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Next generation rare variant discovery in multiplex AD families

ABSTRACT: Next generation rare variant discovery in multiplex AD families

PROVIDER: PRJNA517200 | ENA |

REPOSITORIES: ENA

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Project description:Background: Systemic sclerosis (SSc) is a rare, heterogeneous autoimmune disease characterized by vasculopathy, immune dysregulation, and fibrosis. While typically sporadic, SSc can in rare cases show familial clustering. Family-based genetic studies provide an opportunity to identify and functionally test rare, high-impact variants for potential disease-predisposing effects. These may point to central disease mechanisms and pathways. Methods: We performed whole exome sequencing (WES) on six families, each with two first-degree relatives affected by SSc. Variants were filtered to retain those that are (i) shared by both affected individuals within families, (ii) rare in the general population, and (iii) predicted to affect protein function in silico. Variants in the candidate gene PRKD2 were tested for their effects, in heterozygous knock-in (KI) Jurkat (isogenic human CD4) T cells generated by CRISPR/Cas9-HDR. Wild-type, scrambled guide RNA (scRNA)-targeted, and PRKD2 knock-out (KO) cells were used as controls. We assessed for (basal, T cell receptor stimulated) auto-phosphorylation of PRKD2, the activation of T cell signaling pathways, and cell surface expression of T cell activation markers. RNASeq was performed for a more global picture of the effects of the variants on T cells, in comparison with scRNA and KO controls. Finally, WES was performed on a pan-Belgian series of 279 sporadic SSc patients, to assess for (variant and gene-level) rare variant enrichment of PRKD2 in SSc as compared to gnomAD population controls. Results: WES identified a total of 253 genes with rare, disease co-segregating, predicted-pathogenic variants, across the six SSc families. Of these, we prioritized PRKD2, one of only three genes identified in two families each, for functional testing. PRKD2 codes for the intracellular serine/threonine kinase Protein kinase D2, described notably as a T cell receptor (TCR) signal amplifier. Both of the rare missense variants identified in familial SSc (p.Ser189Arg and p.Pro459Leu) increase PRKD2 autophosphorylation in heterozygous knock-in Jurkat cells, exerting overlapping but not identical activating effects on T cell receptor (TCR) signaling and T cell activation. An additional ten rare missense PRKD2 variants were identified in sporadic SSc, with significant gene-level enrichment as compared to the general population, albeit with highly heterogeneous variant-level effect sizes. Conclusions: We provide genetic and functional in vitro evidence for PRKD2 as a candidate gene contributing to the pathogenesis of SSc.

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Next generation rare variant discovery in multiplex AD families

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