The HLA region in ANCA-associated vasculitis: characterisation of genetic associations in a Scandinavian patient population.

OBJECTIVE: The antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are inflammatory disorders with ANCA autoantibodies recognising either proteinase 3 (PR3-AAV) or myeloperoxidase (MPO-AAV). PR3-AAV and MPO-AAV have been associated with distinct loci in the human leucocyte antigen (HLA) region. While the association between MPO-AAV and HLA has been well characterised in East Asian populations where MPO-AAV is more common, studies in populations of European descent are limited. The aim of this study was to thoroughly characterise associations to the HLA region in Scandinavian patients with PR3-AAV as well as MPO-AAV. METHODS: Genotypes of single-nucleotide polymorphisms (SNPs) located in the HLA region were extracted from a targeted exome-sequencing dataset comprising Scandinavian AAV cases and controls. Classical HLA alleles were called using xHLA. After quality control, association analyses were performed of a joint SNP/classical HLA allele dataset for cases with PR3-AAV (n=411) and MPO-AAV (n=162) versus controls (n=1595). Disease-associated genetic variants were analysed for association with organ involvement, age at diagnosis and relapse, respectively. RESULTS: PR3-AAV was significantly associated with both HLA-DPB1*04:01 and rs1042335 at the HLA-DPB1 locus, also after stepwise conditional analysis. MPO-AAV was significantly associated with HLA-DRB1*04:04. Neither carriage of HLA-DPB1*04:01 alleles in PR3-AAV nor of HLA-DRB1*04:04 alleles in MPO-AAV were associated with organ involvement, age at diagnosis or relapse. CONCLUSIONS: The association to the HLA region was distinct in Scandinavian cases with MPO-AAV compared with cases of East Asian descent. In PR3-AAV, the two separate signals of association to the HLD-DPB1 region mediate potentially different functional effects.

Leveraging base-pair mammalian constraint to understand genetic variation and human disease.

Thousands of genomic regions have been associated with heritable human diseases, but attempts to elucidate biological mechanisms are impeded by an inability to discern which genomic positions are functionally important. Evolutionary constraint is a powerful predictor of function, agnostic to cell type or disease mechanism. Single-base phyloP scores from 240 mammals identified 3.3% of the human genome as significantly constrained and likely functional. We compared phyloP scores to genome annotation, association studies, copy-number variation, clinical genetics findings, and cancer data. Constrained positions are enriched for variants that explain common disease heritability more than other functional annotations. Our results improve variant annotation but also highlight that the regulatory landscape of the human genome still needs to be further explored and linked to disease.

Leveraging Base Pair Mammalian Constraint to Understand Genetic Variation and Human Disease.

Although thousands of genomic regions have been associated with heritable human diseases, attempts to elucidate biological mechanisms are impeded by a general inability to discern which genomic positions are functionally important. Evolutionary constraint is a powerful predictor of function that is agnostic to cell type or disease mechanism. Here, single base phyloP scores from the whole genome alignment of 240 placental mammals identified 3.5% of the human genome as significantly constrained, and likely functional. We compared these scores to large-scale genome annotation, genome-wide association studies (GWAS), copy number variation, clinical genetics findings, and cancer data sets. Evolutionarily constrained positions are enriched for variants explaining common disease heritability (more than any other functional annotation). Our results improve variant annotation but also highlight that the regulatory landscape of the human genome still needs to be further explored and linked to disease.

Identification and functional characterization of a novel susceptibility locus for small vessel vasculitis with MPO-ANCA.

OBJECTIVE: To identify and characterize genetic loci associated with the risk of developing ANCA-associated vasculitides (AAV). METHODS: Genetic association analyses were performed after Illumina sequencing of 1853 genes and subsequent replication with genotyping of selected single nucleotide polymorphisms in a total cohort of 1110 Scandinavian cases with granulomatosis with polyangiitis or microscopic polyangiitis, and 1589 controls. A novel AAV-associated single nucleotide polymorphism was analysed for allele-specific effects on gene expression using luciferase reporter assay. RESULTS: PR3-ANCA+ AAV was significantly associated with two independent loci in the HLA-DPB1/HLA-DPA1 region [rs1042335, P = 6.3 × 10-61, odds ratio (OR) 0.10; rs9277341, P = 1.5 × 10-44, OR 0.22] and with rs28929474 in the SERPINA1 gene (P = 2.7 × 10-10, OR 2.9). MPO-ANCA+ AAV was significantly associated with the HLA-DQB1/HLA-DQA2 locus (rs9274619, P = 5.4 × 10-25, OR 3.7) and with a rare variant in the BACH2 gene (rs78275221, P = 7.9 × 10-7, OR 3.0), the latter a novel susceptibility locus for MPO-ANCA+ granulomatosis with polyangiitis/microscopic polyangiitis. The rs78275221-A risk allele reduced luciferase gene expression in endothelial cells, specifically, as compared with the non-risk allele. CONCLUSION: We identified a novel susceptibility locus for MPO-ANCA+ AAV and propose that the associated variant is of mechanistic importance, exerting a regulatory function on gene expression in specific cell types.

A novel canine reference genome resolves genomic architecture and uncovers transcript complexity.

We present GSD_1.0, a high-quality domestic dog reference genome with chromosome length scaffolds and contiguity increased 55-fold over CanFam3.1. Annotation with generated and existing long and short read RNA-seq, miRNA-seq and ATAC-seq, revealed that 32.1% of lifted over CanFam3.1 gaps harboured previously hidden functional elements, including promoters, genes and miRNAs in GSD_1.0. A catalogue of canine "dark" regions was made to facilitate mapping rescue. Alignment in these regions is difficult, but we demonstrate that they harbour trait-associated variation. Key genomic regions were completed, including the Dog Leucocyte Antigen (DLA), T Cell Receptor (TCR) and 366 COSMIC cancer genes. 10x linked-read sequencing of 27 dogs (19 breeds) uncovered 22.1 million SNPs, indels and larger structural variants. Subsequent intersection with protein coding genes showed that 1.4% of these could directly influence gene products, and so provide a source of normal or aberrant phenotypic modifications.

Targeted sequencing reveals the somatic mutation landscape in a Swedish breast cancer cohort.

Breast cancer (BC) is a genetically heterogeneous disease with high prevalence in Northern Europe. However, there has been no detailed investigation into the Scandinavian somatic landscape. Here, in a homogeneous Swedish cohort, we describe the somatic events underlying BC, leveraging a targeted next-generation sequencing approach. We designed a 20.5 Mb array targeting coding and regulatory regions of genes with a known role in BC (n = 765). The selected genes were either from human BC studies (n = 294) or from within canine mammary tumor associated regions (n = 471). A set of predominantly estrogen receptor positive tumors (ER + 85%) and their normal tissue counterparts (n = 61) were sequenced to ~ 140 × and 85 × mean target coverage, respectively. MuTect2 and VarScan2 were employed to detect single nucleotide variants (SNVs) and copy number aberrations (CNAs), while MutSigCV (SNVs) and GISTIC (CNAs) algorithms estimated the significance of recurrent somatic events. The significantly mutated genes (q ≤ 0.01) were PIK3CA (28% of patients), TP53 (21%) and CDH1 (11%). However, histone modifying genes contained the largest number of variants (KMT2C and ARID1A, together 28%). Mutations in KMT2C were mutually exclusive with PI3KCA mutations (p ≤ 0. 001) and half of these affect the formation of a functional PHD domain. The tumor suppressor CDK10 was deleted in 80% of the cohort while the oncogene MDM4 was amplified. Mutational signature analyses pointed towards APOBEC deaminase activity (COSMIC signature 2) and DNA mismatch repair (COSMIC signature 6). We noticed two significantly distinct patterns related to patient age; TP53 being more mutated in the younger group (29% vs 9% of patients) and CDH23 mutations were absent from the older group. The increased somatic mutation prevalence in the histone modifying genes KMT2C and ARID1A distinguishes the Swedish cohort from previous studies. KMT2C regulates enhancer activation and assists tumor proliferation in a hormone-rich environment, possibly pointing to a role in ER + BC, especially in older cases. Finally, age of onset appears to affect the mutational landscape suggesting that a larger age-diverse population incorporating more molecular subtypes should be studied to elucidate the underlying mechanisms.

SETD2 Is Recurrently Mutated in Whole-Exome Sequenced Canine Osteosarcoma.

Osteosarcoma is a debilitating bone cancer that affects humans, especially children and adolescents. A homologous form of osteosarcoma spontaneously occurs in dogs, and its differential incidence observed across breeds allows for the investigation of tumor mutations in the context of multiple genetic backgrounds. Using whole-exome sequencing and dogs from three susceptible breeds (22 golden retrievers, 21 Rottweilers, and 23 greyhounds), we found that osteosarcoma tumors show a high frequency of somatic copy-number alterations (SCNA), affecting key oncogenes and tumor-suppressor genes. The across-breed results are similar to what has been observed for human osteosarcoma, but the disease frequency and somatic mutation counts vary in the three breeds. For all breeds, three mutational signatures (one of which has not been previously reported) and 11 significantly mutated genes were identified. TP53 was the most frequently altered gene (83% of dogs have either mutations or SCNA in TP53), recapitulating observations in human osteosarcoma. The second most frequently mutated gene, histone methyltransferase SETD2, has known roles in multiple cancers, but has not previously been strongly implicated in osteosarcoma. This study points to the likely importance of histone modifications in osteosarcoma and highlights the strong genetic similarities between human and dog osteosarcoma, suggesting that canine osteosarcoma may serve as an excellent model for developing treatment strategies in both species.Significance: Canine osteosarcoma genomics identify SETD2 as a possible oncogenic driver of osteosarcoma, and findings establish the canine model as a useful comparative model for the corresponding human disease. Cancer Res; 78(13); 3421-31. ©2018 AACR.

Thorough investigation of a canine autoinflammatory disease (AID) confirms one main risk locus and suggests a modifier locus for amyloidosis.

Autoinflammatory disease (AID) manifests from the dysregulation of the innate immune system and is characterised by systemic and persistent inflammation. Clinical heterogeneity leads to patients presenting with one or a spectrum of phenotypic signs, leading to difficult diagnoses in the absence of a clear genetic cause. We used separate genome-wide SNP analyses to investigate five signs of AID (recurrent fever, arthritis, breed specific secondary dermatitis, otitis and systemic reactive amyloidosis) in a canine comparative model, the pure bred Chinese Shar-Pei. Analysis of 255 DNA samples revealed a shared locus on chromosome 13 spanning two peaks of association. A three-marker haplotype based on the most significant SNP (p<2.6×10(-8)) from each analysis showed that one haplotypic pair (H13-11) was present in the majority of AID individuals, implicating this as a shared risk factor for all phenotypes. We also noted that a genetic signature (F ST) distinguishing the phenotypic extremes of the breed specific Chinese Shar-Pei thick and wrinkled skin, flanked the chromosome 13 AID locus; suggesting that breed development and differentiation has played a parallel role in the genetics of breed fitness. Intriguingly, a potential modifier locus for amyloidosis was revealed on chromosome 14, and an investigation of candidate genes from both this and the chromosome 13 regions revealed significant (p<0.05) renal differential expression in four genes previously implicated in kidney or immune health (AOAH, ELMO1, HAS2 and IL6). These results illustrate that phenotypic heterogeneity need not be a reflection of genetic heterogeneity, and that genetic modifiers of disease could be masked if syndromes were not first considered as individual clinical signs and then as a sum of their component parts.