Functional characterization of MLH1 missense variants unveils mechanisms of pathogenicity and clarifies role in cancer

Abstract

Lynch syndrome is a heritable condition caused by a heterozygous germline inactivating mutation of the DNA mismatch repair (MMR) genes, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants, for which the clinical significance is unclear in many cases. We have identified three MLH1 missense alterations (p.(Glu736Lys), p.(Pro640Thr) and p.(Leu73Pro)) in six individuals from large Tunisian families. For none of these alterations, a classification of pathogenicity was available, consequently diagnosis, predictive testing and targeted surveillance in affected families was impossible. We therefore performed functional laboratory testing using a system testing stability as well as catalytic activity that includes clinically validated reference variants. Both p.(Leu73Pro) and p.(Pro640Thr) were found to be non-functional due to severe defects in protein stability and catalytic activity. In contrast, p.(Glu736Lys) was comparable to the wildtype protein and therefore considered a neutral substitution. Analysis of residue conservation and of the structural roles of the substituted residues corroborated these findings. In conjunction with the available clinical data, two variants fulfil classification criteria for class 4 “likely pathogenic”. The findings of this work clarify the mechanism of pathogenicity of two unclear MLH1 variants and enables predictive testing and targeted surveillance in members of carrier families worldwide.

Constitutional mismatch repair deficiency syndrome with atypical features caused by a homozygous MLH1 missense variant (c.1918C>A, p.(Pro640Thr)): a case report

“…MLH1 and PMS2 are known to act as partners in the repair process, and so functional defects in MLH1 can lead to the instability of both MLH1 and PMS2. Therefore, the IHC results were consistent with the functional analyses of Mahdouani et al., who found the missense variant p.(Pro640Thr) reduced MLH1 protein expression ( 6 ). MSI was identified in both non-neoplastic and neoplastic colorectal tissue, and found MLH1 p.(Pro640Thr) caused reduced MMR activity by CIMRA ( 8 ).…”

“…After its first description, more than a hundred cases of biallelic germline variants in one of the MMR genes, including PMS2 , MSH2 , MSH6 , and MLH1 , have been identified ( 11 ). The c.1918C>A; p.(Pro640Thr) variant of the MLH1 gene identified in our patient has been previously detected in LS ( 6 ). However, to our knowledge, a biallelic case of MLH1 c.1918C>A; p.(Pro640Thr) has never been reported in CMMRD syndrome.…”

“…This, the functional results of Mahdouani et al. ( 6 ), and the atypical phenotype of the proband suggest the MLH1 c.1918C>A; p.(Pro640Thr) could also be considered hypomorphic. Alternatively, the relatively mild phenotype of the proband may be explained by other genetic or environmental factors, and the effective management of each tumor, including radical treatment of his cancers and prophylactic proctectomy to avoid additional rectal malignancies.…”

“…They compared protein stability and catalytic activity between the MLH1 p.(Pro640Thr) variant and a validated reference pathogenic variant MLH1 p.(Ala681Thr). The p.(Pro640Thr) variant results in severe defects in protein stability and minimal reduction in catalytic activity ( 6 ) (PS3_Strong). However, due to conflicting functional results from Takahashi et al., we conducted further assessment of the functional impact of the variant.…”

“…MLH1 c.1918C>A has recently been observed as a heterozygous variant in several families with an LS phenotype. It was also shown to disrupt protein expression in a transfected HEK293T cell line model, but had minimal effect on MMR activity in vitro ( 6 ). In contrast, Takahashi et al.…”