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Open Access Highly Accessed Research article

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

Sanne M Petersen1, Mette Dandanell1, Lene J Rasmussen2, Anne-Marie Gerdes3, Lotte N Krogh4, Inge Bernstein5, Henrik Okkels6, Friedrik Wikman7, Finn C Nielsen1 and Thomas v O Hansen1*

  • * Corresponding author: Thomas v O Hansen

Author Affiliations

1 Center for Genomic Medicine, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, Denmark

2 Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark

3 Department of Clinical Genetics, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

4 Department of Clinical Genetics, Odense University Hospital, Odense, Denmark

5 The Danish HNPCC register, Department of Gastroenterology and Clinical Research, University of Copenhagen, Hvidovre, Denmark

6 Section of Molecular Diagnostics, Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark

7 Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark

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BMC Medical Genetics 2013, 14:103  doi:10.1186/1471-2350-14-103

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.

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