colorectal cancer ( crc ) is one of the most common human malignancies in western countries .
colorectal adenomas have high malignancy potential when they are large in diameter and/or present with severe dysplasia and/or a villous component .
colonoscopic polypectomy has been documented to significantly reduce the incidence of colorectal cancer [ 3 , 4 ] .
therefore , the identification of factors associated with the development of colorectal adenoma represents a major goal in colorectal cancer prevention .
they could indeed allow the selection of individuals at risk of crc who may benefit from a screening by colonoscopy .
the adenoma - carcinoma sequence suggests that colorectal adenomas and adenocarcinomas share common environmental and genetic risk factors .
an increased risk of colorectal tumors has been found in relatives of patients with large adenomas [ 5 , 6 ] .
a case - control study had suggested that family history of colorectal cancer influenced only the growth of adenomas or their malignant transformation . however , relatively few epidemiologic studies explored genetic risk factors in colorectal adenomas .
we investigated , through a case - control study , the relation between polymorphisms within a series of candidate genes involved in colorectal tumorigenesis and putatively in the formation and the development of colorectal adenomas such carcinogen metabolism enzymes , methylation enzymes , dna repair genes , oncogenes and tumor suppressor genes .
the genetics of adenomas ( geade ) study is a case - control and family study of patients with high - risk adenomas ( 10 mm ) .
the data were obtained from 18 participating gastroenterology units of general hospitals in france . from september 1995 to march 2000 , 306 consecutive patients with newly diagnosed colorectal large adenoma ( la ) were enrolled in the study .
patients with personal cancer history , familial adenomatous polyposis , established hereditary nonpolyposis colorectal cancer or inflammatory bowel disease were excluded . to distinguish genetic factors involved in the occurrence of adenomas or in their growth , 307 cases with small adenomas ( with a diameter smaller than 0.5 cm ) ( sa ) and 572 polyp - free controls ( with normal colonoscopy ) ( pf )
reason for referral , family history of crc , completeness of colonoscopy were registered for all patients and controls .
two pf per la cases were selected as controls within over 2000 pf for matching on age , gender , and geographic area .
blood specimens were obtained at time of colonoscopy and those patients who presented with a polyp were included only when histological examination revealed the adenomatous nature of the lesion . as polyps were totally removed during colonoscopy , their natural evolution could not be scored .
after longitudinally section , half of the tumor material was fixed for histologic analysis and half was frozen for molecular characterization .
twenty individuals had to be excluded because of insufficient tumor material : 11 patients with la , 5 with sa , and 4 pf .
the final groups contained 295 patients with la , 302 with sa , and 568 pf as controls .
all patients and controls signed an informed consent after approval of the study by an ethic committee for biomedical research ( le kremlin - bictre ) and the database was declared to the national committee commission nationale de linformatique et des liberts ( cnil ) .
genes have been selected for their role in colorectal tumorigenesis and for the presence of frequent neutral polymorphisms .
the polymorphisms of mmp1 , mmp3 , and mmp7 gene promoters , that are responsible for the degradation of extracellular matrix components , had been previously studied and were not considered in the present analysis .
three genes , ts , ugt1a1 , and mdr1 , are implicated in folates , bilirubin metabolism , and transport of xenobiotic respectively .
all these pathways are suspected to play a role in cancer occurrence or in the transformation of benign lesions , folates by interfering with dna synthesis and repair , bilirubin by its known antioxydant properties , and transport of xenobiotic by its detoxication function .
case - control reports   exemplified the relevance of these three genes in addition to hras1 .
three additional pathways have been secondary studied including the wnt pathway ( apc , ctnnb1 , axin2 ) , the p53 pathway ( tp53 , bax ) , the tgfb pathway ( tgfbr2 ) , the main mmr and ber genes ( msh2 , mlh1 , myh ) , and card15 as part of the family with lrr domains .
all these genes have been strongly assessed for their major role in oncogenesis , cycle cell control , apoptosis regulation , cell adhesion and migration , proliferation , dna repair as their main functions .
the vntr of hras1 was studied by conventional electrophoresis after pcr amplification on 1.2% agarose gels at 2 volts / cm for 1518 hours .
hardy - weinberg proportions were tested for each polymorphism . linkage disequilibrium ( ld ) between pairwise loci
genotype - specific odds ratio ( or ) and 95% confidence intervals ( cis ) were computed using unconditional logistic regression adjusted on matching factors ; wald test was used to assess the global effect of each polymorphism .
the homozygous genotype for the more frequent allele among controls was set as the reference class .
the association was further examined using the combination test , that allows the analysis of all possible combinations of snps within a given gene or tightly linked genes to test their association with the disease .
for each snps combination , the method computes a statistic test contrasting the genotypic ( or haplotypic ) distribution between cases and controls .
because all these tests are correlated ( many of them are nested in each other and the snps are likely to be in ld ) , a permutation procedure has been implemented which displays a significance level adequately adjusted for multiple testing .
first , we used the famhap12 software to apply this method by performing haplotypic tests .
then the combintest ( jannot , personal communication ) was used to perform genotypic tests .
the method was extended to the test of combined polymorphisms on different genes that may act interactively .
to avoid multiple tests biases , we chose a three - step strategy that minimizes the number of comparisons .
first , we considered two - by - two combinations of polymorphisms chosen on their plausibility to interact with each other , for instance , apc and myh both responsible , when mutated , for adenomatous polyposis .
finally , as a combination of polymorphisms may have an effect even if the genes are not suspected to interact with each other and are not involved in the same pathway , all possible combinations were considered if the first two steps did not reveal any significant association .
when several combinations of polymorphisms were significant within a given test , it was possible to test nested combinations using chi - square calculation to determine whether a given polymorphism adds a significant contribution to the association found .
the average age was very similar in patients ( 62 years for la and 61 years for sa ) and in pf controls ( 61 years ) .
the sex ratio ( male / female ) was quite similar in la ( 1.7 ) and in pf controls ( 1.5 ) , slightly lower in sa ( 1.2 ) because of the absence of matching . 

table 1 describes the characteristics of the polymorphisms studied and the allele frequencies in controls .
the distribution of genotypes in controls was consistent with hardy - weinberg proportions for all polymorphisms except for tp53.3 ( p = .0013 without correction for multiple testing ) . a similar departure from h - w proportions (
p = .010 ) was found in the hapmap european population ( http://www.ncbi.nlm.nih.gov/projects/snp/ ) for this polymorphism .
all polymorphisms within a same gene were moderately or not found in ld except myh ; myh.1 and myh.2 are in quasicomplete disequilibrium ( d = 1 and r = 0.99 ) .
analysis of single polymorphisms for the different comparisons , la versus pf , la versus sa , and sa versus pf did not reveal any association for most of the genes studied .
the p - values found to be less than .05 were obtained for apc.1 , mdr1.2 , and axin2.9 .
the results of the haplotypic and genotypic combination tests performed for each gene or gene combination are shown in table 4 .
when considering each gene separately , only apc displayed a significant difference between small and large adenomas ( p = .014 in haplotypic analysis and p = .07 in genotypic analysis ) .
as apc.1 alone and the combination apc.1-apc.2 appeared both significant , we tested whether apc.2 provided a significant contribution to the association , which was not the case (  = 2.12 , 1df ) showing that the association was solely due to the apc.1 ( p.glu1317gln ) polymorphism .
the combination apc - myh appeared also significant with a global p - value of .045 .
however , the only significant combination was apc.1-apc.2 already found , showing that myh does not provide any additional contribution to the association between apc and adenomas .
no difference was found for the other two comparisons , that is , sa versus pf and la versus pf . finally , the analysis of all possible combinations did not provide any significant result .
we investigated the role of candidate gene polymorphisms in a case - control study of patients with large adenomatous polyps ( n = 295 ) , compared to patients with small adenomatous polyps ( n = 302 ) and polyp - free controls ( n = 568 ) .
the 38 polymorphisms studied belonged to 14 genes possibly involved in increasing of colorectal cancer risk .
the reason for comparing these different groups of patients was that different genes might be involved in the different steps of carcinogenesis . using this case - control study
, we had shown that mmp polymorphism was most probably involved in the occurrence , but not in the growth of polyps . using the combination test
, we had shown that the effect was due to a specific combination of mmp1-mmp3 polymorphisms which was not found using logistic regression .
indeed , a major property of this test is that it allows the detection of polymorphisms with low marginal effects .
multiple testing may be a limitation of studies in which several polymorphisms in several genes are tested .
the combination test allows a correction for multiple tests on tightly linked markers , which other methods of correction such as bonferroni , benjamini - hochberg , or fdr ( false discovery rate ) do not . in the present study
, we could show that the positive results obtained for axin2 and mdr1 when analyzing single polymorphisms were false positives as these results were not confirmed when using the combination test . for independent markers , there was no need for correcting for multiple testing as our results were essentially negative .
our study did not give evidence for an effect of any of the gene polymorphisms studied except the p.glu1317gly variant of the apc gene .
some studies reported an effect of this variant in patients with multiple adenomas or colorectal cancer [ 1517 ] , and some others found an effect in adenomas but not in cancer [ 18 , 19 ] .
more recently , hahnloser et al .   reported an effect of this variant in a group of patients with a small number of adenomas ( 1 to 3 lifetime adenomas ) and in a group of crc cases .
none of these studies considered the size of adenomas and could differentiate adenomas at high - risk from those at low - risk of cancer .
our results favour the hypothesis that the p.glu1317gly variant would influence the growth and not the occurrence of adenomas , and would have thus indirectly an influence on colorectal cancer risk .
nevertheless , although the three groups are paired on gender and geographic origin , it is not stated if other known risk factors would influence adenoma growth .
it is thus important to plan a specific multivariate analysis when building a replication sample . regarding the ts polymorphisms
chen et al .   found a significant effect of the 3r allele of ts.2 , particularly of the 3r/3r genotype ( or = 3.3 ) . in our study ,
the or associated with 3r/3r genotype was 1.43 ( ci 0.942.16 ) for la versus pf and 1.10 ( ci 0.681.78 ) for la versus sa .
on the other hand , ulrich et al .   found no marginal effect with any of these two polymorphisms but found a significant interaction of ts.2 with folate intake .
similarly , the most recent studies [ 23 , 24 ] found no marginal effect of either polymorphism but a slight interaction with folate or vitamin b6 intake . the groups ' sizes in our study were of the same order of magnitude as in the study of chen et al . ,
but the three other studies which did not show any marginal effect included larger groups ( more than 500 individuals in each group ) . given the size of our population , we could a priori detect an or of 1.7 with a power of at least 80% .
it is highly probable that the ts gene , which produces a key enzyme in folate metabolism , has an effect only in interaction with dietary exposures , which would explain why this effect was not found in our study .
moreover a new snp has been recently identified in the second repeat of the 3r allele leading to split the allele 3r in 3rc and 3rg alleles as a tri - allelic polymorphism .
this variant could explain these discrepant results since patients with 3rc/3rc genotype have a transcriptional activity of ts comparable to those with 2r/2r genotype . in our study ,
the problem of multiple testing was adequately controlled for each test performed by the permutation procedure implemented in the combination test . in the last analysis step ,
the tremendous number of comparisons resulted in a drastic correction of each p - value , which considerably lowered the testing power .
thus , our negative results certainly do not definitively demonstrate the absence of specific combination influencing colorectal tumorigenesis , but more probably that , if it exists , this ( of these ) effect(s ) is ( are ) low and could be found through the initial ( and further ) genome - wide association studies of colorectal cancer testing much larger numbers of patients and controls [ 26 , 27 ] .
 predisposition to sporadic colorectal tumours is influenced by genes with minor phenotypic effects . 
 a case - control study was set up on 295 patients treated for a large adenoma matched with polyp - free individuals on gender , age , and geographic origin in a 1 : 2 proportion . 
 a second group of 302 patients treated for a small adenoma was also characterized to distinguish effects on adenoma occurrence and growth . 
 we focussed the study on 38 single nucleotide polymorphisms ( snps ) encompassing 14 genes involved in colorectal carcinogenesis . 
 effect of snps was tested using unconditional logistic regression . 
 comparisons were made for haplotypes within a given gene and for biologically relevant genes combinations using the combination test . 
 the apc p.glu1317gly variant appeared to influence the adenoma growth ( p = .04 , exact test ) but not its occurrence . 
 this result needs to be replicated and genome - wide association studies may be necessary to fully identify low - penetrance alleles involved in early stages of colorectal tumorigenesis . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion