Candidate Causative Mutation Research Articles

A candidate loss-of-function variant in SGIP1 causes synaptic dysfunction and recessive parkinsonism

Synaptic dysfunction is recognized as an early step in the pathophysiology of parkinsonism. Several genetic mutations affecting the integrity of synaptic proteins cause or increase the risk of developing disease. We have identified a candidate causative mutation in synaptic "SH3GL2 Interacting Protein 1" (SGIP1), linked to early-onset parkinsonism in a consanguineous Arab family. Additionally, affected siblings display intellectual, cognitive, and behavioral dysfunction. Metabolic network analysis of [18F]-fluorodeoxyglucose positron emission tomography scans shows patterns very similar to those of idiopathic Parkinson's disease. We show that the identified SGIP1 mutation causes a loss of protein function, and analyses in newly created Drosophila models reveal movement defects, synaptic transmission dysfunction, and neurodegeneration, including dopaminergic synapse loss. Histology and correlative light and electron microscopy reveal the absence of synaptic multivesicular bodies and the accumulation of degradative organelles. This research delineates a putative form of recessive parkinsonism, converging on defective synaptic proteostasis and opening avenues for diagnosis, genetic counseling, and treatment.

Correction: A 20 bp Duplication in Exon 2 of the Aristaless-Like Homeobox 4 Gene (ALX4) Is the Candidate Causative Mutation for Tibial Hemimelia Syndrome in Galloway Cattle

Correction: A 20 bp Duplication in Exon 2 of the Aristaless-Like Homeobox 4 Gene (ALX4) Is the Candidate Causative Mutation for Tibial Hemimelia Syndrome in Galloway Cattle

Early detection of highly transmissible viral variants using phylogenomics.

As demonstrated by the SARS-CoV-2 pandemic, the emergence of novel viral strains with increased transmission rates poses a serious threat to global health. Statistical models of genome sequence evolution may provide a critical tool for early detection of these strains. Using a novel stochastic model that links transmission rates to the entire viral genome sequence, we study the utility of phylogenetic methods that use a phylogenetic tree relating viral samples versus count-based methods that use case counts of variants over time exclusively to detect increased transmission rates and identify candidate causative mutations. We find that phylogenies in particular can detect novel transmission-enhancing variants very soon after their origin and may facilitate the development of early detection systems for outbreak surveillance.

Genome-wide detection of selection signatures in Jianli pigs reveals novel cis-regulatory haplotype in EDNRB associated with two-end black coat color

BackgroundJianli pig, a renowned indigenous breed in China, has the characteristics of a two-end black (TEB) coat color, excellent meat quality, strong adaptability and increased prolificacy. However, there is limited information available regarding the genetic diversity, population structure and genomic regions under selection of Jianli pig. On the other hand, the genetic mechanism of TEB coat color has remained largely unknown.ResultsIn this study, the whole genome resequencing of 30 Jianli pigs within a context of 153 individuals representing 13 diverse breeds was performed. The population structure analysis revealed that Jianli pigs have close genetic relationships with the Tongcheng pig breed, their geographical neighbors. Three methods (observed heterozygosity, expected heterozygosity, and runs of homozygosity) implied a relatively high level of genetic diversity and, a low inbreeding coefficient in Jianli compared with other pigs. We used Fst and XP-EHH to detect the selection signatures in Jianli pigs compared with Asian wild boar. A total of 451 candidate genes influencing meat quality (CREBBP, ADCY9, EEPD1 and HDAC9), reproduction (ESR1 and FANCA), and coat color (EDNRB, MITF and MC1R), were detected by gene annotation analysis. Finally, to fine-map the genomic region for the two-end black (TEB) coat color phenotype in Jianli pigs, we performed three signature selection methods between the TEB coat color and no-TEB coat color pig breeds. The current study, further confirmed that the EDNRB gene is a candidate gene for TEB color phenotype found in Chinese pigs, including Jinhua pigs, and the haplotype harboring 25 SNPs in the EDNRB gene may promote the formation of TEB coat color. Further ATAC-seq and luciferase reporter assays of these regions suggest that the 25-SNPs region was a strong candidate causative mutation that regulates the TEB coat color phenotype by altering enhancer function.ConclusionOur results advanced the understanding of the genetic mechanism behind artificial selection, and provided further resources for the protection and breeding improvement of Jianli pigs.

Biallelic COQ4 Variants in Hereditary Spastic Paraplegia: Clinical and Molecular Characterization.

Hereditary spastic paraplegias (HSP) are neurologic disorders characterized by progressive lower-extremity spasticity. Despite the identification of several HSP-related genes, many patients lack a genetic diagnosis. The aims were to confirm the pathogenic role of biallelic COQ4 mutations in HSP and elucidate the clinical, genetic, and functional molecular features of COQ4-associated HSP. Whole exome sequences of 310 index patients with HSP of unknown cause from three distinct populations were analyzed to identify potential HSP causal genes. Clinical data obtained from patients harboring candidate causal mutations were examined. Functional characterization of COQ4 variants was performed using bioinformatic tools, single-cell RNA sequencing, biochemical assays in cell lines, primary fibroblasts, induced pluripotent stem cell-derived pyramidal neurons, and zebrafish. Compound heterozygous variants in COQ4, which cosegregated with HSP in pedigrees, were identified in 7 patients from six unrelated families. Patients from four of the six families presented with pure HSP, whereas probands of the other two families exhibited complicated HSP with epilepsy or with cerebellar ataxia. In patient-derived fibroblasts and COQ4 knockout complementation lines, stable expression of these missense variants exerted loss-of-function effects, including mitochondrial reactive oxygen species accumulation, decreased mitochondrial membrane potential, and lower ubiquinone biosynthesis. Whereas differentiated pyramidal neurons expressed high COQ4 levels, coq4 knockdown zebrafish displayed severe motor dysfunction, reflecting motor neuron dysregulation. Our study confirms that loss-of-function, compound heterozygous, pathogenic COQ4 variants are causal for autosomal recessive pure and complicated HSP. Moreover, reduced COQ4 levels attributable to variants correspond with decreased ubiquinone biosynthesis, impaired mitochondrial function, and higher phenotypic disease severity. © 2023 International Parkinson and Movement Disorder Society.

Candidate signatures of positive selection for environmental adaptation in indigenous African cattle: A review.

Environmental adaptation traits of indigenous African cattle are increasingly being investigated to respond to the need for sustainable livestock production in the context of unpredictable climatic changes. Several studies have highlighted genomic regions under positive selection probably associated with adaptation to environmental challenges (e.g. heat stress, trypanosomiasis, tick and tick-borne diseases). However, little attention has focused on pinpointing the candidate causative variant(s) controlling the traits. This review compiled information from 22 studies on signatures of positive selection in indigenous African cattle breeds to identify regions under positive selection. We highlight some key candidate genome regions and genes of relevance to the challenges of living in extreme environments (high temperature, high altitude, high infectious disease prevalence). They include candidate genes involved in biological pathways relating to innate and adaptive immunity (e.g. BoLAs, SPAG11, IL1RL2 and GFI1B), heat stress (e.g. HSPs, SOD1 and PRLH) and hypoxia responses (e.g. BDNF and INPP4A). Notably, the highest numbers of candidate regions are found on BTA3, BTA5 and BTA7. They overlap with genes playing roles in several biological functions and pathways. These include but are not limited to growth and feed intake, cell stability, protein stability and sweat gland development. This review may further guide targeted genome studies aiming to assess the importance of candidate causative mutations, within regulatory and protein-coding genome regions, to further understand the biological mechanisms underlying African cattle's unique adaption.

Mapping Maize Mutants Using Bulked-Segregant Analysis and Next-Generation Sequencing.

Forward genetics is used to identify the genetic basis for a phenotype. The approach involves identifying a mutant organism exhibiting a phenotype of interest and then mapping the causative locus or gene. Bulked-segregant analysis (BSA) is a quick and effective approach to map mutants using pools of mutants and wild-type plants from a segregating population to identify linkage of the mutant phenotype, and this approach has been successfully used in plants. Traditional linkage mapping approaches are outdated and time intensive, and can be very difficult. With the highly evolved development and reduction in cost of high-throughput sequencing, this new approach combined with BSA has become extremely effective in multiple plant species, including Zea mays (maize). While the approach is incredibly powerful, careful experimental design, bioinformatic mapping techniques, and interpretation of results are important to obtain the desired results in an effective and timely manner. Poor design of a mapping population, limitations in bioinformatic experience, and inadequate understanding of sequence data are limitations of these approaches for the researcher. Here, we describe a straightforward protocol for mapping mutations responsible for a phenotype of interest in maize, using high-throughput sequencing and BSA. Specifically, we discuss relevant aspects of developing a mutant mapping population. This is followed by a detailed protocol for DNA preparation and analysis of short-read sequences to map and identify candidate causative mutations responsible for the mutant phenotype of interest. We provide command-line and perl scripts to complete the bioinformatic analysis of the mutant sequence data. This protocol lays out the design of the BSA, bioinformatic approaches, and interpreting the sequencing data. These methods are very adaptable to any forward genetics experiment and provide a step-by-step approach to identifying the genetic basis of a maize mutant phenotype. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Bulked-segregant analysis and high-throughput sequencing to map maize mutants.

Improved Upland Cotton Germplasm for Multiple Fiber Traits Mediated by Transferring and Pyramiding Novel Alleles From Ethyl Methanesulfonate-Generated Mutant Lines Into Elite Genotypes.

Ethyl methanesulfonate (EMS) mutagenesis offers important advantages for improving crops, such as cotton, with limited diversity in elite gene pools. EMS-induced point mutations are less frequently associated with deleterious traits than alleles from wild or exotic germplasm. From 157 mutant lines that have significantly improved fiber properties, we focused on nine mutant lines here. A total of eight populations were developed by crossing mutant lines in different combinations into GA230 (GA2004230) background. Multiple lines in each population were significantly improved for the fiber trait that distinguished the donor parent(s), demonstrating that an elite breeding line (GA230) could be improved for fiber qualities using the mutant lines. Genotypes improved for multiple fiber traits of interest suggesting that allele pyramiding is possible. Compared to midparent values, individual progeny in the population conferred fiber quality improvements of as much as 31.7% (in population O) for micronaire (MIC), 16.1% (in population P) for length, 22.4% (in population K) for strength, 4.1% (in population Q) for uniformity, 45.8% (in population N) for elongation, and 13.9% (in population O) for lint percentage (lint%). While further testing for stability of the phenotype and estimation of yield potential is necessary, mutation breeding shows promise as an approach to reduce the problem of the genetic bottleneck of upland cotton. The populations developed here may also contribute to identifying candidate genes and causal mutations for fiber quality improvement.

Pervasive occurrence of splice-site-creating mutations and their possible involvement in genetic disorders

The search for causative mutations in human genetic disorders has mainly focused on mutations that disrupt coding regions or splice sites. Recently, however, it has been reported that mutations creating splice sites can also cause a range of genetic disorders. In this study, we identified 5656 candidate splice-site-creating mutations (SCMs), of which 3942 are likely to be pathogenic, in 4054 genes responsible for genetic disorders. Reanalysis of exome data obtained from ciliopathy patients led us to identify 38 SCMs as candidate causative mutations. We estimate that, by focusing on SCMs, the increase in diagnosis rate is approximately 5.9–8.5% compared to the number of already known pathogenic variants. This finding suggests that SCMs are mutations worth focusing on in the search for causative mutations of genetic disorders.

Non-additive QTL mapping of lactation traits in 124,000 cattle reveals novel recessive loci

BackgroundDeleterious recessive conditions have been primarily studied in the context of Mendelian diseases. Recently, several deleterious recessive mutations with large effects were discovered via non-additive genome-wide association studies (GWAS) of quantitative growth and developmental traits in cattle, which showed that quantitative traits can be used as proxies of genetic disorders when such traits are indicative of whole-animal health status. We reasoned that lactation traits in cattle might also reflect genetic disorders, given the increased energy demands of lactation and the substantial stresses imposed on the animal. In this study, we screened more than 124,000 cows for recessive effects based on lactation traits.ResultsWe discovered five novel quantitative trait loci (QTL) that are associated with large recessive impacts on three milk yield traits, with these loci presenting missense variants in the DOCK8, IL4R, KIAA0556, and SLC25A4 genes or premature stop variants in the ITGAL, LRCH4, and RBM34 genes, as candidate causal mutations. For two milk composition traits, we identified several previously reported additive QTL that display small dominance effects. By contrasting results from milk yield and milk composition phenotypes, we note differing genetic architectures. Compared to milk composition phenotypes, milk yield phenotypes had lower heritabilities and were associated with fewer additive QTL but had a higher non-additive genetic variance and were associated with a higher proportion of loci exhibiting dominance.ConclusionsWe identified large-effect recessive QTL which are segregating at surprisingly high frequencies in cattle. We speculate that the differences in genetic architecture between milk yield and milk composition phenotypes derive from underlying dissimilarities in the cellular and molecular representation of these traits, with yield phenotypes acting as a better proxy of underlying biological disorders through presentation of a larger number of major recessive impacts.

Integrated analysis of whole genome and transcriptome sequencing reveals a frameshift mutation associated with recessive embryonic lethality in Holstein cattle.

Embryo loss is an important factor affecting fertility in dairy production. HH2 was identified as a haplotype on chromosome 1 associated with embryonic lethality in Holstein cattle. In the current study, both short- and long-read WGS was performed on four carriers and four non-carriers of HH2 to screen for variants in concordance with HH2 haplotype status. Sequence variation analysis revealed five putative functional variants of protein-coding genes, including a frameshift mutation (g.107172616delT) in intraflagellar transport protein 80 (IFT80) gene. Transcriptome analysis of whole blood indicated that no gene exhibited significantly differential expression or allele-specific expression between carriers and non-carriers in the candidate region. This evidence points to g.107172616delT as the highest priority causative mutation for HH2. Protein prediction reveals that the frameshift mutation results in a premature stop codon to reduce the peptide chain from 760 to 383 amino acids and greatly alters the structure and function of IFT80 protein. Our results demonstrate that the use of a combination of multiple high-throughput sequencing technologies is an efficient strategy to screen for the candidate causative mutations responsible for Mendelian traits, including genetic disorders.

Identification of Essential Genes in Caenorhabditis elegans with Lethal Mutations Maintained by Genetic Balancers.

Genetic balancer systems, which allow effective capture and maintenance of lethal mutations stably, play an important role in identifying essential genes. Whole-genome sequencing (WGS) followed by bioinformatics analysis, combined with genetic mapping data analysis, allows for an efficient and economical means of identifying genomic mutations in essential genes. Using this approach, we successfully identified 104 essential genes on ChrI, ChrIII, and ChrV in C. elegans. In this report, we described a protocol that sequences the genome of prebalanced Caenorhabditis elegans (C. elegans) strains to carry lethal mutations and identifies candidate causal mutations and candidate essential genes using a robust bioinformatics procedure.

Identification of a Mosaic Activating Mutation in GNA11 in Atypical Sturge-Weber Syndrome

Identification of a Mosaic Activating Mutation in GNA11 in Atypical Sturge-Weber Syndrome

Meta-analysis for milk fat and protein percentage using imputed sequence variant genotypes in 94,321 cattle from eight cattle breeds

BackgroundSequence-based genome-wide association studies (GWAS) provide high statistical power to identify candidate causal mutations when a large number of individuals with both sequence variant genotypes and phenotypes is available. A meta-analysis combines summary statistics from multiple GWAS and increases the power to detect trait-associated variants without requiring access to data at the individual level of the GWAS mapping cohorts. Because linkage disequilibrium between adjacent markers is conserved only over short distances across breeds, a multi-breed meta-analysis can improve mapping precision.ResultsTo maximise the power to identify quantitative trait loci (QTL), we combined the results of nine within-population GWAS that used imputed sequence variant genotypes of 94,321 cattle from eight breeds, to perform a large-scale meta-analysis for fat and protein percentage in cattle. The meta-analysis detected (p ≤ 10−8) 138 QTL for fat percentage and 176 QTL for protein percentage. This was more than the number of QTL detected in all within-population GWAS together (124 QTL for fat percentage and 104 QTL for protein percentage). Among all the lead variants, 100 QTL for fat percentage and 114 QTL for protein percentage had the same direction of effect in all within-population GWAS. This indicates either persistence of the linkage phase between the causal variant and the lead variant across breeds or that some of the lead variants might indeed be causal or tightly linked with causal variants. The percentage of intergenic variants was substantially lower for significant variants than for non-significant variants, and significant variants had mostly moderate to high minor allele frequencies. Significant variants were also clustered in genes that are known to be relevant for fat and protein percentages in milk.ConclusionsOur study identified a large number of QTL associated with fat and protein percentage in dairy cattle. We demonstrated that large-scale multi-breed meta-analysis reveals more QTL at the nucleotide resolution than within-population GWAS. Significant variants were more often located in genic regions than non-significant variants and a large part of them was located in potentially regulatory regions.

1660-P: Exome Sequencing in a Family with Multiple Cases of Early-Onset Diabetes Reveals a Candidate Causative Mutation in the PTF1A Gene

Maturity-onset diabetes of the young (MODY) is characterized by early-onset diabetes with dominant inheritance. To date, 14 causative genes have been proposed for MODY, however the disease-causing genetic variants in numerous cases of early-onset diabetes with features of MODY remain unknown. The aim of this study was to investigate the genetic background of a family with multiple cases of early-onset diabetes using exome sequencing to identify a causative gene. The Japanese family that we examined included eight members with diabetes, three of whom were diagnosed with the disease before the age of 35. All three of these individuals were lean (BMI <25 kg/m2). Serum C-peptide was detectable and GAD antibody was negative in all three cases. We first performed exome sequencing for two of these three members with early-onset diabetes. No mutations of the 14 known MODY genes were detected in these two family members. We therefore predicted that the familial aggregation of early-onset diabetes in this family was due to a rare non-synonymous variant in genes other than 14 MODY genes. We selected non-synonymous variants identified with exome sequencing and then excluded common variants with minor allele frequency of >1% in the 1000 genomes project or the Japanese exome database. As a result, we identified rare 399 non-synonymous variants shared by two members sequenced. Among the variants, we found a heterozygous S18F mutation in PTF1A [rs761787095, chr10:23192583C>T(GRCh38)]. Following this, we genotyped the remaining family member with early-onset diabetes, who was finally confirmed to share the heterozygous S18F mutation in PTF1A. Interestingly, the PTF1A gene has been implicated in pancreas development. Thus, the S18F mutation in PTF1A may be a causative mutation for early-onset diabetes. Family-based comprehensive exome sequencing is a promising strategy to elucidate the complex genetic background of diabetes. Disclosure D. Tanaka: None. S. Okamoto: None. Y. Liu: None. K. Iizuka: Speaker’s Bureau; Self; Kowa Company, Ltd., Novo Nordisk Inc. Y. Hamamoto: Research Support; Self; Sumitomo Dainippon Pharma Co., Ltd. Speaker’s Bureau; Self; Novo Nordisk Inc. Y. Horikawa: None. D. Yabe: Research Support; Self; ARKRAY, Astellas Pharma Inc., AstraZeneca K.K., Eli Lilly Japan K.K., Japan Agency for Medical Research and Development, Japan Society for the Promotion of Science, Kowa Company, Ltd., Nippon Boehringer Ingelheim Co. Ltd., Sanofi K.K., Sanwa Kagaku Kenkyusho, Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker’s Bureau; Self; Abbott, ARKRAY, Astellas Pharma Inc., AstraZeneca K.K., Bayer Yakuhin, Ltd., Daiichi Sankyo, Eli Lilly Japan K.K., Fukuda Denshi, Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., LifeScan, Inc., Medtronic, Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Novo Nordisk Inc., Novo Nordisk Inc., Sanofi K.K., Sanwa Kagaku Kenkyusho, Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited. Other Relationship; Self; Japan Diabetes Society. N. Inagaki: Research Support; Self; Astellas Pharma Inc., Drawbridge Health, Japan Tobacco Inc., Kissei Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Life Scan Japan, Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Novo Nordisk Pharma Ltd.,, Sanofi K.K., Sanwa Kagaku Kenkyusho, Terumo Medical Corporation. Speaker’s Bureau; Self; Astellas Pharma Inc., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Funding Japan Society for the Promotion of Science (18K08509)

Mutations Upstream of the TBX5 and PITX1 Transcription Factor Genes Are Associated with Feathered Legs in the Domestic Chicken.

Feathered leg is a trait in domestic chickens that has undergone intense selection by fancy breeders. Previous studies have shown that two major loci controlling feathered leg are located on chromosomes 13 and 15. Here, we present genetic evidence for the identification of candidate causal mutations at these loci. This was accomplished by combining classical linkage mapping using an experimental cross segregating for feathered leg and high-resolution identical-by-descent mapping using whole-genome sequence data from 167 samples of chicken with or without feathered legs. The first predicted causal mutation is a single-base change located 25 kb upstream of the gene for the forelimb-specific transcription factor TBX5 on chromosome 15. The second is a 17.7-kb deletion located ∼200 kb upstream of the gene for the hindlimb-specific transcription factor PITX1 on chromosome 13. These mutations are predicted to activate TBX5 and repress PITX1 expression, respectively. The study reveals a remarkable convergence in the evolution of the feathered-leg phenotype in domestic chickens and domestic pigeons, as this phenotype is caused by noncoding mutations upstream of the same two genes. Furthermore, the PITX1 causal variants are large overlapping deletions, 17.7 kb in chicken and 44 kb in pigeons. The results of the present study are consistent with the previously proposed model for pigeon that feathered leg is caused by reduced PITX1 expression and ectopic expression of TBX5 in hindlimb buds resulting in a shift of limb identity from hindlimb to more forelimb-like identity.

A homozygous RPL10L missense mutation associated with male factor infertility and severe oligozoospermia

To identify the genetic cause of male factor infertility characterized by severe oligozoospermia. Genetic studies. Medical university. Two infertile brothers with severe oligozoospermia in a consanguineous Han Chinese family, 414 additional patients with oligo-/azoospermia, and 223 fertile (control) subjects. None. Genetic analyses using whole-exome and Sanger sequencing were performed for two brothers with severe oligozoospermia. The effects of an identified candidate causative mutation were investigated in silico and invitro. Whole-exome sequencing screening for the candidate mutation was conducted in 414 patients with oligo-/azoospermia and 223 fertile subjects. A homozygous missense variant (NM_080746:c.A257C: p.H86P) in RPL10L was identified in the two affected brothers and shown to cosegregate with the severe oligozoospermia phenotype. The mutation was absent in public databases, including the 1000 Genomes Project and the Exome Aggregation Consortium. All queried databases predicted the mutation to be damaging, consistent with the fact that it decreased protein levels invitro. Subsequent mutation screening identified three additional heterozygous RPL10L mutations in three of 414 subjects with oligo-/azoospermia, but no RPL10L mutations among 223 fertile subjects. Our findings implicate RPL10L as a novel candidate gene in the pathogenesis of human male factor infertility and severe oligozoospermia.

Differential Allele-Specific Expression Uncovers Breast Cancer Genes Dysregulated by Cis Noncoding Mutations.

Identifying cancer-relevant mutations in noncoding regions is challenging due to the large numbers of such mutations, their low levels of recurrence, and difficulties in interpreting their functional impact. To uncover genes that are dysregulated due to somatic mutations in cis, we build upon the concept of differential allele-specific expression (ASE) and introduce methods to identify genes within an individual's cancer whose ASE differs from what is found in matched normal tissue. When applied to breast cancer tumor samples, our methods detect the known allele-specific effects of copy number variation and nonsense-mediated decay. Further, genes that are found to recurrently exhibit differential ASE across samples are cancer relevant. Genes with cis mutations are enriched for differential ASE, and we find 147 potentially functional noncoding mutations cis to genes that exhibit significant differential ASE. We conclude that differential ASE is a promising means for discovering gene dysregulation due to cis noncoding mutations.

Identification of potential functional variants underlying ovine resistance to gastrointestinal nematode infection by using RNA-Seq.

In dairy sheep flocks from Mediterranean countries, replacement and adult ewes are the animals most affected by gastrointestinal nematode (GIN) infections. In this study, we have exploited the information derived from an RNA-Seq experiment with the aim of identifying potential causal mutations related to GIN resistance in sheep. Considering the RNA-Seq samples from 12 ewes previously classified as six resistant and six susceptible animals to experimental infection by Teladorsagia circumcincta, we performed a variant calling analysis pipeline using two different types of software, gatk version 3.7 and Samtools version 1.4. The variants commonly identified by the two packages (high-quality variants) within two types of target regions - (i) QTL regions previously reported in sheep for parasite resistance based on SNP-chip or sequencing technology studies and (ii) functional candidate genes selected from gene expression studies related to GIN resistance in sheep - were further characterised to identify mutations with a potential functional impact. Among the genes harbouring these potential functional variants (930 and 553 respectively for the two types of regions), we identified 111 immune-related genes in the QTL regions and 132 immune-related genes from the initially selected candidate genes. For these immune-related genes harbouring potential functional variants, the enrichment analyses performed highlighted significant GO terms related to apoptosis, adhesion and inflammatory response, in relation to the QTL related variants, and significant disease-related terms such as inflammation, adhesion and necrosis, in relation to the initial candidate gene list. Overall, the study provides a valuable list of potential causal mutations that could be considered as candidate causal mutations in relation to GIN resistance in sheep. Future studies should assess the role of these suggested mutations with the aim of identifying genetic markers that could be directly implemented in sheep breeding programmes considering not only production traits, but also functional traits such as resistance to GIN infections.

Genome-wide association analysis reveals QTL and candidate mutations involved in white spotting in cattle

BackgroundWhite spotting of the coat is a characteristic trait of various domestic species including cattle and other mammals. It is a hallmark of Holstein–Friesian cattle, and several previous studies have detected genetic loci with major effects for white spotting in animals with Holstein–Friesian ancestry. Here, our aim was to better understand the underlying genetic and molecular mechanisms of white spotting, by conducting the largest mapping study for this trait in cattle, to date.ResultsUsing imputed whole-genome sequence data, we conducted a genome-wide association analysis in 2973 mixed-breed cows and bulls. Highly significant quantitative trait loci (QTL) were found on chromosomes 6 and 22, highlighting the well-established coat color genes KIT and MITF as likely responsible for these effects. These results are in broad agreement with previous studies, although we also report a third significant QTL on chromosome 2 that appears to be novel. This signal maps immediately adjacent to the PAX3 gene, which encodes a known transcription factor that controls MITF expression and is the causal locus for white spotting in horses. More detailed examination of these loci revealed a candidate causal mutation in PAX3 (p.Thr424Met), and another candidate mutation (rs209784468) within a conserved element in intron 2 of MITF transcripts expressed in the skin. These analyses also revealed a mechanistic ambiguity at the chromosome 6 locus, where highly dispersed association signals suggested multiple or multiallelic QTL involving KIT and/or other genes in this region.ConclusionsOur findings extend those of previous studies that reported KIT as a likely causal gene for white spotting, and report novel associations between candidate causal mutations in both the MITF and PAX3 genes. The sizes of the effects of these QTL are substantial, and could be used to select animals with darker, or conversely whiter, coats depending on the desired characteristics.