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Principal Investigator: Pravitt Gourh
Organization: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
Fiscal Year: 2024
Award: $955,026
Funding agency: National Institute of Arthritis and Musculoskeletal and Skin Diseases
We have established a large multi-center collaboration designated as GRASP (Genome Research in African American Scleroderma Patients), comprising 25 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 from 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. 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. 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. After stratifying the SSc samples by autoantibodies, very strong and specific HLA allele associations were identified. 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 will use the meta-SKAT test to perform a gene-level 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 the 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,
i. Vasculopathy is central to the pathogenesis of SSc and using a gene-based testing approach, we have identified variants in the NOTCH4 gene increasing SSc susceptibility. These variants were enriched in the vascular subsets of SSc and increased NOTCH4 expression and signaling. This leads to endothelial to mesenchymal transition and dysregulated angiogenesis. Currently, we are testing an FDA-approved NOTCH inhibitor drug, Nirogacestat, in a Notch4 overexpression mouse model.
ii. Admixture mapping can identify disease-associated variants that differ substantially in frequency between ancestral populations. We hypothesize that African American SSc patients will be enriched for African ancestry variants and carry a higher proportion of risk alleles at loci associated with SSc. We have performed admixture mapping analysis to identify genes contained within the admixed regions followed by gene-based testing for enrichment of missense or loss of function (LoF) variants in these genes. Admixture mapping of SSc identified one genome-wide significant and thirteen suggestive regions. Stratified analysis on SSc autoantibody subsets of SSc identified one genome-wide significant and several suggestive regions. Gene-based testing within these admixed regions identified enrichment of rare variants in twenty-seven genes in overall SSc and one, six, and three genes in the AFA, ATA, and ACA subsets, respectively. Four genes (IL13, SCL29A2, OR10C1, and GDF9) were significant in both overall SSc and an autoantibody subset.
iii. Given the importance of class II HLA alleles in increasing SSc risk, especially in the autoantibody subsets of SSs, we wanted to explore the role of HLA alleles in the clinical subsets of SSc. For the first time, a protective effect of HLA-DRB4*01:01 was identified in both the limited cutaneous SSc and the diffuse cutaneous SSc subsets. While certain class II HLA alleles and amino acids increase risk for multiple clinical phenotypes, others can be used, in conjunction with autoantibody status, to predict the development of a particular clinical complication.
iv. HLA genes play a pivotal role in SSc pathogenesis and the TCR repertoire is HLA-restricted, antigen-specific, and recognizes antigens presented by HLA proteins. We hypothesize that the TCR sequences favored by the SSc-associated HLA alleles are involved in autoantigen recognition and autoimmunity. We have performed TCR sequencing in SSc patients and controls and plan to examine the specific sequences and amino acid composition of the TCRs. We have identified unique TCR clones forming a motif in groups of SSc patients. Additional TCR sequence analysis identified five clones that were present in 28% of samples and were also present in the SSc skin. Remarkably, these clones had homology to clones seen in SARS-CoV-2 suggesting a potential role for molecular mimicry.
v. Interleukin-1 receptor accessory protein (IL1RAP) is a co-receptor for the Interleukin-1 (IL-1) family of cytokines. It plays an essential role in the signaling of IL-1, IL-33, and IL-36, which are known to mediate inflammation and fibrosis. In this study, we examined the association of rare variants in the IL-1 family of ligands and receptors with systemic sclerosis. All three gene-based tests identified the IL1RAP gene to be enriched for rare variants in African Americans with SSc in the discovery and combined cohorts. Diffuse skin involvement was present in 15 out of 17 patients (88.2%) with IL1RAP rare variants, whereas only 58.4% of the overall cohort had diffuse skin involvement. The IL1RAP association was even stronger in the diffuse skin subset, whereas no association was seen in the limited skin subset. This study, for the first time, identifies association of IL1RAP with SSc.
vi. Our major findings from the SSc GWAS were the variants identified in the HLA and IFT43 gene regions. Functional work to understand the role of the variants in transcriptional regulation of genes is ongoing.
vii. Calcinosis, deposition of insoluble calcium deposits in the dermis and subcutaneous tissues, contributes to morbidity in SSc patients, leading to a decreased quality of life. We have identified rare variants in pathways affecting calcinosis in SSc patients with severe calcinosis. Besides finding variants in genes known to cause Mendelian forms of calcinosis disorders, we have also identified variants in genes leading to dysregulated phosphate metabolism. We also have identified, for the first time, HLA-DRB4 as a novel susceptibility gene in patients with SSc and severe calcinosis. Further analysis of the remaining samples is ongoing.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Affect><African><African American><African American group><African American individual><African American people><African American population><African Americans><African ancestry><African descent><Afro American><Afroamerican><Alleles><Allelomorphs><American><Amino Acids><Anti-Smith><Antigen Presentation><Antigens><Antinuclear Antibodies><Antinuclear Factors><Autoantibodies><Autoantigens><Autoimmune Status><Autoimmunity><Autologous Antigens><Biological Markers><Blood Vessels><COVID-19 virus><COVID19 virus><Calcinosis><Calcium><Cell Communication and Signaling><Cell Signaling><Chronic><Clinical><CoV-2><CoV2><Code><Coding System><Collaborations><Collection><Complication><Corium><Cutaneous><Cutis><Cytokine Signal Transduction><Cytokine Signaling><DNA><Data><Deoxyribonucleic Acid><Deposit><Deposition><Dermis><Development><Dideoxy Chain Termination DNA Sequencing><Diffuse><Disease><Disorder><Drosophila Homolog of NOTCH 4><Endothelium><Enrollment><European><Exons><FDA approved><Family><Fibrosis><Frequencies><GDF-9><GDF9><GDF9 gene><GWA study><GWAS><Gene Action Regulation><Gene Expression Regulation><Gene Proteins><Gene Regulation><Gene Regulation Process><Genes><Genetic><Genetic predisposing factor><Genetic study><Genome><Genotype><Goals><Growth Differentiation Factor 9 Gene><HL-A Antigens><HLA Antigens><HLA-DP1B><HLA-DPB1><HLA-DPB1 gene><HLADPB1><High Prevalence><Human Leukocyte Antigens><Hypodermis><IL-1><IL-1 Receptors><IL-13><IL-1RAcP><IL1><IL1 Receptors><IL13><INT3><Inflammation><Inflammatory><Interleukin I><Interleukin-1><Interleukin-1 Receptors><Interleukin-13><Intermediary Metabolism><Intracellular Communication and Signaling><Leukocyte Antigens><Ligands><Lung><Lung Respiratory System><Lymphocyte-Stimulating Hormone><Macrophage Cell Factor><Mediating><Mesenchymal><Metabolic Processes><Metabolism><Micro RNA><MicroRNAs><Molecular><Molecular Mimicry><Morbidity><Morbidity - 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