Multivariate analysis as a method for evaluating the pathogenicity of novel genetic MLH1 variants in patients with colorectal cancer and microsatellite instability

Abstract

Loss of function of mismatch repair (MMR) genes, mainly MLH1 and MSH2, manifests as high levels of microsatellite instability (MSI) that occurs in >90% of carcinomas in patients with Lynch syndrome (LS). The MSI-high status has also been described in sporadic colorectal cancer (CRC) associated with BRAF gene mutation (V600E); this mutation was not present in LS-associated cancers. The present study performed MSI analysis on 39 CRC patients selected according to Bethesda guidelines, and BRAF V600E genotyping was performed in 26 cases classified as MSI-high or MSI-low (15 MSI-H and 11 MSI-L). These 26 patients were then screened for MLH1 and MSH2 germ-line mutations. Germ-line mutations in these genes were detected in 11/15 patients with MSI-H tumors (73%) and in 1/11 patients with MSI-L tumors (9%). Overall, 11 germ-line mutations in 12/26 analyzed patients (46%) in these genes were identified. Two of these mutations are novel genetic MLH1 variants not previously described in the literature, c.438A>G and c.1844T>C. A combination of computational approaches, co-segregation analysis and RNA assay suggested that these novel mutations, silent and missense, respectively, were probably pathogenic. The findings of the present study further emphasized the requirement for genetic testing in patients with a risk for hereditary CRC and has broadened the spectrum of known mutations of the MLH1 gene.

Two novel sequence variants in MSH2 gene in a patient who underwent cancer genetic counseling for a very early-onset epithelial ovarian cancer

“…The missense mutation (c.1706A > T) should result in an amino acid change at the protein level (p.Glu569Val). A large number of genetic alterations that are still classified as “variants of unknown significance” have pathogenic effects on the mRNA splicing process [13]. In the present case, the missense mutation could activate a cryptic splicing site.…”

Incomplete Segregation of MSH6 Frameshift Variants with Phenotype of Lynch Syndrome

“…For each paraffin block, five 20-μm sections were cut and collected in a 1.5-mL micro-tube. DNA was extracted after deparaffinization according to the protocol described by Duraturo et al, 2015, and using a BACC2 Nucleon kit (Amersham Pharmacia Biotech) [38]. …”

“…We used three complementary algorithms for functional impact prediction of the novel variants: Sorting Intolerant From Tolerant (SIFT) (available online: ) [42], Polymorphism Phenotyping (PolyPhen) (available online: ) [43] and Human Splicing Finder (HSF) (available online: ) [44], as described in our previous studies [7,38]. …”

Promising Colorectal Cancer Biomarkers for Precision Prevention and Therapy

“…MSI accounts for only about 15-22% of sporadic CRCs, while it is a characteristic of the tumor in patients with Lynch syndrome. MSI phenotypes are distinguished based on the degree of instability: MSI-high (MSI-H) and MSI-low (MSI-L); or microsatellite stable (MSS) [9,17,18]. When the percentage of altered monoand di-nucleotide microsatellite markers exceeds 20%, the instability is defined "High" (MSI-High).…”