DOI: 10.5683/sp3/mztwvp
title: Data from thesis entitled "Investigation of causal mutations in livestock and companion animals using whole-genome sequencing (WGS) and bioinformatics "
title: Final Candidate SNPs and Indels data

description: Thesis Abstract
In this thesis project, three studies were conducted, aimed at identifying the causal variants responsible for phenotypic/genotypic sex discordance in cattle, copper toxicosis in Dalmatian dogs, and low fertility/infertility in cattle. In all three studies, whole-genome sequencing (WGS) and bioinformatics were used to detect candidate causal mutations. 
The first study involved samples flagged during a national genetic testing program in Ireland due to the inconsistencies in genotype-test assigned sex compared to farmer-reported phenotypic sex. In all, cows were initially classified as XY male genotypically. Re-testing confirmed this classification, leading to the hypothesis of Swyer syndrome in these cases. First described in humans (Swyer, 1955), individuals with Swyer syndrome possess XY sex chromosomes but develop female reproductive organs. Study samples were characterized by WGS, and data was analyzed alongside fertile XX and XY control animals. The sex chromosome complement was established using WGS data and confirmed that the genotyped cows were XY. Short-sequence read alignments with the reference genome were examined for known differences in sex development (DSD) genes. In seven out of eight affected individuals, an absence of reads aligning to the SRY gene was observed, leading to a designation of SRY-negative and explaining the cause of the DSD. To identify candidate causal mutations for the one SRY-positive affected individual, two variant calling pipelines for SNPs/indels and structural variants (SVs) along with variant filtering and annotation analysis were used. While no candidate causal variants in known DSD genes were identified, a small number (125) of potential causative mutations elsewhere in the genome were found. Due to the lack of known roles established for the genes affected by these variants in sex determination and development, further refinement of this list is needed to pinpoint a true causative mutation, for example, through the characterization of additional samples identified through ongoing genetic testing.
The second study sought to find the cause of an apparent autosomal recessive condition occurring in Dalmatian dogs and in other breeds, termed canine copper toxicosis (CT) (Coronado et al., 2003; Nakaichi et al., 2022). Characterized by excessive copper in the liver, the disease ultimately causes liver damage and if untreated, potentially death (Scheinberg & Sternlieb, 1965). Dalmatian samples (n = 10) identified through pedigree analysis as presumptive cases, carriers, and controls were characterized using WGS. Three samples from Bedlington Terriers were included, two believed to have CT. SNPs, indels, and SVs were identified in all samples and filtered using various criteria, considering pedigree information, predicted functional impact, and overlap with known CT genes. This approach ruled out involvement of the ATP7B or COMMD1 genes, both associated with CT in humans and dogs (Bull et al., 1993; Coronado et al., 2003; Tanzi et al., 1993; van De Sluis et al., 2002). One of the affected Bedlington carriers was confirmed to be missing the COMMD1 gene. Regarding the disease’s cause in Dalmatians, no strong candidate mutations could be proposed from the identified variants. Nevertheless, after applying additional filtering steps to the genotype scenario output, we identified 7 SNPs and 1 indel with predicted functional impacts on genes, alongside 3 indels overlapping with candidate genes. However, due to the limited knowledge of the gene's role in copper metabolism, refining this list to pinpoint a specific causal mutation remains challenging. 
In the third study, we investigated 20 animals with suboptimal fertility using WGS to identify recessive lethal mutations potentially causing embryo loss. We identified SNPs and indels and applied various strategies to isolate sites with heterozygous genotypes in the sequenced animals, never observed in homozygous form in living cattle. Our approach leveraged WGS results from over 2,703 additional cattle collected through the 1000 Bull Genomes Project. Additional filtering based on predicted functional impact led to the identification of 391 candidate causal SNPs and 3,527 candidate causal indels potentially linked to subfertility/infertility in beef cattle. The large number of variants reflects the challenge of distinguishing rare or false-positive variants from true recessive lethals. Further experiments are required to substantiate the true recessive lethality of any identified candidate mutations. Protocol number for this study: AUP00001140.
As a results of this work, updates have been made to the genotyping chip used in genetic evaluations in Ireland to improve sex assignment. Additionally, a follow-up long-read study has begun, using additional Dalmatian samples to enhance the characterization of genomes in affected, carrier, and control animals.