Hereditary non-polyposis colon cancer (HNPCC) is a multi-organ cancer syndrome associated with hereditary defects in DNA mismatch repair (MMR). To date, more than 400 predisposing mutations have been deposited in the ICG-HNPCC mutation database, mostly affecting MLH1 (≈50%), MSH2 (≈40%), and MSH6 (≈10%) (www.nfdht.nl/). Over half of all HNPCC-linked MMR gene mutations consist of nonsense or frameshift changes that result in premature termination codons.¹ Such transcripts are subject to nonsense mediated mRNA decay, a surveillance mechanism whose purpose is to protect the organism against dominant negative or gain of function effects of truncated proteins.² Furthermore, some nonsense as well as missense and even silent changes can alter pre-mRNA splicing by introducing or disrupting exonic splicing enhancer or exonic splicing silencer sequences.^3–5 By doing so, the mutations may promote the skipping or inclusion of exons in which they are located. Evidence in support of this mechanism exists at least for certain nonsense and missense mutations in MLH1.^6,7 Most mutations that affect splicing consist of single nucleotide substitutions in the classical splice sites at intron/exon junctions; such mutations are particularly common in MLH1, constituting one third of all germline mutations in this gene.¹

In this work, a strategy based on RNA was chosen for mutation screening in families with hereditary non-polyposis colorectal cancer, since a previous study on the same population indicated that 75% of MLH1 and MSH2 mutations were detectable as aberrant sized transcripts.⁸ This report focuses on novel splicing associations of the mutations discovered.

MATERIALS AND METHODS

Patient specimens

Families F73, F95, and F65 meeting the Amsterdam I criteria for hereditary non-polyposis colorectal cancer⁹ were screened for predisposing mutations. F73 was part of a population based series, whose clinical and molecular characteristics not related to splicing have been addressed previously.^8,10 Two HNPCC …