90D mutant protein cannot efficiently bind chromophore and is constitutively active. To combat this mutation, we designed and characterized two different hammerhead ribozymes to cleave G90D transcript. In vitro testing showed that the G90D1 ribozyme efficiently and specifically cleaved the mutant transcript while G90D2 cleaved both WT and mutant transcript. AAV-mediated delivery of G90D1 under the control of the mouse opsin promoter (MOP500) to G90D transgenic eyes showed that the ribozyme partially retarded the functional degeneration (as measured by electroretinography [ERG]) associated with this mutation. These results suggest that with additional optimization, ribozymes may be a useful part of the gene therapy knockdown strategy for dominant retinal disease.

TALEN-mediated targeted mutagenesis of fatty acid desaturase 2 (FAD2) in peanut (Arachis hypogaea L.) promotes the accumulation of oleic acid.

PubMed

Wen, Shijie; Liu, Hao; Li, Xingyu; Chen, Xiaoping; Hong, Yanbin; Li, Haifen; Lu, Qing; Liang, Xuanqiang

2018-05-01

A first creation of high oleic acid peanut varieties by using transcription activator-like effecter nucleases (TALENs) mediated targeted mutagenesis of Fatty Acid Desaturase 2 (FAD2). Transcription activator like effector nucleases (TALENs), which allow the precise editing of DNA, have already been developed and applied for genome engineering in diverse organisms. However, they are scarcely used in higher plant study and crop improvement, especially in allopolyploid plants. In the present study, we aimed to create targeted mutagenesis by TALENs in peanut. Targeted mutations in the conserved coding sequence of Arachis hypogaea fatty acid desaturase 2 (AhFAD2) were created by TALENs. Genetic stability of AhFAD2 mutations was identified by DNA sequencing in up to 9.52 and 4.11% of the regeneration plants at two different targeted sites, respectively. Mutation frequencies among AhFAD2 mutant lines were significantly correlated to oleic acid accumulation. Genetically, stable individuals of positive mutant lines displayed a 0.5-2 fold increase in the oleic acid content compared with non-transgenic controls. This finding suggested that TALEN-mediated targeted mutagenesis could increase the oleic acid content in edible peanut oil. Furthermore, this was the first report on peanut genome editing event, and the obtained high oleic mutants could serve for peanut breeding project.

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope.

PubMed

Tharakaraman, Kannan; Watanabe, Satoru; Chan, Kuan Rong; Huan, Jia; Subramanian, Vidya; Chionh, Yok Hian; Raguram, Aditya; Quinlan, Devin; McBee, Megan; Ong, Eugenia Z; Gan, Esther S; Tan, Hwee Cheng; Tyagi, Anu; Bhushan, Shashi; Lescar, Julien; Vasudevan, Subhash G; Ooi, Eng Eong; Sasisekharan, Ram

2018-05-09

Following the recent emergence of Zika virus (ZIKV), many murine and human neutralizing anti-ZIKV antibodies have been reported. Given the risk of virus escape mutants, engineering antibodies that target mutationally constrained epitopes with therapeutically relevant potencies can be valuable for combating future outbreaks. Here, we applied computational methods to engineer an antibody, ZAb_FLEP, that targets a highly networked and therefore mutationally constrained surface formed by the envelope protein dimer. ZAb_FLEP neutralized a breadth of ZIKV strains and protected mice in distinct in vivo models, including resolving vertical transmission and fetal mortality in infected pregnant mice. Serial passaging of ZIKV in the presence of ZAb_FLEP failed to generate viral escape mutants, suggesting that its epitope is indeed mutationally constrained. A single-particle cryo-EM reconstruction of the Fab-ZIKV complex validated the structural model and revealed insights into ZAb_FLEP's neutralization mechanism. ZAb_FLEP has potential as a therapeutic in future outbreaks. Copyright © 2018. Published by Elsevier Inc.

Rapid targeted somatic mutation analysis of solid tumors in routine clinical diagnostics.

PubMed

Magliacane, Gilda; Grassini, Greta; Bartocci, Paola; Francaviglia, Ilaria; Dal Cin, Elena; Barbieri, Gianluca; Arrigoni, Gianluigi; Pecciarini, Lorenza; Doglioni, Claudio; Cangi, Maria Giulia

2015-10-13

Tumor genotyping is an essential step in routine clinical practice and pathology laboratories face a major challenge in being able to provide rapid, sensitive and updated molecular tests. We developed a novel mass spectrometry multiplexed genotyping platform named PentaPanel to concurrently assess single nucleotide polymorphisms in 56 hotspots of the 5 most clinically relevant cancer genes, KRAS, NRAS, BRAF, EGFR and PIK3CA for a total of 221 detectable mutations. To both evaluate and validate the PentaPanel performance, we investigated 1025 tumor specimens of 6 different cancer types (carcinomas of colon, lung, breast, pancreas, and biliary tract, and melanomas), systematically addressing sensitivity, specificity, and reproducibility of our platform. Sanger sequencing was also performed for all the study samples. Our data showed that PentaPanel is a high throughput and robust tool, allowing genotyping for targeted therapy selection of 10 patients in the same run, with a practical turnaround time of 2 working days. Importantly, it was successfully used to interrogate different DNAs isolated from routinely processed specimens (formalin-fixed paraffin embedded, frozen, and cytological samples), covering all the requirements of clinical tests. In conclusion, the PentaPanel platform can provide an immediate, accurate and cost effective multiplex approach for clinically relevant gene mutation analysis in many solid tumors and its utility across many diseases can be particularly relevant in multiple clinical trials, including the new basket trial approach, aiming to identify appropriate targeted drug combination strategies.

Activated Alk triggers prolonged neurogenesis and Ret upregulation providing a therapeutic target in ALK-mutated neuroblastoma

PubMed Central

Cazes, Alex; Lopez-Delisle, Lucille; Tsarovina, Konstantina; Pierre-Eugène, Cécile; De Preter, Katleen; Peuchmaur, Michel; Nicolas, André; Provost, Claire; Louis-Brennetot, Caroline; Daveau, Romain; Kumps, Candy; Cascone, Ilaria; Schleiermacher, Gudrun; Prignon, Aurélie; Speleman, Frank; Rohrer, Hermann; Delattre, Olivier; Janoueix-Lerosey, Isabelle

2014-01-01

Activating mutations of the ALK (Anaplastic lymphoma Kinase) gene have been identified in sporadic and familial cases of neuroblastoma, a cancer of early childhood arising from the sympathetic nervous system (SNS). To decipher ALK function in neuroblastoma predisposition and oncogenesis, we have characterized knock-in (KI) mice bearing the two most frequent mutations observed in neuroblastoma patients. A dramatic enlargement of sympathetic ganglia is observed in AlkF1178L mice from embryonic to adult stages associated with an increased proliferation of sympathetic neuroblasts from E14.5 to birth. In a MYCN transgenic context, the F1178L mutation displays a higher oncogenic potential than the R1279Q mutation as evident from a shorter latency of tumor onset. We show that tumors expressing the R1279Q mutation are sensitive to ALK inhibition upon crizotinib treatment. Furthermore, our data provide evidence that activated ALK triggers RET upregulation in mouse sympathetic ganglia at birth as well as in murine and human neuroblastoma. Using vandetanib, we show that RET inhibition strongly impairs tumor growth in vivo in both MYCN/KI AlkR1279Q and MYCN/KI AlkF1178L mice. Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma. PMID:24811913

A target-unrelated peptide in an M13 phage display library traced to an advantageous mutation in the gene II ribosome-binding site.

PubMed

Brammer, Leighanne A; Bolduc, Benjamin; Kass, Jessica L; Felice, Kristin M; Noren, Christopher J; Hall, Marilena Fitzsimons

2008-02-01

Screening of the commercially available Ph.D.-7 phage-displayed heptapeptide library for peptides that bind immobilized Zn2+ resulted in the repeated selection of the peptide HAIYPRH, although binding assays indicated that HAIYPRH is not a zinc-binding peptide. HAIYPRH has also been selected in several other laboratories using completely different targets, and its ubiquity suggests that it is a target-unrelated peptide. We demonstrated that phage displaying HAIYPRH are enriched after serial amplification of the library without exposure to target. The amplification of phage displaying HAIYPRH was found to be dramatically faster than that of the library itself. DNA sequencing uncovered a mutation in the Shine-Dalgarno (SD) sequence for gIIp, a protein involved in phage replication, imparting to the SD sequence better complementarity to the 16S ribosomal RNA (rRNA). Introducing this mutation into phage lacking a displayed peptide resulted in accelerated propagation, whereas phage displaying HAIYPRH with a wild-type SD sequence were found to amplify normally. The SD mutation may alter gIIp expression and, consequently, the rate of propagation of phage. In the Ph.D.-7 library, the mutation is coincident with the displayed peptide HAIYPRH, accounting for the target-unrelated selection of this peptide in multiple reported panning experiments.

Can non-breeding be a cost of breeding dispersal?

USGS Publications Warehouse

Danchin, E.; Cam, E.

2002-01-01

Breeding habitat selection and dispersal are crucial processes that affect many components of fitness. Breeding dispersal entails costs, one of which has been neglected: dispersing animals may miss breeding opportunities because breeding dispersal requires finding a new nesting site and mate, two time- and energy-consuming activities. Dispersers are expected to be prone to non-breeding. We used the kittiwake (Rissa tridactyla) to test whether breeding dispersal influences breeding probability. Breeding probability was associated with dispersal, in that both were negatively influenced by private information (previous individual reproductive success) and public information (average reproductive success of conspecifics) about patch quality. Furthermore, the probability of skipping breeding was 1.7 times higher in birds that settled in a new patch relative to those that remained on the same patch. Finally, non-breeders that resumed breeding were 4.4 times more likely to disperse than birds that bred in successive years. Although private information may influence breeding probability directly, the link between breeding probability and public information may be indirect, through the influence of public information on breeding dispersal, non-breeding thus being a cost of dispersal. These results support the hypothesis that dispersal may result in not being able to breed. More generally, non-breeding (which can be interpreted as an extreme form of breeding failure) may reveal costs of various previous activities. Because monitoring the non-breeding portion of a population is difficult, non-breeders have been neglected in many studies of reproduction trade-offs.

The clustered regularly interspaced short palindromic repeats/associated proteins system for the induction of gene mutations and phenotypic changes in Bombyx mori.

PubMed

Song, Jia; Che, Jiaqian; You, Zhengying; Ye, Xiaogang; Li, Jisheng; Ye, Lupeng; Zhang, Yuyu; Qian, Qiujie; Zhong, Boxiong

2016-12-01

To probe the general phenomena of gene mutations, Bombyx mori, the lepidopterous model organism, was chosen as the experimental model. To easily detect phenotypic variations, the piggyBac system was utilized to introduce two marker genes into the silkworm, and 23.4% transposition efficiency aided in easily breeding a new strain for the entire experiment. Then, the clustered regularly interspaced short palindromic repeats/an associated protein (Cas9) system was utilized. The results showed that the Cas9 system can induce efficient gene mutations and the base changes could be detected since the G 0 individuals in B. mori; and that the mutation rates on different target sites were diverse. Next, the gRNA2-targeted site that generated higher mutation rate was chosen, and the experimental results were enumerated. First, the mutation proportion in G 1 generation was 30.1%, and some gene mutations were not inherited from the G 0 generation; second, occasionally, base substitutions did not lead to variation in the amino-acid sequence, which decreased the efficiency of phenotypic changes compared with that of genotypic changes. These results laid the foundation for better use of the Cas9 system in silkworm gene editing. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Double muscling in cattle due to mutations in the myostatin gene

PubMed Central

McPherron, Alexandra C.; Lee, Se-Jin

1997-01-01

Myostatin (GDF-8) is a member of the transforming growth factor β superfamily of secreted growth and differentiation factors that is essential for proper regulation of skeletal muscle mass in mice. Here we report the myostatin sequences of nine other vertebrate species and the identification of mutations in the coding sequence of bovine myostatin in two breeds of double-muscled cattle, Belgian Blue and Piedmontese, which are known to have an increase in muscle mass relative to conventional cattle. The Belgian Blue myostatin sequence contains an 11-nucleotide deletion in the third exon which causes a frameshift that eliminates virtually all of the mature, active region of the molecule. The Piedmontese myostatin sequence contains a missense mutation in exon 3, resulting in a substitution of tyrosine for an invariant cysteine in the mature region of the protein. The similarity in phenotypes of double-muscled cattle and myostatin null mice suggests that myostatin performs the same biological function in these two species and is a potentially useful target for genetic manipulation in other farm animals. PMID:9356471

Mitochondria: A Common Target for Genetic Mutations and Environmental Toxicants in Parkinson’s Disease

PubMed Central

Helley, Martin P.; Pinnell, Jennifer; Sportelli, Carolina; Tieu, Kim

2017-01-01

Parkinson’s disease (PD) is a devastating neurological movement disorder. Since its first discovery 200 years ago, genetic and environmental factors have been identified to play a role in PD development and progression. Although genetic studies have been the predominant driving force in PD research over the last few decades, currently only a small fraction of PD cases can be directly linked to monogenic mutations. The remaining cases have been attributed to other risk associated genes, environmental exposures and gene–environment interactions, making PD a multifactorial disorder with a complex etiology. However, enormous efforts from global research have yielded significant insights into pathogenic mechanisms and potential therapeutic targets for PD. This review will highlight mitochondrial dysfunction as a common pathway involved in both genetic mutations and environmental toxicants linked to PD. PMID:29204154

Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data.

PubMed

Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A; Larsen, Martin Jakob

2016-01-01

Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths.

Charge breeding simulations for radioactive ion beam production

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

Variale, V.; Raino, A. C.; Clauser, T.

2012-02-15

The charge breeding technique is used for radioactive ion beam (RIB) production in order of optimizing the re-acceleration of the radioactive element ions produced by a primary beam in a thick target. Charge breeding is achieved by means of a device capable of increasing the ion charge state from 1+ to a desired value n+. In order to get high intensity RIB, experiments with charge breeding of very high efficiency could be required. To reach this goal, the charge breeding simulation could help to optimize the high charge state production efficiency by finding more proper parameters for the radioactive 1+more » ions. In this paper a device based on an electron beam ion source (EBIS) is considered. In order to study that problem, a code already developed for studying the ion selective containment in an EBIS with RF quadrupoles, BRICTEST, has been modified to simulate the ion charge state breeding rate for different 1+ ion injection conditions. Particularly, the charge breeding simulations for an EBIS with a hollow electron beam have been studied.« less

DHPLC technology for high-throughput detection of mutations in a durum wheat TILLING population.

PubMed

Colasuonno, Pasqualina; Incerti, Ornella; Lozito, Maria Luisa; Simeone, Rosanna; Gadaleta, Agata; Blanco, Antonio

2016-02-17

Durum wheat (Triticum turgidum L.) is a cereal crop widely grown in the Mediterranean regions; the amber grain is mainly used for the production of pasta, couscous and typical breads. Single nucleotide polymorphism (SNP) detection technologies and high-throughput mutation induction represent a new challenge in wheat breeding to identify allelic variation in large populations. The TILLING strategy makes use of traditional chemical mutagenesis followed by screening for single base mismatches to identify novel mutant loci. Although TILLING has been combined to several sensitive pre-screening methods for SNP analysis, most rely on expensive equipment. Recently, a new low cost and time saving DHPLC protocol has been used in molecular human diagnostic to detect unknown mutations. In this work, we developed a new durum wheat TILLING population (cv. Marco Aurelio) using 0.70-0.85% ethyl methane sulfonate (EMS). To investigate the efficiency of the mutagenic treatments, a pilot screening was carried out on 1,140 mutant lines focusing on two target genes (Lycopene epsilon-cyclase, ε-LCY, and Lycopene beta-cyclase, β-LCY) involved in carotenoid metabolism in wheat grains. We simplify the heteroduplex detection by two low cost methods: the enzymatic cleavage (CelI)/agarose gel technique and the denaturing high-performance liquid chromatography (DHPLC). The CelI/agarose gel approach allowed us to identify 31 mutations, whereas the DHPLC procedure detected a total of 46 mutations for both genes. All detected mutations were confirmed by direct sequencing. The estimated overall mutation frequency for the pilot assay by the DHPLC methodology resulted to be of 1/77 kb, representing a high probability to detect interesting mutations in the target genes. We demonstrated the applicability and efficiency of a new strategy for the detection of induced variability. We produced and characterized a new durum wheat TILLING population useful for a better understanding of key gene functions

MEN1 mutations and potentially MEN1-targeting miRNAs are responsible for menin deficiency in sporadic and MEN1 syndrome-associated primary hyperparathyroidism.

PubMed

Grolmusz, Vince Kornél; Borka, Katalin; Kövesdi, Annamária; Németh, Kinga; Balogh, Katalin; Dékány, Csaba; Kiss, András; Szentpéteri, Anna; Sármán, Beatrix; Somogyi, Anikó; Csajbók, Éva; Valkusz, Zsuzsanna; Tóth, Miklós; Igaz, Péter; Rácz, Károly; Patócs, Attila

2017-09-01

Inherited, germline mutations of menin-coding MEN1 gene cause multiple endocrine neoplasia type 1 (MEN1), while somatic MEN1 mutations are the sole main driver mutations in sporadic primary hyperparathyroidism (PHPT), suggesting that menin deficiency has a central role in the pathogenesis of PHPT. MiRNAs are small, noncoding RNAs posttranscriptionally regulating gene expression. Our aim was to investigate both the role of MEN1 mutations and potentially MEN1-targeting miRNAs as the underlying cause of menin deficiency in MEN1-associated and sporadic PHPT tissues. Fifty six PHPT tissues, including 16 MEN1-associated tissues, were evaluated. Diagnosis of MEN1 syndrome was based on identification of germline MEN1 mutations. In silico target prediction was used to identify miRNAs potentially targeting MEN1. Menin expression was determined by immunohistochemistry while expression of miRNAs was analyzed by quantitative real-time PCR. Sporadic PHPT tissues were subjected to somatic MEN1 mutation analysis as well. Lack of nuclear menin was identified in all MEN1-associated and in 28% of sporadic PHPT tissues. Somatic MEN1 mutations were found in 25% of sporadic PHPTs. The sensitivity and specificity of menin immunohistochemistry to detect a MEN1 mutation were 86 and 87%, respectively. Expression levels of hsa-miR-24 and hsa-miR-28 were higher in sporadic compared to MEN1-associated PHPT tissues; however, no difference in miRNA levels occurred between menin-positive and menin-negative PHPT tissues. Menin deficiency is the consequence of a MEN1 mutation in most menin-negative PHPT tissues. Elevated expression of hsa-miR-24 and hsa-miR-28 mark the first epigenetic changes observed between sporadic and MEN1-associated PHPT.

Novel compound heterozygous mutations in CNGA1in a Chinese family affected with autosomal recessive retinitis pigmentosa by targeted sequencing.

PubMed

Wang, Min; Gan, Dekang; Huang, Xin; Xu, Gezhi

2016-07-08

About 37 genes have been reported to be involved in autosomal recessive retinitis pigmentosa, a hereditary retinal disease. However, causative genes remain unclear in a lot of cases. Two sibs of a Chinese family with ocular disease were diagnosed in Eye and ENT Hospital of Fudan University. Targeted sequencing performed on proband to screen pathogenic mutations. PCR combined Sanger sequencing then performed on eight family members including two affected and six unaffected individuals to determine whether mutations cosegregate with disease. Two affected members exhibited clinical features that fit the criteria of autosomal recessive retinitis pigmentosa. Two heterozygous mutations (NM000087, p.Y82X and p.L89fs) in CNGA1 were revealed on proband. Affected members were compound heterozygotes for the two mutations whereas unaffected members either had no mutation or were heterozygote carriers for only one of the two mutations. That is, these mutations cosegregate with autosomal recessive retinitis pigmentosa. Compound heterozygous mutations (NM000087, p.Y82X and p.L89fs) in exon 6 of CNGA1are pathogenic mutations in this Chinese family. Of which, p.Y82X is firstly reported in patient with autosomal recessive retinitis pigmentosa.

Convergent Evolution of Slick Coat in Cattle through Truncation Mutations in the Prolactin Receptor.

PubMed

Porto-Neto, Laercio R; Bickhart, Derek M; Landaeta-Hernandez, Antonio J; Utsunomiya, Yuri T; Pagan, Melvin; Jimenez, Esbal; Hansen, Peter J; Dikmen, Serdal; Schroeder, Steven G; Kim, Eui-Soo; Sun, Jiajie; Crespo, Edward; Amati, Norman; Cole, John B; Null, Daniel J; Garcia, Jose F; Reverter, Antonio; Barendse, William; Sonstegard, Tad S

2018-01-01

Evolutionary adaptations are occasionally convergent solutions to the same problem. A mutation contributing to a heat tolerance adaptation in Senepol cattle, a New World breed of mostly European descent, results in the distinct phenotype known as slick, where an animal has shorter hair and lower follicle density across its coat than wild type animals. The causal variant, located in the 11 th exon of prolactin receptor , produces a frameshift that results in a truncated protein. However, this mutation does not explain all cases of slick coats found in criollo breeds. Here, we obtained genome sequences from slick cattle of a geographically distinct criollo breed, namely Limonero, whose ancestors were originally brought to the Americas by the Spanish. These data were used to identify new causal alleles in the 11 th exon of the prolactin receptor , two of which also encode shortened proteins that remove a highly conserved tyrosine residue. These new mutations explained almost 90% of investigated cases of animals that had slick coats, but which also did not carry the Senepol slick allele. These results demonstrate convergent evolution at the molecular level in a trait important to the adaptation of an animal to its environment.

Convergent Evolution of Slick Coat in Cattle through Truncation Mutations in the Prolactin Receptor

PubMed Central

Porto-Neto, Laercio R.; Bickhart, Derek M.; Landaeta-Hernandez, Antonio J.; Utsunomiya, Yuri T.; Pagan, Melvin; Jimenez, Esbal; Hansen, Peter J.; Dikmen, Serdal; Schroeder, Steven G.; Kim, Eui-Soo; Sun, Jiajie; Crespo, Edward; Amati, Norman; Cole, John B.; Null, Daniel J.; Garcia, Jose F.; Reverter, Antonio; Barendse, William; Sonstegard, Tad S.

2018-01-01

Evolutionary adaptations are occasionally convergent solutions to the same problem. A mutation contributing to a heat tolerance adaptation in Senepol cattle, a New World breed of mostly European descent, results in the distinct phenotype known as slick, where an animal has shorter hair and lower follicle density across its coat than wild type animals. The causal variant, located in the 11th exon of prolactin receptor, produces a frameshift that results in a truncated protein. However, this mutation does not explain all cases of slick coats found in criollo breeds. Here, we obtained genome sequences from slick cattle of a geographically distinct criollo breed, namely Limonero, whose ancestors were originally brought to the Americas by the Spanish. These data were used to identify new causal alleles in the 11th exon of the prolactin receptor, two of which also encode shortened proteins that remove a highly conserved tyrosine residue. These new mutations explained almost 90% of investigated cases of animals that had slick coats, but which also did not carry the Senepol slick allele. These results demonstrate convergent evolution at the molecular level in a trait important to the adaptation of an animal to its environment. PMID:29527221

Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes

PubMed Central

Taylor, Benjamin JM; Wu, Yee Ling; Rada, Cristina

2014-01-01

Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. DOI: http://dx.doi.org/10.7554/eLife.03553.001 PMID:25237741

Novel mutations in CRB1 gene identified in a chinese pedigree with retinitis pigmentosa by targeted capture and next generation sequencing

PubMed Central

Lo, David; Weng, Jingning; Liu, xiaohong; Yang, Juhua; He, Fen; Wang, Yun; Liu, Xuyang

2016-01-01

PURPOSE To detect the disease-causing gene in a Chinese pedigree with autosomal-recessive retinitis pigmentosa (ARRP). METHODS All subjects in this family underwent a complete ophthalmic examination. Targeted-capture next generation sequencing (NGS) was performed on the proband to detect variants. All variants were verified in the remaining family members by PCR amplification and Sanger sequencing. RESULTS All the affected subjects in this pedigree were diagnosed with retinitis pigmentosa (RP). The compound heterozygous c.138delA (p.Asp47IlefsX24) and c.1841G>T (p.Gly614Val) mutations in the Crumbs homolog 1 (CRB1) gene were identified in all the affected patients but not in the unaffected individuals in this family. These mutations were inherited from their parents, respectively. CONCLUSION The novel compound heterozygous mutations in CRB1 were identified in a Chinese pedigree with ARRP using targeted-capture next generation sequencing. After evaluating the significant heredity and impaired protein function, the compound heterozygous c.138delA (p.Asp47IlefsX24) and c.1841G>T (p.Gly614Val) mutations are the causal genes of early onset ARRP in this pedigree. To the best of our knowledge, there is no previous report regarding the compound mutations. PMID:27806333

Selection of target mutation in rat gastrointestinal tract E. coli by minute dosage of enrofloxacin.

PubMed

Lin, Dachuan; Chen, Kaichao; Li, Ruichao; Liu, Lizhang; Guo, Jiubiao; Yao, Wen; Chen, Sheng

2014-01-01

It has been suggested that bacterial resistance is selected within a mutation selection window of antibiotics. More recent studies showed that even extremely low concentration of antibiotic could select resistant bacteria in vitro. Yet little is known about the exact antibiotic concentration range that can effectively select for resistant organisms in animal gastrointestinal (GI) tract. In this study, the effect of different dosages of enrofloxacin on resistance and mutation development in rat GI tract E. coli was investigated by determining the number of resistant E. coli recoverable from rat fecal samples. Our data showed that high dose antibiotic treatment could effectively eliminate E. coli with single gyrA mutation in the early course of treatment, yet the eradication effects diminished upon prolonged treatment. Therapeutic and sub-therapeutic dose (1/10 and 1/100 of therapeutic doses) of enrofloxacin could effectively select for mutation in GI tract E. coli at the later course of enrofloxacin treatment and during the cessation periods. Surprisingly, very low dose of enrofloxacin (1/1000 therapeutic dose) could also select for mutation in GI tract E. coli at the later course of enrofloxacin treatment, only with slightly lower efficiency. No enrofloxacin-resistant E. coli could be selected at all test levels of enrofloxacin during long term treatment and the strength of antibiotic treatment does not alter the overall level of E. coli in rat GI tract. This study demonstrated that long term antibiotic treatment seems to be the major trigger for the development of target mutations in GI tract E. coli, which provided insight into the rational use of antibiotics in animal husbandry.

Identification of Genetic Variation on the Horse Y Chromosome and the Tracing of Male Founder Lineages in Modern Breeds

PubMed Central

Wallner, Barbara; Vogl, Claus; Shukla, Priyank; Burgstaller, Joerg P.; Druml, Thomas; Brem, Gottfried

2013-01-01

The paternally inherited Y chromosome displays the population genetic history of males. While modern domestic horses (Equus caballus) exhibit abundant diversity within maternally inherited mitochondrial DNA, no significant Y-chromosomal sequence diversity has been detected. We used high throughput sequencing technology to identify the first polymorphic Y-chromosomal markers useful for tracing paternal lines. The nucleotide variability of the modern horse Y chromosome is extremely low, resulting in six haplotypes (HT), all clearly distinct from the Przewalski horse (E. przewalskii). The most widespread HT1 is ancestral and the other five haplotypes apparently arose on the background of HT1 by mutation or gene conversion after domestication. Two haplotypes (HT2 and HT3) are widely distributed at high frequencies among modern European horse breeds. Using pedigree information, we trace the distribution of Y-haplotype diversity to particular founders. The mutation leading to HT3 occurred in the germline of the famous English Thoroughbred stallion “Eclipse” or his son or grandson and its prevalence demonstrates the influence of this popular paternal line on modern sport horse breeds. The pervasive introgression of Thoroughbred stallions during the last 200 years to refine autochthonous breeds has strongly affected the distribution of Y-chromosomal variation in modern horse breeds and has led to the replacement of autochthonous Y chromosomes. Only a few northern European breeds bear unique variants at high frequencies or fixed within but not shared among breeds. Our Y-chromosomal data complement the well established mtDNA lineages and document the male side of the genetic history of modern horse breeds and breeding practices. PMID:23573227

PROTEIN KINASE C δ IS A THERAPEUTIC TARGET IN MALIGNANT MELANOMA WITH NRAS MUTATION

PubMed Central

Takashima, Asami; English, Brandon; Chen, Zhihong; Cao, Juxiang; Cui, Rutao; Williams, Robert M.; Faller, Douglas V.

2014-01-01

NRAS is the second most frequently mutated gene in melanoma. Previous reports have demonstrated the sensitivity of cancer cell lines carrying KRAS mutations to apoptosis initiated by inhibition of protein kinase C delta (PKCδ). Here, we report that PKCδ inhibition is cytotoxic in melanomas with primary NRAS mutations. Novel small-molecule inhibitors of PKCδ were designed as chimeric hybrids of two naturally-occurring PKCδ inhibitors, staurosporine and rottlerin. The specific hypothesis interrogated and validated is that combining two domains of two naturally-occurring PKCδ inhibitors into a chimeric or hybrid structure retains biochemical and biological activity, and improves PKCδ isozyme selectivity. We have devised a potentially general synthetic protocol to make these chimeric species using Molander trifluorborate coupling chemistry. Inhibition of PKCδ, by siRNA or small molecule inhibitors, suppressed the growth of multiple melanoma cell lines carrying NRAS mutations, mediated via caspase-dependent apoptosis. Following PKCδ inhibition, the stress-responsive JNK pathway was activated, leading to the activation of H2AX. Consistent with recent reports on the apoptotic role of phospho-H2AX, knockdown of H2AX prior to PKCδ inhibition mitigated the induction of caspase-dependent apoptosis. Furthermore, PKCδ inhibition effectively induced cytotoxicity in BRAF-mutant melanoma cell lines that had evolved resistance to a BRAF inhibitor, suggesting the potential clinical application of targeting PKCδ in patients who have relapsed following treatment with BRAF inhibitors. Taken together, the present work demonstrates that inhibition of PKCδ by novel small molecule inhibitors causes caspase-dependent apoptosis mediated via the JNK-H2AX pathway in melanomas with NRAS mutations or BRAF inhibitor-resistance. PMID:24506253

DNA typing of Pakistani cattle breeds Tharparkar and Red Sindhi by microsatellite markers.

PubMed

Azam, Amber; Babar, Masroor Ellahi; Firyal, Sehrish; Anjum, Aftab Ahmad; Akhtar, Nabeela; Asif, Muhammad; Hussain, Tanveer

2012-02-01

Microsatellite markers are used for any individual identity and breed characterization in animals that is an efficient and successful way of investigation. They are used for multiple purposes as genetic detectors including, rapid mutation rate, high level of polymorphism, and range of variety of microsatellite markers available. A panel of 19 microsatellite markers was developed for breed characterization in Tharparkar and Red Sindhi breeds of cattle in Pakistan. Forty four blood samples of cattle (each breed) were collected from Department of Livestock Management, Sindh Agriculture University, Tandojam, Tando Qaiser, Tharparkar Cattle Farm Nabi sar Road, Umer Kot, Sindh, and Govt. Red Sindhi Cattle Breeding Farm, Tando Muhammad Khan Pakistan. Breed characterization was 100% successful. Average PIC, He and Power of Exclusion values were found to be 0.91, 0.62 and 13.28, respectively. Pattern of allelic frequencies of most of the microsatellite markers were clearly distinct between two breeds. As a result of present study a reliable, efficient and very informative panel of microsatellite markers was successfully developed which was capable to interpret individual identity, forensic cases and breed characterization in cattle. This facility is ready to be provided to local cattle breeder at commercial level for DNA testing of cattle. This study will also be highly helpful for breed conservation of cattle. In addition this study can also become a basis to open up new disciplines of animal forensics in Pakistan.

Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data

PubMed Central

Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A.; Larsen, Martin Jakob

2016-01-01

Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths. PMID:27002637

Newly identified mutations at the CSN1S1 gene in Ethiopian goats affect casein content and coagulation properties of their milk.

PubMed

Mestawet, T A; Girma, A; Adnøy, T; Devold, T G; Vegarud, G E

2013-08-01

Very high casein content and good coagulation properties previously observed in some Ethiopian goat breeds led to investigating the αs1-casein (CSN1S1) gene in these breeds. Selected regions of the CSN1S1 gene were sequenced in 115 goats from 5 breeds (2 indigenous: Arsi-Bale and Somali, 1 exotic: Boer, and 2 crossbreeds: Boer × Arsi-Bale and Boer × Somali). The DNA analysis resulted in 35 new mutations: 3 in exons, 3 in the 5' untranslated region (UTR), and 29 in the introns. The mutations in exons that resulted in an amino acid shift were then picked to evaluate their influence on individual casein content (αs1-, αs2-, β-, and κ-CN), micellar size, and coagulation properties in the milk from the 5 goat breeds. A mutation at nucleotide 10657 (exon 10) involved a transversion: CAG→CCG, resulting in an amino acid exchange Gln77→Pro77. This mutation was associated with the indigenous breeds only. Two new mutations, at nucleotide 6072 (exon 4) and 12165 (exon 12), revealed synonymous transitions: GTC→GTT in Val15 and AGA→AGG in Arg100 of the mature protein. Transitions G→A and C→T at nucleotides 1374 and 1866, respectively, occurred in the 5' UTR, whereas the third mutation involved a transversion T→G at nucleotide location 1592. The goats were grouped into homozygote new (CC), homozygote reference (AA), and heterozygote (CA) based on the nucleotide that involved the transversion. The content of αs1-CN (15.32g/kg) in milk samples of goats homozygous (CC) for this newly identified mutation, Gln77→Pro77 was significantly higher than in milks of heterozygous (CA; 9.05g/kg) and reference (AA; 7.61g/kg) genotype animals. The αs2-, β-, and κ-CN contents showed a similar pattern. Milk from goats with a homozygous new mutation had significantly lower micellar size. Milk from both homozygote and heterozygote new-mutation goats had significantly shorter coagulation rate and stronger gel than the reference genotype. Except the transversion, the

Genomic Footprints in Selected and Unselected Beef Cattle Breeds in Korea.

PubMed

Lim, Dajeong; Strucken, Eva M; Choi, Bong Hwan; Chai, Han Ha; Cho, Yong Min; Jang, Gul Won; Kim, Tae-Hun; Gondro, Cedric; Lee, Seung Hwan

2016-01-01

Korean Hanwoo cattle have been subjected to intensive artificial selection over the past four decades to improve meat production traits. Another three cattle varieties very closely related to Hanwoo reside in Korea (Jeju Black and Brindle) and in China (Yanbian). These breeds have not been part of a breeding scheme to improve production traits. Here, we compare the selected Hanwoo against these similar but presumed to be unselected populations to identify genomic regions that have been under recent selection pressure due to the breeding program. Rsb statistics were used to contrast the genomes of Hanwoo versus a pooled sample of the three unselected population (UN). We identified 37 significant SNPs (FDR corrected) in the HW/UN comparison and 21 known protein coding genes were within 1 MB to the identified SNPs. These genes were previously reported to affect traits important for meat production (14 genes), reproduction including mammary gland development (3 genes), coat color (2 genes), and genes affecting behavioral traits in a broader sense (2 genes). We subsequently sequenced (Illumina HiSeq 2000 platform) 10 individuals of the brown Hanwoo and the Chinese Yanbian to identify SNPs within the candidate genomic regions. Based on allele frequency differences, haplotype structures, and literature research, we singled out one non-synonymous SNP in the APP gene (APP: c.569C>T, Ala199Val) and predicted the mutational effect on the protein structure. We found that protein-protein interactions might be impaired due to increased exposed hydrophobic surfaces of the mutated protein. The APP gene has also been reported to affect meat tenderness in pigs and obesity in humans. Meat tenderness has been linked to intramuscular fat content, which is one of the main breeding goals for brown Hanwoo, potentially supporting a causal influence of the herein described nsSNP in the APP gene.

Human Cytochrome P450 2E1 Mutations That Alter Mitochondrial Targeting Efficiency and Susceptibility to Ethanol-induced Toxicity in Cellular Models*

PubMed Central

Bansal, Seema; Anandatheerthavarada, Hindupur K.; Prabu, Govindaswamy K.; Milne, Ginger L.; Martin, Martha V.; Guengerich, F. Peter; Avadhani, Narayan G.

2013-01-01

Human polymorphisms in the 5′-upstream regulatory regions and also protein coding regions of cytochrome P450 2E1 (CYP2E1) are known to be associated with several diseases, including cancer and alcohol liver toxicity. In this study, we report novel mutations in the N-terminal protein targeting regions of CYP2E1 that markedly affect subcellular localization of the protein. Variant W23R/W30R protein (termed W23/30R) is preferentially targeted to mitochondria but very poorly to the endoplasmic reticulum, whereas the L32N protein is preferentially targeted to the endoplasmic reticulum and poorly to mitochondria. These results explain the physiological significance of bimodal CYP targeting to the endoplasmic reticulum and mitochondria previously described. COS-7 cells and HepG2 cells stably expressing W23/30R mutations showed markedly increased alcohol toxicity in terms of increased production of reactive oxygen species, respiratory dysfunction, and loss of cytochrome c oxidase subunits and activity. Stable cells expressing the L32N variant, on the other hand, were relatively less responsive to alcohol-induced toxicity and mitochondrial dysfunction. These results further support our previous data, based on mutational studies involving altered targeting, indicating that mitochondria-targeted CYP2E1 plays an important role in alcohol liver toxicity. The results also provide an interesting new link to genetic variations affecting subcellular distribution of CYP2E1 with alcohol-induced toxicity. PMID:23471973

Novel mutation in the replication focus targeting sequence domain of DNMT1 causes hereditary sensory and autonomic neuropathy IE.

PubMed

Yuan, Junhui; Higuchi, Yujiro; Nagado, Tatsui; Nozuma, Satoshi; Nakamura, Tomonori; Matsuura, Eiji; Hashiguchi, Akihiro; Sakiyama, Yusuke; Yoshimura, Akiko; Takashima, Hiroshi

2013-03-01

DNMT1, encoding DNA methyltransferase 1 (Dnmt1), is a critical enzyme which is mainly responsible for conversion of unmethylated DNA into hemimethylated DNA. To date, two phenotypes produced by DNMT1 mutations have been reported, including hereditary sensory and autonomic neuropathy (HSAN) type IE with mutations in exon 20, and autosomal dominant cerebellar ataxia, deafness, and narcolepsy caused by mutations in exon 21. We report a sporadic case in a Japanese patient with loss of pain and vibration sense, chronic osteomyelitis, autonomic system dysfunctions, hearing loss, and mild dementia, but without definite cerebellar ataxia. Electrophysiological studies revealed absent sensory nerve action potential with nearly normal motor nerve conduction studies. Brain magnetic resonance imaging revealed mild diffuse cerebral and cerebellar atrophy. Using a next-generation sequencing system, 16 candidate genes were analyzed and a novel missense mutation, c.1706A>G (p.His569Arg), was identified in exon 21 of DNMT1. Our findings suggest that mutation in exon 21 of DNMT1 may also produce a HSAN phenotype. Because all reported mutations of DNMT1 are concentrated in exons 20 and 21, which encode the replication focus targeting sequence (RFTS) domain of Dnmt1, the RFTS domain could be a mutation hot spot. © 2013 Peripheral Nerve Society.

A critical functional missense mutation (H173R) in the bovine PROP1 gene significantly affects growth traits in cattle.

PubMed

Pan, Chuanying; Wu, Chongyang; Jia, Wenchao; Xu, Yao; Lei, Chuzhao; Hu, Shenrong; Lan, Xianyong; Chen, Hong

2013-12-01

The PROP1 protein, encoded by the prophet of Pit-1 (PROP1) gene, exhibits both DNA-binding and transcriptional activation abilities. Its expression leads to the ontogenesis of growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH), and pituitary hormone. The missense mutation H173R in PROP1 may result in deficiencies of GH, PRL, TSH, and Pit-1, thereby affecting growth traits. The objective of this study was to characterize the H173R mutation within the PROP1 gene and examine its associations with growth traits in cattle. Accordingly, the H173R mutation was genotyped in 1207 cows belonging to five Chinese native breeds. Three genotypes were identified among the specimens, with genotype AA being the major one. Consequently, the "G" allele was the minor allele. Association testing revealed that the H173R mutation was significantly associated with body weight, average daily weight gain and physical parameters in the analyzed breeds. Interestingly, the cows with genotype AG and/or AA had superior growth traits compared with those expressing the GG genotype, in all tested breeds. These findings revealed that the "A" allele had positive effects on growth traits, which was consistent with the increasing binding ability and enhanced activation capacity associated with the bovine isoform PROP1-173H, representing the "A" allele. Therefore, the H173R mutation can be considered as a DNA marker for selecting individuals with superior growth traits, thereby contributing to research on breeding and genetics in the beef industry. © 2013.

Human deafness mutation E385D disrupts the mechanochemical coupling and subcellular targeting of myosin-1a.

PubMed

Yengo, Christopher M; Ananthanarayanan, Shobana K; Brosey, Chris A; Mao, Suli; Tyska, Matthew J

2008-01-15

Missense mutations in the membrane-binding actin-based motor protein, myosin-1a (Myo1a), have recently been linked to sensorineural deafness in humans. One of these mutations, E385D, impacts a residue in the switch II region of the motor domain that is present in virtually all members of the myosin superfamily. We sought to examine the impact of E385D on the function of Myo1a, both in terms of mechanochemical activity and ability to target to actin-rich microvilli in polarized epithelial cells. While E385D-Myo1a demonstrated actin-activated ATPase activity, the V(MAX) was reduced threefold relative to wild-type. Despite maintaining an active mechanochemical cycle, E385D-Myo1a was unable to move actin in the sliding filament assay. Intriguingly, when an enhanced-green-fluorescent-protein-tagged form of E385D-Myo1a was stably expressed in polarized epithelial cells, this mutation abolished the microvillar targeting normally demonstrated by wild-type Myo1a. Notably, these data are the first to suggest that mechanical activity is essential for proper localization of Myo1a in microvilli. These studies also provide a unique example of how even the most mild substitution of invariant switch II residues can effectively uncouple enzymatic and mechanical activity of the myosin motor domain.

Targeted sequencing identifies associations between IL7R-JAK mutations and epigenetic modulators in T-cell acute lymphoblastic leukemia

PubMed Central

Vicente, Carmen; Schwab, Claire; Broux, Michaël; Geerdens, Ellen; Degryse, Sandrine; Demeyer, Sofie; Lahortiga, Idoya; Elliott, Alannah; Chilton, Lucy; La Starza, Roberta; Mecucci, Cristina; Vandenberghe, Peter; Goulden, Nicholas; Vora, Ajay; Moorman, Anthony V.; Soulier, Jean; Harrison, Christine J.; Clappier, Emmanuelle; Cools, Jan

2015-01-01

T-cell acute lymphoblastic leukemia is caused by the accumulation of multiple oncogenic lesions, including chromosomal rearrangements and mutations. To determine the frequency and co-occurrence of mutations in T-cell acute lymphoblastic leukemia, we performed targeted re-sequencing of 115 genes across 155 diagnostic samples (44 adult and 111 childhood cases). NOTCH1 and CDKN2A/B were mutated/deleted in more than half of the cases, while an additional 37 genes were mutated/deleted in 4% to 20% of cases. We found that IL7R-JAK pathway genes were mutated in 27.7% of cases, with JAK3 mutations being the most frequent event in this group. Copy number variations were also detected, including deletions of CREBBP or CTCF and duplication of MYB. FLT3 mutations were rare, but a novel extracellular mutation in FLT3 was detected and confirmed to be transforming. Furthermore, we identified complex patterns of pairwise associations, including a significant association between mutations in IL7R-JAK genes and epigenetic regulators (WT1, PRC2, PHF6). Our analyses showed that IL7R-JAK genetic lesions did not confer adverse prognosis in T-cell acute lymphoblastic leukemia cases enrolled in the UK ALL2003 trial. Overall, these results identify interconnections between the T-cell acute lymphoblastic leukemia genome and disease biology, and suggest a potential clinical application for JAK inhibitors in a significant proportion of patients with T-cell acute lymphoblastic leukemia. PMID:26206799

Genome characterization of Sugarcane Yellow Leaf Virus with special reference to RNAi based molecular breeding.

PubMed

Khalil, Farghama; Yueyu, Xu; Naiyan, Xiao; Di, Liu; Tayyab, Muhammad; Hengbo, Wang; Islam, Waqar; Rauf, Saeed; Pinghua, Chen

2018-05-04

Sugarcane is an essential crop for sugar and biofuel. Globally, its production is severely affected by sugarcane yellow leaf disease (SCYLD) caused by Sugarcane Yellow Leaf Virus (SCYLV). Many aphid vectors are involved in the spread of the disease which reduced the effectiveness of cultural and chemical management. Empirical methods of plant breeding such as introgression from wild and cultivated germplasm were not possible or at least challenging due to the absence of resistance in cultivated and wild germplasm of sugarcane. RNA interference (RNAi) transformation is an effective method to create virus-resistant varieties. Nevertheless, limited progress has been made due to lack of comprehensive research program on SCYLV based on RNAi technique. In order to show improvement and to propose future strategies for the feasibility of the RNAi technique to cope SCYLV, genome-wide consensus sequences of SCYLV were analyzed through GenBank. The coverage rates of every consensus sequence in SCYLV isolates were calculated to evaluate their practicability. Our analysis showed that single consensus sequence from SCYLV could not work well for RNAi based sugarcane breeding programs. This may be due to high mutation rate and continuous recombination within and between various viral strains. Alternative multi-target RNAi strategy is suggested to combat several strains of the viruses and to reduce the silencing escape. The multi-target small interfering RNA (siRNA) can be used together to construct RNAi plant expression plasmid, and to transform sugarcane tissues to develop new sugarcane varieties resistant to SCYLV. Copyright © 2018 Elsevier Ltd. All rights reserved.

Targeted next-generation sequencing reveals novel USH2A mutations associated with diverse disease phenotypes: implications for clinical and molecular diagnosis.

PubMed

Chen, Xue; Sheng, Xunlun; Liu, Xiaoxing; Li, Huiping; Liu, Yani; Rong, Weining; Ha, Shaoping; Liu, Wenzhou; Kang, Xiaoli; Zhao, Kanxing; Zhao, Chen

2014-01-01

USH2A mutations have been implicated in the disease etiology of several inherited diseases, including Usher syndrome type 2 (USH2), nonsyndromic retinitis pigmentosa (RP), and nonsyndromic deafness. The complex genetic and phenotypic spectrums relevant to USH2A defects make it difficult to manage patients with such mutations. In the present study, we aim to determine the genetic etiology and to characterize the correlated clinical phenotypes for three Chinese pedigrees with nonsyndromic RP, one with RP sine pigmento (RPSP), and one with USH2. Family histories and clinical details for all included patients were reviewed. Ophthalmic examinations included best corrected visual acuities, visual field measurements, funduscopy, and electroretinography. Targeted next-generation sequencing (NGS) was applied using two sequence capture arrays to reveal the disease causative mutations for each family. Genotype-phenotype correlations were also annotated. Seven USH2A mutations, including four missense substitutions (p.P2762A, p.G3320C, p.R3719H, and p.G4763R), two splice site variants (c.8223+1G>A and c.8559-2T>C), and a nonsense mutation (p.Y3745*), were identified as disease causative in the five investigated families, of which three reported to have consanguineous marriage. Among all seven mutations, six were novel, and one was recurrent. Two homozygous missense mutations (p.P2762A and p.G3320C) were found in one individual family suggesting a potential double hit effect. Significant phenotypic divergences were revealed among the five families. Three families of the five families were affected with early, moderated, or late onset RP, one with RPSP, and the other one with USH2. Our study expands the genotypic and phenotypic variability relevant to USH2A mutations, which would help with a clear insight into the complex genetic and phenotypic spectrums relevant to USH2A defects, and is complementary for a better management of patients with such mutations. We have also

Targeted Next-Generation Sequencing Reveals Novel USH2A Mutations Associated with Diverse Disease Phenotypes: Implications for Clinical and Molecular Diagnosis

PubMed Central

Li, Huiping; Liu, Yani; Rong, Weining; Ha, Shaoping; Liu, Wenzhou; Kang, Xiaoli; Zhao, Kanxing; Zhao, Chen

2014-01-01

USH2A mutations have been implicated in the disease etiology of several inherited diseases, including Usher syndrome type 2 (USH2), nonsyndromic retinitis pigmentosa (RP), and nonsyndromic deafness. The complex genetic and phenotypic spectrums relevant to USH2A defects make it difficult to manage patients with such mutations. In the present study, we aim to determine the genetic etiology and to characterize the correlated clinical phenotypes for three Chinese pedigrees with nonsyndromic RP, one with RP sine pigmento (RPSP), and one with USH2. Family histories and clinical details for all included patients were reviewed. Ophthalmic examinations included best corrected visual acuities, visual field measurements, funduscopy, and electroretinography. Targeted next-generation sequencing (NGS) was applied using two sequence capture arrays to reveal the disease causative mutations for each family. Genotype-phenotype correlations were also annotated. Seven USH2A mutations, including four missense substitutions (p.P2762A, p.G3320C, p.R3719H, and p.G4763R), two splice site variants (c.8223+1G>A and c.8559-2T>C), and a nonsense mutation (p.Y3745*), were identified as disease causative in the five investigated families, of which three reported to have consanguineous marriage. Among all seven mutations, six were novel, and one was recurrent. Two homozygous missense mutations (p.P2762A and p.G3320C) were found in one individual family suggesting a potential double hit effect. Significant phenotypic divergences were revealed among the five families. Three families of the five families were affected with early, moderated, or late onset RP, one with RPSP, and the other one with USH2. Our study expands the genotypic and phenotypic variability relevant to USH2A mutations, which would help with a clear insight into the complex genetic and phenotypic spectrums relevant to USH2A defects, and is complementary for a better management of patients with such mutations. We have also

Consequences of splitting whole-genome sequencing effort over multiple breeds on imputation accuracy.

PubMed

Bouwman, Aniek C; Veerkamp, Roel F

2014-10-03

The aim of this study was to determine the consequences of splitting sequencing effort over multiple breeds for imputation accuracy from a high-density SNP chip towards whole-genome sequence. Such information would assist for instance numerical smaller cattle breeds, but also pig and chicken breeders, who have to choose wisely how to spend their sequencing efforts over all the breeds or lines they evaluate. Sequence data from cattle breeds was used, because there are currently relatively many individuals from several breeds sequenced within the 1,000 Bull Genomes project. The advantage of whole-genome sequence data is that it carries the causal mutations, but the question is whether it is possible to impute the causal variants accurately. This study therefore focussed on imputation accuracy of variants with low minor allele frequency and breed specific variants. Imputation accuracy was assessed for chromosome 1 and 29 as the correlation between observed and imputed genotypes. For chromosome 1, the average imputation accuracy was 0.70 with a reference population of 20 Holstein, and increased to 0.83 when the reference population was increased by including 3 other dairy breeds with 20 animals each. When the same amount of animals from the Holstein breed were added the accuracy improved to 0.88, while adding the 3 other breeds to the reference population of 80 Holstein improved the average imputation accuracy marginally to 0.89. For chromosome 29, the average imputation accuracy was lower. Some variants benefitted from the inclusion of other breeds in the reference population, initially determined by the MAF of the variant in each breed, but even Holstein specific variants did gain imputation accuracy from the multi-breed reference population. This study shows that splitting sequencing effort over multiple breeds and combining the reference populations is a good strategy for imputation from high-density SNP panels towards whole-genome sequence when reference

Whole-comparative genomic hybridization in domestic sheep (Ovis aries) breeds.

PubMed

Dávila-Rodríguez, M I; Cortés-Gutiérrez, E I; López-Fernández, C; Pita, M; Mezzanotte, R; Gosálvez, J

2009-01-01

Whole-comparative genomic hybridization (W-CGH) allows identification of chromosomal polymorphisms related to highly repetitive DNA sequences localized in constitutive heterochromatin. Such polymorphisms are detected establishing competition between genomic DNAs in an in situ hybridization environment without subtraction of highly repetitive DNA sequences, when comparing two species from closely related taxa (same species, sub-species, or breeds) or somewhat related taxa. This experimental approach was applied to investigating differences in highly repetitive sequences of three sheep breeds (Castellana, Ojalada, and Assaf). To this end, W-CGH was carried out using mouflon (sheep ancestor) chromosomes as a common target to co-hybridize equimolar quantities of two genomic DNAs obtained from either Castellana, Ojalada or Assaf sheep breeds. The results showed that the amount of constitutive heterochromatin is greater in all pericentromeric heterochromatin regions of acrocentric chromosomes than in metacentric or sex chromosomes. Additionally, when W-CGH was performed using DNAs from the Iberian breeds Castellana and Ojalada, chromosomal pericentromeric regions revealed quantitatively and qualitatively a presence of DNA families similar to that obtained from any of the above-cited breeds. On the contrary, when the DNA used in W-CGH experiments was obtained from Assaf, as compared to either Castellana or Ojalada, two different pericentromeric DNA families of highly repetitive sequences could be detected. Lastly, sex chromosomes were shown to be homogeneous among all breeds and thus revealed no detectable constitutive heterochromatin. W-CGH results were confirmed using DNA breakage detection-FISH experiments (DBD-FISH) carried out on lymphocytes. As a whole, the results showed that two different repetitive DNA families are present in the pericentromeric heterochromatin of the sheep breeds studied here. Additionally, they suggest a differential presence of these distinct

Osimertinib, a third-generation tyrosine kinase inhibitor targeting non-small cell lung cancer with EGFR T790M mutations.

PubMed

McCoach, C E; Jimeno, A

2016-10-01

Oncogenic driver mutations in the epidermal growth factor receptor (EGFR) gene have provided a focus for effective targeted therapy. Unfortunately, all patients eventually develop resistance to frontline therapy with EGFR tyrosine kinase inhibitors (TKIs). The majority of patients develop a large subclonal population of tumor cells with a T790M mutation that renders these cells resistant to first-generation TKIs. Osimertinib is a third-generation EGFR TKI that was designed to overcome resistance from T790M mutations. This agent has demonstrated strong preclinical activity, and in the clinic it has demonstrated a high objective response rate and progression-free survival in patients with EGFR double mutations (L858R/T790M and exon 19 deletion/T790M). It is now approved by the FDA for patients who have a documented T790M mutation and who have progressed on a prior TKI. Osimertinib is also approved in the E.U. and Japan. Copyright 2016 Prous Science, S.A.U. or its licensors. All rights reserved.

A new approach for breeding low-temperature-resistant Volvariella volvacea strains: Genome shuffling in edible fungi.

PubMed

Zhu, Ziping; Wu, Xiao; Lv, Beibei; Wu, Guogan; Wang, Jinbin; Jiang, Wei; Li, Peng; He, Jianhua; Chen, Jianzhong; Chen, Mingjie; Bao, Dapeng; Zhang, Jinsong; Tan, Qi; Tang, Xueming

2016-09-01

Volvariella volvacea is difficult to store fresh because of the lack of low-temperature resistance. Many traditional mutagenic strategies have been applied in order to select out strains resistant to low temperature, but few commercially efficient strains have been produced. In order to break through the bottleneck of traditional breeding and significantly improve low-temperature resistance of the edible fungus V. volvacea, strains resistant to low temperature were constructed by genome shuffling. The optimum conditions of V. volvacea strain mutation, protoplast regeneration, and fusion were determined. After protoplasts were treated with 1% (v/v) ethylmethylsulfonate (EMS), 40 Sec of ultraviolet (UV) irradiation, 600 Gy electron beam implantation, and 750 Gy 60 Co-γ irradiation, separately, the lethality was within 70%-80%, which favored generating protoplasts being used in following forward mutation. Under these conditions, 16 strains of V. volvacea mutated by EMS, electron beam, UV irradiation, and 60 Co-γ irradiation were obtained. The 16 mutated protoplasts were selected to serve as the shuffling pool based on their excellent low-temperature resistance. After four rounds of genome shuffling and low-temperature resistance testing, three strains (VF 1 , VF 2 , and VF 3 ) with high genetic stability were screened. VF 1 , VF 2 , and VF 3 significantly enhanced fruit body shelf life to 20, 28, and 28 H at 10 °C, respectively, which exceeded 25%, 75%, and 75%, respectively, compared with the storage time of V23, the most low-temperature-resistant strain. Genome shuffling greatly improved the low-temperature resistance of V. volvacea, and shortened the course of screening required to generate desirable strains. To our knowledge, this is the first paper to apply genome shuffling to breeding new varieties of mushroom, and offers a new approach for breeding edible fungi with optimized phenotype. © 2015 International Union of Biochemistry and Molecular Biology

Towards social acceptance of plant breeding by genome editing.

PubMed

Araki, Motoko; Ishii, Tetsuya

2015-03-01

Although genome-editing technologies facilitate efficient plant breeding without introducing a transgene, it is creating indistinct boundaries in the regulation of genetically modified organisms (GMOs). Rapid advances in plant breeding by genome-editing require the establishment of a new global policy for the new biotechnology, while filling the gap between process-based and product-based GMO regulations. In this Opinion article we review recent developments in producing major crops using genome-editing, and we propose a regulatory model that takes into account the various methodologies to achieve genetic modifications as well as the resulting types of mutation. Moreover, we discuss the future integration of genome-editing crops into society, specifically a possible response to the 'Right to Know' movement which demands labeling of food that contains genetically engineered ingredients. Copyright © 2015 Elsevier Ltd. All rights reserved.

IQCB1 and PDE6B Mutations Cause Similar Early Onset Retinal Degenerations in Two Closely Related Terrier Dog Breeds

PubMed Central

Goldstein, Orly; Mezey, Jason G.; Schweitzer, Peter A.; Boyko, Adam R.; Gao, Chuan; Bustamante, Carlos D.; Jordan, Julie Ann; Aguirre, Gustavo D.; Acland, Gregory M.

2013-01-01

Purpose. To identify the causative mutations in two early-onset canine retinal degenerations, crd1 and crd2, segregating in the American Staffordshire terrier and the Pit Bull Terrier breeds, respectively. Methods. Retinal morphology of crd1- and crd2-affected dogs was evaluated by light microscopy. DNA was extracted from affected and related unaffected controls. Association analysis was undertaken using the Illumina Canine SNP array and PLINK (crd1 study), or the Affymetrix Version 2 Canine array, the “MAGIC” genotype algorithm, and Fisher's Exact test for association (crd2 study). Positional candidate genes were evaluated for each disease. Results. Structural photoreceptor abnormalities were observed in crd1-affected dogs as young as 11-weeks old. Rod and cone inner segment (IS) and outer segments (OS) were abnormal in size, shape, and number. In crd2-affected dogs, rod and cone IS and OS were abnormal as early as 3 weeks of age, progressing with age to severe loss of the OS, and thinning of the outer nuclear layer (ONL) by 12 weeks of age. Genome-wide association study (GWAS) identified association at the telomeric end of CFA3 in crd1-affected dogs and on CFA33 in crd2-affected dogs. Candidate gene evaluation identified a three bases deletion in exon 21 of PDE6B in crd1-affected dogs, and a cytosine insertion in exon 10 of IQCB1 in crd2-affected dogs. Conclusions. Identification of the mutations responsible for these two early-onset retinal degenerations provides new large animal models for comparative disease studies and evaluation of potential therapeutic approaches for the homologous human diseases. PMID:24045995

Signalling and chemosensitivity assays in melanoma: is mutated status a prerequisite for targeted therapy?

PubMed

Passeron, Thierry; Lacour, Jean-Philippe; Allegra, Maryline; Ségalen, Coralie; Deville, Anne; Thyss, Antoine; Giacchero, Damien; Ortonne, Jean-Paul; Bertolotto, Corine; Ballotti, Robert; Bahadoran, Philippe

2011-12-01

Selection for targeted therapies in melanoma is currently based on the search for mutations in selected genes. We aimed at evaluating the interest of signalling and chemosensitivity studies in addition to genotyping for assessing the best suitable treatment in an individual patient. We extracted genomic DNA and melanoma cells from tumor tissue of a skin metastasis of a 17-year-old woman with stage IV melanoma progressing despite three successive lines of treatment. Despite the absence of mutation in BRAF, NRAS cKIT, the MAPK pathway was activated and a significant response to sorafenib, a mitogen-activated protein kinase (MAPK)/RAF inhibitor, was found in signalling and chemosensitivity assays. A treatment combining sorafenib and dacarbazine produced a partial response for 9 months, with marked necrosis in some lesions. Chemosensitivity assays and signalling pathway studies could be of great value in addition to genotyping for assessing the most appropriate treatment in melanoma. © 2011 John Wiley & Sons A/S.

Chemicals or mutations that target mitochondrial translation can rescue the respiratory deficiency of yeast bcs1 mutants.

PubMed

Panozzo, C; Laleve, A; Tribouillard-Tanvier, D; Ostojić, J; Sellem, C H; Friocourt, G; Bourand-Plantefol, A; Burg, A; Delahodde, A; Blondel, M; Dujardin, G

2017-12-01

Bcs1p is a chaperone that is required for the incorporation of the Rieske subunit within complex III of the mitochondrial respiratory chain. Mutations in the human gene BCS1L (BCS1-like) are the most frequent nuclear mutations resulting in complex III-related pathologies. In yeast, the mimicking of some pathogenic mutations causes a respiratory deficiency. We have screened chemical libraries and found that two antibiotics, pentamidine and clarithromycin, can compensate two bcs1 point mutations in yeast, one of which is the equivalent of a mutation found in a human patient. As both antibiotics target the large mtrRNA of the mitoribosome, we focused our analysis on mitochondrial translation. We found that the absence of non-essential translation factors Rrf1 or Mif3, which act at the recycling/initiation steps, also compensates for the respiratory deficiency of yeast bcs1 mutations. At compensating concentrations, both antibiotics, as well as the absence of Rrf1, cause an imbalanced synthesis of respiratory subunits which impairs the assembly of the respiratory complexes and especially that of complex IV. Finally, we show that pentamidine also decreases the assembly of complex I in nematode mitochondria. It is well known that complexes III and IV exist within the mitochondrial inner membrane as supramolecular complexes III 2 /IV in yeast or I/III 2 /IV in higher eukaryotes. Therefore, we propose that the changes in mitochondrial translation caused by the drugs or by the absence of translation factors, can compensate for bcs1 mutations by modifying the equilibrium between illegitimate, and thus inactive, and active supercomplexes. Copyright © 2017. Published by Elsevier B.V.

Aggressive natural killer-cell leukemia mutational landscape and drug profiling highlight JAK-STAT signaling as therapeutic target.

PubMed

Dufva, Olli; Kankainen, Matti; Kelkka, Tiina; Sekiguchi, Nodoka; Awad, Shady Adnan; Eldfors, Samuli; Yadav, Bhagwan; Kuusanmäki, Heikki; Malani, Disha; Andersson, Emma I; Pietarinen, Paavo; Saikko, Leena; Kovanen, Panu E; Ojala, Teija; Lee, Dean A; Loughran, Thomas P; Nakazawa, Hideyuki; Suzumiya, Junji; Suzuki, Ritsuro; Ko, Young Hyeh; Kim, Won Seog; Chuang, Shih-Sung; Aittokallio, Tero; Chan, Wing C; Ohshima, Koichi; Ishida, Fumihiro; Mustjoki, Satu

2018-04-19

Aggressive natural killer-cell (NK-cell) leukemia (ANKL) is an extremely aggressive malignancy with dismal prognosis and lack of targeted therapies. Here, we elucidate the molecular pathogenesis of ANKL using a combination of genomic and drug sensitivity profiling. We study 14 ANKL patients using whole-exome sequencing (WES) and identify mutations in STAT3 (21%) and RAS-MAPK pathway genes (21%) as well as in DDX3X (29%) and epigenetic modifiers (50%). Additional alterations include JAK-STAT copy gains and tyrosine phosphatase mutations, which we show recurrent also in extranodal NK/T-cell lymphoma, nasal type (NKTCL) through integration of public genomic data. Drug sensitivity profiling further demonstrates the role of the JAK-STAT pathway in the pathogenesis of NK-cell malignancies, identifying NK cells to be highly sensitive to JAK and BCL2 inhibition compared to other hematopoietic cell lineages. Our results provide insight into ANKL genetics and a framework for application of targeted therapies in NK-cell malignancies.

Genetic diversity of mtDNA D-loop sequences in four native Chinese chicken breeds.

PubMed

Guo, H W; Li, C; Wang, X N; Li, Z J; Sun, G R; Li, G X; Liu, X J; Kang, X T; Han, R L

2017-10-01

1. To explore the genetic diversity of Chinese indigenous chicken breeds, a 585 bp fragment of the mitochondrial DNA (mtDNA) region was sequenced in 102 birds from the Xichuan black-bone chicken, Yunyang black-bone chicken and Lushi chicken. In addition, 30 mtDNA D-loop sequences of Silkie fowls were downloaded from NCBI. The mtDNA D-loop sequence polymorphism and maternal origin of 4 chicken breeds were analysed in this study. 2. The results showed that a total of 33 mutation sites and 28 haplotypes were detected in the 4 chicken breeds. The haplotype diversity and nucleotide diversity of these 4 native breeds were 0.916 ± 0.014 and 0.012 ± 0.002, respectively. Three clusters were formed in 4 Chinese native chickens and 12 reference breeds. Both the Xichuan black-bone chicken and Yunyang black-bone chicken were grouped into one cluster. Four haplogroups (A, B, C and E) emerged in the median-joining network in these breeds. 3. It was concluded that these 4 Chinese chicken breeds had high genetic diversity. The phylogenetic tree and median network profiles showed that Chinese native chickens and its neighbouring countries had at least two maternal origins, one from Yunnan, China and another from Southeast Asia or its surrounding area.

Both TALENs and CRISPR/Cas9 directly target the HBB IVS2-654 (C > T) mutation in β-thalassemia-derived iPSCs.

PubMed

Xu, Peng; Tong, Ying; Liu, Xiu-zhen; Wang, Ting-ting; Cheng, Li; Wang, Bo-yu; Lv, Xiang; Huang, Yue; Liu, De-pei

2015-07-09

β-Thalassemia is one of the most common genetic blood diseases and is caused by either point mutations or deletions in the β-globin (HBB) gene. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations may be a potential therapeutic strategy for this disease. Due to the low efficiency of typical homologous recombination, endonucleases, including TALENs and CRISPR/Cas9, have been widely used to enhance the gene correction efficiency in patient-derived iPSCs. Here, we designed TALENs and CRISPR/Cas9 to directly target the intron2 mutation site IVS2-654 in the globin gene. We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor. In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs. Finally, TALENs-corrected iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system and detected relatively higher transcription of HBB than the uncorrected cells. This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases.

Use of mutation spectra analysis software.

PubMed

Rogozin, I; Kondrashov, F; Glazko, G

2001-02-01

The study and comparison of mutation(al) spectra is an important problem in molecular biology, because these spectra often reflect on important features of mutations and their fixation. Such features include the interaction of DNA with various mutagens, the function of repair/replication enzymes, and properties of target proteins. It is known that mutability varies significantly along nucleotide sequences, such that mutations often concentrate at certain positions, called "hotspots," in a sequence. In this paper, we discuss in detail two approaches for mutation spectra analysis: the comparison of mutation spectra with a HG-PUBL program, (FTP: sunsite.unc.edu/pub/academic/biology/dna-mutations/hyperg) and hotspot prediction with the CLUSTERM program (www.itba.mi.cnr.it/webmutation; ftp.bionet.nsc.ru/pub/biology/dbms/clusterm.zip). Several other approaches for mutational spectra analysis, such as the analysis of a target protein structure, hotspot context revealing, multiple spectra comparisons, as well as a number of mutation databases are briefly described. Mutation spectra in the lacI gene of E. coli and the human p53 gene are used for illustration of various difficulties of such analysis. Copyright 2001 Wiley-Liss, Inc.

Detection of EGFR Gene Mutation by Mutation-oriented LAMP Method.

PubMed

Matsumoto, Naoyuki; Kumasaka, Akira; Ando, Tomohiro; Komiyama, Kazuo

2018-04-01

Epidermal growth factor receptor (EGFR) is a target of molecular therapeutics for non-small cell lung cancer. EGFR gene mutations at codons 746-753 promote constitutive EGFR activation and result in worst prognosis. However, these mutations augment the therapeutic effect of EGFR-tyrosine kinase inhibitor. Therefore, the detection of EGFR gene mutations is important for determining treatment planning. The aim of the study was to establish a method to detect EGFR gene mutations at codons 746-753. EGFR gene mutation at codons 746-753 in six cancer cell lines were investigated. A loop-mediated isothermal amplification (LAMP)-based procedure was developed, that employed peptide nucleic acid to suppress amplification of the wild-type allele. This mutation-oriented LAMP can amplify the DNA fragment of the EGFR gene with codons 746-753 mutations within 30 min. Moreover, boiled cells can work as template resources. Mutation oriented-LAMP assay for EGFR gene mutation is sensitive on extracted DNA. This procedure would be capable of detecting EGFR gene mutation in sputum, pleural effusion, broncho-alveolar lavage fluid or trans-bronchial lung biopsy by chair side. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Targeted prostate cancer screening in BRCA1 and BRCA2 mutation carriers: results from the initial screening round of the IMPACT study.

PubMed

Bancroft, Elizabeth K; Page, Elizabeth C; Castro, Elena; Lilja, Hans; Vickers, Andrew; Sjoberg, Daniel; Assel, Melissa; Foster, Christopher S; Mitchell, Gillian; Drew, Kate; Mæhle, Lovise; Axcrona, Karol; Evans, D Gareth; Bulman, Barbara; Eccles, Diana; McBride, Donna; van Asperen, Christi; Vasen, Hans; Kiemeney, Lambertus A; Ringelberg, Janneke; Cybulski, Cezary; Wokolorczyk, Dominika; Selkirk, Christina; Hulick, Peter J; Bojesen, Anders; Skytte, Anne-Bine; Lam, Jimmy; Taylor, Louise; Oldenburg, Rogier; Cremers, Ruben; Verhaegh, Gerald; van Zelst-Stams, Wendy A; Oosterwijk, Jan C; Blanco, Ignacio; Salinas, Monica; Cook, Jackie; Rosario, Derek J; Buys, Saundra; Conner, Tom; Ausems, Margreet G; Ong, Kai-ren; Hoffman, Jonathan; Domchek, Susan; Powers, Jacquelyn; Teixeira, Manuel R; Maia, Sofia; Foulkes, William D; Taherian, Nassim; Ruijs, Marielle; Helderman-van den Enden, Apollonia T; Izatt, Louise; Davidson, Rosemarie; Adank, Muriel A; Walker, Lisa; Schmutzler, Rita; Tucker, Kathy; Kirk, Judy; Hodgson, Shirley; Harris, Marion; Douglas, Fiona; Lindeman, Geoffrey J; Zgajnar, Janez; Tischkowitz, Marc; Clowes, Virginia E; Susman, Rachel; Ramón y Cajal, Teresa; Patcher, Nicholas; Gadea, Neus; Spigelman, Allan; van Os, Theo; Liljegren, Annelie; Side, Lucy; Brewer, Carole; Brady, Angela F; Donaldson, Alan; Stefansdottir, Vigdis; Friedman, Eitan; Chen-Shtoyerman, Rakefet; Amor, David J; Copakova, Lucia; Barwell, Julian; Giri, Veda N; Murthy, Vedang; Nicolai, Nicola; Teo, Soo-Hwang; Greenhalgh, Lynn; Strom, Sara; Henderson, Alex; McGrath, John; Gallagher, David; Aaronson, Neil; Ardern-Jones, Audrey; Bangma, Chris; Dearnaley, David; Costello, Philandra; Eyfjord, Jorunn; Rothwell, Jeanette; Falconer, Alison; Gronberg, Henrik; Hamdy, Freddie C; Johannsson, Oskar; Khoo, Vincent; Kote-Jarai, Zsofia; Lubinski, Jan; Axcrona, Ulrika; Melia, Jane; McKinley, Joanne; Mitra, Anita V; Moynihan, Clare; Rennert, Gad; Suri, Mohnish; Wilson, Penny; Killick, Emma; Moss, Sue; Eeles, Rosalind A

2014-09-01

Men with germline breast cancer 1, early onset (BRCA1) or breast cancer 2, early onset (BRCA2) gene mutations have a higher risk of developing prostate cancer (PCa) than noncarriers. IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in BRCA1/2 mutation carriers and controls) is an international consortium of 62 centres in 20 countries evaluating the use of targeted PCa screening in men with BRCA1/2 mutations. To report the first year's screening results for all men at enrollment in the study. We recruited men aged 40-69 yr with germline BRCA1/2 mutations and a control group of men who have tested negative for a pathogenic BRCA1 or BRCA2 mutation known to be present in their families. All men underwent prostate-specific antigen (PSA) testing at enrollment, and those men with PSA >3 ng/ml were offered prostate biopsy. PSA levels, PCa incidence, and tumour characteristics were evaluated. The Fisher exact test was used to compare the number of PCa cases among groups and the differences among disease types. We recruited 2481 men (791 BRCA1 carriers, 531 BRCA1 controls; 731 BRCA2 carriers, 428 BRCA2 controls). A total of 199 men (8%) presented with PSA >3.0 ng/ml, 162 biopsies were performed, and 59 PCas were diagnosed (18 BRCA1 carriers, 10 BRCA1 controls; 24 BRCA2 carriers, 7 BRCA2 controls); 66% of the tumours were classified as intermediate- or high-risk disease. The positive predictive value (PPV) for biopsy using a PSA threshold of 3.0 ng/ml in BRCA2 mutation carriers was 48%-double the PPV reported in population screening studies. A significant difference in detecting intermediate- or high-risk disease was observed in BRCA2 carriers. Ninety-five percent of the men were white, thus the results cannot be generalised to all ethnic groups. The IMPACT screening network will be useful for targeted PCa screening studies in men with germline genetic risk variants as they are discovered. These preliminary results support the

Targeted Prostate Cancer Screening in BRCA1 and BRCA2 Mutation Carriers: Results from the Initial Screening Round of the IMPACT Study

PubMed Central

Bancroft, Elizabeth K.; Page, Elizabeth C.; Castro, Elena; Lilja, Hans; Vickers, Andrew; Sjoberg, Daniel; Assel, Melissa; Foster, Christopher S.; Mitchell, Gillian; Drew, Kate; Mæhle, Lovise; Axcrona, Karol; Evans, D. Gareth; Bulman, Barbara; Eccles, Diana; McBride, Donna; van Asperen, Christi; Vasen, Hans; Kiemeney, Lambertus A.; Ringelberg, Janneke; Cybulski, Cezary; Wokolorczyk, Dominika; Selkirk, Christina; Hulick, Peter J.; Bojesen, Anders; Skytte, Anne-Bine; Lam, Jimmy; Taylor, Louise; Oldenburg, Rogier; Cremers, Ruben; Verhaegh, Gerald; van Zelst-Stams, Wendy A.; Oosterwijk, Jan C.; Blanco, Ignacio; Salinas, Monica; Cook, Jackie; Rosario, Derek J.; Buys, Saundra; Conner, Tom; Ausems, Margreet G.; Ong, Kai-ren; Hoffman, Jonathan; Domchek, Susan; Powers, Jacquelyn; Teixeira, Manuel R.; Maia, Sofia; Foulkes, William D.; Taherian, Nassim; Ruijs, Marielle; den Enden, Apollonia T. Helderman-van; Izatt, Louise; Davidson, Rosemarie; Adank, Muriel A.; Walker, Lisa; Schmutzler, Rita; Tucker, Kathy; Kirk, Judy; Hodgson, Shirley; Harris, Marion; Douglas, Fiona; Lindeman, Geoffrey J.; Zgajnar, Janez; Tischkowitz, Marc; Clowes, Virginia E.; Susman, Rachel; Ramón y Cajal, Teresa; Patcher, Nicholas; Gadea, Neus; Spigelman, Allan; van Os, Theo; Liljegren, Annelie; Side, Lucy; Brewer, Carole; Brady, Angela F.; Donaldson, Alan; Stefansdottir, Vigdis; Friedman, Eitan; Chen-Shtoyerman, Rakefet; Amor, David J.; Copakova, Lucia; Barwell, Julian; Giri, Veda N.; Murthy, Vedang; Nicolai, Nicola; Teo, Soo-Hwang; Greenhalgh, Lynn; Strom, Sara; Henderson, Alex; McGrath, John; Gallagher, David; Aaronson, Neil; Ardern-Jones, Audrey; Bangma, Chris; Dearnaley, David; Costello, Philandra; Eyfjord, Jorunn; Rothwell, Jeanette; Falconer, Alison; Gronberg, Henrik; Hamdy, Freddie C.; Johannsson, Oskar; Khoo, Vincent; Kote-Jarai, Zsofia; Lubinski, Jan; Axcrona, Ulrika; Melia, Jane; McKinley, Joanne; Mitra, Anita V.; Moynihan, Clare; Rennert, Gad; Suri, Mohnish; Wilson, Penny; Killick, Emma; Moss, Sue; Eeles, Rosalind A.

2014-01-01

Background Men with germline breast cancer 1, early onset (BRCA1) or breast cancer 2, early onset (BRCA2) gene mutations have a higher risk of developing prostate cancer (PCa) than noncarriers. IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in BRCA1/2 mutation carriers and controls) is an international consortium of 62 centres in 20 countries evaluating the use of targeted PCa screening in men with BRCA1/2 mutations. Objective To report the first year's screening results for all men at enrolment in the study. Design, setting and participants We recruited men aged 40–69 yr with germline BRCA1/2 mutations and a control group of men who have tested negative for a pathogenic BRCA1 or BRCA2 mutation known to be present in their families. All men underwent prostate-specific antigen (PSA) testing at enrolment, and those men with PSA >3 ng/ml were offered prostate biopsy. Outcome measurements and statistical analysis PSA levels, PCa incidence, and tumour characteristics were evaluated. The Fisher exact test was used to compare the number of PCa cases among groups and the differences among disease types. Results and limitations We recruited 2481 men (791 BRCA1 carriers, 531 BRCA1 controls; 731 BRCA2 carriers, 428 BRCA2 controls). A total of 199 men (8%) presented with PSA >3.0 ng/ml, 162 biopsies were performed, and 59 PCas were diagnosed (18 BRCA1 carriers, 10 BRCA1 controls; 24 BRCA2 carriers, 7 BRCA2 controls); 66% of the tumours were classified as intermediate- or high-risk disease. The positive predictive value (PPV) for biopsy using a PSA threshold of 3.0 ng/ml in BRCA2 mutation carriers was 48%—double the PPV reported in population screening studies. A significant difference in detecting intermediate- or high-risk disease was observed in BRCA2 carriers. Ninety-five percent of the men were white, thus the results cannot be generalised to all ethnic groups. Conclusions The IMPACT screening network will be useful

[Status of traditional Chinese medicine materials seed and seedling breeding bases].

PubMed

Li, Ying; Huang, Lu-Qi; Zhang, Xiao-Bo; Wang, Hui; Cheng, Meng; Zhang, Tian; Yang, Guang

2017-11-01

Seeds and seedlings are the material basis of traditional Chinese medicine materials production, and the construction of traditional Chinese medicine materials seed and seedling breeding bases is beneficial to the production of high-quality traditional Chinese medicine materials. The construction of traditional Chinese medicine materials seed and seedling breeding bases is one of the major topics of Chinese medica resources census pilot. Targets, tasks of traditional Chinese medicine materials seed and seedling breeding bases based on Chinese medica resources census pilot were expounded.Construction progress including hardware construction, germplasm conservation and breeding, procedures and standardsestablishment, social servicesare presented. Development counter measures were proposed for the next step: perfect the standard and system, maintain and strengthen the breeding function, strengthen the cultivation of multi-level talents, explore market development model, joint efforts to deepen services and development. Copyright© by the Chinese Pharmaceutical Association.

Use of bacterial artificial chromosomes in generating targeted mutations in human and mouse cytomegaloviruses.

PubMed

Borst, Eva Maria; Benkartek, Corinna; Messerle, Martin

2007-05-01

Cloning of cytomegalovirus (CMV) genomes as bacterial artificial chromosomes (BAC) in E. coli and their manipulation using the techniques of bacterial genetics has greatly facilitated the construction of CMV mutants. This unit describes easily applicable procedures that allow rapid introduction of any kind of targeted mutation into BAC-cloned CMV genomes. Protocols for the reconstitution of virus from isolated BAC DNA, preparation of a virus stock, and isolation and characterization of viral DNA are also included. Special emphasis is laid on description of critical steps and thorough characterization of the altered BACs.

HER2 mutations in lung adenocarcinomas: A report from the Lung Cancer Mutation Consortium.

PubMed

Pillai, Rathi N; Behera, Madhusmita; Berry, Lynne D; Rossi, Mike R; Kris, Mark G; Johnson, Bruce E; Bunn, Paul A; Ramalingam, Suresh S; Khuri, Fadlo R

2017-11-01

Human epidermal growth factor receptor 2 (HER2) mutations have been reported in lung adenocarcinomas. Herein, the authors describe the prevalence, clinical features, and outcomes associated with HER2 mutations in 1007 patients in the Lung Cancer Mutation Consortium (LCMC). Patients with advanced-stage lung adenocarcinomas were enrolled to the LCMC. Tumor specimens were assessed for diagnosis and adequacy; multiplexed genotyping was performed in Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories to examine 10 oncogenic drivers. The LCMC database was queried for patients with HER2 mutations to access demographic data, treatment history, and vital status. An exploratory analysis was performed to evaluate the survival of patients with HER2 mutations who were treated with HER2-directed therapies. A total of 920 patients were tested for HER2 mutations; 24 patients (3%) harbored exon 20 insertion mutations (95% confidence interval, 2%-4%). One patient had a concurrent mesenchymal-epithelial transition factor (MET) amplification. The median age of the patients was 62 years, with a slight predominance of females over males (14 females vs 10 males). The majority of the patients were never-smokers (71%) and presented with advanced disease at the time of diagnosis. The median survival for patients who received HER2-targeted therapies (12 patients) was 2.1 years compared with 1.4 years for those who did not (12 patients) (P = .48). Patients with HER2 mutations were found to have inferior survival compared with the rest of the LCMC cohort with other mutations: the median survival was 3.5 years in the LCMC population receiving targeted therapy and 2.4 years for patients not receiving targeted therapy. HER2 mutations were detected in 3% of patients with lung adenocarcinoma in the LCMC. HER2-directed therapies should be investigated in this subgroup of patients. Cancer 2017;123:4099-4105. © 2017 American Cancer Society. © 2017 American Cancer Society.

Functional validation of the oncogenic cooperativity and targeting potential of tuberous sclerosis mutation in medulloblastoma using a MYC-amplified model cell line.

PubMed

Henderson, Jacob J; Wagner, Jacob P; Hofmann, Nicolle E; Eide, Christopher A; Cho, Yoon-Jae; Druker, Brian J; Davare, Monika A

2017-10-01

Medulloblastoma is the most common malignant brain tumor of childhood. To identify targetable vulnerabilities, we employed inhibitor screening that revealed mTOR inhibitor hypersensitivity in the MYC-overexpressing medulloblastoma cell line, D341. Concomitant exome sequencing unveiled an uncharacterized missense mutation, TSC2 A415V , in these cells. We biochemically demonstrate that the TSC2 A415V mutation is functionally deleterious, leading to shortened half-life and proteasome-mediated protein degradation. These data suggest that MYC cooperates with activated kinase pathways, enabling pharmacologic intervention in these treatment refractory tumors. We propose that identification of activated kinase pathways may allow for tailoring targeted therapy to improve survival and treatment-related morbidity in medulloblastoma. © 2017 Wiley Periodicals, Inc.

Breed distribution of the ABCB1-1Delta (multidrug sensitivity) polymorphism among dogs undergoing ABCB1 genotyping.

PubMed

Mealey, Katrina L; Meurs, Kathryn M

2008-09-15

To evaluate the breed distribution of the ABCB1-1Delta polymorphism in a large number of dogs in North America, including dogs of several herding breeds in which this polymorphism has been detected and other breeds in which this polymorphism has not yet been identified. Cross-sectional study. 5,368 dogs from which buccal swab samples were collected for purposes of ABCB1 genotyping. From May 1, 2004, to September 30, 2007, DNA specimens derived from buccal swab samples collected from 5,368 dogs underwent ABCB1 genotyping. These data were reviewed, and results for each dog were recorded in a spreadsheet, along with the dog's breed. The genotypes for each breed were tallied by use of a sorting function. The ABCB1-1Delta allele was identified in 9 breeds of dogs and in many mixed-breed dogs. Breeds that had the ABCB1-1Delta allele included Collie, Longhaired Whippet, Australian Shepherd (standard and miniature), Shetland Sheepdog, Old English Sheepdog, Border Collie, Silken Windhound, and German Shepherd Dog (a breed in which this mutation had not been detected previously). The ABCB1-1Delta polymorphism is associated with increased susceptibility to many adverse drug reactions and with suppression of the hypothalamic-pituitary-adrenal axis and is present in many herding breeds of dog. Veterinarians should be familiar with the breeds that have the ABCB1-1Delta polymorphism to make appropriate pharmacologic choices for these patients.

CREBBP mutations in relapsed acute lymphoblastic leukaemia

PubMed Central

Mullighan, Charles G.; Zhang, Jinghui; Kasper, Lawryn H.; Lerach, Stephanie; Payne-Turner, Debbie; Phillips, Letha A.; Heatley, Sue L.; Holmfeldt, Linda; Collins-Underwood, J. Racquel; Ma, Jing; Buetow, Kenneth H.; Pui, Ching-Hon; Baker, Sharyn D.; Brindle, Paul K.; Downing, James R.

2010-01-01

Relapsed acute lymphoblastic leukaemia (ALL) is a leading cause of death due to disease in young people, but the biologic determinants of treatment failure remain poorly understood. Recent genome-wide profiling of structural DNA alterations in ALL have identified multiple submicroscopic somatic mutations targeting key cellular pathways1,2, and have demonstrated substantial evolution in genetic alterations from diagnosis to relapse3. However, detailed analysis of sequence mutations in ALL has not been performed. To identify novel mutations in relapsed ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including the transcriptional coactivators CREBBP and NCOR1, the transcription factors ERG, SPI1, TCF4 and TCF7L2, components of the Ras signalling pathway, histone genes, genes involved in histone modification (CREBBP and CTCF), and genes previously shown to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 71 diagnosis-relapse cases and 270 acute leukaemia cases that did not relapse found that 18.3% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase (HAT) CREB-binding protein (CBP)4. The mutations were either present at diagnosis or acquired at relapse, and resulted in truncated alleles or deleterious substitutions in conserved residues of the HAT domain. Functionally, the mutations impaired histone acetylation and transcriptional regulation of CREBBP targets, including glucocorticoid responsive genes. Several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations may confer resistance to therapy. These results extend the landscape of genetic alterations in leukaemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism

Target Acquired: Progress and Promise of Targeted Therapeutics in the Treatment of Prostate Cancer.

PubMed

Stuchbery, Ryan; Kurganovs, Natalie J; McCoy, Patrick J; Nelson, Colleen C; Hayes, Vanessa M; Corcoran, Niall M; Hovens, Christopher M

2015-01-01

Cancer is fundamentally a genomic disease caused by mutations or rearrangements in the DNA or epigenetic machinery of a patient. An emerging field in cancer treatment targets key aberrations arising from the mutational landscape of an individual patient's disease rather than employing a cancer-wide cytotoxic therapy approach. In prostate cancer in particular, where there is an observed variation in response to standard treatments between patients with disease of a similar pathological stage and grade, mutationdirected treatment may grow to be a viable tool for clinicians to tailor more effective treatments. This review will describe a number of mutations across multiple forms of cancer that have been successfully antagonised by targeted therapeutics including their identification, the development of targeted compounds to combat them and the development of resistance to these therapies. This review will continue to examine these same mutations in the treatment and management of prostate cancer; the prevalence of targetable mutations in prostate cancer, recent clinical trials of targeted-agents and the potential or limitations for their use.

A large indel mutation of the bovine ADD1/SREBP1c gene and its effects on growth traits in some native cattle breeds from China.

PubMed

Huang, Yong-Zhen; Zhang, En-Ping; Wang, Jing; Huai, Yong-Tao; Ma, Liang; Chen, Fu-Ying; Lan, Xian-Yong; Lei, Chu-Zhao; Fang, Xing-Tang; Wang, Ju-Qiang; Chen, Hong

2011-03-01

Adipocyte determination and differentiation-dependent factor 1/sterol regulatory element-binding protein-1c (ADD1/SREBP1c) is a major determinant of tissue differential lipogenic capacity in mammalian and avian species. The objectives of the present study were to focus on insertion-deletion polymorphism (indel) in the bovine ADD1/SREBP1c gene, and analyzing its effect on growth traits in a sample of 1035 cattle belonging to four Chinese cattle breeds. PCR-SSCP, DNA sequencing and agarose electrophoresis methods were used. The 778 bp PCR products of ADD1/SREBP1c gene exhibited three genotypes and two alleles were revealed: W and D. Frequencies of the W allele varied from 0.8651 to 1.000. The associations of the 84 bp indel mutation of ADD1/SREBP1c gene with growth traits of 265 Nanyang cows were analyzed. The animals with genotype WD had significantly higher birth weight, body weight, average daily gain than those with genotype WW at birth, 6-, 12-, 18-, and 24-month old (P < 0.05 or P < 0.01). These results suggest that the indel mutation of bovine ADD1/SREBP1c gene may influence the growth traits in cattle.

Optimization of routine KRAS mutation PCR-based testing procedure for rational individualized first-line-targeted therapy selection in metastatic colorectal cancer.

PubMed

Chretien, Anne-Sophie; Harlé, Alexandre; Meyer-Lefebvre, Magali; Rouyer, Marie; Husson, Marie; Ramacci, Carole; Harter, Valentin; Genin, Pascal; Leroux, Agnès; Merlin, Jean-Louis

2013-02-01

KRAS mutation detection represents a crucial issue in metastatic colorectal cancer (mCRC). The optimization of KRAS mutation detection delay enabling rational prescription of first-line treatment in mCRC including anti-EGFR-targeted therapy requires robust and rapid molecular biology techniques. Routine analysis of mutations in codons 12 and 13 on 674 paraffin-embedded tissue specimens of mCRC has been performed for KRAS mutations detection using three molecular biology techniques, that is, high-resolution melting (HRM), polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), and allelic discrimination PCR (TaqMan PCR). Discordant cases were assessed with COBAS 4800 KRAS CE-IVD assay. Among the 674 tumor specimens, 1.5% (10/674) had excessive DNA degradation and could not be analyzed. KRAS mutations were detected in 38.0% (256/674) of the analysable specimens (82.4% in codon 12 and 17.6% in codon 13). Among 613 specimens in whom all three techniques were used, 12 (2.0%) cases of discordance between the three techniques were observed. 83.3% (10/12) of the discordances were due to PCR-RFLP as confirmed by COBAS 4800 retrospective analysis. The three techniques were statistically comparable (κ > 0.9; P < 0.001). From these results, optimization of the routine procedure consisted of proceeding to systematic KRAS detection using HRM and TaqMan and PCR-RFLP in case of discordance and allowed significant decrease in delays. The results showed an excellent correlation between the three techniques. Using HRM and TaqMan warrants high-quality and rapid-routine KRAS mutation detection in paraffin-embedded tumor specimens. The new procedure allowed a significant decrease in delays for reporting results, enabling rational prescription of first-line-targeted therapy in mCRC.

A novel mouse model identifies cooperating mutations and therapeutic targets critical for chronic myeloid leukemia progression

PubMed Central

Giotopoulos, George; van der Weyden, Louise; Osaki, Hikari; Rust, Alistair G.; Gallipoli, Paolo; Meduri, Eshwar; Horton, Sarah J.; Chan, Wai-In; Foster, Donna; Prinjha, Rab K.; Pimanda, John E.; Tenen, Daniel G.; Vassiliou, George S.; Koschmieder, Steffen; Adams, David J.

2015-01-01

The introduction of highly selective ABL-tyrosine kinase inhibitors (TKIs) has revolutionized therapy for chronic myeloid leukemia (CML). However, TKIs are only efficacious in the chronic phase of the disease and effective therapies for TKI-refractory CML, or after progression to blast crisis (BC), are lacking. Whereas the chronic phase of CML is dependent on BCR-ABL, additional mutations are required for progression to BC. However, the identity of these mutations and the pathways they affect are poorly understood, hampering our ability to identify therapeutic targets and improve outcomes. Here, we describe a novel mouse model that allows identification of mechanisms of BC progression in an unbiased and tractable manner, using transposon-based insertional mutagenesis on the background of chronic phase CML. Our BC model is the first to faithfully recapitulate the phenotype, cellular and molecular biology of human CML progression. We report a heterogeneous and unique pattern of insertions identifying known and novel candidate genes and demonstrate that these pathways drive disease progression and provide potential targets for novel therapeutic strategies. Our model greatly informs the biology of CML progression and provides a potent resource for the development of candidate therapies to improve the dismal outcomes in this highly aggressive disease. PMID:26304963

Prevalence of the AMHR2 mutation in Miniature Schnauzers and genetic investigation of a Belgian Malinois with persistent Müllerian duct syndrome.

PubMed

Smit, M M; Ekenstedt, K J; Minor, K M; Lim, C K; Leegwater, Paj; Furrow, E

2018-04-01

Persistent Müllerian duct syndrome (PMDS) is a sex-limited disorder in which males develop portions of the female reproductive tract. Important consequences of PMDS are cryptorchidism and its sequelae of infertility and increased risk of testicular cancer. Anti-Müllerian hormone (AMH) and its receptor (AMHR2) induce the regression of the Müllerian ducts in male embryos. In Miniature Schnauzer dogs, the genetic basis has been identified as an autosomal recessive nonsense mutation in AMHR2, but the allele frequency of the mutation is unknown. Thus, the primary objective of this study was to estimate the prevalence of the AMHR2 mutation in North American Miniature Schnauzers, in order to ascertain the value of genetic testing in this breed. An additional objective was to determine whether mutations in AMH or AMHR2 were responsible for PMDS in a Belgian Malinois; this would aid development of a genetic test for the Belgian Malinois breed. Genomic DNA from 216 Miniature Schnauzers (including one known PMDS case) was genotyped for the AMHR2 mutation, and DNA from a single PMDS-affected Belgian Malinois was sequenced for all coding exons of AMH and AMHR2. The Miniature Schnauzer cohort had an AMHR2 mutation allele frequency of 0.16 and a carrier genotypic frequency of 0.27. The genetic basis for PMDS in the Belgian Malinois was not determined, as no coding or splicing mutations were identified in either AMH or AMHR2. These findings support a benefit to AMHR2 mutation testing Miniature Schnauzers used for breeding or with cryptorchidism. © 2017 Blackwell Verlag GmbH.

Translating knowledge about abiotic stress tolerance to breeding programmes.

PubMed

Gilliham, Matthew; Able, Jason A; Roy, Stuart J

2017-06-01

Plant breeding and improvements in agronomic practice are making a consistent contribution to increasing global crop production year upon year. However, the rate of yield improvement currently lags behind the targets set to produce enough food to meet the demands of the predicted global population in 2050. Furthermore, crops that are exposed to harmful abiotic environmental factors (abiotic stresses, e.g. water limitation, salinity, extreme temperature) are prone to reduced yields. Here, we briefly describe the processes undertaken in conventional breeding programmes, which are usually designed to improve yields in near-optimal conditions rather than specifically breeding for improved crop yield stability under stressed conditions. While there is extensive fundamental research activity that examines mechanisms of plant stress tolerance, there are few examples that apply this research to improving commercial crop yields. There are notable exceptions, and we highlight some of these to demonstrate the magnitude of yield gains that could be made by translating agronomic, phenological and genetic solutions focused on improving or mitigating the effect of abiotic stress in the field; in particular, we focus on improvements in crop water-use efficiency and salinity tolerance. We speculate upon the reasons for the disconnect between research and research translation. We conclude that to realise untapped rapid gains towards food security targets new funding structures need to be embraced. Such funding needs to serve both the core and collaborative activities of the fundamental, pre-breeding and breeding research communities in order to expedite the translation of innovative research into the fields of primary producers. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

A missense mutation in MYH1 is associated with susceptibility to immune-mediated myositis in Quarter Horses.

PubMed

Finno, Carrie J; Gianino, Giuliana; Perumbakkam, Sudeep; Williams, Zoë J; Bordbari, Matthew H; Gardner, Keri L; Burns, Erin; Peng, Sichong; Durward-Akhurst, Sian A; Valberg, Stephanie J

2018-03-06

The cause of immune-mediated myositis (IMM), characterized by recurrent, rapid-onset muscle atrophy in Quarter Horses (QH), is unknown. The histopathologic hallmark of IMM is lymphocytic infiltration of myofibers. The purpose of this study was to identify putative functional variants associated with equine IMM. A genome-wide association (GWA) study was performed on 36 IMM QHs and 54 breed matched unaffected QHs from the same environment using the Equine SNP50 and SNP70 genotyping arrays. A mixed model analysis identified nine SNPs within a ~ 2.87 Mb region on chr11 that were significantly (P unadjusted  < 1.4 × 10 - 6 ) associated with the IMM phenotype. Associated haplotypes within this region encompassed 38 annotated genes, including four myosin genes (MYH1, MYH2, MYH3, and MYH13). Whole genome sequencing of four IMM and four unaffected QHs identified a single segregating nonsynonymous E321G mutation in MYH1 encoding myosin heavy chain 2X. Genotyping of additional 35 IMM and 22 unaffected QHs confirmed an association (P = 2.9 × 10 - 5 ), and the putative mutation was absent in 175 horses from 21 non-QH breeds. Lymphocytic infiltrates occurred in type 2X myofibers and the proportion of 2X fibers was decreased in the presence of inflammation. Protein modeling and contact/stability analysis identified 14 residues affected by the mutation which significantly decreased stability. We conclude that a mutation in MYH1 is highly associated with susceptibility to the IMM phenotype in QH-related breeds. This is the first report of a mutation in MYH1 and the first link between a skeletal muscle myosin mutation and autoimmune disease.

Investigation of Prolific Sheep from UK and Ireland for Evidence on Origin of the Mutations in BMP15 (FecXG, FecXB) and GDF9 (FecGH) in Belclare and Cambridge Sheep

PubMed Central

Mullen, Michael P.; Hanrahan, James P.; Howard, Dawn J.

2013-01-01

This paper concerns the likely origin of three mutations with large effects on ovulation rate identified in the Belclare and Cambridge sheep breeds; two in the BMP15 gene (FecXG and FecXB) and the third (FecGH) in GDF9. All three mutations segregate in Belclare sheep while one, FecXB, has not been found in the Cambridge. Both Belclare and Cambridge breeds are relatively recently developed composites that have common ancestry through the use of genetic material from the Finnish Landrace and Lleyn breeds. The development of both composites also involved major contributions from exceptionally prolific ewes screened from flocks in Ireland (Belclare) and Britain (Cambridge) during the 1960s. The objective of the current study was to establish the likely origin of the mutations (FecXG, FecXB and FecGH) through analysis of DNA from Finnish Landrace and Lleyn sheep, and Galway and Texel breeds which contributed to the development of the Belclare breed. Ewes with exceptionally high prolificacy (hyper-prolific ewes) in current flocks on Irish farms were identified to simulate the screening of ewes from Irish flocks in the 1960s. DNA was obtained from: prolific ewes in extant flocks of Lleyn sheep (n = 44) on the Lleyn peninsula in Wales; hyper-prolific ewes (n = 41); prolific Galway (n = 41) ewes; Finnish Landrace (n = 124) and Texel (n = 19) ewes. The FecXG mutation was identified in Lleyn but not in Finnish Landrace, Galway or Texel sheep; FecXB was only found among the hyper-prolific ewes. The FecGH mutation was identified in the sample of Lleyn sheep. It was concluded from these findings that the Lleyn breed was the most likely source of the FecXG and FecGH mutations in Belclare and Cambridge sheep and that the FecXB mutation came from the High Fertility line that was developed using prolific ewes selected from commercial flocks in Ireland in the 1960′s and subsequently used in the genesis of the Belclare. PMID:23301039

High-throughput discovery of mutations in tef semi-dwarfing genes by next-generation sequencing analysis.

PubMed

Zhu, Qihui; Smith, Shavannor M; Ayele, Mulu; Yang, Lixing; Jogi, Ansuya; Chaluvadi, Srinivasa R; Bennetzen, Jeffrey L

2012-11-01

Tef (Eragrostis tef) is a major cereal crop in Ethiopia. Lodging is the primary constraint to increasing productivity in this allotetraploid species, accounting for losses of ∼15-45% in yield each year. As a first step toward identifying semi-dwarf varieties that might have improved lodging resistance, an ∼6× fosmid library was constructed and used to identify both homeologues of the dw3 semi-dwarfing gene of Sorghum bicolor. An EMS mutagenized population, consisting of ∼21,210 tef plants, was planted and leaf materials were collected into 23 superpools. Two dwarfing candidate genes, homeologues of dw3 of sorghum and rht1 of wheat, were sequenced directly from each superpool with 454 technology, and 120 candidate mutations were identified. Out of 10 candidates tested, six independent mutations were validated by Sanger sequencing, including two predicted detrimental mutations in both dw3 homeologues with a potential to improve lodging resistance in tef through further breeding. This study demonstrates that high-throughput sequencing can identify potentially valuable mutations in under-studied plant species like tef and has provided mutant lines that can now be combined and tested in breeding programs for improved lodging resistance.

A COL7A1 Mutation Causes Dystrophic Epidermolysis Bullosa in Rotes Höhenvieh Cattle

PubMed Central

Menoud, Annie; Welle, Monika; Tetens, Jens; Lichtner, Peter; Drögemüller, Cord

2012-01-01

We identified a congenital mechanobullous skin disorder in six calves on a single farm of an endangered German cattle breed in 2010. The condition presented as a large loss of skin distal to the fetlocks and at the mucosa of the muzzle. All affected calves were euthanized on humane grounds due to the severity, extent and progression of the skin and oral lesions. Examination of skin samples under light microscopy revealed detachment of the epidermis from the dermis at the level of the dermo epidermal junction, leading to the diagnosis of a subepidermal bullous dermatosis such as epidermolysis bullosa. The pedigree was consistent with monogenic autosomal recessive inheritance. We localized the causative mutation to an 18 Mb interval on chromosome 22 by homozygosity mapping. The COL7A1 gene encoding collagen type VII alpha 1 is located within this interval and COL7A1 mutations have been shown to cause inherited dystrophic epidermolysis bullosa (DEB) in humans. A SNP in the bovine COL7A1 exon 49 (c.4756C>T) was perfectly associated with the observed disease. The homozygous mutant T/T genotype was exclusively present in affected calves and their parents were heterozygous C/T confirming the assumed recessive mode of inheritance. All known cases and genotyped carriers were related to a single cow, which is supposed to be the founder animal. The mutant T allele was absent in 63 animals from 24 cattle breeds. The identified mutation causes a premature stop codon which leads to a truncated protein representing a complete loss of COL7A1 function (p.R1586*). We thus have identified a candidate causative mutation for this genetic disease using only three cases to unravel its molecular basis. Selection against this mutation can now be used to eliminate the mutant allele from the Rotes Höhenvieh breed. PMID:22715415

Mutation site and context dependent effects of ESR1 mutation in genome-edited breast cancer cell models.

PubMed

Bahreini, Amir; Li, Zheqi; Wang, Peilu; Levine, Kevin M; Tasdemir, Nilgun; Cao, Lan; Weir, Hazel M; Puhalla, Shannon L; Davidson, Nancy E; Stern, Andrew M; Chu, David; Park, Ben Ho; Lee, Adrian V; Oesterreich, Steffi

2017-05-23

Mutations in the estrogen receptor alpha (ERα) 1 gene (ESR1) are frequently detected in ER+ metastatic breast cancer, and there is increasing evidence that these mutations confer endocrine resistance in breast cancer patients with advanced disease. However, their functional role is not well-understood, at least in part due to a lack of ESR1 mutant models. Here, we describe the generation and characterization of genome-edited T47D and MCF7 breast cancer cell lines with the two most common ESR1 mutations, Y537S and D538G. Genome editing was performed using CRISPR and adeno-associated virus (AAV) technologies to knock-in ESR1 mutations into T47D and MCF7 cell lines, respectively. Various techniques were utilized to assess the activity of mutant ER, including transactivation, growth and chromatin-immunoprecipitation (ChIP) assays. The level of endocrine resistance was tested in mutant cells using a number of selective estrogen receptor modulators (SERMs) and degraders (SERDs). RNA sequencing (RNA-seq) was employed to study gene targets of mutant ER. Cells with ESR1 mutations displayed ligand-independent ER activity, and were resistant to several SERMs and SERDs, with cell line and mutation-specific differences with respect to magnitude of effect. The SERD AZ9496 showed increased efficacy compared to other drugs tested. Wild-type and mutant cell co-cultures demonstrated a unique evolution of mutant cells under estrogen deprivation and tamoxifen treatment. Transcriptome analysis confirmed ligand-independent regulation of ERα target genes by mutant ERα, but also identified novel target genes, some of which are involved in metastasis-associated phenotypes. Despite significant overlap in the ligand-independent genes between Y537S and D538G, the number of mutant ERα-target genes shared between the two cell lines was limited, suggesting context-dependent activity of the mutant receptor. Some genes and phenotypes were unique to one mutation within a given cell line

[A study on space mutation of Streptomyces fradiae].

PubMed

Fang, Xiao-mei; Zhao, Zhi-jia; Gu, Hai-ke

2005-04-01

To study the rule of mutation of Streptomyces fradiae during spaceflight, and to select efficient tylosin producing strains for industrial production. Streptomyces fradiae 9940S(+)-86 were carried on-board spaceship "Shenzhou" I, "Shenzhou" III and "Shenzhou" IV sequentially to achieve spaceflight mutation breeding experiment. After space experiments and the screening tests in the lab, 48 strains were obtained which promoted production by +20% or more at shaker level. And the highest production of a strain was 14950 micrograms/ml, which means an increase of 91.5%. Comparing the results of three tests, it is found that the outer space environment can lead to a cumulative mutation. After the medium scale tests and production experiments, strain T1-156-84-23 was finally selected to be used for sample production. And its output was increased by 18%.

A Novel GUSB Mutation in Brazilian Terriers with Severe Skeletal Abnormalities Defines the Disease as Mucopolysaccharidosis VII

PubMed Central

Hytönen, Marjo K.; Arumilli, Meharji; Lappalainen, Anu K.; Kallio, Heli; Snellman, Marjatta; Sainio, Kirsi; Lohi, Hannes

2012-01-01

Hundreds of different human skeletal disorders have been characterized at molecular level and a growing number of resembling dysplasias with orthologous genetic defects are being reported in dogs. This study describes a novel genetic defect in the Brazilian Terrier breed causing a congenital skeletal dysplasia. Affected puppies presented severe skeletal deformities observable within the first month of life. Clinical characterization using radiographic and histological methods identified delayed ossification and spondyloepiphyseal dysplasia. Pedigree analysis suggested an autosomal recessive disorder, and we performed a genome-wide association study to map the disease locus using Illumina’s 22K SNP chip arrays in seven cases and eleven controls. A single association was observed near the centromeric end of chromosome 6 with a genome-wide significance after permutation (pgenome  = 0.033). The affected dogs shared a 13-Mb homozygous region including over 200 genes. A targeted next-generation sequencing of the entire locus revealed a fully segregating missense mutation (c.866C>T) causing a pathogenic p.P289L change in a conserved functional domain of β-glucuronidase (GUSB). The mutation was confirmed in a population of 202 Brazilian terriers (p = 7,71×10−29). GUSB defects cause mucopolysaccharidosis VII (MPS VII) in several species and define the skeletal syndrome in Brazilian Terriers. Our results provide new information about the correlation of the GUSB genotype to phenotype and establish a novel canine model for MPS VII. Currently, MPS VII lacks an efficient treatment and this model could be utilized for the development and validation of therapeutic methods for better treatment of MPS VII patients. Finally, since almost one third of the Brazilian terrier population carries the mutation, breeders will benefit from a genetic test to eradicate the detrimental disease from the breed. PMID:22815736

A study of therapy targeted EGFR/ALK mutations in Indian patients with lung adenocarcinoma: A clinical and epidemiological study.

PubMed

Rana, Vandana; Ranjan, Praveer; Jagani, Rajat; Rathi, K R; Kumar, Dharmesh; Khera, Anurag

2018-04-01

Established predictive biomarkers for Non-Small Cell Lung Carcinoma (NSCLC) include sensitizing Epidermal Growth Factor Receptor (EGFR) mutations and Anaplastic Lymphoma Kinase (ALK) fusion oncogene. The primary aim of the study is to ascertain the prevalence of EGFR mutation and ALK gene rearrangement in patients of lung adenocarcinoma in Indian population and the second objective is to impress upon the importance of adequate processing of limited tissue samples. Histopathologically confirmed cases of lung adenocarcinoma, whose tumour had been tested for both EGFR and ALK gene mutations, were included in this study. The EGFR mutations were analyzed using PCR and Gene Sequencing. ALK fusion oncogene was found by Fluorescence In Situ Hybridization (FISH) technique using kit of Vysis LSI ALK Dual colour Break Apart Rearrangement probe. A total of 152 cases of lung adenocarcinoma were included. Out of which, 92 (60.5%) were male and 60 (39.5%) were female. After exclusion of 17 cases due to unsatisfactory result, EGFR mutations were found positive in 35.5% cases (48/135). ALK gene rearrangement was found in 7.6% (10/131) after excluding 21 cases with unsatisfactory result. EGFR mutations and ALK gene rearrangement was found to be mutually exclusive. Incidence of EGFR mutations (35.5%) is much higher in Indian population than in Caucasians (13%) and is close to the incidence in East Asian countries. The 7.6% incidence of ALK fusion oncogene in Indian patients establishes the importance of molecular studies to give maximum benefit of targeted therapy to the patients.

Neuronal ceroid lipofuscinosis associated with an MFSD8 mutation in Chihuahuas.

PubMed

Ashwini, Akanksha; D'Angelo, Antonio; Yamato, Osamu; Giordano, Cristina; Cagnotti, Giulia; Harcourt-Brown, Tom; Mhlanga-Mutangadura, Tendai; Guo, Juyuan; Johnson, Gary S; Katz, Martin L

2016-08-01

The neuronal ceroid lipofuscinoses (NCLs) are hereditary neurodegenerative disorders characterized by progressive declines in neurological functions, seizures, and premature death. NCLs result from mutations in at least 13 different genes. Canine versions of the NCLs can serve as important models in developing effective therapeutic interventions for these diseases. NCLs have been described in a number of dog breeds, including Chihuahuas. Studies were undertaken to further characterize the pathology of Chihuahua NCL and to verify its molecular genetic basis. Four unrelated client owned Chihuahuas from Japan, Italy and England that exhibited progressive neurological signs consistent with a diagnosis of NCL underwent neurological examinations. Brain and in some cases also retinal and heart tissues were examined postmortem for the presence of lysosomal storage bodies characteristic of NCL. The affected dogs exhibited massive accumulation of autofluorescent lysosomal storage bodies in the brain, retina and heart accompanied by brain atrophy and retinal degeneration. The dogs were screened for known canine NCL mutations previously reported in a variety of dog breeds. All 4 dogs were homozygous for the MFSD8 single base pair deletion (MFSD8:c.843delT) previously associated with NCL in a Chinese Crested dog and in 2 affected littermate Chihuahuas from Scotland. The dogs were all homozygous for the normal alleles at the other genetic loci known to cause different forms of canine NCL. The MFSD8:c.843delT mutation was not present in 57 Chihuahuas that were either clinically normal or suffered from unrelated diseases or in 1761 unaffected dogs representing 186 other breeds. Based on these data it is almost certain that the MFSD8:c.843delT mutation is the cause of NCL in Chihuahuas. Because the disorder occurred in widely separated geographic locations or in unrelated dogs from the same country, it is likely that the mutant allele is widespread among Chihuahuas. Genetic testing

Whole-genome sequencing reveals mutational landscape underlying phenotypic differences between two widespread Chinese cattle breeds.

PubMed

Xu, Yao; Jiang, Yu; Shi, Tao; Cai, Hanfang; Lan, Xianyong; Zhao, Xin; Plath, Martin; Chen, Hong

2017-01-01

Whole-genome sequencing provides a powerful tool to obtain more genetic variability that could produce a range of benefits for cattle breeding industry. Nanyang (Bos indicus) and Qinchuan (Bos taurus) are two important Chinese indigenous cattle breeds with distinct phenotypes. To identify the genetic characteristics responsible for variation in phenotypes between the two breeds, in the present study, we for the first time sequenced the genomes of four Nanyang and four Qinchuan cattle with 10 to 12 fold on average of 97.86% and 98.98% coverage of genomes, respectively. Comparison with the Bos_taurus_UMD_3.1 reference assembly yielded 9,010,096 SNPs for Nanyang, and 6,965,062 for Qinchuan cattle, 51% and 29% of which were novel SNPs, respectively. A total of 154,934 and 115,032 small indels (1 to 3 bp) were found in the Nanyang and Qinchuan genomes, respectively. The SNP and indel distribution revealed that Nanyang showed a genetically high diversity as compared to Qinchuan cattle. Furthermore, a total of 2,907 putative cases of copy number variation (CNV) were identified by aligning Nanyang to Qinchuan genome, 783 of which (27%) encompassed the coding regions of 495 functional genes. The gene ontology (GO) analysis revealed that many CNV genes were enriched in the immune system and environment adaptability. Among several CNV genes related to lipid transport and fat metabolism, Lepin receptor gene (LEPR) overlapping with CNV_1815 showed remarkably higher copy number in Qinchuan than Nanyang (log2 (ratio) = -2.34988; P value = 1.53E-102). Further qPCR and association analysis investigated that the copy number of the LEPR gene presented positive correlations with transcriptional expression and phenotypic traits, suggesting the LEPR CNV may contribute to the higher fat deposition in muscles of Qinchuan cattle. Our findings provide evidence that the distinct phenotypes of Nanyang and Qinchuan breeds may be due to the different genetic variations including SNPs, indels

Whole-genome sequencing reveals mutational landscape underlying phenotypic differences between two widespread Chinese cattle breeds

PubMed Central

Jiang, Yu; Shi, Tao; Cai, Hanfang; Lan, Xianyong; Zhao, Xin; Plath, Martin; Chen, Hong

2017-01-01

Whole-genome sequencing provides a powerful tool to obtain more genetic variability that could produce a range of benefits for cattle breeding industry. Nanyang (Bos indicus) and Qinchuan (Bos taurus) are two important Chinese indigenous cattle breeds with distinct phenotypes. To identify the genetic characteristics responsible for variation in phenotypes between the two breeds, in the present study, we for the first time sequenced the genomes of four Nanyang and four Qinchuan cattle with 10 to 12 fold on average of 97.86% and 98.98% coverage of genomes, respectively. Comparison with the Bos_taurus_UMD_3.1 reference assembly yielded 9,010,096 SNPs for Nanyang, and 6,965,062 for Qinchuan cattle, 51% and 29% of which were novel SNPs, respectively. A total of 154,934 and 115,032 small indels (1 to 3 bp) were found in the Nanyang and Qinchuan genomes, respectively. The SNP and indel distribution revealed that Nanyang showed a genetically high diversity as compared to Qinchuan cattle. Furthermore, a total of 2,907 putative cases of copy number variation (CNV) were identified by aligning Nanyang to Qinchuan genome, 783 of which (27%) encompassed the coding regions of 495 functional genes. The gene ontology (GO) analysis revealed that many CNV genes were enriched in the immune system and environment adaptability. Among several CNV genes related to lipid transport and fat metabolism, Lepin receptor gene (LEPR) overlapping with CNV_1815 showed remarkably higher copy number in Qinchuan than Nanyang (log2 (ratio) = -2.34988; P value = 1.53E-102). Further qPCR and association analysis investigated that the copy number of the LEPR gene presented positive correlations with transcriptional expression and phenotypic traits, suggesting the LEPR CNV may contribute to the higher fat deposition in muscles of Qinchuan cattle. Our findings provide evidence that the distinct phenotypes of Nanyang and Qinchuan breeds may be due to the different genetic variations including SNPs, indels

A novel unstable duplication upstream of HAS2 predisposes to a breed-defining skin phenotype and a periodic fever syndrome in Chinese Shar-Pei dogs.

PubMed

Olsson, Mia; Meadows, Jennifer R S; Truvé, Katarina; Rosengren Pielberg, Gerli; Puppo, Francesca; Mauceli, Evan; Quilez, Javier; Tonomura, Noriko; Zanna, Giordana; Docampo, Maria José; Bassols, Anna; Avery, Anne C; Karlsson, Elinor K; Thomas, Anne; Kastner, Daniel L; Bongcam-Rudloff, Erik; Webster, Matthew T; Sanchez, Armand; Hedhammar, Ake; Remmers, Elaine F; Andersson, Leif; Ferrer, Lluis; Tintle, Linda; Lindblad-Toh, Kerstin

2011-03-01

Hereditary periodic fever syndromes are characterized by recurrent episodes of fever and inflammation with no known pathogenic or autoimmune cause. In humans, several genes have been implicated in this group of diseases, but the majority of cases remain unexplained. A similar periodic fever syndrome is relatively frequent in the Chinese Shar-Pei breed of dogs. In the western world, Shar-Pei have been strongly selected for a distinctive thick and heavily folded skin. In this study, a mutation affecting both these traits was identified. Using genome-wide SNP analysis of Shar-Pei and other breeds, the strongest signal of a breed-specific selective sweep was located on chromosome 13. The same region also harbored the strongest genome-wide association (GWA) signal for susceptibility to the periodic fever syndrome (p(raw) = 2.3 × 10⁻⁶, p(genome) = 0.01). Dense targeted resequencing revealed two partially overlapping duplications, 14.3 Kb and 16.1 Kb in size, unique to Shar-Pei and upstream of the Hyaluronic Acid Synthase 2 (HAS2) gene. HAS2 encodes the rate-limiting enzyme synthesizing hyaluronan (HA), a major component of the skin. HA is up-regulated and accumulates in the thickened skin of Shar-Pei. A high copy number of the 16.1 Kb duplication was associated with an increased expression of HAS2 as well as the periodic fever syndrome (p < 0.0001). When fragmented, HA can act as a trigger of the innate immune system and stimulate sterile fever and inflammation. The strong selection for the skin phenotype therefore appears to enrich for a pleiotropic mutation predisposing these dogs to a periodic fever syndrome. The identification of HA as a major risk factor for this canine disease raises the potential of this glycosaminoglycan as a risk factor for human periodic fevers and as an important driver of chronic inflammation.

A Novel Unstable Duplication Upstream of HAS2 Predisposes to a Breed-Defining Skin Phenotype and a Periodic Fever Syndrome in Chinese Shar-Pei Dogs

PubMed Central

Olsson, Mia; Mauceli, Evan; Quilez, Javier; Tonomura, Noriko; Zanna, Giordana; Docampo, Maria José; Bassols, Anna; Avery, Anne C.; Karlsson, Elinor K.; Thomas, Anne; Kastner, Daniel L.; Bongcam-Rudloff, Erik; Webster, Matthew T.; Sanchez, Armand; Hedhammar, Åke; Remmers, Elaine F.; Andersson, Leif; Ferrer, Lluis; Tintle, Linda; Lindblad-Toh, Kerstin

2011-01-01

Hereditary periodic fever syndromes are characterized by recurrent episodes of fever and inflammation with no known pathogenic or autoimmune cause. In humans, several genes have been implicated in this group of diseases, but the majority of cases remain unexplained. A similar periodic fever syndrome is relatively frequent in the Chinese Shar-Pei breed of dogs. In the western world, Shar-Pei have been strongly selected for a distinctive thick and heavily folded skin. In this study, a mutation affecting both these traits was identified. Using genome-wide SNP analysis of Shar-Pei and other breeds, the strongest signal of a breed-specific selective sweep was located on chromosome 13. The same region also harbored the strongest genome-wide association (GWA) signal for susceptibility to the periodic fever syndrome (praw = 2.3×10−6, pgenome = 0.01). Dense targeted resequencing revealed two partially overlapping duplications, 14.3 Kb and 16.1 Kb in size, unique to Shar-Pei and upstream of the Hyaluronic Acid Synthase 2 (HAS2) gene. HAS2 encodes the rate-limiting enzyme synthesizing hyaluronan (HA), a major component of the skin. HA is up-regulated and accumulates in the thickened skin of Shar-Pei. A high copy number of the 16.1 Kb duplication was associated with an increased expression of HAS2 as well as the periodic fever syndrome (p<0.0001). When fragmented, HA can act as a trigger of the innate immune system and stimulate sterile fever and inflammation. The strong selection for the skin phenotype therefore appears to enrich for a pleiotropic mutation predisposing these dogs to a periodic fever syndrome. The identification of HA as a major risk factor for this canine disease raises the potential of this glycosaminoglycan as a risk factor for human periodic fevers and as an important driver of chronic inflammation. PMID:21437276

"Boldness" in the domestic dog differs among breeds and breed groups.

PubMed

Starling, Melissa J; Branson, Nicholas; Thomson, Peter C; McGreevy, Paul D

2013-07-01

"Boldness" in dogs is believed to be one end of the shy-bold axis, representing a super-trait. Several personality traits fall under the influence of this super-trait. Previous studies on boldness in dogs have found differences among breeds, but grouping breeds on the basis of behavioural similarities has been elusive. This study investigated differences in the expression of boldness among dog breeds, kennel club breed groups, and sub-groups of kennel club breed groups by way of a survey on dog personality circulated among Australian dog-training clubs and internet forums and lists. Breed had a significant effect on boldness (F=1.63, numDF=111, denDF=272, p<0.001), as did breed group (F=10.66, numDF=8, denDF=772, p<0.001). Herding and gundog groups were broken into sub-groups based on historic breed purpose. Retrievers were significantly bolder than flushing and pointing breeds (Reg. Coef.=2.148; S.E.=0.593; p<0.001), and tending and loose-eyed herding breeds were bolder than heading and cattle-herding breeds (Reg. Coef.=1.744; S.E.=0.866; p=0.045 and Reg. Coef.=1.842; S.E.=0.693; p=0.0084, respectively). This study supports the existence of the shy-bold continuum in dogs. Differences in boldness among groups and sub-groups suggest that behavioural tendencies may be influenced by historical purpose regardless of whether that purpose still factors in selective breeding. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of allele-specific primer PCR for a swine TLR2 SNP and comparison of the frequency among several pig breeds of Japan and the Czech Republic.

PubMed

Muneta, Yoshihiro; Minagawa, Yu; Kusumoto, Masahiro; Shinkai, Hiroki; Uenishi, Hirohide; Splichal, Igor

2012-05-01

In the present study, we have developed an allele-specific primer-polymerase chain reaction (ASP-PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 2 (TLR2) (C406G), which is related to the prevalence of pneumonia caused by Mycoplasma hyopneumoniae. We also compared the allele frequency among several pig breeds of Japan and the Czech Republic. Allele-specific primers were constructed by introducing 1-base mismatch sequence before the SNP site. The swine TLR2 C406G mutation was successfully determined by the ASP-PCR using genomic DNA samples in Japan as previously genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from pig blood obtained from 110 pigs from 7 different breeds in the Czech Republic were genotyped by the ASP-PCR. The genotyping results from the ASP-PCR were completely matched with the results from the sequencing method. The allele frequency of the swine TLR2 C406G mutation was 27.5% in the Czech Republic and 3.6% in Japan. The C406G mutation was only found in the Landrace breed in Japan, and was almost exclusively found in the Landrace breed in the Czech Republic as well. These results indicated the usefulness of ASP-PCR for detecting a specific SNP for swine TLR2.

An Inversion Disrupting FAM134B Is Associated with Sensory Neuropathy in the Border Collie Dog Breed

PubMed Central

Forman, Oliver P.; Hitti, Rebekkah J.; Pettitt, Louise; Jenkins, Christopher A.; O’Brien, Dennis P.; Shelton, G. Diane; De Risio, Luisa; Quintana, Rodrigo Gutierrez; Beltran, Elsa; Mellersh, Cathryn

2016-01-01

Sensory neuropathy in the Border Collie is a severe neurological disorder caused by the degeneration of sensory and, to a lesser extent, motor nerve cells with clinical signs starting between 2 and 7 months of age. Using a genome-wide association study approach with three cases and 170 breed matched controls, a suggestive locus for sensory neuropathy was identified that was followed up using a genome sequencing approach. An inversion disrupting the candidate gene FAM134B was identified. Genotyping of additional cases and controls and RNAseq analysis provided strong evidence that the inversion is causal. Evidence of cryptic splicing resulting in novel exon transcription for FAM134B was identified by RNAseq experiments. This investigation demonstrates the identification of a novel sensory neuropathy associated mutation, by mapping using a minimal set of cases and subsequent genome sequencing. Through mutation screening, it should be possible to reduce the frequency of or completely eliminate this debilitating condition from the Border Collie breed population. PMID:27527794

Plant Glandular Trichomes as Targets for Breeding or Engineering of Resistance to Herbivores

PubMed Central

Glas, Joris J.; Schimmel, Bernardus C. J.; Alba, Juan M.; Escobar-Bravo, Rocío; Schuurink, Robert C.; Kant, Merijn R.

2012-01-01

Glandular trichomes are specialized hairs found on the surface of about 30% of all vascular plants and are responsible for a significant portion of a plant’s secondary chemistry. Glandular trichomes are an important source of essential oils, i.e., natural fragrances or products that can be used by the pharmaceutical industry, although many of these substances have evolved to provide the plant with protection against herbivores and pathogens. The storage compartment of glandular trichomes usually is located on the tip of the hair and is part of the glandular cell, or cells, which are metabolically active. Trichomes and their exudates can be harvested relatively easily, and this has permitted a detailed study of their metabolites, as well as the genes and proteins responsible for them. This knowledge now assists classical breeding programs, as well as targeted genetic engineering, aimed to optimize trichome density and physiology to facilitate customization of essential oil production or to tune biocide activity to enhance crop protection. We will provide an overview of the metabolic diversity found within plant glandular trichomes, with the emphasis on those of the Solanaceae, and of the tools available to manipulate their activities for enhancing the plant’s resistance to pests. PMID:23235331

Potential of gene drives with genome editing to increase genetic gain in livestock breeding programs.

PubMed

Gonen, Serap; Jenko, Janez; Gorjanc, Gregor; Mileham, Alan J; Whitelaw, C Bruce A; Hickey, John M

2017-01-04

This paper uses simulation to explore how gene drives can increase genetic gain in livestock breeding programs. Gene drives are naturally occurring phenomena that cause a mutation on one chromosome to copy itself onto its homologous chromosome. We simulated nine different breeding and editing scenarios with a common overall structure. Each scenario began with 21 generations of selection, followed by 20 generations of selection based on true breeding values where the breeder used selection alone, selection in combination with genome editing, or selection with genome editing and gene drives. In the scenarios that used gene drives, we varied the probability of successfully incorporating the gene drive. For each scenario, we evaluated genetic gain, genetic variance [Formula: see text], rate of change in inbreeding ([Formula: see text]), number of distinct quantitative trait nucleotides (QTN) edited, rate of increase in favourable allele frequencies of edited QTN and the time to fix favourable alleles. Gene drives enhanced the benefits of genome editing in seven ways: (1) they amplified the increase in genetic gain brought about by genome editing; (2) they amplified the rate of increase in the frequency of favourable alleles and reduced the time it took to fix them; (3) they enabled more rapid targeting of QTN with lesser effect for genome editing; (4) they distributed fixed editing resources across a larger number of distinct QTN across generations; (5) they focussed editing on a smaller number of QTN within a given generation; (6) they reduced the level of inbreeding when editing a subset of the sires; and (7) they increased the efficiency of converting genetic variation into genetic gain. Genome editing in livestock breeding results in short-, medium- and long-term increases in genetic gain. The increase in genetic gain occurs because editing increases the frequency of favourable alleles in the population. Gene drives accelerate the increase in allele frequency

Diagnostic algorithm for detection of targetable driver mutations in lung adenocarcinomas: Comprehensive analyses of 205 cases with immunohistochemistry, real-time PCR and fluorescence in situ hybridization methods.

PubMed

Kao, Hua-Lin; Yeh, Yi-Chen; Lin, Chin-Hsuan; Hsu, Wei-Fang; Hsieh, Wen-Yu; Ho, Hsiang-Ling; Chou, Teh-Ying

2016-11-01

Analysis of the targetable driver mutations is now recommended in all patients with advanced lung adenocarcinoma. Molecular-based methods are usually adopted, however, along with the implementation of highly sensitive and/or mutation-specific antibodies, immunohistochemistry (IHC) has been considered an alternative method for identifying driver mutations in lung adenocarcinomas. A total of 205 lung adenocarcinomas were examined for EGFR mutations and ALK and ROS1 rearrangements using real-time PCR, fluorescence in situ hybridization (FISH) and IHC in parallel. The performance of different commercially available IHC antibody clones toward targetable driver mutations was evaluated. The association between these driver mutations and clinicopathological characteristics was also analyzed. In 205 cases we studied, 58.5% were found to harbor EGFR mutations, 6.3% ALK rearrangements and 1.0% ROS1 rearrangements. Compared to molecular-based methods, IHC of EGFR mutations showed an excellent specificity but the sensitivity is suboptimal, while IHC of ALK and ROS1 rearrangements demonstrated high sensitivity and specificity. No significant difference regarding the performance of different antibody clones toward these driver mutations was observed, except that clone SP125 showed a higher sensitivity than 43B2 in the detection of p.L858R of EGFR. In circumstances such as poor quality of nucleic acids or low content of tumor cells, IHC of EGFR mutation-specific antibodies could be used as an alternative method. Patients negative for EGFR mutations are subjected to further analysis on ALK and ROS1 rearrangements using IHC methods. Herein, we proposed a lung adenocarcinoma testing algorithm for the application of IHC in therapeutic diagnosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Reverse breeding: a novel breeding approach based on engineered meiosis.

PubMed

Dirks, Rob; van Dun, Kees; de Snoo, C Bastiaan; van den Berg, Mark; Lelivelt, Cilia L C; Voermans, William; Woudenberg, Leo; de Wit, Jack P C; Reinink, Kees; Schut, Johan W; van der Zeeuw, Eveline; Vogelaar, Aat; Freymark, Gerald; Gutteling, Evert W; Keppel, Marina N; van Drongelen, Paul; Kieny, Matthieu; Ellul, Philippe; Touraev, Alisher; Ma, Hong; de Jong, Hans; Wijnker, Erik

2009-12-01

Reverse breeding (RB) is a novel plant breeding technique designed to directly produce parental lines for any heterozygous plant, one of the most sought after goals in plant breeding. RB generates perfectly complementing homozygous parental lines through engineered meiosis. The method is based on reducing genetic recombination in the selected heterozygote by eliminating meiotic crossing over. Male or female spores obtained from such plants contain combinations of non-recombinant parental chromosomes which can be cultured in vitro to generate homozygous doubled haploid plants (DHs). From these DHs, complementary parents can be selected and used to reconstitute the heterozygote in perpetuity. Since the fixation of unknown heterozygous genotypes is impossible in traditional plant breeding, RB could fundamentally change future plant breeding. In this review, we discuss various other applications of RB, including breeding per chromosome.

Contrasting skeletal phenotypes in mice with an identical mutation targeted to thyroid hormone receptor alpha1 or beta.

PubMed

O'Shea, Patrick J; Bassett, J H Duncan; Sriskantharajah, Srividya; Ying, Hao; Cheng, Sheue-yann; Williams, Graham R

2005-12-01

Thyroid hormone (T(3)) regulates bone turnover and mineralization in adults and is essential for skeletal development. Surprisingly, we identified a phenotype of skeletal thyrotoxicosis in T(3) receptor beta(PV) (TRbeta(PV)) mice in which a targeted frameshift mutation in TRbeta results in resistance to thyroid hormone. To characterize mechanisms underlying thyroid hormone action in bone, we analyzed skeletal development in TRalpha1(PV) mice in which the same PV mutation was targeted to TRalpha1. In contrast to TRbeta(PV) mice, TRalpha1(PV) mutants exhibited skeletal hypothyroidism with delayed endochondral and intramembranous ossification, severe postnatal growth retardation, diminished trabecular bone mineralization, reduced cortical bone deposition, and delayed closure of the skull sutures. Skeletal hypothyroidism in TRalpha1(PV) mutants was accompanied by impaired GH receptor and IGF-I receptor expression and signaling in the growth plate, whereas GH receptor and IGF-I receptor expression and signaling were increased in TRbeta(PV) mice. These data indicate that GH receptor and IGF-I receptor are physiological targets for T(3) action in bone in vivo. The divergent phenotypes observed in TRalpha1(PV) and TRbeta(PV) mice arise because the pituitary gland is a TRbeta-responsive tissue, whereas bone is TRalpha responsive. These studies provide a new understanding of the complex relationship between central and peripheral thyroid status.

Targeted Exome Sequencing of Krebs Cycle Genes Reveals Candidate Cancer-Predisposing Mutations in Pheochromocytomas and Paragangliomas.

PubMed

Remacha, Laura; Comino-Méndez, Iñaki; Richter, Susan; Contreras, Laura; Currás-Freixes, María; Pita, Guillermo; Letón, Rocío; Galarreta, Antonio; Torres-Pérez, Rafael; Honrado, Emiliano; Jiménez, Scherezade; Maestre, Lorena; Moran, Sebastian; Esteller, Manel; Satrústegui, Jorgina; Eisenhofer, Graeme; Robledo, Mercedes; Cascón, Alberto

2017-10-15

Purpose: Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes. Experimental Design: We used downregulation and methylation of RBP1, as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases. Results: One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in IDH1 A variant in GOT2 , c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in SDHC in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline IDH3B mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio. Conclusions: This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol. Clin Cancer Res; 23(20); 6315-24. ©2017 AACR . ©2017 American Association for Cancer Research.

Both TALENs and CRISPR/Cas9 directly target the HBB IVS2–654 (C > T) mutation in β-thalassemia-derived iPSCs

PubMed Central

Xu, Peng; Tong, Ying; Liu, Xiu-zhen; Wang, Ting-ting; Cheng, Li; Wang, Bo-yu; Lv, Xiang; Huang, Yue; Liu, De-pei

2015-01-01

β-Thalassemia is one of the most common genetic blood diseases and is caused by either point mutations or deletions in the β-globin (HBB) gene. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations may be a potential therapeutic strategy for this disease. Due to the low efficiency of typical homologous recombination, endonucleases, including TALENs and CRISPR/Cas9, have been widely used to enhance the gene correction efficiency in patient-derived iPSCs. Here, we designed TALENs and CRISPR/Cas9 to directly target the intron2 mutation site IVS2-654 in the globin gene. We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor. In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs. Finally, TALENs-corrected iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system and detected relatively higher transcription of HBB than the uncorrected cells. This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases. PMID:26156589

Targeted exome sequencing identifies novel compound heterozygous mutations in P3H1 in a fetus with osteogenesis imperfecta type VIII.

PubMed

Huang, Yanru; Mei, Libin; Lv, Weigang; Li, Haoxian; Zhang, Rui; Pan, Qian; Tan, Hu; Guo, Jing; Luo, Xiaomei; Chen, Chen; Liang, Desheng; Wu, Lingqian

2017-01-01

Osteogenesis imperfecta (OI) is a highly clinically and genetically heterogeneous group of disorders. It is difficult to identify severe OI in the perinatal period. Here, a Chinese woman with a suspected history of fetal OI was referred to our institution at 19weeks of gestation, due to ultrasound inspection during antenatal screening, which revealed bulbous metaphyses, short humeri, and short thick bent femora in the fetus. Using targeted exome sequencing of 248 genes known to be involved in skeletal system diseases, we identified novel compound heterozygous mutation in the P3H1 gene in the fetus with OI type VIII: c.105_120del (p.D36Rfs*16) and c.2164C>T (p.Q722*). These two mutations were inherited from the father and mother, respectively. The mRNA level of P3H1 wasn't changed suggested that mRNA with this mutation escaped from nonsense-mediated RNA decay. Besides, the level of P3H1 was absence while the CRTAP was mildly decreased. In conclusion, our findings imply this novel compound heterozygous mutation as the molecular pathogenetic in a Chinese fetus with OI type VIII, and demonstrate that targeted next-generation sequencing (NGS) is an accurate, rapid, and cost-effective method in the genetic diagnosis of fetal skeletal dysplasia with genetic and clinical heterogeneity, especially for autosomal recessive skeletal disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

New autosomal recessive mutations in aquaporin-2 causing nephrogenic diabetes insipidus through deficient targeting display normal expression in Xenopus oocytes

PubMed Central

Leduc-Nadeau, Alexandre; Lussier, Yoann; Arthus, Marie-Françoise; Lonergan, Michèle; Martinez-Aguayo, Alejandro; Riveira-Munoz, Eva; Devuyst, Olivier; Bissonnette, Pierre; Bichet, Daniel G

2010-01-01

Aquaporin-2 (AQP2), located at the luminal side of the collecting duct principal cells, is a water channel responsible for the final concentration of urine. Lack of function, often occurring through mistargeting of mutated proteins, induces nephrogenic diabetes insipidus (NDI), a condition characterized by large urinary volumes. In the present study, two new mutations (K228E and V24A) identified in NDI-affected individuals from distinct families along with the already reported R187C were analysed in comparison to the wild-type protein (AQP2-wt) using Xenopus laevis oocytes and a mouse collecting duct cell-line (mIMCD-3). Initial data in oocytes showed that all mutations were adequately expressed at reduced levels when compared to AQP2-wt. K228E and V24A were found to be properly targeted at the plasma membrane and exhibited adequate functionality similar to AQP2-wt, as opposed to R187C which was retained in internal stores and was thus inactive. In coexpression studies using oocytes, R187C impeded the functionality of all other AQP2 variants while combinations with K228E, V24A and AQP2-wt only showed additive functionalities. When expressed in mIMCD-3 cells, forskolin treatment efficiently promoted the targeting of AQP2-wt at the plasma membrane (>90%) while K228E only weakly responded to the same treatment (∼20%) and both V24A and R187C remained completely insensitive to the treatment. We concluded that both V24A and K228E are intrinsically functional water channels that lack a proper response to vasopressin, which leads to NDI as found in both compound mutations studied (K228E + R187C and V24A + R187C). The discrepancies in plasma membrane targeting response found in both expression systems stress the need to evaluate such data using mammalian cell systems. PMID:20403973

Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding.

PubMed

Podevin, Nancy; Davies, Howard V; Hartung, Frank; Nogué, Fabien; Casacuberta, Josep M

2013-06-01

Conventional plant breeding exploits existing genetic variability and introduces new variability by mutagenesis. This has proven highly successful in securing food supplies for an ever-growing human population. The use of genetically modified plants is a complementary approach but all plant breeding techniques have limitations. Here, we discuss how the recent evolution of targeted mutagenesis and DNA insertion techniques based on tailor-made site-directed nucleases (SDNs) provides opportunities to overcome such limitations. Plant breeding companies are exploiting SDNs to develop a new generation of crops with new and improved traits. Nevertheless, some technical limitations as well as significant uncertainties on the regulatory status of SDNs may challenge their use for commercial plant breeding. Copyright © 2013 Elsevier Ltd. All rights reserved.

On the distance of genetic relationships and the accuracy of genomic prediction in pig breeding.

PubMed

Meuwissen, Theo H E; Odegard, Jorgen; Andersen-Ranberg, Ina; Grindflek, Eli

2014-08-01

With the advent of genomic selection, alternative relationship matrices are used in animal breeding, which vary in their coverage of distant relationships due to old common ancestors. Relationships based on pedigree (A) and linkage analysis (GLA) cover only recent relationships because of the limited depth of the known pedigree. Relationships based on identity-by-state (G) include relationships up to the age of the SNP (single nucleotide polymorphism) mutations. We hypothesised that the latter relationships were too old, since QTL (quantitative trait locus) mutations for traits under selection were probably more recent than the SNPs on a chip, which are typically selected for high minor allele frequency. In addition, A and GLA relationships are too recent to cover genetic differences accurately. Thus, we devised a relationship matrix that considered intermediate-aged relationships and compared all these relationship matrices for their accuracy of genomic prediction in a pig breeding situation. Haplotypes were constructed and used to build a haplotype-based relationship matrix (GH), which considers more intermediate-aged relationships, since haplotypes recombine more quickly than SNPs mutate. Dense genotypes (38 453 SNPs) on 3250 elite breeding pigs were combined with phenotypes for growth rate (2668 records), lean meat percentage (2618), weight at three weeks of age (7387) and number of teats (5851) to estimate breeding values for all animals in the pedigree (8187 animals) using the aforementioned relationship matrices. Phenotypes on the youngest 424 to 486 animals were masked and predicted in order to assess the accuracy of the alternative genomic predictions. Correlations between the relationships and regressions of older on younger relationships revealed that the age of the relationships increased in the order A, GLA, GH and G. Use of genomic relationship matrices yielded significantly higher prediction accuracies than A. GH and G, differed not significantly

Reverse breeding: a novel breeding approach based on engineered meiosis

PubMed Central

Dirks, Rob; van Dun, Kees; de Snoo, C Bastiaan; van den Berg, Mark; Lelivelt, Cilia L C; Voermans, William; Woudenberg, Leo; de Wit, Jack P C; Reinink, Kees; Schut, Johan W; van der Zeeuw, Eveline; Vogelaar, Aat; Freymark, Gerald; Gutteling, Evert W; Keppel, Marina N; van Drongelen, Paul; Kieny, Matthieu; Ellul, Philippe; Touraev, Alisher; Ma, Hong; de Jong, Hans; Wijnker, Erik

2009-01-01

Reverse breeding (RB) is a novel plant breeding technique designed to directly produce parental lines for any heterozygous plant, one of the most sought after goals in plant breeding. RB generates perfectly complementing homozygous parental lines through engineered meiosis. The method is based on reducing genetic recombination in the selected heterozygote by eliminating meiotic crossing over. Male or female spores obtained from such plants contain combinations of non-recombinant parental chromosomes which can be cultured in vitro to generate homozygous doubled haploid plants (DHs). From these DHs, complementary parents can be selected and used to reconstitute the heterozygote in perpetuity. Since the fixation of unknown heterozygous genotypes is impossible in traditional plant breeding, RB could fundamentally change future plant breeding. In this review, we discuss various other applications of RB, including breeding per chromosome. PMID:19811618

A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

PubMed

Thierry, Alain R

2016-01-01

Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics

A network-based drug repositioning infrastructure for precision cancer medicine through targeting significantly mutated genes in the human cancer genomes.

PubMed

Cheng, Feixiong; Zhao, Junfei; Fooksa, Michaela; Zhao, Zhongming

2016-07-01

Development of computational approaches and tools to effectively integrate multidomain data is urgently needed for the development of newly targeted cancer therapeutics. We proposed an integrative network-based infrastructure to identify new druggable targets and anticancer indications for existing drugs through targeting significantly mutated genes (SMGs) discovered in the human cancer genomes. The underlying assumption is that a drug would have a high potential for anticancer indication if its up-/down-regulated genes from the Connectivity Map tended to be SMGs or their neighbors in the human protein interaction network. We assembled and curated 693 SMGs in 29 cancer types and found 121 proteins currently targeted by known anticancer or noncancer (repurposed) drugs. We found that the approved or experimental cancer drugs could potentially target these SMGs in 33.3% of the mutated cancer samples, and this number increased to 68.0% by drug repositioning through surveying exome-sequencing data in approximately 5000 normal-tumor pairs from The Cancer Genome Atlas. Furthermore, we identified 284 potential new indications connecting 28 cancer types and 48 existing drugs (adjusted P < .05), with a 66.7% success rate validated by literature data. Several existing drugs (e.g., niclosamide, valproic acid, captopril, and resveratrol) were predicted to have potential indications for multiple cancer types. Finally, we used integrative analysis to showcase a potential mechanism-of-action for resveratrol in breast and lung cancer treatment whereby it targets several SMGs (ARNTL, ASPM, CTTN, EIF4G1, FOXP1, and STIP1). In summary, we demonstrated that our integrative network-based infrastructure is a promising strategy to identify potential druggable targets and uncover new indications for existing drugs to speed up molecularly targeted cancer therapeutics. © The Author 2016. Published by Oxford University Press on behalf of the American Medical Informatics Association. All

Transcription factor mutations in myelodysplastic/myeloproliferative neoplasms

PubMed Central

Ernst, Thomas; Chase, Andrew; Zoi, Katerina; Waghorn, Katherine; Hidalgo-Curtis, Claire; Score, Joannah; Jones, Amy; Grand, Francis; Reiter, Andreas; Hochhaus, Andreas; Cross, Nicholas C.P.

2010-01-01

Background Aberrant activation of tyrosine kinases, caused by either mutation or gene fusion, is of major importance for the development of many hematologic malignancies, particularly myeloproliferative neoplasms. We hypothesized that hitherto unrecognized, cytogenetically cryptic tyrosine kinase fusions may be common in non-classical or atypical myeloproliferative neoplasms and related myelodysplastic/myeloproliferative neoplasms. Design and Methods To detect genomic copy number changes associated with such fusions, we performed a systematic search in 68 patients using custom designed, targeted, high-resolution array comparative genomic hybridization. Arrays contained 44,000 oligonucleotide probes that targeted 500 genes including all 90 tyrosine kinases plus downstream tyrosine kinase signaling components, other translocation targets, transcription factors, and other factors known to be important for myelopoiesis. Results No abnormalities involving tyrosine kinases were detected; however, nine cytogenetically cryptic copy number imbalances were detected in seven patients, including hemizygous deletions of RUNX1 or CEBPA in two cases with atypical chronic myeloid leukemia. Mutation analysis of the remaining alleles revealed non-mutated RUNX1 and a frameshift insertion within CEBPA. A further mutation screen of 187 patients with myelodysplastic/myeloproliferative neoplasms identified RUNX1 mutations in 27 (14%) and CEBPA mutations in seven (4%) patients. Analysis of other transcription factors known to be frequently mutated in acute myeloid leukemia revealed NPM1 mutations in six (3%) and WT1 mutations in two (1%) patients with myelodysplastic/myeloproliferative neoplasms. Univariate analysis indicated that patients with mutations had a shorter overall survival (28 versus 44 months, P=0.019) compared with patients without mutations, with the prognosis for cases with CEBPA, NPM1 or WT1 mutations being particularly poor. Conclusions We conclude that mutations of

Comparative Aspects of BRAF Mutations in Canine Cancers

PubMed Central

Mochizuki, Hiroyuki; Breen, Matthew

2015-01-01

Activating mutations of the BRAF gene lead to constitutive activation of the MAPK pathway. The characterization and discovery of BRAF mutations in a variety of human cancers has led to the development of specific inhibitors targeting the BRAF/MAPK pathway and dramatically changed clinical outcomes in BRAF-mutant melanoma patients. Recent discovery of BRAF mutation in canine cancers underscores the importance of MAPK pathway activation as an oncogenic molecular alteration evolutionarily conserved between species. A comparative approach using the domestic dog as a spontaneous cancer model will provide new insights into the dysregulation of BRAF/MAPK pathway in carcinogenesis and facilitate in vivo studies to evaluate therapeutic strategies targeting this pathway’s molecules for cancer therapy. The BRAF mutation in canine cancers may also represent a molecular marker and therapeutic target in veterinary oncology. This review article summarizes the current knowledge on BRAF mutations in human and canine cancers and discusses the potential applications of this abnormality in veterinary oncology. PMID:29061943

Frequency and distribution of 152 genetic disease variants in over 100,000 mixed breed and purebred dogs.

PubMed

Donner, Jonas; Anderson, Heidi; Davison, Stephen; Hughes, Angela M; Bouirmane, Julia; Lindqvist, Johan; Lytle, Katherine M; Ganesan, Balasubramanian; Ottka, Claudia; Ruotanen, Päivi; Kaukonen, Maria; Forman, Oliver P; Fretwell, Neale; Cole, Cynthia A; Lohi, Hannes

2018-04-01

Knowledge on the genetic epidemiology of disorders in the dog population has implications for both veterinary medicine and sustainable breeding. Limited data on frequencies of genetic disease variants across breeds exists, and the disease heritage of mixed breed dogs remains poorly explored to date. Advances in genetic screening technologies now enable comprehensive investigations of the canine disease heritage, and generate health-related big data that can be turned into action. We pursued population screening of genetic variants implicated in Mendelian disorders in the largest canine study sample examined to date by examining over 83,000 mixed breed and 18,000 purebred dogs representing 330 breeds for 152 known variants using a custom-designed beadchip microarray. We further announce the creation of MyBreedData (www.mybreeddata.com), an online updated inherited disorder prevalence resource with its foundation in the generated data. We identified the most prevalent, and rare, disease susceptibility variants across the general dog population while providing the first extensive snapshot of the mixed breed disease heritage. Approximately two in five dogs carried at least one copy of a tested disease variant. Most disease variants are shared by both mixed breeds and purebreds, while breed- or line-specificity of others is strongly suggested. Mixed breed dogs were more likely to carry a common recessive disease, whereas purebreds were more likely to be genetically affected with one, providing DNA-based evidence for hybrid vigor. We discovered genetic presence of 22 disease variants in at least one additional breed in which they were previously undescribed. Some mutations likely manifest similarly independently of breed background; however, we emphasize the need for follow up investigations in each case and provide a suggested validation protocol for broader consideration. In conclusion, our study provides unique insight into genetic epidemiology of canine disease risk

The contribution of de novo coding mutations to autism spectrum disorder

PubMed Central

Iossifov, Ivan; O’Roak, Brian J.; Sanders, Stephan J.; Ronemus, Michael; Krumm, Niklas; Levy, Dan; Stessman, Holly A.; Witherspoon, Kali; Vives, Laura; Patterson, Karynne E.; Smith, Joshua D.; Paeper, Bryan; Nickerson, Deborah A.; Dea, Jeanselle; Dong, Shan; Gonzalez, Luis E.; Mandell, Jefferey D.; Mane, Shrikant M.; Murtha, Michael T.; Sullivan, Catherine A.; Walker, Michael F.; Waqar, Zainulabedin; Wei, Liping; Willsey, A. Jeremy; Yamrom, Boris; Lee, Yoon-ha; Grabowska, Ewa; Dalkic, Ertugrul; Wang, Zihua; Marks, Steven; Andrews, Peter; Leotta, Anthony; Kendall, Jude; Hakker, Inessa; Rosenbaum, Julie; Ma, Beicong; Rodgers, Linda; Troge, Jennifer; Narzisi, Giuseppe; Yoon, Seungtai; Schatz, Michael C.; Ye, Kenny; McCombie, W. Richard; Shendure, Jay; Eichler, Evan E.; State, Matthew W.; Wigler, Michael

2015-01-01

We sequenced exomes from more than 2,500 simplex families each having a child with an autistic spectrum disorder (ASD). By comparing affected to unaffected siblings, we estimate that 13% of de novo (DN) missense mutations and 42% of DN likely gene-disrupting (LGD) mutations contribute to 12% and 9% of diagnoses, respectively. Including copy number variants, coding DN mutations contribute to about 30% of all simplex and 45% of female diagnoses. Virtually all LGD mutations occur opposite wild-type alleles. LGD targets in affected females significantly overlap the targets in males of lower IQ, but neither overlaps significantly with targets in males of higher IQ. We estimate that LGD mutation in about 400 genes can contribute to the joint class of affected females and males of lower IQ, with an overlapping and similar number of genes vulnerable to causative missense mutation. LGD targets in the joint class overlap with published targets for intellectual disability and schizophrenia, and are enriched for chromatin modifiers, FMRP-associated genes and embryonically expressed genes. Virtually all significance for the latter comes from affected females. PMID:25363768

Identifying pathways affected by cancer mutations.

PubMed

Iengar, Prathima

2017-12-16

Mutations in 15 cancers, sourced from the COSMIC Whole Genomes database, and 297 human pathways, arranged into pathway groups based on the processes they orchestrate, and sourced from the KEGG pathway database, have together been used to identify pathways affected by cancer mutations. Genes studied in ≥15, and mutated in ≥10 samples of a cancer have been considered recurrently mutated, and pathways with recurrently mutated genes have been considered affected in the cancer. Novel doughnut plots have been presented which enable visualization of the extent to which pathways and genes, in each pathway group, are targeted, in each cancer. The 'organismal systems' pathway group (including organism-level pathways; e.g., nervous system) is the most targeted, more than even the well-recognized signal transduction, cell-cycle and apoptosis, and DNA repair pathway groups. The important, yet poorly-recognized, role played by the group merits attention. Pathways affected in ≥7 cancers yielded insights into processes affected. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparison of molecular breeding values based on within- and across-breed training in beef cattle.

PubMed

Kachman, Stephen D; Spangler, Matthew L; Bennett, Gary L; Hanford, Kathryn J; Kuehn, Larry A; Snelling, Warren M; Thallman, R Mark; Saatchi, Mahdi; Garrick, Dorian J; Schnabel, Robert D; Taylor, Jeremy F; Pollak, E John

2013-08-16

Although the efficacy of genomic predictors based on within-breed training looks promising, it is necessary to develop and evaluate across-breed predictors for the technology to be fully applied in the beef industry. The efficacies of genomic predictors trained in one breed and utilized to predict genetic merit in differing breeds based on simulation studies have been reported, as have the efficacies of predictors trained using data from multiple breeds to predict the genetic merit of purebreds. However, comparable studies using beef cattle field data have not been reported. Molecular breeding values for weaning and yearling weight were derived and evaluated using a database containing BovineSNP50 genotypes for 7294 animals from 13 breeds in the training set and 2277 animals from seven breeds (Angus, Red Angus, Hereford, Charolais, Gelbvieh, Limousin, and Simmental) in the evaluation set. Six single-breed and four across-breed genomic predictors were trained using pooled data from purebred animals. Molecular breeding values were evaluated using field data, including genotypes for 2227 animals and phenotypic records of animals born in 2008 or later. Accuracies of molecular breeding values were estimated based on the genetic correlation between the molecular breeding value and trait phenotype. With one exception, the estimated genetic correlations of within-breed molecular breeding values with trait phenotype were greater than 0.28 when evaluated in the breed used for training. Most estimated genetic correlations for the across-breed trained molecular breeding values were moderate (> 0.30). When molecular breeding values were evaluated in breeds that were not in the training set, estimated genetic correlations clustered around zero. Even for closely related breeds, within- or across-breed trained molecular breeding values have limited prediction accuracy for breeds that were not in the training set. For breeds in the training set, across- and within-breed trained

Comparison of molecular breeding values based on within- and across-breed training in beef cattle

PubMed Central

2013-01-01

Background Although the efficacy of genomic predictors based on within-breed training looks promising, it is necessary to develop and evaluate across-breed predictors for the technology to be fully applied in the beef industry. The efficacies of genomic predictors trained in one breed and utilized to predict genetic merit in differing breeds based on simulation studies have been reported, as have the efficacies of predictors trained using data from multiple breeds to predict the genetic merit of purebreds. However, comparable studies using beef cattle field data have not been reported. Methods Molecular breeding values for weaning and yearling weight were derived and evaluated using a database containing BovineSNP50 genotypes for 7294 animals from 13 breeds in the training set and 2277 animals from seven breeds (Angus, Red Angus, Hereford, Charolais, Gelbvieh, Limousin, and Simmental) in the evaluation set. Six single-breed and four across-breed genomic predictors were trained using pooled data from purebred animals. Molecular breeding values were evaluated using field data, including genotypes for 2227 animals and phenotypic records of animals born in 2008 or later. Accuracies of molecular breeding values were estimated based on the genetic correlation between the molecular breeding value and trait phenotype. Results With one exception, the estimated genetic correlations of within-breed molecular breeding values with trait phenotype were greater than 0.28 when evaluated in the breed used for training. Most estimated genetic correlations for the across-breed trained molecular breeding values were moderate (> 0.30). When molecular breeding values were evaluated in breeds that were not in the training set, estimated genetic correlations clustered around zero. Conclusions Even for closely related breeds, within- or across-breed trained molecular breeding values have limited prediction accuracy for breeds that were not in the training set. For breeds in the training

High resolution melting curve analysis targeting the HBB gene mutational hot-spot offers a reliable screening approach for all common as well as most of the rare beta-globin gene mutations in Bangladesh.

PubMed

Islam, Md Tarikul; Sarkar, Suprovath Kumar; Sultana, Nusrat; Begum, Mst Noorjahan; Bhuyan, Golam Sarower; Talukder, Shezote; Muraduzzaman, A K M; Alauddin, Md; Islam, Mohammad Sazzadul; Biswas, Pritha Promita; Biswas, Aparna; Qadri, Syeda Kashfi; Shirin, Tahmina; Banu, Bilquis; Sadya, Salma; Hussain, Manzoor; Sarwardi, Golam; Khan, Waqar Ahmed; Mannan, Mohammad Abdul; Shekhar, Hossain Uddin; Chowdhury, Emran Kabir; Sajib, Abu Ashfaqur; Akhteruzzaman, Sharif; Qadri, Syed Saleheen; Qadri, Firdausi; Mannoor, Kaiissar

2018-01-02

Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh. Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result. Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and

Can functional hologenomics aid tackling current challenges in plant breeding?

PubMed

Nogales, Amaia; Nobre, Tânia; Valadas, Vera; Ragonezi, Carla; Döring, Matthias; Polidoros, Alexios; Arnholdt-Schmitt, Birgit

2016-07-01

Molecular plant breeding usually overlooks the genetic variability that arises from the association of plants with endophytic microorganisms, when looking at agronomic interesting target traits. This source of variability can have crucial effects on the functionality of the organism considered as a whole (the holobiont), and therefore can be selectable in breeding programs. However, seeing the holobiont as a unit for selection and improvement in breeding programs requires novel approaches for genotyping and phenotyping. These should not focus just at the plant level, but also include the associated endophytes and their functional effects on the plant, to make effective desirable trait screenings. The present review intends to draw attention to a new research field on functional hologenomics that if associated with adequate phenotyping tools could greatly increase the efficiency of breeding programs. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Economic Evaluation of Companion Diagnostic Testing for EGFR Mutations and First-Line Targeted Therapy in Advanced Non-Small Cell Lung Cancer Patients in South Korea.

PubMed

Lim, Eun-A; Lee, Haeyoung; Bae, Eunmi; Lim, Jaeok; Shin, Young Kee; Choi, Sang-Eun

2016-01-01

As targeted therapy becomes increasingly important, diagnostic techniques for identifying targeted biomarkers have also become an emerging issue. The study aims to evaluate the cost-effectiveness of treating patients as guided by epidermal growth factor receptor (EGFR) mutation status compared with a no-testing strategy that is the current clinical practice in South Korea. A cost-utility analysis was conducted to compare an EGFR mutation testing strategy with a no-testing strategy from the Korean healthcare payer's perspective. The study population consisted of patients with stage 3b and 4 lung adenocarcinoma. A decision tree model was employed to select the appropriate treatment regimen according to the results of EGFR mutation testing and a Markov model was constructed to simulate disease progression of advanced non-small cell lung cancer. The length of a Markov cycle was one month, and the time horizon was five years (60 cycles). In the base case analysis, the testing strategy was a dominant option. Quality-adjusted life-years gained (QALYs) were 0.556 and 0.635, and total costs were $23,952 USD and $23,334 USD in the no-testing and testing strategy respectively. The sensitivity analyses showed overall robust results. The incremental cost-effectiveness ratios (ICERs) increased when the number of patients to be treated with erlotinib increased, due to the high cost of erlotinib. Treating advanced adenocarcinoma based on EGFR mutation status has beneficial effects and saves the cost compared to no testing strategy in South Korea. However, the cost-effectiveness of EGFR mutation testing was heavily affected by the cost-effectiveness of the targeted therapy.

Is Mutation Random or Targeted?: No Evidence for Hypermutability in Snail Toxin Genes.

PubMed

Roy, Scott W

2016-10-01

Ever since Luria and Delbruck, the notion that mutation is random with respect to fitness has been foundational to modern biology. However, various studies have claimed striking exceptions to this rule. One influential case involves toxin-encoding genes in snails of the genus Conus, termed conotoxins, a large gene family that undergoes rapid diversification of their protein-coding sequences by positive selection. Previous reconstructions of the sequence evolution of conotoxin genes claimed striking patterns: (1) elevated synonymous change, interpreted as being due to targeted "hypermutation" in this region; (2) elevated transversion-to-transition ratios, interpreted as reflective of the particular mechanism of hypermutation; and (3) much lower rates of synonymous change in the codons encoding several highly conserved cysteine residues, interpreted as strong position-specific codon bias. This work has spawned a variety of studies on the potential mechanisms of hypermutation and on causes for cysteine codon bias, and has inspired hypermutation hypotheses for various other fast-evolving genes. Here, I show that all three findings are likely to be artifacts of statistical reconstruction. First, by simulating nonsynonymous change I show that high rates of dN can lead to overestimation of dS. Second, I show that there is no evidence for any of these three patterns in comparisons of closely related conotoxin sequences, suggesting that the reported findings are due to breakdown of statistical methods at high levels of sequence divergence. The current findings suggest that mutation and codon bias in conotoxin genes may not be atypical, and that random mutation and selection can explain the evolution of even these exceptional loci. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Instability of the insertional mutation in CftrTgH(neoim)Hgu cystic fibrosis mouse model

PubMed Central

Charizopoulou, Nikoletta; Jansen, Silke; Dorsch, Martina; Stanke, Frauke; Dorin, Julia R; Hedrich, Hans-Jürgen; Tümmler, Burkhard

2004-01-01

Background A major boost to the cystic fibrosis disease research was given by the generation of various mouse models using gene targeting in embryonal stem cells. Moreover, the introduction of the same mutation on different inbred strains generating congenic strains facilitated the search for modifier genes. From the original CftrTgH(neoim)Hgu CF mouse model we have generated using strict brother × sister mating two inbred CftrTgH(neoim)Hgu mouse lines (CF/1 and CF/3). Thereafter, the insertional mutation was introgressed from CF/3 into three inbred backgrounds (C57BL/6, BALB/c, DBA/2J) generating congenic animals. In every backcross cycle germline transmission of the insertional mutation was monitored by direct probing the insertion via Southern RFLP. In order to bypass this time consuming procedure we devised an alternative PCR based protocol whereby mouse strains are differentiated at the Cftr locus by Cftr intragenic microsatellite genotypes that are tightly linked to the disrupted locus. Results Using this method we were able to identify animals carrying the insertional mutation based upon the differential haplotypic backgrounds of the three inbred strains and the mutant CftrTgH(neoim)Hgu at the Cftr locus. Moreover, this method facilitated the identification of the precise vector excision from the disrupted Cftr locus in two out of 57 typed animals. This reversion to wild type status took place without any loss of sequence revealing the instability of insertional mutations during the production of congenic animals. Conclusions We present intragenic microsatellite markers as a tool for fast and efficient identification of the introgressed locus of interest in the recipient strain during congenic animal breeding. Moreover, the same genotyping method allowed the identification of a vector excision event, posing questions on the stability of insertional mutations in mice. PMID:15102331

Novel recurrently mutated genes in African American colon cancers.

PubMed

Guda, Kishore; Veigl, Martina L; Varadan, Vinay; Nosrati, Arman; Ravi, Lakshmeswari; Lutterbaugh, James; Beard, Lydia; Willson, James K V; Sedwick, W David; Wang, Zhenghe John; Molyneaux, Neil; Miron, Alexander; Adams, Mark D; Elston, Robert C; Markowitz, Sanford D; Willis, Joseph E

2015-01-27

We used whole-exome and targeted sequencing to characterize somatic mutations in 103 colorectal cancers (CRC) from African Americans, identifying 20 new genes as significantly mutated in CRC. Resequencing 129 Caucasian derived CRCs confirmed a 15-gene set as a preferential target for mutations in African American CRCs. Two predominant genes, ephrin type A receptor 6 (EPHA6) and folliculin (FLCN), with mutations exclusive to African American CRCs, are by genetic and biological criteria highly likely African American CRC driver genes. These previously unsuspected differences in the mutational landscapes of CRCs arising among individuals of different ethnicities have potential to impact on broader disparities in cancer behaviors.

Molecular breeding in Brassica for salt tolerance: importance of microsatellite (SSR) markers for molecular breeding in Brassica.

PubMed

Kumar, Manu; Choi, Ju-Young; Kumari, Nisha; Pareek, Ashwani; Kim, Seong-Ryong

2015-01-01

Salinity is one of the important abiotic factors for any crop management in irrigated as well as rainfed areas, which leads to poor harvests. This yield reduction in salt affected soils can be overcome by improving salt tolerance in crops or by soil reclamation. Salty soils can be reclaimed by leaching the salt or by cultivation of salt tolerance crops. Salt tolerance is a quantitative trait controlled by several genes. Poor knowledge about mechanism of its inheritance makes slow progress in its introgression into target crops. Brassica is known to be a good reclamation crop. Inter and intra specific variation within Brassica species shows potential of molecular breeding to raise salinity tolerant genotypes. Among the various molecular markers, SSR markers are getting high attention, since they are randomly sparsed, highly variable and show co-dominant inheritance. Furthermore, as sequencing techniques are improving and softwares to find SSR markers are being developed, SSR markers technology is also evolving rapidly. Comparative SSR marker studies targeting Arabidopsis thaliana and Brassica species which lie in the same family will further aid in studying the salt tolerance related QTLs and subsequent identification of the "candidate genes" and finding out the origin of important QTLs. Although, there are a few reports on molecular breeding for improving salt tolerance using molecular markers in Brassica species, usage of SSR markers has a big potential to improve salt tolerance in Brassica crops. In order to obtain best harvests, role of SSR marker driven breeding approaches play important role and it has been discussed in this review especially for the introgression of salt tolerance traits in crops.

Molecular breeding in Brassica for salt tolerance: importance of microsatellite (SSR) markers for molecular breeding in Brassica

PubMed Central

Kumar, Manu; Choi, Ju-Young; Kumari, Nisha; Pareek, Ashwani; Kim, Seong-Ryong

2015-01-01

Salinity is one of the important abiotic factors for any crop management in irrigated as well as rainfed areas, which leads to poor harvests. This yield reduction in salt affected soils can be overcome by improving salt tolerance in crops or by soil reclamation. Salty soils can be reclaimed by leaching the salt or by cultivation of salt tolerance crops. Salt tolerance is a quantitative trait controlled by several genes. Poor knowledge about mechanism of its inheritance makes slow progress in its introgression into target crops. Brassica is known to be a good reclamation crop. Inter and intra specific variation within Brassica species shows potential of molecular breeding to raise salinity tolerant genotypes. Among the various molecular markers, SSR markers are getting high attention, since they are randomly sparsed, highly variable and show co-dominant inheritance. Furthermore, as sequencing techniques are improving and softwares to find SSR markers are being developed, SSR markers technology is also evolving rapidly. Comparative SSR marker studies targeting Arabidopsis thaliana and Brassica species which lie in the same family will further aid in studying the salt tolerance related QTLs and subsequent identification of the “candidate genes” and finding out the origin of important QTLs. Although, there are a few reports on molecular breeding for improving salt tolerance using molecular markers in Brassica species, usage of SSR markers has a big potential to improve salt tolerance in Brassica crops. In order to obtain best harvests, role of SSR marker driven breeding approaches play important role and it has been discussed in this review especially for the introgression of salt tolerance traits in crops. PMID:26388887

Broad Resistance to ACCase Inhibiting Herbicides in a Ryegrass Population Is Due Only to a Cysteine to Arginine Mutation in the Target Enzyme

PubMed Central

Kaundun, Shiv Shankhar; Hutchings, Sarah-Jane; Dale, Richard Paul; McIndoe, Eddie

2012-01-01

Background The design of sustainable weed management strategies requires a good understanding of the mechanisms by which weeds evolve resistance to herbicides. Here we have conducted a study on the mechanism of resistance to ACCase inhibiting herbicides in a Lolium multiflorum population (RG3) from the UK. Methodology/Principal Findings Analysis of plant phenotypes and genotypes showed that all the RG3 plants (72%) that contained the cysteine to arginine mutation at ACCase codon position 2088 were resistant to ACCase inhibiting herbicides. Whole plant dose response tests on predetermined wild and mutant 2088 genotypes from RG3 and a standard sensitive population indicated that the C2088R mutation is the only factor conferring resistance to all ten ACCase herbicides tested. The associated resistance indices ranged from 13 for clethodim to over 358 for diclofop-methyl. Clethodim, the most potent herbicide was significantly affected even when applied on small mutant plants at the peri-emergence and one leaf stages. Conclusion/Significance This study establishes the clear and unambiguous importance of the C2088R target site mutation in conferring broad resistance to ten commonly used ACCase inhibiting herbicides. It also demonstrates that low levels “creeping”, multigenic, non target site resistance, is not always selected before single gene target site resistance appears in grass weed populations subjected to herbicide selection pressure. PMID:22768118

Targeting RAF kinases for cancer therapy: BRAF mutated melanoma and beyond

PubMed Central

Holderfield, Matthew; Deuker, Marian M.; McCormick, Frank; McMahon, Martin

2014-01-01

The identification of mutationally activated BRAF in many cancers altered our conception of the role played by the RAF family of protein kinases in oncogenesis. In this review we describe the development of BRAF inhibitors and the results that have emerged from their analysis in both the laboratory and the clinic. We discuss the spectrum of RAF mutations in human cancer and the complex interplay between tissue of origin and response to RAF inhibition. Finally, we enumerate mechanisms of resistance to BRAF inhibition that have been characterized and postulate how strategies of RAF pathway inhibition may be extended in scope to benefit, not only the thousands of patients diagnosed annually with BRAF-mutated metastatic melanoma, but also the larger patient population with malignancies harboring mutationally activated RAF genes that is ineffectively treated with the current generation of BRAF kinase inhibitors. PMID:24957944

Genome-Wide Specific Selection in Three Domestic Sheep Breeds.

PubMed

Wang, Huihua; Zhang, Li; Cao, Jiaxve; Wu, Mingming; Ma, Xiaomeng; Liu, Zhen; Liu, Ruizao; Zhao, Fuping; Wei, Caihong; Du, Lixin

2015-01-01

Commercial sheep raised for mutton grow faster than traditional Chinese sheep breeds. Here, we aimed to evaluate genetic selection among three different types of sheep breed: two well-known commercial mutton breeds and one indigenous Chinese breed. We first combined locus-specific branch lengths and di statistical methods to detect candidate regions targeted by selection in the three different populations. The results showed that the genetic distances reached at least medium divergence for each pairwise combination. We found these two methods were highly correlated, and identified many growth-related candidate genes undergoing artificial selection. For production traits, APOBR and FTO are associated with body mass index. For meat traits, ALDOA, STK32B and FAM190A are related to marbling. For reproduction traits, CCNB2 and SLC8A3 affect oocyte development. We also found two well-known genes, GHR (which affects meat production and quality) and EDAR (associated with hair thickness) were associated with German mutton merino sheep. Furthermore, four genes (POL, RPL7, MSL1 and SHISA9) were associated with pre-weaning gain in our previous genome-wide association study. Our results indicated that combine locus-specific branch lengths and di statistical approaches can reduce the searching ranges for specific selection. And we got many credible candidate genes which not only confirm the results of previous reports, but also provide a suite of novel candidate genes in defined breeds to guide hybridization breeding.

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT.

PubMed

Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

2016-01-01

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors' sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib.

A Novel SLC27A4 Splice Acceptor Site Mutation in Great Danes with Ichthyosis.

PubMed

Metzger, Julia; Wöhlke, Anne; Mischke, Reinhard; Hoffmann, Annalena; Hewicker-Trautwein, Marion; Küch, Eva-Maria; Naim, Hassan Y; Distl, Ottmar

2015-01-01

Ichthyoses are a group of various different types of hereditary disorders affecting skin cornification. They are characterized by hyperkeratoses of different severity levels and are associated with a dry and scaling skin. Genome-wide association analysis of nine affected and 13 unaffected Great Danes revealed a genome-wide significant peak on chromosome 9 at 57-58 Mb in the region of SLC27A4. Sequence analysis of genomic DNA of SLC27A4 revealed the non-synonymous SNV SLC27A4:g.8684G>A in perfect association with ichthyosis-affection in Great Danes. The mutant transcript of SLC27A4 showed an in-frame loss of 54 base pairs in exon 8 probably induced by a new splice acceptor site motif created by the mutated A- allele of the SNV. Genotyping 413 controls from 35 different breeds of dogs and seven wolves revealed that this mutation could not be found in other populations except in Great Danes. Affected dogs revealed high amounts of mutant transcript but only low levels of the wild type transcript. Targeted analyses of SLC27A4 protein from skin tissues of three affected and two unaffected Great Danes indicated a markedly reduced or not detectable wild type and truncated protein levels in affected dogs but a high expression of wild type SLC27A4 protein in unaffected controls. Our data provide evidence of a new splice acceptor site creating SNV that results in a reduction or loss of intact SLC27A4 protein and probably explains the severe skin phenotype in Great Danes. Genetic testing will allow selective breeding to prevent ichthyosis-affected puppies in the future.

Genomic deletion of CNGB3 is identical by descent in multiple canine breeds and causes achromatopsia

PubMed Central

2013-01-01

Background Achromatopsia is an autosomal recessive disease characterized by the loss of cone photoreceptor function that results in day-blindness, total colorblindness, and decreased central visual acuity. The most common causes for the disease are mutations in the CNGB3 gene, coding for the beta subunit of the cyclic nucleotide-gated channels in cones. CNGB3-achromatopsia, or cone degeneration (cd), is also known to occur in two canine breeds, the Alaskan malamute (AM) and the German shorthaired pointer. Results Here we report an in-depth characterization of the achromatopsia phenotype in a new canine breed, the miniature Australian shepherd (MAS). Genotyping revealed that the dog was homozygous for a complete genomic deletion of the CNGB3 gene, as has been previously observed in the AM. Identical breakpoints on chromosome 29 were identified in both the affected AM and MAS with a resulting deletion of 404,820 bp. Pooled DNA samples of unrelated purebred Australian shepherd, MAS, Siberian husky, Samoyed and Alaskan sled dogs were screened for the presence of the affected allele; one Siberian husky and three Alaskan sled dogs were identified as carriers. The affected chromosomes from the AM, MAS, and Siberian husky were genotyped for 147 SNPs in a 3.93 Mb interval within the cd locus. An identical shared affected haplotype, 0.5 Mb long, was observed in all three breeds and defined the minimal linkage disequilibrium (LD) across breeds. This supports the idea that the mutated allele was identical by descent (IBD). Conclusion We report the occurrence of CNGB3-achromatopsia in a new canine breed, the MAS. The CNGB3-deletion allele previously described in the AM was also observed in a homozygous state in the affected MAS, as well as in a heterozygous carrier state in a Siberian husky and Alaskan sled dogs. All affected alleles were shown to be IBD, strongly suggesting an affected founder effect. Since the MAS is not known to be genetically related to the AM, other

Characterization of sulfonylurea-resistant Schoenoplectus juncoides having a target-site Asp(376)Glu mutation in the acetolactate synthase.

PubMed

Sada, Yoshinao; Ikeda, Hajime; Yamato, Seiji; Kizawa, Satoru

2013-09-01

Schoenoplectus juncoides, a noxious weed for paddy rice, is known to become resistant to sulfonylurea (SU) herbicides by a target-site mutation in either of the two acetolactate synthase (ALS) genes (ALS1 and ALS2). SU-resistant S. juncoides plants having an Asp376Glu mutation in ALS2 were found from a paddy rice field in Japan, but their resistance profile has not been quantitatively investigated. In this study, dose-response of the SU-resistant accession was compared with that of a SU-susceptible accession at in vivo whole-plant level as well as at in vitro enzymatic level. In whole-plant tests, resistance factors (RFs) based on 50% growth reduction (GR50) for imazosulfuron (ISF), bensulfuron-methyl (BSM), metsulfuron-methyl (MSM), bispyribac-sodium (BPS), and imazaquin (IMQ) were 176, 40, 14, 5.2 and 1.5, respectively. Thus, the accession having an Asp376Glu mutation in ALS2 was highly resistant to the three SU herbicides and moderately resistant to BPS, but was not substantially resistant to IMQ. This is slightly different from the earlier results reported from other weeds with an Asp376Glu mutation, in which the mutation confers resistance to broadly all the chemical classes of ALS-inhibiting herbicides. In enzymatic tests, ALS2 of S. juncoides was expressed in E. coli; the resultant ALS2 was subjected to an in vitro assay. RFs of the mutated ALS2 based on 50% enzymatic inhibition (I50) for ISF, BSM, MSM, BPS, and IMQ were 3699, 2438, 322, 80, and 4.8, respectively. The RFs of ALS2 were highly correlated with those of the whole-plant; this suggests that the Asp376Glu mutation in ALS2 is a molecular basis for the whole-plant resistance. The presence of two ALS genes in S. juncoides can at least partially explain why the whole-plant RFs were less than those of the expressed ALS2 enzymes. Copyright © 2013 Elsevier Inc. All rights reserved.

AIP mutations and gigantism.

PubMed

Rostomyan, Liliya; Potorac, Iulia; Beckers, Pablo; Daly, Adrian F; Beckers, Albert

2017-06-01

AIP mutations are rare in sporadic acromegaly but they are seen at a higher frequency among certain specific populations of pituitary adenoma patients (pituitary gigantism cases, familial isolated pituitary adenoma (FIPA) kindreds, and patients with macroadenomas who are diagnosed ≤30 years). AIP mutations are most prevalent in patients with pituitary gigantism (29% of this group were found to have mutations in AIP gene). These data support targeted genetic screening for AIP mutations/deletions in these groups of pituitary adenoma patients. Earlier diagnosis of AIP-related acromegaly-gigantism cases enables timely clinical evaluation and treatment, thereby improving outcomes in terms of excessive linear growth and acromegaly comorbidities. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Mutations in MexB that affect the efflux of antibiotics with cytoplasmic targets.

PubMed

Ohene-Agyei, Thelma; Lea, Jon D; Venter, Henrietta

2012-08-01

Drug efflux pumps such as MexAB-OprM from Pseudomonas aeruginosa confer resistance to a wide range of chemically different compounds. Within the tripartite assembly, the inner membrane protein MexB is mainly responsible for substrate recognition. Recently, considerable advances have been made in elucidating the drug efflux pathway through the large periplasmic domains of resistance-nodulation-division (RND) transporters. However, little is known about the role of amino acids in other parts of the protein. We have investigated the role of two conserved phenylalanine residues that are aligned around the cytoplasmic side of the central cavity of MexB. The two conserved phenylalanine residues have been mutated to alanine residues (FAFA MexB). The interaction of the wild-type and mutant proteins with a variety of drugs from different classes was investigated by assays of cytotoxicity and drug transport. The FAFA mutation affected the efflux of compounds that have targets inside the cell, but antibiotics that act on cell wall synthesis and membrane probes were unaffected. Combined, our results indicate the presence of a hitherto unidentified cytoplasmic-binding site in RND drug transporters and enhance our understanding of the molecular mechanisms that govern drug resistance in Gram-negative pathogens. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Genomic evaluation of regional dairy cattle breeds in single-breed and multibreed contexts.

PubMed

Jónás, D; Ducrocq, V; Fritz, S; Baur, A; Sanchez, M-P; Croiseau, P

2017-02-01

An important prerequisite for high prediction accuracy in genomic prediction is the availability of a large training population, which allows accurate marker effect estimation. This requirement is not fulfilled in case of regional breeds with a limited number of breeding animals. We assessed the efficiency of the current French routine genomic evaluation procedure in four regional breeds (Abondance, Tarentaise, French Simmental and Vosgienne) as well as the potential benefits when the training populations consisting of males and females of these breeds are merged to form a multibreed training population. Genomic evaluation was 5-11% more accurate than a pedigree-based BLUP in three of the four breeds, while the numerically smallest breed showed a < 1% increase in accuracy. Multibreed genomic evaluation was beneficial for two breeds (Abondance and French Simmental) with maximum gains of 5 and 8% in correlation coefficients between yield deviations and genomic estimated breeding values, when compared to the single-breed genomic evaluation results. Inflation of genomic evaluation of young candidates was also reduced. Our results indicate that genomic selection can be effective in regional breeds as well. Here, we provide empirical evidence proving that genetic distance between breeds is only one of the factors affecting the efficiency of multibreed genomic evaluation. © 2016 Blackwell Verlag GmbH.

AG-221, a First-in-Class Therapy Targeting Acute Myeloid Leukemia Harboring Oncogenic IDH2 Mutations.

PubMed

Yen, Katharine; Travins, Jeremy; Wang, Fang; David, Muriel D; Artin, Erin; Straley, Kimberly; Padyana, Anil; Gross, Stefan; DeLaBarre, Byron; Tobin, Erica; Chen, Yue; Nagaraja, Raj; Choe, Sung; Jin, Lei; Konteatis, Zenon; Cianchetta, Giovanni; Saunders, Jeffrey O; Salituro, Francesco G; Quivoron, Cyril; Opolon, Paule; Bawa, Olivia; Saada, Véronique; Paci, Angelo; Broutin, Sophie; Bernard, Olivier A; de Botton, Stéphane; Marteyn, Benoît S; Pilichowska, Monika; Xu, YingXia; Fang, Cheng; Jiang, Fan; Wei, Wentao; Jin, Shengfang; Silverman, Lee; Liu, Wei; Yang, Hua; Dang, Lenny; Dorsch, Marion; Penard-Lacronique, Virginie; Biller, Scott A; Su, Shin-San Michael

2017-05-01

Somatic gain-of-function mutations in isocitrate dehydrogenases ( IDH ) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite ( R )-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate-dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation-positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2 R140Q -mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation-positive advanced hematologic malignancies. Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478-93. ©2017 AACR. See related commentary by Thomas and Majeti, p. 459 See related article by Shih et al., p. 494 This article is highlighted in the In This Issue feature, p. 443 . ©2017 American Association for Cancer Research.

Target-site mutations conferring resistance to glyphosate in feathertop Rhodes grass (Chloris virgata) populations in Australia.

PubMed

Ngo, The D; Krishnan, Mahima; Boutsalis, Peter; Gill, Gurjeet; Preston, Christopher

2018-05-01

Chloris virgata is a warm-season, C 4 , annual grass weed affecting field crops in northern Australia that has become an emerging weed in southern Australia. Four populations with suspected resistance to glyphosate were collected in South Australia, Queensland and New South Wales, Australia, and compared with one susceptible (S) population to confirm glyphosate resistance and elucidate possible mechanisms of resistance. Based on the rate of glyphosate required to kill 50% of treated plants (LD 50 ), glyphosate resistance (GR) was confirmed in four populations of C. virgata (V12, V14.2, V14.16 and V15). GR plants were 2-9.7-fold more resistant and accumulated less shikimate after glyphosate treatment than S plants. GR and S plants did not differ in glyphosate absorption and translocation. Target-site EPSPS mutations corresponding to Pro-106-Leu (V14.2) and Pro-106-Ser (V15, V14.16 and V12) substitutions were found in GR populations. The population with Pro-106-Leu substitution was 2.9-4.9-fold more resistant than the three other populations with Pro-106-Ser substitution. This report confirms glyphosate resistance in C. virgata and shows that target-site EPSPS mutations confer resistance to glyphosate in this species. The evolution of glyphosate resistance in C. virgata highlights the need to identify alternative control tactics. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Identification of MicroRNAs and their Targets Associated with Embryo Abortion during Chrysanthemum Cross Breeding via High-Throughput Sequencing.

PubMed

Zhang, Fengjiao; Dong, Wen; Huang, Lulu; Song, Aiping; Wang, Haibin; Fang, Weimin; Chen, Fadi; Teng, Nianjun

2015-01-01

MicroRNAs (miRNAs) are important regulators in plant development. They post-transcriptionally regulate gene expression during various biological and metabolic processes by binding to the 3'-untranslated region of target mRNAs to facilitate mRNA degradation or inhibit translation. Chrysanthemum (Chrysanthemum morifolium) is one of the most important ornamental flowers with increasing demand each year. However, embryo abortion is the main reason for chrysanthemum cross breeding failure. To date, there have been no experiments examining the expression of miRNAs associated with chrysanthemum embryo development. Therefore, we sequenced three small RNA libraries to identify miRNAs and their functions. Our results will provide molecular insights into chrysanthemum embryo abortion. Three small RNA libraries were built from normal chrysanthemum ovules at 12 days after pollination (DAP), and normal and abnormal chrysanthemum ovules at 18 DAP. We validated 228 miRNAs with significant changes in expression frequency during embryonic development. Comparative profiling revealed that 69 miRNAs exhibited significant differential expression between normal and abnormal embryos at 18 DAP. In addition, a total of 1037 miRNA target genes were predicted, and their annotations were defined by transcriptome data. Target genes associated with metabolic pathways were most highly represented according to the annotation. Moreover, 52 predicted target genes were identified to be associated with embryonic development, including 31 transcription factors and 21 additional genes. Gene ontology (GO) annotation also revealed that high-ranking miRNA target genes related to cellular processes and metabolic processes were involved in transcription regulation and the embryo developmental process. The present study generated three miRNA libraries and gained information on miRNAs and their targets in the chrysanthemum embryo. These results enrich the growing database of new miRNAs and lay the foundation

An Inversion Disrupting FAM134B Is Associated with Sensory Neuropathy in the Border Collie Dog Breed.

PubMed

Forman, Oliver P; Hitti, Rebekkah J; Pettitt, Louise; Jenkins, Christopher A; O'Brien, Dennis P; Shelton, G Diane; De Risio, Luisa; Quintana, Rodrigo Gutierrez; Beltran, Elsa; Mellersh, Cathryn

2016-09-08

Sensory neuropathy in the Border Collie is a severe neurological disorder caused by the degeneration of sensory and, to a lesser extent, motor nerve cells with clinical signs starting between 2 and 7 months of age. Using a genome-wide association study approach with three cases and 170 breed matched controls, a suggestive locus for sensory neuropathy was identified that was followed up using a genome sequencing approach. An inversion disrupting the candidate gene FAM134B was identified. Genotyping of additional cases and controls and RNAseq analysis provided strong evidence that the inversion is causal. Evidence of cryptic splicing resulting in novel exon transcription for FAM134B was identified by RNAseq experiments. This investigation demonstrates the identification of a novel sensory neuropathy associated mutation, by mapping using a minimal set of cases and subsequent genome sequencing. Through mutation screening, it should be possible to reduce the frequency of or completely eliminate this debilitating condition from the Border Collie breed population. Copyright © 2016 Forman et al.

Comprehensive mutation profiling of mucinous gastric carcinoma.

PubMed

Rokutan, Hirofumi; Hosoda, Fumie; Hama, Natsuko; Nakamura, Hiromi; Totoki, Yasushi; Furukawa, Eisaku; Arakawa, Erika; Ohashi, Shoko; Urushidate, Tomoko; Satoh, Hironori; Shimizu, Hiroko; Igarashi, Keiko; Yachida, Shinichi; Katai, Hitoshi; Taniguchi, Hirokazu; Fukayama, Masashi; Shibata, Tatsuhiro

2016-10-01

Mucinous gastric carcinoma (MGC) is a unique subtype of gastric cancer with a poor survival outcome. Comprehensive molecular profiles and putative therapeutic targets of MGC remain undetermined. We subjected 16 tumour-normal tissue pairs to whole-exome sequencing (WES) and an expanded set of 52 tumour-normal tissue pairs to subsequent targeted sequencing. The latter focused on 114 genes identified by WES. Twenty-two histologically differentiated MGCs (D-MGCs) and 46 undifferentiated MGCs (U-MGCs) were analysed. Chromatin modifier genes, including ARID1A (21%), MLL2 (19%), MLL3 (15%), and KDM6A (7%), were frequently mutated (47%) in MGC. We also identified mutations in potential therapeutic target genes, including MTOR (9%), BRCA2 (9%), BRCA1 (7%), and ERBB3 (6%). RHOA mutation was detected only in 4% of U-MGCs and in no D-MGCs. MYH9 was recurrently (13%) mutated in MGC, with all these being of the U-MGC subtype (p = 0.023). Three U-MGCs harboured MYH9 nonsense mutations. MYH9 knockdown enhanced cell migration and induced intracytoplasmic mucin and cellular elongation. BCOR mutation was associated with improved survival. In U-MGCs, the MLH1 expression status and combined mutation status (TP53/BCL11B or TP53/MLL2) were prognostic factors. A comparative analysis of driver genes revealed that the mutation profile of D-MGC was similar to that of intestinal-type gastric cancer, whereas U-MGC was a distinct entity, harbouring a different mutational profile to intestinal- and diffuse-type gastric cancers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Drug Resistance Missense Mutations in Cancer Are Subject to Evolutionary Constraints

PubMed Central

Friedman, Ran

2013-01-01

Several tumour types are sensitive to deactivation of just one or very few genes that are constantly active in the cancer cells, a phenomenon that is termed ‘oncogene addiction’. Drugs that target the products of those oncogenes can yield a temporary relief, and even complete remission. Unfortunately, many patients receiving oncogene-targeted therapies relapse on treatment. This often happens due to somatic mutations in the oncogene (‘resistance mutations’). ‘Compound mutations’, which in the context of cancer drug resistance are defined as two or more mutations of the drug target in the same clone may lead to enhanced resistance against the most selective inhibitors. Here, it is shown that the vast majority of the resistance mutations occurring in cancer patients treated with tyrosin kinase inhibitors aimed at three different proteins follow an evolutionary pathway. Using bioinformatic analysis tools, it is found that the drug-resistance mutations in the tyrosine kinase domains of Abl1, ALK and exons 20 and 21 of EGFR favour transformations to residues that can be identified in similar positions in evolutionary related proteins. The results demonstrate that evolutionary pressure shapes the mutational landscape in the case of drug-resistance somatic mutations. The constraints on the mutational landscape suggest that it may be possible to counter single drug-resistance point mutations. The observation of relatively many resistance mutations in Abl1, but not in the other genes, is explained by the fact that mutations in Abl1 tend to be biochemically conservative, whereas mutations in EGFR and ALK tend to be radical. Analysis of Abl1 compound mutations suggests that such mutations are more prevalent than hitherto reported and may be more difficult to counter. This supports the notion that such mutations may provide an escape route for targeted cancer drug resistance. PMID:24376513

THE EFFECT OF TARGETED KNOCKOUT MUTATION ON THE TRANSCRIPTIONAL PROFILE OF THE KIDNEY IN TSC2 MUTANT LONG-EVANS (EKER) RATS.

EPA Science Inventory

The effect of a targeted knockout mutation on the transcriptional profile of the kidney in
Tsc2 mutant Long-Evans (Eker) rats.

Renal cell carcinoma (RCC) is the most common tumor of the adult kidney, accounting
for up to 80% of malignant renal neoplasms. Hereditary...

Actionable mutations in canine hemangiosarcoma.

PubMed

Wang, Guannan; Wu, Ming; Maloneyhuss, Martha A; Wojcik, John; Durham, Amy C; Mason, Nicola J; Roth, David B

2017-01-01

Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma. Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage. Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

Succinate dehydrogenase subunit D and succinate dehydrogenase subunit B mutation analysis in canine phaeochromocytoma and paraganglioma.

PubMed

Holt, D E; Henthorn, P; Howell, V M; Robinson, B G; Benn, D E

2014-07-01

Phaeochromocytomas (PCs) are tumours of the adrenal medulla chromaffin cells. Paragangliomas (PGLs) arise in sympathetic ganglia (previously called extra-adrenal PCs) or in non-chromaffin parasympathetic ganglia cells that are usually non-secretory. Parenchymal cells from these tumours have a common embryological origin from neural crest ectoderm. Several case series of canine PCs and PGLs have been published and a link between the increased incidence of chemoreceptor neoplasia in brachycephalic dog breeds and chronic hypoxia has been postulated. A similar link to hypoxia in man led to the identification of germline heterozygous mutations in the gene encoding succinate dehydrogenase subunit D (SDHD) and subsequently SDHA, SDHB and SDHC in similar tumours. We investigated canine PCs (n = 6) and PGLs (n = 2) for SDHD and SDHB mutations and in one PGL found a somatic SDHD mutation c.365A>G (p.Lys122Arg) in exon 4, which was not present in normal tissue from this brachycephalic dog. Two PCs were heterozygous for both c.365A>G (p.Lys122Arg) mutation and an exon 3 silent variant c.291G>A. We also identified the heterozygous SDHB exon 2 mutation c.113G>A (p.Arg38Gln) in a PC. These results illustrate that genetic mutations may underlie tumourigenesis in canine PCs and PGLs. The spontaneous nature of these canine diseases and possible association of PGLs with hypoxia in brachycephalic breeds may make them an attractive model for studying the corresponding human tumours. Copyright © 2014 Elsevier Ltd. All rights reserved.

Investigation of prolific sheep from UK and Ireland for evidence on origin of the mutations in BMP15 (FecX(G), FecX(B)) and GDF9 (FecG(H)) in Belclare and Cambridge sheep.

PubMed

Mullen, Michael P; Hanrahan, James P; Howard, Dawn J; Powell, Richard

2013-01-01

This paper concerns the likely origin of three mutations with large effects on ovulation rate identified in the Belclare and Cambridge sheep breeds; two in the BMP15 gene (FecX(G) and FecX(B)) and the third (FecG(H)) in GDF9. All three mutations segregate in Belclare sheep while one, FecX(B), has not been found in the Cambridge. Both Belclare and Cambridge breeds are relatively recently developed composites that have common ancestry through the use of genetic material from the Finnish Landrace and Lleyn breeds. The development of both composites also involved major contributions from exceptionally prolific ewes screened from flocks in Ireland (Belclare) and Britain (Cambridge) during the 1960s. The objective of the current study was to establish the likely origin of the mutations (FecX(G), FecX(B) and FecG(H)) through analysis of DNA from Finnish Landrace and Lleyn sheep, and Galway and Texel breeds which contributed to the development of the Belclare breed. Ewes with exceptionally high prolificacy (hyper-prolific ewes) in current flocks on Irish farms were identified to simulate the screening of ewes from Irish flocks in the 1960s. DNA was obtained from: prolific ewes in extant flocks of Lleyn sheep (n = 44) on the Lleyn peninsula in Wales; hyper-prolific ewes (n = 41); prolific Galway (n = 41) ewes; Finnish Landrace (n = 124) and Texel (n = 19) ewes. The FecX(G) mutation was identified in Lleyn but not in Finnish Landrace, Galway or Texel sheep; FecX(B) was only found among the hyper-prolific ewes. The FecG(H) mutation was identified in the sample of Lleyn sheep. It was concluded from these findings that the Lleyn breed was the most likely source of the FecX(G) and FecG(H) mutations in Belclare and Cambridge sheep and that the FecX(B) mutation came from the High Fertility line that was developed using prolific ewes selected from commercial flocks in Ireland in the 1960's and subsequently used in the genesis of the Belclare.

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT

PubMed Central

Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

2016-01-01

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors’ sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib. PMID:27467080

An International Genetic Survey of Breed-Specific Diseases in Working Dogs from the United States, Israel, and Poland.

PubMed

Shaffer, Lisa G; Ramirez, Christina J; Phelps, Patricia; Aviram, Maya; Walczak, Marta; Bar-Gal, Gila Kahila; Ballif, Blake C

2017-01-01

Genetic diseases occur in breeds used for law enforcement. As important team members, dogs are expected to operate at peak performance for several years and are significant investments for both the initial purchase and extensive, specialized training. Previous studies have not focused on causes for retirement or euthanasia as genetic (inherited) versus acquired (environmental). We performed direct mutational analysis for breed-specific conditions on samples from 304 dogs including 267 law enforcement (122 US, 87 Israeli, and 58 Polish) and 37 search and rescue dogs. Genetic testing identified 29% (n = 89) of the dogs tested to be carriers of a genetic mutation and 6% (n = 19) to be at risk for a debilitating inherited condition that may eventually impair the dog's ability to work. At-risk dogs included Labrador Retrievers (n = 4) with exercise-induced collapse, Bloodhounds (n = 2) with degenerative myelopathy (DM), and German Shepherd dogs with DM (n = 12) or leukocyte adhesion deficiency, type III (n = 1). A substantial number of working dogs were shown to be at risk for genetic conditions that may shorten the dog's career. The loss of dogs, due to early retirement or euthanasia, as a result of preventable genetic conditions has an emotional cost to handlers and financial cost to service organizations that can be avoided with genetic screening prior to breeding, buying, or training. © 2018 S. Karger AG, Basel.

A Pathway-Centered Analysis of Pig Domestication and Breeding in Eurasia

PubMed Central

Leno-Colorado, Jordi; Hudson, Nick J.; Reverter, Antonio; Pérez-Enciso, Miguel

2017-01-01

Ascertaining the molecular and physiological basis of domestication and breeding is an active area of research. Due to the current wide distribution of its wild ancestor, the wild boar, the pig (Sus scrofa) is an excellent model to study these processes, which occurred independently in East Asia and Europe ca. 9000 yr ago. Analyzing genome variability patterns in terms of metabolic pathways is attractive since it considers the impact of interrelated functions of genes, in contrast to genome-wide scans that treat genes or genome windows in isolation. To that end, we studied 40 wild boars and 123 domestic pig genomes from Asia and Europe when metabolic pathway was the unit of analysis. We computed statistical significance for differentiation (Fst) and linkage disequilibrium (nSL) statistics at the pathway level. In terms of Fst, we found 21 and 12 pathways significantly differentiated at a q-value < 0.05 in Asia and Europe, respectively; five were shared across continents. In Asia, we found six significant pathways related to behavior, which involved essential neurotransmitters like dopamine and serotonin. Several significant pathways were interrelated and shared a variable percentage of genes. There were 12 genes present in >10 significant pathways (in terms of Fst), comprising genes involved in the transduction of a large number of signals, like phospholipase PCLB1, which is expressed in the brain, or ITPR3, which has an important role in taste transduction. In terms of nSL, significant pathways were mainly related to reproductive performance (ovarian steroidogenesis), a similarly important target trait during domestication and modern animal breeding. Different levels of recombination cannot explain these results, since we found no correlation between Fst and recombination rate. However, we did find an increased ratio of deleterious mutations in domestic vs. wild populations, suggesting a relaxed functional constraint associated with the domestication and breeding

A Pathway-Centered Analysis of Pig Domestication and Breeding in Eurasia.

PubMed

Leno-Colorado, Jordi; Hudson, Nick J; Reverter, Antonio; Pérez-Enciso, Miguel

2017-07-05

Ascertaining the molecular and physiological basis of domestication and breeding is an active area of research. Due to the current wide distribution of its wild ancestor, the wild boar, the pig ( Sus scrofa ) is an excellent model to study these processes, which occurred independently in East Asia and Europe ca. 9000 yr ago. Analyzing genome variability patterns in terms of metabolic pathways is attractive since it considers the impact of interrelated functions of genes, in contrast to genome-wide scans that treat genes or genome windows in isolation. To that end, we studied 40 wild boars and 123 domestic pig genomes from Asia and Europe when metabolic pathway was the unit of analysis. We computed statistical significance for differentiation (Fst) and linkage disequilibrium (nSL) statistics at the pathway level. In terms of Fst, we found 21 and 12 pathways significantly differentiated at a q -value < 0.05 in Asia and Europe, respectively; five were shared across continents. In Asia, we found six significant pathways related to behavior, which involved essential neurotransmitters like dopamine and serotonin. Several significant pathways were interrelated and shared a variable percentage of genes. There were 12 genes present in >10 significant pathways (in terms of Fst), comprising genes involved in the transduction of a large number of signals, like phospholipase PCLB1, which is expressed in the brain, or ITPR3, which has an important role in taste transduction. In terms of nSL, significant pathways were mainly related to reproductive performance (ovarian steroidogenesis), a similarly important target trait during domestication and modern animal breeding. Different levels of recombination cannot explain these results, since we found no correlation between Fst and recombination rate. However, we did find an increased ratio of deleterious mutations in domestic vs. wild populations, suggesting a relaxed functional constraint associated with the domestication and

Exome Sequencing Identifies Potentially Druggable Mutations in Nasopharyngeal Carcinoma.

PubMed

Chow, Yock Ping; Tan, Lu Ping; Chai, San Jiun; Abdul Aziz, Norazlin; Choo, Siew Woh; Lim, Paul Vey Hong; Pathmanathan, Rajadurai; Mohd Kornain, Noor Kaslina; Lum, Chee Lun; Pua, Kin Choo; Yap, Yoke Yeow; Tan, Tee Yong; Teo, Soo Hwang; Khoo, Alan Soo-Beng; Patel, Vyomesh

2017-03-03

In this study, we first performed whole exome sequencing of DNA from 10 untreated and clinically annotated fresh frozen nasopharyngeal carcinoma (NPC) biopsies and matched bloods to identify somatically mutated genes that may be amenable to targeted therapeutic strategies. We identified a total of 323 mutations which were either non-synonymous (n = 238) or synonymous (n = 85). Furthermore, our analysis revealed genes in key cancer pathways (DNA repair, cell cycle regulation, apoptosis, immune response, lipid signaling) were mutated, of which those in the lipid-signaling pathway were the most enriched. We next extended our analysis on a prioritized sub-set of 37 mutated genes plus top 5 mutated cancer genes listed in COSMIC using a custom designed HaloPlex target enrichment panel with an additional 88 NPC samples. Our analysis identified 160 additional non-synonymous mutations in 37/42 genes in 66/88 samples. Of these, 99/160 mutations within potentially druggable pathways were further selected for validation. Sanger sequencing revealed that 77/99 variants were true positives, giving an accuracy of 78%. Taken together, our study indicated that ~72% (n = 71/98) of NPC samples harbored mutations in one of the four cancer pathways (EGFR-PI3K-Akt-mTOR, NOTCH, NF-κB, DNA repair) which may be potentially useful as predictive biomarkers of response to matched targeted therapies.

Exome Sequencing Identifies Potentially Druggable Mutations in Nasopharyngeal Carcinoma

PubMed Central

Chow, Yock Ping; Tan, Lu Ping; Chai, San Jiun; Abdul Aziz, Norazlin; Choo, Siew Woh; Lim, Paul Vey Hong; Pathmanathan, Rajadurai; Mohd Kornain, Noor Kaslina; Lum, Chee Lun; Pua, Kin Choo; Yap, Yoke Yeow; Tan, Tee Yong; Teo, Soo Hwang; Khoo, Alan Soo-Beng; Patel, Vyomesh

2017-01-01

In this study, we first performed whole exome sequencing of DNA from 10 untreated and clinically annotated fresh frozen nasopharyngeal carcinoma (NPC) biopsies and matched bloods to identify somatically mutated genes that may be amenable to targeted therapeutic strategies. We identified a total of 323 mutations which were either non-synonymous (n = 238) or synonymous (n = 85). Furthermore, our analysis revealed genes in key cancer pathways (DNA repair, cell cycle regulation, apoptosis, immune response, lipid signaling) were mutated, of which those in the lipid-signaling pathway were the most enriched. We next extended our analysis on a prioritized sub-set of 37 mutated genes plus top 5 mutated cancer genes listed in COSMIC using a custom designed HaloPlex target enrichment panel with an additional 88 NPC samples. Our analysis identified 160 additional non-synonymous mutations in 37/42 genes in 66/88 samples. Of these, 99/160 mutations within potentially druggable pathways were further selected for validation. Sanger sequencing revealed that 77/99 variants were true positives, giving an accuracy of 78%. Taken together, our study indicated that ~72% (n = 71/98) of NPC samples harbored mutations in one of the four cancer pathways (EGFR-PI3K-Akt-mTOR, NOTCH, NF-κB, DNA repair) which may be potentially useful as predictive biomarkers of response to matched targeted therapies. PMID:28256603

Miniaturized GPS Tags Identify Non-breeding Territories of a Small Breeding Migratory Songbird.

PubMed

Hallworth, Michael T; Marra, Peter P

2015-06-09

For the first time, we use a small archival global positioning system (GPS) tag to identify and characterize non-breeding territories, quantify migratory connectivity, and identify population boundaries of Ovenbirds (Seiurus aurocapilla), a small migratory songbird, captured at two widely separated breeding locations. We recovered 15 (31%) GPS tags with data and located the non-breeding territories of breeding Ovenbirds from Maryland and New Hampshire, USA (0.50 ± 0.15 ha, mean ± SE). All non-breeding territories had similar environmental attributes despite being distributed across parts of Florida, Cuba and Hispaniola. New Hampshire and Maryland breeding populations had non-overlapping non-breeding population boundaries that encompassed 114,803 and 169,233 km(2), respectively. Archival GPS tags provided unprecedented pinpoint locations and associated environmental information of tropical non-breeding territories. This technology is an important step forward in understanding seasonal interactions and ultimately population dynamics of populations throughout the annual cycle.

DNA mutations of the cat: the good, the bad and the ugly.

PubMed

Lyons, Leslie A

2015-03-01

The health of the cat is a complex interaction between its environment (nurture) and its genetics (nature). Over 70 genetic mutations (variants) have been defined in the cat, many involving diseases, structural abnormalities and clinically relevant health concerns. As more of the cat's genome is deciphered, less commonly will the term 'idiopathic' be used regarding the diagnosis of diseases and unique health conditions. State-of-the-art health care will include DNA profiling of the individual cat, and perhaps its tumor, to establish the best treatment approaches. Genetic testing and eventually whole genome sequencing should become routine diagnostics for feline health care. Cat breeds have disseminated around the world. Thus, practitioners should be aware of the breeds common to their region and the mutations found in those regional populations. Specific random-bred populations can also have defined genetic characteristics and mutations. This review of 'the good, the bad and the ugly' DNA variants provides the current state of knowledge for genetic testing and genetic health management for cats. It is aimed at feline and general practitioners wanting to update and review the basics of genetics, what tests are available for cats and sources for genetic testing. The tables are intended to be used as references in the clinic. Practitioners with a high proportion of cat breeder clientele will especially benefit from the review. The data presented is extracted from peer-reviewed publications pertaining to mutation identification, and relevant articles concerning the heritable trait and/or disease. The author also draws upon personal experience and expertise in feline genetics. © ISFM and AAFP 2015.

Frequency and distribution of 152 genetic disease variants in over 100,000 mixed breed and purebred dogs

PubMed Central

Anderson, Heidi; Davison, Stephen; Hughes, Angela M.; Bouirmane, Julia; Lindqvist, Johan; Lytle, Katherine M.; Ganesan, Balasubramanian; Ottka, Claudia; Ruotanen, Päivi; Forman, Oliver P.; Fretwell, Neale; Cole, Cynthia A.; Lohi, Hannes

2018-01-01

Knowledge on the genetic epidemiology of disorders in the dog population has implications for both veterinary medicine and sustainable breeding. Limited data on frequencies of genetic disease variants across breeds exists, and the disease heritage of mixed breed dogs remains poorly explored to date. Advances in genetic screening technologies now enable comprehensive investigations of the canine disease heritage, and generate health-related big data that can be turned into action. We pursued population screening of genetic variants implicated in Mendelian disorders in the largest canine study sample examined to date by examining over 83,000 mixed breed and 18,000 purebred dogs representing 330 breeds for 152 known variants using a custom-designed beadchip microarray. We further announce the creation of MyBreedData (www.mybreeddata.com), an online updated inherited disorder prevalence resource with its foundation in the generated data. We identified the most prevalent, and rare, disease susceptibility variants across the general dog population while providing the first extensive snapshot of the mixed breed disease heritage. Approximately two in five dogs carried at least one copy of a tested disease variant. Most disease variants are shared by both mixed breeds and purebreds, while breed- or line-specificity of others is strongly suggested. Mixed breed dogs were more likely to carry a common recessive disease, whereas purebreds were more likely to be genetically affected with one, providing DNA-based evidence for hybrid vigor. We discovered genetic presence of 22 disease variants in at least one additional breed in which they were previously undescribed. Some mutations likely manifest similarly independently of breed background; however, we emphasize the need for follow up investigations in each case and provide a suggested validation protocol for broader consideration. In conclusion, our study provides unique insight into genetic epidemiology of canine disease risk

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.

Molecular identification of livestock breeds: a tool for modern conservation biology.

PubMed

Yaro, Mohammed; Munyard, Kylie A; Stear, Michael J; Groth, David M

2017-05-01

Global livestock genetic diversity includes all of the species, breeds and strains of domestic animals, and their variations. Although a recent census indicated that there were 40 species and over 8000 breeds of domestic animals; for the purpose of conservation biology the diversity between and within breeds rather than species is regarded to be of crucial importance. This domestic animal genetic diversity has developed through three main evolutionary events, from speciation (about 3 million years ago) through domestication (about 12000 years ago) to specialised breeding (starting about 200 years ago). These events and their impacts on global animal genetic resources have been well documented in the literature. The key importance of global domestic animal resources in terms of economic, scientific and cultural heritage has also been addressed. In spite of their importance, there is a growing number of reports on the alarming erosion of domestic animal genetic resources. This erosion of is happening in spite of several global conservation initiatives designed to mitigate it. Herein we discuss these conservation interventions and highlight their strengths and weaknesses. However, pivotal to the success of these conservation initiatives is the reliability of the genetic assignment of individual members to a target breed. Finally, we discuss the prospect of using improved breed identification methodologies to develop a reliable breed-specific molecular identification tool that is easily applicable to populations of livestock breeds in various ecosystems. These identification tools, when developed, will not only facilitate the regular monitoring of threatened or endangered breed populations, but also enhance the development of more efficient and sustainable livestock production systems. © 2016 Cambridge Philosophical Society.

Genome editing in livestock: Are we ready for a revolution in animal breeding industry?

PubMed

Ruan, Jinxue; Xu, Jie; Chen-Tsai, Ruby Yanru; Li, Kui

2017-12-01

Genome editing is a powerful technology that can efficiently alter the genome of organisms to achieve targeted modification of endogenous genes and targeted integration of exogenous genes. Current genome-editing tools mainly include ZFN, TALEN and CRISPR/Cas9, which have been successfully applied to all species tested including zebrafish, humans, mice, rats, monkeys, pigs, cattle, sheep, goats and others. The application of genome editing has quickly swept through the entire biomedical field, including livestock breeding. Traditional livestock breeding is associated with rate limiting issues such as long breeding cycle and limitations of genetic resources. Genome editing tools offer solutions to these problems at affordable costs. Generation of gene-edited livestock with improved traits has proven feasible and valuable. For example, the CD163 gene-edited pig is resistant to porcine reproductive and respiratory syndrome (PRRS, also referred to as "blue ear disease"), and a SP110 gene knock-in cow less susceptible to tuberculosis. Given the high efficiency and low cost of genome editing tools, particularly CRISPR/Cas9, it is foreseeable that a significant number of genome edited livestock animals will be produced in the near future; hence it is imperative to comprehensively evaluate the pros and cons they will bring to the livestock breeding industry. Only with these considerations in mind, we will be able to fully take the advantage of the genome editing era in livestock breeding.

The contribution of de novo coding mutations to autism spectrum disorder.

PubMed

Iossifov, Ivan; O'Roak, Brian J; Sanders, Stephan J; Ronemus, Michael; Krumm, Niklas; Levy, Dan; Stessman, Holly A; Witherspoon, Kali T; Vives, Laura; Patterson, Karynne E; Smith, Joshua D; Paeper, Bryan; Nickerson, Deborah A; Dea, Jeanselle; Dong, Shan; Gonzalez, Luis E; Mandell, Jeffrey D; Mane, Shrikant M; Murtha, Michael T; Sullivan, Catherine A; Walker, Michael F; Waqar, Zainulabedin; Wei, Liping; Willsey, A Jeremy; Yamrom, Boris; Lee, Yoon-ha; Grabowska, Ewa; Dalkic, Ertugrul; Wang, Zihua; Marks, Steven; Andrews, Peter; Leotta, Anthony; Kendall, Jude; Hakker, Inessa; Rosenbaum, Julie; Ma, Beicong; Rodgers, Linda; Troge, Jennifer; Narzisi, Giuseppe; Yoon, Seungtai; Schatz, Michael C; Ye, Kenny; McCombie, W Richard; Shendure, Jay; Eichler, Evan E; State, Matthew W; Wigler, Michael

2014-11-13

Whole exome sequencing has proven to be a powerful tool for understanding the genetic architecture of human disease. Here we apply it to more than 2,500 simplex families, each having a child with an autistic spectrum disorder. By comparing affected to unaffected siblings, we show that 13% of de novo missense mutations and 43% of de novo likely gene-disrupting (LGD) mutations contribute to 12% and 9% of diagnoses, respectively. Including copy number variants, coding de novo mutations contribute to about 30% of all simplex and 45% of female diagnoses. Almost all LGD mutations occur opposite wild-type alleles. LGD targets in affected females significantly overlap the targets in males of lower intelligence quotient (IQ), but neither overlaps significantly with targets in males of higher IQ. We estimate that LGD mutation in about 400 genes can contribute to the joint class of affected females and males of lower IQ, with an overlapping and similar number of genes vulnerable to contributory missense mutation. LGD targets in the joint class overlap with published targets for intellectual disability and schizophrenia, and are enriched for chromatin modifiers, FMRP-associated genes and embryonically expressed genes. Most of the significance for the latter comes from affected females.

Schemes for Oestrus Synchronization Protocols and Controlled Breeding Programs in Cattle

NASA Astrophysics Data System (ADS)

Sabo, Y. G.; Sandabe, U. K.; Maina, V. A.; Balla, H. G.

Today prostaglandin and progesterone has been found widely used in several schemes of oestrus synchronization and controlled breeding program. Several controlled breeding program, have been developed for synchronizing groups of all open or lactating cows within a breeding group with or without ovarian palpation. Such programs are reviewed in this article which involves extending the luteal phase by treatment with exogenous progesterone such as: progesterone treatment regimes using syncro-mate-B, progesterone releasing intravaginal device, melengesterol acetate-select and melegestrol acetate plus prostaglandin. Also reviewed in the program is the termination of the luteal phase by treatment with prostaglandin or its analogues. These includes, controlled breeding without ovarian palpation such as, the 7-days program; 11-days program, target breeding, ovsynch program, Heat synch, Cosynch and pre synch-ovsynch program. In our opinion full potential of progesterone and prostaglandin for the detection of oestrus and timed artificial insemination should be utilized. This reduces the much labour input employed in previous years. The practitioner of the livestock herd health must-develop strategies for the delivery of this technology to livestock farmers, its use and limitations.

Breeding of ozone resistant rice: relevance, approaches and challenges.

PubMed

Frei, Michael

2015-02-01

Tropospheric ozone concentrations have been rising across Asia, and will continue to rise during the 21st century. Ozone affects rice yields through reductions in spikelet number, spikelet fertility, and grain size. Moreover, ozone leads to changes in rice grain and straw quality. Therefore the breeding of ozone tolerant rice varieties is warranted. The mapping of quantitative trait loci (QTL) using bi-parental populations identified several tolerance QTL mitigating symptom formation, grain yield losses, or the degradation of straw quality. A genome-wide association study (GWAS) demonstrated substantial natural genotypic variation in ozone tolerance in rice, and revealed that the genetic architecture of ozone tolerance in rice is dominated by multiple medium and small effect loci. Transgenic approaches targeting tolerance mechanisms such as antioxidant capacity are also discussed. It is concluded that the breeding of ozone tolerant rice can contribute substantially to the global food security, and is feasible using different breeding approaches. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cereal phytochromes: targets of selection, targets for manipulation?

PubMed

Sawers, Ruairidh J H; Sheehan, Moira J; Brutnell, Thomas P

2005-03-01

Plants respond to shading through an adaptive syndrome termed shade avoidance. In high-density crop plantings, shade avoidance generally increases extension growth at the expense of yield and can be at odds with the agronomic performance of the crop as a whole. Studies in Arabidopsis are beginning to reveal the essential role phytochromes play in regulating this process and to identify genes underlying the response. In this article, we focus on how phytochrome signaling networks have been targeted in cereal breeding programs in the past and discuss the potential to alter these pathways through breeding and transgenic manipulation to develop crops that perform better under typical high density conditions.

Possible introgression of the VRTN mutation increasing vertebral number, carcass length and teat number from Chinese pigs into European pigs

PubMed Central

Yang, Jie; Huang, Lusheng; Yang, Ming; Fan, Yin; Li, Lin; Fang, Shaoming; Deng, Wenjiang; Cui, Leilei; Zhang, Zhen; Ai, Huashui; Wu, Zhenfang; Gao, Jun; Ren, Jun

2016-01-01

Vertnin (VRTN) variants have been associated with the number of thoracic vertebrae in European pigs, but the association has not been evidenced in Chinese indigenous pigs. In this study, we first performed a genome-wide association study in Chinese Erhualian pigs using one VRTN candidate causative mutation and the Illumina Porcine 60K SNP Beadchips. The VRTN mutation is significantly associated with thoracic vertebral number in this population. We further show that the VRTN mutation has pleiotropic and desirable effects on teat number and carcass (body) length across four diverse populations, including Erhualian, White Duroc × Erhualian F2 population, Duroc and Landrace pigs. No association was observed between VRTN genotype and growth and fatness traits in these populations. Therefore, testing for the VRTN mutation in pig breeding schemes would not only increase the number of vertebrae and nipples, but also enlarge body size without undesirable effects on growth and fatness traits, consequently improving pork production. Further, by using whole-genome sequence data, we show that the VRTN mutation was possibly introgressed from Chinese pigs into European pigs. Our results provide another example showing that introgressed Chinese genes greatly contributed to the development and production of modern European pig breeds. PMID:26781738

Conceptual framework for drought phenotyping during molecular breeding.

PubMed

Salekdeh, Ghasem Hosseini; Reynolds, Matthew; Bennett, John; Boyer, John

2009-09-01

Drought is a major threat to agricultural production and drought tolerance is a prime target for molecular approaches to crop improvement. To achieve meaningful results, these approaches must be linked with suitable phenotyping protocols at all stages, such as the screening of germplasm collections, mutant libraries, mapping populations, transgenic lines and breeding materials and the design of OMICS and quantitative trait loci (QTLs) experiments. Here we present a conceptual framework for molecular breeding for drought tolerance based on the Passioura equation of expressing yield as the product of water use (WU), water use efficiency (WUE) and harvest index (HI). We identify phenotyping protocols that address each of these factors, describe their key features and illustrate their integration with different molecular approaches.

Breeding potential of elite Pee Dee germplasm in Upland cotton breeding programs

USDA-ARS?s Scientific Manuscript database

Successful plant breeding programs begin with parental line selection. Effective parental line selection is facilitated when the breeding potential of candidate parental lines is known. Using topcross families involving germplasm representing eight US public cotton breeding programs, we evaluated th...

Microsatellites as targets of natural selection.

PubMed

Haasl, Ryan J; Payseur, Bret A

2013-02-01

The ability to survey polymorphism on a genomic scale has enabled genome-wide scans for the targets of natural selection. Theory that connects patterns of genetic variation to evidence of natural selection most often assumes a diallelic locus and no recurrent mutation. Although these assumptions are suitable to selection that targets single nucleotide variants, fundamentally different types of mutation generate abundant polymorphism in genomes. Moreover, recent empirical results suggest that mutationally complex, multiallelic loci including microsatellites and copy number variants are sometimes targeted by natural selection. Given their abundance, the lack of inference methods tailored to the mutational peculiarities of these types of loci represents a notable gap in our ability to interrogate genomes for signatures of natural selection. Previous theoretical investigations of mutation-selection balance at multiallelic loci include assumptions that limit their application to inference from empirical data. Focusing on microsatellites, we assess the dynamics and population-level consequences of selection targeting mutationally complex variants. We develop general models of a multiallelic fitness surface, a realistic model of microsatellite mutation, and an efficient simulation algorithm. Using these tools, we explore mutation-selection-drift equilibrium at microsatellites and investigate the mutational history and selective regime of the microsatellite that causes Friedreich's ataxia. We characterize microsatellite selective events by their duration and cost, note similarities to sweeps from standing point variation, and conclude that it is premature to label microsatellites as ubiquitous agents of efficient adaptive change. Together, our models and simulation algorithm provide a powerful framework for statistical inference, which can be used to test the neutrality of microsatellites and other multiallelic variants.

Microsatellites as Targets of Natural Selection

PubMed Central

Haasl, Ryan J.; Payseur, Bret A.

2013-01-01

The ability to survey polymorphism on a genomic scale has enabled genome-wide scans for the targets of natural selection. Theory that connects patterns of genetic variation to evidence of natural selection most often assumes a diallelic locus and no recurrent mutation. Although these assumptions are suitable to selection that targets single nucleotide variants, fundamentally different types of mutation generate abundant polymorphism in genomes. Moreover, recent empirical results suggest that mutationally complex, multiallelic loci including microsatellites and copy number variants are sometimes targeted by natural selection. Given their abundance, the lack of inference methods tailored to the mutational peculiarities of these types of loci represents a notable gap in our ability to interrogate genomes for signatures of natural selection. Previous theoretical investigations of mutation-selection balance at multiallelic loci include assumptions that limit their application to inference from empirical data. Focusing on microsatellites, we assess the dynamics and population-level consequences of selection targeting mutationally complex variants. We develop general models of a multiallelic fitness surface, a realistic model of microsatellite mutation, and an efficient simulation algorithm. Using these tools, we explore mutation-selection-drift equilibrium at microsatellites and investigate the mutational history and selective regime of the microsatellite that causes Friedreich’s ataxia. We characterize microsatellite selective events by their duration and cost, note similarities to sweeps from standing point variation, and conclude that it is premature to label microsatellites as ubiquitous agents of efficient adaptive change. Together, our models and simulation algorithm provide a powerful framework for statistical inference, which can be used to test the neutrality of microsatellites and other multiallelic variants. PMID:23104080

Targeting Nonsense Mutations in Diseases with Translational Read-Through-Inducing Drugs (TRIDs).

PubMed

Nagel-Wolfrum, Kerstin; Möller, Fabian; Penner, Inessa; Baasov, Timor; Wolfrum, Uwe

2016-04-01

In recent years, remarkable advances in the ability to diagnose genetic disorders have been made. The identification of disease-causing genes allows the development of gene-specific therapies with the ultimate goal to develop personalized medicines for each patient according to their own specific genetic defect. In-depth genotyping of many different genes has revealed that ~12% of inherited genetic disorders are caused by in-frame nonsense mutations. Nonsense (non-coding) mutations are caused by point mutations, which generate premature termination codons (PTCs) that cause premature translational termination of the mRNA, and subsequently inhibit normal full-length protein expression. Recently, a gene-based therapeutic approach for genetic diseases caused by nonsense mutations has emerged, namely the so-called translational read-through (TR) therapy. Read-through therapy is based on the discovery that small molecules, known as TR-inducing drugs (TRIDs), allow the translation machinery to suppress a nonsense codon, elongate the nascent peptide chain, and consequently result in the synthesis of full-length protein. Several TRIDs are currently under investigation and research has been performed on several genetic disorders caused by nonsense mutations over the years. These findings have raised hope for the usage of TR therapy as a gene-based pharmacogenetic therapy for nonsense mutations in various genes responsible for a variety of genetic diseases.

A New Targeted CFTR Mutation Panel Based on Next-Generation Sequencing Technology.

PubMed

Lucarelli, Marco; Porcaro, Luigi; Biffignandi, Alice; Costantino, Lucy; Giannone, Valentina; Alberti, Luisella; Bruno, Sabina Maria; Corbetta, Carlo; Torresani, Erminio; Colombo, Carla; Seia, Manuela

2017-09-01

Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing-based method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Exploiting Genomic Knowledge in Optimising Molecular Breeding Programmes: Algorithms from Evolutionary Computing

PubMed Central

O'Hagan, Steve; Knowles, Joshua; Kell, Douglas B.

2012-01-01

Comparatively few studies have addressed directly the question of quantifying the benefits to be had from using molecular genetic markers in experimental breeding programmes (e.g. for improved crops and livestock), nor the question of which organisms should be mated with each other to best effect. We argue that this requires in silico modelling, an approach for which there is a large literature in the field of evolutionary computation (EC), but which has not really been applied in this way to experimental breeding programmes. EC seeks to optimise measurable outcomes (phenotypic fitnesses) by optimising in silico the mutation, recombination and selection regimes that are used. We review some of the approaches from EC, and compare experimentally, using a biologically relevant in silico landscape, some algorithms that have knowledge of where they are in the (genotypic) search space (G-algorithms) with some (albeit well-tuned ones) that do not (F-algorithms). For the present kinds of landscapes, F- and G-algorithms were broadly comparable in quality and effectiveness, although we recognise that the G-algorithms were not equipped with any ‘prior knowledge’ of epistatic pathway interactions. This use of algorithms based on machine learning has important implications for the optimisation of experimental breeding programmes in the post-genomic era when we shall potentially have access to the full genome sequence of every organism in a breeding population. The non-proprietary code that we have used is made freely available (via Supplementary information). PMID:23185279

Novel Insight into Mutational Landscape of Head and Neck Squamous Cell Carcinoma

PubMed Central

Gaykalova, Daria A.; Mambo, Elizabeth; Choudhary, Ashish; Houghton, Jeffery; Buddavarapu, Kalyan; Sanford, Tiffany; Darden, Will; Adai, Alex; Hadd, Andrew; Latham, Gary; Danilova, Ludmila V.; Bishop, Justin; Li, Ryan J.; Westra, William H.; Hennessey, Patrick; Koch, Wayne M.; Ochs, Michael F.; Califano, Joseph A.; Sun, Wenyue

2014-01-01

Development of head and neck squamous cell carcinoma (HNSCC) is characterized by accumulation of mutations in several oncogenes and tumor suppressor genes. We have formerly described the mutation pattern of HNSCC and described NOTCH signaling pathway alterations. Given the complexity of the HNSCC, here we extend the previous study to understand the overall HNSCC mutation context and to discover additional genetic alterations. We performed high depth targeted exon sequencing of 51 highly actionable cancer-related genes with a high frequency of mutation across many cancer types, including head and neck. DNA from primary tumor tissues and matched normal tissues was analyzed for 37 HNSCC patients. We identified 26 non-synonymous or stop-gained mutations targeting 11 of 51 selected genes. These genes were mutated in 17 out of 37 (46%) studied HNSCC patients. Smokers harbored 3.2-fold more mutations than non-smokers. Importantly, TP53 was mutated in 30%, NOTCH1 in 8% and FGFR3 in 5% of HNSCC. HPV negative patients harbored 4-fold more TP53 mutations than HPV positive patients. These data confirm prior reports of the HNSCC mutational profile. Additionally, we detected mutations in two new genes, CEBPA and FES, which have not been previously reported in HNSCC. These data extend the spectrum of HNSCC mutations and define novel mutation targets in HNSCC carcinogenesis, especially for smokers and HNSCC without HPV infection. PMID:24667986

High-density marker imputation accuracy in sixteen French cattle breeds.

PubMed

Hozé, Chris; Fouilloux, Marie-Noëlle; Venot, Eric; Guillaume, François; Dassonneville, Romain; Fritz, Sébastien; Ducrocq, Vincent; Phocas, Florence; Boichard, Didier; Croiseau, Pascal

2013-09-03

improvement was observed when multi-breed imputation was performed. In all breeds, imputation accuracy was higher than 97%, which indicates that imputation to the high-density chip was accurate. Imputation accuracy depends mainly on the size of the reference population and the relationship between reference and target populations.

Breed-specific reference intervals for assessing thyroid function in seven dog breeds.

PubMed

Hegstad-Davies, Rebecca L; Torres, Sheila M F; Sharkey, Leslie C; Gresch, Sarah C; Muñoz-Zanzi, Claudia A; Davies, Peter R

2015-11-01

Thyroxine (T4), free T4 (FT4), and thyrotropin (TSH) concentrations were measured in serum from 693 healthy representatives from 7 dog breeds (Alaskan Malamute, Collie, English Setter, Golden Retriever, Keeshond, Samoyed, or Siberian Husky) to determine whether breed-specific reference intervals (RIs) are warranted. Veterinarians reviewed the health history, performed a physical examination, and approved laboratory data for the enrolled dogs. Many purebred dogs had T4 and FT4 concentrations that were at, or below, the lower limits previously determined for non-breed-specific RIs. Mean concentrations of T4, FT4, and TSH varied significantly among breeds. The range of mean concentration of T4 (19.7 nmol/L [1.53 µg/dL] in English Setters to 29.0 nmol/L [2.25 µg/dL] in Keeshonds) and FT4 (12.6 pmol/L [0.98 ng/dL] in English Setters to 20.2 pmol/L [1.57 ng/dL] in Samoyeds) was considerable. Median TSH values ranged from 6.10 mIU/L (0.07 ng/mL; Alaskan Malamute and Golden Retriever) to 17.6 mIU/L (0.26 ng/mL; Collie). Mean T4 and FT4 concentrations were higher in females. Increasing age was associated with decreasing T4 and FT4, and increasing TSH concentration. The substantial ranges across breeds of measures of central tendency (mean, median) for all hormones indicate that breed-specific RIs are warranted. RIs encompassing the central 95% of reference values for all breeds combined, and for individual breeds, were calculated using nonparametric (TSH) and robust (T4, FT4) methods. Use of breed-specific RIs in combination with careful attention to the potential for pre-analytical and analytical variability in test results will improve thyroid function assessment in these breeds. © 2015 The Author(s).

Actionable mutations in canine hemangiosarcoma

PubMed Central

Wang, Guannan; Wu, Ming; Maloneyhuss, Martha A.; Wojcik, John; Durham, Amy C.; Mason, Nicola J.

2017-01-01

Background Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma. Methods Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage. Results and conclusions Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease. PMID:29190660

Effective immuno-targeting of the IDH1 mutation R132H in a murine model of intracranial glioma.

PubMed

Pellegatta, Serena; Valletta, Lorella; Corbetta, Cristina; Patanè, Monica; Zucca, Ileana; Riccardi Sirtori, Federico; Bruzzone, Maria Grazia; Fogliatto, Gianpaolo; Isacchi, Antonella; Pollo, Bianca; Finocchiaro, Gaetano

2015-01-21

The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas.Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than glioblastomas. Using site-directed mutagenesis we introduced the R132H mutation into the murine glioma cell line GL261,creating mIDH1-GL261. Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant. In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261). After intracranial injection, MRI suggested that the initial growth rate was slower in mIDH1-GL261 than p-GL261 gliomas but overall survival was similar. mIDH1-GL261 gliomas showed evidence of R132H expression and of intratumoral 2HG production (evaluated by MRS and LC-MS/MS). Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF. Control mice were injected with four ovalbumin peptides or vehicle. Mice with mIDH1-GL261 but not p-GL261 gliomas treated with mIDH1 peptides survived longer than controls; 25% of them were cured. Immunized mice showed higher amounts of peripheral CD8+ T cells, higher production of IFN-γ, and evidence of anti-mIDH1 antibodies.Immunizations led to intratumoral up-regulation of IFN-γ, granzyme-b and perforin-1 and down-regulation of TGF-β2 and IL-10. These results support the translational potential of immunotherapeutic targeting of gliomas carrying IDH1 mutations.

Breed and trait preferences of Sheko cattle keepers in southwestern Ethiopia.

PubMed

Desta, Takele Taye; Ayalew, Workneh; Hegde, B P

2011-04-01

Like their smallholder subsistence counterparts in developing countries, breed and trait preferences of Sheko cattle keepers have broad perspectives. Our study has documented breed and trait preferences of Sheko cattle keepers in southwestern Ethiopia--the natural breeding tract of Sheko cattle. Our results showed that due to their multifunctionality, cattle are the most preferred livestock species. Overall, farmers showed slightly more preference to local Zebus over Sheko breed. This is due to voracious feeding behavior of Sheko cattle, which make them less preferable in the face of worsening feed shortage, and due to aggressive temperament of Sheko cattle. This is despite Sheko's outperforming potential over local Zebus in their milk production, draft power, and hardiness. At trait level, overall milk production was consistently reported as the most preferred trait followed by fertility and traction. This trait preference rank has matched with the reported frequency count ranks for Sheko cattle use. However, breed preference rank has not matched with reported trait preference ranks because Sheko excels local Zebus in all the three most preferred traits, but it was ranked second. Therefore, to minimize these conflicting interests, breed management plans for Sheko cattle should target on strategies that help to solve feed shortage problem and to improve feeding practices, and on selection of less aggressive Sheko cattle. Therefore, these strategies should be considered in line with Sheko cattle conservation and genetic improvement programs.

Optimum allocation of test resources and comparison of breeding strategies for hybrid wheat.

PubMed

Longin, C Friedrich H; Mi, Xuefei; Melchinger, Albrecht E; Reif, Jochen C; Würschum, Tobias

2014-10-01

The use of a breeding strategy combining the evaluation of line per se with testcross performance maximizes annual selection gain for hybrid wheat breeding. Recent experimental studies confirmed a high commercial potential for hybrid wheat requiring the design of optimum breeding strategies. Our objectives were to (1) determine the optimum allocation of the type and number of testers, the number of test locations and the number of doubled haploid lines for different breeding strategies, (2) identify the best breeding strategy and (3) elaborate key parameters for an efficient hybrid wheat breeding program. We performed model calculations using the selection gain for grain yield as target variable to optimize the number of lines, testers and test locations in four different breeding strategies. A breeding strategy (BS2) combining the evaluation of line per se performance and general combining ability (GCA) had a far larger annual selection gain across all considered scenarios than a breeding strategy (BS1) focusing only on GCA. In the combined strategy, the production of testcross seed conducted in parallel with the first yield trial for line per se performance (BS2rapid) resulted in a further increase of the annual selection gain. For the current situation in hybrid wheat, this relative superiority of the strategy BS2rapid amounted to 67 % in annual selection gain compared to BS1. Varying a large number of parameters, we identified the high costs for hybrid seed production and the low variance of GCA in hybrid wheat breeding as key parameters limiting selection gain in BS2rapid.

Mitochondrial targeting of recombinant RNAs modulates the level of a heteroplasmic mutation in human mitochondrial DNA associated with Kearns Sayre Syndrome

PubMed Central

Comte, Caroline; Tonin, Yann; Heckel-Mager, Anne-Marie; Boucheham, Abdeldjalil; Smirnov, Alexandre; Auré, Karine; Lombès, Anne; Martin, Robert P.; Entelis, Nina; Tarassov, Ivan

2013-01-01

Mitochondrial mutations, an important cause of incurable human neuromuscular diseases, are mostly heteroplasmic: mutated mitochondrial DNA is present in cells simultaneously with wild-type genomes, the pathogenic threshold being generally >70% of mutant mtDNA. We studied whether heteroplasmy level could be decreased by specifically designed oligoribonucleotides, targeted into mitochondria by the pathway delivering RNA molecules in vivo. Using mitochondrially imported RNAs as vectors, we demonstrated that oligoribonucleotides complementary to mutant mtDNA region can specifically reduce the proportion of mtDNA bearing a large deletion associated with the Kearns Sayre Syndrome in cultured transmitochondrial cybrid cells. These findings may be relevant to developing of a new tool for therapy of mtDNA associated diseases. PMID:23087375

Improved health-span and lifespan in mtDNA mutator mice treated with the mitochondrially targeted antioxidant SkQ1

PubMed Central

Shabalina, Irina G.; Vyssokikh, Mikhail Yu.; Gibanova, Natalia; Csikasz, Robert I.; Edgar, Daniel; Hallden-Waldemarson, Anne; Rozhdestvenskaya, Zinaida; Bakeeva, Lora E.; Vays, Valeria B.; Pustovidko, Antonina V.; Skulachev, Maxim V.; Cannon, Barbara; Skulachev, Vladimir P.; Nedergaard, Jan

2017-01-01

MtDNA mutator mice exhibit marked features of premature aging. We find that these mice treated from age of ≈100 days with the mitochondria-targeted antioxidant SkQ1 showed a delayed appearance of traits of aging such as kyphosis, alopecia, lowering of body temperature, body weight loss, as well as ameliorated heart, kidney and liver pathologies. These effects of SkQ1 are suggested to be related to an alleviation of the effects of an enhanced reactive oxygen species (ROS) level in mtDNA mutator mice: the increased mitochondrial ROS released due to mitochondrial mutations probably interact with polyunsaturated fatty acids in cardiolipin, releasing malondialdehyde and 4-hydroxynonenal that form protein adducts and thus diminishes mitochondrial functions. SkQ1 counteracts this as it scavenges mitochondrial ROS. As the results, the normal mitochondrial ultrastructure is preserved in liver and heart; the phosphorylation capacity of skeletal muscle mitochondria as well as the thermogenic capacity of brown adipose tissue is also improved. The SkQ1-treated mice live significantly longer (335 versus 290 days). These data may be relevant in relation to treatment of mitochondrial diseases particularly and the process of aging in general. PMID:28209927

BSE Case Associated with Prion Protein Gene Mutation

PubMed Central

Richt, Jürgen A.; Hall, S. Mark

2008-01-01

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) of cattle and was first detected in 1986 in the United Kingdom. It is the most likely cause of variant Creutzfeldt-Jakob disease (CJD) in humans. The origin of BSE remains an enigma. Here we report an H-type BSE case associated with the novel mutation E211K within the prion protein gene (Prnp). Sequence analysis revealed that the animal with H-type BSE was heterozygous at Prnp nucleotides 631 through 633. An identical pathogenic mutation at the homologous codon position (E200K) in the human Prnp has been described as the most common cause of genetic CJD. This finding represents the first report of a confirmed case of BSE with a potential pathogenic mutation within the bovine Prnp gene. A recent epidemiological study revealed that the K211 allele was not detected in 6062 cattle from commercial beef processing plants and 42 cattle breeds, indicating an extremely low prevalence of the E211K variant (less than 1 in 2000) in cattle. PMID:18787697

Generation of novel resistance genes using mutation and targeted gene editing

USDA-ARS?s Scientific Manuscript database

Classical breeding for virus resistance is a lengthy process and is restricted by the availability of resistance genes. Precise genome editing is a "dream technology" to improve plants for virus resistance and these tools have opened new and very promising ways to generate virus resistant plants by ...

Adaptation to climate change--exploring the potential of locally adapted breeds.

PubMed

Hoffmann, Irene

2013-06-01

The livestock sector and agriculture as a whole face unprecedented challenges to increase production while improving the environment. On the basis of a literature review, the paper first discusses challenges related to climate change, food security and other drivers of change in livestock production. On the basis of a recent discourse in ecology, a framework for assessing livestock species' and breeds' vulnerability to climate change is presented. The second part of the paper draws on an analysis of data on breed qualities obtained from the Food and Agriculture Organization's Domestic Animal Diversity Information System (DAD-IS) to explore the range of adaptation traits present in today's breed diversity. The analysis produced a first mapping of a range of ascribed adaptation traits of national breed populations. It allowed to explore what National Coordinators understand by 'locally adapted' and other terms that describe general adaptation, to better understand the habitat, fodder and temperature range of each species and to shed light on the environments in which targeted search for adaptation traits could focus.

Identification of single nucleotide polymorphisms in the agouti signaling protein (ASIP) gene in some goat breeds in tropical and temperate climates.

PubMed

Adefenwa, Mufliat A; Peters, Sunday O; Agaviezor, Brilliant O; Wheto, Matthew; Adekoya, Khalid O; Okpeku, Moses; Oboh, Bola; Williams, Gabriel O; Adebambo, Olufunmilayo A; Singh, Mahipal; Thomas, Bolaji; De Donato, Marcos; Imumorin, Ikhide G

2013-07-01

The agouti-signaling protein (ASIP) plays a major role in mammalian pigmentation as an antagonist to melanocortin-1 receptor gene to stimulate pheomelanin synthesis, a major pigment conferring mammalian coat color. We sequenced a 352 bp fragment of ASIP gene spanning part of exon 2 and part of intron 2 in 215 animals representing six goat breeds from Nigeria and the United States: West African Dwarf, predominantly black; Red Sokoto, mostly red; and Sahel, mostly white from Nigeria; black and white Alpine, brown and white Spanish and white Saanen from the US. Twenty haplotypes from nine mutations representing three intronic, one silent and five missense (p.S19R, p.N35K, p.L36V, p.M42L and p.L45W) mutations were identified in Nigerian goats. Approximately 89 % of Nigerian goats carry haplotype 1 (TGCCATCCG) which seems to be the wild type configuration of mutations in this region of the gene. Although we found no association between these polymorphisms in the ASIP gene and coat color in Nigerian goats, in-silico functional analysis predicts putative deleterious functional impact of the p.L45W mutation on the basic amino-terminal domain of ASIP. In the American goats, two intronic mutations, g.293G>A and g.327C>A, were identified in the Alpine breed, although the g.293G>A mutation is common to American and Nigerian goat populations. All Sannen and Sahel goats in this study belong to haplotypes 1 of both populations which seem to be the wild-type composite ASIP haplotype. Overall, there was no clear association of this portion of the ASIP gene interrogated in this study with coat color variation. Therefore, additional genomic analyses of promoter sequence, the entire coding and non-coding regions of the ASIP gene will be required to obtain a definite conclusion.

Evaluation of the phospholamban gene in purebred large-breed dogs with dilated cardiomyopathy.

PubMed

Stabej, Polona; Leegwater, Peter A; Stokhof, Arnold A; Domanjko-Petric, Aleksandra; van Oost, Bernard A

2005-03-01

To evaluate the role of the phospholamban gene in purebred large-breed dogs with dilated cardiomyopathy (DCM). 6 dogs with DCM, including 2 Doberman Pinschers, 2 Newfoundlands, and 2 Great Danes. All dogs had clinical signs of congestive heart failure, and a diagnosis of DCM was made on the basis of echocardiographic findings. Blood samples were collected from each dog, and genomic DNA was isolated by a salt extraction method. Specific oligonucleotides were designed to amplify the promoter, exon 1, the 5'-part of exon 2 including the complete coding region, and part of intron 1 of the canine phospholamban gene via polymerase chain reaction procedures. These regions were screened for mutations in DNA obtained from the 6 dogs with DCM. No mutations were identified in the promoter, 5' untranslated region, part of intron 1, part of the 3' untranslated region, and the complete coding region of the phospholamban gene in dogs with DCM. Results indicate that mutations in the phospholamban gene are not a frequent cause of DCM in Doberman Pinschers, Newfoundlands, and Great Danes.

Prevalence of inherited disorders among mixed-breed and purebred dogs: 27,254 cases (1995-2010).

PubMed

Bellumori, Thomas P; Famula, Thomas R; Bannasch, Danika L; Belanger, Janelle M; Oberbauer, Anita M

2013-06-01

To determine the proportion of mixed-breed and purebred dogs with common genetic disorders. Case-control study. 27,254 dogs with an inherited disorder. Electronic medical records were reviewed for 24 genetic disorders: hemangiosarcoma, lymphoma, mast cell tumor, osteosarcoma, aortic stenosis, dilated cardiomyopathy, hypertrophic cardiomyopathy, mitral valve dysplasia, patent ductus arteriosus, ventricular septal defect, hyperadrenocorticism, hypoadrenocorticism, hypothyroidism, elbow dysplasia, hip dysplasia, intervertebral disk disease, patellar luxation, ruptured cranial cruciate ligament, atopy or allergic dermatitis, bloat, cataracts, epilepsy, lens luxation, and portosystemic shunt. For each disorder, healthy controls matched for age, body weight, and sex to each affected dog were identified. Genetic disorders differed in expression. No differences in expression of 13 genetic disorders were detected between purebred dogs and mixed-breed dogs (ie, hip dysplasia, hypo- and hyperadrenocorticism, cancers, lens luxation, and patellar luxation). Purebred dogs were more likely to have 10 genetic disorders, including dilated cardiomyopathy, elbow dysplasia, cataracts, and hypothyroidism. Mixed-breed dogs had a greater probability of ruptured cranial cruciate ligament. Prevalence of genetic disorders in both populations was related to the specific disorder. Recently derived breeds or those from similar lineages appeared to be more susceptible to certain disorders that affect all closely related purebred dogs, whereas disorders with equal prevalence in the 2 populations suggested that those disorders represented more ancient mutations that are widely spread through the dog population. Results provided insight on how breeding practices may reduce prevalence of a disorder.

Urolithiasis in dogs. II: Breed prevalence, and interrelations of breed, sex, age, and mineral composition.

PubMed

Ling, G V; Franti, C E; Ruby, A L; Johnson, D L

1998-05-01

To analyze selected breed-related data for canine urinary calculi. 11,000 specimens: 5,781 from female dogs, 5,215 from males, and 4 from dogs of unrecorded sex. Information was compiled for all canine urinary calculi submitted between July 1981 and January 1994. Results for a mixed-breed group and 26 of the most common breeds of stone-forming dogs were analyzed. Interrelations of breed, sex, and age of affected dogs and mineral composition of the specimens were determined. Prevalence of 5 specific mineral types was significantly correlated between the sexes of 27 common breed groups: struvite, calcium phosphate (apatite), calcium oxalate, brushite, and urate. Struvite-containing calculi were seen in high proportions in both sexes of 7 breeds, and in low proportions in both sexes of 7 other breeds. Male and female Lhasa Apsos, Cairn Terriers, and 5 other breeds had high proportions of oxalate-containing calculi; values in males were substantially higher. Low numbers of oxalate-containing calculi were seen in both sexes of 7 breeds; Dalmatians had the lowest numbers. Males and females of 6 breeds had high numbers of urate-containing calculi, Dalmatians and English Bulldogs had the highest numbers. Low amounts of urate were found in calculi from males and females of 6 breeds, Samoyeds had the lowest numbers. Highest proportions of cystine-containing calculi were seen in male Dachshunds, English Bulldogs, and Chihuahuas. Males of 8 breeds had no specimens that contained cystine; only 2 such specimens were obtained from females. Prevalence of uroliths differs among breed, age, and sex of affected dogs. Breed, sex, and age of dogs; mineral types of calculi in males versus females; and their anatomic location within the tract are important considerations for clinicians when evaluating risk in dogs with urolithiasis and in identifying areas that need further in-depth applied or clinical investigation, or both.

The 80-kb DNA duplication on BTA1 is the only remaining candidate mutation for the polled phenotype of Friesian origin

PubMed Central

2014-01-01

Background The absence of horns, called polled phenotype, is the favored trait in modern cattle husbandry. To date, polled cattle are obtained primarily by dehorning calves. Dehorning is a practice that raises animal welfare issues, which can be addressed by selecting for genetically hornless cattle. In the past 20 years, there have been many studies worldwide to identify unique genetic markers in complete association with the polled trait in cattle and recently, two different alleles at the POLLED locus, both resulting in the absence of horns, were reported: (1) the Celtic allele, which is responsible for the polled phenotype in most breeds and for which a single candidate mutation was detected and (2) the Friesian allele, which is responsible for the polled phenotype predominantly in the Holstein-Friesian breed and in a few other breeds, but for which five candidate mutations were identified in a 260-kb haplotype. Further studies based on genome-wide sequencing and high-density SNP (single nucleotide polymorphism) genotyping confirmed the existence of the Celtic and Friesian variants and narrowed down the causal Friesian haplotype to an interval of 145 kb. Results Almost 6000 animals were genetically tested for the polled trait and we detected a recombinant animal which enabled us to reduce the Friesian POLLED haplotype to a single causal mutation, namely a 80-kb duplication. Moreover, our results clearly disagree with the recently reported perfect co-segregation of the POLLED mutation and a SNP at position 1 390 292 bp on bovine chromosome 1 in the Holstein-Friesian population. Conclusion We conclude that the 80-kb duplication, as the only remaining variant within the shortened Friesian haplotype, represents the most likely causal mutation for the polled phenotype of Friesian origin. PMID:24993890

Lung tumors with distinct p53 mutations respond similarly to p53 targeted therapy but exhibit genotype-specific statin sensitivity

PubMed Central

Turrell, Frances K.; Kerr, Emma M.; Gao, Meiling; Thorpe, Hannah; Doherty, Gary J.; Cridge, Jake; Shorthouse, David; Speed, Alyson; Samarajiwa, Shamith; Hall, Benjamin A.; Griffiths, Meryl; Martins, Carla P.

2017-01-01

Lung adenocarcinoma accounts for ∼40% of lung cancers, the leading cause of cancer-related death worldwide, and current therapies provide only limited survival benefit. Approximately half of lung adenocarcinomas harbor mutations in TP53 (p53), making these mutants appealing targets for lung cancer therapy. As mutant p53 remains untargetable, mutant p53-dependent phenotypes represent alternative targeting opportunities, but the prevalence and therapeutic relevance of such effects (gain of function and dominant-negative activity) in lung adenocarcinoma are unclear. Through transcriptional and functional analysis of murine KrasG12D-p53null, -p53R172H (conformational), and -p53R270H (contact) mutant lung tumors, we identified genotype-independent and genotype-dependent therapeutic sensitivities. Unexpectedly, we found that wild-type p53 exerts a dominant tumor-suppressive effect on mutant tumors, as all genotypes were similarly sensitive to its restoration in vivo. These data show that the potential of p53 targeted therapies is comparable across all p53-deficient genotypes and may explain the high incidence of p53 loss of heterozygosity in mutant tumors. In contrast, mutant p53 gain of function and their associated vulnerabilities can vary according to mutation type. Notably, we identified a p53R270H-specific sensitivity to simvastatin in lung tumors, and the transcriptional signature that underlies this sensitivity was also present in human lung tumors, indicating that this therapeutic approach may be clinically relevant. PMID:28790158

Simulated Breeding

NASA Astrophysics Data System (ADS)

Unemi, Tatsuo

This chapter describes a basic framework of simulated breeding, a type of interactive evolutionary computing to breed artifacts, whose origin is Blind Watchmaker by Dawkins. These methods make it easy for humans to design a complex object adapted to his/her subjective criteria, just similarly to agricultural products we have been developing over thousands of years. Starting from randomly initialized genome, the solution candidates are improved through several generations with artificial selection. The graphical user interface helps the process of breeding with techniques of multifield user interface and partial breeding. The former improves the diversity of individuals that prevents being trapped at local optimum. The latter makes it possible for the user to fix features he/she already satisfied. These methods were examined through artistic applications by the author: SBART for graphics art and SBEAT for music. Combining with a direct genome editor and exportation to another graphical or musical tool on the computer, they can be powerful tools for artistic creation. These systems may contribute to the creation of a type of new culture.

All known patient mutations in the ASH-RhoGAP domains of OCRL affect targeting and APPL1 binding

PubMed Central

McCrea, Heather J.; Paradise, Summer; Tomasini, Livia; Addis, Maria; Melis, Maria Antonietta; De Matteis, Maria Antonietta; De Camilli, Pietro

2008-01-01

Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, an X-linked disorder characterized by bilateral cataracts, mental retardation, neonatal hypotonia, and renal Fanconi syndrome, and for Dent disease, another X-linked condition characterized by kidney reabsorption defects. We have previously described an interaction of OCRL with the endocytic adaptor APPL1 that links OCRL to protein networks involved in the disease phenotype. Here we provide new evidence showing that among the interactions which target OCRL to membranes of the endocytic pathway, binding to APPL1 is the only one abolished by all known disease-causing missense mutations in the ASH-RhoGAP domains of the protein. Furthermore, we demonstrate that APPL1 and rab5 independently contribute to recruit OCRL to enlarged endosomes induced by the expression of constitutively active Rab5. Thus, binding to APPL1 helps localize OCRL at specific cellular sites, and disruption of this interaction may play a role in disease. PMID:18307981

The SWI/SNF Subunit INI1 Contains an N-Terminal Winged Helix DNA Binding Domain that Is a Target for Mutations in Schwannomatosis

PubMed Central

Allen, Mark D.; Freund, Stefan M.V.; Zinzalla, Giovanna; Bycroft, Mark

2015-01-01

Summary SWI/SNF complexes use the energy of ATP hydrolysis to remodel chromatin. In mammals they play a central role in regulating gene expression during differentiation and proliferation. Mutations in SWI/SNF subunits are among the most frequent gene alterations in cancer. The INI1/hSNF5/SMARCB1 subunit is mutated in both malignant rhabdoid tumor, a highly aggressive childhood cancer, and schwannomatosis, a tumor-predisposing syndrome characterized by mostly benign tumors of the CNS. Here, we show that mutations in INI1 that cause schwannomatosis target a hitherto unidentified N-terminal winged helix DNA binding domain that is also present in the BAF45a/PHF10 subunit of the SWI/SNF complex. The domain is structurally related to the SKI/SNO/DAC domain, which is found in a number of metazoan chromatin-associated proteins. PMID:26073604

Cancer-Associated Mutations in Endometriosis without Cancer

PubMed Central

Anglesio, M.S.; Papadopoulos, N.; Ayhan, A.; Nazeran, T.M.; Noë, M.; Horlings, H.M.; Lum, A.; Jones, S.; Senz, J.; Seckin, T.; Ho, J.; Wu, R.-C.; Lac, V.; Ogawa, H.; Tessier-Cloutier, B.; Alhassan, R.; Wang, A.; Wang, Y.; Cohen, J.D.; Wong, F.; Hasanovic, A.; Orr, N.; Zhang, M.; Popoli, M.; McMahon, W.; Wood, L.D.; Mattox, A.; Allaire, C.; Segars, J.; Williams, C.; Tomasetti, C.; Boyd, N.; Kinzler, K.W.; Gilks, C.B.; Diaz, L.; Wang, T.-L.; Vogelstein, B.; Yong, P.J.; Huntsman, D.G.; Shih, I.-M.

2017-01-01

BACKGROUND Endometriosis, defined as the presence of ectopic endometrial stroma and epithelium, affects approximately 10% of reproductive-age women and can cause pelvic pain and infertility. Endometriotic lesions are considered to be benign inflammatory lesions but have cancerlike features such as local invasion and resistance to apoptosis. METHODS We analyzed deeply infiltrating endometriotic lesions from 27 patients by means of exomewide sequencing (24 patients) or cancer-driver targeted sequencing (3 patients). Mutations were validated with the use of digital genomic methods in micro-dissected epithelium and stroma. Epithelial and stromal components of lesions from an additional 12 patients were analyzed by means of a droplet digital polymerase-chain-reaction (PCR) assay for recurrent activating KRAS mutations. RESULTS Exome sequencing revealed somatic mutations in 19 of 24 patients (79%). Five patients harbored known cancer driver mutations in ARID1A, PIK3CA, KRAS, or PPP2R1A, which were validated by Safe-Sequencing System or immunohistochemical analysis. The likelihood of driver genes being affected at this rate in the absence of selection was estimated at P = 0.001 (binomial test). Targeted sequencing and a droplet digital PCR assay identified KRAS mutations in 2 of 3 patients and 3 of 12 patients, respectively, with mutations in the epithelium but not the stroma. One patient harbored two different KRAS mutations, c.35G→T and c.35G→C, and another carried identical KRAS c.35G→A mutations in three distinct lesions. CONCLUSIONS We found that lesions in deep infiltrating endometriosis, which are associated with virtually no risk of malignant transformation, harbor somatic cancer driver mutations. Ten of 39 deep infiltrating lesions (26%) carried driver mutations; all the tested somatic mutations appeared to be confined to the epithelial compartment of endometriotic lesions. PMID:28489996

Addition of a breeding database in the Genome Database for Rosaceae.

PubMed

Evans, Kate; Jung, Sook; Lee, Taein; Brutcher, Lisa; Cho, Ilhyung; Peace, Cameron; Main, Dorrie

2013-01-01

Breeding programs produce large datasets that require efficient management systems to keep track of performance, pedigree, geographical and image-based data. With the development of DNA-based screening technologies, more breeding programs perform genotyping in addition to phenotyping for performance evaluation. The integration of breeding data with other genomic and genetic data is instrumental for the refinement of marker-assisted breeding tools, enhances genetic understanding of important crop traits and maximizes access and utility by crop breeders and allied scientists. Development of new infrastructure in the Genome Database for Rosaceae (GDR) was designed and implemented to enable secure and efficient storage, management and analysis of large datasets from the Washington State University apple breeding program and subsequently expanded to fit datasets from other Rosaceae breeders. The infrastructure was built using the software Chado and Drupal, making use of the Natural Diversity module to accommodate large-scale phenotypic and genotypic data. Breeders can search accessions within the GDR to identify individuals with specific trait combinations. Results from Search by Parentage lists individuals with parents in common and results from Individual Variety pages link to all data available on each chosen individual including pedigree, phenotypic and genotypic information. Genotypic data are searchable by markers and alleles; results are linked to other pages in the GDR to enable the user to access tools such as GBrowse and CMap. This breeding database provides users with the opportunity to search datasets in a fully targeted manner and retrieve and compare performance data from multiple selections, years and sites, and to output the data needed for variety release publications and patent applications. The breeding database facilitates efficient program management. Storing publicly available breeding data in a database together with genomic and genetic data will

High-density marker imputation accuracy in sixteen French cattle breeds

PubMed Central

2013-01-01

high-density. No improvement was observed when multi-breed imputation was performed. Conclusion In all breeds, imputation accuracy was higher than 97%, which indicates that imputation to the high-density chip was accurate. Imputation accuracy depends mainly on the size of the reference population and the relationship between reference and target populations. PMID:24004563

Allele-specific primer polymerase chain reaction for a single nucleotide polymorphism (C1205T) of swine toll-like receptor 5 and comparison of the allelic frequency among several pig breeds in Japan and the Czech Republic.

PubMed

Muneta, Yoshihiro; Minagawa, Yu; Kusumoto, Masahiro; Shinkai, Hiroki; Uenishi, Hirohide; Splichal, Igor

2012-06-01

In the present study, an allele-specific primer-polymerase chain reaction (ASP-PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 5 (TLR5) (C1205T; P402L) that is related to the impaired recognition of Salmonella enterica serovar Choleraesuis (SC) was developed. The allele frequencies in several pig breeds in Japan and the Czech Republic were also compared. The swine TLR5 C1205T mutation was successfully determined by ASP-PCR using genomic DNA samples in Japan that had previously been genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from blood obtained from 110 pigs from seven different breeds in the Czech Republic were genotyped by the ASP-PCR. The genotyping results from the ASP-PCR completely matched the results from the sequencing method. The allele frequency of the swine TLR5 C1205T mutation was 27.5% in the Landrace breed of the Czech Republic compared with 50.0% in Japanese Landrace. In Japan, the C1205T mutation was found only in the Landrace breed, whereas in the Czech Republic it was found in both the Landrace and Piétrain breeds. These results indicate the usefulness of ASP-PCR for detecting a specific SNP for swine TLR5 affecting ligand recognition. They also suggest the possibility of genetically improving pigs to enhance their resistance against SC infection by eliminating or selecting this specific SNP of swine TLR5. © 2012 The Societies and Blackwell Publishing Asia Pty Ltd.

Immunotherapy Targets Common Cancer Mutation

Cancer.gov

In a study of an immune therapy for colorectal cancer that involved a single patient, researchers identified a method for targeting the cancer-causing protein produced by a mutant form of the KRAS gene.

HER2 missense mutations have distinct effects on oncogenic signaling and migration

PubMed Central

Zabransky, Daniel J.; Yankaskas, Christopher L.; Cochran, Rory L.; Wong, Hong Yuen; Croessmann, Sarah; Chu, David; Kavuri, Shyam M.; Red Brewer, Monica; Rosen, D. Marc; Dalton, W. Brian; Cimino-Mathews, Ashley; Cravero, Karen; Button, Berry; Kyker-Snowman, Kelly; Cidado, Justin; Erlanger, Bracha; Parsons, Heather A.; Manto, Kristen M.; Bose, Ron; Lauring, Josh; Arteaga, Carlos L.; Konstantopoulos, Konstantinos; Park, Ben Ho

2015-01-01

Recurrent human epidermal growth factor receptor 2 (HER2) missense mutations have been reported in human cancers. These mutations occur primarily in the absence of HER2 gene amplification such that most HER2-mutant tumors are classified as “negative” by FISH or immunohistochemistry assays. It remains unclear whether nonamplified HER2 missense mutations are oncogenic and whether they are targets for HER2-directed therapies that are currently approved for the treatment of HER2 gene-amplified breast cancers. Here we functionally characterize HER2 kinase and extracellular domain mutations through gene editing of the endogenous loci in HER2 nonamplified human breast epithelial cells. In in vitro and in vivo assays, the majority of HER2 missense mutations do not impart detectable oncogenic changes. However, the HER2 V777L mutation increased biochemical pathway activation and, in the context of a PIK3CA mutation, enhanced migratory features in vitro. However, the V777L mutation did not alter in vivo tumorigenicity or sensitivity to HER2-directed therapies in proliferation assays. Our results suggest the oncogenicity and potential targeting of HER2 missense mutations should be considered in the context of cooperating genetic alterations and provide previously unidentified insights into functional analysis of HER2 mutations and strategies to target them. PMID:26508629

In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics

PubMed Central

Grundberg, Ida; Kiflemariam, Sara; Mignardi, Marco; Imgenberg-Kreuz, Juliana; Edlund, Karolina; Micke, Patrick; Sundström, Magnus; Sjöblom, Tobias

2013-01-01

Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of genetically normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy. PMID:24280411

Degenerative myelopathy in German Shepherd Dog: comparison of two molecular assays for the identification of the SOD1:c.118G>A mutation.

PubMed

Capucchio, Maria Teresa; Spalenza, Veronica; Biasibetti, Elena; Bottero, Maria Teresa; Rasero, Roberto; Dalmasso, Alessandra; Sacchi, Paola

2014-02-01

Degenerative myelopathy (DM) is a late-onset, slowly progressive degeneration of spinal cord white matter which is reported primarily in large breed dogs. The missense mutation SOD1:c.118G>A is associated with this pathology in several dog breeds, including the German Shepherd Dog (GSD). The aims of the present study were to develop a tool for the rapid screening of the SOD1 mutation site in dogs and to evaluate the association of the polymorphism with DM in the German Shepherd breed. Two different techniques were compared: a minisequencing test and a real-time pcr allelic discrimination assay. Both approaches resulted effective and efficient. A sample of 47 dogs were examined. Ten subjects presented the symptoms of the illness; for one of them the diagnosis was confirmed by postmortem investigations and it resulted to be an A/A homozygote. In another clinically suspected dog, heterozygote A/G, the histopathological examination of the medulla showed moderate axon and myelin degenerative changes. GSD shows a frequency of the mutant allele equal to 0.17, quite high being a high-risk allele. Because canine DM has a late onset in adulthood and homozygous mutant dogs are likely as fertile as other genotypes, the natural selection is mild and the mutant allele may reach high frequencies. A diagnostic test, easy to implement, may contribute to control the gene diffusion in populations. The SOD1:c.118G>A mutation could be a useful marker for breeding strategies intending to reduce the incidence of DM.

Reduced nighttime transpiration is a relevant breeding target for high water-use efficiency in grapevine

PubMed Central

Coupel-Ledru, Aude; Lebon, Eric; Christophe, Angélique; Gallo, Agustina; Gago, Pilar; Pantin, Florent; Doligez, Agnès; Simonneau, Thierry

2016-01-01

Increasing water scarcity challenges crop sustainability in many regions. As a consequence, the enhancement of transpiration efficiency (TE)—that is, the biomass produced per unit of water transpired—has become crucial in breeding programs. This could be achieved by reducing plant transpiration through a better closure of the stomatal pores at the leaf surface. However, this strategy generally also lowers growth, as stomatal opening is necessary for the capture of atmospheric CO2 that feeds daytime photosynthesis. Here, we considered the reduction in transpiration rate at night (En) as a possible strategy to limit water use without altering growth. For this purpose, we carried out a genetic analysis for En and TE in grapevine, a major crop in drought-prone areas. Using recently developed phenotyping facilities, potted plants of a cross between Syrah and Grenache cultivars were screened for 2 y under well-watered and moderate soil water deficit scenarios. High genetic variability was found for En under both scenarios and was primarily associated with residual diffusion through the stomata. Five quantitative trait loci (QTLs) were detected that underlay genetic variability in En. Interestingly, four of them colocalized with QTLs for TE. Moreover, genotypes with favorable alleles on these common QTLs exhibited reduced En without altered growth. These results demonstrate the interest of breeding grapevine for lower water loss at night and pave the way to breeding other crops with this underexploited trait for higher TE. PMID:27457942

Reduced nighttime transpiration is a relevant breeding target for high water-use efficiency in grapevine.

PubMed

Coupel-Ledru, Aude; Lebon, Eric; Christophe, Angélique; Gallo, Agustina; Gago, Pilar; Pantin, Florent; Doligez, Agnès; Simonneau, Thierry

2016-08-09

Increasing water scarcity challenges crop sustainability in many regions. As a consequence, the enhancement of transpiration efficiency (TE)-that is, the biomass produced per unit of water transpired-has become crucial in breeding programs. This could be achieved by reducing plant transpiration through a better closure of the stomatal pores at the leaf surface. However, this strategy generally also lowers growth, as stomatal opening is necessary for the capture of atmospheric CO2 that feeds daytime photosynthesis. Here, we considered the reduction in transpiration rate at night (En) as a possible strategy to limit water use without altering growth. For this purpose, we carried out a genetic analysis for En and TE in grapevine, a major crop in drought-prone areas. Using recently developed phenotyping facilities, potted plants of a cross between Syrah and Grenache cultivars were screened for 2 y under well-watered and moderate soil water deficit scenarios. High genetic variability was found for En under both scenarios and was primarily associated with residual diffusion through the stomata. Five quantitative trait loci (QTLs) were detected that underlay genetic variability in En Interestingly, four of them colocalized with QTLs for TE. Moreover, genotypes with favorable alleles on these common QTLs exhibited reduced En without altered growth. These results demonstrate the interest of breeding grapevine for lower water loss at night and pave the way to breeding other crops with this underexploited trait for higher TE.

Efficiency of multi-breed genomic selection for dairy cattle breeds with different sizes of reference population.

PubMed

Hozé, C; Fritz, S; Phocas, F; Boichard, D; Ducrocq, V; Croiseau, P

2014-01-01

Single-breed genomic selection (GS) based on medium single nucleotide polymorphism (SNP) density (~50,000; 50K) is now routinely implemented in several large cattle breeds. However, building large enough reference populations remains a challenge for many medium or small breeds. The high-density BovineHD BeadChip (HD chip; Illumina Inc., San Diego, CA) containing 777,609 SNP developed in 2010 is characterized by short-distance linkage disequilibrium expected to be maintained across breeds. Therefore, combining reference populations can be envisioned. A population of 1,869 influential ancestors from 3 dairy breeds (Holstein, Montbéliarde, and Normande) was genotyped with the HD chip. Using this sample, 50K genotypes were imputed within breed to high-density genotypes, leading to a large HD reference population. This population was used to develop a multi-breed genomic evaluation. The goal of this paper was to investigate the gain of multi-breed genomic evaluation for a small breed. The advantage of using a large breed (Normande in the present study) to mimic a small breed is the large potential validation population to compare alternative genomic selection approaches more reliably. In the Normande breed, 3 training sets were defined with 1,597, 404, and 198 bulls, and a unique validation set included the 394 youngest bulls. For each training set, estimated breeding values (EBV) were computed using pedigree-based BLUP, single-breed BayesC, or multi-breed BayesC for which the reference population was formed by any of the Normande training data sets and 4,989 Holstein and 1,788 Montbéliarde bulls. Phenotypes were standardized by within-breed genetic standard deviation, the proportion of polygenic variance was set to 30%, and the estimated number of SNP with a nonzero effect was about 7,000. The 2 genomic selection (GS) approaches were performed using either the 50K or HD genotypes. The correlations between EBV and observed daughter yield deviations (DYD) were computed

Targeted next-generation sequencing identifies a homozygous nonsense mutation in ABHD12, the gene underlying PHARC, in a family clinically diagnosed with Usher syndrome type 3.

PubMed

Eisenberger, Tobias; Slim, Rima; Mansour, Ahmad; Nauck, Markus; Nürnberg, Gudrun; Nürnberg, Peter; Decker, Christian; Dafinger, Claudia; Ebermann, Inga; Bergmann, Carsten; Bolz, Hanno Jörn

2012-09-02

Usher syndrome (USH) is an autosomal recessive genetically heterogeneous disorder with congenital sensorineural hearing impairment and retinitis pigmentosa (RP). We have identified a consanguineous Lebanese family with two affected members displaying progressive hearing loss, RP and cataracts, therefore clinically diagnosed as USH type 3 (USH3). Our study was aimed at the identification of the causative mutation in this USH3-like family. Candidate loci were identified using genomewide SNP-array-based homozygosity mapping followed by targeted enrichment and next-generation sequencing. Using a capture array targeting the three identified homozygosity-by-descent regions on chromosomes 1q43-q44, 20p13-p12.2 and 20p11.23-q12, we identified a homozygous nonsense mutation, p.Arg65X, in ABHD12 segregating with the phenotype. Mutations of ABHD12, an enzyme hydrolyzing an endocannabinoid lipid transmitter, cause PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and early-onset cataract). After the identification of the ABHD12 mutation in this family, one patient underwent neurological examination which revealed ataxia, but no polyneuropathy. ABHD12 is not known to be related to the USH protein interactome. The phenotype of our patient represents a variant of PHARC, an entity that should be taken into account as differential diagnosis for USH3. Our study demonstrates the potential of comprehensive genetic analysis for improving the clinical diagnosis.

Potential Use of a Weak Ethylene Receptor Mutant, Sletr1-2, as Breeding Material To Extend Fruit Shelf Life of Tomato.

PubMed

Mubarok, Syariful; Okabe, Yoshihiro; Fukuda, Naoya; Ariizumi, Tohru; Ezura, Hiroshi

2015-09-16

Mutations in the ethylene receptor gene (SlETR1), Sletr1-1 and Sletr1-2, are effective in reducing ethylene sensitivity and improving fruit shelf life. In this study the effect of Sletr1-1 and Sletr1-2 mutations was investigated in F1 hybrid lines. These two mutants and control were crossed with four commercial pure-line tomatoes. The Sletr1-1 mutation showed undesirable pleiotropic effects in the F1 hybrid lines. The Sletr1-2 mutation was effective in improving fruit shelf life of F1 hybrid lines for 4-5 days longer. It was also effective in improving fruit firmness without change in fruit size, ethylene production, respiration rate, and total soluble solids or a great reduction in fruit color, lycopene, and β-carotene, although the titratable acidity was increased by Sletr1-2 mutation. These results indicate that the Sletr1-2 mutant allele has the potential to improve fruit shelf life via incorporation in tomato breeding programs.

Novel recurrently mutated genes and a prognostic mutation signature in colorectal cancer.

PubMed

Yu, Jun; Wu, William K K; Li, Xiangchun; He, Jun; Li, Xiao-Xing; Ng, Simon S M; Yu, Chang; Gao, Zhibo; Yang, Jie; Li, Miao; Wang, Qiaoxiu; Liang, Qiaoyi; Pan, Yi; Tong, Joanna H; To, Ka F; Wong, Nathalie; Zhang, Ning; Chen, Jie; Lu, Youyong; Lai, Paul B S; Chan, Francis K L; Li, Yingrui; Kung, Hsiang-Fu; Yang, Huanming; Wang, Jun; Sung, Joseph J Y

2015-04-01

Characterisation of colorectal cancer (CRC) genomes by next-generation sequencing has led to the discovery of novel recurrently mutated genes. Nevertheless, genomic data has not yet been used for CRC prognostication. To identify recurrent somatic mutations with prognostic significance in patients with CRC. Exome sequencing was performed to identify somatic mutations in tumour tissues of 22 patients with CRC, followed by validation of 187 recurrent and pathway-related genes using targeted capture sequencing in additional 160 cases. Seven significantly mutated genes, including four reported (APC, TP53, KRAS and SMAD4) and three novel recurrently mutated genes (CDH10, FAT4 and DOCK2), exhibited high mutation prevalence (6-14% for novel cancer genes) and higher-than-expected number of non-silent mutations in our CRC cohort. For prognostication, a five-gene-signature (CDH10, COL6A3, SMAD4, TMEM132D, VCAN) was devised, in which mutation(s) in one or more of these genes was significantly associated with better overall survival independent of tumor-node-metastasis (TNM) staging. The median survival time was 80.4 months in the mutant group versus 42.4 months in the wild type group (p=0.0051). The prognostic significance of this signature was successfully verified using the data set from the Cancer Genome Atlas study. The application of next-generation sequencing has led to the identification of three novel significantly mutated genes in CRC and a mutation signature that predicts survival outcomes for stratifying patients with CRC independent of TNM staging. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Nucleotide Substitution in 3' Arm of Bovine MIR-2467 in Five Cattle Breeds.

PubMed

Łukaszewicz, Aneta; Basiak, Szymon; Proskura, Witold Stanisław; Dybus, Andrzej

2015-01-01

The T > C single nucleotide polymorphism (SNP) in the MIR2467 gene was investigated in order to confirm its presence in cattle genome and to check for possible differences in its genotype distribution among different breeds. Additional purpose of the study was to investigate in silico potential effect of that substitution on the structure and stability of precursor mir-2467. The study involved 634 individuals of five cattle breeds: Angus, Hereford, Holstein-Friesian, Jersey, and Limousin, which were genotyped using PCR-RFLP assay. In this study, the presence of T > C polymorphism at position 24 was observed in all the cattle breeds excepting Hereford. In addition, the differences in the genotype distribution among analyzed breeds were indicated. On the basis of minimum free energy structure prediction, the C allele was indicated to have possible impact on decreasing the stability of the pre-mir-2467, thus altering its ability to regulate target genes expression.

Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice.

PubMed

Safdar, Adeel; Khrapko, Konstantin; Flynn, James M; Saleem, Ayesha; De Lisio, Michael; Johnston, Adam P W; Kratysberg, Yevgenya; Samjoo, Imtiaz A; Kitaoka, Yu; Ogborn, Daniel I; Little, Jonathan P; Raha, Sandeep; Parise, Gianni; Akhtar, Mahmood; Hettinga, Bart P; Rowe, Glenn C; Arany, Zoltan; Prolla, Tomas A; Tarnopolsky, Mark A

2016-01-01

Human genetic disorders and transgenic mouse models have shown that mitochondrial DNA (mtDNA) mutations and telomere dysfunction instigate the aging process. Epidemiologically, exercise is associated with greater life expectancy and reduced risk of chronic diseases. While the beneficial effects of exercise are well established, the molecular mechanisms instigating these observations remain unclear. Endurance exercise reduces mtDNA mutation burden, alleviates multisystem pathology, and increases lifespan of the mutator mice, with proofreading deficient mitochondrial polymerase gamma (POLG1). We report evidence for a POLG1-independent mtDNA repair pathway mediated by exercise, a surprising notion as POLG1 is canonically considered to be the sole mtDNA repair enzyme. Here, we show that the tumor suppressor protein p53 translocates to mitochondria and facilitates mtDNA mutation repair and mitochondrial biogenesis in response to endurance exercise. Indeed, in mutator mice with muscle-specific deletion of p53, exercise failed to prevent mtDNA mutations, induce mitochondrial biogenesis, preserve mitochondrial morphology, reverse sarcopenia, or mitigate premature mortality. Our data establish a new role for p53 in exercise-mediated maintenance of the mtDNA genome and present mitochondrially targeted p53 as a novel therapeutic modality for diseases of mitochondrial etiology.

Breeding season survival and breeding incidence of female Mottled Ducks on the upper Texas gulf coast

USGS Publications Warehouse

Rigby, Elizabeth A.; Haukos, David A.

2012-01-01

Previous Mottled Duck (Anas fulvigula) studies suggested that high female breeding season survival may be caused by low nesting effort, but few breeding season estimates of survival associated with nesting effort exist on the western Gulf Coast. Here, breeding season survival (N = 40) and breeding incidence (N = 39) were estimated for female Mottled Ducks on the upper Texas coast, 2006–2008. Females were fitted with backpack radio transmitters and visually relocated every 3–4 days. Weekly survival was estimated using the Known Fate procedure of program MARK with breeding incidence estimated as the annual proportion of females observed nesting or with broods. The top-ranked survival model included a body mass covariate and held weekly female survival constant across weeks and years (SW = 0.986, SE = 0.006). When compared to survival across the entire year estimated from previous band recovery and age ratio analysis, survival rate during the breeding season did not differ. Breeding incidence was well below 100% in all years and highly variable among years (15%–63%). Breeding season survival and breeding incidence were similar to estimates obtained with implant transmitters from the mid-coast of Texas. The greatest breeding incidence for both studies occurred when drought indices indicated average environmental moisture during the breeding season. The observed combination of low breeding incidence and high breeding season survival support the hypothesis of a trade-off between the ecological cost of nesting effort and survival for Mottled Duck females. Habitat cues that trigger nesting are unknown and should be investigated.

Landscape of somatic mutations and clonal evolution in mantle cell lymphoma.

PubMed

Beà, Sílvia; Valdés-Mas, Rafael; Navarro, Alba; Salaverria, Itziar; Martín-Garcia, David; Jares, Pedro; Giné, Eva; Pinyol, Magda; Royo, Cristina; Nadeu, Ferran; Conde, Laura; Juan, Manel; Clot, Guillem; Vizán, Pedro; Di Croce, Luciano; Puente, Diana A; López-Guerra, Mónica; Moros, Alexandra; Roue, Gael; Aymerich, Marta; Villamor, Neus; Colomo, Lluís; Martínez, Antonio; Valera, Alexandra; Martín-Subero, José I; Amador, Virginia; Hernández, Luis; Rozman, Maria; Enjuanes, Anna; Forcada, Pilar; Muntañola, Ana; Hartmann, Elena M; Calasanz, María J; Rosenwald, Andreas; Ott, German; Hernández-Rivas, Jesús M; Klapper, Wolfram; Siebert, Reiner; Wiestner, Adrian; Wilson, Wyndham H; Colomer, Dolors; López-Guillermo, Armando; López-Otín, Carlos; Puente, Xose S; Campo, Elías

2013-11-05

Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.

[Mechanisms of endogenous drug resistance acquisition by spontaneous chromosomal gene mutation].

PubMed

Fukuda, H; Hiramatsu, K

1997-05-01

Endogenous resistance in bacteria is caused by a change or loss of function and generally genetically recessive. However, this type of resistance acquisition are now prevalent in clinical setting. Chromosomal genes that afford endogenous resistance are the genes correlated with the target of the drug, the drug inactivating enzymes, and permeability of the molecules including the antibacterial agents. Endogenous alteration of the drug target are mediated by the spontaneous mutation of their structural gene. This mutation provides much lower affinity of the drugs for the target. Gene expression of the inactivating enzymes, such as class C beta-lactamase, is generally regulated by regulatory genes. Spontaneous mutations in the regulatory genes cause constitutive enzyme production and provides the resistant to the agent which is usually stable for such enzymes. Spontaneous mutation in the structural gene gives the enzyme extra-spectrum substrate specificity, like ESBL (Extra-Spectrum-beta-Lactamase). Expression of structural genes encoding the permeability systems are also regulated by some regulatory genes. The spontaneous mutation of the regulatory genes reduce an amount of porin protein. This mutation causes much lower influx of the drug in the cell. Spontaneous mutation in promoter region of the structural gene of efflux protein was observed. This mutation raised the gene transcription and overproduced efflux protein. This protein progresses the drug efflux from the cell.

Whole-genome sequencing reveals a potential causal mutation for dwarfism in the Miniature Shetland pony.

PubMed

Metzger, Julia; Gast, Alana Christina; Schrimpf, Rahel; Rau, Janina; Eikelberg, Deborah; Beineke, Andreas; Hellige, Maren; Distl, Ottmar

2017-04-01

The Miniature Shetland pony represents a horse breed with an extremely small body size. Clinical examination of a dwarf Miniature Shetland pony revealed a lowered size at the withers, malformed skull and brachygnathia superior. Computed tomography (CT) showed a shortened maxilla and a cleft of the hard and soft palate which protruded into the nasal passage leading to breathing difficulties. Pathological examination confirmed these findings but did not reveal histopathological signs of premature ossification in limbs or cranial sutures. Whole-genome sequencing of this dwarf Miniature Shetland pony and comparative sequence analysis using 26 reference equids from NCBI Sequence Read Archive revealed three probably damaging missense variants which could be exclusively found in the affected foal. Validation of these three missense mutations in 159 control horses from different horse breeds and five donkeys revealed only the aggrecan (ACAN)-associated g.94370258G>C variant as homozygous wild-type in all control samples. The dwarf Miniature Shetland pony had the homozygous mutant genotype C/C of the ACAN:g.94370258G>C variant and the normal parents were heterozygous G/C. An unaffected full sib and 3/5 unaffected half-sibs were heterozygous G/C for the ACAN:g.94370258G>C variant. In summary, we could demonstrate a dwarf phenotype in a miniature pony breed perfectly associated with a missense mutation within the ACAN gene.

20 ans après: a second mutation in MAOA identified by targeted high-throughput sequencing in a family with altered behavior and cognition

PubMed Central

Piton, Amélie; Poquet, Hélène; Redin, Claire; Masurel, Alice; Lauer, Julia; Muller, Jean; Thevenon, Julien; Herenger, Yvan; Chancenotte, Sophie; Bonnet, Marlène; Pinoit, Jean-Michel; Huet, Frédéric; Thauvin-Robinet, Christel; Jaeger, Anne-Sophie; Le Gras, Stéphanie; Jost, Bernard; Gérard, Bénédicte; Peoc'h, Katell; Launay, Jean-Marie; Faivre, Laurence; Mandel, Jean-Louis

2014-01-01

Intellectual disability (ID) is characterized by an extraordinary genetic heterogeneity, with >250 genes that have been implicated in monogenic forms of ID. Because this complexity precluded systematic testing for mutations and because clinical features are often non-specific, for some of these genes only few cases or families have been unambiguously documented. It is the case of the X-linked gene encoding monoamine oxidase A (MAOA), for which only one nonsense mutation has been identified in Brunner syndrome, characterized in a single family by mild non-dysmorphic ID and impulsive, violent and aggressive behaviors. We have performed targeted high-throughput sequencing of 220 genes, including MAOA, in patients with undiagnosed ID. We identified a c.797_798delinsTT (p.C266F) missense mutation in MAOA in a boy with autism spectrum disorder, attention deficit and autoaggressive behavior. Two maternal uncles carry the mutation and have severe ID, with a history of maltreatment in early childhood. This novel missense mutation decreases MAOA enzymatic activity, leading to abnormal levels of urinary monoamines. The identification of this new point mutation confirms, for the first time since 1993, the monogenic implication of the MAOA gene in ID of various degrees, autism and behavioral disturbances. The variable expressivity of the mutation observed in male patients of this family may involve gene–environment interactions, and the identification of a perturbation in monoamine metabolism should be taken into account when prescribing psychoactive drugs in such patients. PMID:24169519

20 ans après: a second mutation in MAOA identified by targeted high-throughput sequencing in a family with altered behavior and cognition.

PubMed

Piton, Amélie; Poquet, Hélène; Redin, Claire; Masurel, Alice; Lauer, Julia; Muller, Jean; Thevenon, Julien; Herenger, Yvan; Chancenotte, Sophie; Bonnet, Marlène; Pinoit, Jean-Michel; Huet, Frédéric; Thauvin-Robinet, Christel; Jaeger, Anne-Sophie; Le Gras, Stéphanie; Jost, Bernard; Gérard, Bénédicte; Peoc'h, Katell; Launay, Jean-Marie; Faivre, Laurence; Mandel, Jean-Louis

2014-06-01

Intellectual disability (ID) is characterized by an extraordinary genetic heterogeneity, with >250 genes that have been implicated in monogenic forms of ID. Because this complexity precluded systematic testing for mutations and because clinical features are often non-specific, for some of these genes only few cases or families have been unambiguously documented. It is the case of the X-linked gene encoding monoamine oxidase A (MAOA), for which only one nonsense mutation has been identified in Brunner syndrome, characterized in a single family by mild non-dysmorphic ID and impulsive, violent and aggressive behaviors. We have performed targeted high-throughput sequencing of 220 genes, including MAOA, in patients with undiagnosed ID. We identified a c.797_798delinsTT (p.C266F) missense mutation in MAOA in a boy with autism spectrum disorder, attention deficit and autoaggressive behavior. Two maternal uncles carry the mutation and have severe ID, with a history of maltreatment in early childhood. This novel missense mutation decreases MAOA enzymatic activity, leading to abnormal levels of urinary monoamines. The identification of this new point mutation confirms, for the first time since 1993, the monogenic implication of the MAOA gene in ID of various degrees, autism and behavioral disturbances. The variable expressivity of the mutation observed in male patients of this family may involve gene-environment interactions, and the identification of a perturbation in monoamine metabolism should be taken into account when prescribing psychoactive drugs in such patients.

Improving breeding efficiency in potato using molecular and quantitative genetics.

PubMed

Slater, Anthony T; Cogan, Noel O I; Hayes, Benjamin J; Schultz, Lee; Dale, M Finlay B; Bryan, Glenn J; Forster, John W

2014-11-01

Potatoes are highly heterozygous and the conventional breeding of superior germplasm is challenging, but use of a combination of MAS and EBVs can accelerate genetic gain. Cultivated potatoes are highly heterozygous due to their outbreeding nature, and suffer acute inbreeding depression. Modern potato cultivars also exhibit tetrasomic inheritance. Due to this genetic heterogeneity, the large number of target traits and the specific requirements of commercial cultivars, potato breeding is challenging. A conventional breeding strategy applies phenotypic recurrent selection over a number of generations, a process which can take over 10 years. Recently, major advances in genetics and molecular biology have provided breeders with molecular tools to accelerate gains for some traits. Marker-assisted selection (MAS) can be effectively used for the identification of major genes and quantitative trait loci that exhibit large effects. There are also a number of complex traits of interest, such as yield, that are influenced by a large number of genes of individual small effect where MAS will be difficult to deploy. Progeny testing and the use of pedigree in the analysis can provide effective identification of the superior genetic factors that underpin these complex traits. Recently, it has been shown that estimated breeding values (EBVs) can be developed for complex potato traits. Using a combination of MAS and EBVs for simple and complex traits can lead to a significant reduction in the length of the breeding cycle for the identification of superior germplasm.

The SWI/SNF Subunit INI1 Contains an N-Terminal Winged Helix DNA Binding Domain that Is a Target for Mutations in Schwannomatosis.

PubMed

Allen, Mark D; Freund, Stefan M V; Zinzalla, Giovanna; Bycroft, Mark

2015-07-07

SWI/SNF complexes use the energy of ATP hydrolysis to remodel chromatin. In mammals they play a central role in regulating gene expression during differentiation and proliferation. Mutations in SWI/SNF subunits are among the most frequent gene alterations in cancer. The INI1/hSNF5/SMARCB1 subunit is mutated in both malignant rhabdoid tumor, a highly aggressive childhood cancer, and schwannomatosis, a tumor-predisposing syndrome characterized by mostly benign tumors of the CNS. Here, we show that mutations in INI1 that cause schwannomatosis target a hitherto unidentified N-terminal winged helix DNA binding domain that is also present in the BAF45a/PHF10 subunit of the SWI/SNF complex. The domain is structurally related to the SKI/SNO/DAC domain, which is found in a number of metazoan chromatin-associated proteins. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Recurrent PTPRB and PLCG1 mutations in angiosarcoma.

PubMed

Behjati, Sam; Tarpey, Patrick S; Sheldon, Helen; Martincorena, Inigo; Van Loo, Peter; Gundem, Gunes; Wedge, David C; Ramakrishna, Manasa; Cooke, Susanna L; Pillay, Nischalan; Vollan, Hans Kristian M; Papaemmanuil, Elli; Koss, Hans; Bunney, Tom D; Hardy, Claire; Joseph, Olivia R; Martin, Sancha; Mudie, Laura; Butler, Adam; Teague, Jon W; Patil, Meena; Steers, Graham; Cao, Yu; Gumbs, Curtis; Ingram, Davis; Lazar, Alexander J; Little, Latasha; Mahadeshwar, Harshad; Protopopov, Alexei; Al Sannaa, Ghadah A; Seth, Sahil; Song, Xingzhi; Tang, Jiabin; Zhang, Jianhua; Ravi, Vinod; Torres, Keila E; Khatri, Bhavisha; Halai, Dina; Roxanis, Ioannis; Baumhoer, Daniel; Tirabosco, Roberto; Amary, M Fernanda; Boshoff, Chris; McDermott, Ultan; Katan, Matilda; Stratton, Michael R; Futreal, P Andrew; Flanagan, Adrienne M; Harris, Adrian; Campbell, Peter J

2014-04-01

Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionizing radiation or chronic lymphoedema. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signaling genes, as a key driver of angiosarcoma. Here we employed whole-genome, whole-exome and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harbored predominantly truncating mutations in 10 of 39 tumors (26%). PLCG1, a signal transducer of tyrosine kinases, encoded a recurrent, likely activating p.Arg707Gln missense variant in 3 of 34 cases (9%). Overall, 15 of 39 tumors (38%) harbored at least one driver mutation in angiogenesis signaling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signaling in angiosarcoma.

Analysis of breed effects on semen traits in light horse, warmblood, and draught horse breeds.

PubMed

Gottschalk, Maren; Sieme, Harald; Martinsson, Gunilla; Distl, Ottmar

2016-05-01

In the present study, systematic effects on semen quality traits were investigated in 381 stallions representing 22 breeds. All stallions were used for AI either at the Lower Saxon National Stud Celle or the North Rhine-Westphalian National Stud Warendorf. A total of 71,078 fresh semen reports of the years 2001 to 2014 were edited for analysis of gel-free volume, sperm concentration, total number of sperm, progressive motility, and total number of progressively motile sperm. Breed differences were studied for warmblood and light horse breeds of both national studs (model I) and for warmblood breeds and the draught horse breed Rhenish German Coldblood from the North Rhine-Westphalian National stud (model II) using mixed model procedures. The fixed effects of age class, year, and month of semen collection had significant influences on all semen traits in both analyses. A significant influence of the horse breed was found for all semen traits but gel-free volume in both statistical models. Comparing warmblood and light horse stallions of both national studs, we observed highest sperm concentrations, total numbers of sperm, and total numbers of progressively motile sperm in Anglo-Arabian stallions. The draught horse breed Rhenish German Coldblood had the highest least squares means for gel-free volume, whereas all other investigated semen traits were significantly lower in this breed compared to the warmblood stallions under study. The variance components among stallions within breeds were significant for all semen traits and accounted for 40% to 59% of the total variance. The between-breed-variance among stallions was not significant underlining the similar size of the random stallion effect in each of the horse breeds analyzed here. In conclusion, breed and stallion are accounting for a significant proportion of the variation in semen quality. Copyright © 2016 Elsevier Inc. All rights reserved.

First report of the East African kdr mutation in an Anopheles gambiae mosquito in Côte d'Ivoire.

PubMed

Chouaïbou, Mouhamadou; Kouadio, Fodjo Behi; Tia, Emmanuel; Djogbenou, Luc

2017-02-09

Background . The intensive use of insecticides in public health and agriculture has led to the development of insecticide resistances in malaria vectors across sub-Saharan Africa countries in the last two decades. The kdr target site point mutation which is among the best characterised resistance mechanisms seems to be changing its distribution patterns on the African continent. The 1014F  kdr mutation originally described only in West Africa is spreading to East Africa while the 1014S  kdr mutation originally described in East Africa, is spreading to West and Central Africa. However, the East- kdr mutation has not been reported in Côte d'Ivoire so far. Methods . Immature stages of Anopheles gambiae s.l. were collected from breeding sites at the outskirts of Yamoussoukro, Côte d'Ivoire. Emerging 3-5 day old adult female mosquitoes were tested for susceptibility to deltamethrin 0.05%, malathion 5%, bendiocarb 1% and dichlorodiphenyltrichloroethane (DDT) 4% according to WHO standard procedures. A total of 50 An. gambiae s.l. specimens were drawn at random for DNA extraction and identification down to the species level. A subsample of 30 mosquitoes was tested for the East-African kdr mutation using a Taqman assay. Results . The tested mosquito population appeared to be strongly resistant to deltamethrin (1.03% mortality), bendiocarb (38.46% mortality) and DDT (0% mortality) with probable resistance observed for malathion (92.47%). Among the 41 mosquitoes that were successfully characterized, An. coluzzii was predominant (68.3%) followed by An. gambiae   s.s. (19.5%) and a few hybrids (7.3%). Out of 30 specimens genotyped for East- kdr , a single hybrid mosquito appeared to be heterozygous for the mutation. Conclusion . The present study revealed the presence of the East- kdr mutation in Côte d'Ivoire for the first time in An. gambiae and highlights the urgent need to start monitoring the allele and genotype frequencies.

Canine gastrointestinal physiology: Breeds variations that can influence drug absorption.

PubMed

Oswald, Hayley; Sharkey, Michele; Pade, Devendra; Martinez, Marilyn N

2015-11-01

Although all dogs belong to Canis lupus familiaris, the physiological diversity resulting from selective breeding can lead to wide interbreed variability in drug pharmacokinetics (PK) or in oral drug product performance. It is important to understand this diversity in order to predict the impact of drug product formulation attributes on in vivo dissolution and absorption characteristics across the canine population when the dog represents the targeted patient population. Based upon published information, this review addresses breed differences in gastrointestinal (GI) physiology and discusses the in vivo implications of these differences. In addition to the importance of such information for understanding the variability that may exist in the performance of oral dosage forms in dogs for the purpose of developing canine therapeutics, an appreciation of breed differences in GI physiology can improve our prediction of oral drug formulation performance when we extrapolate bioavailability results from the dog to the humans, and vice versa. In this literature review, we examine reports of breed associated diversity in GI anatomy and morphology, gastric emptying time (GET), oro-cecal transit time (OCTT), small intestinal transit time (SITT), large intestinal transit time (LITT), intestinal permeability, sodium/potassium fecal concentrations, intestinal flora, and fecal moisture content. Published by Elsevier B.V.

Canine Breed-Specific Hepatopathies.

PubMed

Watson, Penny

2017-05-01

Canine hepatopathies, both congenital and acquired, arise from an interaction between genes and environment. Many show increased breed prevalences. This article reviews the current understanding on breed predispositions for congenital portosystemic shunts; microvascular dysplasia and portal vein hypoplasia; ductal plate abnormalities (congenital hepatic fibrosis and Caroli disease); chronic hepatitis (both copper associated and idiopathic); vacuolar hepatopathies; and gallbladder mucocele. Although all these diseases can occur in many breeds and crossbreeds, understanding breed predispositions helps recognition and will guide future research to improve understanding of causes and treatments. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

Induced Mutations for Improving Production on Bread and Durum Wheat

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

Stamo, Ilirjana; Ylli, Ariana; Dodbiba, Andon

Wheat is a very important crop and has been bred for food and its improvement is continuous from cross-breeding. Radiation and chemically induced mutations have provided variability in selection for novel varieties. Four bread and one durum wheat cultivars were exposed to gamma rays, Cs 137 with doses 10, 15 and 20 krad (2000 seeds of each dose and cultivars). We have isolated mutant plants with height reduced and on cv Progress spike without chaff.

Induced Mutations for Improving Production on Bread and Durum Wheat

NASA Astrophysics Data System (ADS)

Stamo, Ilirjana; Ylli, Ariana; Dodbiba, Andon

2007-04-01

Wheat is a very important crop and has been bred for food and its improvement is continuous from cross-breeding. Radiation and chemically induced mutations have provided variability in selection for novel varieties. Four bread and one durum wheat cultivars were exposed to gamma rays, Cs 137 with doses 10, 15 and 20 krad (2000 seeds of each dose and cultivars). We have isolated mutant plants with height reduced and on cv Progress spike without chaff.

Pulmonary Sarcomatoid Carcinomas Commonly Harbor Either Potentially Targetable Genomic Alterations or High Tumor Mutational Burden as Observed by Comprehensive Genomic Profiling.

PubMed

Schrock, Alexa B; Li, Shuyu D; Frampton, Garrett M; Suh, James; Braun, Eduardo; Mehra, Ranee; Buck, Steven C; Bufill, Jose A; Peled, Nir; Karim, Nagla Abdel; Hsieh, K Cynthia; Doria, Manuel; Knost, James; Chen, Rong; Ou, Sai-Hong Ignatius; Ross, Jeffrey S; Stephens, Philip J; Fishkin, Paul; Miller, Vincent A; Ali, Siraj M; Halmos, Balazs; Liu, Jane J

2017-06-01

Pulmonary sarcomatoid carcinoma (PSC) is a high-grade NSCLC characterized by poor prognosis and resistance to chemotherapy. Development of targeted therapeutic strategies for PSC has been hampered because of limited and inconsistent molecular characterization. Hybrid capture-based comprehensive genomic profiling was performed on DNA from formalin-fixed paraffin-embedded sections of 15,867 NSCLCs, including 125 PSCs (0.8%). Tumor mutational burden (TMB) was calculated from 1.11 megabases (Mb) of sequenced DNA. The median age of the patients with PSC was 67 years (range 32-87), 58% were male, and 78% had stage IV disease. Tumor protein p53 gene (TP53) genomic alterations (GAs) were identified in 74% of cases, which had genomics distinct from TP53 wild-type cases, and 62% featured a GA in KRAS (34%) or one of seven genes currently recommended for testing in the National Comprehensive Cancer Network NSCLC guidelines, including the following: hepatocyte growth factor receptor gene (MET) (13.6%), EGFR (8.8%), BRAF (7.2%), erb-b2 receptor tyrosine kinase 2 gene (HER2) (1.6%), and ret proto-oncogene (RET) (0.8%). MET exon 14 alterations were enriched in PSC (12%) compared with non-PSC NSCLCs (∼3%) (p < 0.0001) and were more prevalent in PSC cases with an adenocarcinoma component. The fraction of PSC with a high TMB (>20 mutations per Mb) was notably higher than in non-PSC NSCLC (20% versus 14%, p = 0.056). Of nine patients with PSC treated with targeted or immunotherapies, three had partial responses and three had stable disease. Potentially targetable GAs in National Comprehensive Cancer Network NSCLC genes (30%) or intermediate or high TMB (43%, >10 mutations per Mb) were identified in most of the PSC cases. Thus, the use of comprehensive genomic profiling in clinical care may provide important treatment options for a historically poorly characterized and difficult to treat disease. Copyright © 2017 International Association for the Study of Lung Cancer. Published

Speed breeding is a powerful tool to accelerate crop research and breeding.

PubMed

Watson, Amy; Ghosh, Sreya; Williams, Matthew J; Cuddy, William S; Simmonds, James; Rey, María-Dolores; Asyraf Md Hatta, M; Hinchliffe, Alison; Steed, Andrew; Reynolds, Daniel; Adamski, Nikolai M; Breakspear, Andy; Korolev, Andrey; Rayner, Tracey; Dixon, Laura E; Riaz, Adnan; Martin, William; Ryan, Merrill; Edwards, David; Batley, Jacqueline; Raman, Harsh; Carter, Jeremy; Rogers, Christian; Domoney, Claire; Moore, Graham; Harwood, Wendy; Nicholson, Paul; Dieters, Mark J; DeLacy, Ian H; Zhou, Ji; Uauy, Cristobal; Boden, Scott A; Park, Robert F; Wulff, Brande B H; Hickey, Lee T

2018-01-01

The growing human population and a changing environment have raised significant concern for global food security, with the current improvement rate of several important crops inadequate to meet future demand 1 . This slow improvement rate is attributed partly to the long generation times of crop plants. Here, we present a method called 'speed breeding', which greatly shortens generation time and accelerates breeding and research programmes. Speed breeding can be used to achieve up to 6 generations per year for spring wheat (Triticum aestivum), durum wheat (T. durum), barley (Hordeum vulgare), chickpea (Cicer arietinum) and pea (Pisum sativum), and 4 generations for canola (Brassica napus), instead of 2-3 under normal glasshouse conditions. We demonstrate that speed breeding in fully enclosed, controlled-environment growth chambers can accelerate plant development for research purposes, including phenotyping of adult plant traits, mutant studies and transformation. The use of supplemental lighting in a glasshouse environment allows rapid generation cycling through single seed descent (SSD) and potential for adaptation to larger-scale crop improvement programs. Cost saving through light-emitting diode (LED) supplemental lighting is also outlined. We envisage great potential for integrating speed breeding with other modern crop breeding technologies, including high-throughput genotyping, genome editing and genomic selection, accelerating the rate of crop improvement.

Detection of haplotypes associated with prenatal death in dairy cattle and identification of deleterious mutations in GART, SHBG and SLC37A2.

PubMed

Fritz, Sébastien; Capitan, Aurelien; Djari, Anis; Rodriguez, Sabrina C; Barbat, Anne; Baur, Aurélia; Grohs, Cécile; Weiss, Bernard; Boussaha, Mekki; Esquerré, Diane; Klopp, Christophe; Rocha, Dominique; Boichard, Didier

2013-01-01

The regular decrease of female fertility over time is a major concern in modern dairy cattle industry. Only half of this decrease is explained by indirect response to selection on milk production, suggesting the existence of other factors such as embryonic lethal genetic defects. Genomic regions harboring recessive deleterious mutations were detected in three dairy cattle breeds by identifying frequent haplotypes (>1%) showing a deficit in homozygotes among Illumina Bovine 50k Beadchip haplotyping data from the French genomic selection database (47,878 Holstein, 16,833 Montbéliarde, and 11,466 Normande animals). Thirty-four candidate haplotypes (p<10(-4)) including previously reported regions associated with Brachyspina, CVM, HH1, and HH3 in Holstein breed were identified. Haplotype length varied from 1 to 4.8 Mb and frequencies from 1.7 up to 9%. A significant negative effect on calving rate, consistent in heifers and in lactating cows, was observed for 9 of these haplotypes in matings between carrier bulls and daughters of carrier sires, confirming their association with embryonic lethal mutations. Eight regions were further investigated using whole genome sequencing data from heterozygous bull carriers and control animals (45 animals in total). Six strong candidate causative mutations including polymorphisms previously reported in FANCI (Brachyspina), SLC35A3 (CVM), APAF1 (HH1) and three novel mutations with very damaging effect on the protein structure, according to SIFT and Polyphen-2, were detected in GART, SHBG and SLC37A2 genes. In conclusion, this study reveals a yet hidden consequence of the important inbreeding rate observed in intensively selected and specialized cattle breeds. Counter-selection of these mutations and management of matings will have positive consequences on female fertility in dairy cattle.

Detection of Haplotypes Associated with Prenatal Death in Dairy Cattle and Identification of Deleterious Mutations in GART, SHBG and SLC37A2

PubMed Central

Fritz, Sébastien; Capitan, Aurelien; Djari, Anis; Rodriguez, Sabrina C.; Barbat, Anne; Baur, Aurélia; Grohs, Cécile; Weiss, Bernard; Boussaha, Mekki; Esquerré, Diane; Klopp, Christophe; Rocha, Dominique; Boichard, Didier

2013-01-01

The regular decrease of female fertility over time is a major concern in modern dairy cattle industry. Only half of this decrease is explained by indirect response to selection on milk production, suggesting the existence of other factors such as embryonic lethal genetic defects. Genomic regions harboring recessive deleterious mutations were detected in three dairy cattle breeds by identifying frequent haplotypes (>1%) showing a deficit in homozygotes among Illumina Bovine 50k Beadchip haplotyping data from the French genomic selection database (47,878 Holstein, 16,833 Montbéliarde, and 11,466 Normande animals). Thirty-four candidate haplotypes (p<10−4) including previously reported regions associated with Brachyspina, CVM, HH1, and HH3 in Holstein breed were identified. Haplotype length varied from 1 to 4.8 Mb and frequencies from 1.7 up to 9%. A significant negative effect on calving rate, consistent in heifers and in lactating cows, was observed for 9 of these haplotypes in matings between carrier bulls and daughters of carrier sires, confirming their association with embryonic lethal mutations. Eight regions were further investigated using whole genome sequencing data from heterozygous bull carriers and control animals (45 animals in total). Six strong candidate causative mutations including polymorphisms previously reported in FANCI (Brachyspina), SLC35A3 (CVM), APAF1 (HH1) and three novel mutations with very damaging effect on the protein structure, according to SIFT and Polyphen-2, were detected in GART, SHBG and SLC37A2 genes. In conclusion, this study reveals a yet hidden consequence of the important inbreeding rate observed in intensively selected and specialized cattle breeds. Counter-selection of these mutations and management of matings will have positive consequences on female fertility in dairy cattle. PMID:23762392

Detecting novel SNPs and breed-specific haplotypes at calpastatin gene in Iranian fat- and thin-tailed sheep breeds and their effects on protein structure.

PubMed

Aali, Mohsen; Moradi-Shahrbabak, Mohammad; Moradi-Shahrbabak, Hosein; Sadeghi, Mostafa

2014-03-01

Calpastatin has been introduced as a potential candidate gene for growth and meat quality traits. In this study, genetic variability was investigated in the exon 6 and its intron boundaries of ovine CAST gene by PCR-SSCP analysis and DNA sequencing. Also a protein sequence and structural analysis were performed to predict the possible impact of amino acid substitutions on physicochemical properties and structure of the CAST protein. A total of 487 animals belonging to four ancient Iranian sheep breeds with different fat metabolisms, Lori-Bakhtiari and Chall (fat-tailed), Zel-Atabay cross-bred (medium fat-tailed) and Zel (thin-tailed), were analyzed. Eight unique SSCP patterns, representing eight different sequences or haplotypes, CAST-1, CAST-2 and CAST-6 to CAST-11, were identified. Haplotypes CAST-1 and CAST-2 were most common with frequency of 0.365 and 0.295. The novel haplotype CAST-8 had considerable frequency in Iranian sheep breeds (0.129). All the consensus sequences showed 98-99%, 94-98%, 92-93% and 82-83% similarity to the published ovine, caprine, bovine and porcine CAST locus sequences, respectively. Sequence analysis revealed four SNPs in intron 5 (C24T, G62A, G65T and T69-) and three SNPs in exon 6 (c.197A>T, c.282G>T and c.296C>G). All three SNPs in exon 6 were missense mutations which would result in p.Gln 66 Leu, p.Glu 94 Asp and p.Pro 99 Arg substitutions, respectively, in CAST protein. All three amino acid substitutions affected the physicochemical properties of ovine CAST protein including hydrophobicity, amphiphilicity and net charge and subsequently might influence its structure and effect on the activity of Ca2+ channels; hence, they might regulate calpain activity and afterwards meat tenderness and growth rate. The Lori-Bakhtiari population showed the highest heterozygosity in the ovine CAST locus (0.802). Frequency difference of haplotypes CAST-10 and CAST-8 between Lori-Bakhtiari (fat-tailed) and Zel (thin-tailed) breeds was highly

Targeted next generation sequencing identified a novel mutation in MYO7A causing Usher syndrome type 1 in an Iranian consanguineous pedigree.

PubMed

Kooshavar, Daniz; Razipour, Masoumeh; Movasat, Morteza; Keramatipour, Mohammad

2018-01-01

Usher syndrome (USH) is characterized by congenital hearing loss and retinitis pigmentosa (RP) with a later onset. It is an autosomal recessive trait with clinical and genetic heterogeneity which makes the molecular diagnosis much difficult. In this study, we introduce a pedigree with two affected members with USH type 1 and represent a cost and time effective approach for genetic diagnosis of USH as a genetically heterogeneous disorder. Target region capture in the genes of interest, followed by next generation sequencing (NGS) was used to determine the causative mutations in one of the probands. Then segregation analysis in the pedigree was conducted using PCR-Sanger sequencing. Targeted NGS detected a novel homozygous nonsense variant c.4513G > T (p.Glu1505Ter) in MYO7A. The variant is segregating in the pedigree with an autosomal recessive pattern. In this study, a novel stop gained variant c.4513G > T (p.Glu1505Ter) in MYO7A was found in an Iranian pedigree with two affected members with USH type 1. Bioinformatic as well as pedigree segregation analyses were in line with pathogenic nature of this variant. Targeted NGS panel was showed to be an efficient method for mutation detection in hereditary disorders with locus heterogeneity. Copyright © 2017 Elsevier B.V. All rights reserved.

Mutational spectrum in a worldwide study of 29,700 families with BRCA1 or BRCA2 mutations.

PubMed

Rebbeck, Timothy R; Friebel, Tara M; Friedman, Eitan; Hamann, Ute; Huo, Dezheng; Kwong, Ava; Olah, Edith; Olopade, Olufunmilayo I; Solano, Angela R; Teo, Soo-Hwang; Thomassen, Mads; Weitzel, Jeffrey N; Chan, T L; Couch, Fergus J; Goldgar, David E; Kruse, Torben A; Palmero, Edenir Inêz; Park, Sue Kyung; Torres, Diana; van Rensburg, Elizabeth J; McGuffog, Lesley; Parsons, Michael T; Leslie, Goska; Aalfs, Cora M; Abugattas, Julio; Adlard, Julian; Agata, Simona; Aittomäki, Kristiina; Andrews, Lesley; Andrulis, Irene L; Arason, Adalgeir; Arnold, Norbert; Arun, Banu K; Asseryanis, Ella; Auerbach, Leo; Azzollini, Jacopo; Balmaña, Judith; Barile, Monica; Barkardottir, Rosa B; Barrowdale, Daniel; Benitez, Javier; Berger, Andreas; Berger, Raanan; Blanco, Amie M; Blazer, Kathleen R; Blok, Marinus J; Bonadona, Valérie; Bonanni, Bernardo; Bradbury, Angela R; Brewer, Carole; Buecher, Bruno; Buys, Saundra S; Caldes, Trinidad; Caliebe, Almuth; Caligo, Maria A; Campbell, Ian; Caputo, Sandrine M; Chiquette, Jocelyne; Chung, Wendy K; Claes, Kathleen B M; Collée, J Margriet; Cook, Jackie; Davidson, Rosemarie; de la Hoya, Miguel; De Leeneer, Kim; de Pauw, Antoine; Delnatte, Capucine; Diez, Orland; Ding, Yuan Chun; Ditsch, Nina; Domchek, Susan M; Dorfling, Cecilia M; Velazquez, Carolina; Dworniczak, Bernd; Eason, Jacqueline; Easton, Douglas F; Eeles, Ros; Ehrencrona, Hans; Ejlertsen, Bent; Engel, Christoph; Engert, Stefanie; Evans, D Gareth; Faivre, Laurence; Feliubadaló, Lidia; Ferrer, Sandra Fert; Foretova, Lenka; Fowler, Jeffrey; Frost, Debra; Galvão, Henrique C R; Ganz, Patricia A; Garber, Judy; Gauthier-Villars, Marion; Gehrig, Andrea; Gerdes, Anne-Marie; Gesta, Paul; Giannini, Giuseppe; Giraud, Sophie; Glendon, Gord; Godwin, Andrew K; Greene, Mark H; Gronwald, Jacek; Gutierrez-Barrera, Angelica; Hahnen, Eric; Hauke, Jan; Henderson, Alex; Hentschel, Julia; Hogervorst, Frans B L; Honisch, Ellen; Imyanitov, Evgeny N; Isaacs, Claudine; Izatt, Louise; Izquierdo, Angel; Jakubowska, Anna; James, Paul; Janavicius, Ramunas; Jensen, Uffe Birk; John, Esther M; Vijai, Joseph; Kaczmarek, Katarzyna; Karlan, Beth Y; Kast, Karin; Investigators, KConFab; Kim, Sung-Won; Konstantopoulou, Irene; Korach, Jacob; Laitman, Yael; Lasa, Adriana; Lasset, Christine; Lázaro, Conxi; Lee, Annette; Lee, Min Hyuk; Lester, Jenny; Lesueur, Fabienne; Liljegren, Annelie; Lindor, Noralane M; Longy, Michel; Loud, Jennifer T; Lu, Karen H; Lubinski, Jan; Machackova, Eva; Manoukian, Siranoush; Mari, Véronique; Martínez-Bouzas, Cristina; Matrai, Zoltan; Mebirouk, Noura; Meijers-Heijboer, Hanne E J; Meindl, Alfons; Mensenkamp, Arjen R; Mickys, Ugnius; Miller, Austin; Montagna, Marco; Moysich, Kirsten B; Mulligan, Anna Marie; Musinsky, Jacob; Neuhausen, Susan L; Nevanlinna, Heli; Ngeow, Joanne; Nguyen, Huu Phuc; Niederacher, Dieter; Nielsen, Henriette Roed; Nielsen, Finn Cilius; Nussbaum, Robert L; Offit, Kenneth; Öfverholm, Anna; Ong, Kai-Ren; Osorio, Ana; Papi, Laura; Papp, Janos; Pasini, Barbara; Pedersen, Inge Sokilde; Peixoto, Ana; Peruga, Nina; Peterlongo, Paolo; Pohl, Esther; Pradhan, Nisha; Prajzendanc, Karolina; Prieur, Fabienne; Pujol, Pascal; Radice, Paolo; Ramus, Susan J; Rantala, Johanna; Rashid, Muhammad Usman; Rhiem, Kerstin; Robson, Mark; Rodriguez, Gustavo C; Rogers, Mark T; Rudaitis, Vilius; Schmidt, Ane Y; Schmutzler, Rita Katharina; Senter, Leigha; Shah, Payal D; Sharma, Priyanka; Side, Lucy E; Simard, Jacques; Singer, Christian F; Skytte, Anne-Bine; Slavin, Thomas P; Snape, Katie; Sobol, Hagay; Southey, Melissa; Steele, Linda; Steinemann, Doris; Sukiennicki, Grzegorz; Sutter, Christian; Szabo, Csilla I; Tan, Yen Y; Teixeira, Manuel R; Terry, Mary Beth; Teulé, Alex; Thomas, Abigail; Thull, Darcy L; Tischkowitz, Marc; Tognazzo, Silvia; Toland, Amanda Ewart; Topka, Sabine; Trainer, Alison H; Tung, Nadine; van Asperen, Christi J; van der Hout, Annemieke H; van der Kolk, Lizet E; van der Luijt, Rob B; Van Heetvelde, Mattias; Varesco, Liliana; Varon-Mateeva, Raymonda; Vega, Ana; Villarreal-Garza, Cynthia; von Wachenfeldt, Anna; Walker, Lisa; Wang-Gohrke, Shan; Wappenschmidt, Barbara; Weber, Bernhard H F; Yannoukakos, Drakoulis; Yoon, Sook-Yee; Zanzottera, Cristina; Zidan, Jamal; Zorn, Kristin K; Hutten Selkirk, Christina G; Hulick, Peter J; Chenevix-Trench, Georgia; Spurdle, Amanda B; Antoniou, Antonis C; Nathanson, Katherine L

2018-05-01

The prevalence and spectrum of germline mutations in BRCA1 and BRCA2 have been reported in single populations, with the majority of reports focused on White in Europe and North America. The Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) has assembled data on 18,435 families with BRCA1 mutations and 11,351 families with BRCA2 mutations ascertained from 69 centers in 49 countries on six continents. This study comprehensively describes the characteristics of the 1,650 unique BRCA1 and 1,731 unique BRCA2 deleterious (disease-associated) mutations identified in the CIMBA database. We observed substantial variation in mutation type and frequency by geographical region and race/ethnicity. In addition to known founder mutations, mutations of relatively high frequency were identified in specific racial/ethnic or geographic groups that may reflect founder mutations and which could be used in targeted (panel) first pass genotyping for specific populations. Knowledge of the population-specific mutational spectrum in BRCA1 and BRCA2 could inform efficient strategies for genetic testing and may justify a more broad-based oncogenetic testing in some populations. © 2018 Wiley Periodicals, Inc.