Skip to main content
NCBI home page
American Journal of Human Genetics logoLink to American Journal of Human Genetics
letter
. 2003 Nov;73(5):1208–1212. doi: 10.1086/379285

Meta-Analysis and a Large Association Study Confirm a Role for Calpain-10 Variation in Type 2 Diabetes Susceptibility

Michael N Weedon 1, Peter E H Schwarz 2, Yukio Horikawa 3, Naoko Iwasaki 4, Thomas Illig 5, Rolf Holle 6, Wolfgang Rathmann 7, Thomas Selisko 2, Jan Schulze 2, Katherine R Owen 1, Julie Evans 1, Laura del Bosque-Plata 8, Graham Hitman 9, Mark Walker 10, Jonathan C Levy 11, Mike Sampson 14, Graeme I Bell 8, Mark I McCarthy 12,13, Andrew T Hattersley 1, Timothy M Frayling 1
PMCID: PMC1180500  PMID: 14574648

To the Editor:

Variation in the calpain-10 gene (CAPN10 [MIM 605286]) was recently linked and associated with type 2 diabetes mellitus (T2DM) susceptibility (Horikawa et al. 2000). The initial linkage of T2DM to chromosome 2 was found in a population of Mexican Americans from Starr County, Texas (Hanis et al. 1996). Specific combinations of three intronic variants, designated “SNP-43,” “SNP-19,” and “SNP-63,” that capture most of the haplotype diversity at CAPN10 were associated with a three-fold increased risk of T2DM in this population and could account for the observed linkage (Horikawa et al. 2000). Subsequent association and linkage studies of these three polymorphisms in other populations have produced conflicting results, with association being observed in some populations (Baier et al. 2000 [Pima Indian]; Cassell et al. 2002 [South Indian]; Garant et al. 2002 [African American]; Malecki et al. 2002 [Polish]; Orho-Melander et al. 2002 [Finnish/Botnia]), but not others (Evans et al. 2001 [British]; Hegele et al. 2001 [Oji-Cree Indians]; Tsai et al. 2001 [Samoan]; Xiang et al. 2001 [Chinese]; Daimon et al. 2002 [Japanese]; Elbein et al. 2002 [whites from Utah]; Fingerlin et al. 2002 [Finnish]; Rasmussen et al. 2002 [Danish and Swedish]; Horikawa et al. 2003 [Japanese]).

We previously reported that another variant, SNP-44 (designated “CAPN10-g4841T→C”; minor allele frequency 16%), located in intron 3 and 11 bp from SNP-43, was independently associated with T2DM in whites from the United Kingdom (Evans et al. 2001). Further studies have provided tentative support for a role of SNP-44 in T2DM and related traits: associations with polycystic ovary syndrome (Gonzalez et al. 2002) and with measures of oral glucose tolerance (Wang et al. 2002; Tschritter et al. 2003) have been reported. Functional studies suggest that SNP-44 is located in an enhancer element and might affect CAPN10 expression (Horikawa et al. 2000). Also, in the U.K., German, Japanese, and South Indian populations, SNP-44 is in perfect linkage disequilibrium (r2=1) with a missense mutation Thr504Ala (SNP-110) and two polymorphisms in the 5′-UTR (SNP-134 and SNP-135) (Evans et al. 2001; Cassell et al. 2002; Y. Horikawa and P. E. Schwarz, unpublished data).

To assess the association of SNP-44 with T2DM more comprehensively, we performed a meta-analysis of all published SNP-44/T2DM association study data. To identify all relevant published studies, we searched PubMed using the keywords “calpain 10,” “diabetes,” “44,” “SNP 44,” “CAPN10,” and “type 2,” in different combinations. When necessary, authors were contacted to obtain exact genotype numbers, so that precise odds ratios (ORs) from each study could be calculated. Our search identified 10 published case/control studies, consisting of 3,303 subjects. The studies were spread across a number of ethnic groups: British (three studies, Evans et al. 2001); Chinese (Wang et al. 2002); Japanese (Daimon et al. 2002; Horikawa et al. 2003); Finnish/Botnia (two studies, Orho-Melander et al. 2002); South Indian (Cassell et al. 2002); and Mexican American (Horikawa et al. 2000). The frequency of the T2DM-associated SNP-44 C allele (allele 2) ranged from 6% in Mexican Americans to 25% in the Botnia I control population. There was no evidence for OR heterogeneity (Q test P=.27), and, although these studies are only a small sample from the many existing T2DM genetic resources, a funnel-plot analysis (Egger et al. 1997) suggested an absence of publication bias (P=.44). A Mantel-Haenszel meta-analysis of these studies showed that the C allele was associated with increased risk of T2DM (OR 1.17 [1.02–1.34], P=.02).

Three transmission/disequilibrium tests (TDT) had been performed (Evans et al. 2001; Cassell et al. 2002; Orho-Melander et al. 2002). The combined TDT results demonstrated that the C allele was significantly overtransmitted (117 transmitted vs. 77 not transmitted, P=.004) from heterozygous parents to diabetic offspring. Although this result cannot be considered independent replication, as proband data was included in the case/control meta-analysis from two of the TDT studies (Evans et al. 2001; Cassell et al. 2002), it provides evidence that the association is not due to population stratification. Of the 10 studies in the meta-analysis, only 1 reported a significant (P<.05) association (Evans et al. 2001). However, these studies were small and the mean power to detect an OR of 1.17 at P<.05 was ∼11% (range 5%–14%).

In the context of genetic association studies, which test many polymorphisms in numerous candidate genes, a P value of .02 can only be considered evidence suggestive of a real association. We therefore genotyped SNP-44 in an additional 4,213 subjects: 3,274 white European subjects from four case/control studies (one British, two German, and one Czech); 691 Japanese subjects from two case/control studies; and 248 Mexican (mestizo) subjects from Mexico City and Orizaba City from one case/control study. Overall, this provided 2,056 subjects with T2DM and 2,157 controls, and a power of ∼80% to detect an OR of 1.17. Clinical details of the study subjects are presented in table 1; further details are available as supplementary information from the authors. All studies were approved by the relevant ethics committee, and all subjects gave their informed consent.

Table 1.

Clinical Characteristics of Subjects in Study[Note]

Finding in Study
UK4
 German1
 German2
 Czech
 Japanese3
 Japanese4
 Mexican(Mestizo)
Characteristic ControlEFSa ControlECACCb W2 T2DcProbands YT2Dd Control T2DM Control T2DM Control T2DM Control T2DM Control T2DM Control T2DM
SNP-44 minor allele frequency .16 .16 .20 .19 .11 .14 .16 .17 .l2 .14 .09 .06 .12 .14 .04 .09
N [% males] 994 [50] 335 [56] 399 [54] 297 [55] 73 [35] 308 [38] 235 [56] 244 [59] 110 [69] 279 [36] 206 [67] 206 [59] 90 [33] 189 [57] 114 [50] 134 [42]
Average age±SD (years) 32.5±5.5 NA 70.6±8.6 NA 50.8±11.9 61.8±11.3 65±5.4 65.0±5.1 18.1±2.3 58.5±7.4 67.9±5.5 59.1±13.0 68.2±5.8 61.7±12.8 49.9±14.4 55.1±9.8
Age at diagnosis±SD (years) NA NA 55.2±8.5 40.5±10.0 NA 49.2±12.4 NA NA NA 49.6±8.7 NA 45.8±12.8 NA 50.3±12.9 NA 44.8±7.5
BMI (kg/m2)±SD 26.7±3.8e NA 28.9±5.4 30.9±4.5 24.9±4.4 28.7±4.8 27.5±3.8 31.0±4.7 24.3±4.0 30.1±5.3 23.0±2.5 23.5±3.6 23.0±2.9 23.7±4.7 26.5±3.8 27.4±4.1
% Receiving treatment:
 Diet NA NA 16 9 NA 13 NA 56 NA 26 NA 20 NA 12 NA NA
 OHAf NA NA 70 38 NA 33 NA 28 NA 58 NA 40 NA 53 NA NA
 Insulin NA NA 14 53 NA 53 NA 16 NA 16 NA 40 NA 35 NA NA
Open in a new tab

Note.— Continuous variables are presented as mean±SD. NA=not applicable or not available.

a

EFS=Exeter Family Study.

b

ECACC=European Collection of Cell Cultures.

c

W2 T2D=Warren 2 Type 2 Diabetes Collection.

d

YT2D=Young-Onset Type 2 Diabetes Collection.

e

Males only, as females were pregnant.

f

OHA=oral hypoglycemia agents.

When all the studies were combined, there was no evidence for between-studies OR heterogeneity (Q test P=.23); a Mantel-Haenszel fixed-effects model was therefore used for subsequent analysis. Meta-analysis of the new studies gave an OR for the SNP-44 C allele of 1.18 (1.04–1.34), P=.01 (fig. 1). A combined meta-analysis of all previously published data and our new data gave an OR of 1.17 (1.07–1.29), P=.0007. All study populations were in Hardy-Weinberg equilibrium except the T2DM cohort of Horikawa et al. 2003 (P=.005) and the control population of the third Japanese study (P=.02). Although these deviations may be due to random fluctuation and multiple-hypothesis testing, they contributed a large amount to heterogeneity (27% of the Q statistic); excluding these studies, the SNP-44 C allele OR for the new studies was 1.23 (1.07–1.40), P=.003; the overall OR was 1.19 (1.08–1.31), P=.0005. This OR is of similar magnitude to that of E23K (Gloyn et al. 2003; Love-Gregory et al. 2003; Nielsen et al. 2003) and Pro12Ala (Altshuler et al. 2000), the other common variants confirmed as T2DM-susceptibility polymorphisms. An OR of 1.17 is low and may help explain why there is little evidence for linkage of the CAPN10 region to T2DM in most populations. The haplotypes responsible for the CAPN10 linkage seen in the Mexican American population were associated with a higher T2DM OR (∼3.0) and were more likely to be detected by linkage analysis (Horikawa et al. 2000). These haplotypes are less common in other populations.

Figure 1.

Figure 1

Open in a new tab

Mantel-Haenszel OR meta-analysis plot (fixed effects) for SNP-44 association with T2DM. Point estimates and 95% CLs for each previously published, new, and combined case/control study.

SNP-44 is in perfect linkage disequilibrium (r2=1) with the missense mutation, Thr504Ala, and two SNPs (SNP-134 and SNP-135) in the 5′-UTR and therefore may not be the causal variant. Further haplotype and functional analyses are required to confirm which of these polymorphisms contribute to T2DM susceptibility.

In conclusion, our results have confirmed that a CAPN10 haplotype defined by the SNP-44 polymorphism predisposes to T2DM. Meta-analyses of published genetic associations, combined with large replication studies, are a powerful approach to detecting susceptibility variants in common disease.

Acknowledgments

This work was principally funded by Diabetes UK and the Warren 2 bequest. The funding for the Exeter Family Study of Childhood was provided by the South and West NHS Research Directorate for the UK4 normal population samples. This work was also supported by the Technical University, Dresden, funding grant MeDDrive and by the KORA study group: A. Döring, H. Löwel, C. Meisinger, B. Thorand, H. E. Wichmann (GSF National Research Center for Environment and Health, Institute of Epidemiology), and J. John (GSF National Research Center for Environment and Health, Institute of Health Economics and Health Care Management). Grants from the German National Genome Research Net (NGFN) platform 6 and from the GSF Research Center, as well as United States Public Health Service grants DK-20595 and DK-47486, also supported this work. T.M.F. is a career scientist of the South and West NHS Research Directorate. A.T.H. is a Wellcome Trust Career Leave Research Fellow.

Electronic-Database Information

Accession numbers and URLs for data presented herein are as follows:

  1. Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for calpain-10, KCNJ11, and PPARγ)
  2. PubMed, http://www.ncbi.nlm.nih.gov/PubMed/

References

  1. Altshuler D, Hirschhorn JN, Klannemark M, Lindgren CM, Vohl M-C, Nemesh J, Lane CR, Schaffner F, Bolk S, Brewer C, Tuomi T, Gaudet D, Hudson TJ, Daly M, Groop L, Lander ES (2000) The common PPARγ Pro12Ala polymorphism is associated with decreased risk of type 2 diabetes. Nat Genet 26:76–80 [DOI] [PubMed] [Google Scholar]
  2. Baier LJ, Permana PA, Yang X, Pratley RE, Hanson RL, Shen GQ, Mott D, Knowler WC, Cox NJ, Horikawa Y, Oda N, Bell GI, Bogardus C (2000) A calpain-10 gene polymorphism is associated with reduced muscle mRNA levels and insulin resistance. J Clin Invest 106:R69–R73 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cassell PG, Jackson AE, North BV, Evans JC, Syndercombe-Court D, Phillips C, Ramachandran A, Snehalatha C, Gelding SV, Vijayaravaghan S, Curtis D, Hitman GA (2002) Haplotype combinations of calpain 10 gene polymorphisms associate with increased risk of impaired glucose tolerance and type 2 diabetes in South Indians. Diabetes 51:1622–1628 [DOI] [PubMed] [Google Scholar]
  4. Daimon M, Oizumi T, Saitoh T, Kameda W, Yamaguchi H, Ohnuma H, Igarashi M, Manaka H, Kato T (2002) Calpain 10 gene polymorphisms are related, not to type 2 diabetes, but to increased serum cholesterol in Japanese. Diabetes Res Clin Pract 56:147–152 [DOI] [PubMed] [Google Scholar]
  5. Egger M, Davey Smith G, Schneider M, Minder C (1997) Bias in meta-analysis detected by a simple, graphical test. Bmj 315:629–634 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Elbein SC, Chu W, Ren Q, Hemphill C, Schay J, Cox NJ, Hanis CL, Hasstedt SJ (2002) Role of calpain-10 gene variants in familial type 2 diabetes in Caucasians. J Clin Endocrinol Metab 87:650–654 [DOI] [PubMed] [Google Scholar]
  7. Evans JC, Frayling TM, Cassell PG, Saker PJ, Hitman GA, Walker M, Levy JC, et al (2001) Studies of association between the gene for calpain-10 and type 2 diabetes mellitus in the United Kingdom. Am J Hum Genet 69:544–552 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fingerlin TE, Erdos MR, Watanabe RM, Wiles KR, Stringham HM, Mohlke KL, Silander K, Valle TT, Buchanan TA, Tuomilehto J, Bergman RN, Boehnke M, Collins FS (2002) Variation in three single nucleotide polymorphisms in the calpain-10 gene not associated with type 2 diabetes in a large Finnish cohort. Diabetes 51:1644–1648 [DOI] [PubMed] [Google Scholar]
  9. Garant MJ, Kao WH, Brancati F, Coresh J, Rami TM, Hanis CL, Boerwinkle E, Shuldiner AR (2002) SNP43 of CAPN10 and the risk of type 2 diabetes in African-Americans: the atherosclerosis risk in communities study. Diabetes 51:231–237 [DOI] [PubMed] [Google Scholar]
  10. Gloyn AL, Weedon MN, Owen KR, Turner MJ, Knight BA, Hitman G, Walker M, Levy JC, Sampson M, Halford S, McCarthy MI, Hattersley AT, Frayling TM (2003) Large-scale association studies of variants in genes encoding the pancreatic beta-cell KATP channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) confirm that the KCNJ11 E23K variant is associated with type 2 diabetes. Diabetes 52:568–572 [DOI] [PubMed] [Google Scholar]
  11. Gonzalez A, Abril E, Roca A, Aragon MJ, Figueroa MJ, Velarde P, Royo JL, Real LM, Ruiz A (2002) Comment: CAPN10 alleles are associated with polycystic ovary syndrome. J Clin Endocrinol Metab 87:3971–3976 [DOI] [PubMed] [Google Scholar]
  12. Hanis CL, Boerwinkle E, Chakraborty R, Ellsworth DL, Concannon P, Stirling B, Morrison VA, et al (1996) A genome wide search for human non-insulin dependent (type 2) diabetes genes reveals a major susceptibility locus on chromosome 2. Nat Genet 13:161–166 [DOI] [PubMed] [Google Scholar]
  13. Hegele RA, Harris SB, Zinman B, Hanley AJ, Cao H (2001) Absence of association of type 2 diabetes with CAPN10 and PC-1 polymorphisms in Oji-Cree. Diabetes Care 24:1498–1499 [DOI] [PubMed] [Google Scholar]
  14. Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner TH, Mashima H, Schwarz PE, del Bosque-Plata L, Oda Y, Yoshiuchi I, Colilla S, Polonsky KS, Wei S, Concannon P, Iwasaki N, Schulze J, Baier LJ, Bogardus C, Groop L, Boerwinkle E, Hanis CL, Bell GI (2000) Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nat Genet 26:163–175 [DOI] [PubMed] [Google Scholar]
  15. Horikawa Y, Oda N, Yu L, Imamura S, Fujiwara K, Makino M, Seino Y, Itoh M, Takeda J (2003) Genetic variations in calpain-10 gene are not a major factor in the occurrence of type 2 diabetes in Japanese. J Clin Endocrinol Metab 88:244–247 [DOI] [PubMed] [Google Scholar]
  16. Love-Gregory L, Wasson J, Lin J, Skolnick G, Suarez B, Permutt MA (2003) E23K single nucleotide polymorphism in the islet ATP-sensitive potassium channel gene (Kir6.2) contributes as much to the risk of type II diabetes in Caucasians as the PPARγ Pro12Ala variant. Diabetologia 46:136–137 [DOI] [PubMed] [Google Scholar]
  17. Malecki MT, Moczulski DK, Klupa T, Wanic K, Cyganek K, Frey J, Sieradzki J (2002) Homozygous combination of calpain 10 gene haplotypes is associated with type 2 diabetes mellitus in a Polish population. Eur J Endocrinol 146:695–699 [DOI] [PubMed] [Google Scholar]
  18. Nielsen EM, Hansen L, Carstensen B, Echwald SM, Drivsholm T, Glumer C, Thorsteinsson B, Borch-Johnsen K, Hansen T, Pedersen O (2003) The E23K variant of Kir6.2 associates with impaired post-OGTT serum insulin response and increased risk of type 2 diabetes. Diabetes 52:573–577 [DOI] [PubMed] [Google Scholar]
  19. Orho-Melander M, Klannemark M, Svensson MK, Ridderstrale M, Lindgren CM, Groop L (2002) Variants in the calpain-10 gene predispose to insulin resistance and elevated free fatty acid levels. Diabetes 51:2658–2664 [DOI] [PubMed] [Google Scholar]
  20. Rasmussen SK, Urhammer SA, Berglund L, Jensen JN, Hansen L, Echwald SM, Borch-Johnsen K, Horikawa Y, Mashima H, Lithell H, Cox NJ, Hansen T, Bell GI, Pedersen O (2002) Variants within the calpain-10 gene on chromosome 2q37 (NIDDM1) and relationships to type 2 diabetes, insulin resistance, and impaired acute insulin secretion among Scandinavian Caucasians. Diabetes 51:3561–3567 [DOI] [PubMed] [Google Scholar]
  21. Tsai HJ, Sun G, Weeks DE, Kaushal R, Wolujewicz M, McGarvey ST, Tufa J, Viali S, Deka R (2001) Type 2 diabetes and three calpain-10 gene polymorphisms in Samoans: no evidence of association. Am J Hum Genet 69:1236–1244 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tschritter O, Fritsche A, Shirkavand F, Machicao F, Haring H, Stumvoll M (2003) Assessing the shape of the glucose curve during an oral glucose tolerance test. Diabetes Care 26:1026–1033 [DOI] [PubMed] [Google Scholar]
  23. Wang Y, Xiang K, Zheng T, Jia W, Shen K, Li J (2002) [The UCSNP44 variation of calpain 10 gene on NIDDM1 locus and its impact on plasma glucose levels in type 2 diabetic patients.] Zhonghua Yi Xue Za Zhi 82:613–616 [PubMed] [Google Scholar]
  24. Xiang K, Fang Q, Zheng T, Jia W, Wang Y, Zhang R, Li J, Shen K (2001) [The impact of calpain-10 gene combined-SNP variation on type 2 diabetes mellitus and its related metabolic traits.] Zhonghua Yi Xue Yi Chuan Xue Za Zhi 18:426–430 [PubMed] [Google Scholar]

Articles from American Journal of Human Genetics are provided here courtesy of American Society of Human Genetics

RESOURCES