Case-control study for colorectal cancer genetic susceptibility in EPICOLON: previously identified variants and mucins

  • Anna Abulí1, 2,

    Affiliated with

    • Ceres Fernández-Rozadilla3,

      Affiliated with

      • Virginia Alonso-Espinaco1,

        Affiliated with

        • Jenifer Muñoz1,

          Affiliated with

          • Victoria Gonzalo1,

            Affiliated with

            • Xavier Bessa2,

              Affiliated with

              • Dolors González4,

                Affiliated with

                • Joan Clofent5,

                  Affiliated with

                  • Joaquin Cubiella6,

                    Affiliated with

                    • Juan D Morillas7,

                      Affiliated with

                      • Joaquim Rigau8,

                        Affiliated with

                        • Mercedes Latorre9,

                          Affiliated with

                          • Fernando Fernández-Bañares10,

                            Affiliated with

                            • Elena Peña11,

                              Affiliated with

                              • Sabino Riestra12,

                                Affiliated with

                                • Artemio Payá13,

                                  Affiliated with

                                  • Rodrigo Jover13,

                                    Affiliated with

                                    • Rosa M Xicola14,

                                      Affiliated with

                                      • Xavier Llor14,

                                        Affiliated with

                                        • Luis Carvajal-Carmona15,

                                          Affiliated with

                                          • Cristina M Villanueva16,

                                            Affiliated with

                                            • Victor Moreno17,

                                              Affiliated with

                                              • Josep M Piqué1,

                                                Affiliated with

                                                • Angel Carracedo3,

                                                  Affiliated with

                                                  • Antoni Castells1,

                                                    Affiliated with

                                                    • Montserrat Andreu2,

                                                      Affiliated with

                                                      • Clara Ruiz-Ponte3,

                                                        Affiliated with

                                                        • Sergi Castellví-Bel1Email author and

                                                          Affiliated with

                                                          • for the Gastrointestinal Oncology Group of the Spanish Gastroenterological Association18

                                                            Affiliated with

                                                            BMC Cancer201111:339

                                                            DOI: 10.1186/1471-2407-11-339

                                                            Received: 22 February 2011

                                                            Accepted: 5 August 2011

                                                            Published: 5 August 2011

                                                            Abstract

                                                            Background

                                                            Colorectal cancer (CRC) is the second leading cause of cancer death in developed countries. Familial aggregation in CRC is also important outside syndromic forms and, in this case, a polygenic model with several common low-penetrance alleles contributing to CRC genetic predisposition could be hypothesized. Mucins and GALNTs (N-acetylgalactosaminyltransferase) are interesting candidates for CRC genetic susceptibility and have not been previously evaluated. We present results for ten genetic variants linked to CRC risk in previous studies (previously identified category) and 18 selected variants from the mucin gene family in a case-control association study from the Spanish EPICOLON consortium.

                                                            Methods

                                                            CRC cases and matched controls were from EPICOLON, a prospective, multicenter, nationwide Spanish initiative, comprised of two independent stages. Stage 1 corresponded to 515 CRC cases and 515 controls, whereas stage 2 consisted of 901 CRC cases and 909 controls. Also, an independent cohort of 549 CRC cases and 599 controls outside EPICOLON was available for additional replication. Genotyping was performed for ten previously identified SNPs in ADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11, and 18 selected variants in the mucin gene family.

                                                            Results

                                                            None of the 28 SNPs analyzed in our study was found to be associated with CRC risk. Although four SNPs were significant with a P-value < 0.05 in EPICOLON stage 1 [rs698 in ADH1C (OR = 1.63, 95% CI = 1.06-2.50, P-value = 0.02, recessive), rs1800795 in IL6 (OR = 1.62, 95% CI = 1.10-2.37, P-value = 0.01, recessive), rs3803185 in ARL11 (OR = 1.58, 95% CI = 1.17-2.15, P-value = 0.007, codominant), and rs2102302 in GALNTL2 (OR = 1.20, 95% CI = 1.00-1.44, P-value = 0.04, log-additive 0, 1, 2 alleles], only rs3803185 achieved statistical significance in EPICOLON stage 2 (OR = 1.34, 95% CI = 1.06-1.69, P-value = 0.01, recessive). In the joint analysis for both stages, results were only significant for rs3803185 (OR = 1.12, 95% CI = 1.00-1.25, P-value = 0.04, log-additive 0, 1, 2 alleles) and borderline significant for rs698 and rs2102302. The rs3803185 variant was not significantly associated with CRC risk in an external cohort (MCC-Spain), but it still showed some borderline significance in the pooled analysis of both cohorts (OR = 1.08, 95% CI = 0.98-1.18, P-value = 0.09, log-additive 0, 1, 2 alleles).

                                                            Conclusions

                                                            ARL11, ADH1C, GALNTL2 and IL6 genetic variants may have an effect on CRC risk. Further validation and meta-analyses should be undertaken in larger CRC studies.

                                                            Colorectal Neoplasms Genetic Predisposition to Disease Single Nucleotide Polymorphism Mucins Genetic Association Studies

                                                            Background

                                                            Colorectal cancer (CRC) is the second leading cause of cancer death in developed countries [1]. Familial adenomatous polyposis and Lynch syndrome are the most frequent hereditary CRC syndromes with a more aggressive presentation, earlier onset and strong familial aggregation. However, they only correspond to a minority of the total CRC burden (~5%). Most genetic components involved in these less frequent hereditary forms were successfully identified in the past two decades and they correspond to rare, highly penetrant alleles that predispose to CRC. Genetic association analyses have been the strategy to identify predisposing CRC alleles in the last decade, firstly by studying a small number of such variants or single nucleotide polymorphisms (SNP) using candidate-gene approaches [2], and lately with an unbiased strategy by genome-wide association studies (GWAS) [3, 4].

                                                            From the EPICOLON consortium [5], several genetic association candidate-gene efforts have been pursued since 2005 aiming to identify genetic susceptibility variants for CRC. In this manner, SNPs/genes were selected to be studied from the previously identified category (variants linked to CRC risk in previous studies), from human syntenic CRC susceptibility regions identified in mouse, from the CRC carcinogenesis-related pathways Wnt and BMP, from regions 9q22 and 3q22 with positive linkage in CRC families, and from the mucin gene family [68]

                                                            SNPs in genes selected in the previously identified category (ADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11) have been analyzed in previous independent genetic association studies and they are a priori attractive candidates for genetic susceptibility to CRC [6, 924]. The expression of most genes in this category are altered in CRC and they appear to be involved in important processes for CRC risk such as hereditary CRC (APC), alcohol metabolism (ADH1C), inflammation (IL6, IL8), cell cycle regulation (CCDN1), energy balance (IRS1, PPARG), methylation (MTHFR), vitamin D (VDR), and the RAS superfamily (ARL11). On the other hand, mucins are protein constituents of the mucous barrier that protects human epithelia from adverse conditions, and they are highly glycosylated by GALNT proteins. Mucins and GALNTs, members of the mucin gene family, can be found deregulated in CRC and other neoplasms and, although also interesting candidates, they have not been previously evaluated for CRC genetic susceptibility [25].

                                                            Here, we report results from a case-control association study for CRC risk in the EPICOLON cohort for previously identified SNPs in ADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11, and selected variants within the mucin gene family.

                                                            Methods

                                                            Study populations

                                                            Studied subjects were mainly from EPICOLON, a prospective, multicenter, nationwide Spanish initiative http://​www.​aegastro.​es/​aeg/​ctl_​servlet?​_​f=​16&​grupo=​4[5], comprised of two stages (2000-2001 and 2006-2008), where CRC cases and matched healthy controls were collected. DNA samples were extracted as previously described [6, 7]. EPICOLON stage 1 corresponded to 515 CRC cases and 515 controls, whereas EPICOLON stage 2 consisted of 901 CRC cases and 909 controls. Additionally, an independent cohort of 549 CRC cases and 599 controls (MCC-Spain; http://​www.​creal.​cat) was available for further replication of putative positive hits in EPICOLON. Cases and controls were matched for sex and age (± 5 years) and controls were negative for personal and family cancer history. All samples were obtained with informed consent reviewed by the ethical board of the corresponding hospital.

                                                            Gene and SNP selection

                                                            Selected SNPs were included in the previously identified category if they corresponded to genetic variants linked to CRC risk by previously published independent studies, or in the mucin gene family if located on genes encoding for mucins and GALNT proteins. Mucins are protein constituents of the mucous barrier highly glycosylated by GALNT proteins. One relevant previously identified SNP was studied in each of the following genes: ADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11. For the mucin gene family, 1-2 SNPs were selected from each of the following genes: MUC7, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19, MUC21, GALNT1, GALNTL2, GALNT10, GALNT14, GALNT4 and OVGP1. Mucin gene selection did not intentionally include GALTN12. This gene is located on the 9q22 region and it was studied in our candidate-gene approach for regions with positive linkage in CRC families (Abulí et al., manuscript in preparation). SNP selection in the mucin gene family was performed using Pupasuite, a web tool for the selection of genetic variants with potential phenotypic effect (pupasuite.bioinfo.cipf.es) [26]. SNPs were always prioritized if they were coding, evolutionary conserved in mouse and SNP minor allele frequency (MAF) was above 5%. Other selected SNPs with a putative regulatory effect were in promoter, intronic or 3'-UTR regions. A complete list of SNP and genes analyzed in the present study is detailed in Table 1.
                                                            Table 1

                                                            SNPs genotyped in EPICOLON from the previously identified category and the mucin gene family to evaluate their implication in CRC genetic susceptibility.

                                                            Gene

                                                            SNP ID

                                                            Chr

                                                            Position*

                                                            Category

                                                            SNP type

                                                            Alleles

                                                            MTHFR

                                                            rs1801133

                                                            1

                                                            11856378

                                                            Previously identified

                                                            Missense (A222V)

                                                            C/T

                                                            IRS1

                                                            rs1801278

                                                            2

                                                            227660544

                                                            Previously identified

                                                            Missense (G971R)

                                                            A/G

                                                            PPARG

                                                            rs1801282

                                                            3

                                                            12393125

                                                            Previously identified

                                                            Missense (P12A)

                                                            C/G

                                                            IL8

                                                            rs4073

                                                            4

                                                            74606024

                                                            Previously identified

                                                            Promoter

                                                            A/T

                                                            ADH1C

                                                            rs698

                                                            4

                                                            100260789

                                                            Previously identified

                                                            Missense (I350V)

                                                            A/G

                                                            APC

                                                            rs459552

                                                            5

                                                            112176756

                                                            Previously identified

                                                            Missense (V1822D)

                                                            A/T

                                                            IL6

                                                            rs1800795

                                                            7

                                                            22766645

                                                            Previously identified

                                                            Promoter

                                                            C/G

                                                            CCDN1

                                                            rs9344

                                                            11

                                                            69462910

                                                            Previously identified

                                                            Synonymous (P241P)

                                                            A/G

                                                            VDR

                                                            rs2228570

                                                            12

                                                            48272895

                                                            Previously identified

                                                            Missense (M1T)

                                                            C/T

                                                            ARL11

                                                            rs3803185

                                                            13

                                                            50205025

                                                            Previously identified

                                                            Missense (C158R)

                                                            A/G

                                                            OVGP1

                                                            rs10067

                                                            1

                                                            111957311

                                                            Mucin family

                                                            Missense (H604Q)

                                                            C/G

                                                            GALNT14

                                                            rs2288101

                                                            2

                                                            31135184

                                                            Mucin family

                                                            Missense (Q469K)

                                                            A/C

                                                            GALNT14

                                                            rs11676188

                                                            2

                                                            31352788

                                                            Mucin family

                                                            Intronic

                                                            C/G

                                                            GALNTL2

                                                            rs2102302

                                                            3

                                                            16215650

                                                            Mucin family

                                                            Promoter

                                                            A/G

                                                            MUC13

                                                            rs12732

                                                            3

                                                            124624568

                                                            Mucin family

                                                            3'-UTR

                                                            C/T

                                                            MUC13

                                                            rs4679392

                                                            3

                                                            124646594

                                                            Mucin family

                                                            Missense (I99T)

                                                            A/G

                                                            MUC7

                                                            rs6826961

                                                            4

                                                            71346701

                                                            Mucin family

                                                            Missense (N80K)

                                                            C/G

                                                            GALNT10

                                                            rs6580076

                                                            5

                                                            153783753

                                                            Mucin family

                                                            Synonymous (A382A)

                                                            C/T

                                                            GALNT10

                                                            rs2277937

                                                            5

                                                            153799165

                                                            Mucin family

                                                            3'-UTR

                                                            C/T

                                                            MUC21

                                                            rs1634730

                                                            6

                                                            30954245

                                                            Mucin family

                                                            Missense (V98A)

                                                            C/T

                                                            MUC12

                                                            rs11766125

                                                            7

                                                            100648117

                                                            Mucin family

                                                            Missense (T4758R)

                                                            C/G

                                                            MUC17

                                                            rs4729656

                                                            7

                                                            100701610

                                                            Mucin family

                                                            3'-UTR

                                                            A/T

                                                            MUC15

                                                            rs15783

                                                            11

                                                            26586801

                                                            Mucin family

                                                            Missense (T229I)

                                                            A/G

                                                            MUC15

                                                            rs11029621

                                                            11

                                                            26596998

                                                            Mucin family

                                                            3' near gene

                                                            A/G

                                                            MUC19

                                                            rs2933353

                                                            12

                                                            40857943

                                                            Mucin family

                                                            Missense (G1803A)

                                                            A/C

                                                            GALNT4

                                                            rs2230283

                                                            12

                                                            89916811

                                                            Mucin family

                                                            Missense (V506I)

                                                            A/G

                                                            GALNT1

                                                            rs17647532

                                                            18

                                                            33234072

                                                            Mucin family

                                                            Promoter

                                                            C/T

                                                            MUC16

                                                            rs1862458

                                                            19

                                                            9069792

                                                            Mucin family

                                                            Missense (S5885F)

                                                            A/G

                                                            SNP ID, single nucleotide polymorphism identification.

                                                            Chr: Chromosome; UTR: untranslated region. *According to NCBI build 37.1 available at http://​www.​ncbi.​nlm.​nih.​gov/​sites/​entrez?​db=​snp.

                                                            High-throughput genotyping in EPICOLON cohorts was performed according to manufacturer's instructions with the TaqMan allelic discrimination and SNPlex™ systems (Applied Biosystems, Foster City, USA), and single-base primer extension chemistry matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) genotyping platform (Sequenom Inc., San Diego, USA). Genotyping in the MCC-Spain cohort was performed using the VeraCode technology (Illumina, San Diego, USA). Genotyping was performed at the Santiago de Compostela and Barcelona nodes of the Spanish National Genotyping Centre http://​www.​cegen.​org, and at the Genome Analysis Platform of the CIC-BioGUNE http://​www.​cicbiogune.​es.

                                                            Statistical methods

                                                            As quality control, genotyping success was set above 90% for SNPs. Allelic frequency description and Hardy-Weinberg equilibrium test were performed using SNPator, a web-based tool offered by the Bioinformatics division of the Spanish National Genotyping Centre (bioinformatica.cegen.upf.es) [27]. All SNPs analyzed had a genotype success rate > 90%. The genotype frequencies of all variants in the control population fitted the Hardy-Weinberg equilibrium (P > 0.01), supporting absence of genotyping artifacts. There was no sign of underlying population stratification in EPICOLON as tested by an independent study [7]. Genotype analysis was carried out using the SNPassoc R library [28]. Inter-group comparisons of genotype frequency differences were performed by regression analysis for codominant, dominant, recessive and log-additive models of inheritance. We estimated the crude odds ratio (OR) and their 95% confidence intervals (95% CIs). As expected, results did not change after sex and age adjustment. The best genetic or inheritance model was selected using the Akaike information criteria. To address the issue of multiple testing, we used Bonferroni correction (P = 0.0125 for four SNPs). Study power was estimated with CaTS software [29].

                                                            Results

                                                            Twenty-eight SNPs, ten from the previously identified category and 18 from the mucin gene family, were successfully genotyped in EPICOLON stage 1. Results in EPICOLON stage 1 are shown in Additional File 1. Four SNPs were significant with a P-value < 0.05 in any of the tested inheritance models: rs698 in ADH1C (OR = 1.63, 95% CI = 1.06-2.50, P-value = 0.02, recessive, AA-AG vs GG), rs1800795 in IL6 (OR = 1.62, 95% CI = 1.10-2.37, P-value = 0.01, recessive, GG-GC vs CC), rs3803185 in ARL11 (OR = 1.58, 95% CI = 1.17-2.15, P-value = 0.007, codominant, GG vs AG vs GG), and rs2102302 in GALNTL2 (OR = 1.20, 95% CI = 1.00-1.44, P-value = 0.04, log-additive 0, 1, 2 alleles).

                                                            In the EPICOLON stage 1 cohort, a liberal P-value threshold (P-value < 0.05) was used to avoid false-negative results. We then validated statistically-significant stage 1 results by replicating them in another independent CRC cohort (EPICOLON stage 2). Further replication in stage 2 was performed only for significant SNPs in stage 1. Results for rs698, rs1800795, rs3803185, and rs2102302 in EPICOLON stage 2 are shown in Additional File 2. Only rs3803185 maintained statistical significance in stage 2 (OR = 1.34, 95% CI = 1.06-1.69, P-value = 0.01, recessive model, AA-AG vs GG). In order to improve statistical power, results for EPICOLON stages 1 and 2 were also analyzed jointly for these four SNPs. Results were only significant for rs3803185 (OR = 1.12, 95% CI = 1.00-1.25, P-value = 0.04, log-additive 0, 1, 2 alleles) and borderline significant for rs698 and rs2102302 (Table 2).
                                                            Table 2

                                                            SNPassoc results for previously identified and mucin SNPs in EPICOLON cohorts.

                                                            EPICOLON stages 1+2 (1,416 CRC cases and 1,424 controls)

                                                            ADH1C

                                                                    

                                                            rs698

                                                            Controls

                                                            %

                                                            Cases

                                                            %

                                                            OR

                                                            lower

                                                            upper

                                                            P-value

                                                            Codominant

                                                                    

                                                            A/A

                                                            629

                                                            46.1

                                                            616

                                                            44.2

                                                            1.00

                                                              

                                                            0.13565

                                                            A/G

                                                            610

                                                            44.7

                                                            620

                                                            44.4

                                                            1.04

                                                            0.89

                                                            1.22

                                                             

                                                            G/G

                                                            125

                                                            9.2

                                                            159

                                                            11.4

                                                            1.30

                                                            1.00

                                                            1.69

                                                             

                                                            Dominant

                                                                    

                                                            A/A

                                                            629

                                                            46.1

                                                            616

                                                            44.2

                                                            1.00

                                                              

                                                            0.29181

                                                            A/G-G/G

                                                            735

                                                            53.9

                                                            779

                                                            55.8

                                                            1.08

                                                            0.93

                                                            1.26

                                                             

                                                            Recessive

                                                                    

                                                            A/A-A/G

                                                            1239

                                                            90.8

                                                            1236

                                                            88.6

                                                            1.00

                                                              

                                                            0.05243

                                                            G/G

                                                            125

                                                            9.2

                                                            159

                                                            11.4

                                                            1.28

                                                            1.00

                                                            1.63

                                                             

                                                            Log-Additive

                                                                    

                                                            0, 1, 2

                                                            1364

                                                            49.4

                                                            1395

                                                            50.6

                                                            1.10

                                                            0.98

                                                            1.24

                                                            0.09043

                                                            IL6

                                                                    

                                                            rs1800795

                                                            Controls

                                                            %

                                                            Cases

                                                            %

                                                            OR

                                                            lower

                                                            upper

                                                            P-value

                                                            Codominant

                                                                    

                                                            G/G

                                                            593

                                                            42.7

                                                            586

                                                            41.7

                                                            1.00

                                                              

                                                            0.7950

                                                            G/C

                                                            623

                                                            44.9

                                                            635

                                                            45.2

                                                            1.03

                                                            0.88

                                                            1.21

                                                             

                                                            C/C

                                                            172

                                                            12.4

                                                            184

                                                            13.1

                                                            1.08

                                                            0.85

                                                            1.37

                                                             

                                                            Dominant

                                                                    

                                                            G/G

                                                            593

                                                            42.7

                                                            586

                                                            41.7

                                                            1.00

                                                              

                                                            0.5862

                                                            G/C-C/C

                                                            795

                                                            57.3

                                                            819

                                                            58.3

                                                            1.04

                                                            0.90

                                                            1.21

                                                             

                                                            Recessive

                                                                    

                                                            G/G-G/C

                                                            1216

                                                            87.6

                                                            1221

                                                            86.9

                                                            1.00

                                                              

                                                            0.5763

                                                            C/C

                                                            172

                                                            12.4

                                                            184

                                                            13.1

                                                            1.07

                                                            0.85

                                                            1.33

                                                             

                                                            log-Additive

                                                                    

                                                            0, 1, 2

                                                            1388

                                                            49.7

                                                            1405

                                                            50.3

                                                            1.04

                                                            0.93

                                                            1.16

                                                            0.5034

                                                            ARL11

                                                                    

                                                            rs3803185

                                                            Controls

                                                            %

                                                            Cases

                                                            %

                                                            OR

                                                            lower

                                                            upper

                                                            P-value

                                                            Codominant

                                                                    

                                                            A/A

                                                            359

                                                            26.9

                                                            301

                                                            23.7

                                                            1.00

                                                              

                                                            0.12263

                                                            A/G

                                                            686

                                                            51.4

                                                            666

                                                            52.4

                                                            1.15

                                                            0.95

                                                            1.39

                                                             

                                                            G/G

                                                            289

                                                            21.7

                                                            304

                                                            23.9

                                                            1.26

                                                            1.01

                                                            1.57

                                                             

                                                            Dominant

                                                                    

                                                            A/A

                                                            359

                                                            26.9

                                                            301

                                                            23.7

                                                            1.00

                                                              

                                                            0.06417

                                                            A/G-G/G

                                                            975

                                                            73.1

                                                            970

                                                            76.3

                                                            1.18

                                                            0.99

                                                            1.41

                                                             

                                                            Recessive

                                                                    

                                                            A/A-A/G

                                                            1045

                                                            78.3

                                                            967

                                                            76.1

                                                            1.00

                                                              

                                                            0.15480

                                                            G/G

                                                            289

                                                            21.7

                                                            304

                                                            23.9

                                                            1.14

                                                            0.95

                                                            1.37

                                                             

                                                            log-Additive

                                                                    

                                                            0, 1, 2

                                                            1334

                                                            51.2

                                                            1271

                                                            48.8

                                                            1.12

                                                            1.00

                                                            1.25

                                                            0.04338

                                                            GALNTL2

                                                                    

                                                            rs2102302

                                                            Controls

                                                            %

                                                            Cases

                                                            %

                                                            OR

                                                            lower

                                                            upper

                                                            P-value

                                                            Codominant

                                                                    

                                                            A/A

                                                            579

                                                            42.1

                                                            538

                                                            39.0

                                                            1.00

                                                              

                                                            0.16056

                                                            A/G

                                                            607

                                                            44.1

                                                            624

                                                            45.3

                                                            1.11

                                                            0.94

                                                            1.30

                                                             

                                                            G/G

                                                            189

                                                            13.7

                                                            217

                                                            15.7

                                                            1.23

                                                            0.98

                                                            1.55

                                                             

                                                            Dominant

                                                                    

                                                            A/A

                                                            579

                                                            42.1

                                                            538

                                                            39.0

                                                            1.00

                                                              

                                                            0.0971

                                                            A/G-G/G

                                                            796

                                                            57.9

                                                            841

                                                            61.0

                                                            1.14

                                                            0.98

                                                            1.32

                                                             

                                                            Recessive

                                                                    

                                                            A/A-A/G

                                                            1186

                                                            86.3

                                                            1162

                                                            84.3

                                                            1.00

                                                              

                                                            0.14317

                                                            G/G

                                                            189

                                                            13.7

                                                            217

                                                            15.7

                                                            1.17

                                                            0.95

                                                            1.45

                                                             

                                                            log-Additive

                                                                    

                                                            0, 1, 2

                                                            1375

                                                            49.9

                                                            1379

                                                            50.1

                                                            1.11

                                                            1.00

                                                            1.24

                                                            0.0558

                                                            Significant P-values (< 0.05) are highlighted in bold.

                                                            OR, odds ratio

                                                            In order to further investigate rs3803185, we were able to genotype it additionally outside EPICOLON in a cohort from a Spanish multicase-control population study for common neoplasms (MCC-Spain). This variant was not significantly associated with CRC risk in this independent cohort. Pooled analysis for rs3803185 in the EPICOLON and MCC-Spain cohorts still showed borderline significance (OR = 1.08, 95% CI = 0.98-1.18, P-value = 0.09, log-additive 0, 1, 2 alleles).

                                                            Associations of rs698, rs1800795, rs3803185 and rs2102302 were also evaluated in 2 cohorts described in a previous GWAS [30], either by checking the original variant or a proxy SNP highly correlated with it (r2 > 0.7) (Table 3). Interestingly, rs3803185 showed again significance in one of the GWAS (P = 0.03). However, it should be commented that weak associations observed in our study would have not been present if Bonferroni correction for multiple testing was applied and, therefore, they should be considered as not statistically significant.
                                                            Table 3

                                                            Association results for four selected SNPs evaluated in two external GWAS (CORGI and VQ58), either by checking the original variant or a proxy SNP highly correlated with it (r2 > 0

                                                             

                                                            Present in I5?

                                                            Present in I3?

                                                            Proxy

                                                            r2 and D'

                                                            P-value (CORGI)

                                                            P-value (VQ58)

                                                            rs698

                                                            no

                                                            no

                                                            rs1789924

                                                            1.000 1.000

                                                            0.1806

                                                            0.1658

                                                            rs1800795

                                                            no

                                                            no

                                                            rs1554606

                                                            0.868 0.932

                                                            0.1743

                                                            0.3346

                                                            rs3803185

                                                            no

                                                            no

                                                            rs4942859

                                                            0.716 1.000

                                                            0.5943

                                                            0.03525

                                                            rs2102302

                                                            no

                                                            no

                                                            rs2730351

                                                            0.853 1.000

                                                            0.1447

                                                            0.4666

                                                            Significant P-values (< 0.05) are highlighted in bold.

                                                            I5, Illumina HumanHap550; I3 Illumina HumanHap300.

                                                            Discussion

                                                            Ten previously identified SNPs in ADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11, and 18 selected variants in the mucin gene family were evaluated with a genetic association strategy. CRC cases and matched controls collected in two independent stages within the EPICOLON consortium were genotyped in order to evaluate its potential association with CRC risk. Mucins and GALNTs, members of the mucin gene family, have not been previously evaluated for CRC genetic susceptibility.

                                                            Significant results for previously identified SNPs in independent cohorts regarding CRC risk were previously reported for APC [11, 12], CCDN1 [13, 14], IL6 and IL8 [15, 16], IRS1 [17], MTHFR [1820], PPARG [21], and VDR [22, 23]. Remarkably, variants in MTHFR and CCDN1 were additionally supported by meta-analyses of several individual studies [31]. However, none of them was confirmed in our cohort.

                                                            In our study, four SNPs were significant in EPICOLON stage 1 (rs698 in ADH1C, rs1800795 in IL6, rs3803185 in ARL11, and rs2102302 in GALNTL2), but only rs3803185 achieved statistical significance in EPICOLON stage 2. In the joint analysis for both stages, results were only significant for rs3803185 and borderline significant for rs698 and rs2102302. The rs3803185 variant was not significantly associated with CRC risk in an external cohort (MCC-Spain), but it still showed some borderline significance in the pooled analysis of both cohorts.

                                                            ARL11, also known as ARLTS1 (ADP-ribosylation factor-like tumor suppressor gene 1), is a tumor suppressor gene that belongs to the ARF family of the Ras superfamily of small GTPases that are known to be involved in multiple regulatory pathways altered in human carcinogenesis. It has been suggested that ARL11 SNPs, especially rs3803185, may act as low penetrance variants in several neoplasms including CRC [6, 24, 32]. The ADH1C gene encodes for class I alcohol dehydrogenase, gamma subunit, which is a member of the alcohol dehydrogenase family. Members of this enzyme family metabolize a wide variety of substrates, including ethanol. There is a noticeable association between alcohol consumption and CRC risk [33], and it is plausible that this association could also be modified by germline variants in enzymes that metabolize ethanol. GALNTL2 (UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-like 2), also known as GALNT15, is ubiquitously expressed in human tissues [34]. This gene has never been investigated in CRC susceptibility and it has only been reported to be studied as a genetic factor involved in longevity [35]. Chronic inflammation is in the etiology of CRC and release of large amount of cytokines and growth factors may influence the carcinogenesis process. The IL6 (interleukin 6) gene, among others, has been analyzed in previous independent genetic association studies and it is an a priori attractive candidate for genetic susceptibility to CRC [15, 16].

                                                            Finally, as limitations of our study, it should be commented that our cohort sample size may probably be not large enough to reach stronger conclusions for the analyzed variants. However, our study (1,416 CRC cases and 1,424 controls for the EPICOLON cohorts) had an estimated 80% power to detect an OR as low as 1.3 with a MAF of 0.30, and 1.34 for a MAF down to 0.20, assuming a log-additive model and α = 0.05. Also, since we did not follow a GWAS strategy, our results apply only for the selected SNPs. Nevertheless, gene/SNP selection was biased to include those with previously published positive association results for CRC risk or those mucin SNPs with a putative functional effect.

                                                            In summary, none of the 28 SNPs analyzed in our study could be associated with CRC risk. However, variants in ARL11, ADH1C, GALNTL2 and IL6 may have an effect on CRC risk. Mucins and GALNTs, included in the mucin gene family, have been found deregulated in CRC and other neoplasms, and they are interesting candidates for CRC genetic susceptibility [27]. Since they have not been previously evaluated and despite our mostly negative results, we consider that genetic variation in the mucin gene family should be further explored in larger CRC cohorts in order to draw more solid conclusions. Also, additional case-control studies in larger CRC cohorts and meta-analyses could be useful to confirm or refute the role of ARL11, ADH1C, GALNTL2 and IL6 variants in CRC susceptibility.

                                                            Conclusions

                                                            None of the 28 SNPs analyzed in our study could be associated with CRC risk. However, ARL11, ADH1C, GALNTL2 and IL6 genetic variants may have an effect on CRC risk. Further validation and meta-analyses should be undertaken in larger CRC cohorts.

                                                            Abbreviations

                                                            CRC: 

                                                            colorectal cancer

                                                            SNP: 

                                                            single nucleotide polymorphism

                                                            GWAS: 

                                                            genome-wide association study

                                                            GALNT: 

                                                            N-acetylgalactosaminyltransferase

                                                            MCC: 

                                                            multicase-control

                                                            MAF: 

                                                            minor allele frequency

                                                            MALDI-TOF MS: 

                                                            matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

                                                            95% CI: 

                                                            95% confidence interval

                                                            OR: 

                                                            odds ratio

                                                            ARLTS1: 

                                                            ADP-ribosylation factor-like tumor suppressor gene 1

                                                            GALNTL2: 

                                                            UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-like 2

                                                            Chr: 

                                                            Chromosome

                                                            UTR: 

                                                            untranslated region

                                                            I5: 

                                                            Illumina HumanHap550

                                                            I3: 

                                                            Illumina HumanHap300

                                                            AIC: 

                                                            Akaike information content.

                                                            Declarations

                                                            Acknowledgements and Funding

                                                            We are sincerely grateful to all patients participating in this study who were recruited in 25 (EPICOLON 1) and 14 (EPICOLON 2) Spanish hospitals as part of the EPICOLON project (a list of members is provided below). We are also grateful to the Spanish National Genotyping Center (CEGEN-ISCIII)-USC and UPF nodes, and the Genome Analysis Platform of the CIC-BioGUNE. The work was carried out (in part) at the Esther Koplowitz Centre, Barcelona. SCB is supported by a contract from the Fondo de Investigación Sanitaria (CP 03-0070). CF has obtained a FPU Fellowship from the Ministerio de Educacion. VAE and JM are supported by a contract from the CIBERehd. CIBERehd, CIBERER and CIBERESP are funded by the Instituto de Salud Carlos III. This work was supported by grants from the Fondo de Investigación Sanitaria/FEDER (06/1384, 08/0024, 08/0533, 08/1276, 10/00918), Instituto de Salud Carlos III (Acción Transversal de Cáncer), Ministerio de Ciencia e Innovación (SAF2010-19273), Asociación Española contra el Cáncer (Fundación Científica y Junta de Barcelona), Fundació Olga Torres (SCB and CRP), and EU FP7 (FOOD-CT-2006-036224, CMV and CHIBCHA Consortium, LCC, ACar and SCB).

                                                            Members of the EPICOLON Consortium (Gastrointestinal Oncology Group of the Spanish Gastroenterological Association)

                                                            Hospital 12 de Octubre, Madrid: Juan Diego Morillas (local coordinator), Raquel Muñoz, Marisa Manzano, Francisco Colina, Jose Díaz, Carolina Ibarrola, Guadalupe López, Alberto Ibáñez; Hospital Clínic, Barcelona: Antoni Castells (local coordinator), Virgínia Piñol, Sergi Castellví-Bel, Francesc Balaguer, Victoria Gonzalo, Teresa Ocaña, María Dolores Giráldez, Maria Pellisé, Anna Serradesanferm, Leticia Moreira, Miriam Cuatrecasas, Josep M. Piqué; Hospital Clínico Universitario, Zaragoza: Ángel Lanas (local coordinator), Javier Alcedo, Javier Ortego; Hospital Cristal-Piñor, Complexo Hospitalario de Ourense: Joaquin Cubiella (local coordinator), Mª Soledad Díez, Mercedes Salgado, Eloy Sánchez, Mariano Vega; Hospital del Mar, Barcelona: Montserrat Andreu (local coordinator), Anna Abuli, Xavier Bessa, Mar Iglesias, Agustín Seoane, Felipe Bory, Gemma Navarro, Beatriz Bellosillo; Josep Mª Dedeu, Cristina Álvarez, Begoña Gonzalez; Hospital San Eloy, Baracaldo and Hospital Donostia, CIBERehd, University of Country Basque, San Sebastián: Luis Bujanda (local coordinator) Ángel Cosme, Inés Gil, Mikel Larzabal, Carlos Placer, María del Mar Ramírez, Elisabeth Hijona, Jose M. Enríquez-Navascués y Jose L. Elosegui; Hospital General Universitario de Alicante: Artemio payá (EPICOLON I local coordinator), Rodrigo Jover (EPICOLON II local coordinator), Cristina Alenda, Laura Sempere, Nuria Acame, Estefanía Rojas, Lucía Pérez-Carbonell; Hospital General de Granollers: Joaquim Rigau (local coordinator), Ángel Serrano, Anna Giménez; Hospital General de Vic: Joan Saló (local coordinator), Eduard Batiste-Alentorn, Josefina Autonell, Ramon Barniol; Hospital General Universitario de Guadalajara and Fundación para la Formación e Investigación Sanitarias Murcia: Ana María García (local coordinator), Fernando Carballo, Antonio Bienvenido, Eduardo Sanz, Fernando González, Jaime Sánchez, Akiko Ono; Hospital General Universitario de Valencia: Mercedes Latorre (local coordinator), Enrique Medina, Jaime Cuquerella, Pilar Canelles, Miguel Martorell, José Ángel García, Francisco Quiles, Elisa Orti; CHUVI-Hospital Meixoeiro, Vigo: EPICOLON I: Juan Clofent (local coordinator), Jaime Seoane, Antoni Tardío, Eugenia Sanchez. EPICOLON II Mª Luisa de Castro (local coordinator), Antoni Tardío, Juan Clofent, Vicent Hernández; Hospital Universitari Germans Trias i Pujol, Badalona and Section of Digestive Diseases and Nutrition, University of Illinois at Chicago, IL, USA.: Xavier Llor (local coordinator), Rosa M. Xicola, Marta Piñol, Mercè Rosinach, Anna Roca, Elisenda Pons, José M. Hernández, Miquel A. Gassull; Hospital Universitari Mútua de Terrassa: Fernando Fernández-Bañares (local coordinator), Josep M. Viver, Antonio Salas, Jorge Espinós, Montserrat Forné, Maria Esteve; Hospital Universitari Arnau de Vilanova, Lleida: Josep M. Reñé (local coordinator), Carmen Piñol, Juan Buenestado, Joan Viñas; Hospital Universitario de Canarias: Enrique Quintero (local coordinator), David Nicolás, Adolfo Parra, Antonio Martín; Hospital Universitario La Fe, Valencia: Lidia Argüello (local coordinator), Vicente Pons, Virginia Pertejo, Teresa Sala; Hospital Sant Pau, Barcelona: Dolors Gonzalez (local coordinator) Eva Roman, Teresa Ramon, Maria Poca, Mª Mar Concepción, Marta Martin, Lourdes Pétriz; Hospital Xeral Cies, Vigo: Daniel Martinez (local coordinator); Fundacion Publica Galega de Medicina Xenomica (FPGMX), CIBERER, Genomic Medicine Group-University of Santiago de Compostela, Santiago de Compostela, Galicia, Spain: Ángel Carracedo (local coordinator), Clara Ruiz-Ponte, Ceres Fernández-Rozadilla, Mª Magdalena Castro; Hospital Universitario Central de Asturias: Sabino Riestra (local coordinator), Luis Rodrigo; Hospital de Galdácano, Vizcaya: Javier Fernández (local coordinator), Jose Luis Cabriada; Fundación Hospital de Calahorra (La Rioja) La Rioja: Luis Carreño (local coordinator), Susana Oquiñena, Federico Bolado; Hospital Royo Villanova, Zaragoza: Elena Peña (local coordinator), José Manuel Blas, Gloria Ceña, Juan José Sebastián; Hospital Universitario Reina Sofía, Córdoba: Antonio Naranjo (local coordinator).

                                                            Authors’ Affiliations

                                                            (1)
                                                            Department of Gastroenterology, IDIBAPS, Hospital Clínic, CIBERehd, University of Barcelona
                                                            (2)
                                                            Gastroenterology Department, Parc de Salut Mar, Institut Municipal d’Investigació Mèdica (IMIM), Pompeu Fabra University
                                                            (3)
                                                            Galician Public Foundation of Genomic Medicine, CIBERER, Genomics Medicine Group, Hospital Clínico, Santiago de Compostela, University of Santiago de Compostela
                                                            (4)
                                                            Hospital Sant Pau
                                                            (5)
                                                            Department of Gastroenterology, Hospital Meixoeiro
                                                            (6)
                                                            Department of Gastroenterology, Hospital de Ourense
                                                            (7)
                                                            Department of Gastroenterology, Hospital 12 de Octubre
                                                            (8)
                                                            Department of Medicine, Hospital General de Granollers
                                                            (9)
                                                            Department of Gastroenterology, Hospital General Universitario de Valencia
                                                            (10)
                                                            Department of Gastroenterology, Hospital Mutua de Terrassa
                                                            (11)
                                                            Department of Gastroenterology, Hospital Royo Villanova
                                                            (12)
                                                            Department of Gastroenterology, Hospital Central de Asturias
                                                            (13)
                                                            Department of Pathology and Gastroenterology, Hospital General d’Alacant
                                                            (14)
                                                            Section of Digestive Diseases and Nutrition and Cancer Center, University of Illinois at Chicago
                                                            (15)
                                                            Wellcome Trust Centre for Human Genetics, University of Oxford
                                                            (16)
                                                            Centre for Research in Environmental Epidemiology (CREAL), IMIM (Hospital del Mar Research Institute). CIBER Epidemiología y Salud Pública (CIBERESP)
                                                            (17)
                                                            IDIBELL-Institut Català d’Oncologia (ICO), CIBER Epidemiología y Salud Pública (CIBERESP), University of Barcelona, L’Hospitalet de Llobregat
                                                            (18)
                                                            All authors are listed in the Acknowledgements section

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                                                            36. Pre-publication history

                                                              1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2407/​11/​339/​prepub

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                                                            © Abulí et al; licensee BioMed Central Ltd. 2011

                                                            This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.