Background: Systemic sclerosis (SSc) is a rare (prevalence ±20/100,000), chronic condition characterized by vascular, immunological and connective tissue abnormalities. Genetic factors contribute to disease, as evidenced by a 15-fold increased relative risk in relatives of patients. Case-control studies have identified numerous associated loci; the identification and functional validation of risk alleles has however been elusive. No monogenic or oligogenic causes of SSc have been demonstrated, likely due to the infrequency of familial clustering (≤1.6% of cases) in this disease. The identification of disease-causative or strongly predisposing genes may reveal central pathogenic mechanisms in SSc.

Objectives: The goal of our research is to identify and functionally validate genes that drive SSc pathogenesis in rare families, thereby shedding light on fundamental disease mechanisms.

Methods: Whole exome sequencing (WES) was performed on blood-DNA from twelve affected individuals, from six families with SSc. We filtered for variants that are (a) shared by both affected individuals within each family, (b) absent-to-rare in the general population, and (c) predicted to affect protein function by multiple in silico tools. We prioritized candidate genes shared by more than one family. The effects of the variants identified were tested in isogenic, CRISPR/Cas9-HDR engineered heterozygous and homozygous “knock-in” cell-lines with endogenous expression of the gene-of-interest.

Results: WES yielded 31-47 genes per family with variants that satisfied the filtering criteria described. Of these, only three genes were common to more than one family: TTN , COL12A1 and PRKD2. Mutations in TTN and COL12A1 cause autosomal dominant and recessive cardiac and/or skeletal muscle myopathies. We prioritized PRKD2 (encoding Protein Kinase D2, and intracellular serine threonine kinase), based on its expression pattern and described functions. PRKD2 participates in T cell receptor (TCR) signaling, thymic selection of T cells, and T-dependent B cell responses and autoantibody production. Loss of PRKD2 has also been reported to decrease bleomycin-induced fibrosis in mice. Data from heterozygous knock-in Jurkat (CD4 ⁺ T) cell-lines suggest that both of the (heterozygous) variants identified in SSc families result in increased NF-kB activation and expression of T cell activation markers (such as CD69 and PD1) upon TCR stimulation. One of the two variants also seems to increase calcium flux upon TCR stimulation, potentially increasing NFAT signalling. The second variant instead decreases calcium flux. These data support a pathogenic role for PRKD2 in T cells in SSc, with functional differences between the two variants identified possibly contributing to phenotypic differences between the patients in these families. Alternatively, these phenotypic differences may be potentiated by ‘’partner gene’’ variants identified in these families.

Conclusion: We report, for the first time, WES analysis of a familial SSc series followed by functional testing of potentially pathogenic variants in the PRKD2 gene, identified in two out of the six families. These data may point to a role for PRKD2 and, more broadly, underline the importance of dysregulated T cell function in the pathogenesis of SSc.

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.