Molecular analysis of hereditary nonpolyposis colorectal cancer in the United States: high mutation detection rate among clinically selected families and characterization of an American founder genomic deletion of the MSH2 gene

Abstract

The identification of germline mutations in families with HNPCC is hampered by genetic heterogeneity and clinical variability. In previous studies, MSH2 and MLH1 mutations were found in approximately two-thirds of the Amsterdam-criteria-positive families and in much lower percentages of the Amsterdam-criteria-negative families. Therefore, a considerable proportion of HNPCC seems not to be accounted for by the major mismatch repair (MMR) genes. Does the latter result from a lack of sensitivity of mutation detection techniques, or do additional genes underlie the remaining cases? In this study we address these questions by thoroughly investigating a cohort of clinically selected North American families with HNPCC. We analyzed 59 clinically well-defined U.S. families with HNPCC for MSH2, MLH1, and MSH6 mutations. To maximize mutation detection, different techniques were employed, including denaturing gradient gel electrophoresis, Southern analysis, microsatellite instability, immunohistochemistry, and monoallelic expression analysis. In 45 (92%) of the 49 Amsterdam-criteria-positive families and in 7 (70%) of the 10 Amsterdam-criteria-negative families, a mutation was detected in one of the three analyzed MMR genes. Forty-nine mutations were in MSH2 or MLH1, and only three were in MSH6. A considerable proportion (27%) of the mutations were genomic rearrangements (12 in MSH2 and 2 in MLH1). Notably, a deletion encompassing exons 1-6 of MSH2 was detected in seven apparently unrelated families (12% of the total cohort) and was subsequently proven to be a founder. Screening of a second U.S. cohort with HNPCC from Ohio allowed the identification of two additional kindreds with the identical founder deletion. In the present study, we show that optimal mutation detection in HNPCC is achieved by combining accurate and expert clinical selection with an extensive mutation detection strategy. Notably, we identified a common North American deletion in MSH2, accounting for approximately 10% of our cohort. Genealogical, molecular, and haplotype studies showed that this deletion represents a North American founder mutation that could be traced back to the 19th century.

Figure 1

A, Southern blot analysis of genomic DNA samples from North American individuals with HNPCC, revealing a common rearrangement. Total genomic DNA was digested with EcoRI and was hybridized with the MSH2 exon 7–specific probe. An aberrant ∼14-kb band was observed in lanes 7, 8, 9, 10, 14, 17, and 18, corresponding to individuals from families 122, 123, 124, 130, 148, 152, and 155, respectively. Aberrant fragments of different sizes are also observed in lanes 4 (family 108, carrying a similar but distinct MSH2 exon 1–6 deletion) and 20 (a previously identified carrier of an MSH2 exon 6 deletion, employed here as positive control). Also note that the presence of additional bands is accompanied by a decreased intensity of the ∼9-kb wild-type fragments encompassing exon 7. The latter was confirmed by hybridizations with exon 1–6 probes and by BclI digestion (data not shown). B, Genomic map of the normal and deleted MSH2 gene. For the sake of clarity, only the EcoRI restriction sites, the Alu repeats involved in the recombination (shaded boxes), and the relevant MSH2 exons (blackened boxes) are depicted. The normal (∼9-kb) and aberrant (∼14-kb) EcoRI restriction fragments also shown in panel A are indicated, as well as the primers (R3, R2, and F3) employed to selectively amplify the deletion breakpoint by PCR. C, Nucleotide sequence of the founder deletion breakpoint. Three distinct sequences are reported from top to bottom: the normal sequence flanking MSH2 at the 5′ side, the deletion breakpoint, and the normal sequence of MSH2 intron 6. The shaded boxes indicate the nucleotide homology between the two Alu repeats present in the 5′ flanking sequence and within intron 6 of MSH2. Because of the extensive homology, it is difficult to pinpoint the exact deletion breakpoint. The arrow indicates its most likely position on the basis of nucleotide homology.

Figure 2

Haplotype analysis of the founder MSH2 deletion chromosome. A total of seven polymorphic markers flanking MSH2 on both the telomeric and centromeric side were employed. The haplotype encompassing the founder MSH2 deletion could be reconstructed in most families, with the exception of family 124 from the Lynch cohort and kindred CG336 from the Ohio cohort, because of a lack of samples from informative relatives. The haplotype of family 726NM was derived from a somatic cell hybrid containing the isolated deletion chromosome in a rodent background. Shaded boxes indicate the alleged founder haplotype. Both alleles are indicated in those cases where the exact phase could not be derived.