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

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

doi:10.1186/1471-2350-14-103

Sanne M Petersen, Lotte N Krogh + Show 8 more

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

Highlights

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)
Lynch syndrome, 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 [1]
The MLH1, MSH2, and MSH6 proteins are involved in the repair of single base mismatches and short insertiondeletion loops that arise during DNA replication [3]

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Introduction

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. 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 [4,5,6,7]. 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

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