Genomic deletions of MSH2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach

Abstract

Hereditary non-polyposis colorectal cancer is an autosomal dominant condition due to germline mutations in DNA-mismatch-repair genes, in particular MLH1, MSH2 and MSH6. Here we describe the application of a novel technique for the detection of genomic deletions in MLH1 and MSH2. This method, called multiplex ligation-dependent probe amplification, is a quantitative multiplex PCR approach to determine the relative copy number of each MLH1 and MSH2 exon. Mutation screening of genes was performed in 126 colorectal cancer families selected on the basis of clinical criteria and in addition, for a subset of families, the presence of microsatellite instability (MSI-high) in tumours. Thirty-eight germline mutations were detected in 37 (29.4%) of these kindreds, 31 of which have a predicted pathogenic effect. Among families with MSI-high tumours 65.7% harboured germline gene defects. Genomic deletions accounted for 54.8% of the pathogenic mutations. A complete deletion of the MLH1 gene was detected in two families. The multiplex ligation-dependent probe amplification approach is a rapid method for the detection of genomic deletions in MLH1 and MSH2. In addition, it reveals alterations that might escape detection using conventional diagnostic techniques. Multiplex ligation-dependent probe amplification might be considered as an early step in the molecular diagnosis of hereditary non-polyposis colorectal cancer.

Figure 1

(A) Multiplex Ligation-dependent Probe Amplification (MLPA). Denatured genomic DNA (50–500 ng) is hybridised with a mixture of 42 probes. Each MLPA probe consists of two oligonucleotides. The two parts of each probe hybridise to adjacent target sequences and are ligated by a thermostable ligase. All probe ligation products are amplified simultaneously by PCR using a single primer pair. The amplification product of each probe has a unique length (130–472 bp). Amplification products are separated by capillary electrophoresis (ABI model 310 or ABI 3700). Relative amounts of probe amplification products reflect the relative copy number of target sequences. (B) Deletion of the entire MLH1 gene detected by MLPA. Normalised MLPA peak pattern from the index patient of family C149 (red) and from control DNA (blue) plotted in one figure for easy comparison. MLH1 peaks are labelled with their exon numbers. Unlabelled peaks represent MSH2 exons and control genes.

Figure 2

(A) Family C149. The index patient had a double colonic tumour at age 32, his father had colonic cancer preceded by skin tumours including Muir–Torre syndrome skin lesions (sebaceous gland adenoma, kerato-acanthoma, planocellular cancer). Patient 1-2 had primary cancers of the vulva and stomach. (B) Family C293. The index patient had colonic cancer at the age of 21 years. His unaffected mother had a tubular adenoma on colonoscopic screening, his maternal grandmother had late-onset colonic cancer. Family members I-2 (C149) and I-1 (C293) have a common ancestor.