CHEK2 germline variants identified in familial nonmedullary thyroid cancer lead to impaired protein structure and function

Abstract

Approximately 5-15% of non-medullary thyroid cancers (NMTC) present in a familial form (FNMTC). Unlike sporadic NMTC, the genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic, therapeutic and follow-up decisions. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer, suggesting a role in FNMTC etiology. Here, two FNMTC families were studied, each with two members affected with thyroid cancer. Ninety-four hereditary cancer predisposition genes were analysed through next-generation sequencing, revealing two germline missense variants in CHEK2 (c.962A>C, p.E321A and c.470T>C, p.I157T), which segregated with thyroid cancer in each FNMTC family. p.E321A, located in the CHK2 protein kinase domain, is a rare variant, previously unreported in the literature. Conversely, p.I157T, located in CHK2 forkhead-associated domain, has been extensively described, having conflicting interpretations of pathogenicity. CHK2 proteins (wild type and variants) were characterized using biophysical methods, molecular dynamics simulations, and immunohistochemistry. Overall, biophysical characterization of these CHK2 variants showed that they have compromised structural and conformational stability and impaired kinase activity, compared to the wild-type protein. Additionally, CHK2 appears to aggregate into amyloid-like fibrils in vitro, which opens future perspectives towards positioning CHK2 in cancer pathophysiology. CHK2 variants exhibited higher propensity for this conformational change, also displaying higher expression in thyroid tumors. The present findings support the utility of complementary biophysical and in silico approaches towards understanding the impact of genetic variants in protein structure and function, improving the current knowledge on CHEK2 variants’ role in FNMTC genetic basis, with prospective clinical translation.