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Disentangling the mutational effects on protein stability and interaction of human MLH1

ABSTRACT: Missense mutations can have diverse effects on proteins, depending on their location within the protein and the specific amino acid substitution. Mutations in the DNA mismatch repair gene MLH1 are associated with Lynch syndrome, yet the underlying mechanism of most disease-causing mutations remain elusive. To address this gap, we aim to disentangle the mutational effects on two essential properties for MLH1 function: protein stability and protein-protein interaction. We systematically examine the cellular abundance and interaction with PMS2 of 4839 (94%) MLH1 variants in the C-terminal domain. Our combined data shows that most MLH1 variants lose interaction with PMS2 due to reduced cellular abundance. However, substitutions to charged residues in the canonical interface lead to reduced interaction with PMS2. Unexpectedly, we also identify a distal region in the C-terminal domain of MLH1 where substitutions cause both decreased and increased binding with PMS2. Our data successfully distinguish benign from pathogenic MLH1 variants and correlate with thermodynamic stability predictions and evolutionary conservation. This work provides mechanistic insights into variant consequences and may help interpret MLH1 variants.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE273652 | GEO | 2024/08/01

REPOSITORIES: GEO

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The MLH1 c.-27C>A and c.85G>T variants are borne on a European ancestral haplotype which underlies an autosomal dominant form of MLH1 epimutation.

Project description:Lynch syndrome, caused by germline heterozygous mutations of the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2, or deletions affecting the EPCAM gene upstream of MSH2, is characterized by a predisposition to early-onset colorectal and additional extracolonic cancers. An alternative but rare cause of Lynch syndrome is a constitutional epimutation of MLH1, which is characterized by promoter methylation and transcriptional silencing of a single allele in normal tissues. Worldwide, five families with autosomal dominant transmission of a constitutional MLH1 epimutation linked to an MLH1 haplotype with two single nucleotide variants (c.-27C>A and c.85G>T) have been identified. Array-based genotyping using Affymetrix SNP 6.0 data in four of these families revealed a shared haplotype extending across a ≤2.6 Mb region of chromosome 3p22 encompassing MLH1 and additional flanking genes, indicating common ancestry. Genomic DNA from 5 carriers of the c.-27C>A and c.85G>T variants was hybridized on Affymetrix SNP6.0 array according to manufacturer's procedures

2013-04-01 | E-GEOD-45149 | biostudies-arrayexpress

The MLH1 c.-27C>A and c.85G>T variants are borne on a European ancestral haplotype which underlies an autosomal dominant form of MLH1 epimutation.

2013-04-01 | GSE45149 | GEO

Disentangling the mutational effects on protein stability and interaction of human MLH1

Project description:Disentangling the mutational effects on protein stability and interaction of human MLH1

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DNA mismatch repair activity of MutLα is regulated by CK2-dependent phosphorylation of MLH1 (S477)

Project description:MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player of DNA mismatch repair (MMR), yet little is known about its regulation. In this study, we used mass spectrometry to identify phosphorylated residues within MLH1 and PMS2. The most frequently detected phosphorylated amino acid was serine 477 of MLH1. Isolation of nuclear and cytoplasmic fractions showed that p-MLH1S477 remains predominantly in the cytoplasm. Pharmacological treatment indicates that Casein Kinase II (CK2) could be responsible for the phosphorylation of MLH1 at serine 477 in vivo. In vitro kinase assay verified MLH1 as a substrate of CK2. Most importantly, using in vitro MMR assay we could demonstrate that p-MLH1S477 lost MMR activity while the activity of MLH1S477A, which could not be phosphorylated at position S477, was not modified. In summary, we identified that post-translational phosphorylation of MLH1 by CK2 at amino acid position 477 can switch off MMR activity in vitro. Since CK2 is overexpressed in many tumors and is able to inactivate MMR by phosphorylation of MLH1, the new mechanism here described could have an important impact on tumors overactive in CK2.

2018-08-28 | PXD009524 | Pride

DNA mismatch repair activity of MutLalpha is regulated by CK2-dependent phosphorylation of MLH1

Project description:Mass spectrometry based PTM phosphorylation analysis to study regulation mechanism of MUTL alpha, a heterodimer consisting of MLH1 and PMS2 and a key player in DNA mismatch repair (MMR). It could be demonstrated that phosphorylation of MLH1 by Casein Kinase II (CK2) at amino acid position 477 can switch off MMR activity in vitro.

2018-08-29 | PXD009026 | Pride

Dominant MLH1 epimutation linked to 5'UTR variant c.-27C>A

Project description:Constitutional epimutations of tumor suppressor genes manifest as promoter methylation and transcriptional silencing of a single allele in normal somatic tissues, thereby predisposing to cancer. Constitutional MLH1 epimutations occur in individuals with young-onset cancer and demonstrate non-Mendelian inheritance through their reversal in the germline. We report a cancer-affected family showing dominant transmission of soma-wide highly mosaic MLH1 methylation and transcriptional repression linked to a particular genetic haplotype. The epimutation was erased in spermatozoa but reinstated in the somatic cells of the next generation. The affected haplotype harbored two single nucleotide substitutions in tandem: c.-27C>A located near the transcription initiation site and c.85G>T. The c.-27C>A variant significantly reduced transcriptional activity in reporter assays and is the probable cause of this epimutation. Five members of a three-generation Caucasian Lynch syndrome family with an autosomal dominant MLH1 epimutation linked to a single nucleotide variant (c.-27C>A) within the MLH1 5'UTR were examined for copy number variations and retention of heterozygosity on chromosome 3. These five carriers of constitutional MLH1 methylation and the c.-27C>A variant were compared with 300 healthy Caucasian controls from the Wellcome Trust Case Control Consortium using three algorithms (QuantiSNP, PennCNV, COKGEN) to detect any copy number variants. The five family members studied were female (the proband II5, her affected mother I1, and three asymptomatic relatives II2, II4 and III2) are labeled according to the pedigree in Figure 3 of the associated publication (Hitchins et al., Cancer Cell, 2011). The supplementary file 'GSE30348_gw6.lrr_baf.txt' contains log R ratio and B-allele frequency values in a tab-delimited format with one marker per row.

2011-07-01 | E-GEOD-30348 | biostudies-arrayexpress

Primary constitutional MLH1 epimutations: a focal epigenetic event [blood data sets]

Project description:Background: Constitutional MLH1 epimutations are characterized by monoallelic methylation of the MLH1 promoter throughout normal tissues, accompanied by allele-specific silencing. The mechanism underlying primary MLH1 epimutations is currently unknown. The aim of this study was to perform an in-depth characterization of constitutional MLH1 epimutations targeting the aberrantly methylated region around MLH1 and other genomic loci. Methods: Twelve MLH1 epimutation carriers, 61 Lynch syndrome patients and 41 healthy controls, were analyzed by Infinium Human Methylation 450K beadchip, and targeted molecular techniques were used to characterize the MLH1 epimutation in carriers and their inheritance pattern. Results: No nucleotide or structural variants were identified in-cis on the epimutated allele in ten carriers, in which intergenerational methylation erasure was demonstrated in two, suggesting primary type of epimutation. CNVs outside the MLH1 locus were found in two cases. EPM2AIP1-MLH1 CpG island was identified as the sole differentially methylated region in MLH1 epimutation carriers compared to controls. Conclusion: Primary constitutional MLH1 epimutations arise as a focal epigenetic event at the EPM2AIP1-MLH1 CpG island in the absence of cis-acting genetic variants. Further molecular characterization is needed to elucidate the mechanistic basis of MLH1 epimutations and their heritability/reversibility.

2017-11-28 | GSE107351 | GEO

Primary constitutional MLH1 epimutations: a focal epigenetic event [FFPE data sets]

2017-11-28 | GSE107352 | GEO

Dominant MLH1 epimutation linked to 5'UTR variant c.-27C>A

2011-07-01 | GSE30348 | GEO

Highly sensitive MLH1 methylation analysis in blood allows the identification of low-level epigenetic mosaicism

Project description:Constitutional MLH1 epimutations are a rare cause of Lynch syndrome. Low methylation levels (≤10%) have been occasionally described. The aim of this study was the identification of patients with low levels of epigenetic mosaicism in MLH1 gene. Eighteen patients with MLH1 hypermethylated tumors and undetectable methylation in blood as assessed by Methylation-Specific Multiplex Ligation-Dependent Probe Amplification were included (MS-MLPA). Highly sensitive MS-Melting Curve Analysis (MS-MCA) at MLH1 promoter was used to screen for epigenetic mosaicism. Constitutional methylation was confirmed by other methods. Mutational analysis of hereditary cancer genes including MLH1 was performed. MS-MCA analysis identified one case (5.6%) with low levels of methylation (1-2%) in blood DNA. The patient had developed 3 gastrointestinal tumors at ages 22, 24 and 25, sharing MLH1 promoter hypermethylation and loss of heterozygosity associated with c.655A allele. The presence of low MLH1 methylation levels was confirmed by clonal bisulfite sequencing, evidencing the association with c.-93G allele (in phase with c.655G). The extension of the hypermethylated region overlaps with the reported in constitutional MLH1 epimutation carriers. No rare germline variants were identified. The use of highly sensitive techniques such as MS-MCA has demonstrated to be useful for the detection of low MLH1 methylation levels in blood.

2019-05-22 | GSE131541 | GEO

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