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1.  C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins 
PLoS ONE  2012;7(2):e31863.
The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.
PMCID: PMC3279419  PMID: 22348133
2.  Human MutL homolog (MLH1) function in DNA mismatch repair: a prospective screen for missense mutations in the ATPase domain 
Nucleic Acids Research  2004;32(18):5321-5338.
Germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1 are responsible for the majority of hereditary non-polyposis colorectal cancer (HNPCC), an autosomal-dominant early-onset cancer syndrome. Genetic testing of both MSH2 and MLH1 from individuals suspected of HNPCC has revealed a considerable number of missense codons, which are difficult to classify as either pathogenic mutations or silent polymorphisms. To identify novel MLH1 missense codons that impair MMR activity, a prospective genetic screen in the yeast Saccharomyces cerevisiae was developed. The screen utilized hybrid human-yeast MLH1 genes that encode proteins having regions of the yeast ATPase domain replaced by homologous regions from the human protein. These hybrid MLH1 proteins are functional in MMR in vivo in yeast. Mutagenized MLH1 fragments of the human coding region were synthesized by error-prone PCR and cloned directly in yeast by in vivo gap repair. The resulting yeast colonies, which constitute a library of hybrid MLH1 gene variants, were initially screened by semi-quantitative in vivo MMR assays. The hybrid MLH1 genes were recovered from yeast clones that exhibited a MMR defect and sequenced to identify alterations in the mutagenized region. This investigation identified 117 missense codons that conferred a 2-fold or greater decreased efficiency of MMR in subsequent quantitative MMR assays. Notably, 10 of the identified missense codons were equivalent to codon changes previously observed in the human population and implicated in HNPCC. To investigate the effect of all possible codon alterations at single residues, a comprehensive mutational analysis of human MLH1 codons 43 (lysine-43) and 44 (serine-44) was performed. Several amino acid replacements at each residue were silent, but the majority of substitutions at lysine-43 (14/19) and serine-44 (18/19) reduced the efficiency of MMR. The assembled data identifies amino acid substitutions that disrupt MLH1 structure and/or function, and should assist the interpretation of MLH1 genetic tests.
PMCID: PMC524276  PMID: 15475387
3.  The G67E mutation in hMLH1 is associated with an unusual presentation of Lynch syndrome 
British Journal of Cancer  2009;100(2):376-380.
Germline mutations in the mismatch repair (MMR) genes are associated with Lynch syndrome, also known as hereditary non-polyposis colorectal cancer (HNPCC) syndrome. Here, we characterise a variant of hMLH1 that confers a loss-of-function MMR phenotype. The mutation changes the highly conserved Gly67 residue to a glutamate (G67E) and is reminiscent of the hMLH1-p.Gly67Arg mutation, which is present in several Lynch syndrome cohorts. hMLH1-Gly67Arg has previously been shown to confer loss-of-function (Shimodaira et al, 1998), and two functional assays suggest that the hMLH1-Gly67Glu protein fails to sustain normal MMR functions. In the first assay, hMLH1-Gly67Glu abolishes the protein's ability to interfere with MMR in yeast. In the second assay, mutation of the analogous residue in yMLH1 (yMLH1-Gly64Glu) causes a loss-of-function mutator phenotype similar to yMLH1-Gly64Arg. Despite these molecular similarities, an unusual spectrum of tumours is associated with hMLH1-Gly67Glu, which is not typical of those associated with Lynch syndrome and differs from those found in families carrying the hMLH1-Gly67Arg allele. This suggests that hMLH1 may have different functions in certain tissues and/or that additional factors may modify the influence of hMLH1 mutations in causing Lynch syndrome.
PMCID: PMC2634701  PMID: 19142183
G67E; HNPCC; hMLH1; breast; prostate
4.  Complex genetic predisposition to cancer in an extended HNPCC family with an ancestral hMLH1 mutation. 
Journal of Medical Genetics  1996;33(8):636-640.
Hereditary non-polyposis colorectal cancer (HNPCC) is characterised by a genetic predisposition to develop colorectal cancer at an early age and, to a lesser degree, cancer of the endometrium, ovaries, urinary tract, and organs of the gastrointestinal tract other than the colon. In the majority of families the disease is linked to mutations in one of the two mismatch repair genes, hMSH2 or hMLH1. We have found a novel hMLH1 nonsense mutation in a Swiss family with Lynch syndrome, which has been transmitted through at least nine generations. A different tumour spectrum of neoplasms of the skin, soft palate, breast, duodenum, and pancreas was observed in three branches of this family, where there was a virtual absence of colonic tumours. The hMLH1 mutation could not be detected in members of these branches suggesting that at least a second genetic defect predisposing to cancer is segregating in part of the kindred.
PMCID: PMC1050695  PMID: 8863153
5.  DNA binding by yeast Mlh1 and Pms1: implications for DNA mismatch repair 
Nucleic Acids Research  2003;31(8):2025-2034.
The yeast Mlh1–Pms1 heterodimer required for mismatch repair (MMR) binds to DNA. Here we map DNA binding to N-terminal fragments of Mlh1 and Pms1. We demonstrate that Mlh1 and Pms1 N-terminal domains (NTDs) independently bind to double-stranded and single-stranded DNA, in the absence of dimerization and with different affinities. Full-length Mlh1p alone, which can homodimerize, also binds to DNA. Substituting conserved positively charged amino acids in Mlh1 produces mutator phenotypes in a haploid yeast strain characteristic of reduced MMR. These substitutions strongly reduce DNA binding by the Mlh1 NTD and, to a lesser extent, they also reduce DNA binding by full-length Mlh1 and the Mlh1–Pms1 heterodimer. Replacement of a homologous Pms1 residue has a much smaller effect on mutation rate and does not reduce DNA binding. The results demonstrate that NTDs of yeast Mlh1 and Pms1 contain independent DNA binding sites and they suggest that the C-terminal region of Mlh1p may also contribute to DNA binding. The differential mutator effects and binding properties observed here further suggest that Mlh1 and Pms1 differ in their interactions with DNA. Finally, the results are consistent with the hypothesis that DNA binding by Mlh1 is important for MMR.
PMCID: PMC153752  PMID: 12682353
6.  Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients 
BMC Medical Genetics  2013;14:103.
Germ-line mutations in the DNA mismatch repair genes MLH1, MSH2, and MSH6 predispose to the development of colorectal cancer (Lynch syndrome or hereditary nonpolyposis colorectal cancer). These mutations include disease-causing frame-shift, nonsense, and splicing mutations as well as large genomic rearrangements. However, a large number of mutations, including missense, silent, and intronic variants, are classified as variants of unknown clinical significance.
Intronic MLH1, MSH2, or MSH6 variants were investigated using in silico prediction tools and mini-gene assay to asses the effect on splicing.
We describe in silico and in vitro characterization of nine intronic MLH1, MSH2, or MSH6 mutations identified in Danish colorectal cancer patients, of which four mutations are novel. The analysis revealed aberrant splicing of five mutations (MLH1 c.588 + 5G > A, MLH1 c.677 + 3A > T, MLH1 c.1732-2A > T, MSH2 c.1276 + 1G > T, and MSH2 c.1662-2A > C), while four mutations had no effect on splicing compared to wild type (MLH1 c.117-34A > T, MLH1 c.1039-8 T > A, MSH2 c.2459-18delT, and MSH6 c.3439-16C > T).
In conclusion, we classify five MLH1/MSH2 mutations as pathogenic, whereas four MLH1/MSH2/MSH6 mutations are classified as neutral. This study supports the notion that in silico prediction tools and mini-gene assays are important for the classification of intronic variants, and thereby crucial for the genetic counseling of patients and their family members.
PMCID: PMC3850734  PMID: 24090359
Colorectal cancer; HNPCC; Lynch syndrome; Mini-gene assay; Mismatch repair genes MLH1, MSH2, and MSH6; Splicing defect
7.  Comprehensive molecular analysis of mismatch repair gene defects in suspected Lynch Syndrome (Hereditary Non-polyposis Colorectal Cancer) cases 
Cancer research  2009;69(17):7053-7061.
An accurate algorithm is essential for effective molecular diagnosis of hereditary colorectal cancer. Here we have extended the analysis of 71 colorectal cancer cases suspected to be Lynch Syndrome cases for MSH2, MLH1, MSH6 and PMS2 gene defects. All cases were screened for mutations in MSH2, MLH1 and MSH6 and all cases where tumors were available were screened for microsatellite instability and expression of MSH2 and MLH1. Subsequently, mutation negative cases were screened for MLH1 methylation and mutations in PMS2. Of the MSI-H cases, 96% had a mismatch repair gene defect, mostly involving MSH2 or MLH1; 1 PMS2 mutation, 1 MLH1 epimutation, and no MSH6 mutations were found. Four of the 28 MSI-H cases, including 1 Amsterdam criteria case, had biallelic tumor MLH1 methylation indicating that sporadic cases can be admixed in with Lynch Syndrome cases even those meeting the strongest criteria for Lynch Syndrome. Mismatch repair gene defects were found in similar frequency in cases where tumors were and were not available. One MLH1 and 1 MSH2 deletion mutation were found in MSI-S/L cases indicating that microsatellite instability testing can exclude cases with pathogenic mutations. Our analysis support a diagnostic algorithm where cases are selected for analysis based on clinical criteria or prediction models; isolated sporadic young-onset cases can be pre-screened by tumor testing whereas familial cases may be directly subjected to molecular analysis for mutations in mismatch repair genes followed by microsatellite instability, protein expression and DNA methylation analysis to aid in the resolution of mutation negative cases.
PMCID: PMC2761236  PMID: 19690142
8.  Mutations in the MutSα interaction interface of MLH1 can abolish DNA mismatch repair 
Nucleic Acids Research  2006;34(22):6574-6586.
MutLα, a heterodimer of MLH1 and PMS2, plays a central role in human DNA mismatch repair. It interacts ATP-dependently with the mismatch detector MutSα and assembles and controls further repair enzymes. We tested if the interaction of MutLα with DNA-bound MutSα is impaired by cancer-associated mutations in MLH1, and identified one mutation (Ala128Pro) which abolished interaction as well as mismatch repair activity. Further examinations revealed three more residues whose mutation interfered with interaction. Homology modelling of MLH1 showed that all residues clustered in a small accessible surface patch, suggesting that the major interaction interface of MutLα for MutSα is located on the edge of an extensive β-sheet that backs the MLH1 ATP binding pocket. Bioinformatic analysis confirmed that this patch corresponds to a conserved potential protein–protein interaction interface which is present in both human MLH1 and its E.coli homologue MutL. MutL could be site-specifically crosslinked to MutS from this patch, confirming that the bacterial MutL–MutS complex is established by the corresponding interface in MutL. This is the first study that identifies the conserved major MutLα–MutSα interaction interface in MLH1 and demonstrates that mutations in this interface can affect interaction and mismatch repair, and thereby can also contribute to cancer development.
PMCID: PMC1747184  PMID: 17135187
9.  Interrelationship between microsatellite instability and microRNA in gastrointestinal cancer 
There is an increasing understanding of the roles that microsatellite instability (MSI) plays in Lynch syndrome (by mutations) and sporadic (by mainly epigenetic changes) gastrointestinal (GI) and other cancers. Deficient DNA mismatch repair (MMR) results in the strong mutator phenotype known as MSI, which is the hallmark of cancers arising within Lynch syndrome. MSI is characterized by length alterations within simple repeated sequences called microsatellites. Lynch syndrome occurs primarily because of germline mutations in one of the MMR genes, mainly MLH1 or MSH2, less frequently MSH6, and rarely PMS2. MSI is also observed in about 15% of sporadic colorectal, gastric, and endometrial cancers and in lower frequencies in a minority of other cancers where it is often associated with the hypermethylation of the MLH1 gene. miRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level and are critical in many biological processes and cellular pathways. There is accumulating evidence to support the notion that the interrelationship between MSI and miRNA plays a key role in the pathogenesis of GI cancer. As a possible new mechanism underlying MSI, overexpression of miR-155 has been shown to downregulate expression of MLH1, MSH2, and MSH6. Thus, a subset of MSI-positive (MSI+) cancers without known MMR defects may result from miR-155 overexpression. Target genes of frameshift mutation for MSI are involved in various cellular functions, such as DNA repair, cell signaling, and apoptosis. A novel class of target genes that included not only epigenetic modifier genes, such as HDAC2, but also miRNA processing machinery genes, including TARBP2 and XPO5, were found to be mutated in MSI+ GI cancers. Thus, a subset of MSI+ colorectal cancers (CRCs) has been proposed to exhibit a mutated miRNA machinery phenotype. Genetic, epigenetic, and transcriptomic differences exist between MSI+ and MSI− cancers. Molecular signatures of miRNA expression apparently have the potential to distinguish between MSI+ and MSI− CRCs. In this review, we summarize recent advances in the MSI pathogenesis of GI cancer, with the focus on its relationship with miRNA as well as on the potential to use MSI and related alterations as biomarkers and novel therapeutic targets.
PMCID: PMC3374977  PMID: 22719182
Microsatellite instability; MicroRNA; DNA mismatch repair; Frameshift mutation; MicroRNA processing
10.  Screening of the DNA mismatch repair genes MLH1, MSH2 and MSH6 in a Greek cohort of Lynch syndrome suspected families 
BMC Cancer  2010;10:544.
Germline mutations in the DNA mismatch repair genes predispose to Lynch syndrome, thus conferring a high relative risk of colorectal and endometrial cancer. The MLH1, MSH2 and MSH6 mutational spectrum reported so far involves minor alterations scattered throughout their coding regions as well as large genomic rearrangements. Therefore, a combination of complete sequencing and a specialized technique for the detection of genomic rearrangements should be conducted during a proper DNA-testing procedure. Our main goal was to successfully identify Lynch syndrome families and determine the spectrum of MLH1, MSH2 and MSH6 mutations in Greek Lynch families in order to develop an efficient screening protocol for the Greek colorectal cancer patients' cohort.
Forty-two samples from twenty-four families, out of which twenty two of Greek, one of Cypriot and one of Serbian origin, were screened for the presence of germline mutations in the major mismatch repair genes through direct sequencing and MLPA. Families were selected upon Amsterdam criteria or revised Bethesda guidelines.
Ten deleterious alterations were detected in twelve out of the twenty-four families subjected to genetic testing, thus our detection rate is 50%. Four of the pathogenic point mutations, namely two nonsense, one missense and one splice site change, are novel, whereas the detected genomic deletion encompassing exon 6 of the MLH1 gene has been described repeatedly in the LOVD database. The average age of onset for the development of both colorectal and endometrial cancer among mutation positive families is 43.2 years.
The mutational spectrum of the MMR genes investigated as it has been shaped by our analysis is quite heterogeneous without any strong indication for the presence of a founder effect.
PMCID: PMC2976752  PMID: 20937110
11.  Dominant Mutations in S. cerevisiae PMS1 Identify the Mlh1-Pms1 Endonuclease Active Site and an Exonuclease 1-Independent Mismatch Repair Pathway 
PLoS Genetics  2013;9(10):e1003869.
Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.
Author Summary
Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. In addition to these genes, various DNA replication factors and the excision factor EXO1 function in the repair of damaged DNA by the MMR pathway. Although EXO1 is considered to be the major repair nuclease functioning in mismatch repair, the relatively low mutation rates caused by an exo1 deletion suggest otherwise. Here we used genetics, microscopy and protein biochemistry to analyze the model organism Saccharomyces cerevisiae to further characterize a poorly understood mismatch repair pathway that functions in the absence of EXO1 that is highly dependent on the Mlh1-Pms1 complex. Surprisingly, we found that the highly conserved metal binding site that is critical for the endonuclease activity of the Mlh1-Pms1 heterodimer is required for MMR in the absence of Exo1 to a much greater extent than in the presence of Exo1. Thus, this work establishes that there are at least two different polynucleotide excision pathways that function in MMR.
PMCID: PMC3814310  PMID: 24204293
12.  The MLH1 c.1852_1853delinsGC (p.K618A) Variant in Colorectal Cancer: Genetic Association Study in 18,723 Individuals 
PLoS ONE  2014;9(4):e95022.
Colorectal cancer is one of the most frequent neoplasms and an important cause of mortality in the developed world. Mendelian syndromes account for about 5% of the total burden of CRC, being Lynch syndrome and familial adenomatous polyposis the most common forms. Lynch syndrome tumors develop mainly as a consequence of defective DNA mismatch repair associated with germline mutations in MLH1, MSH2, MSH6 and PMS2. A significant proportion of variants identified by screening these genes correspond to missense or noncoding changes without a clear pathogenic consequence, and they are designated as “variants of uncertain significance”, being the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene a clear example. The implication of this variant as a low-penetrance risk variant for CRC was assessed in the present study by performing a case-control study within a large cohort from the COGENT consortium-COST Action BM1206 including 18,723 individuals (8,055 colorectal cancer cases and 10,668 controls) and a case-only genotype-phenotype correlation with several clinical and pathological characteristics restricted to the Epicolon cohort. Our results showed no involvement of this variant as a low-penetrance variant for colorectal cancer genetic susceptibility and no association with any clinical and pathological characteristics including family history for this neoplasm or Lynch syndrome.
PMCID: PMC3990597  PMID: 24743384
13.  Contribution of Large Genomic Rearrangements in Italian Lynch Syndrome Patients: Characterization of a Novel Alu-Mediated Deletion 
BioMed Research International  2012;2013:219897.
Lynch syndrome is associated with germ-line mutations in the DNA mismatch repair (MMR) genes, mainly MLH1 and MSH2. Most of the mutations reported in these genes to date are point mutations, small deletions, and insertions. Large genomic rearrangements in the MMR genes predisposing to Lynch syndrome also occur, but the frequency varies depending on the population studied on average from 5 to 20%. The aim of this study was to examine the contribution of large rearrangements in the MLH1 and MSH2 genes in a well-characterised series of 63 unrelated Southern Italian Lynch syndrome patients who were negative for pathogenic point mutations in the MLH1, MSH2, and MSH6 genes. We identified a large novel deletion in the MSH2 gene, including exon 6 in one of the patients analysed (1.6% frequency). This deletion was confirmed and localised by long-range PCR. The breakpoints of this rearrangement were characterised by sequencing. Further analysis of the breakpoints revealed that this rearrangement was a product of Alu-mediated recombination. Our findings identified a novel Alu-mediated rearrangement within MSH2 gene and showed that large deletions or duplications in MLH1 and MSH2 genes are low-frequency mutational events in Southern Italian patients with an inherited predisposition to colon cancer.
PMCID: PMC3591251  PMID: 23484096
14.  Patients with an unexplained microsatellite instable tumour have a low risk of familial cancer 
British Journal of Cancer  2007;96(10):1605-1612.
The cancer risk is unknown for those families in which a microsatellite instable tumour is neither explained by MLH1 promoter methylation nor by a germline mutation in a mismatch repair (MMR) gene. Such information is essential for genetic counselling. Families suspected of Lynch syndrome (n=614) were analysed for microsatellite instability, MLH1 promoter methylation and/or germline mutations in MLH1, MSH2, MSH6, and PMS2. Characteristics of the 76 families with a germline mutation (24 MLH1, 2 PMS2, 32 MSH2, and 18 MSH6) were compared with those of 18 families with an unexplained microsatellite instable tumour. The mean age at diagnosis of the index patients in both groups was comparable at 44 years. Immunohistochemistry confirmed the loss of an MMR protein. Together this suggests germline inactivation of a known gene. The Amsterdam II criteria were fulfilled in 50/75 families (66%) that carried a germline mutation in an MMR gene and in only 2/18 families (11%) with an unexplained microsatellite instable tumour (P<0.0001). Current diagnostic strategies can detect almost all highly penetrant MMR gene mutations. Patients with an as yet unexplained microsatellite instable tumour likely carry a different type of mutation that confers a lower risk of cancer for relatives.
PMCID: PMC2359954  PMID: 17453009
colorectal neoplasms; hereditary nonpolyposis; microsatellite instability; DNA mismatch repair; DNA methylation
15.  Mlh2 Is an Accessory Factor for DNA Mismatch Repair in Saccharomyces cerevisiae 
PLoS Genetics  2014;10(5):e1004327.
In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I-SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-Msh3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1.
Author Summary
Lynch syndrome (hereditary nonpolyposis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. In this syndrome, predisposition to cancer results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the human mismatch repair genes MLH1, MSH2, MSH6 or PMS2. In addition to these genes, various DNA replication factors and the excision factor EXO1 function in the repair of damaged DNA by the MMR pathway. In Saccharomyces cerevisiae, the MLH2 gene encodes a MutL homolog protein whose role in DNA mismatch repair has been unclear. Here, we used phylogenetic analysis to demonstrate that the S. cerevisiae Mlh2 protein and the mammalian Pms1 protein are homologs. A combination of genetics, biochemistry and imaging studies were used to demonstrate that the Mlh1-Mlh2 complex is recruited to mispair-containing DNA by the Msh2-Msh6 and Msh2-Msh3 mispair recognition complexes where it forms foci that colocalize with Mlh1-Pms1 foci (note that scPms1 is the homolog of hPms2) and augments the function of the Mlh1-Pms1 complex. Thus, this work establishes the Mlh1-Mlh2 complex as a non-essential accessory factor that functions in MMR.
PMCID: PMC4014439  PMID: 24811092
Immunohistochemistry for mismatch repair proteins has shown utility in the identification of Lynch syndrome, but majority of tumours with loss MLH1 expression are due to sporadic hypermethylation of the MLH1 promoter. These tumours can also show epigenetic silencing of other genes, such as p16. The aim of our study is to evaluate the utility of p16 immunohistochemistry in the prediction of MLH1 germline mutations.
p16 immunohistochemistry was appropriately evaluated in 79 colorectal cancers with loss of MLH1 expression. Methylation of MLH1 and p16 were quantitatively studied using real time PCR assay Methylight. BRAF V600E mutation in tumour tissue was also investigated. Genetic testing for germline mutation of MLH1 was made on 52 patients.
Loss of p16 expression was seen in 21 out of 79 samples (26,6%). There was found statistically significant association between p16 expression and p16 methylation (p<0.001), MLH1 methylation (p<0.001) and BRAF mutation (p<0.005). All tumours with loss of p16 expression showed hypermethylation of p16 (21/21), 95.2% (20/21) showed MLH1 methylation and 71.4% (15/21) were mutated for BRAF V600E Mutational analysis showed pathogenic germline mutations in 8 of the patients, harbouring 10 tumours. All 10 of these tumours showed normal staining of p16 in the immunochemical analysis.
p16 immunohistochemistry is a good surrogate marker for p16 and MLH1 epigenetic silencing due to hypermethylation, and is useful as screening tool in the selection of patients for genetic testing in Lynch syndrome.
PMCID: PMC2825754  PMID: 19383812
colorectal cancer; Lynch syndrome; p16; immunohistochemistry; diagnosis
17.  MLH1 methylation screening is effective in identifying epimutation carriers 
European Journal of Human Genetics  2012;20(12):1256-1264.
Recently, constitutional MLH1 epimutations have been identified in a subset of Lynch syndrome (LS) cases. The aim of this study was the identification of patients harboring constitutional MLH1 epimutations in a set of 34 patients with a clinical suspicion of LS, MLH1-methylated tumors and non-detected germline mutations in mismatch repair (MMR) genes. MLH1 promoter methylation was analyzed in lymphocyte DNA samples by MS-MLPA (Methylation-specific multiplex ligation-dependent probe amplification). Confirmation of MLH1 constitutional methylation was performed by MS-MCA (Methylation-specific melting curve analysis), bisulfite sequencing and pyrosequencing in different biological samples. Allelic expression was determined using heterozygous polymorphisms. Vertical transmission was evaluated by MS-MLPA and haplotype analyses. MS-MLPA analysis detected constitutional MLH1 methylation in 2 of the 34 individuals whose colorectal cancers showed MLH1 methylation (5.9%). These results were confirmed by bisulfite-based methods. Both epimutation carriers had developed metachronous early-onset LS tumors, with no family history of LS-associated cancers in their first-degree relatives. In one of the cases, the identified MLH1 constitutional methylation was monoallelic and results in MLH1 and EPM2AIP1 allele-specific transcriptional silencing. It was present in normal somatic tissues and absent in spermatozoa. The methylated MLH1 allele was maternally transmitted and methylation was reversed in a daughter who inherited the same allele. MLH1 methylation screening in lymphocyte DNA from patients with early-onset MLH1-methylated LS-associated tumors allows the identification of epimutation carriers. The present study adds further evidence to the emerging entity of soma-wide MLH1 epimutation and its heritability.
PMCID: PMC3499751  PMID: 22763379
Lynch syndrome; constitutional epimutation; MLH1; methylation; MS-MLPA; pyrosequencing
18.  MLH1 promoter hypermethylation in the analytical algorithm of Lynch syndrome: a cost-effectiveness study 
The analytical algorithm of Lynch syndrome (LS) is increasingly complex. BRAF V600E mutation and MLH1 promoter hypermethylation have been proposed as a screening tool for the identification of LS. The aim of this study was to assess the clinical usefulness and cost-effectiveness of both somatic alterations to improve the yield of the diagnostic algorithm of LS. A total of 122 colorectal tumors from individuals with family history of colorectal cancer that showed microsatellite instability and/or loss of mismatch repair (MMR) protein expression were studied. MMR germline mutations were detected in 57 cases (40 MLH1, 15 MSH2 and 2 MSH6). BRAF V600E mutation was assessed by single-nucleotide primer extension. MLH1 promoter hypermethylation was assessed by methylation-specific multiplex ligation-dependent probe amplification in a subset of 71 cases with loss of MLH1 protein. A decision model was developed to estimate the incremental costs of alternative case-finding methods for detecting MLH1 mutation carriers. One-way sensitivity analysis was performed to assess robustness of estimations. Sensitivity of the absence of BRAF mutations for depiction of LS patients was 96% (23/24) and specificity was 28% (13/47). Specificity of MLH1 promoter hypermethylation for depiction of sporadic tumors was 66% (31/47) and sensitivity of 96% (23/24). The cost per additional mutation detected when using hypermethylation analysis was lower when compared with BRAF study and germinal MLH1 mutation study. Somatic hypermethylation of MLH1 is an accurate and cost-effective pre-screening method in the selection of patients that are candidates for MLH1 germline analysis when LS is suspected and MLH1 protein expression is absent.
PMCID: PMC3376264  PMID: 22274583
Lyncg Syndrome; MLH1 promoter hypermethylation; BRAF V600E mutation; MS-MLPA; cost-effectiveness
19.  Germline deletions in the EPCAM gene as a cause of Lynch syndrome – literature review 
Lynch syndrome (clinically referred to as HNPCC – Hereditary Non-Polyposis Colorectal Cancer) is a frequent, autosomal, dominantly-inherited cancer predisposition syndrome caused by various germline alterations that affect DNA mismatch repair genes, mainly MLH1 and MSH2. Patients inheriting this predisposition are susceptible to colorectal, endometrial and other extracolonic tumors. It has recently been shown that germline deletions of the last few exons of the EPCAM gene are involved in the etiology of Lynch syndrome. Such constitutional mutations lead to subsequent epigenetic silencing of a neighbouring gene, here, MSH2, causing Lynch syndrome. Thus, deletions of the last few exons of EPCAM constitute a distinct class of mutations associated with HNPCC. Worldwide, several investigators have reported families with EPCAM 3’end deletions. The risk of colorectal cancer in carriers of EPCAM deletions is comparable to situations when patients are MSH2 mutation carriers, and is associated with high expression levels of EPCAM in colorectal cancer stem cells. A lower risk of endometrial cancer was also reported. Until now the standard diagnostic tests for Lynch syndrome have contained analyses such as immunohistochemistry and tests for microsatellite instability of mismatch repair genes. The identification of EPCAM deletions or larger EPCAM-MSH2 deletions should be included in routine mutation screening, as this has implications for cancer predisposition.
PMCID: PMC3765447  PMID: 23938213
Lynch syndrome; EPCAM gene; Colon cancer; MSH2 hypermethylation
20.  Systematic Study on Genetic and Epimutational Profile of a Cohort of Amsterdam Criteria-Defined Lynch Syndrome in Singapore 
PLoS ONE  2014;9(4):e94170.
Germline defects of mismatch repair (MMR) genes underlie Lynch Syndrome (LS). We aimed to gain comprehensive genetic and epigenetic profiles of LS families in Singapore, which will facilitate efficient molecular diagnosis of LS in Singapore and the region.
Fifty nine unrelated families were studied. Mutations in exons, splice-site junctions and promoters of five MMR genes were scanned by high resolution melting assay followed by DNA sequencing, large fragment deletions/duplications and promoter methylation in MLH1, MSH2, MSH6 and PMS2 were evaluated by multiplex ligation-dependent probe amplification. Tumor microsatellite instability (MSI) was assessed with five mononucleotide markers and immunohistochemical staining (IHC) was also performed.
Pathogenic defects, all confined to MLH1 and MSH2, were identified in 17 out of 59 (28.8%) families. The mutational spectrum was highly heterogeneous and 28 novel variants were identified. One recurrent mutation in MLH1 (c.793C>T) was also observed. 92.9% sensitivity for indication of germline mutations conferred by IHC surpassed 64.3% sensitivity by MSI. Furthermore, 15.6% patients with MSS tumors harbored pathogenic mutations.
Among major ethnic groups in Singapore, all pathogenic germline defects were confined to MLH1 and MSH2. Caution should be applied when the Amsterdam criteria and consensus microsatellite marker panel recommended in the revised Bethesda guidelines are applied to the local context. We recommend a screening strategy for the local LS by starting with tumor IHC and the hotspot mutation testing at MLH1 c.793C>T followed by comprehensive mutation scanning in MLH1 and MSH2 prior to proceeding to other MMR genes.
PMCID: PMC3978005  PMID: 24710284
21.  Aberrant DNA Methylation in Hereditary Non-Polyposis Colorectal Cancer without Mismatch Repair Deficiency 
Gastroenterology  2010;138(5):1854-1862.
Background & Aims
Approximately half of the families that fulfill Amsterdam criteria for Lynch syndrome or hereditary non-polyposis colorectal cancer (HNPCC) do not have evidence of the germline mismatch repair (MMR) gene mutations that define this syndrome and result in microsatellite instability. The carcinogenic pathways and the best diagnostic approaches to detect microsatellite stable (MSS) HNPCC tumors are unclear. We investigated the contribution of epigenetic alterations to development of MSS HNPCC tumors.
Colorectal cancers were divided in four groups: 1. Microsatellite stable, Amsterdam positive (MSS HNPCC) (N=22); 2. Lynch syndrome cancers (identified mismatch repair mutations) (N=21); 3. Sporadic MSS (N=92); 4. Sporadic MSI (N=46). Methylation status was evaluated for CACNAG1, SOCS1, RUNX3, NEUROG1, MLH1, and LINE-1. KRAS and BRAF mutations status was analyzed.
MSS HNPCC tumors displayed a significantly lower degree of LINE-1 methylation, marker for global methylation, than any other group. Whereas most MSS HNPCC tumors had some degree of CpG island methylation, none presented a high index of methylation. MSS HNPCC tumors had KRAS mutations exclusively in codon 12, but none harbored V600E BRAF mutations.
Tumors from Amsterdam-positive patients without mismatch repair deficiency (MSS HNPCC) have certain molecular features, including global hypomethylation that distinguish them from all other colorectal cancers. These characteristics could have an important impact on tumor behavior or treatment response. Studies are underway to further assess the cause and effects of these features.
PMCID: PMC2859993  PMID: 20102720
Colorectal cancer; Microsatellite stable Hereditary Non-Polyposis Colorectal Cancer; Non-Lynch HNPCC; DNA Methylation; hypomethylation
22.  Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast 
BMC Cancer  2009;9:382.
Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells.
Yeast MLH1 and PMS1 genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested.
The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic.
Results of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene.
PMCID: PMC2773791  PMID: 19863800
23.  De novo constitutional MLH1 epimutations confer early-onset colorectal cancer in two new sporadic Lynch syndrome cases, with derivation of the epimutation on the paternal allele in one 
Lynch syndrome is an autosomal dominant cancer predisposition syndrome classically caused by germline mutations of the mismatch repair genes, MLH1, MSH2, MSH6 and PMS2. Constitutional epimutations of the MLH1 gene, characterized by soma-wide methylation of a single allele of the promoter and allelic transcriptional silencing, have been identified in a subset of Lynch syndrome cases lacking a sequence mutation in MLH1. We report two individuals with no family history of colorectal cancer who developed that disease at age 18 and 20 years. In both cases, cancer had arisen because of the de novo occurrence of a constitutional MLH1 epimutation and somatic loss-of-heterozygosity of the functional allele in the tumors. We show for the first time that the epimutation in one case arose on the paternally inherited allele. Analysis of 13 tumors from seven individuals with constitutional MLH1 epimutations showed eight tumors had lost the second MLH1 allele, two tumors had a novel pathogenic missense mutation and three had retained heterozygosity. Only 1 of 12 tumors demonstrated the BRAF V600E mutation and 3 of 11 tumors harbored a mutation in KRAS. The finding that epimutations can originate on the paternal allele provides important new insights into the mechanism of origin of epimutations. It is clear that the second hit in MLH1 epimutation-associated tumors typically has a genetic not epigenetic basis. Individuals with mismatch repair–deficient cancers without the BRAF V600E mutation are candidates for germline screening for sequence or methylation changes in MLH1.
PMCID: PMC3794437  PMID: 20473912
colorectal cancer; Lynch syndrome; MLH1 epimutation; microsatellite instability; BRAF
24.  Analysis of Families with Lynch Syndrome Complicated by Advanced Serrated Neoplasia: The Importance of Pathology Review and Pedigree Analysis 
Familial cancer  2009;8(4):313-323.
The identification of Lynch syndrome has been greatly assisted by the advent of tumour immunohistochemistry (IHC) for mismatch repair (MMR) proteins, and by the recognition of the role of acquired somatic BRAF mutation in sporadic MMR-deficient colorectal cancer (CRC). However, somatic BRAF mutation may also be present in the tumours in families with a predisposition to develop serrated polyps in the colorectum. In a subgroup of affected members in these families, CRCs emerge which demonstrate clear evidence of MMR deficiency with absent MLH1 staining and high-level microsatellite instability (MSI). This may result in these families being erroneously classified as Lynch syndrome or, conversely, an individual is considered “sporadic” due to the presence of a somatic BRAF mutation in a tumour. In this report, we describe two Lynch syndrome families who demonstrated several such inconsistencies. In one family, IHC deficiency of both MSH2 and MLH1 was demonstrated in tumours from different affected family members, presenting a confusing diagnostic picture. In the second family, MLH1 loss was observed in the lesions of both MLH1 mutation carriers and those who showed normal MLH1 germline sequence. Both families had Lynch syndrome complicated by an independently segregating serrated neoplasia phenotype, suggesting that in families such as these, tumour and germline studies of several key members, rather than of a single proband, are indicated to clarify the spectrum of risk.
PMCID: PMC2783449  PMID: 19241144
25.  Molecular signaling mechanisms of apoptosis in hereditary non-polyposis colorectal cancer 
Colorectal cancer is the second most leading cause of cancer related deaths in the western countries. One of the forms of colorectal cancer is hereditary non-polyposis colorectal cancer (HNPCC), also known as “Lynch syndrome”. It is the most common hereditary form of cancer accounting for 5%-10% of all colon cancers. HNPCC is a dominant autosomal genetic disorder caused by germ line mutations in mismatch repair genes. Human mismatch repair genes play a crucial role in genetic stability of DNA, the inactivation of which results in an increased rate of mutation and often a loss of mismatch repair function. Recent studies have shown that certain mismatch repair genes are involved in the regulation of key cellular processes including apoptosis. Thus, differential expression of mismatch repair genes particularly the contributions of MLH1 and MSH2 play important roles in therapeutic resistance to certain cytotoxic drugs such as cisplatin that is used normally as chemoprevention. An understanding of the role of mismatch repair genes in molecular signaling mechanism of apoptosis and its involvement in HNPCC needs attention for further work into this important area of cancer research, and this review article is intended to accomplish that goal of linkage of apoptosis with HNPCC. The current review was not intended to provide a comprehensive enumeration of the entire body of literature in the area of HNPCC or mismatch repair system or apoptosis; it is rather intended to focus primarily on the current state of knowledge of the role of mismatch repair proteins in molecular signaling mechanism of apoptosis as it relates to understanding of HNPCC.
PMCID: PMC3382705  PMID: 22737591
Colorectal cancer; Hereditary non-polyposis colorectal cancer; Apoptosis; Molecular signaling mechanisms; DNA mismatch repair proteins

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