Large genomic alterations in hMSH2 and hMLH1 in early‐onset colorectal cancer: identification of a large complex de novo hMLH1 alteration

To the Editor: The Victorian Colorectal Cancer Family Study is a population-based, case-family study of earlyonset colorectal cancer conducted in Australia during 1993–1997 (1). Eligible case patients comprised adult men and women living in the Melbourne metropolitan area who were under the age of 45 years when diagnosed with a histologically confirmed first primary adenocarcinoma of the colon or rectum (ICD-9 rubrics 153 and 154, respectively) who were identified through the population-wide Victorian Cancer Registry. Invasive tumour samples of primary colorectal adenocarcinoma were obtained from hospitals and private pathology laboratories for 118 case patients (90% of those recruited). The tumour protein expression of MLH1, MSH2, MSH6 and PMS2 was assessed as described and reported elsewhere (2). Microsatellite instability (MSI) was assessed utilizing a five-microsatellite marker panel (3, 4). All exonic and flanking intronic sequences of hMLH1, hMSH2, hMSH6 and hPMS2were screened for germline mutations using sequencing approaches, except for exon 4 of hMSH6 that was screened in eight overlapping fragments using denaturing high-performance liquid chromatography (DHPLC) (2). The multiplex ligation-dependent probe amplification (MLPA) assay (MRC-Holland, Amsterdam, The Netherlands) (5) was used to detect large genomic alterations in hMLH1 and hMSH2 and was performed on samples from 10 case patients that had tumours lacking expression of at least one mismatch repair (MMR) protein and for which no mutation had been identified byDNA-sequencing methods or DHPLC. Breakpoints were characterized by performing a long-range PCR encompassing the predicted genomic alteration, cloning and sequencing. PCR primer sequences and PCR product sizes are given in Table 1 panel a. The germline DNA from two individuals were found to have MLPA profiles consistent with having a large deletion in hMLH1. No alterations were identified in hMSH2. Case 1 was a male diagnosed with cancer of the transverse colon at the age of 24 years, with no reported family history of colorectal cancer in any firstor second-degree relative. The endoscopic biopsywasMSI high, displaying instability in four of the five markers tested (Bat25, Bat26, D5S345 and D17S250). The tumour expressed MSH2 and MSH6 protein, lacked the expression of PMS2, and some focal cells were negative for MLH1 as determined by immuno-histo-chemistry (IHC). MLPA analysis suggested that exon 15 of hMLH1 was deleted in the germline. Long-range PCR confirmed the deletion by identifying the breakpoint, a deletion of 1444 bp and a 264–269 bp insertion. Specifically, the genomic change was hMLH1, 50334del1444 and 50334ins264–269 (reference sequence accessionnumberAY217549). The insertion of 264–269 bp between the breakpoints identifiedhas99%homologyto theAlu-repeat subfamily Sb2 (accession number U14570). This complex mutation is illustrated in Fig. 1(a). A specific test was devised utilizing long-range PCR over the breakpoint region, and other participating family members were screened for hMLH1, 50334del1444ins264–269 (Table 1 panel a). Neither parent was found to carry this mutation. Two microsatellite markers, UT5013 and UT1699 (6), confirmed parentage, implying that thismutationwas de novo. Case 2 was a male diagnosed with cancer of the ascending colon at the age of 42 years, with a brother diagnosed with cancer of the caecum at age 36, a sister diagnosed with cancer of the colon at age 41, and father diagnosed with cancer of the stomach at age 67. This family history was sufficient to meet the Amsterdam II criteria for hereditary non-polyposis colorectal cancer