Conversion analysis for mutation detection in MLH1 and MSH2 in patients with colorectal cancer.

Department of Cancer Biology, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio 44195, USA.
JAMA The Journal of the American Medical Association (Impact Factor: 30.39). 03/2005; 293(7):799-809. DOI: 10.1001/jama.293.7.799
Source: PubMed

ABSTRACT The accurate identification and interpretation of germline mutations in mismatch repair genes in colorectal cancer cases is critical for clinical management. Current data suggest that mismatch repair mutations are highly heterogeneous and that many mutations are not detected when conventional DNA sequencing alone is used.
To evaluate the potential of conversion analysis compared with DNA sequencing alone to detect heterogeneous germline mutations in MLH1, MSH2, and MSH6 in colorectal cancer patients.
Multicenter study with patients who participate in the Colon Cancer Family Registry. Mutation analyses were performed in participant samples determined to have a high probability of carrying mismatch repair germline mutations. Samples from a total of 64 hereditary nonpolyposis colorectal cancer cases, 8 hereditary nonpolyposis colorectal cancer-like cases, and 17 cases diagnosed prior to age 50 years were analyzed from June 2002 to June 2003.
Classification of family members as carriers or noncarriers of germline mutations in MLH1, MSH2, or MSH6; mutation data from conversion analysis compared with genomic DNA sequencing.
Genomic DNA sequencing identified 28 likely deleterious exon mutations, 4 in-frame deletion mutations, 16 missense changes, and 22 putative splice site mutations. Conversion analysis identified all mutations detected by genomic DNA sequencing--plus an additional exon mutation, 12 large genomic deletions, and 1 exon duplication mutation--yielding an increase of 33% (14/42) in diagnostic yield of deleterious mutations. Conversion analysis also showed that 4 of 16 missense changes resulted in exon skipping in transcripts and that 17 of 22 putative splice site mutations affected splicing or mRNA transcript stability. Conversion analysis provided an increase of 56% (35/63) in the diagnostic yield of genetic testing compared with genomic DNA sequencing alone.
The data confirm the heterogeneity of mismatch repair mutations and reveal that many mutations in colorectal cancer cases would be missed using conventional genomic DNA sequencing alone. Conversion analysis substantially increases the diagnostic yield of genetic testing for mismatch repair mutations in patients diagnosed as having colorectal cancer.

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    • "This should lead to an increase in demand for mutation screening (Terdiman, 2005). The situation is complicated by the realization that a substantial proportion (30%) of mutations may not be detectable using routine sequencing (Casey et al., 2005), while epigenetic inactivation of one allele can cause cancer predisposition and may even be transmissible through the germline (Suter et al., 2004; Hitchins et al., 2007; Valle et al., 2007). Approximately 50% of known mutations in MLH1 and MSH2 result in premature termination of the encoded proteins , and may trigger nonsense mediated decay (NMD), the preferential degradation of mRNA molecules containing a mutation that leads to premature termination (Weischenfeldt et al., 2005; Amrani et al., 2006; Behm-Ansmant & Izaurralde, 2006). "
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    ABSTRACT: Germline defects in the MLH1 gene are associated with Lynch syndrome. A substantial proportion of these mutations leads to premature termination codons and can induce nonsense mediated decay (NMD) of the corresponding transcript. Resulting allelic expression differences represent a fast and inexpensive method to identify patients carrying MLH1 mutations. In patients and controls, we show that allelic expression imbalance (AEI) can be readily detected in RNA extracted from whole blood from patients carrying mutations expected to elicit NMD using mass spectrometry. Mutations closer to the 5' end of the gene tend to show smaller imbalances. AEI can also be detected in normal controls. Analysis of allelic expression in controls and individuals with mutations not expected to exhibit NMD revealed that MLH1 expression is influenced by sequence variation acting in cis. A maximum likelihood framework was used to identify two SNPs, rs1799977 (c.655G>A; p.I219V) and rs1800734 (c.-93 G>A) that are independently associated with expression. These influences are, however, small compared to the differences associated with pathological variants.
    Annals of Human Genetics 11/2010; 74(6):479-88. DOI:10.1111/j.1469-1809.2010.00603.x · 1.93 Impact Factor
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    • "For years it is known that oncogenic mutations and suppression can manipulate the development of cancer. Specific mutations can be identified using large-scale sequencing of the genome in many cancers (Capella et al., 1991; Casey et al., 2005; Frank et al., 1999; Li et al., 1998). The type of mutation in a tumor can be a better source to have a choice of treatment. "
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    ABSTRACT: The completion of human genome project has evolved many techniques used to locate the human genes. The focus is mainly on the genome, transcriptome or proteome to recognise distinctive characteristics that may explain the basis of human disease and potentially envisage prospect outcomes. Cancer is one of the recent deadliest diseases. Various cancer types root problems in the generalised dealing. The objective of these investigative pursuits is to ultimately individualize treatment for each patient based on their exclusive gene expression prototypes.
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    • "The single report for MLH1 c.116+5 G>C used conversion analysis to identify a 227 nucleotide inclusion of intron 1 (Casey, et al., 2005), as we have done here using relatively straightforward experimental methods. The single report for MLH1 c.208-3C>G provided evidence from functional analysis of LCL-derived cDNA for an in-frame deletion of exon 3 (Otway, et al., 2005), confirmed in this study. "
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    ABSTRACT: Reliable methods for predicting functional consequences of variants in disease genes would be beneficial in the clinical setting. This study was undertaken to predict, and confirm in vitro, splicing aberrations associated with mismatch repair (MMR) variants identified in familial colon cancer patients. Six programs were used to predict the effect of 13 MLH1 and 6 MSH2 gene variants on pre-mRNA splicing. mRNA from cycloheximide-treated lymphoblastoid cell lines of variant carriers was screened for splicing aberrations. Tumors of variant carriers were tested for microsatellite instability and MMR protein expression. Variant segregation in families was assessed using Bayes factor causality analysis. Amino acid alterations were examined for evolutionary conservation and physicochemical properties. Splicing aberrations were detected for 10 variants, including a frameshift as a minor cDNA product, and altered ratio of known alternate splice products. Loss of splice sites was well predicted by splice-site prediction programs SpliceSiteFinder (90%) and NNSPLICE (90%), but consequence of splice site loss was less accurately predicted. No aberrations correlated with ESE predictions for the nine exonic variants studied. Seven of eight missense variants had normal splicing (88%), but only one was a substitution considered neutral from evolutionary/physicochemical analysis. Combined with information from tumor and segregation analysis, and literature review, 16 of 19 variants were considered clinically relevant. Bioinformatic tools for prediction of splicing aberrations need improvement before use without supporting studies to assess variant pathogenicity. Classification of mismatch repair gene variants is assisted by a comprehensive approach that includes in vitro, tumor pathology, clinical, and evolutionary conservation data.
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