Compound heterozygosity for two MSH6 mutations in a patient with early onset colorectal cancer, vitiligo and systemic lupus erythematosus.

Lynch syndrome (hereditary non-polyposis colorectal cancer, HNPCC) is an autosomal dominant condition caused by heterozygous germline mutations in the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2. Rare cases have been reported of an inherited bi-allelic deficiency of MMR genes, associated with multiple café-au-lait spots, early onset CNS tumors, hematological malignancies, and early onset gastrointestinal neoplasia. We report on a patient with vitiligo in segments of the integument who developed systemic lupus erythematosus (SLE) at the age of 16, and four synchronous colorectal cancers at age 17 years. Examination of the colorectal cancer tissue showed high microsatellite instability (MSI-H) and an exclusive loss of expression of the MSH6 protein. Immunohistochemical analysis of normal colon tissue also showed loss of MSH6, pointing to a bi-allelic MSH6 mutation. Sequencing of the MSH6 gene showed the two germline mutations; c.1806_1809delAAAG;p.Glu604LeufsX5 and c.3226C > T;p.Arg1076Cys. We confirmed that the two mutations are on two different alleles by allele-specific PCR. To our knowledge, neither parent is clinically affected. They did not wish to be tested for the mutations identified in their daughter. These data suggest that bi-allelic mutations of one of the MMR genes should be considered in patients who develop early-onset multiple HNPCC-associated tumors and autoimmune disorders, even in absence of either hematological malignancies or brain tumors.

Aberrant splicing in MLH1 and MSH2 due to exonic and intronic variants.

Single base substitutions in DNA mismatch repair genes which are predicted to lead either to missense or silent mutations, or to intronic variants outside the highly conserved splicing region are often found in hereditary nonpolyposis colorectal cancer (HNPCC) families. In order to use the variants for predictive testing in persons at risk, their pathogenicity has to be evaluated. There is growing evidence that some substitutions have a detrimental influence on splicing. We examined 19 unclassified variants (UVs) detected in MSH2 or MLH1 genes in patients suspected of HNPCC for expression at RNA level. We demonstrate that 10 of the 19 UVs analyzed affect splicing. For example, the substitution MLH1,c.2103G > C in the last position of exon 18 does not result in a missense mutation as theoretically predicted (p.Gln701His), but leads to a complete loss of exon 18. The substitution MLH1,c.1038G > C (predicted effect p.Gln346His) leads to complete inactivation of the mutant allele by skipping of exons 10 and 11, and by activation of a cryptic intronic splice site. Similarly, the intronic variant MLH1,c.306+2dupT results in loss of exon 3 and a frameshift mutation due to a new splice donor site 5 bp upstream. Furthermore, we confirmed complete exon skipping for the mutations MLH1,c.1731G > A and MLH1,c.677G > A. Partial exon skipping was demonstrated for the mutations MSH2,c.1275A > G, MLH1,c.588+5G > A, MLH1,c.790+4A > G and MLH1,c.1984A > C. In contrast, five missense mutations (MSH2,c.4G > A, MSH2,c.2123T > A, MLH1,c.464T > G, MLH1,c.875T > C and MLH1,c.2210A > T) were found in similar proportions in the mRNA as in the genomic DNA. We conclude that the mRNA examination should precede functional tests at protein level.

Hereditary nonpolyposis colorectal cancer: pitfalls in deletion screening in MSH2 and MLH1 genes.

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by a deficiency in DNA mismatch repair in consequence of germline mutations mainly in the genes MSH2 and MLH1. Around 10% of patients suspected of HNPCC are identified with large genomic deletions that cannot be detected by conventional methods of mutation screening. The recently developed multiplex ligation-dependent probe amplification (MLPA) proved to be an easy to perform method for deletion detection and is reliable when more than one exon is deleted. We show that, in some cases, apparent deletions of single exons may actually result from single base substitutions or small insertions/deletions in the hybridisation sequence of MLPA probes. We conclude that single exon deletions, detected by MLPA or multiplex PCR, should be validated with additional methods.

A modified multiplex PCR assay for detection of large deletions in MSH2 and MLH1.

A method for detection of large genomic deletions in the MSH2 and MLH1 genes based on multiplex PCR and quantitative evaluation of PCR products is presented. All 35 exons of MSH2 and MLH1 were screened simultaneously in seven PCR reactions, each of them including primers for both genes. The method is reliable for uncovering large genomic deletions in patients suspected of HNPCC. With this method, six novel deletions were identified, two in MSH2: EX1_10del and EX1_16del (representing deletion of the entire MSH2 gene); and four in MLH1: EX1_10del in two unrelated patients, EX3_5del, and EX4del. The deletions were detected in 18 unrelated patients in whom no germline mutation had been identified by SSCP and DHPLC. These results indicate that our modified multiplex PCR assay is suited for the detection of large deletions both in the MSH2 and MLH1 gene and therefore represents an additional valuable tool for mutation screening in HNPCC families.