Metallothionein genes: no association with Crohn's disease in a New Zealand population

  • Angharad R Morgan1, 2Email author,

    Affiliated with

    • Alan G Fraser2, 3 and

      Affiliated with

      • Lynnette R Ferguson1, 2

        Affiliated with

        Journal of Negative Results in BioMedicine201211:8

        DOI: 10.1186/1477-5751-11-8

        Received: 23 November 2011

        Accepted: 28 January 2012

        Published: 28 January 2012

        Abstract

        Metallothioneins (MTs) are excellent candidate genes for Inflammatory Bowel Disease (IBD) and have previously been shown to have altered expression in both animal and human studies of IBD. This is the first study to examine genetic variants within the MT genes and aims to determine whether such genetic variants have an important role in this disease. 28 tag SNPs in genes MT1 (subtypes A, B, E, F, G, H, M, X), MT2, MT3 and MT4 were selected for genotyping in a well-characterized New Zealand dataset consisting of 406 patients with Crohn's Disease and 638 controls. We did not find any evidence of association for MT genetic variation with CD. The lack of association indicates that genetic variants in the MT genes do not play a significant role in predisposing to CD in the New Zealand population.

        Keywords

        Crohn's disease Metallothioneins genetic association

        Background

        Inflammatory bowel disease (IBD) is a disorder characterised by chronic, relapsing inflammation of the gastrointestinal tract and is characterized by the presence of an increased level of reactive oxygen species in the mucosal intestinal tissue as well as oxidative DNA and protein damage, defective host-microbe interactions, immune cell infiltration, and disturbed T cell apoptosis. Metallothioneins (MTs) are able to affect all of these processes, making them good candidates for IBD. MTs are ubiquitous metal-binding proteins that have been highly conserved throughout evolution and are rapidly upregulated in response to an inflammatory stimulus. There are four main isoforms expressed in humans: MT1 (subtypes A, B, E, F, G, H, M, X), MT2, MT3 and MT4. These genes cluster together on a single locus on chromosome 16 (16q13) [1].

        Support for the hypothesis of MTs playing an important role in the pathogenesis of IBD has come from various reports demonstrating altered MT expression in IBD. However these reports have been contradictory (for a summary see Waeytens et al., 2009 [2]) with some studies reporting MT upregulation in IBD [3, 4] and others demonstrating a downregulation [511]. The inconsistencies between the various studies may be explained by differences in patients samples such as age, medication, disease activity, zinc status, as well as from where the tissue was sampled (ileum or colon).

        Whilst there have been several studies investigating expression of MTs in IBD, there have been no studies published to date investigating genetic variants in MTs and IBD. The aim of this study is to identify SNPs across these genes and to genotype these in a well-characterised Caucasian New Zealand IBD dataset and to examine the results for evidence of genetic association between any of the MT genes and IBD.

        Methods

        Samples

        A total of 1044 subjects from New Zealand were included in the study: 406 CD patients and 638 age and sex matched controls. All participants self-reported European ancestry.

        Clinical records were analysed to confirm diagnosis, and IBD status was defined using standard diagnostic criteria [12]. Cases were phenotyped according to the Montreal Classification systems. Clinical characteristics of the CD patients are shown in table 1.
        Table 1

        Summary of clinical data of CD patients.

          

        CD

        Gender

        F

        265 (65.6)

         

        M

        139 (34.4)

        Age at Diagnosis

        < 17

        46 (12.6)

         

        17 to 40

        257 (70.2)

         

        40 <

        63 (17.2)

        CD Behaviour

        Inflammatory

        201 (55.1)

         

        Stricturing

        118 (32.3)

         

        Penetrating

        46 (12.6)

        CD Location

        Ileal

        136 (37.2)

         

        Colonic

        119 (32.5)

         

        Ileocolonic

        111 (30.3)

        Bowel resection

        N

        270 (66.7)

         

        Y

        135 (33.3)

        Other IBD family

        N

        330 (89.7)

         

        Y

        38 (10.3)

        EIM

        N

        301 (81.8)

         

        Y

        67 (18.2)

        Perianal disease

        N

        329 (85.7)

         

        Y

        55 (14.3)

        Participants consented to collection of peripheral blood or a buccal swab for DNA extraction and genotyping, and DNA was extracted from the blood/buccal samples using Qiagen's DNA extraction kit and following the manufacturer's instructions.

        The study was conducted under ethical protocol MEC/04/12/011, authorised through the New Zealand Multi-Region Human Ethics Committee. All study subjects gave informed consent.

        Snp selection

        Tag SNPs in genes MT1 (subtypes A, B, E, F, G, H, M, X), MT2, MT3 and MT4 were selected using Hapmap release 28, and the tagger functionality within Haploview with pairwise tagging to identify SNPs using an r2 > 0.8 and a minor allele frequency > 5%. As a result 28 tag SNPs were selected for genotyping.

        Genotyping

        Genotyping was performed with the MassARRAY and iPlex systems of the Sequenom genotyping platform (Sequenom, San Diego, CA), which uses the MALDI-TOF primer extension assay [13, 14], according to manufacturers' recommendations.

        Assays were optimized in 24 samples consisting of 20 reference Centre d'Etude du Polymorphisme Humain (CEPH) samples and 4 blanks.

        All sample plates contained cases, controls, blanks, CEPH and duplicate samples. Quality control measures included independent double genotyping and, where available, comparison of our CEPH genotypes to those in the Hapmap database http://​www.​hapmap.​org.

        Statistical analysis

        SNPs were tested for deviation from HWE in both cases and controls using a chi-square goodness-of-fit test. To determine if there were differences between cases and controls, genotype and allele frequencies for each SNP were analyzed by 2 × 3 and 2 × 2 Chi-square tables respectively

        Genotype and phenotype associations were assessed by comparing allele frequencies between controls and patient subgroups defined using the clinical characteristics. These analyses were carried out using R (R: A language and environment for statistical computing, R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://​www.​R-project.​org/​) and SAS (V9.1 SAS Institute., Cary, NC, USA).

        To determine linkage disequilibrium (LD) between SNPs and to define haplotype blocks, we uploaded our data into Haploview [15]. Haplotype blocks were defined using the default algorithm which uses confidence intervals [16]. Haplotype analysis was carried using HAPLO.SCORE in R to test for association of these haplotypes with CD.

        For all analyses we considered a p values less than 0.05 to indicate statistical significance.

        The false discovery rate (FDR) was used to correct for multiple testing [17, 18].

        Results and discussion

        Three SNPs did not fit into the Sequenom multiplexes: rs1827210 (MT1M), rs11076161 (MT1A), rs2298846 (MT1G). Two SNPs failed to be successfully genotyped: rs1827208 (MT1M) and rs12448654 (MT1G). One SNP was out of HWE in the control samples and so was excluded from further analysis: rs12051311 (MT1B). Thus there were 22 SNPs with genotype data for analysis. Genotype and allele counts/frequencies and p-values are shown in table 2. One SNP rs4784708 (MT1H) was found to be associated with CD (genotypic p = 0.0197, allelic p = 0.016) but after applying multiple testing correction using FDR the result is no longer statistically significant. Thus we can conclude that individual SNPs in the MT genes are not associated with CD.
        Table 2

        Genotype and allele counts (and frequencies) in CD patients and in controls

        SNP

        Location

        gene

         

        CASE

        CONTROL

        p

         

        CASE

        CONTROL

        p

        rs182221

        56, 599, 221

        MT4

        G/G

        262 (0.66)

        398 (0.64)

        0.87

        G

        646 (0.81)

        995 (0.80)

        0.72

           

        C/G

        122 (0.31)

        199 (0.32)

         

        C

        154 (0.19)

        247 (0.20)

         
           

        C/C

        16 (0.04)

        24 (0.04)

             

        rs666636

        56, 601, 720

        MT4

        G/G

        386 (0.95)

        599 (0.95)

        0.70*

        G

        791 (0.98)

        1229 (0.97)

        0.55

           

        A/G

        19 (0.05)

        31 (0.05)

         

        A

        19 (0.02)

        35 (0.03)

         
           

        A/A

        0 (0.00)

        2 (0.00)

             

        rs666647

        56, 601, 722

        MT4

        C/C

        386 (0.95)

        605 (0.95)

        0.73*

        C

        791 (0.98)

        1241 (0.97)

        0.58

           

        C/T

        19 (0.05)

        31 (0.05)

         

        T

        19 (0.02)

        35 (0.03)

         
           

        T/T

        0 (0.00)

        2 (0.00)

             

        rs669293

        56, 602, 352

        MT4

        T/T

        293 (0.73)

        428 (0.67)

        0.16

        T

        686 (0.85)

        1039 (0.82)

        0.051

           

        C/T

        100 (0.25)

        283 (0.29)

         

        C

        120 (0.15)

        231 (0.18)

         
           

        C/C

        10 (0.02)

        24 (0.04)

             

        rs762604

        56, 602, 671

        MT4

        C/C

        183 (0.45)

        302 (0.48)

        0.18

        C

        538 (0.67)

        878 (0.70)

        0.15

           

        C/T

        172 (0.43)

        274 (0.44)

         

        T

        268 (0.33)

        380 (0.30)

         
           

        T/T

        48 (0.12)

        53 (0.08)

             

        rs11643815

        56, 602, 798

        MT4

        G/G

        307 (0.76)

        482 (0.76)

        0.53

        G

        700 (0.86)

        1102 (0.87)

        0.61

           

        A/G

        86 (0.21)

        138 (0.22)

         

        A

        110 (0.14)

        162 (0.13)

         
           

        A/A

        12 (0.03)

        12 (0.02)

             

        rs11644094

        56, 624, 079

        MT3

        A/A

        134 (0.34)

        220 (0.35)

        0.90

        A

        460 (0.58)

        736 (0.58)

        0.79

           

        A/G

        192 (0.48)

        295 (0.47)

         

        G

        338 (0.42)

        527 (0.42)

         
           

        G/G

        73 (0.18)

        116 (0.18)

             

        rs10636

        56, 643, 343

        MT2A

        G/G

        228 (0.57)

        358 (0.57)

        0.66

        G

        598 (0.74)

        946 (0.75)

        0.67

           

        C/G

        142 (0.35)

        230 (0.37)

         

        C

        206 (0.26)

        312 (0.25)

         
           

        C/C

        32 (0.08)

        41 (0.07)

             

        rs2070836

        56, 660, 120

        MT1E

        G/G

        346 (0.85)

        530 (0.84)

        0.56

        G

        746 (0.92)

        1156 (0.92)

        0.69

           

        C/G

        54 (0.13)

        96 (0.15)

         

        C

        64 (0.08)

        106 (0.08)

         
           

        C/C

        5 (0.01)

        5 (0.01)

             

        rs708274

        56, 660, 906

        MT1E

        G/G

        310 (0.78)

        482 (0.77)

        0.22

        G

        701 (0.88)

        1100 (0.88)

        0.80

           

        G/T

        81 (0.20)

        136 (0.22)

         

        T

        95 (0.12)

        144 (0.12)

         
           

        T/T

        7 (0.02)

        4 (0.01)

             

        rs2270836

        56, 667, 614

        MT1M

        C/C

        169 (0.42)

        231 (0.37)

        0.19

        C

        516 (0.64)

        764 (0.61)

        0.083

           

        C/T

        178 (0.44)

        302 (0.48)

         

        T

        286 (0.36)

        498 (0.39)

         
           

        T/T

        54 (0.13)

        98 (0.16)

             

        rs9936741

        56, 667, 785

        MT1M

        T/T

        387 (0.96)

        610 (0.95)

        0.94*

        T

        792 (0.98)

        1249 (0.98)

        0.94

           

        C/T

        18 (0.04)

        29 (0.05)

         

        C

        18 (0.02)

        29 (0.02)

         
           

        C/C

        0 (0.00)

        0 (0.00)

             

        rs7190725

        56, 673, 290

        MT1A

        G/G

        308 (0.76)

        483 (0.76)

        0.99

        G

        706 (0.87)

        1109 (0.87)

        0.94

           

        G/T

        90 (0.22)

        143 (0.22)

         

        T

        104 (0.13)

        165 (0.13)

         
           

        T/T

        7 (0.02)

        11 (0.02)

             

        rs964372

        56, 686, 030

        MT1B

        G/G

        317 (0.78)

        514 (0.81)

        0.65

        G

        714 (0.88)

        1142 (0.89)

        0.34

           

        C/G

        80 (0.20)

        114 (0.18)

         

        C

        96 (0.12)

        134 (0.11)

         
           

        C/C

        8 (0.02)

        10 (0.02)

             

        rs2070839

        56, 686, 242

        MT1B

        C/C

        152 (0.38)

        256 (0.40)

        0.17

        C

        503 (0.63)

        792 (0.62)

        0.89

           

        C/T

        199 (0.50)

        280 (0.44)

         

        T

        301 (0.37)

        480 (0.38)

         
           

        T/T

        51 (0.13)

        100 (0.16)

             

        rs2291956

        56, 692, 218

        MT1F

        C/C

        308 (0.76)

        481 (0.76)

        0.97

        C

        705(0.87)

        1104 (0.87)

        0.97

           

        C/T

        89 (0.22)

        142 (0.22)

         

        T

        103 (0.13)

        162 (0.13)

         
           

        T/T

        7 (0.02)

        10 (0.02)

             

        rs2298847

        56, 701, 294

        MT1G

        T/T

        310 (0.77)

        495 (0.79)

        0.28

        T

        709 (0.88)

        1107 (0.89)

        0.58

           

        A/T

        89 (0.22)

        117 (0.19)

         

        A

        99 (0.12)

        143 (0.11)

         
           

        A/A

        5 (0.01)

        13 (0.02)

             

        rs4784708

        56, 703, 914

        MT1H

        G/G

        376 (0.94)

        565 (0.89)

        0.0197*

        G

        778 (0.97)

        1196 (0.94)

        0.016

           

        C/G

        26 (0.06)

        66 (0.10)

         

        C

        26 (0.03)

        70 (0.06)

         
           

        C/C

        0 (0.00)

        2 (0.00)

             

        rs2062545

        56, 704, 125

        MT1H

        A/A

        364 (0.90)

        580 (0.91)

        0.43*

        A

        768 (0.95)

        1214 (0.96)

        0.42

           

        A/G

        40 (0.10)

        54 (0.09)

         

        G

        42 (0.05)

        56 (0.04)

         
           

        G/G

        1 (0.00)

        1 (0.00)

             

        rs2062546

        56, 704, 217

        MT1H

        T/T

        353 (0.87)

        566 (0.89)

        0.35*

        T

        757 (0.94)

        1200 (0.95)

        0.36

           

        A/T

        51 (0.13)

        68 (0.11)

         

        A

        51 (0.06)

        68 (0.05)

         
           

        A/A

        0 (0.00)

        0 (0.00)

             

        rs2301234

        56, 716, 982

        MT1X

        G/G

        173 (0.43)

        248 (0.39)

        0.41

        G

        517 (0.64)

        791 (0.62)

        0.35

           

        G/T

        171 (0.43)

        395 (0.46)

         

        T

        287 (0.36)

        479 (0.38)

         
           

        T/T

        58 (0.14)

        92 (0.14)

             

        *p value calculated from comparing rare homozygote + heterozygote group against common homozygote group

        Calculations of statistical power using PS 2.1.31 [19] show that using our case-control sample set although we have good power to detect an odds ratio of 1.3 for common SNPs (for example for a SNP with MAF 0.42 (e.g., rs11644094) we have 86.5% power) the power is considerably lower for SNPs with low MAF. For example for a SNP with MAF of 0.02 (e.g., rs9936741) we have 16.1% power. Thus, an association of metallothionein genes with CD cannot be entirely ruled out as it may simply be that we do not have the power in our study to detect such an association. A replication study in a larger sample set is encouraged.

        There are 5 haplotype blocks in this region (Figure 1) but examining the different haplotypes we did not find any associations with CD (data not shown but available on request).
        http://static-content.springer.com/image/art%3A10.1186%2F1477-5751-11-8/MediaObjects/12952_2011_111_Fig1_HTML.jpg
        Figure 1

        LD plot

        We also undertook a phenotype analysis and after multiple testing corrections none of the SNPs were associated with any of the different clinical characteristics of CD (data not shown but available on request).

        This is the first study to investigate genetic association with MT SNPs and IBD. In summary although MT genes are good candidates for CD, the present study failed to detect any significant associations. The lack of association indicates that genetic variants in the MT genes do not play a significant role in predisposing to CD in the New Zealand population.

        Declarations

        Acknowledgements

        We would like to thank the many patients with IBD who participated in this study, and also the control subjects.

        We thank Wen-Jiun Lam for assistance with DNA extraction and preparation.

        Nutrigenomics New Zealand is collaboration between AgResearch Ltd., Plant & Food Research and The University of Auckland with funding through the Ministry of Science and Innovation (MSI).

        Authors’ Affiliations

        (1)
        Discipline of Nutrition, FMHS, The University of Auckland
        (2)
        Nutrigenomics New Zealand
        (3)
        Department of Medicine, FMHS, The University of Auckland

        References

        1. Karin M, Eddy RL, Henry WM, Haley LL, Byers MG, Shows TB: Human metallothionein genes are clustered on chromosome 16. Proceedings of the National Academy of Sciences of the United States of America 1984,81(17):5494–5498.PubMedView Article
        2. Waeytens A, De Vos M, Laukens D: Evidence for a potential role of metallothioneins in inflammatory bowel diseases. Mediators Inflamm 2009, 729172.
        3. Bruwer M, Schmid KW, Metz KA, et al.: Increased expression of metallothionein in inflammatory bowel disease. Inflammation Research 2001,50(60):289–293.PubMedView Article
        4. Dooley TP, Curto EV, Reddy SP, et al.: Regulation of gene expression in inflammatory bowel disease and correlation with IBD drugs: screening by DNA microarrays. Inflammatory Bowel Diseases 2004,10(1):1–14.PubMedView Article
        5. Clarkson JP, Elmes ME, Jasani B, Webb M: Histological demonstration of immunoreactive zinc metallothionein in liver and ileum of rat and man. Histochem J 1985,17(3):343–352.PubMedView Article
        6. Elmes ME, Clarkson JP, Jasani B: Histological demonstration of immunoreactive metallothionein in rat and human tissues. Experientia Suppl 1987, 52:533–537.PubMed
        7. Mulder TP, Verspaget HW, Janssens AR, et al.: Decrease in two intestinal copper/zinc containing proteins with antioxidant function in inflammatory bowel disease. Gut 1991,32(10):1146–1150.PubMedView Article
        8. Sturniolo GC, Mestriner C, Lecis PE, et al.: Altered plasma and mucosal concentrations of trace elements and antioxidants in active ulcerative colitis. Scand J Gastroenterol 1998,33(6):644–649.PubMedView Article
        9. Lawrance IC, Fiocchi C, Chakravarti S: Ulcerative colitis and Crohn's disease: distinctive gene expression profiles and novel susceptibility candidate genes. Hum Mol Genet 2001,10(5):445–456.PubMedView Article
        10. Ioachim E, Michael M, Katsanos C, et al.: The immunohistochemical expression of metallothionein in inflammatory bowel disease. Correlation with HLA-DR antigen expression, lymphocyte subpopulations and proliferation-associated indices. Histol Histopathol 2003,18(1):75–82.PubMed
        11. Kruidenier L, Kuiper I, Van Duijn W, et al.: Imbalanced secondary mucosal antioxidant response in inflammatory bowel disease. J Pathol 2003,201(1):17–27.PubMedView Article
        12. Lennard-Jones JE: Classification of inflammatory bowel disease. Scandinavian Journal of Gastroenterology 1989, 170:2–6.View Article
        13. Jurinke C, van der Boom D, Cantor CR, Köster H: The use of massARRAY technology for high throughput genotyping. Adv Biochem Eng Biotechnol 2002, 77:57–74.PubMed
        14. Storm N, Darnhofer-Patel B, van der Boom D, Rodi CP: MALDI-TOF mass spectrometry-based SNP genotyping. Methods Molecular Biology 2003, 212:241–262.
        15. Barrett JC, Fry B, Maller J, Daly MJ: Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005,21(2):263–265.PubMedView Article
        16. Gabriel SB, Schaffner SF, Nguyen H, et al.: The structure of haplotype blocks in the human genome. Science 2002,296(5576):2225–2229.PubMedView Article
        17. Bender R, Lange S: Adjusting for multiple testing--when and how? Journal of Clinical Epidemiology 2001, 54:343–349.PubMedView Article
        18. Jones HE, Ohlssen DI, Spiegelhalter DJ: Use of the false discovery rate when comparing multiple health care providers. Journal of Clinical Epidemiology 2008, 61:232–240.PubMedView Article
        19. Dupont WD, Plummer WD Jr: Power and Sample Size Calculations for Studies Involving Linear Regression. Controlled Clinical Trials 1998, 19:589–601.PubMedView Article

        Copyright

        © Morgan et al; licensee BioMed Central Ltd. 2012

        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.