在 RECQL4 遗传性疾病相关骨肉瘤中鉴定的 RECQL4 错义变异的功能评估

Pathogenic germline mutations in hRECQL4 , a member of the highly conserved family of RecQ DNA helicases, are associated with three rare genetic disorders: Rothmund-Thomson Syndrome type II (RTS type II), Baller-Gerold Syndrome (BGS), and RAPADILLINO. All three disorders predispose patients to an array of cancers including osteosarcoma, breast, ovarian, and lymphoma. While most known pathogenic mutations found in RECQL4 -associated disorders lead to premature protein termination, a subset of patients also display missense variants, making their clinical classification more challenging. Moreover, some missense variants of unknown significance (VUS), are present in the general population, however, their contribution to disease prevalence has remained mostly unexplored. To assess the functional impact of RECQL4 missense VUS, we selected 15 missense variants identified in RECQL4 -associated disorders from the ClinVar and genomAD databases. Our dataset includes three known benign, three known pathogenic, nine VUS, and the helicase-dead K508M variant. Here, we utilize immunochemical and fluorescence microscopy techniques to assess the functional impact of these 16 RECQL4 missense variants. Using a pcDNA5 - FRT - eGFP - RECQL4 construct, we generated missense variants by site-directed mutagenesis followed by transient transfection into endogenous RECQL4 -silenced U2OS osteosarcoma cells in triplicate. Missense variants were analyzed by Western blotting for alterations in protein expression compared to the wildtype. While the benign variants p.(E71G) and p.(E267D) displayed expression like the wildtype as expected, pathogenic variants p.(P466L) and p.(S1079I) showed markedly reduced expression levels. Interestingly, VUS p.(L566P), p.(V768A), and p.(R1058G) also displayed reduced protein expression as compared to the wildtype, indicating a possible mechanism of pathogenicity. Furthermore, we performed fluorescence microscopy to assess the effect of the variants on subcellular localization under basal conditions and in the presence of the oxidative DNA damaging agent Streptonigrin. The pathogenic p.(P466L) variant, which is located in the nuclear localization signal (NLS), failed to localize to the nucleus, however, VUS p.(L566P), which is located downstream of the NLS, also impaired nuclear localization. Even under oxidative DNA damage conditions, which targets RECQL4 to the nucleolus, neither pathogenic p.(P466L) nor VUS p.(L566P) were capable of nuclear localization. In summary, we have successfully generated 16 RECQL4 missense variant constructs and evaluated them for both protein expression and subcellular localization under normal and oxidative DNA damaging conditions, identifying a VUS that behaves similarly to a known pathogenic variant.