Evaluation of Lynch syndrome modifier genes in 748 MMR mutation carriers

Abstract Several studies have reported that, in Lynch syndrome resulting from mutations of the mismatch repair (MMR) genes, a CA repeat ≤17 within the IGF1 promoter, SNPs within the xenobiotic metabolizing enzyme gene CYP1A1 and SNPs on 8q23.3 and 11q23.1 modify colorectal cancer (CRC) risk in MMR mutation carriers. We analysed the impact of these polymorphisms on CRC risk in 748 French MMR mutation carriers derived from 359 families. We also analysed the effect of the Novel 1 SNP (18q21), which has recently been shown to increase CRC risk in the general population. We observed a significant difference in the CRC-free survival time between males and females, between MSH2 and MSH6 mutation carriers and between MLH1 and MSH6, indicating that this series is representative of Lynch syndrome. In contrast, the univariate log-rank test, as well as multivariate Cox model analysis controlling for familial aggregation and mutated MMR gene, year of birth and gender showed that the polymorphic alleles tested were not associated with a significant CRC risk increase, neither on the entire sample nor among males and females. This discrepancy with previous reports might be explained both by the genetic heterogeneity between the different populations analysed and the allelic heterogeneity of the MMR mutations. We conclude that genotyping of these polymorphisms is not useful to evaluate CRC risk in MMR mutation carriers and to optimize their clinical follow-up. Introduction Lynch syndrome or hereditary non-polyposis colorectal cancer, the most common form of inherited colorectal cancer (CRC), results from germline mutations within the genes of the mismatch repair (MMR) system, MSH2, MLH1, MSH6 and PMS2 (for a review, see Lynch et al1). In MMR mutation carriers, the main tumour risks are colorectal and endometrial cancers. The cumulative risk at 70 years of CRC has been estimated to be 47–78% in males2, 3, 4 and 30–57% in females,2, 3, 4 and the risk of endometrial cancer to be 25–61%.2, 3 Like in other Mendelian forms of cancer characterized by an incomplete penetrance, one of the main challenges is to identify modifier genetic factors that can modulate the mutation penetrance. Characterization of validated modifier genetic factors should have important clinical consequences in the future since it should then be possible to adapt the follow-up of MMR gene mutation carriers, in terms of nature and timing of investigations, according to modifier alleles. The modifier genes that have been reported so far in Lynch syndrome have been characterized according to two strategies: several studies have been focused on genes whose implication in CRC development had previously been suggested. One of the first modifier genetic factors identified in Lynch syndrome corresponds to a CA repeat polymorphism present within the IGF1 promoter, 1 kb upstream from the transcriptional initiation site.5, 6 In a study performed in 121 MMR mutation carriers from 59 families, mainly of Caucasian origin, Zecevic et al5 reported that a CA repeat ≤17 was significantly associated with a higher CRC risk and an earlier age of tumour onset. This result was confirmed by an independent study performed in 443 Australian and Polish MMR mutation carriers originated from 269 distinct families.6 Another class of modifier genes reported in Lynch syndrome corresponds to genes encoding xenobiotic metabolizing enzymes involved in environmental carcinogen metabolism. In 129 subjects of South African origin and harbouring the same MLH1 missense mutation, males harbouring the null genotype for the GSTT1 and GSTM1 genes developed cancer earlier than the males harbouring the other genotypes.7 This effect of the GSTT1 and GSTM1 null genotypes was not confirmed in a second study including 257 MMR mutation carriers from 130 families.8 Nevertheless, the authors reported that subjects heterozygous for the CYP1A1 rs1048943 SNP (c.1384A>G; p.Ile462Val) developed CRC earlier than individuals with the homozygous wild-type genotype and that subjects heterozygous for this polymorphism and an additional SNP rs4646903 (Msp1; g.6235T>C) had an increased CRC risk.8 The second strategy, which has recently allowed the detection of modifier genes in Lynch patients, originated from the numerous genome-wide association studies reporting SNPs associated with CRC risk in the general population. Starting from the hypothesis that SNPs acting as risk factors for CRC in the general population might act as risk modifiers in patients harbouring a highly penetrant mutation, Wijnen et al9 recently reported a significant association of CRC risk with rs16892766 (8q23.3) and rs3802842 (11q23.1) in 675 Dutch MMR mutation carriers from 127 families. In this study, we investigated the impact of these different genetic factors on CRC risk in a large series of French MMR mutation carriers. Patients and methods Patients The study included 748 unselected patients (Table 1) derived from 359 families with Lynch syndrome and recruited from Rouen (n=494) and Lille (n=254) university hospitals in France. All these individuals were confirmed carriers of a deleterious mutation of MSH2, MLH1 or MSH6 and were Caucasian. Among these 748 mutation carriers, 329 (44%) had been diagnosed with CRC prior to inclusion in the study, with a mean age of CRC onset of 43 years (range: 18–82 years). Additionally, 51 mutation carriers (6.8%) had been diagnosed with another tumour belonging to the Lynch syndrome spectrum (endometrial carcinoma, tumour of the urinary tract, ovarian carcinoma, cancer of the stomach and of the small intestine) and 368 (49.2%) had developed no tumour at the time of inclusion. Table 1: Clinical characteristics of MMR mutation carriers Full size table Genotyping The IGF1 CA repeat was PCR-amplified using dye-labelled primers and the length of the PCR products was determined after migration on an Applied Biosystems model 3130 Genetic Analyser (PE Applied Biosystems, Foster City, CA, USA) and analysed using the GeneMapper Analysis Software version 4.0 (Applied Biosystems). To calibrate the results, we sequenced, after cloning into a plasmid, the most frequent allele (n=19) and used the corresponding genomic DNA as a reference DNA. The rs16892766 (8q23.3), rs3802842 (11q23.1) SNPs and the rs1048943 (c.1384A>G; p.Ile462Val) and rs4646903 SNPs, both located within the CYP1A1 gene (15q24.1), were genotyped using SNaPshot multiplex assays based on primer extension with dye-labelled dideoxynucleotides (ABI PRISM SNaPshot Multiplex kit, Applied Biosystems). We also analysed, by SNaPshot multiplex analysis, the Novel 1 SNP located on 18q21, which had been shown to be associated with CRC risk in the general population, by altering SMAD7 expression.10 Labelled products were separated using a 25-min run on an ABI Prism 3100 DNA sequencer and data were analysed using the GeneMapper Analysis Software version 4.0 (Applied Biosystems). Primer sequences used for genotyping (...truncated)