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© 2002 American Society for Clinical Oncology
Multimodal Molecular Screening Is Required to Improve the Sensitivity of MLH1 and MSH2 Mutation Analysis
McGill University Health Centre, Montreal, Quebec, Canada To the Editor:In a recent article published in the Journal of Clinical Oncology, Percesepe et al1 concluded that the population-based prevalence of molecularly confirmed hereditary nonpolyposis colorectal cancer (HNPCC) is lower than that observed in previous studies. We suggest that the prevalence of mismatch repair (MMR) gene mutations in unselected series of individuals with colorectal cancer is considerably higher than has been reported here and elsewhere,1-3 and the low yields reported in these series are to a large extent explained by the laboratory techniques used. Of the 391 unselected patients with colorectal cancer recruited to the study of Percesepe et al,1 336 were screened for microsatellite instability, of which 28 were positive (8.3%). Twelve of the 28 cases were studied further, as were three microsatellite instability–negative cases that fulfilled the Amsterdam criteria (AC) for HNPCC. The mutation detection methods used to analyze MLH1 and MSH2 in these 15 cases were exon-by-exon sequencing (n = 10), single-strand conformation polymorphism (n = 5), and Southern blotting (n = 2). Altogether, only one germ-line mutation was identified (MSH2, exon 16, 2647delA), accounting for 0.3% (95% confidence interval [CI], 0.0075% to 1.6%) of the study population (n = 336), and 16.6% (95% CI, 0.4% to 64%) of cases fulfilling AC (n = 6). We agree with the authors that the low yield of mutations obtained in their study is likely due to their use of techniques that are unable to detect large deletions. We consider that their reported frequency of germ-line MMR mutations (0.3%) is a substantial underestimation of the actual mutation prevalence in unselected populations of colorectal cancer cases, and complementary methods for MMR gene mutation analysis may be required to provide a more accurate estimate. Our own experience with mutation identification in 19 HNPCC families fulfilling AC and ascertained between 1997 and 2001 is relevant to this discussion. Starting with both RNA and DNA and using established protocols, we searched for mutations using both the protein truncation test (PTT) and direct DNA sequencing. We identified 14 mutations (74%; 95% CI, 49% to 91%) (Table 1). Nine mutations were detected in MLH1, including four separate cDNA deletions of entire exons, three frameshift mutations, and two intron splice site mutations resulting in exon deletions. Five mutations were detected in MSH2, including two cDNA deletions of entire exons, two nonsense mutations, and one missense mutation. Overall, eight (57%) of the 14 mutations would have been detected by direct sequencing or single-strand conformation polymorphism alone, whereas all but one of the mutations were detected by PTT alone, indicating that for our study population, direct sequencing by itself would not have provided a high detection rate (Table 1).
Our findings support the growing literature that germ-line deletions are a frequent cause of HNPCC.4,5 These observations, taken together, suggest that whatever the study design, relying on exon-by-exon genomic DNA analysis1-3 will result in an underestimation of the frequency of disease-causing alterations in MMR genes in individuals with colorectal cancer. We therefore propose that traditional genomic DNA-based mutation detection methods should be complemented by PTT (also known as in vitro synthesized protein assay), semi-quantitative polymerase chain reaction using genomic DNA,5 or other related methods. In this way, alterations from subtle point mutations to large deletions will be detected. Multimodal mutation detection should become the gold standard in clinical genetic testing until such time as a single test is able to detect all the different types of MMR gene mutations. REFERENCES
1.
Percesepe A, Borghi F, Menigatti M, et al: Molecular screening for hereditary nonpolyposis colorectal cancer: A prospective, population based study. J Clin Oncol 19: 3944-3950, 2001
2.
Peel D, Ziogas A, Fox E, et al: Characterization of hereditary nonpolyposis colorectal cancer families from a population-based series of cases. J Natl Cancer Inst 92: 1517-1522, 2000 3. Cunningham J, Kim C-Y, Christensen E, et al: The frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas. Am J Hum Genet 69: 780-790, 2001[CrossRef][Medline] 4. Wijnen J, van der Klift H, Vasen H, et al: MSH2 genomic deletions are a frequent cause of HNPCC. Nat Genet 20: 326-328, 1998[CrossRef][Medline]
5.
Charbonnier F, Raux G, Wang Q, et al: Detection of exon deletions and duplications of the mismatch repair genes in hereditary nonpolyposis colorectal cancer families using multiplex polymerase chain reaction of short fluorescent fragments. Cancer Res 60: 2760-2763, 2000
Response
University of Modena, Modena, Italy In Reply:We would like to thank Dr Andermann et al for their comments and interest in our study. It gives us the opportunity to re-emphasize the risks for underestimating the frequency of hereditary nonpolyposis colorectal cancer (HNPCC), as thoroughly discussed in our article,1 and to share the point raised that a multitechnical approach to the molecular diagnosis of HNPCC is most desirable. In fact, we, too, applied a second method, namely Southern blot hybridization, for selected cases in our study, with the aim to detect large genomic deletions that would have been missed by exon-specific sequencing or single-strand conformational polymorphism. The authors also present their personal experience in mutation screening with an RNA-based technique (in vitro synthesized protein assay) together with direct DNA sequencing and propose this combined approach as the gold standard for mutation detection in HNPCC. The authors found six cDNA deletions (four in MLH1 and two in MSH2) that would have escaped detection by direct exon-specific sequencing. Although we agree that in vitro synthesized protein assay is useful as a rapid screening method, we would like to stress the importance of validating all cDNA changes at the genomic level. Apart from genomic deletion, cDNA changes could represent alternatively spliced isoforms, as previously demonstrated for both MSH2 and MLH1,2,3 and therefore, confirmation on genomic DNA is required, for example, for a mutation to be included in the ICG-HNPCC mutation database.4 Whereas technical shortcomings may result in the failure to detect some mutations, we would like to point out that a clear reduction of mutation detection rates is also seen when comparing HNPCC families not meeting the Amsterdam criteria with those that meet these criteria,5,6 suggesting that the prevalence of germline mutations in the mismatch repair genes strongly depends on the clinical presentation and method of ascertainment of the cases. REFERENCES 1. Percesepe A, Borghi F, Menigatti M, et al: Molecular screening for hereditary nonpolyposis colorectal cancer: A prospective, population-based study. J Clin Oncol 19: 3944-3950, 2001
2.
Charbonnier F, Martin C, Scotte M, et al: Alternative splicing of MLH1 messenger RNA in human normal cells. Cancer Res 55: 1839-1841, 1995 3. Genuardi M, Viel A, Bonora D, et al: Characterization of MLH1 and MSH2 alternative splicing and its relevance to molecular testing of colorectal cancer susceptibility. Hum Genet 102: 15-20, 1998[CrossRef][Medline] 4. Peltomäki P, Vasen HF: Mutations predisposing to hereditary nonpolyposis colorectal cancer: Database and results of a collaborative study—The International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer. Gastroenterology 113: 1146-1158, 1997[CrossRef][Medline]
5.
Nystrom-Lahti M, Wu Y, Moisio AL, et al: DNA mismatch repair gene mutations in 55 kindreds with verified or putative hereditary non-polyposis colorectal cancer. Hum Mol Genet 5: 763-769, 1996 6. Wijnen J, Khan PM, Vasen H, et al: Hereditary nonpolyposis colorectal cancer families not complying with the Amsterdam criteria show extremely low frequency of mismatch-repair-gene mutations. Am J Hum Genet 61: 329-335, 1997[Medline]
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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