Locating a novel autosomal recessive genetic condition using only WGS data from three cases and six controls; a case study of a new variant in the cattle glucokinase gene

New Mendelian genetic conditions, which adversely affect livestock, arise all the time. To manage them effectively, some methods need to be devised that are quick and accurate. Until recently, finding the causal genomic site of a new autosomal recessive genetic disease has required a two-stage approach using single-nucleotide polymorphism (SNP) chip genotyping to locate the region containing the new variant. This region is then explored using fine-mapping methods to locate the actual site of the new variant. This study explores bioinformatic methods that can be used to identify the causative variants of recessive genetic disorders with full penetrance with just nine whole genome-sequenced animals to simplify and expedite the process to a one-step procedure. Using whole genome sequencing of only three cases and six carriers, the site of a novel variant causing perinatal mortality in Irish moiled calves was located. Four methods were used to interrogate the variant call format (VCF) data file of these nine animals, they are genotype criteria (GCR), autozygosity-by-difference (ABD), variant prediction scoring, and registered SNP information. From more than nine million variants in the VCF file, only one site was identified by all four methods (Chr4: g.77173487A>T (ARS-UCD1.2 (GCF_002263795.1)). This site was a splice acceptor variant located in the glucokinase gene (GCK). It was verified on an independent sample of animals from the breed using genotyping by polymerase chain reaction at the candidate site and autozygosity-by-difference using SNP-chips. Both methods confirmed the candidate site. Investigation of the GCR method found that sites meeting the GCR were not evenly spread across the genome but concentrated in regions of long runs of homozygosity. Locating GCR sites was best performed using two carriers to every case, and the carriers should be distantly related to the cases, within the breed concerned. Fewer than 20 animals need to be sequenced when using the GCR and ABD methods together. The genomic site of novel autosomal recessive Mendelian genetic diseases can be located using fewer than 20 animals combined with two bioinformatic methods, autozygosity-by-difference, and genotype criteria. In many instances it may also be confirmed with variant prediction scoring. This should speed-up and simplify the management of new genetic diseases to a single-step process

A Nonsynonymous Change in Adhesion G Protein–Coupled Receptor L3 Associated With Risk for Equine Degenerative Myeloencephalopathy in the Caspian Horse

Equine degenerative myeloencephalopathy (EDM), a neurological disease of young horses, causes progressive development of symmetric ataxia predominantly in the pelvic limbs. Equine degenerative myeloencephalopathy is likely inherited and with no known treatment affected horses frequently need euthanasia. Alpha-tocopherol deficiency during early life appears to contribute to the phenotype. This study sought to identify any genetic variants correlated with EDM in Caspian foals. Two half-sibling EDM-diagnosed cases were genotyped at 52,063 loci and evaluated by the Autozygosity by Difference statistic. Additional horses not affected by EDM were used for genetic comparison to identify regions unique to the case phenotype. The associated region on chromosome 3 contains only one gene encoding adhesion G protein–coupled receptor L3 (ADGRL3). Adhesion G protein–coupled receptor L3 is a member of the latrophilin subfamily of G protein–coupled receptors and may contribute to attention deficit/hyperactivity disorder in humans and hyperactive motor function in mice and zebrafish. Analysis of the predicted coding regions for Equine ADGRL3 in affected horses revealed a nonsynonymous single nucleotide polymorphism at Chr3:71,917,591 bp. Caspian and Caspian cross-relatives (n = 81) of the two initial cases and unrelated horses from similar breeds (n = 130, including Arabians, American Miniatures, and Shetlands) possessed this allele at 5% frequency, with no homozygotes observed within the non-Caspian breeds. This study suggests that a polymorphism in ADGRL3 could contribute to a genetic predisposition to Caspian horse EDM

Blunted cardiomyocyte remodeling response in exercise-resistant rats

Increasing a subject’s aerobic exercise capacity with training decreases cardiovascular morbidity and mortality. Of major concern is the key observation that up to 20% of subjects demonstrate little or no change in maximal oxygen consumption (VO2max) with exercise training (1) and can be considered exercise resistant. Our goal with the current research was to test the hypothesis that variation in training response is associated with cardiomyocyte functional response to training

Polymorphisms in the selectin gene cluster are associated with fertility and survival time in a population of Holstein Friesian cows

Selectins are adhesion molecules, which mediate attachment between leucocytes and endothelium. They aid extravasation of leucocytes from blood into inflamed tissue during the mammary gland’s response to infection. Selectins are also involved in attachment of the conceptus to the endometrium and subsequent placental development. Poor fertility and udder health are major causes for culling dairy cows. The three identified bovine selectin genes SELP, SELL and SELE are located in a gene cluster. SELP is the most polymorphic of these genes. Several SNP in SELP and SELE are associated with human vascular disease, while SELP SNP rs6127 has been associated with recurrent pregnancy loss in women. This study describes the results of a gene association study for SNP in SELP (n = 5), SELL (n = 2) and SELE (n = 1) with fertility, milk production and longevity traits in a population of 337 Holstein Friesian dairy cows. Blood samples for PCR-RFLP were collected at 6 months of age and animals were monitored until either culling or 2,340 days from birth. Three SNP in SELPEx4-6 formed a haplotype block containing a Glu/Ala substitution at rs42312260. This region was associated with poor fertility and reduced survival times. SELPEx8 (rs378218397) coded for a Val475Met variant locus in the linking region between consensus repeats 4 and 5, which may influence glycosylation. The synonymous SNP rs110045112 in SELEEx14 deviated from Hardy Weinberg equilibrium. For both this SNP and rs378218397 there were too few AA homozygotes present in the population and AG heterozygotes had significantly worse fertility than GG homozygotes. Small changes in milk production associated with some SNP could not account for the reduced fertility and only SELPEx6 showed any association with somatic cell count. These results suggest that polymorphisms in SELP and SELE are associated with the likelihood of successful pregnancy, potentially through compromised implantation and placental development

Bovine P-selectin mediates leukocyte adhesion and is highly polymorphic in dairy breeds

Bovine P-selectin (SELP) mediates leukocyte rolling and primes leukocyte adhesion to endothelium, both essential for leukocyte recruitment to an infection site. We investigated SELP-mediated adhesion between bovine peripheral blood leukocytes (PBLs) and cultured bovine aortic endothelial cells pre-activated with lipopolysaccharide (LPS). We examined gene polymorphism for bovine selectins SELP, l-selectin (SELL) and E-selectin (SELE) and compared their SNP frequency between five dairy breeds (Holstein, Friesian, Jersey, Ayrshire and Brown Swiss). LPS treatment caused a rapid (10 min) and slower (4 h) enhancement of PBL adhesion (P < 0.01). Antibody blocking of SELP inhibited LPS induced cell adhesion. SELP was highly polymorphic, with 9 of the 13 SNPs in its exons, whereas only three synonymous SNPs in SELL and one in SELE. The resulting amino acid changes for the three missense SELP SNP were located in the lectin domain and in two consensus repeat (CR) regions, CR2 and CR5. The Val475Met variant locus in the CR4 and CR5 linking region was very close to a predicted N-acetyl-d-glucosamine glycosylation site, which is likely to influence SELP function. The AA genotype was under-represented, only being found in 1% of 373 heifers genotyped from the 5 breeds (P = 0.056), suggesting that AA homozygous animals carrying the Val475Met substitution for SELP may have compromised development. Our study thus confirmed that SELP mediates the attachment of PBL to endothelium and provides novel evidence that its high polymorphism is likely to affect biological function. This may potentially influence leukocyte migration and fertility, both key to successful performance in dairy cows

Kaplan—Meier analysis showing the proportion of animals from surviving from birth through to 2,340 d.

<p>A population of 336 Holstein Friesian cows were genotyped for the SELP_Ex4 SNP rs110033243. Survival time was significantly lower for animals with the TT genotype (P < 0.05).</p

Basic statistics of the traits analysed in this study.

<p>Basic statistics of the traits analysed in this study.</p

Associations between polymorphisms in selectin genes with fertility traits in a population of Holstein cows.

<p>Associations between polymorphisms in selectin genes with fertility traits in a population of Holstein cows.</p