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: 35.29). 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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Available from: Daniela Seminara, Sep 26, 2015
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    Journal of Translational Medicine 06/2012; 10(1):128. DOI:10.1186/1479-5876-10-128 · 3.93 Impact Factor
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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 · 2.21 Impact Factor
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    • "RT-PCR analysis of the MLH1, c.116+5G > C mutation, which is located in intron 1, results in a 227-base pair insertion taking place between exons 1 and 2 (data not shown). The specific mutation has been reported twice in the literature, while both bioinformatic prediction tools and in vitro techniques (conversion/cDNA analysis) demonstrated that it activates a cryptic splice site [16,17]. On the other hand, the MLH1, c.790+1G > A mutation, which is located in intron 9, induces skipping of exons 9 and 10 [18]. "
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