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Open Access 01-12-2013 | Research article

Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients

Authors: Sanne M Petersen, Mette Dandanell, Lene J Rasmussen, Anne-Marie Gerdes, Lotte N Krogh, Inge Bernstein, Henrik Okkels, Friedrik Wikman, Finn C Nielsen, Thomas v O Hansen

Published in: BMC Medical Genetics | Issue 1/2013

Abstract

Background

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.

Methods

Intronic MLH1, MSH2, or MSH6 variants were investigated using in silico prediction tools and mini-gene assay to asses the effect on splicing.

Results

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).

Conclusions

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.

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Lynch HT, de la Chapelle A: Hereditary colorectal cancer. N Engl J Med. 2003, 348 (10): 919-932. 10.1056/NEJMra012242.CrossRefPubMed

Stoffel E, Mukherjee B, Raymond VM, Tayob N, Kastrinos F, Sparr J, Wang F, Bandipalliam P, Syngal S, Gruber SB: Calculation of risk of colorectal and endometrial cancer among patients with Lynch syndrome. Gastroenterology. 2009, 137 (5): 1621-1627. 10.1053/j.gastro.2009.07.039.CrossRefPubMedPubMedCentral

Hsieh P, Yamane K: DNA mismatch repair: molecular mechanism, cancer, and ageing. Mech Ageing Dev. 2008, 129 (7–8): 391-407.CrossRefPubMedPubMedCentral

Bisgaard ML, Jager AC, Myrhoj T, Bernstein I, Nielsen FC: Hereditary non-polyposis colorectal cancer (HNPCC): phenotype-genotype correlation between patients with and without identified mutation. Hum Mutat. 2002, 20 (1): 20-27. 10.1002/humu.10083.CrossRefPubMed

Jager AC, Bisgaard ML, Myrhoj T, Bernstein I, Rehfeld JF, Nielsen FC: Reduced frequency of extracolonic cancers in hereditary nonpolyposis colorectal cancer families with monoallelic hMLH1 expression. Am J Hum Genet. 1997, 61 (1): 129-138. 10.1086/513896.CrossRefPubMedPubMedCentral

Jager AC, Rasmussen M, Bisgaard HC, Singh KK, Nielsen FC, Rasmussen LJ: HNPCC mutations in the human DNA mismatch repair gene hMLH1 influence assembly of hMutLalpha and hMLH1-hEXO1 complexes. Oncogene. 2001, 20 (27): 3590-3595. 10.1038/sj.onc.1204467.CrossRefPubMed

Nilbert M, Wikman FP, Hansen TV, Krarup HB, Orntoft TF, Nielsen FC, Sunde L, Gerdes AM, Cruger D, Timshel S, et al: Major contribution from recurrent alterations and MSH6 mutations in the Danish Lynch syndrome population. Fam Cancer. 2009, 8 (1): 75-83. 10.1007/s10689-008-9199-3.CrossRefPubMed

Rasmussen LJ, Heinen CD, Royer-Pokora B, Drost M, Tavtigian S, Hofstra RM, de Wind N: Pathological assessment of mismatch repair gene variants in Lynch syndrome: Past, present, and future. Hum Mutat. 2012, 33 (12): 1617-1625. 10.1002/humu.22168.CrossRefPubMed

Thery JC, Krieger S, Gaildrat P, Revillion F, Buisine MP, Killian A, Duponchel C, Rousselin A, Vaur D, Peyrat JP, et al: Contribution of bioinformatics predictions and functional splicing assays to the interpretation of unclassified variants of the BRCA genes. Eur J Hum Genet. 2011, 19 (10): 1052-1058. 10.1038/ejhg.2011.100.CrossRefPubMedPubMedCentral

10.

Hansen TV, Steffensen AY, Jonson L, Andersen MK, Ejlertsen B, Nielsen FC: The silent mutation nucleotide 744 G - > A, Lys172Lys, in exon 6 of BRCA2 results in exon skipping. Breast Cancer Res Treat. 2010, 119 (3): 547-550. 10.1007/s10549-009-0359-4.CrossRefPubMed

11.

Cartegni L, Chew SL, Krainer AR: Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet. 2002, 3 (4): 285-298. 10.1038/nrg775.CrossRefPubMed

12.

Baralle D, Baralle M: Splicing in action: assessing disease causing sequence changes. J Med Genet. 2005, 42 (10): 737-748. 10.1136/jmg.2004.029538.CrossRefPubMedPubMedCentral

13.

Bonnet C, Krieger S, Vezain M, Rousselin A, Tournier I, Martins A, Berthet P, Chevrier A, Dugast C, Layet V, et al: Screening BRCA1 and BRCA2 unclassified variants for splicing mutations using reverse transcription PCR on patient RNA and an ex vivo assay based on a splicing reporter minigene. J Med Genet. 2008, 45 (7): 438-446. 10.1136/jmg.2007.056895.CrossRefPubMed

14.

Schneider B, Koppius A, Sedlmeier R: Use of an exon-trapping vector for the evaluation of splice-site mutations. Mamm Genome. 2007, 18 (9): 670-676. 10.1007/s00335-007-9047-z.CrossRefPubMed

15.

Tournier I, Vezain M, Martins A, Charbonnier F, Baert-Desurmont S, Olschwang S, Wang Q, Buisine MP, Soret J, Tazi J, et al: A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects. Hum Mutat. 2008, 29 (12): 1412-1424. 10.1002/humu.20796.CrossRefPubMed

16.

Betz B, Theiss S, Aktas M, Konermann C, Goecke TO, Moslein G, Schaal H, Royer-Pokora B: Comparative in silico analyses and experimental validation of novel splice site and missense mutations in the genes MLH1 and MSH2. J Cancer Res Clin Oncol. 2010, 136 (1): 123-134. 10.1007/s00432-009-0643-z.CrossRefPubMed

17.

Pagenstecher C, Wehner M, Friedl W, Rahner N, Aretz S, Friedrichs N, Sengteller M, Henn W, Buettner R, Propping P, et al: Aberrant splicing in MLH1 and MSH2 due to exonic and intronic variants. Hum Genet. 2006, 119 (1–2): 9-22.CrossRefPubMed

18.

Kruger S, Bier A, Plaschke J, Hohl R, Aust DE, Kreuz FR, Pistorius SR, Saeger HD, Rothhammer V, Al-Taie O, et al: Ten novel MSH2 and MLH1 germline mutations in families with HNPCC. Hum Mutat. 2004, 24 (4): 351-352.CrossRefPubMed

19.

Kruger S, Plaschke J, Jeske B, Gorgens H, Pistorius SR, Bier A, Kreuz FR, Theissig F, Aust DE, Saeger HD, et al: Identification of six novel MSH2 and MLH1 germline mutations in HNPCC. Hum Mutat. 2003, 21 (4): 445-446.CrossRefPubMed

20.

Wijnen J, Khan PM, Vasen H, Menko F, van der Klift H, van den Broek M, van Leeuwen-Cornelisse I, Nagengast F, Meijers-Heijboer EJ, Lindhout D, et al: Majority of hMLH1 mutations responsible for hereditary nonpolyposis colorectal cancer cluster at the exonic region 15–16. Am J Hum Genet. 1996, 58 (2): 300-307.PubMedPubMedCentral

21.

Mangold E, Pagenstecher C, Friedl W, Fischer HP, Merkelbach-Bruse S, Ohlendorf M, Friedrichs N, Aretz S, Buettner R, Propping P, et al: Tumours from MSH2 mutation carriers show loss of MSH2 expression but many tumours from MLH1 mutation carriers exhibit weak positive MLH1 staining. J Pathol. 2005, 207 (4): 385-395. 10.1002/path.1858.CrossRefPubMed

22.

Perez-Cabornero L, Velasco E, Infante M, Sanz D, Lastra E, Hernandez L, Miner C, Duran M: A new strategy to screen MMR genes in Lynch Syndrome: HA-CAE, MLPA and RT-PCR. Eur J Cancer. 2009, 45 (8): 1485-1493. 10.1016/j.ejca.2009.01.030.CrossRefPubMed

23.

Pinto C, Veiga I, Pinheiro M, Mesquita B, Jeronimo C, Sousa O, Fragoso M, Santos L, Moreira-Dias L, Baptista M, et al: MSH6 germline mutations in early-onset colorectal cancer patients without family history of the disease. Br J Cancer. 2006, 95 (6): 752-756. 10.1038/sj.bjc.6603318.CrossRefPubMedPubMedCentral

24.

Sanchez de Abajo A, de la Hoya M, Tosar A, Godino J, Fernandez JM, Asenjo JL, Villamil BP, Segura PP, Diaz-Rubio E, Caldes T: Low prevalence of germline hMSH6 mutations in colorectal cancer families from Spain. World J Gastroenterol. 2005, 11 (37): 5770-5776.CrossRefPubMed

The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/14/103/prepub

Title: Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients
Authors: Sanne M Petersen
Mette Dandanell
Lene J Rasmussen
Anne-Marie Gerdes
Lotte N Krogh
Inge Bernstein
Henrik Okkels
Friedrik Wikman
Finn C Nielsen
Thomas v O Hansen
Publication date: 01-12-2013
Publisher: BioMed Central
Published in: BMC Medical Genetics / Issue 1/2013
Electronic ISSN: 1471-2350
DOI: https://doi.org/10.1186/1471-2350-14-103

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Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients

Abstract

Background

Methods

Results

Conclusions

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

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