Frequent genomic disorganisation of MLH1 in hereditary non-polyposis colorectal cancer (HNPCC) screened by RT-PCR on puromycin treated samples.

Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited autosomal disease presumed to comprise at least 5-8% of all colorectal cancer (CRC) cases. HNPCC is characterised by the early onset of colon tumours mostly located proximal to the splenic flexure and extracolonic tumours. HNPCC families segregate germline mutations in one of the several DNA mismatch repair genes, such as MSH2, MLH1, PMS1, PMS2, and MSH6. The MSH2 protein is a homologue of the bacterial mutS that recognises mismatched base pairs, and the MLH1 protein is a homologue of the bacterial mutL that interacts with mutS to recruit proteins required for DNA mismatch repair. 3 Disruption of the mismatch repair system results in genomic instability in the microsatellite repeats, that is, microsatellite instability (MSI). Detection of germline mutations in the mismatch repair genes confirms the diagnosis genetically for HNPCC kindreds who are at risk for developing CRCs and extracolonic tumours of the endometrium, small intestine, hepatobiliary tract, kidney, and ovary. 4 Mutations of the mismatch repair genes such as MSH2 or MLH1 have so far been reported at most in 45-86% of the HNPCC kindreds fulfilling the Amsterdam criteria. The presence of mutation negative cases raised the question that they harboured mutations in other genes responsible for HNPCC or that mutations are missing simply because of technical failure. PCR based approaches, such as exon by exon genomic DNA amplification, would be unsuitable to detect relatively large genomic alterations such as deletions or rearrangements. On the other hand, RNA based analysis for screening germline mutations was suggested to be disadvantageous owing to the appearance of normal splicing variants and nonsense mediated mRNA decay (NMD), which resulted in the reduction of mRNA from the mutated allele carrying deleterious mutations. Previously, we have reported the application of reverse transcriptase (RT)-PCR/ direct sequencing from RNA samples extracted after puromycin treatment. The translation inhibitor puromycin suppresses NMD caused by nonsense and other deleterious mutations of MLH1. 10 This method is simple and efficient for screening MLH1 abnormalities such as nonsense mutations, frameshift mutations, and splicing variants. We report here three cases of novel MLH1 genomic disorganisation, two genomic deletions and one partial duplication, screened by the appearance of the splicing variant, which was enhanced and easily detectable by RT-PCR from puromycin treated samples.