Abstract for presentation at 11th International Congress of Human Genetics

Mutations in the DNA mismatch repair gene MLH1 are a major cause of hereditary nonpolyposis colorectal cancer (HNPCC). The phenotype is normal until a second hit inactivates the wild type (wt) allele in a target tissue, initiating multi-step carcinogenesis.
We addressed loss of heterozygosity (LOH) as a putative second hit, by examining 26 colorectal (CRC) and 33 endometrial cancers (EC) from carriers of three MLH1 founding mutations. LOH was studied by a quantitative method, Matrix Assisted Laser Desorption Ionisation – Time-of-Flight (MALDI-TOF), using four intragenic single nucleotide polymorphisms and mutations. We further analyzed the extent of LOH by six flanking microsatellite markers.
LOH was observed in over half of Mut1-3 carriers (see table), more frequently in CRC (16/26, 61.5%) than in EC (17/33, 51.5%). The difference between CRC and EC was statistically significant for Mut1 (p=0.001). Irrespective of the predisposing mutation, LOH affected more frequently the wt than the mutant allele (24/59 vs. 9/59, p=0.004). ECs from Mut2 and Mut3 carriers harbored more frequent LOH than those from Mut1 carriers (15/20 vs. 2/13, p=0.001). Moreover, while LOH primarily affected the wt allele in the tumors, ECs from Mut2 and Mut3 families harbored mutant allele LOH relatively more frequently when compared to Mut1 ECs (7/20 vs. 0/13, p=0.03).
LOH extended beyond MLH1 (up to 2.5Mb) in 6/25 (24.0%) informative EC or CRC tumors. MLH1 promoter methylation was observed in 9/25 (36.0%) tumors with LOH, coexisting with mutant allele LOH in 6/9 (66.7%) and with wt allele LOH in 3/9 (33.3%) tumors. The MALDI-TOF results were in good accordance with LOH data obtained by a semiquantitative primer extension method (SNuPE).
We conclude that: (1) Interesting mutation specific differences were observed in the frequencies and allele-specificity (wt vs. mutant) of LOH in CRCs vs. ECs, which correlated with the predominant tumor spectrum of the families. (2) In most cases, LOH was observed with intragenic markers only (and not with flanking microsatellites), suggesting a locus-restricted mechanism (e.g. gene conversion). (3) MALDI-TOF provides a novel and reliable approach for the detection and quantification of LOH.