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-mismatchrepair 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.

Distinct patterns of germ-line deletions inMLH1 andMSH2: the implication of Alu repetitive element in the genetic etiology of Lynch syndrome (HNPCC)

“…The role of genomic rearrangements in the etiology of HNPCC has been under-investigated, due to the lack of technically simple and reliable approaches for detecting this type of mutation. Recently, with the development of PCR-based semi-quantitative approaches for detecting gene dosage, it has become apparent that a substantial proportion of HNPCC cases are associated with germ-line genomic rearrangements in the MMR genes, mainly MLH1and MSH2 (Papadopoulos et al, 1994;Nystrom-Lahti et al, 1995;Mauillon et al, 1996;Wijnen et al, 1998;Charbonnier et al, 2000;Charbonnier et al, 2002;Gille et al, 2002;Viel et al, 2002;Wang et al, 2002;Nakagawa et al, 2003;Plaschke et al, 2003;Pyatt et al, 2003;Taylor et al, 2003;Wang et al, 2003;Di Fiore et al, 2004;Miyaki et al, 2004;Thiffault et al, 2004;Casey et al, 2005). A germ-line genomic deletion in HNPCC was first identified as a founder mutation at MLH1 among the Finnish population (Nystrom-Lahti et al, 1995).…”

A founder MLH1 mutation in Lynch syndrome families from Piedmont, Italy, is associated with an increased risk of pancreatic tumours and diverse immunohistochemical patterns

“…PCR products with heteroduplex profiles were sequenced on an ABI 3100 Avant sequencer (Applied Biosystems, Foster City, CA, USA). Large deletions were screened using SALSA MLPA KIT P003 MLH1/MSH2 [28], according to the manufacturer's instructions (MRC-Holland, Amsterdam, The Netherlands). Genotyping of the 278 controls was performed by DHPLC analysis.…”

High‐Resolution Analysis of Genomic Copy Number Changes

“…These include detection of trisomies [35], Duchene and Becker muscular dystrophy [36], centrifugal muscular dystrophy and also detection of deletions and duplications of one or more exons of the BRCA1 [37] and the MLH1 /MSH2 genes [32]. However, the protocols described in this chapter are robust and specific enough to be used as a routine diagnostic assay.…”

“…MLPA is a multiplex technique for determining copy numbers of genomic DNA sequences [32] and promoter methylation status [33], as well as mRNA profiling [34]. While genomic profiling is becoming increasingly important in both the research and diagnostic setting, routine detection methods for exon deletions and duplications are still lacking.…”

“…MLPA kits for the most common hereditary genetic disorders are already available, such as: Down's syndrome (see Figure 1.4), breast (BRCA1 ) [37] and colon cancer [32] (MSH2 and MLH1 ), Duchenne [36], cystic fibrosis, centrifugal muscular atrophy, sickle cell anemia and Marfan syndrome, to name but a few. However, future MLPA kits will focus more on the detection of aberrations of genes playing roles in tumorigenesis, apoptosis, angiogenesis and pharmacogenetics.…”