N-Terminal Variant p.Ala2Val in X-Linked Dyskeratosis Congenita Gene (DKC1) Disrupts Its Post-Translational Modification and Nucleolar Localization.

backgroundDyskeratosis congenita (DKC) is typically characterized by the triad of abnormal cutaneous pigmentation, nail dystrophy, and oral mucosal leukoplakia. Progressive bone marrow failure develops in over 80% of cases and constitutes the leading cause of early mortality. Pathogenic variants in DKC1 have been associated with a high risk of developing aplastic anemia, myelodysplastic syndrome, leukemia, and solid tumors. The DKC1 missense variant, c.5C>T, leading to p.(Ala2Val), has been reported in families affected with dyskeratosis congenita. However, due to lack of functional analysis, it remains a variant of uncertain significance (VUS). Here, we performed functional analysis of the DKC1 gene product to investigate the pathophysiology of DKC1-related dyskeratosis congenita.

methodsWe performed co-localization studies of HEK293T cells transfected with GFP-tagged constructs encoding either a wild-type (WT) or p.(Ala2Val) human DKC1 protein, along with Liquid Chromatography Mass Spectrometry (LCMS) of immunoprecipitated (IP) WT or p.(Ala2Val) DKC1 protein to study post-translation modification status.

resultsWe have shown that p.(Ala2Val) DKC1 leads to mis-localization of the protein from the nucleolus to the nucleoplasm. Also, LCMS showed that N-terminal methionine cleavage occurs for both, but N-acetylation occurs for WT but not for p.(Ala2Val) DKC1.

conclusionThe mis-localization and altered post-translational modification of p.(Ala2Val) DKC1 provides functional evidence to clinical laboratories to reclassify p.(Ala2Val) variant as "pathogenic" as per American College of Medical Genetics (ACMG) variant interpretation guidelines.