Fine mapping for Weaver syndrome in Brown Swiss cattle and the identification of 41 concordant mutations across NRCAM, PNPLA8 and CTTNBP2.
ABSTRACT: Bovine Progressive Degenerative Myeloencephalopathy (Weaver Syndrome) is a recessive neurological disease that has been observed in the Brown Swiss cattle breed since the 1970's in North America and Europe. Bilateral hind leg weakness and ataxia appear in afflicted animals at 6 to 18 months of age, and slowly progresses to total loss of hind limb control by 3 to 4 years of age. While Weaver has previously been mapped to Bos taurus autosome (BTA) 4?46-56 Mb and a diagnostic test based on the 6 microsatellite (MS) markers is commercially available, neither the causative gene nor mutation has been identified; therefore misdiagnosis can occur due to recombination between the diagnostic MS markers and the causative mutation. Analysis of 34,980 BTA 4 SNPs genotypes derived from the Illumina BovineHD assay for 20 Brown Swiss Weaver carriers and 49 homozygous normal bulls refined the Weaver locus to 48-53 Mb. Genotyping of 153 SNPs, identified from whole genome sequencing of 10 normal and 10 carrier animals, across a validation set of 841 animals resulted in the identification of 41 diagnostic SNPs that were concordant with the disease. Except for one intergenic SNP all are associated with genes expressed in nervous tissues: 37 distal to NRCAM, one non-synonymous (serine to asparagine) in PNPLA8, one synonymous and one non-synonymous (lysine to glutamic acid) in CTTNBP2. Haplotype and imputation analyses of 7,458 Brown Swiss animals with Illumina BovineSNP50 data and the 41 diagnostic SNPs resulted in the identification of only one haplotype concordant with the Weaver phenotype. Use of this haplotype and the diagnostic SNPs more accurately identifies Weaver carriers in both Brown Swiss purebred and influenced herds.
Project description:Bovine progressive degenerative myeloencephalopathy (Weaver syndrome) is a neurodegenerative disorder in Brown Swiss cattle that is characterized by progressive hind leg weakness and ataxia, while sensorium and spinal reflexes remain unaffected. Although the causal mutation has not been identified yet, an indirect genetic test based on six microsatellite markers and consequent exclusion of Weaver carriers from breeding have led to the complete absence of new cases for over two decades. Evaluation of disease status by imputation of 41 diagnostic single nucleotide polymorphisms (SNPs) and a common haplotype published in 2013 identified several suspected carriers in the current breeding population, which suggests a higher frequency of the Weaver allele than anticipated. In order to prevent the reemergence of the disease, this study aimed at mapping the gene that underlies Weaver syndrome and thus at providing the basis for direct genetic testing and monitoring of today's Braunvieh/Brown Swiss herds.Combined linkage/linkage disequilibrium mapping on Bos taurus chromosome (BTA) 4 based on Illumina Bovine SNP50 genotypes of 43 Weaver-affected, 31 Weaver carrier and 86 Weaver-free animals resulted in a maximum likelihood ratio test statistic value at position 49,812,384 bp. The confidence interval (0.853 Mb) determined by the 2-LOD drop-off method was contained within a 1.72-Mb segment of extended homozygosity. Exploitation of whole-genome sequence data from two official Weaver carriers and 1145 other bulls that were sequenced in Run4 of the 1000 bull genomes project showed that only a non-synonymous SNP (rs800397662) within the PNPLA8 gene at position 49,878,773 bp was concordant with the Weaver carrier status. Targeted SNP genotyping confirmed this SNP as a candidate causal mutation for Weaver syndrome. Genotyping for the candidate causal mutation in a random sample of 2334 current Braunvieh animals suggested a frequency of the Weaver allele of 0.26 %.Through combined use of exhaustive sequencing data and SNP genotyping results, we were able to provide evidence that supports the non-synonymous mutation at position 49,878,773 bp as the most likely causal mutation for Weaver syndrome. Further studies are needed to uncover the exact mechanisms that underlie this syndrome.
Project description:BACKGROUND:Little is known about the genetic architecture of economically important traits in Brown Swiss cattle because only few genome-wide association studies (GWAS) have been carried out in this breed. Moreover, most GWAS have been performed for single traits, thus not providing detailed insights into potentially existing pleiotropic effects of trait-associated loci. RESULTS:To compile a comprehensive catalogue of large-effect quantitative trait loci (QTL) segregating in Brown Swiss cattle, we carried out association tests between partially imputed genotypes at 598,016 SNPs and daughter-derived phenotypes for more than 50 economically important traits, including milk production, growth and carcass quality, body conformation, reproduction and calving traits in 4578 artificial insemination bulls from two cohorts of Brown Swiss cattle (Austrian-German and Swiss populations). Across-cohort multi-trait meta-analyses of the results from the single-trait GWAS revealed 25 quantitative trait loci (QTL; P?<?8.36?×?10-?8) for economically relevant traits on 17 Bos taurus autosomes (BTA). Evidence of pleiotropy was detected at five QTL located on BTA5, 6, 17, 21 and 25. Of these, two QTL at BTA6:90,486,780 and BTA25:1,455,150 affect a diverse range of economically important traits, including traits related to body conformation, calving, longevity and milking speed. Furthermore, the QTL at BTA6:90,486,780 seems to be a target of ongoing selection as evidenced by an integrated haplotype score of 2.49 and significant changes in allele frequency over the past 25?years, whereas either no or only weak evidence of selection was detected at all other QTL. CONCLUSIONS:Our findings provide a comprehensive overview of QTL segregating in Brown Swiss cattle. Detected QTL explain between 2 and 10% of the variation in the estimated breeding values and thus may be considered as the most important QTL segregating in the Brown Swiss cattle breed. Multi-trait association testing boosts the power to detect pleiotropic QTL and assesses the full spectrum of phenotypes that are affected by trait-associated variants.
Project description:The recent discovery of bovine haplotypes with negative effects on fertility in the Brown Swiss, Holstein, and Jersey breeds has allowed producers to identify carrier animals using commercial single nucleotide polymorphism (SNP) genotyping assays. This study was devised to identify the causative mutations underlying defective bovine embryo development contained within three of these haplotypes (Brown Swiss haplotype 1 and Holstein haplotypes 2 and 3) by combining exome capture with next generation sequencing. Of the 68,476,640 sequence variations (SV) identified, only 1,311 genome-wide SNP were concordant with the haplotype status of 21 sequenced carriers. Validation genotyping of 36 candidate SNP identified only 1 variant that was concordant to Holstein haplotype 3 (HH3), while no variants located within the refined intervals for HH2 or BH1 were concordant. The variant strictly associated with HH3 is a non-synonymous SNP (T/C) within exon 24 of the Structural Maintenance of Chromosomes 2 (SMC2) on Chromosome 8 at position 95,410,507 (UMD3.1). This polymorphism changes amino acid 1135 from phenylalanine to serine and causes a non-neutral, non-tolerated, and evolutionarily unlikely substitution within the NTPase domain of the encoded protein. Because only exome capture sequencing was used, we could not rule out the possibility that the true causative mutation for HH3 might lie in a non-exonic genomic location. Given the essential role of SMC2 in DNA repair, chromosome condensation and segregation during cell division, our findings strongly support the non-synonymous SNP (T/C) in SMC2 as the likely causative mutation. The absence of concordant variations for HH2 or BH1 suggests either the underlying causative mutations lie within a non-exomic region or in exome regions not covered by the capture array.
Project description:Cattle are ideally suited to investigate the genetics of male reproduction, because semen quality and fertility are recorded for all ejaculates of artificial insemination bulls. We analysed 26,090 ejaculates of 794 Brown Swiss bulls to assess ejaculate volume, sperm concentration, sperm motility, sperm head and tail anomalies and insemination success. The heritability of the six semen traits was between 0 and 0.26. Genome-wide association testing on 607,511 SNPs revealed a QTL on bovine chromosome 6 that was associated with sperm motility (P = 2.5 x 10-27), head (P = 2.0 x 10-44) and tail anomalies (P = 7.2 x 10-49) and insemination success (P = 9.9 x 10-13). The QTL harbors a recessive allele that compromises semen quality and male fertility. We replicated the effect of the QTL on fertility (P = 7.1 x 10-32) in an independent cohort of 2481 Brown Swiss bulls. The analysis of whole-genome sequencing data revealed that a synonymous variant (BTA6:58373887C>T, rs474302732) in WDR19 encoding WD repeat-containing protein 19 was in linkage disequilibrium with the fertility-associated haplotype. WD repeat-containing protein 19 is a constituent of the intraflagellar transport complex that is essential for the physiological function of motile cilia and flagella. Bioinformatic and transcription analyses revealed that the BTA6:58373887 T-allele activates a cryptic exonic splice site that eliminates three evolutionarily conserved amino acids from WDR19. Western blot analysis demonstrated that the BTA6:58373887 T-allele decreases protein expression. We make the remarkable observation that, in spite of negative effects on semen quality and bull fertility, the BTA6:58373887 T-allele has a frequency of 24% in the Brown Swiss population. Our findings are the first to uncover a variant that is associated with quantitative variation in semen quality and male fertility in cattle.
Project description:Background:Impaired fertility in cattle limits the efficiency of livestock production systems. Unraveling the genetic architecture of fertility traits would facilitate their improvement by selection. In this study, we characterized SNP chip haplotypes at QTL blocks then used whole-genome sequencing to fine map genomic regions associated with reproduction in a population of Nellore (Bos indicus) heifers. Methods:The dataset comprised of 1337 heifers genotyped using a GeneSeek® Genomic Profiler panel (74677 SNPs), representing the daughters from 78 sires. After performing marker quality control, 64800 SNPs were retained. Haplotypes carried by each sire at six previously identified QTL on BTAs 5, 14 and 18 for heifer pregnancy and BTAs 8, 11 and 22 for antral follicle count were constructed using findhap software. The significance of the contrasts between the effects of every two paternally-inherited haplotype alleles were used to identify sires that were heterozygous at each QTL. Whole-genome sequencing data localized to the haplotypes from six sires and 20 other ancestors were used to identify sequence variants that were concordant with the haplotype contrasts. Enrichment analyses were applied to these variants using KEGG and MeSH libraries. Results:A total of six (BTA 5), six (BTA 14) and five (BTA 18) sires were heterozygous for heifer pregnancy QTL whereas six (BTA 8), fourteen (BTA 11), and five (BTA 22) sires were heterozygous for number of antral follicles' QTL. Due to inadequate representation of many haplotype alleles in the sequenced animals, fine mapping analysis could only be reliably performed for the QTL on BTA 5 and 14, which had 641 and 3733 concordant candidate sequence variants, respectively. The KEGG "Circadian rhythm" and "Neurotrophin signaling pathway" were significantly associated with the genes in the QTL on BTA 5 whereas 32 MeSH terms were associated with the QTL on BTA 14. Among the concordant sequence variants, 0.2% and 0.3% were classified as missense variants for BTAs 5 and 14, respectively, highlighting the genes MTERF2, RTMB, ENSBTAG00000037306 (miRNA), ENSBTAG00000040351, PRKDC, and RGS20. The potential causal mutations found in the present study were associated with biological processes such as oocyte maturation, embryo development, placenta development and response to reproductive hormones. Conclusions:The identification of heterozygous sires by positionally phasing SNP chip data and contrasting haplotype effects for previously detected QTL can be used for fine mapping to identify potential causal mutations and candidate genes. Genomic variants on genes MTERF2, RTBC, miRNA ENSBTAG00000037306, ENSBTAG00000040351, PRKDC, and RGS20, which are known to have influence on reproductive biological processes, were detected.
Project description:Tyrolean Grey cattle represent a local breed with a population size of ?5000 registered cows. In 2003, a previously unknown neurological disorder was recognized in Tyrolean Grey cattle. The clinical signs of the disorder are similar to those of bovine progressive degenerative myeloencephalopathy (weaver syndrome) in Brown Swiss cattle but occur much earlier in life. The neuropathological investigation of an affected calf showed axonal degeneration in the central nervous system (CNS) and femoral nerve. The pedigrees of the affected calves suggested a monogenic autosomal recessive inheritance. We localized the responsible mutation to a 1.9 Mb interval on chromosome 16 by genome-wide association and haplotype mapping. The MFN2 gene located in this interval encodes mitofusin 2, a mitochondrial membrane protein. A heritable human axonal neuropathy, Charcot-Marie-Tooth disease-2A2 (CMT2A2), is caused by MFN2 mutations. Therefore, we considered MFN2 a positional and functional candidate gene and performed mutation analysis in affected and control Tyrolean Grey cattle. We did not find any non-synonymous variants. However, we identified a perfectly associated silent SNP in the coding region of exon 20 of the MFN2 gene. This SNP is located within a putative exonic splice enhancer (ESE) and the variant allele leads to partial retention of the entire intron 19 and a premature stop codon in the aberrant MFN2 transcript. Thus we have identified a highly unusual splicing defect, where an exonic single base exchange leads to the retention of the preceding intron. This splicing defect represents a potential explanation for the observed degenerative axonopathy. Marker assisted selection can now be used to eliminate degenerative axonopathy from Tyrolean Grey cattle.
Project description:Subclinical mastitis (SM) is one of the most common diseases of cows in milk production herds caused by contagious and/or environmental pathogens. Since there are no visible abnormalities in the milk or udder, the detection of SM requires special diagnostic tests. Somatic cell count (SCC) is the most common test used to detect changes in milk due to the inflammatory process. Previously, we developed somatic cell count index (SCCI), a new method for the accurate prediction of milk yield losses caused by elevated SCC. The aim of this study was to identify new candidate genetic markers for SCCI in the Slovenian population of Brown Swiss (BS) cattle. For that purpose, we analyzed samples of BS cows, which were genotyped using single-nucleotide polymorphism (SNP) microarray ICBF International Dairy and Beef v3 (ICBF, Ireland) for a total of 53,262 SNP markers. After quality control, the set of 18,136 SNPs was used in association analysis. Our association analysis revealed that 130 SNPs were associated with SCCI, which were used for haplotype and overlap analysis. Haplotypes generated from the genotyped data for those 130 SNPs revealed 10 haplotype blocks among 22 SNPs. Additionally, all 130 SNPs, mastitis-related quantitative trait loci, and protein-coding genes are shown on the bovine genome. Overlap analysis shows that the majority of significantly associated SNPs (70) are intergenic, while 60 SNPs are mapped within, upstream, or downstream of the protein-coding genes. However, those genes can serve as strong candidate genes for the marker-assisted selection programs in our and possibly other populations of cattle.
Project description:<h4>Background</h4>It has been known for almost a century that the belted phenotype in cattle follows a pattern of dominant inheritance. In 2009, the approximate position of the belt locus in Brown Swiss cattle was mapped to a 922-kb interval on bovine chromosome 3 and, subsequently, assigned to a 336-kb haplotype block based on an animal set that included, Brown Swiss, Dutch Belted (Lakenvelder) and Belted Galloway individuals. A possible candidate gene in this region i.e. HES6 was investigated but the causal mutation remains unknown. Thus, to elucidate the causal mutation of this prominent coat color phenotype, we decided to remap the belted phenotype in an independent animal set of several European bovine breeds, i.e. Gurtenvieh (belted Brown Swiss), Dutch Belted and Belted Galloway and to systematically scan the candidate region. We also checked the presence of the detected causal mutation in the genome of belted individuals from a Siberian cattle breed.<h4>Results</h4>A combined linkage disequilibrium and linkage analysis based on 110 belted and non-belted animals identified a candidate interval of 2.5 Mb. Manual inspection of the haplotypes in this region identified four candidate haplotypes that consisted of five to eight consecutive SNPs. One of these haplotypes overlapped with the initial 922-kb interval, whereas two were positioned proximal and one was positioned distal to this region. Next-generation sequencing of one heterozygous and two homozygous belted animals identified only one private belted candidate allele, i.e. a multiplication event that is located between 118,608,000 and 118,614,000 bp. Targeted locus amplification and quantitative real-time PCR confirmed an increase in copy number of this region in the genomes of both European (Belted Galloway, Dutch Belted and Gurtenvieh) and Siberian (Yakutian cattle) breeds. Finally, using nanopore sequencing, the exact breakpoints were determined at 118,608,362 and 118,614,132 bp. The closest gene to the candidate causal mutation (16 kb distal) is TWIST2.<h4>Conclusions</h4>Based on our findings and those of a previously published study that identified the same multiplication event, a quadruplication on bovine chromosome 3 between positions 118,608,362 and 118,614,132 bp is the most likely candidate causal mutation for the belted phenotype in cattle.
Project description:The detection of quantitative trait loci has accelerated with recent developments in genomics. The introduction of genomic selection in combination with sequencing efforts has made a large amount of genotypic data available. Functional traits such as fertility and calving traits have been included in routine genomic estimation of breeding values making large quantities of phenotypic data available for these traits. This data was used to investigate the genetics underlying fertility and calving traits and to identify potentially causative genomic regions and variants. We performed genome-wide association studies for 13 functional traits related to female fertility as well as for direct and maternal calving ease based on imputed whole-genome sequences. Deregressed breeding values from ~1000-5000 bulls per trait were used to test for associations with approximately 10 million imputed sequence SNPs.We identified a QTL on BTA17 associated with non-return rate at 56 days and with interval from first to last insemination. We found two significantly associated non-synonymous SNPs within this QTL region. Two more QTL for fertility traits were identified on BTA25 and 29. A single QTL was identified for maternal calving traits on BTA13 whereas three QTL on BTA19, 21 and 25 were identified for direct calving traits. The QTL on BTA19 co-localizes with the reported BH2 haplotype. The QTL on BTA25 is concordant for fertility and calving traits and co-localizes with a QTL previously reported to influence stature and related traits in Brown Swiss dairy cattle.The detection of QTL and their causative variants remains challenging. Combining comprehensive phenotypic data with imputed whole genome sequences seems promising. We present a QTL on BTA17 for female fertility in dairy cattle with two significantly associated non-synonymous SNPs, along with five additional QTL for fertility traits and calving traits. For all of these we fine mapped the regions and suggest candidate genes and candidate variants.
Project description:BACKGROUND:Red deer with very pale coat colour are observed sporadically. In the red deer (Cervus elaphus) population of Reinhardswald in Germany, about 5% of animals have a white coat colour that is not associated with albinism. In order to facilitate the conservation of the animals, it should be determined whether and to what extent brown animals carry the white gene. For this purpose, samples of one white hind and her brown calf were available for whole genome sequencing to identify the single nucleotide polymorphism(s) responsible for the white phenotype. Subsequently, samples from 194 brown and 11 white animals were genotyped. RESULTS:Based on a list of colour genes of the International Federation of Pigment Cell Societies, a non-synonymous mutation with exchange of a glycine residue at position 291 of the tyrosinase protein by arginine was identified as the cause of dilution of the coat colour. A gene test led to exactly matching genotypes in all examined animals. The study showed that 14% of the brown animals carry the white gene. This provides a simple and reliable way of conservation for the white animals. However, results could not be transferred to another, unrelated red deer population with white animals. Although no brown animals with a white tyrosinase genotype were detected, the cause for the white colouring in this population was different. CONCLUSIONS:A gene test for the conservation of white red deer is available for the population of the Reinhardswald. While mutations in the tyrosinase are commonly associated with oculocutaneous albinism type 1, the amino acid exchange at position 291 was found to be associated with coat colour dilution in Cervus elaphus.