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.
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.
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
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.
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.
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.
Project description: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:Genome-wide DNA methylation analysis of colorectal tumour samples from 44 FFPE tumour and 15 FFPE normal mucosa samples was performed to invesgitate genome-wide DNA methylation signatures that can distinguish MLH1 epimutation carrier CRCs. This was further applied to resolve clinically challenging CRCs including MLH1 promoter VUS carriers and MLH1 methylated EOCRCs
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.