SNP43 of CAPN10 and the Risk of Type 2 Diabetes in African-Americans The Atherosclerosis Risk in Communities Study Michael J. Garant,1,2 W.H. Linda Kao,3 Frederick Brancati,3,4 Josef Coresh,3,4 Tejal M. Rami,3 Craig L. Hanis,5 Eric Boerwinkle,5 and Alan R. Shuldiner1,2

Recently, an A-to-G variant in intron 3 (SNP43) of the calcium-activated neutral 10 (CAPN10) was identified as a possible type 2 diabetes susceptibil- ype 2 diabetes (formerly known as non–insulin- ity gene through positional cloning in Mexican-Ameri- dependent diabetes) is a heterogeneous disorder cans. We conducted cross-sectional and prospective in which both genetic and nongenetic influences studies to evaluate the relation between SNP43 and contribute to disease risk (1). The genetic con- type 2 diabetes and related traits in middle-aged Afri- T tribution is likely to comprise several gene variants, each can-American participants of the Atherosclerosis Risk in Communities Study, a population-based longitudinal with relatively modest effect, which act in combination study. At baseline, 269 prevalent diabetes cases and with each other and with environmental provocations to 1,159 nondiabetic control subjects were studied. Those cause the disease. Genome wide scans have led to the with the G/G genotype were more likely to have diabetes chromosomal localization of susceptibility loci for type 2 than those with the A/G or A/A genotype (odds ratio diabetes in the Pima Indians of North America (2), in In the prospec- African-Americans, Caucasians, and Japanese Americans .(0.05 ؍ OR] 1.41, 95% CI 1.00–1.99, P] tive study, 166 of the control subjects developed inci- from the American Diabetes Association Genetics of dent diabetes over 9 years of follow-up. The incidence of NIDDM (GENNID) Study (3), in Mexican-Americans (3–5), diabetes for individuals with the G/G genotype did not and in Caucasians from Finland (6–9), France (10), Utah differ significantly from those with at least one copy of (U.S.) (11), and the U.K. (12). The first type 2 diabetes ؍ the A allele (23.3 vs. 19.5 per 1,000 person years, P 0.29). Pooling prevalent and incident diabetic cases susceptibility locus, NIDDM1, was discovered in Mexican- together, individuals with the G/G genotype were ϳ40% Americans from Starr County, TX, and was localized to the more likely to have diabetes than those without (OR D2S125-D2S140 region on two (4). Linkage in Because of the high the region of NIDDM1 has also been observed in French .(0.03 ؍ CI 1.04–1.83, P 95% ,1.38 frequency of the G allele (0.88), ϳ25% of the suscepti- families in some studies (13) but not in other studies bility to type 2 diabetes in African-Americans may be (5,14–16). Through positional cloning, Horikawa et al. (17) attributed to the G/G genotype at SNP43 of CAPN10, recently identified NIDDM1 as 10 (CAPN10). A although most of the subjects with the G/G genotype did common A-to-G single nucleotide polymorphism in intron not develop diabetes over the 9 years of follow-up. We 3 (SNP-43) of CAPN10 was recessively associated with conclude from this large prospective study that the G type 2 diabetes in a Mexican-American population from allele of SNP43 of CAPN10 or another allele or gene that is in linkage disequilibrium with it increases sus- Starr County, TX (17). , or calcium-activated ceptibility to type 2 diabetes in African-Americans. neutral , are intracellular nonlysosomal cysteine Diabetes 51:231–237, 2002 proteases, which contribute to diverse physiological cell functions (18–21). Despite these diverse functions, cal- pains have not previously been implicated in pathways that regulate glucose homeostasis, and thus, the mecha- nism whereby this variant contributes to diabetes risk is unknown. Furthermore, because SNP43 is in the noncod- ing region of CAPN10, it is not known how this polymor- From the 1Division of Endocrinology, Diabetes and Nutrition, Department of phism affects CAPN10 activity. Given the unclear role of Medicine, University of Maryland, Baltimore, Maryland; the 2Baltimore Veter- ans Administration Geriatric Research and Education Clinical Center, Balti- CAPN10 in diabetes pathogenesis, defining whether more, Maryland; the 3Department of Epidemiology, Johns Hopkins University SNP43 modifies diabetes risk in other populations is of School of Hygiene and Public Health, Baltimore, Maryland; the 4Department of critical importance. Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; and the 5Human Genetics Center, University of Texas Houston Health Science African-Americans are at increased risk for the develop- Center, Houston, Texas. ment of type 2 diabetes (22). However, few studies have Address correspondence and reprint requests to Alan R. Shuldiner, Depart- ment of Medicine, University of Maryland, 660 West Redwood St., Room 494, investigated the genetic underpinnings of type 2 diabetes Baltimore, MD 21201. E-mail. [email protected]. in African-Americans. To define the relevance of SNP43 of Received for publication 17 May 2001 and accepted in revised form 12 CAPN10 in this ethnic group, we examined its relation to October 2001. ARIC, Atherosclerosis Risk in Communities; OR, odds ratio; PAR, popula- type 2 diabetes risk in a large population-based prospec- tion attributable risk. tive study of middle-aged African-American participants of

DIABETES, VOL. 51, JANUARY 2002 231 CAPN10 AND TYPE 2 DIABETES IN AFRICAN-AMERICANS

ϭ the Atherosclerosis Risk in Communities (ARIC) Study, insulin ln[insulin at V4] – ln[insulin at V1], which becomes ln(insulin at V /insulin at V ). who were characterized with respect to diabetes preva- 4 1 Effect modifications of the association between SNP43 genotype and type lence and incidence. 2 diabetes by physical activity levels (categorized into quartiles) and total energy intake (categorized into quartiles) were assessed by fitting interaction terms into the multiple logistic regression model. In addition, we examined RESEARCH DESIGN AND METHODS potential interactions between diet or physical activity and genotype on the continuous variables, BMI, and glucose using analysis of covariance. Study participants. Subjects of the present analyses were selected from the 4,266 African-American participants of the ARIC Study. The ARIC Study is a prospective epidemiological study that examines clinical and subclinical RESULTS atherosclerotic disease, characteristics of which have been previously re- Allele frequency and genotype distribution by type 2 ported (23). The present analyses were based on information obtained at baseline and after 9 years of follow-up, consisting of a total of four clinic visits diabetes status. At baseline, 269 prevalent diabetes cases

(V1 through V4) scheduled 3 years apart. African-American individuals were and 1,159 control subjects were randomly selected from excluded from the sampling frame of the present analyses if they had missing 3,268 African-American ARIC participants to be included demographic, clinical, dietary, or laboratory data at baseline (n ϭ 998), in the present study, with 86% of the participants from resulting in a sampling frame of 3,268 individuals (2,006 women and 1,262 Jackson, MS. The baseline characteristics of the partici- men). Subjects who were excluded because of incomplete data did not differ significantly with respect to demographics. Using sex-stratified random sam- pants were compared between those with and without pling to maintain a female:male ratio of 3:2, as in the original cohort, 1,441 diabetes in Table 1. In the cross-sectional analysis at individuals were selected for the present analyses (sampling fraction 44%). baseline, the allele frequency of the G allele was slightly Study participants were not known to be first-degree relatives of one another. higher in subjects with diabetes (n ϭ 269) than in control Diabetic case subjects were defined as individuals with any one of the ϭ ϭ Ն subjects (n 1,159) (90.0 vs. 87.2%, P 0.08). The following characteristics at V1 through V4 of the study: 1) fasting glucose 7.0 mmol/l (126 mg/dl), 2) nonfasting glucose Ն11.1 mmol/l (200 mg/dl), 3) genotype frequencies were in accordance with Hardy- current use of medication to treat diabetes, or 4) a positive response to the Weinberg equilibrium in both strata, with 2.2% of the question “Has a doctor ever told you that you had diabetes (sugar in the participants homozygous for the A allele (A/A), 77.7% blood)?” homozygous for the G allele (G/G), and 20.1% heterozy- Details of baseline and follow-up examinations of ARIC study subjects have been reported elsewhere. For the purposes of this study, information gous (A/G). Based on studies in Mexican-Americans from included age, sex, race, personal and family history of diabetes, anthropom- Starr County, TX, we assumed a recessive mode of inher- etry (height, weight, waist, hip, and subscapular and triceps skinfolds), and itance in which the more common G allele is the “at risk” fasting blood (glucose, insulin, total cholesterol, HDL cholesterol, and triglyc- allele (17). Participants with the G/G genotype were more erides). Physical activity during leisure time was assessed by a modified likely to have diabetes than those with at least one copy of version of the questionnaire developed by Baecke et al. (24). Dietary intake ϭ was assessed by a modified version of the 61-item food frequency question- the A allele (OR 1.41, 95% CI 1.00–1.99, P 0.05) (Table 2). naire developed by Willett et al. (25). Adjusting the OR for BMI did not change the results, nor Detection of the SNP43 of the CAPN10 gene. A polymerase chain reaction were there any effects of age or sex on the association of was performed (AmpliTaq Gold with GeneAmp; Perkin Elmer Biosystems, the G/G genotype with diabetes. Branchburg, NJ) with 20 ng DNA using an upstream primer of 5Ј-CACGCTT GCTGTGAAGTAATGC-3Ј and a downstream primer of 5Ј-CTCTGATTCCCAT In prospective analysis, 166 of the individuals who were GGTCTGTAG-3Ј in the presence of 8.5% dimethylsulfoxide (Sigma Chemical, nondiabetic at baseline developed incident diabetes during St. Louis, MO). The resulting 144-bp product was digested with Nsi I (New the 9 years of follow-up, leaving 993 participants free of England Biolabs, Beverly, MA) at 37°C for 15–18 h. The digested products incident type 2 diabetes. The incidence of diabetes for were subjected to electrophoresis through a 4% agarose gel (MetaPhor individuals with the G/G genotype did not differ signifi- agarose; FMC Bioproducts, Rockland, ME) containing 250 nmol/l ethidium bromide. The homozygous G/G product remained 144 bp after digestion with cantly from those with at least one copy of the A allele Nsi I, whereas the homozygous A/A product was cleaved into two products of (23.3 vs. 19.5 per 1,000 person years, P ϭ 0.29). Pooling all 121 and 23 bp. The products were visualized in the gel under UV translumi- prevalent and incident diabetic cases together, individuals nation. Genotypes were scored by two independent observers. The accuracy with the G/G genotype were significantly more likely to rate based on blind repeats (n ϭ 86) was 96%. Statistical analysis. All statistical analyses were performed using the SAS have or to develop diabetes than those who remained free statistical package (Cary, NC). Means Ϯ SD and frequencies of baseline of diabetes throughout the study (OR 1.38, 95% CI 1.04– characteristics were calculated. Fasting serum insulin and triglyceride levels 1.83, P ϭ 0.03) (Table 2). Association between the G/G were not normally distributed; therefore, those values were natural logarithm- genotype and diabetes was independent of age, sex, and transformed. Allele frequencies were calculated, and a two-sample test for BMI. binomial proportions was used to assess differences in allele frequencies between diabetic and nondiabetic individuals. The ␹2 goodness-of-fit test was SNP43 genotype distribution, obesity, and diabetes- used to assess deviation from Hardy-Weinberg equilibrium of the genotypic related quantitative traits at baseline. To discern frequency by calculating expected frequencies of genotypes. potential mechanisms whereby SNP43 genotype might Based on studies in Mexican-Americans in Starr County, TX, we assumed increase diabetes risk, three different analyses were per- a recessive mode of inheritance in which the more common genotype G/G is the “at risk” genotype (17). Multiple logistic regression was used to obtain formed. First, we examined diabetes- and obesity-related odds ratios (ORs) for diabetes (with 95% CI) after adjustment for age, sex, and traits at baseline in G/G homozygotes compared with G/A other potential covariates. To assess the effect of adiposity on serotypically and A/A genotypes (Table 3). There were no significant conferred diabetes risk, stratification analyses were repeated after stratifica- differences in subjects with or without diabetes in any of tion for adiposity, according to NHLBI criteria: nonoverweight (BMI Ͻ25 the diabetes- or obesity-related traits (BMI, waist-to-hip kg/m2), overweight (BMI 25–29.9 kg/m2), and obese (BMI Ն30 kg/m2). Analysis of variance was used to test for association between each continuous ratio, waist circumference, subscapular and triceps skin- phenotype and SNP43 genotype. fold, and glucose) at baseline between the participants Diabetes-related quantitative traits were measured at each of the four visits carrying at least one copy of the A allele and those of the study. To assess prospective changes in these traits, we used multiple homozygous for the G allele at baseline. Similarly, there linear regression to look at changes in quantitative traits. Because fasting were no significant differences in the cardiovascular risk serum insulin levels were only measured at V1 and V4 of the study, analysis of insulin levels were assessed by examining genotypic influences on prospective factors (HDL and LDL cholesterol, triglycerides, and blood changes in insulin between V1 and V4, calculated as the difference between pressure) between genotypes, except for systolic blood

232 DIABETES, VOL. 51, JANUARY 2002 M.J. GARANT AND ASSOCIATES

TABLE 1 Baseline characteristics of 1,428 African-American ARIC participants stratified by diabetes status Subjects without diabetes Subjects with diabetes P n 1,159 269 Age (years) 53.0 Ϯ 5.7 54.9 Ϯ 5.8 Ͻ0.001 Sex (% male) 40.9 39.8 0.74 Education (%) Յ11 years 39.3 47.6 High school graduate 28.9 29.4 0.01‡ Attended college 31.8 23.0 ARIC Study site (% at Jackson, MS) 85.6 87.4 0.52 Parental history of diabetes (% yes) 23.3 42.8 Ͻ0.001 BMI (kg/m2) 28.6 Ϯ 5.9 31.3 Ϯ 5.6 Ͻ0.001 Waist-to-hip ratio 0.909 Ϯ 0.074 0.954 Ϯ 0.063 Ͻ0.001 Waist circumference (cm) 96.6 Ϯ 14.6 105.2 Ϯ 12.8 Ͻ0.001 Mean subscapular skinfold (mm) 29.8 Ϯ 13.6 36.0 Ϯ 12.3 Ͻ0.001 Mean triceps skinfold (mm) 25.9 Ϯ 12.2 28.4 Ϯ 11.7 0.002 Total caloric intake (kcal/day) 1663 Ϯ 798 1461 Ϯ 692 Ͻ0.001 Physical activity Sports related 2.17 Ϯ 0.70 2.14 Ϯ 0.70 0.56 Leisure related 2.10 Ϯ 0.57 2.00 Ϯ 0.50 0.01 Fasting serum glucose (mmol/l) 5.47 Ϯ 0.55 *NR — Ln(fasting insulin) (pmol/l) 4.31 Ϯ 0.67 *NR — HDL cholesterol (mmol/l) 1.47 Ϯ 0.47 1.29 Ϯ 0.36† Ͻ0.001 LDL cholesterol (mmol/l) 3.53 Ϯ 1.12 3.77 Ϯ 1.23† 0.005 Ln(triglycerides) (mmol/l) 0.03 Ϯ 0.44 0.26 Ϯ .21† Ͻ0.001 Systolic blood pressure (mmHg) 126.7 Ϯ 20.0 133.7 Ϯ 21.4 Ͻ0.001 Diastolic blood pressure (mmHg) 79.7 Ϯ 12.2 78.9 Ϯ 12.3 0.32 Data are means Ϯ SE unless otherwise indicated. *NR, not reported because diabetes and its treatment may confound glucose and insulin levels; †analysis limited to 212 diabetic individuals who fasted Ͼ8h;‡significance assessed by ␹2 test of homogeneity. pressure, which was significantly higher in nondiabetic more in G/G homozygotes, suggesting the development of subjects homozygous for the G allele compared with those greater insulin resistance. with at least one copy of the A allele (127.5 vs. 124.3 Third, among subjects who developed diabetes during mmHg, P ϭ 0.02). the study (incident cases), we examined associations of Second, we examined changes in diabetes- and obesity- genotype with phenotypes measured at the visit just related phenotypes in nondiabetic subjects over the 9 before the development of diabetes. There were no signif- years of follow-up (Table 4). There were no significant icant differences in any of the obesity or diabetic measures differences between SNP43 genotypes with respect to between G/G homozygotes and subjects carrying at least changes in any of the obesity-, diabetes-, or cardiovascular- one copy of the A allele before diagnosis. (Table 5). related traits that were examined over the 9 years of Similarly, there were no differences in any of these traits at follow-up except for triglycerides, in which there were the time of diabetes diagnosis (data not shown). greater increases in the subjects homozygous for the G The availability of data on physical activity and dietary allele (Table 4). Fasting insulin levels tended to increase intake provided the opportunity to examine potential

TABLE 2 Prevalence and incidence rates over 9 years of follow-up of type 2 diabetes in African-American ARIC participants by CAPN10 SNP43 genotype SNP43 genotype A/A A/G G/G

Prevalent type 2 diabetes (at V1) 7 40 222 Nondiabetic control subjects 25 247 887 OR (95% CI)* 1.00 (reference) 1.41† (1.00–1.99) Age of diabetes onset 49.8 Ϯ 8.3 (n ϭ 37) 50.7 Ϯ 8.3 (n ϭ 167)

Incident type 2 diabetes (V2 to V4) 2 31 133 Nondiabetic control subjects 23 216 754 Incident rate ratio (95% CI)* 1.00 (reference) 1.21† (0.86–1.27) Age of diabetes onset 57.1 Ϯ 5.9 (n ϭ 32) 57.7 Ϯ 5.6 (n ϭ 114)

All type 2 diabetes (V1 to V4) 9 71 355 Nondiabetic control subjects 23 216 754 OR (95% CI)* 1.00 (reference) 1.38‡ (1.04–1.83) Age of diabetes onset 53.2 Ϯ 8.1 (n ϭ 69) 53.5 Ϯ 7.6 (n ϭ 281) Ϯ Data are means SE unless otherwise indicated. ORs were calculated using a recessive model (i.e., A/X vs. G/G). *Adjusted for age at V1 and sex; †P ϭ 0.05; ‡P ϭ 0.03.

DIABETES, VOL. 51, JANUARY 2002 233 CAPN10 AND TYPE 2 DIABETES IN AFRICAN-AMERICANS

TABLE 3 Selected characteristics by SNP43 genotype in diabetic and nondiabetic African-American ARIC participants Subjects with diabetes Subjects without diabetes A/A or A/G G/G P A/A or A/G G/G P n 47 222 272 887 BMI (kg/m2) 31.5 31.3 0.79 28.5 28.7 0.71 Waist-to-hip ratio 0.955 0.953 0.88 0.904 0.910 0.23 Waist circumference (cm) 106.1 104.9 0.58 95.9 96.8 0.40 Subscapular skinfold (mm) 37.2 35.7 0.43 29.6 29.9 0.74 Triceps skinfold (mm) 29.7 28.1 0.31 25.7 26.0 0.60 Glucose (mmol/l) *NR *NR — 5.49 5.46 0.58 Ln(insulin) (pmol/l) *NR *NR — 4.30 4.31 0.77 HDL cholesterol (mmol/l) 1.28 1.29 0.79 1.49 1.46 0.32 LDL cholesterol (mmol/l) 3.92 3.74 0.45 3.46 3.56 0.21 Ln(triglycerides) (mmol/l) 0.28 0.25 0.73 0.003 0.0034 0.31 Systolic blood pressure (mmHg) 131.3 134.2 0.40 124.3 127.5 0.02 Diastolic blood pressure (mmHg) 78.2 79.1 0.63 78.7 80.1 0.11

All analyses adjusted for age at V1 and sex. *NR, not reported because diabetes and its treatment may confound glucose and insulin levels; †analyses of lipid traits limited to 212 diabetic individuals who fasted Ͼ8h(n ϭ 34 for A/A or A/G, n ϭ 178 G/G). interactions between these extrinsic factors and SNP43 crepant findings is that Horikawa et al. (17) showed that a genotype on diabetes risk. No statistically significant in- combination of two different haplotypes (each containing teractions (multiplicative or additive) were detected (data the G allele at SNP43) confers greater risk of type 2 not shown). diabetes. Although haplotypes were not examined directly in this study, the “at risk” haplotypes are much more DISCUSSION common in Mexican-Americans, Native Americans, and The main finding of this study is that among African- African-Americans than in Caucasians (17; C.L.H., per- Americans, homozygosity for the common G allele of sonal communication). SNP43 of the CAPN10 gene is associated with a modest This well-characterized cohort provided the opportunity increase in risk of type 2 diabetes. These results are in to examine potential interactions of extrinsic factors that agreement of Horikawa et al. (17), who showed that are known to influence diabetes risk with the SNP43 SNP43 of CAPN10 is associated with susceptibility to type genotype. We did not detect any interactions between 2 diabetes in Mexican-Americans from Starr County, TX. SNP43 genotype and leisure time physical activity or Association of the G allele of SNP43 of CAPN10 with type dietary intake, suggesting that these extrinsic risk factors 2 diabetes in African-Americans persisted when including do not play a major role in altering the diabetes risk both prevalent cases and incident cases over the 9 years of associated with G/G homozygosity. follow-up. Despite this association with type 2 diabetes, This study design has several strengths. First, it is a there were no associations of the G/G genotype with other relatively large and well-characterized study of African- quantitative traits related to diabetes, such as glucose or Americans. Second, sampling was population-based, thus insulin levels, as was shown by Baier et al. (26) in Pima reducing potential biases that may result from clinic- or Indians. Our results are in contrast to two recent negative hospital-based ascertainment. However, we cannot ex- studies of SNP43 in large case-control studies, one of clude the possibility that other factors (e.g., selection bias Caucasians from the U.K. (27) and another of Oji-Cree or preferential attrition rate) may have resulted in a study Native Canadians (28). A possible reason for these dis- population that differed from the general African-Amer-

TABLE 4

Change between V1 and V4 of selected characteristics by SNP43 genotype in African-American ARIC participants SNP43 Genotype A/A or A/G G/G ⌬ ⌬ (V4 to V1) (V4 to V1) P BMI* (kg/m2) 0.948 1.025 0.71 Waist-to-hip ratio 0.031 0.027 0.44 Waist circumference (cm) 5.59 5.50 0.88 Fasting glucose (mmol/l)* 0.102 0.177 0.15 Ln(fasting insulin) (pmol/l)* 0.047 0.193 0.16 HDL cholesterol (mmol/l) Ϫ0.08 Ϫ0.07 0.80 LDL cholesterol (mmol/l) Ϫ0.27 Ϫ0.34 0.29 Ln(triglycerides) (mmol/l) 0.02 0.08 0.05 Systolic blood pressure (mmHg) 8.57 7.73 0.61 Diastolic blood pressure (mmHg) Ϫ2.88 Ϫ3.54 0.49

Data are means. All analyses adjusted for age at V1 and sex. * Analysis limited to 589 individuals who were nondiabetic at baseline and remained nondiabetic through V4.

234 DIABETES, VOL. 51, JANUARY 2002 M.J. GARANT AND ASSOCIATES

TABLE 5 Characteristics by SNP43 genotype in 161 incident diabetic African-American ARIC participants for the visit prior to their diagnosis of type 2 diabetes Visit of prediagnosis A/A or A/G G/G P BMI (kg/m2) 33.5 Ϯ 1.1 31.5 Ϯ 0.6 0.12 Waist-to-Hip ratio 0.954 Ϯ 0.012 0.941 Ϯ 0.006 0.34 Waist circumference (cm) 109.4 Ϯ 2.8 105.2 Ϯ 1.4 0.17 Fasting glucose (mmol/l)* 6.22 Ϯ 0.09 6.04 Ϯ 0.05 0.09 Ln(fasting insulin) (pmol/l)† 4.90 Ϯ 0.15 4.58 Ϯ 0.07 0.09 HDL cholesterol (mmol/l)* 1.37 Ϯ 0.07 1.33 Ϯ 0.04 0.64 LDL cholesterol (mmol/l)* 3.61 Ϯ 0.17 3.41 Ϯ 0.09 0.31 Ln(triglycerides) (mmol/l)* 0.23 Ϯ 0.08 0.20 Ϯ 0.04 0.69 Systolic blood pressure (mmHg) 126.9 Ϯ 3.5 128.6 Ϯ 1.8 0.67 Diastolic blood pressure (mmHg) 77.5 Ϯ 2.2 77.3 Ϯ 0.3 0.94 Ϯ Data are means SE. All analyses adjusted for age at V1 and sex. *Analysis limited to 151 individuals who had fasted for at least 8 h; †analysis limited to 83 individuals who had fasted for at least 8 h and for whom insulin data was available. ican population. Third, using 9 years of follow-up data, the ϳ40% more likely to have diabetes than those individuals present study examined the associations between SNP43 with at least one copy of the A allele, there was no and changes in BMI, waist-to-hip ratio, and fasting serum association of SNP43 genotype with any diabetes-related glucose and insulin levels in order to examine potential traits (e.g., BMI, fasting insulin) or with changes in these mechanisms whereby this variant increases diabetes risk. diabetes-related traits over time, leaving further questions Finally, the large sample size of the present study and the regarding a potential mechanism whereby this variant may availability of information on leisure time physical activity increase diabetes risk. Calpains are cysteine proteases, and dietary intake provide good power to detect modest which process specific substrates at a limited number of gene-environment interactions and provide relatively nar- sites, causing activation or inactivation of (33). row confidence intervals when no significant associations Calpains have been implicated in adipocyte differentiation were detected. in 3T3-L1 cells (34). Calpain 10 is expressed in liver, Despite these strengths, several limitations of the muscle, and pancreatic islets (17). Thus, altered calpain 10 present study may have influenced the results. First, expression and/or function may have effects in several characterization of body composition and insulin resis- tissues important for glucose homeostasis (17,35–38). tance were limited to anthropometric measures and fast- Studies suggest that the G allele is associated with altered ing serum insulin, respectively. Anthropometric measures binding of nuclear proteins and decreased transcription such as BMI and waist-to-hip ratio represent relatively (17). Indeed, Pima Indians with the G/G SNP43 genotype crude markers for adiposity. Similarly, there is a large had decreased expression of calpain 10 in skeletal muscle degree of variation in dietary and physical activity ques- and reduced rates of postabsorptive and insulin-stimulated tionnaires as assessments of caloric intake and expendi- glucose turnover (17), and calpain inhibitors may decrease ture, respectively. The inconsistency between greater BMI insulin secretion in isolated islets (39). and lower caloric intake in subjects with diabetes is likely The high G allele frequency of SNP43 of CAPN10 in to reflect systematic under-reporting of caloric intake in African-Americans suggests that it has a major public subjects with diabetes compared with control subjects health impact. A typical approach to quantify public health (29). Although fasting serum insulin is only a marker of impact is calculating population attributable risk (PAR). insulin sensitivity, it has been demonstrated in previous PAR calculations are generally confined to relations with studies to correlate reasonably well with more sensitive strong evidence of causality, and association studies can- measures assessed by the euglycemic-hyperinsulinemic not prove causality, especially in light of the fact that clamp or frequently sampled intravenous glucose toler- SNP43 is in the noncoding region of CAPN10. Nonethe- ance tests (30). Second, positive associations reported by less, preliminary calculations are useful to provide context the present study may be the result of a type 1 error, for the research, especially in community-based studies. although this is mitigated by a strong a priori hypothesis Assuming causality of SNP43 or a variant in linkage and confirmatory results in other studies (17,31,32). Third, disequilibrium with SNP43 and using Levin’s formula for the association between SNP43 and diabetes could be the population attributable risk (40), we found that ϳ25% of result of linkage disequilibrium between the candidate the susceptibility to type 2 diabetes in African-Americans gene variant and the true disease-causing variant. Fourth, may be attributed to the G/G genotype at SNP43. However, the lack of changes in diabetes- or obesity-related traits homozygosity for the G allele constitutes 78% of this over time in nondiabetic individuals (Table 4) may be African-American sample, which is much greater than the caused by the absence of other susceptibility gene variants prevalence of type 2 diabetes in this population. These or the presence of additional gene variants that are pro- findings suggest that this allele increases diabetes risk on tective from the development of diabetes. Finally, we a population-wide basis, but has poor positive predictive cannot exclude the possibility of false positive or negative value. Possibly, the additive effects of other unlinked associations caused by population stratification. yet-to-be-determined gene variants with SNP43 of CAPN10 Although individuals with the G/G SNP43 genotype were may better define individual risk. Indeed, there is evidence

DIABETES, VOL. 51, JANUARY 2002 235 CAPN10 AND TYPE 2 DIABETES IN AFRICAN-AMERICANS that a locus on chromosome 15 may interact with CAPN10 K, Parkkonen M, Reeve-Daly MP, Weaver A, Brettin T, Duyk G, Lander ES, or with another gene variant within the NIDDM1 locus Groop LC: Mapping of a gene for type 2 diabetes associated with an insulin secretion defect by a genome scan in Finnish families. Nat Genet 14:90–94, (41). Alternatively, SNP43 may be in linkage disequilibrium 1996 with a less common variant that might better predict 7. Ghosh S, Watanabe RM, Hauser ER, Valle T, Magnuson VL, Erdos MR, and/or define diabetes risk. In Mexican-Americans, haplo- Langefeld CD, Balow J Jr, Ally DS, Kohtamaki K, Chines P, Birznieks G, type analysis suggests that the combination of two differ- Kaleta HS, Musick A, Te C, Tannenbaum J, Eldridge W, Shapiro S, Martin ent CAPN 10 haplotypes, both of which include the G C, Witt A, So A, Chang J, Shurtleff B, Porter R, Boehnke M, et al: Type 2 allele of SNP43, are required to confer increased diabetes diabetes: evidence for linkage on chromosome 20 in 716 Finnish affected sib pairs. Proc Natl Acad SciUSA, 96:2198–203, 1999 risk. We cannot rule this out as a possibility in African- 8. Ghosh S, Watanabe RM, Valle TT, Hauser ER, Magnuson VL, Langefeld CD, Americans because haplotypes in this population-based Ally DS, Mohlke KL, Silander K, Kohtamaki K, Chines P, Balow J Jr, study have not yet been examined. Birznieks G, Chang J, Eldridge W, Erdos MR, Karanjawala ZE, Knapp JI, In summary, results from the present study indicate a Kudelko K, Martin C, Morales-Mena A, Musick A, Musick T, Pfahl C, Porter significant association between homozygosity for the com- R, Rayman JB: The Finland-United States investigation of non-insulin- dependent diabetes mellitus genetics (FUSION) study. I. An autosomal mon G allele of SNP43 of CAPN10 and typical type 2 genome scan for that predispose to type 2 diabetes. Am J Hum diabetes in a large population-based longitudinal study of Genet, 67:1174–1185, 2000 African-Americans. If this genetic variant is the causative 9. Watanabe RM, Ghosh S, Langefeld CD, Valle TT, Hauser ER, Magnuson VL, allele, it may account for as much as 25% of the attribut- Mohlke KL, Silander K, Ally DS, Chines P, Blaschak-Harvan J, Douglas JA, able risk of diabetes in this population. Further studies are Duren WL, Epstein MP, Fingerlin TE, Kaleta HS, Lange EM, Li C, McEachin RC, Stringham HM, Trager E, White PP, Balow J Jr, Birznieks G, Chang J, warranted to define the role of this variant in other Eldridge W: The Finland-United States investigation of non-insulin-depen- populations on diabetes risk and to investigate the patho- dent diabetes mellitus genetics (FUSION) study. II. An autosomal genome physiological, cellular, and molecular mechanisms that scan for diabetes-related quantitative-trait loci. Am J Hum Genet, 67:1186– underlie these associations. 200, 2000 10. Vionnet N, Hani El-H, Dupont S, Gallina S, Francke S, Dotte S, De Matos F, Durand E, Lepretre F, Lecoeur C, Gallina P, Zekiri L, Dina C, Froguel P: ACKNOWLEDGMENTS Genomewide search for type 2 diabetes-susceptibility genes in French This research was supported by National Institutes of whites: evidence for a novel susceptibility locus for early-onset diabetes on Health (NIH) Grant 1R01DK53959-01. F.L.B. was sup- chromosome 3q27-qter and independent replication of a type 2-diabetes ported by an Established Investigator grant from the locus on chromosome 1q21–q24. Am J Hum Genet, 67:1470–1480, 2000 11. Elbein SC, Hoffman MD, Teng K, Leppert MF, Hasstedt SJ: A genome-wide American Heart Association, Dallas, TX; W.H.L.K. was search for type 2 diabetes susceptibility genes in Utah Cacausians. supported by NIH Training Grant T32HL077024-23; A.R.S. Diabetes 68:1175–1182, 1999 is supported by NIH K24 Grant DK02673-01A1; and C.L.H. 12. Wiltshire S, Hattersley AT, Hitman GA, Walker M, Levy JC, Sampson M, was supported by NIH grant DK47487. O’Rahilly S, Frayling TM, Bell JI, Lathrop GM, Bennett A, Dhillon R, The ARIC Study is carried out as a collaborative study Fletcher C, Groves CJ, Jones E, Prestwich P, Simecek N, Subba Rao PV, Marie Wishart, Foxon R, Howell S, Smedley D, Cardon LR, Menzel S, supported by contracts N01-HC-55015, N01-HC-555016, McCarthy MI: A genomewide scan for loci predisposing to type 2 diabetes N01-HC-55018, N01-HC-55019, N01-HC-55020, N01-HC- in a U.K. population (The Diabetes U.K. Warren 2 Repository): analysis of 55021, and N01-HC-55022 from the National Heart, Lung, 573 pedigrees provides independent replication of a susceptibility locus on and Blood Institute. chromosome 1q. Am J Hum Genet 69:553–569, 2001 The authors thank the staff and participants in the ARIC 13. Hani EH, Hager J, Philippi A, Demenais F, Froguel P, Vionnet N: Mapping NIDDM susceptibility loci in French families: studies with markers in the Study for their important contributions and Demian G. region of NIDDM1 on chromosome 2q. Diabetes 46:1225–1226, 1997 Lewis for his assistance with SNP43 genotyping. 14. Ghosh S, Hauser ER, Magnuson VL, Valle T, Ally DS, Karanjawala ZE, Rayman JB, Knapp JI, Musick A, Tannenbaum J, Te C, Eldridge W, Shapiro REFERENCES S, Musick T, Martin C, So A, Witt A, Harvan JB, Watanabe RM, Hagopian W, Eriksson J, Nylund SJ, Kohtamaki K, Tuomilehto-Wolf E, Boehnke M, et 1. Elbein SC: The genetics of human noninsulin-dependent (type 2) diabetes al.: A large sample of Finnish diabetic sib-pairs reveals no evidence for a mellitus. J Nutr 127:1891S–1896S, 1997 non-insulin-dependent diabetes mellitus susceptibility locus at 2qter. 2. Hanson RL, Ehm MG, Pettitt DJ, Prochazka M, Thompson DB, Timberlake J Clin Invest 102:704–709, 1998 D, Foroud T, Kobes S, Baier L, Burns DK, Almasy L, Blangero J, Garvey 15. Ciccarese M, Tonolo G, Delin I, Wong FK, Holm P, Atzeni MM, Lichtenstein WT, Bennett PH, Knowler WC: An autosomal genomic scan for loci linked to type II diabetes mellitus and body-mass index in Pima Indians. Am J P, Kockum I, Maiolo M, Luthman H: Preliminary data on a genome search Hum Genet 63:1130–1138, 1998 in NIDDM siblings: the NIDDM1 locus on is not linked to 3. Ehm MG, Karnoub MC, Sakul H, Gottschalk K, Holt DC, Weber JL, Vaske NIDDM in the Sardinian population: Study Group for the Genetics of D, Briley D, Briley L, Kopf J, McMillen P, Nguyen Q, Reisman M, Lai EH, Diabetes in Sardinia. Diabetolgia, 40:1366–1367, 1997 Joslyn G, Shepherd NS, Bell C, Wagner MJ, Burns DK, the American 16. Thomas AW, Sherratt EJ, Gagg Jw, Davies SA, Majid A, Alcolado JC: Diabetes Association GENNID Study Group: Genomewide search for type Genetic linkage study of a major susceptibility locus (D2S125) in a British 2 diabetes susceptibility genes in four American populations. Am J Hum population of non-insulin dependent diabetic sib-pairs using a simple Genet, 66:1871–1881, 2000 non-isotopic screening method. Hum Genet, 101:212–213, 1997 4. Hanis CL, Boerwinkle E, Chakraborty R, Ellsworth DL, Cocannon P, 17. Horikawa Y, Oda N, Cox NJ, Li X, Orhu-Melander M, Hara M, Hinokio Y, Stirling B, Morrison VA, Wapelhorst B, Spielman RS, Gogolin-Ewens KJ, Linder TH, Mashima H, Schwarz PEH, del Bosque-Plata L, Oda Y, Yoshiu- Shepard JM, Williams SR, Risch N, Hinds D, Iwasaki N, Ogata M, Omori Y, chi I, Colilla S, Polonsky KS, Wei S, Concannon P, Iwasaki N, Schulze, Petzold C, Rietzch H, Schroder HE, Schulze J, Cox NJ, Menzel S, Bororaj Baier LJ, Bogardus C, Groop L, Boerwinkle E, Hanis CL, Bell GI: Genetic VV, Chen X, et al: A genome-wide search for human non-insulin-dependent variation in the calpain 10 gene (CAPN10) is associated with type 2 (type 2) diabetes genes reveals a major susceptibility locus on chromo- diabetes mellitus. Nat Genet 26:163–175, 2000 some 2. Nat Genet 13:161–166, 1996 18. Johnson DE: Noncaspase proteases in apoptosis. Leukemia 14:1695–1703, 5. Duggirala R, Blangero J, Almasy L, Dyer TD, Williams KL, Leach RJ, 2000 O’Connell P, Stern MP: Linkage of type 2 diabetes mellitus and age onset 19. Chen M, Durr J, Fernandez HL: Possible role of calpain in normal to a location on chromosome 10q in Mexican-Americans. Am J Hum Genet processing of beta-amyloid precursor in human platelets. Biochem 64:1127–1140, 1999 Biophys Res Commun 273:170–175, 2000 6. Mahtani MM, Widen E, Lehto M, Thomas J, McCarthy M, Brayer J, Bryant 20. Bushby KM: The limb-girdle muscular dystrophies-multiple genes, multiple B, Chan G, Daly M, Forsblom C, Kanninen T, Kirby A, Kruglyak L Munnelly mechanisms. Hum Mol Genet 8:1875–1882, 1999

236 DIABETES, VOL. 51, JANUARY 2002 M.J. GARANT AND ASSOCIATES

21. Santella L: The role of calcium in the cell cycle: facts and hypotheses. 30. Lillioja S, Bogardus C: Obesity and insulin resistance: lessons learned from Biochem Biophys Res Commun 244:317–324, 1998 the Pima Indians. Diabete Metab Rev 4:517–540, 1988 22. Brancati FL, Kao WH, Folsom AR, Watson RI, Szklo M: Incident type 2 31. Frayling TM, Evans JC, Ellard S, Hattersley AT, Study Group-BDA Warren diabetes mellitus in African-Americans and white adults: the Atheroscle- 2 Consortium: Transmission disequilibrium at the calpain10/NIDDM1 gene rosis Risk in Communities Study. JAMA 283:2253–2259, 2000 in U.K. caucasians type 2 diabetes parent-offspring trios (Abstract). 23. Lei HH, Coresh J, Shuldiner AR, Boerwinkle E, Brancati FL: Variants of the Diabetes 49 (Suppl. 1):A8, 2000 insulin receptor substrate-1 and fatty acid binding protein 2 genes and the 32. Ren Q, Hasstedt S, Hanis C, Elbein SC: Increased transmission of NIDDM1 risk of type 2 diabetes, obesity, and hyperinsulinemia in African-Ameri- variant in caucasian familial type 2 diabetes (Abstract). Diabetes 49 (Suppl. cans: the Atherosclerosis Risk in Communities Study. Diabetes, 48:1868– 1):A200, 2000 1872, 1999 33. Carafoli E, Molinari M: Calpain: a protease in search of a function. 24. Baecke JA, Burema J, Frijters JE: A short questionnaire for the measure- Biochem Biophys Res Commun 247:197–203, 1998 ment of habitual physical activity in epidemiological studies. Am J Clin 34. Patel YM, Lane MD: Role of calpain in adipocyte differentiation. Proc Natl Nutr 36:936–942, 1982 Acad SciUSA96:1279–1284, 1999 35. Sologub LI, Kusen SI, Pashkovskaia IS, Gurskaia NI, Senkus IT, Antoniak 25. Willett WC, Sampson L, Stampfer MJ, Rosner B, Bain C, Witschi J, GL: Insulin-degrading protease from plasma membranes of rat liver cells Hennekens CH, Speizer FE: Reproducibility and validity of a semi- and erythrocytes. WMJ 61:28–33, 1989 quantitative food frequency questionnaire. Am J Epidemiol 122:51–65, 36. Smith LK, Rice KM, Garner CW: The insulin-induced downregulation of 1985 IRS-1 in 3T3–L1 adipocytes is mediated by a calcium-dependent thiol 26. Baier LJ, Permana PA, Yang X, Pratley RE, Hanson RL, Shen G-Q, Mott D, protease. Mol Cell Endocrinol 122:81–92, 1996 Knowler WC, Cox NJ, Horikawa Y, Oda N, Bell GI, Bogardus C: A 37. Folli F, Saad MJ, Backer MJ, Kahn CR: Insulin stimulation of phosphati- calpain-10 gene polymorphism is associated with reduced muscle mRNA dylinositol 3-kinase activity and association with insulin receptor substrate levels and insulin resistance. J Clin Invest 106:R69–73, 2000 1 in liver and muscle of the intact rat. J Biol Chem 267:22171–22177, 1992 27. Evans JC, Frayling TM, Cassell PG, Saker PJ, Hitman GA, Walker M, Levy 38. Yamauchi T, Tobe K, Tamemoto H, Ueki K, Kaburagi Y, Yamamoto-Honda JC, O’Rahilly S, PVS Rao, Bennet AJ, Jones EC, Menzel S, Prestwich P, R, Takahashi Y, Yoshizawa F, Aizawa S, Akanuma Y, Sonenberg N, Yazaki Simecek N, Wishart M, Dhillon R, Fletcher C, Millward A, Demaine A, Y, Kadowaki T: Insulin signaling and insulin actions in the muscles and Wilkin T, Horikawa Y, Cox NJ, Bell GI, Ellard S, McCarthy MI, Hattersley livers of insulin-resistant, insulin receptor substrate1-deficient mice. Mol AT: Studies of association between the gene for calpain-10 and type 2 Cell Biol 16:3074–3084, 1996 diabetes mellitus in the United Kingdon. Am J Hum Genet 69:544–552, 39. Zhou YP, Sreenan S, Bindokas VP, Pan CY, Currie KPM, Lee JP, Fox AP, 2001 Miller RJ, Cox NJ, Polonsky KS: Calpain inhibitors impair insulin secretion 28. Hegele RA, Harris SB, Zinman B, Hanley AJG, Cao H: Absence of after 48-hours: a model for 32 ␤-cell dysfunction in type 2 diabetes association of type 2 diabetes with CAPN10 and PC-1 polymorphisms in (Abstract)? Diabetes 49 (Suppl. 1):A80, 2000 Oji-Cree (Letter). Diabetes Care 24:1498–1499, 2001 40. Levin ML: The occurrence of lung cancer in man. Acta Intern Cancer 9:531, 29. Briefel RR, McDowell A, Alaimo K, Caughman CR, Bischof AL, Carroll MD, 1953 Johnson CL: Total energy intake of the US population: the third National 41. Cox NJ, Frigge M, Nicolae DL, Concannon P, Hanis CL, Bell GI, Kong A: Health and Nutrition Examination Survey, 1988–1991. Am J Clin Nutr 62 Loci on 2 (NIDDM1) and 15 interact to increase suscepti- (Suppl.):1072S–1080S, 1995 bility to diabetes in Mexican-Americans. Nat Genet, 21:213–215, 1999

DIABETES, VOL. 51, JANUARY 2002 237