1657: Extreme Phenotype Approach Identifies Rare Variants in Systemic Sclerosis and Dermatomyositis Patients with Severe Calcinosis

Srijana Davuluri¹, Urvashi Kaundal², Christian Lood³, Puneet Kapoor¹, Yumeko Kawano⁴, Stefania Dell'Orso⁵, Zuoming Deng⁶, Zsuzsanna McMahan⁷, Ami Shah⁷, Laura Hummers⁸, Daniel Kastner⁹, Fredrick Wigley¹⁰, David Fiorentino¹¹, Pravitt Gourh¹² and Lorinda Chung¹¹, ¹Stanford University School of Medicine, Palo Alto, CA, ²National Institutes of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH), Bethesda, MD, ³Division of Rheumatology, University of Washington, Seattle, WA, ⁴Brigham and Women's Hospital, Boston, MA, ⁵NIH, Bethesda, MD, ⁶National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, ⁷Johns Hopkins Rheumatology, Baltimore, MD, ⁸Johns Hopkins Univerisity, Baltimore, MD, ⁹National Human Genome Research Institute, Bethesda, MD, ¹⁰Johns Hopkins University, Baltimore, MD, ¹¹Stanford University, Stanford, CA, ¹²National Institutes of Health, Bethesda, MD

Background/Purpose: Calcinosis, deposition of insoluble calcium salts in skin and subcutaneous tissues, affects up to 40% of systemic sclerosis (SSc) patients, and up to 20% of adult dermatomyositis (DM) patients. It contributes to a significant disease burden as patients suffer from pain, ulceration, and secondary infections. The genetics of calcinosis in SSc and DM has not been investigated and there are no effective therapies to treat calcinosis. In a recent report, lower levels of circulating inorganic pyrophosphate (PPi), an inhibitor of mineralization, was observed in SSc patients as compared to healthy controls¹. We hypothesized that rare, protein-coding variants are driving the severe calcinosis phenotype in both SSc and DM patients, and identifying these gene variants would provide us with more insight into the pathogenesis of calcinosis. The goal of this study was to perform genome sequencing of individuals with severe calcinosis, an extreme phenotype, to identify rare variants in genes previously reported in ectopic mineralization pathways as well as novel gene pathways.

Methods: We enrolled 31 SSc and 19 DM patients with severe calcinosis from Stanford and NIH (all met the EULAR/ACR classification criteria) (Table 1). Severe calcinosis was defined as the presence of calcinosis at three or more sites, simultaneously. Genome sequencing was performed using the Illumina platform and variants were called using the standard Genome Analysis Tool Kit pipeline.

Results: Genome sequencing had an average 30X coverage. Using an in-house computational pipeline, we filtered variants with minor allele frequency (MAF) < 0.01 and combined annotation dependent depletion (CADD) score > 15. Thirty-nine rare, coding, singleton variants among 24 genes involved in ectopic calcification pathways were identified (Table 2). Out of these 39 variants, 5 were present in the ABCC6 gene (4 in SSc and 1 in DM) and 2 in the ENPP1 gene.

To identify ultra-rare variants, MAF< 0.0001 and CADD >15 were used which led to the identification of 9 variants: 6 of these were observed in SSc and 3 in DM patients. Two of the variants were seen in GALNT3 and KL genes, and 1 loss of function variant was observed in SAMD9 gene. Lastly, we analyzed genome-wide, ultra-rare variants and identified 54 genes with at least 3 or more variants in each gene.

Conclusion: This is the first study to use an extreme phenotype approach to identify variants in severe calcinosis in SSc and adult DM patients. ABCC6 and ENPP1 are known genes involved in ectopic calcification and have reduced PPi levels (Figure 1). ;GALNT3 and KL genes are implicated in familial tumoral calcinosis and SAMD9 is implicated in normophosphatemic familial tumoral calcinosis. SSc and DM variants in the ABCC6 and ENPP1 genes may contribute to dysregulated phosphate metabolism, reduced PPi and subsequent calcification. Functional studies to elucidate the role of these variants in this pathway are currently underway. Our findings may be of particular importance given that clinical trials exploring PPi supplementation for the treatment of calcinosis are in development.

References-

1) Hsu, Vivien M et al. Rheumatology (Oxford, England), 2022. 61(3):p. 1158-1165.
Table 1- Demographic and Clinical Features of Systemic sclerosis (SSc) and Dermatomyositis (DM) patients with severe calcinosis.
*- Duration from the onset of Raynaud’s symptoms until sample collection.
**- Duration from the onset of disease or first non-Raynaud’s symptoms until sample collection.
***- Anatomic areas based on mRSS.


Table 2- Coding variants in genes involved in calcification pathways in severe calcinosis patients

Figure 1- ABCC6 (member of ATP binding cassette protein coded by ABCC6 gene) is a transmembrane efflux transporter expressed mainly in the liver and mediates the release of ATP into the bloodstream. ENPP1 gene (ectonucleotide pyrophosphatase) encodes a transmembrane glycoprotein that cleaves ATP to give inorganic pyrophosphate (PPi). PPi has inhibitory effects on mineralization.
Disclosures: S. Davuluri, None; U. Kaundal, None; C. Lood, Eli Lilly, Gilead Sciences, Pfizer, Bristol-Myers Squibb(BMS), Redd Pharma, Horizon Diagnostic, Exagen Diagnostic; P. Kapoor, None; Y. Kawano, None; S. Dell'Orso, None; Z. Deng, None; Z. McMahan, None; A. Shah, Arena Pharmaceuticals, Medpace/Eicos, Kadmon Corporation; L. Hummers, Boehringer-Ingelheim, Corbus Pharmaceuticals, Cumberland Pharmaceuticals, Kadmon Corporation, Medpace, CSL Behring, Mitsubishi Tanabe, Horizon Pharmaceuticals; D. Kastner, None; F. Wigley, None; D. Fiorentino, None; P. Gourh, None; L. Chung, Kyverna, Mitsubishi Tanabe, Eicos, Boehringer-Ingelheim, Jasper, Genentech.