Genetics and Pathogenesis of Scleroderma

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

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Abstract

We have established a large multi-center collaboration designated as GRASP (Genome Research in African American Scleroderma Patients), comprising 24 centers outside of the NIH and have enrolled the largest cohort of African American scleroderma (systemic sclerosis, SSc) patients. Currently, we have collected DNA samples from 1300 African American SSc patients and have received DNA samples of 1200 controls from Charles Rotimi at NHGRI. 1039 sera samples from controls have been tested for anti-nuclear antibody (ANA) and ANA negative control DNA samples have been used for further genetic studies. 1008 SSc patients and 1008 controls have been genotyped on the Illumina Multi-Ethnic Global Array (MEGA) that contains 1.7 million markers and a second custom Illumina OmniExpressExome array that contains 1 million markers to conduct genome wide association study (GWAS). We will genotype an additional 432 samples (both patients and controls) on the MEGA array for replication and joint analysis. Whole exome sequencing (WES) has been performed on 400 SSc patients and 482 controls. Based on our preliminary analysis, 392 genes from WES and 44 genes from GWAS have undergone targeted resequencing in another 600 SSc patients and 360 controls. After quality control filtering on the Illumina MEGA array data, 934 patients and 946 controls remained and were included in the analysis. High quality variants from the MEGA array were imputed into the 1000 Genomes Phase 3 v5 reference panel. GWAS of the MEGA array identified class II Human leukocyte antigen (HLA) genes as the strongest risk factor in SSc susceptibility in the African American population. We are analyzing the HLA region using two different approaches: classical HLA allele association and single nucleotide polymorphism (SNP) association. On imputing classical HLA types, the most significantly SSc-associated HLA type was a predominantly African allele, HLA-DRB1*08:04, with odds ratio 2.95, 95%CI 2.26-3.85. Regression analysis conditioning on the disease-associated alleles identified another African DRB1 allele, *11:02, as well as HLA-DPB1*13:01, and HLA-DRB4*01:01 as independent contributors to disease risk. Compared to 16.3% of controls, 34.6% of African American patients carry the African ancestry HLA alleles DRB1*08:04 or *11:02. After stratifying the SSc samples by autoantibodies, very strong and specific HLA allele associations were identified with HLA-DRB1*0804, increasing risk by 7.2-fold in an anti-fibrillarin antibody subset of African American SSc and HLA-DPB1*1301, increasing risk by 4.1-fold in an anti-topoisomerase I antibody subset of African American SSc. HLA-DRB1*0804 is associated with a specific amino acid change in the peptide binding groove of the DRB1 molecule at position 74 and this change could lead to recognition of a specific self-antigen (i.e. fibrillarin). HLA-DPB1*1301 is associated with a specific amino acid change in the peptide binding groove of the DPB1 molecule at position 76 and this change could lead to recognition of a specific self-antigen (i.e. topoisomerase I). The top SNP in the GWAS was rs35915063 near HLA-DQB1 gene with a P=2.2x10-17 and OR=1.96 (95%CI 1.7-2.3). Two non-HLA, African ancestry specific loci IFT43/TGFB3 and FSD2/HOMER2 were also identified. We have performed WES on 400 patients and 482 controls using a Nimblegen capture kit that targets 64 Mb of coding exons and miRNA regions, plus 32 Mb of untranslated regions. We divided the exomic variants into three categories based on frequency and deleteriousness. Next, we performed gene-level tests for association in the discovery series using Combined Multivariate and Collapsing (CMC), C-alpha, sequence kernel association test-optimized (SKAT-O), and kernel-based adaptive cluster (KBAC) tests for all 3 category variants. Our preliminary analysis of the WES data for overall SSc using the 3 categories of variants identified 392 genes at p-value 0.005 by SKAT-O. Using a Nimblegen capture-based target enrichment kit, 436 genes selected from WES and GWAS have been sequenced in 600 SSc patients and 360 controls. We will use the meta-SKAT test to perform a gene-level meta-analysis. This approach will be taken for overall SSc as well as clinical and autoantibody subsets of SSc. Samples with genes identified to be enriched in rare and low frequency variants will be confirmed by Sanger sequencing. We will also search for presence of rare, homozygous coding variants in SSc patients and analyze them for aggregation in a gene or pathway. Each of these genes enriched in rare and low frequency variants will have their own unique story and may involve fibrosis, cytokine signaling, inflammatory pathways or epithelial to mesenchymal transition. These genes could also be part of a common pathway and studying the dysregulation of that specific pathway may yield greater insight into SSc pathogenesis. During the current reporting period we focused on, 1) the functional characterization of the IFT43 intronic variants identified from the GWAS. To generate fibroblasts with the prioritized IFT43 variants, we used lymphoblastoid cell lines (LCLs) from the 1000 Genome Project that were homozygous and wildtype (WT) for IFT43 variants, reprogrammed them into induced pluripotent stem cells (iPSC), and then differentiated them into fibroblasts. RNA-seq analysis revealed higher expression of TGFB3 in LCL-iPSC-derived fibroblasts homozygous for the variants. ATAC-seq analysis revealed that the homozygous sample had accessibility at the site of the top variant, which was intriguingly inaccessible in WT samples, meaning the variant creates a site that may interact with transcription factors (TF) to regulate the gene expression of neighboring genes. Using a TF binding database, we have identified GATA2 as the TF that binds only when the alternate allele is present. TGFB3 stimulation of normal human dermal fibroblasts resulted in increased expression of previously reported pro-fibrotic genes including SERPINE1, COL1A1, and COMP. TGFB3 is upregulated in SSc skin and could be a potential therapeutic target. 2) Identification and characterization of functional variants in the NOTCH4 gene. Functional variants in the NOTCH4 gene are associated with African American SSc patients and are independent of the HLA genes. Increased Notch signaling in endothelial cells can induce endothelial-to-mesenchymal transition and increase ACTA2 expression as well as neoangiogenic circulation with sparse branching and dilated capillaries. SSc associated variants are linked to increased NOTCH4 expression and constitutively increased NOTCH4 signaling. 3) T cell receptor (TCR) repertoire in African American SSc patients. We explored the diversity of the hypervariable complementarity determining region 3 (CDR3) and TCR repertoire in SSc patients. We also examined the role of HLA-II alleles, especially HLA-DPB1*13:01, in the selection of specific sequence and amino acid composition of the TCR in SSc patients. We identified differential V, J genes and FG-loop amino acid usages that correlated with HLA-II allele presence. Overall amino acid usage of the FG loop (antigen binding region) of the TCR identified increased usage of the hydrophobic amino acids leucine and valine, and reduced usage of negatively charged glutamine in SSc patients as compared to controls. These results emphasize the important role HLA-II alleles and T cells play in SSc pathogenesis.