This article has Open Peer Review reports available.
Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients
© Petersen et al.; licensee BioMed Central Ltd. 2013
Received: 11 March 2013
Accepted: 25 September 2013
Published: 3 October 2013
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.
Intronic MLH1, MSH2, or MSH6 variants were investigated using in silico prediction tools and mini-gene assay to asses the effect on splicing.
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).
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.
Lynch syndrome, also called hereditary nonpolyposis colorectal cancer (HNPCC), is an autosomal dominantly inherited cancer predisposition syndrome primarily associated with germ-line mutations in the MLH1 (MIM# 120436), MSH2 (MIM# 609309), and MSH6 (MIM# 600678) genes . Mutation carriers have an increased risk of several specific cancers, in particular colorectal, endometrial, small bowel, and ovarian cancer as well as uroepithelial tumors. The estimated lifetime risk of developing colorectal cancer with a pathogenic mutation in one of these genes is up to 70% , depending on the mutated mismatch repair gene and the gender of the patient.
The MLH1, MSH2, and MSH6 proteins are involved in the repair of single base mismatches and short insertion-deletion loops that arise during DNA replication . Mutations in MLH1, MSH2, and MSH6 are scattered throughout the genes (http://chromium.liacs.nl/LOVD2/colon_cancer/) and include frame-shift, nonsense, missense, and splice site mutations as well as large genomic rearrangements, of which several have been identified in Danish Lynch syndrome families [4–7]. However, a large number of MLH1, MSH2, and MSH6 missense, silent, and intronic mutations are of unknown clinical significance. It is clinically important to optimize the classification of these mutations into pathogenic mutations or benign polymorphisms in order to provide affected families with a more accurate risk assessment but also to offer predictive (presymptomatic) genetic testing to family members. The classification can be facilitated by performing functional assays (reviewed by ). In this study, we performed in silico analysis and functional examinations of nine intronic MLH1, MSH2, and MSH6 variants identified in Danish colorectal cancer patients enabling us to classify five mutations as pathogenic and four variants as neutral/polymorphisms.
Patients and clinical data
Following verbal and written consent blood samples were collected from the probands (all adults) and genetic screening was performed. Since the study is part of normal diagnostic procedures no ethical approval was obtained (H-4-2013-FSP-082). Clinical data regarding family phenotype, individual phenotype, cancer diagnosis, age at onset, adenomas, and age at adenomas (See Additional file 1) were obtained from the Danish HNPCC register. The study was conducted in accordance with the Helsinki Declaration.
MLH1, MSH2, and MSH6screening
Genomic DNA was purified from whole blood using Qiagen’s QIAamp DNA mini kit or Promega’s Maxwell DNA purification system according to the accompanying instructions. MLH1, MSH2, and MSH6 were amplified using intronic primer pairs flanking each exon. PCR products were sequenced using an ABI3730 DNA analyzer (Applied Biosystems). Moreover, genomic DNA was examined by MLPA analysis using kit P003 and P072 (MRC-Holland). Sequence variations, except well-known polymorphisms, were verified in a new blood sample. MLH1, MSH2, and MSH6 variants are numbered according to GenBank accession numbers NM_000249, NM_000251, and NM_000179, respectively. The nomenclature guidelines of the Human Genome Variation Society (http://www.hgvs.org/mutnomen) were used in all cases.
The following five splice site prediction programs were used to predict the effect of mutations on the efficiency of splicing: Splice Site Finder (http://www.interactive-biosoftware.com); GeneSplicer (http://www.cbcb.umd.edu/software/GeneSplicer); Splice Site Prediction by Neural Network (http://www.fruitfly.org/seq_tools/splice.html); MaxEntScan (http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html); and Human Splicing Finder (http://www.umd.be/HSF/). The analysis was performed by the integrated software Alamut V.2.2.1 (http://www.interactive-biosoftware.com). The genomic sequence spanning the individual mutations and nearby exons was submitted according to the guidelines of each program and default settings were used in all predictions. A variation of more than 10% in at least two algorithms was considered as having an effect on splicing .
Wild type exons along with at least 200 bp of 5′ and 3′ intronic sequences from MLH1, MSH2, and MSH6 were PCR amplified from human genomic DNA using Pwo DNA polymerase (Roche) and forward and reverse primers carrying restriction sites for BamHI or XhoI (primer sequences are available on request). PCR products were subcloned into the pSPL3 vector and all constructs were verified by sequencing. Single nucleotide substitutions or deletions were introduced using Finnzymes’ Phusion site-directed mutagenesis kit or Stratagene’s QuikChange II site-directed mutagenesis kit with PfuUltra high-fidelity DNA polymerase according to the accompanying instructions. Wild type and mutant constructs were transfected in duplicate into COS-7 cells as recently described . Cells were harvested after 48 hours and total RNA was extracted using NucleoSpin RNA/protein kits for total RNA and protein isolation (Macherey-Nagel). cDNA was synthesized using 1 μg/μl of RNA, M-MuLV reverse transcriptase polymerase (New England Biolabs), and 0.5 μg/μl of nucleotide oligo(dT)15 primer. cDNA was amplified with Pwo DNA polymerase using the primers dUSD2 (5′-TCTGAGTCACCTGGACAACC-3′) and dUSA4 (5′-ATCTCAGTGGTATTTGTGAGC-3′). PCR products were separated by electrophoresis on a 1% agarose gel containing ethidium bromide. Each DNA band was gel purified using GE Healthcare’s Illustra GFX PCR DNA and gel band purification kit and sequenced with dUSD2 and dUSA4 primers.
In silico prediction of the effect of mutations on splice donor (SD) or splice acceptor (SA) sites
c.117-34A > T
c.588 + 5G > A
c.677 + 3A > T
c.1039-8 T > A
c.1732-2A > T
c.1276 + 1G > T
c.1662-2A > C
c.3439-16C > T
Mutations located in the introns of mismatch repair genes can interfere with splicing and cause aberrant spliced mRNA transcripts leading to non-functional mismatch repair proteins. Several cis-acting elements, including the donor splice site, the acceptor splice site, the branch point, the polypyrimidine tract, and exonic/intronic splicing enhancers and silencers, are crucial for the splicing mechanism. The donor splice site consists of the conserved dinucleotides GT, whereas the acceptor splice site consists of three regions: the conserved dinucleotides AG, the polypyrimidine tract, and the branch point . Mutations in splicing motifs can lead to partial or complete skipping of the neighboring exon or inclusion of intronic sequence. Moreover, a mutation can create an ectopic splice site or activate a cryptic splice site, both of which are usually weak and only used when a mutation disrupts the normal splice site.
Ideally RNA from a patient should be examined by RT-PCR analysis to establish if a mutation has an effect on splicing. However, in many cases, RNA is not available from the patient. Alternatively, the mutation can be examined by mini-gene analysis . In fact, a high concordance between RT-PCR analysis and mini-gene assay has previously been observed [9, 13–15]. As an indicative examination prior to the mini-gene assay, several in silico prediction tools can be used to indicate which variants require further analysis.
In this study, we examined the effect on splicing of nine intronic variants identified in Danish colorectal cancer families by in silico analysis and in vitro using a mini-gene assay. The in silico analysis predicted altered splicing for MLH1 c.588 + 5G > A, MLH1 c.677 + 3A > T, MLH1 c.1039-8 T > A, MLH1 c.1732-2A > T, MSH2 c.1276 + 1G > T, MSH2 c.1662-2A > C, and MSH2 c.2459-18delT, whereas MLH1 c.117-34A > T and MSH6 c.3439-16C > T were predicted to have no effect on splicing. It should be noted that three mutations in our study (MLH1 c.1732-2A > T, MSH2 c.1276 + 1G > T, and MSH2 c.1662-2A > C) are located in the highly conserved donor and acceptor splice sites and hence they are easily predicted by in silico programs. However, mini-gene analysis revealed that the two mutations MLH1 c.1039-8 T > A and MSH2 c.2459-18delT had no effect on splicing, suggesting that the employed criterion (>10% difference between wild type and mutant scores in at least two programs) results in false-positive predictions as previously shown .
The effect on splicing determined by mini-gene assays and an overview of the mutations listed in the literature
Frequency in the ESP database (Eur. Am.)
Described in the literature
c.117-34A > T
No effect on splicing
c.588 + 5G > A
Out-of-frame skipping of exon 7
Pagenstecher; partial deletion of exon 7 
Tournier; deletion of exon 7 and exons 7–8 
c.677 + 3A > T
Out-of-frame skipping of exon 8
c.1039-8 T > A
No effect on splicing
Betz; No effect on splicing 
c.1732-2A > T
In-frame skipping of exon 16
c.1276 + 1G > T
In-frame exclusion of 48 bp of exon 7
c.1662-2A > C
Out-of-frame skipping of exon 11
No effect on splicing
c.3439-16C > T
No effect on splicing
Sanchez de Abajo* 
Overall, in all Amsterdam positive families - except one (H229) - a pathogenic mutation was identified. The index individual in family H229 had rectum cancer at age 58 and transverse colon cancer at age 66. His sister and two maternal cousins all had adenomas, while his mother has caecum cancer at age 48. Moreover his maternal aunt had transverse colon cancer at age 69 and his maternal grandmother had ascending colon cancer. The lack of a pathogenic mutation in this family could be due to an unidentified mutation in regions not previously examined, including the promoter region, the untranslated regions (UTRs) or deep intron sequences in the MLH1, MSH2 or MSH6 genes, or due to a mutation in other genes like PMS2. Future studies using exome sequencing might help identifying a putative pathogenic mutation in this family.
In conclusion, we have examined nine MLH1/MSH2/MSH6 intronic mutations by in silico and functional assays, thus enabling us to classify five mutations as pathogenic and four variants as neutral/polymorphisms. This study supports the notion that in silico prediction tools and mini-gene assays are important for the assessment of the pathogenicity of intronic variants, together with clinical data, IHC and MSI.
We thank Stine Østergaard, Dorte Pedersen, Merete Overgaard and Aseeba Ajub for technical assistance.
- Lynch HT, de la Chapelle A: Hereditary colorectal cancer. N Engl J Med. 2003, 348 (10): 919-932. 10.1056/NEJMra012242.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedPubMed CentralGoogle Scholar
- Hsieh P, Yamane K: DNA mismatch repair: molecular mechanism, cancer, and ageing. Mech Ageing Dev. 2008, 129 (7–8): 391-407.View ArticlePubMedPubMed CentralGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedPubMed CentralGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedPubMed CentralGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedPubMed CentralGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.PubMedPubMed CentralGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedPubMed CentralGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/14/103/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.