Three-step site-directed mutagenesis screen identifies pathogenic MLH1 variants associated with Lynch syndrome.

BACKGROUND: Inactivating mutations in the MLH1 DNA mismatch repair (MMR) gene underlie 42% of Lynch syndrome (LS) cases. LS is a cancer predisposition causing early onset colorectal and endometrial cancer. Nonsense and frameshift alterations unambiguously cause LS. The phenotype of missense mutations that only alter a single amino acid is often unclear. These variants of uncertain significance (VUS) hinder LS diagnosis and family screening and therefore functional tests are urgently needed. We developed a functional test for MLH1 VUS termed 'oligonucleotide-directed mutation screening' (ODMS). METHODS: The MLH1 variant was introduced by oligonucleotide-directed gene modification in mouse embryonic stem cells that were subsequently exposed to the guanine analogue 6-thioguanine to determine whether the variant abrogated MMR. RESUTS: In a proof-of-principle analysis, we demonstrate that ODMS can distinguish pathogenic and non-pathogenic MLH1 variants with a sensitivity of >95% and a specificity of >91%. We subsequently applied the screen to 51 MLH1 VUS and identified 31 pathogenic variants. CONCLUSION: ODMS is a reliable tool to identify pathogenic MLH1 variants. Implementation in clinical diagnostics will improve clinical care of patients with suspected LS and their relatives.

Suspected Lynch syndrome associated MSH6 variants: A functional assay to determine their pathogenicity.

Lynch syndrome (LS) is a hereditary cancer predisposition caused by inactivating mutations in DNA mismatch repair (MMR) genes. Mutations in the MSH6 DNA MMR gene account for approximately 18% of LS cases. Many LS-associated sequence variants are nonsense and frameshift mutations that clearly abrogate MMR activity. However, missense mutations whose functional implications are unclear are also frequently seen in suspected-LS patients. To conclusively diagnose LS and enroll patients in appropriate surveillance programs to reduce morbidity as well as mortality, the functional consequences of these variants of uncertain clinical significance (VUS) must be defined. We present an oligonucleotide-directed mutagenesis screen for the identification of pathogenic MSH6 VUS. In the screen, the MSH6 variant of interest is introduced into mouse embryonic stem cells by site-directed mutagenesis. Subsequent selection for MMR-deficient cells using the DNA damaging agent 6-thioguanine (6TG) allows the identification of MMR abrogating VUS because solely MMR-deficient cells survive 6TG exposure. We demonstrate the efficacy of the genetic screen, investigate the phenotype of 26 MSH6 VUS and compare our screening results to clinical data from suspected-LS patients carrying these variant alleles.

Extracellular loops matter - subcellular location and function of the lysine transporter Lyp1 from Saccharomyces cerevisiae.

Yeast amino acid transporters of the APC superfamily are responsible for the proton motive force-driven uptake of amino acids into the cell, which for most secondary transporters is a reversible process. The l-lysine proton symporter Lyp1 of Saccharomyces cerevisiae is special in that the Michaelis constant from out-to-in transport ( Kmout→in ) is much lower than Kmin→out , which allows accumulation of l-lysine to submolar concentration. It has been proposed that high intracellular lysine is part of the antioxidant mechanism of the cell. The molecular basis for the unique kinetic properties of Lyp1 is unknown. We compared the sequence of Lyp1 with APC para- and orthologues and find structural features that set Lyp1 apart, including differences in extracellular loop regions. We screened the extracellular loops by alanine mutagenesis and determined Lyp1 localization and activity and find positions that affect either the localization or activity of Lyp1. Half of the affected mutants are located in the extension of extracellular loop 3 or in a predicted α-helix in extracellular loop 4. Our data indicate that extracellular loops not only connect the transmembrane helices but also serve functionally important roles.