Brief Report Common Variation in the LMNA Gene (Encoding Lamin A/C) and Type 2 Diabetes Association Analyses in 9,518 Subjects Katharine R. Owen, 1 Christopher J. Groves, 1 Robert L. Hanson, 2 William C. Knowler, 2 Alan R. Shuldiner, 3 Steven C. Elbein, 4,5 Braxton D. Mitchell, 3 Philippe Froguel, 6,7 Maggie C.Y. Ng, 8,9 Juliana C. Chan, 9 Weiping Jia, 10 Panos Deloukas, 11 Graham A. Hitman, 12 Mark Walker, 13 Timothy M. Frayling, 14 Andrew T. Hattersley, 14 Eleftheria Zeggini, 1,15 and Mark I. McCarthy 1,15 for the International Type 2 Diabetes 1q Consortium* Mutations in the LMNA gene (encoding lamin A/C) under- lie familial partial lipodystrophy, a syndrome of monogenic insulin resistance and diabetes. LMNA maps to the well- replicated diabetes-linkage region on chromosome 1q, and there are reported associations between LMNA single nucleotide polymorphisms (SNPs) (particularly rs4641; H566H) and metabolic syndrome components. We exam- ined the relationship between LMNA variation and type 2 diabetes (using six tag SNPs capturing >90% of common variation) in several large datasets. Analysis of 2,490 U.K. diabetic case and 2,556 control subjects revealed no signif- icant associations at either genotype or haplotype level: the minor allele at rs4641 was no more frequent in case subjects (allelic odds ratio [OR] 1.07 [95% CI 0.98 –1.17], P � 0.15). In 390 U.K. trios, family-based association analyses revealed nominally significant overtransmission of the major allele at rs12063564 (P � 0.01), which was not corroborated in other samples. Finally, genotypes for 2,817 additional subjects from the International 1q Consortium revealed no consistent case-control or family-based asso- ciations with LMNA variants. Across all our data, the OR for the rs4641 minor allele approached but did not attain significance (1.07 [0.99 –1.15], P � 0.08). Our data do not therefore support a major effect of LMNA variation on diabetes risk. However, in a meta-analysis including other available data, there is evidence that rs4641 has a modest effect on diabetes susceptibility (1.10 [1.04 –1.16], P � 0.001). Diabetes 56:879 – 883, 2007 O nly a limited number of genes with reproduc- ible evidence of association with type 2 diabe- tes have been described. One emerging theme is the frequency with which rare mutations in these same genes display causal involvement in mono- genic forms of diabetes or insulin resistance (1). Conse- quently, there are good grounds for considering genes causing monogenic forms of disease as especially promis- ing candidates with regard to susceptibility to common forms of type 2 diabetes. Mutations in the LMNA gene cause one form of familial partial lipodystrophy (FPLD) (2), a monogenic syndrome of extreme insulin resistance characterized by abnormal fat distribution, dyslipidemia, hypertension, hepatic ste- atosis, and diabetes. LMNA codes (by alternate splicing) for two major protein products, lamin A and C. As constit- uents of the nuclear envelope, these have both structural and regulatory functions (3). LMNA mutations (at sites other than those underlying FPLD) are responsible for a range of pathologies (the “laminopathies”) affecting mul- tiple cell types (4). The structure-function relationships underlying these diverse phenotypes are unclear. Equally, the mechanisms whereby LMNA mutations lead to FPLD are not understood, though loss of LMNA binding to the sterol responsive element binding protein 1 may explain the disturbed adipocyte differentiation and development (5). Consequent diversion of dietary-derived triglycerides into ectopic sites (liver and skeletal muscle) likely under- lies the profound insulin resistance. Similar mechanisms From the 1Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, U.K.; the 2Phoenix Epidemiology and Clinical Research Section, National Institute of Diabetes and Digestive and Kidney Diseases, Phoenix, Arizona; the 3Division of Endocrinology, Diabetes and Nutrition, University of Maryland School of Medicine, Baltimore, Maryland; the 4Endocrinology Section, Medical Service, Central Arkansas Veterans Healthcare System, Little Rock, Arkansas; the 5Division of Endocrinology and Metabolism, Department of Internal Medicine, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas; the 6CNRS UMR 8090, Institut de Biologie de Lille, Lille, France; the 7Faculty of Life Sciences, Imperial College, London, U.K.; the 8Department of Medicine, University of Chicago, Chicago, Illinois; the 9Department of Medicine and Therapeutics, Chinese University of Hong Kong, Shatin, Hong Kong SAR; the 10Shanghai Diabetes Institute, Department of Endocrinology and Metabolism, Shanghai Jiaotong University No. 6 People’s Hospital, Shanghai, China; the 11Wellcome Trust Sanger Institute, Hinxton, U.K.; the 12Centre for Diabetes and Metabolic Medicine, Bart’s and the London Queen Mary’s School of Medicine and Dentistry, London, U.K.; the 13Department of Medicine, University of New- castle, Newcastle, U.K.; the 14Institute of Clinical and Biomedical Science, Peninsula Medical School, Exeter, U.K.; and the 15Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, U.K. Address correspondence and reprint requests to Katharine Owen, Clinical Lecturer, Oxford Centre for Diabetes, Endocrinology and Metabolism, Uni- versity of Oxford, Churchill Hospital, Old Road, Headington, Oxford OX3 7LJ, U.K. E-mail: katharine.owen@drl.ox.ac.uk. Received for publication 6 July 2006 and accepted in revised form 15 November 2006. *A complete list of the International Type 2 Diabetes 1q Consortium is available in the online appendix. Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db06-0930. FPLD, familial partial lipodystrophy; HRC, Human Random Control; MAF, minor allele frequency; SNP, single nucleotide polymorphism. DOI: 10.2337/db06-0930 © 2007 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. See accompanying Brief Reports, p. 694 and 884. DIABETES, VOL. 56, MARCH 2007 879 are increasingly implicated in the pathogenesis of insulin resistance, which characterizes type 2 diabetes (6). LMNA’s credentials as a type 2 diabetes candidate are enhanced by prior genetic data. LMNA maps within the well-replicated area of type 2 diabetes linkage on chromo- some 1q21–24, which has generated powerful signals in European, East-Asian, and Native-American pedigrees (7,8). Additionally, there have been several recent associ- ation studies, most concentrating on a coding variant in exon 10 (rs4641; H566H). As this codon is directly adjacent to the lamin A/C alternate splice site, even synonymous DNA sequence variation has the potential to modulate relative expression of LMNA products. Initial reports in indigenous North American popula- tions (9,10) suggested the minor allele of rs4641 was associated with increased BMI and central obesity. How- ever, the largest published study of this variant (11) (1,338 Pima Indians, 60% with diabetes) detected no association with diabetes, BMI, lipid parameters, insulin sensitivity, or �-cell function. Subsequent data from the same group indicated a possible association with abdominal adipocyte size (12). Likewise, a small Japanese study found no association between rs4641 and diabetes (13). A more extensive survey of common variation within LMNA (six tag single nucleotide polymorphisms [SNPs] including rs4641) in the Amish Family Study (n � 971, 10% with type 2 diabetes) reported that rs4641 was associated with metabolic syndrome and triglyceride levels but not diabe- tes (14). Most recently, analyses of appropriately large Danish samples (15) have provided the most convincing evidence yet that the minor allele at rs4641 is associated with type 2 diabetes and that other LMNA variants show (at least nominally) significant associations with metabolic and anthropometric traits. The present study sought to exam- ine these interesting, but inconsistent, findings with re- spect to type 2 diabetes susceptibility in analyses of 6,701 U.K. subjects and, through the International 1q Consor- tium, a further 2,817 samples from populations with the strongest evidence of linkage to the LMNA region. First, we performed a large-scale case-control analysis in 5,046 U.K. samples (Table 1). We included as case subjects 571 probands, all ascertained for positive family history, from the Diabetes U.K. Warren 2 sibpair collec- tion; 1,569 type 2 diabetic subjects from the MRC/Diabetes U.K. case resource, ascertained for type 2 diabetes diag- nosed before age 65 years; and 350 exclusively British/ Irish probands from the Warren 2 trios resource. As control subjects, we examined 539 U.K. subjects (Human Random Control [HRC]�), 472 from the HRC resource plus 67 non-HRC samples from the same source (ECACC, Salisbury, U.K.), and 2,017 from the British Birth Cohort of 1958. All cases were diagnosed with diabetes based on biochemical evidence of hyperglycemia and/or require- ment for oral agents or insulin. Subtypes other than type 2 diabetes were excluded using clinical, genetic, and immu- nological criteria (all are GAD antibody negative). Glucose tolerance status is not known for any of the control subjects. All subjects were unrelated and of British/Irish- European origin. Further details of ascertainment, subject characteristics, and validation of these samples are pro- vided in the online appendix (available at http://dx.doi.org/ 10.2337/db06-0930). Using pairwise tag selection approaches (16) applied to U.K. control genotype data for LMNA-region SNPs (minor allele frequency [MAF] �1%) generated by the 1q Consor- tium (see below), we prioritized six tag SNPs (threshold r2 � 0.8) for genotyping. Three mapped upstream of the LMNA coding region (rs12063564 [MAF 0.15], rs6661281 [MAF 0.39], and rs955383 [MAF 0.24]), one in the large first intron (rs693671 [MAF 0.04]), and two were synonymous SNPs (rs505058 [D446D] in exon 7 [MAF 0.06] and rs4641 [H566H] in exon 10 [MAF 0.30]). rs12063564 was included as a proxy for a 1q Consortium SNP (rs4661146), which failed assay redesign (mutual r2 of one in the CEU component of HapMap). SNP positions and linkage dis- equilibrium relationships are summarized in online appen- dix Figure A. Using HapMap phase 2 data where available (rs12063564, rs6661281, and rs955383) plus HapMap prox- ies for rs693671 and rs505058 (identified using the 1q Consortium genotypes), we estimate that these SNPs capture �90% of common variation at an r2 � 0.8 across the 83-kb region (containing 43 HapMap SNPs), which spans LMNA and its putative regulatory regions. TABLE 1 Characteristics of the U.K. subjects studied Case samples Control samples Probands from sibpair families Warren2 case subjects Probands from parent-offspring trios* 1958 Birth Cohort HRC resource n 571 1,569 390 2,017 539 Male (%) 54.4 59.9 59.4 50.1 49.4 Age at examination (years) 64.1 � 8.1 60.2 � 8.2 46.3 � 7.1 Not available Not known Age at diagnosis (years) 55.3 � 8.4 51.4 � 7.5 40.3 � 7.7 Not applicable Not applicable BMI (kg/m2) 28.4 (24.0–33.7) 31.5 (26.1–37.9) 32.3 (26.2–39.8) Not available Not known Waist-to-hip ratio (males) 0.95 (0.89–1.03) 0.98 (0.92–1.06) 0.98 (0.91–1.05) Not available Not known Waist-to-hip ratio (females) 0.87 (0.80–0.93) 0.91 (0.84–0.98) 0.89 (0.81–0.98) Not available Not known Treatment (ins/OHA/diet)† (%) 16/69/15 8/62/31 18/63/19 Not applicable Not applicable Data are mean � SD or geometric mean (SD range). *Results given for all trios probands (n � 390). Of these, 350 were of British/Irish origin (60% male; age at diagnosis 40.3 � 7.4 years; BMI 32.3 kg/m2 �28.4 –37.3�). †Treatment at the time of ascertainment. ins, insulin; OHA, oral hypoglycemic agent. COMMON VARIATION IN LMNA AND TYPE 2 DIABETES 880 DIABETES, VOL. 56, MARCH 2007 Genotyping was performed at KBiosciences (Hoddes- don, U.K.) using a fluorescence-based competitive allele- specific (KASPar) assay (details available from the authors upon request). Call rates for all SNPs exceeded 95% overall (with no SNP in any sample �90%). Genotyping perfor- mance was evaluated against stringent quality control criteria, including a discrepancy rate on duplicate geno- typing �0.5%; there were no Mendelian inconsistencies observed in 963 families and no departure from Hardy- Weinberg equilibrium (all P � 0.05) in control subjects. Genotype counts by subgroup are shown in online appendix Table B. In the absence of heterogeneity be- tween case and control subgroups (P � 0.01), our primary analyses used pooled case and control data. Analyses were conducted with both inclusion (to maximize power) and exclusion (to preserve the independence of the family- based analyses) of the 350 British/Irish Warren 2 trio probands. Genotype frequency comparisons were imple- mented in StatXact 6 (Cytel Corporation, Cambridge, MA) using the Cochran-Armitage trend test (additive model) supplemented by recessive analyses where the MAF was �20%. In the case-control study (Table 2), only a single SNP, rs12063564, displayed nominal evidence (uncorrected P � 0.05) of association with type 2 diabetes (odds ratio [OR] per additional copy of allele C: 1.13 [95% CI 1.01–1.27], Cochran-Armitage test, P � 0.039). However, inclusion of the Warren 2 trio probands rendered this association nonsignificant (P � 0.098). Notably, the minor allele of rs4641 showed no significant association with type 2 diabetes (all case vs. all control subjects: 1.07 [0.98 –1.17], P � 0.15). Stratification by sex did not alter the findings for any SNP. LMNA haplotypes were inferred using the expectation- maximization algorithm implemented in HelixTree (Boze- man, MT) (online appendix Table C). Haplotype trend regression (17) revealed no evidence for haplotypic asso- ciations (P � 0.20). Family-based association tests (Table 3) were per- formed in all 1,170 members of the full set of 390 parent- offspring trio pedigrees (see online appendix). The transmission disequilibrium test, implemented in UN- PHASED (18), indicated overtransmission of the common allele (T) at rs12063564 (P � 0.01) but no evidence of departure from expectation for any other allele or haplo- type. Estimates of overall significance (i.e., global tests of whether any of the individual SNPs or haplotypes showed transmission disequilibrium, based on 10,000 permuta- tions) were not significant for either single-point (P � 0.054) or haplotypic (0.74) analyses. Using LMNA genotypes from all 1,406 members of the 573 Warren 2 sibpair families, there was no indication that TABLE 2 Case-control analysis of combined groups SNP NCBI build 36 position Genotype Combined case subjects W2SP � W2C (n � 2,140) W2TP (n � 350) Combined control subjects (n � 2,556) Case-control: W2SP � W2C vs. controls Case-control: including W2TP Cochran Armitage test Recessive test* Cochran Armitage test Recessive test* rs12063564 154321809 TT 1,435 (70.8) 240 (74.5) 1,745 (72.6) 0.039 0.16 0.098 0.30 TC 530 (26.1) 77 (23.9) 614 (25.6) CC 63 (3.1) 5 (1.6) 43 (1.8) rs6661281 154341469 TT 740 (36.1) 117 (36.4) 843 (34.4) 0.76 — 0.89 — TC 941 (45.9) 153 (47.7) 1,224 (49.9) CC 371 (18.1) 51 (15.9) 386 (15.7) rs955383 154348654 AA 1,188 (57.5) 174 (54.7) 1,344 (55.5) 0.23 — 0.41 — AG 751 (36.4) 116 (36.5) 924 (38.2) GG 126 (6.1) 28 (8.8) 153 (6.3) rs693671 154359819 TT 1,889 (91.5) 274 (87.5) 2,296 (91.7) 0.68 0.83 0.26 0.39 TC 168 (8.1) 37 (11.8) 205 (8.2) CC 7 (0.3) 2 (0.6) 3 (0.1) rs505058 154372809 TT 1,814 (87.3) 272 (84.7) 2,110 (87.8) 0.39 0.60 0.22 0.37 TC 250 (12.0) 47 (14.6) 287 (11.9) CC 14 (0.7) 2 (0.6) 6 (0.2) rs4641 154374158 CC 1,072 (51.6) 157 (50.0) 1,316 (53.8) 0.21 — 0.15 — CT 851 (40.9) 132 (42.0) 948 (38.8) TT 156 (7.5) 25 (8.0) 181 (7.4) Data are n (%). *Recessive test used where MAF was �20%; considers common allele as recessive. NCBI, National Center for Biotechnology Information; W2C, Warren2 case subjects; W2SP, Warren2 sibpair probands; W2TP, Warren2 trio probands. TABLE 3 Family-based association in 390 U.K. trios rs12063564 rs6661281 rs955383 rs693671 rs505058 rs4641 Minor allele C C G C C T T/NT* 63/95 148/149 127/117 36/37 41/48 129/119 P 0.01 0.95 0.52 0.91 0.46 0.52 Analyses are by single-point transmission disequilibrium test. The global P value (P � 0.054) addresses the null hypothesis that there is no departure from expectation across the set of six single-point tests. *Transmission/nontransmission (T/NT) of minor allele to offspring. Permutation P � 0.054. K.R. OWEN AND ASSOCIATES DIABETES, VOL. 56, MARCH 2007 881 common variants were contributing to the 1q linkage signal previously observed in these pedigrees (P � 0.8, using the program LAMP, which tests the extent to which associated SNPs can account for regional linkage [19]). In addition, using ANOVA approaches (SPSS version 14) in the case samples, we found no evidence that LMNA SNPs were associated with age of diagnosis of diabetes, BMI, or waist-to-hip ratio, after logarithmic transformation to nor- mality where appropriate (see online appendix Table D for rs4641 data; other data not shown). Next, genotype data gathered by the International Type 2 Diabetes 1q Consortium (see online appendix), in the course of efforts to map susceptibility variants within the replicated linkage region on chromosome 1q, allowed us to extend our LMNA analysis in 3,707 samples from the 1q case-control study (2,084 non-U.K.; 890 U.K.) and Pima family study (n � 733). The 1q Consortium has to date attempted genotyping of 20 SNPs (online appendix Figure A and Table E) spanning the LMNA region in these samples. The 1q case-control study includes some of the U.K. samples included in the analyses described above (Warren 2 sibpair probands and HRC�: these were the only U.K. samples typed for more than the six tag SNPs) and Amish and Pima samples included in previous publi- cations (11,14). Genotypes were gathered as part of three 1536-plex Illumina Golden Gate bundles (20). Single-point (Cochran-Armitage test using Stata version 8) and haplo- type-based (haplotype trend regression using HelixTree) analyses of these data revealed no consistent associations between LMNA SNPs and type 2 diabetes (data not shown). Analyses of the tag SNPs typed in the 1q Consor- tium samples (rs6661281, rs955383, rs693671, rs505058, rs4641, and rs4661146) confirmed no association with type 2 diabetes in the Amish, Pima, U.K., Shanghai, or Hong Kong case-control datasets. Nominal associations for rs6661281 in the Utah sample (P � 0.015), and for rs693671 (P � 0.003) and rs505058 (P � 0.002) in the French (additive model), were not substantiated in other samples. Combined analysis of information from all seven datasets (using the Mantel-Haenszel meta-analysis method under recessive, dominant, and additive models) showed no convincing association of LMNA tag SNPs with type 2 diabetes (online appendix Table F). Notably, rs4641 was not associated with type 2 diabetes in any of the samples (online appendix Table G). Finally, a further 733 Pima samples from the original linkage pedigrees (21 and online appendix data) were, with 99 individuals from the case-control sample, analyzed using family-based association methods. These 832 Pima samples included 570 type 2 diabetic subjects (diagnosed �45 years), 104 nondiabetic siblings (aged �45 years), and 158 parents (to reconstruct family relationships). Family- based association analyses under the additive model were performed using binomial generalized estimating equa- tions to control for family membership (22). Again, no LMNA SNPs were associated with type 2 diabetes (all P � 0.3). For reasons stated earlier, LMNA is a logical choice of candidate to investigate for association with multifactorial type 2 diabetes. In this study, we have been unable to show any compelling evidence of association with any of the SNPs typed. It is noteworthy that the nominally significant results at rs12063564 in the case-control and family-based analyses lie in the opposite direction. The estimate of the combined OR (including all the nonoverlapping data re- ported in the present study), was calculated using the inverse variance method (23) to allow proper adjustment for nonindependence in some of the datasets (e.g., Amish). In this meta-analysis, the effect of rs4641 on diabetes risk approached but did not attain nominal significance: allelic OR 1.07 (95% CI 0.99 –1.15), P � 0.08. The strongest evidence supporting an association be- tween the minor allele of rs4641 and type 2 diabetes risk comes from a large study of Danish subjects (15). In comparison of 1,324 case and 4,386 control subjects, the observed OR was 1.14 (95% CI 1.03–1.26). While our study fails to replicate this association, the OR estimates from the two studies show substantial overlap in their CIs. Ascertainment effects, as well as sampling error, may have contributed to modest differences in the effect size esti- mates. Many of the U.K. case subjects were selected for positive family history and/or early disease onset, maneu- vers expected to boost effect size estimates compared with the less-selective Danish case ascertainment. However, differences in control ascertainment may have had a small effect in the opposite direction. The Danish control sub- jects are confirmed as normoglycemic, while glycemic status is unknown for the U.K. control subjects. However, given the relatively low prevalence of diabetes in middle- aged U.K. subjects (24), the magnitude of the dilution of effect size engendered by such misclassification can be shown to be extremely modest (25). Meta-analysis provides one route to improved specifica- tion of true effect sizes. Combining all the case-control data in the present study with the previous Japanese report (13) (using inverse variance method, not including the previous Amish and Pima data, given overlap with the current study), the per-allele OR for the minor allele at rs4641 reaches 1.08 (95% CI 1.01–1.16), P � 0.04. Further, if the Danish case-control data (15) are included (contrib- uting 42% of the total 13,694 genotypes), the evidence in favor of a type 2 diabetes susceptibility effect at rs4641 increases substantially (1.10 [1.04 –1.16], P � 0.001). While our data cannot be considered to provide replication (P � 0.05) of the association reported by Wegner et al. (15), the fact that this combined analysis generates a more signifi- cant result than that seen in either study alone indicates that the U.K. data provides some support for the Danish findings, particularly when one factors in the strong bio- logical candidacy of LMNA. These data again illustrate the tremendous difficulties that exist in the detection, replication, and interpretation of association analyses for variants with modest suscepti- bility effects. If the true effect size of rs4641 is an OR of 1.1, then even a study of 2,500 case-control pairs has only 57% power (given a liberal � 0.05). Indeed, reaching strin- gent genome-wide significance (P � 5 10�8) for such a variant would require analysis of �25,000 case-control pairs. In addition, such modest effects need to be distin- guished from spurious association signals on a similar scale that may be generated as a result of artifact (e.g., informative missingness) or biological effects such as cryptic population stratification. ACKNOWLEDGMENTS This study was funded by Diabetes U.K. (collection of U.K. samples and U.K. genotyping) and supported by grants U01-DK58026, R01-DK073490, R01-DK54261, K24-DK2673, and R01-DK39311 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK); grant T32- AG00219 from the National Institute of Aging (NIA); and COMMON VARIATION IN LMNA AND TYPE 2 DIABETES 882 DIABETES, VOL. 56, MARCH 2007 intramural funds. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIDDK and NIA. E.Z. is a Wellcome Trust Research Development Fellow (Wellcome Trust 079557). We thank Oluf Pedersen, Torben Hansen, and col- leagues for sharing data prepublication. 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