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

BMC Medical Genetics

Thomas v O Hansen⁷  Finn C Nielsen⁷  Friedrik Wikman⁴  Henrik Okkels³  Inge Bernstein²  Lotte N Krogh¹  Anne-Marie Gerdes⁶  Lene J Rasmussen⁵  Mette Dandanell⁷  Sanne M Petersen⁷ 

[1] Department of Clinical Genetics, Odense University Hospital, Odense, Denmark;The Danish HNPCC register, Department of Gastroenterology and Clinical Research, University of Copenhagen, Hvidovre, Denmark;Section of Molecular Diagnostics, Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark;Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark;Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark;Department of Clinical Genetics, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark;Center for Genomic Medicine, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, Denmark

关键词: Splicing defect;    Mismatch repair genes MLH1, MSH2, and MSH6;    Mini-gene assay;    Lynch syndrome;    HNPCC;    Colorectal cancer;   

Others  :  1122614
DOI  :  10.1186/1471-2350-14-103

 received in 2013-03-11, accepted in 2013-09-25,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Petersen et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

【 参考文献 】

• [1]Lynch HT, de la Chapelle A: Hereditary colorectal cancer. N Engl J Med 2003, 348(10):919-932.
• [2]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.
• [3]Hsieh P, Yamane K: DNA mismatch repair: molecular mechanism, cancer, and ageing. Mech Ageing Dev 2008, 129(7–8):391-407.
• [4]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.
• [5]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.
• [6]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.
• [7]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.
• [8]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.
• [9]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]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.
• [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.
• [12]Baralle D, Baralle M: Splicing in action: assessing disease causing sequence changes. J Med Genet 2005, 42(10):737-748.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.
• [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.