Whole-genome genotyping and resequencing reveal the association of a deletion in the complex interferon alpha gene cluster with hypothyroidism in dogs

Background: Hypothyroidism is a common complex endocrinopathy that typically has an autoimmune etiology, and it affects both humans and dogs. Genetic and environmental factors are both known to play important roles in the disease development. In this study, we sought to identify the genetic risk factors potentially involved in the susceptibility to the disease in the high-risk Giant Schnauzer dog breed. Results: By employing genome-wide association followed by fine-mapping (top variant p-value=5.7x10(-6)), integrated with whole-genome resequencing and copy number variation analysis, we detected a similar to 8.9 kbp deletion strongly associated (p-value=0.0001) with protection against development of hypothyroidism. The deletion is located between two predicted Interferon alpha (IFNA) genes and it may eliminate functional elements potentially involved in the transcriptional regulation of these genes. Remarkably, type I IFNs have been extensively associated to human autoimmune hypothyroidism and general autoimmunity. Nonetheless, the extreme genomic complexity of the associated region on CFA11 warrants further long-read sequencing and annotation efforts in order to ascribe functions to the identified deletion and to characterize the canine IFNA gene cluster in more detail. Conclusions: Our results expand the current knowledge on genetic determinants of canine hypothyroidism by revealing a significant link with the human counterpart disease, potentially translating into better diagnostic tools across species, and may contribute to improved canine breeding strategies

Multiple Changes of Gene Expression and Function Reveal Genomic and Phenotypic Complexity in SLE-like Disease

The complexity of clinical manifestations commonly observed in autoimmune disorders poses a major challenge to genetic studies of such diseases. Systemic lupus erythematosus (SLE) affects humans as well as other mammals, and is characterized by the presence of antinuclear antibodies (ANA) in patients' sera and multiple disparate clinical features. Here we present evidence that particular sub-phenotypes of canine SLE-related disease, based on homogenous (ANA(H)) and speckled ANA (ANA(S)) staining pattern, and also steroid-responsive meningitis-arteritis (SRMA) are associated with different but overlapping sets of genes. In addition to association to certain MHC alleles and haplotypes, we identified 11 genes (WFDC3, HOMER2, VRK1, PTPN3, WHAMM, BANK1, AP3B2, DAPP1, LAM-TOR3, DDIT4L and PPP3CA) located on five chromosomes that contain multiple risk haplotypes correlated with gene expression and disease sub-phenotypes in an intricate manner. Intriguingly, the association of BANK1 with both human and canine SLE appears to lead to similar changes in gene expression levels in both species. Our results suggest that molecular definition may help unravel the mechanisms of different clinical features common between and specific to various autoimmune disease phenotypes in dogs and humans.Peer reviewe

Multiple Changes of Gene Expression and Function Reveal Genomic and Phenotypic Complexity in SLE-like Disease

The complexity of clinical manifestations commonly observed in autoimmune disorders poses a major challenge to genetic studies of such diseases. Systemic lupus erythematosus (SLE) affects humans as well as other mammals, and is characterized by the presence of antinuclear antibodies (ANA) in patients' sera and multiple disparate clinical features. Here we present evidence that particular sub-phenotypes of canine SLE-related disease, based on homogenous (ANA(H)) and speckled ANA (ANA(S)) staining pattern, and also steroid-responsive meningitis-arteritis (SRMA) are associated with different but overlapping sets of genes. In addition to association to certain MHC alleles and haplotypes, we identified 11 genes (WFDC3, HOMER2, VRK1, PTPN3, WHAMM, BANK1, AP3B2, DAPP1, LAM-TOR3, DDIT4L and PPP3CA) located on five chromosomes that contain multiple risk haplotypes correlated with gene expression and disease sub-phenotypes in an intricate manner. Intriguingly, the association of BANK1 with both human and canine SLE appears to lead to similar changes in gene expression levels in both species. Our results suggest that molecular definition may help unravel the mechanisms of different clinical features common between and specific to various autoimmune disease phenotypes in dogs and humans.Peer reviewe

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

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

Identification of Genomic Regions Associated with Phenotypic Variation between Dog Breeds using Selection Mapping

Peer reviewe

Molecular coat colour genetics

Coat colour, being sufficiently easy to observe and score, is a model phenotype for studying gene action and interaction. Farm animals provide us a valuable resource for identifying genes behind such phenotypic traits, because they show a remarkably higher diversity in coat colour compared to natural populations. This thesis concerns two different farm animal coat colour phenotypes. First, the dominant white phenotype in domestic pigs is studied. It has been previously determined that white coat colour in domestic pigs is caused by two mutations in the KIT gene – a gene duplication and a splice mutation in one of the copies. The genetic analysis of such a locus with many alleles is complicated. Pyrosequencing and minisequencing are the methods applied in present studies for quantitative analysis of the splice mutation and unexpectedly high allelic diversity at the porcine KIT locus was revealed. Furthermore, a method based on pyrosequencing for analysing gene copy numbers, using this locus as a model, was developed. This method is based on a competitive amplification of the breakpoint regions from duplicated and normal copies of KIT and quantification of the products. This strategy, analysing genes with multiple copies, could have a much broader use than just analysing porcine KIT locus. For instance, several human genetic diseases are caused by changes in gene copy number and analysis of these genes has been quite complicated. The second part of the thesis is summarising the greying with age phenotype in horses. These horses are born coloured, turn progressively grey and most of them develop melanoma. The locus responsible for this phenotype has been mapped to horse chromosome 25, homologous to human chromosome 9. Two Swedish Warmblood horse families were used in present studies to perform a comparative linkage mapping of the Grey locus. The identified interval region has no obvious candidate genes affecting coat colour or melanoma development. Since this mutation occurred most probably only once, thousands of years ago, the identification of a minimum shared haplotype among individuals from different breeds should reveal the gene and the mutation behind this fascinating phenotype

A Sensitive Method for Detecting Variation in Copy Numbers of Duplicated Genes

Gene duplications are common in the vertebrate genome, and duplicated loci often show a variation in copy number that may have important phenotypic effects. Here we describe a powerful method for quantification of duplicated copies based on pyrosequencing. A reliable quantification was obtained by amplification of the duplication break-point and a corresponding nonduplicated sequence in a competitive PCR assay. A comparison with an independent method for quantification based on the Invader technology revealed an excellent correlation between the two methods. The pyrosequencing-based method was evaluated by analyzing variation in copy number at the duplicated KIT/Dominant white locus in pigs. We were able to distinguish haplotypes at this locus by combining the duplication breakpoint test with a diagnostic test for a functionally important splice mutation in the duplicated gene. An extensive allelic variation, including the presence of a new allele carrying a single KIT copy expected to encode a truncated KIT receptor, was revealed when analyzing white pigs from commercial lines