Publications (4)14.68 Total impact
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Article: Biochemical characterization of MLH3 missense mutations does not reveal an apparent role of MLH3 in Lynch syndrome.
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ABSTRACT: So far 18 MLH3 germline mutations/variants have been identified in familial colorectal cancer cases. Sixteen of these variants are amino acid substitutions of which the pathogenic nature is still unclear. These substitutions are known as unclassified variants or UVs. To clarify a possible role for eight of these MLH3 UVs identified in suspected Lynch syndrome patients, we performed several biochemical tests. We determined the protein expression and stability, protein localization and interaction of the mutant MLH3 proteins with wildtype MLH1. All eight MLH3 UVs gave protein expression levels comparable with wildtype MLH3. Furthermore, the UV-containing proteins, in contrast to previous studies, were all localized normally in the nucleus and they interacted normally with wildtype MLH1. Our different biochemical assays yielded no evidence that the eight MLH3 UVs tested are the cause of hereditary colorectal cancer, including Lynch syndrome.Genes Chromosomes and Cancer 02/2009; 48(4):340-50. · 3.31 Impact Factor -
Article: A database to support the interpretation of human mismatch repair gene variants.
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ABSTRACT: Germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2 can cause Lynch syndrome. This syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC), is an autosomal dominantly-inherited disorder predominantly characterized by colorectal and endometrial cancer. Truncating MMR gene mutations generally offer a clear handle for genetic counseling and allow for presymptomatic testing. In contrast, the clinical implications of most missense mutations and small in-frame deletions detected in patients suspected of having Lynch syndrome are unclear. We have constructed an online database, the Mismatch Repair Gene Unclassified Variants Database (www.mmruv.info), for information on the results of functional assays and other findings that may help in classifying these MMR gene variants. Ideally, such mutations should be clinically classified by a broad expert panel rather than by the individual database curators. In addition, the different MMR gene mutation databases could be interlinked or combined to increase user-friendliness and avoid unnecessary overlap between them. Both activities are presently being organized by the International Society for Gastrointestinal Hereditary Tumours (InSiGHT; www.insight-group.org).Human Mutation 11/2008; 29(11):1337-41. · 5.69 Impact Factor -
Article: Functional analysis helps to clarify the clinical importance of unclassified variants in DNA mismatch repair genes.
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ABSTRACT: Hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome is caused by DNA variations in the DNA mismatch repair (MMR) genes MSH2, MLH1, MSH6, and PMS2. Many of the mutations identified result in premature termination of translation and thus in loss-of-function of the encoded mutated protein. These DNA variations are thought to be pathogenic mutations. However, some patients carry other DNA mutations, referred to as unclassified variants (UVs), which do not lead to such a premature termination of translation; it is not known whether these contribute to the disease phenotype or merely represent rare polymorphisms. This is a major problem which has direct clinical consequences. Several criteria can be used to classify these UVs, such as: whether they segregate with the disease within pedigrees, are absent in control individuals, show a change of amino acid polarity or size, provoke an amino acid change in a domain that is evolutionary conserved and/or shared between proteins belonging to the same protein family, or show altered function in an in vitro assay. In this review we discuss the various functional assays reported for the HNPCC-associated MMR proteins and the outcomes of these tests on UVs identified in patients diagnosed with or suspected of having HNPCC. We conclude that a large proportion of MMR UVs are likely to be pathogenic, suggesting that missense variants of MMR proteins do indeed play a role in HNPCC.Human Mutation 12/2007; 28(11):1047-54. · 5.69 Impact Factor -
Article: Functional analysis and carrier detection of mismatch repair gene mutations
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ABSTRACT: In this thesis we present a number of studies related to the classification of missense mutations in Lynch syndrome and we tested an alternative, tumorindependent approach for the identification of mismatch repair (MMR) mutation carriers. Identification of MMR mutation carriers. Identification of MMR-gene mutation carriers and enrolling mutation carriers in surveillance programs for the early detection of tumors has been shown to remarkably reduce the risk of colon cancer in the identified mutation carriers and of mortality due to colon cancer. Identification of mutation carriers, therefore, has been given considerable attention. However, as mutation analysis of all MMR genes is timeconsuming and costly, several clinical selection criteria and laboratory prescreening methods have been developed which help to decide whether or not a patient should be screened for mutations. The Dutch guidelines for the selection of patients for mutation analysis are based on the age at diagnosis, microsatellite instability screening, immunohistochemical screening of the MMR proteins in the tumor, methylation screening of the MLH1 promoter and BRAF mutation screening. When a tumor is microsatellite instable (MSI-H) and/or immunohistochemistry is negative for MSH2 or MSH6, mutation analysis will be performed. When MLH1 is negative by immunohistochemistry, tumors are first tested for a BRAF mutation and MLH1 promoter hypermethylation. Tumors with MLH1 promoter hypermethylation and a BRAF mutation will not be tested for germline mutations as this is evidence that points towards sporadic cases. Tumors without MLH1 promoter hypermethylation and without a BRAF mutation will be screened for a MMR mutation. When no tumor material (and or no DNA) from a patient or another affected family member is available one generally needs to screen all Lynch associated MMR genes for mutations often in multiple family members. We worked on an approach for which no tumor material is necessary and which is based on the expression levels of the MMR proteins/genes in leukocytes. What we were hoping to develop was a method that could detect the loss of the mutated MMR allele (haploinsuffiency) based on gene- or protein-expression levels. Haploinsuffiency would point directly to the mutated gene (in the germline) thereby saving time and money. We show (Chapter 2) that MMR gene expression in blood leukocytes is extremely low and that a direct analysis on blood leukocyte samples is therefore not possible. However, after a short-term culture of leukocytes the expression level of the MMR genes is boosted making it possible to measure MMR protein or mRNA levels. Unfortunately, large deviations in expression levels, both at the RNA as well as at the protein level were detected in proven mutation carriers and healthy controls. We therefore had to conclude that this method could not be used for the accurate detection of MMR mutation carriers. Classification of missense mutations in Lynch syndrome Finding a DNA variant is one issue, deciding whether a DNA variant is pathogenic, in other words whether it is contributing to the disease phenotype is another. This decision is easily made for mutations that result in premature termination of translation and thus in loss of function. For mutations that do not lead to a truncated protein this decision is much more difficult. This type of DNA variants, consisting mostly of missense mutations, is usually called unclassified variant (UV). These UVs form a significant proportion of the DNA variants found in Lynch syndrome (suspected) patients. They consist of DNA variants that give rise to single amino acid substitutions or small in-frame deletions (~10% of both MSH2 and MLH1 mutations). The question whether a UV contributes to the disease phenotype or merely represents a rare polymorphism, constitutes a major problem with obvious direct clinical consequences. In practice, it is difficult to determine pathogenecity. Most evidence is indirect such as segregation data, MSI, immunohistochemistry or absence of the UV in controls. We therefore believe that most proof for pathogenicity should come from functional assays. In chapter 3 all published functional assays applied to studying MMR variants and the test outcome are reviewed. This information has been stored in an online database (www.mmrmissense.info) (see chapter 4). Although the database contains data on over a 100 functionally tested UVs, a much larger set of UVs has never been tested. One subset of mutations never tested are those identified in MLH3. Until now, 18 MLH3 germline mutations/UVs been identified in colorectal cancer cases suspected of Lynch syndrome. Sixteen of these are UVs, all single amino acid substitutions, for which the pathogenic nature is yet unclear. In chapter 5, we functionally tested 8 MLH3 missense variants that we had previously identified in Lynch syndrome suspected patients. We determined protein expression and stability, protein localization and interaction of the mutant MLH3 proteins with MLH1. All 8 mutant MLH3 proteins were expressed at levels comparable to wild type MLH3. Furthermore, the mutant proteins all localized normally to the nucleus and they interacted normally with wild type MLH1. We therefore have found no proof that the 8 missense variants tested are involved in colon cancer and thus in Lynch syndrome. Another subgroup of UVs that is underrepresented in the set of functionally tested UVs are those in MSH6. In chapter 6, functional assays have been performed to evaluate the pathogenicity of 5 of such inherited MSH6 UVs found in patients suspected of Lynch syndrome. The mutated MSH6 proteins, all containing single amino acid substitutions, were tested for expression and stability, interaction with MSH2, and for the subcellular localization. It was shown that protein expression of 4 of the 5 MSH6 mutants (S144I, A1021D, A326V, and T1219I) was significantly decreased after transfection when compared with expression of the wild type MSH6. Quantitative PCR analyses of hMSH6 mRNA after transfection showed that possibly this could be due to lower mRNA levels. No effects were observed on MSH2-MSH6 protein-protein interaction and the subcellular localization was normal for all 5 MSH6 missense mutants. Our data show that 4 of the 5 tested MSH6 UVs seem to have an influence on gene expression and thereby on protein translation. Why the expression of these 4 UVs in vivo is reduced is yet unknown. These UVs might therefore be pathogenic. Further studies are needed to confirm our findings.
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2007–2009
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University of Groningen
- Department of Genetics
Groningen, Province of Groningen, Netherlands
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