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

Dyskeratosis 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.

We 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.

We 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.

The 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.