Calpain-10 gene polymorphisms in type 2 diabetes and its micro- and macrovascular complications

Journal of Diabetes and Its Complications 27 (2013) 54–58

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Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M

Calpain-10 gene polymorphisms in type 2 diabetes and its micro- and macrovascular complications☆,☆☆ Monika Buraczynska a,⁎, Piotr Wacinski b, Anna Stec a, Agata Kuczmaszewska a a b

Laboratory for DNA Analysis and Molecular Diagnostics, Department of Nephrology, Medical University of Lublin, Dr K. Jaczewskiego 8, 20–954 Lublin, Poland Department of Cardiology, Medical University of Lublin, Lublin, Poland

a r t i c l e

i n f o

Article history: Received 9 December 2011 Received in revised form 13 July 2012 Accepted 25 July 2012 Available online 26 September 2012 Keywords: Type 2 diabetes Calpain 10 Single nucleotide polymorphism Genotype Cardiovascular disease

a b s t r a c t Genetic variations in the calpain 10 gene (CAPN10) were previously implicated with increased risk of type 2 diabetes (T2DM). We studied the association of single nucleotide polymorphisms in the CAPN10 gene, SNP -43, SNP -19 and SNP -63, with T2DM and its complications. Overall, we examined 1440 individuals: 880 patients with diabetes and 560 healthy subjects, all Caucasians of Polish origin. All subjects were genotyped for the CAPN10 SNPs by polymerase chain reaction (PCR). The frequencies of alleles, genotypes and haplotypes at three studied loci were similar between the groups. However, the -43 SNP was significantly more frequent in T2DM patients with coexisting cardiovascular disease (CVD) than in patients without CVD (p=0.001). The -43 SNP was still significantly associated with the risk of CVD after adjusting for potential risk factors including male gender, age, BMI, dyslipidemia and hypertension. The odds ratio for G allele for CVD+ versus CVD- patients was 1.89, 95% CI 1.52–2.35. None of the studied SNPs was significantly associated with microvascular diabetic complications. There was a tendency to increased frequency of SNP -43 1/1 homozygotes in patients with diabetic retinopathy (p=0.057). The homozygous haplotype combination 121/121 was more frequent in T2DM patients than in non-diabetic controls (18.4% vs 10.5%, p=0.019). In conclusion, the results of our study suggest the significant association of SNP -43 with the risk of CVD coexisting with T2DM. We also observed that 121/121 haplotype was associated with T2DM in the studied population. © 2013 Elsevier Inc. All rights reserved.

1. Introduction A combination of multiple genetic and environmental factors contributes to the pathogenesis of type 2 diabetes (T2DM) (McCarthy, 2004; O'Rahilly, Barroso, & Wareham, 2005). Type 2 diabetes is associated with increased mortality and morbidity from cardiovascular disease (CVD) (Tang, Maroo, & Young, 2004; Winer & Sowers, 2004; Yamagishi, Nakamura, Matsui, Ueda, & Imaizumi, 2007). Epidemiologic evidence suggests that diabetes and CVD may share common genetic factors (Mitchell & Imumorin, 2002). The identification of causative genes predisposing to T2DM, its micro- and macrovascular complications and comorbidities could provide means to better understanding the pathogenesis of the disease and result in a better prevention, diagnosis and treatment.

☆ Conflict of interest: The authors declare no conflict of interest associated with this manuscript. ☆☆ This study was supported by the grant from National Research Center No. NN402 522940 (MB) and in part by the grant DS 379/11 (MB) from Medical University of Lublin. ⁎ Corresponding author. Tel.: +48 81 7244 716; fax: +48 81 7244 357. E-mail address: [email protected] (M. Buraczynska). 1056-8727/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jdiacomp.2012.07.005

The gene encoding calpain 10 (CAPN10) is an example of positional cloning of a candidate gene for T2DM (Ridderstrale, Parikh, & Groop, 2005). CAPN10 was shown to function as intracellular calcium-dependent nonlysosomal cysteine protease in calciumregulated signaling pathways (Dear & Boehm, 2001). It is expressed in several tissue types, especially involved in the regulation of glucose homeostasis, such as pancreatic β islet cells, liver, skeletal muscle and adipocytes (Carlsson et al., 2005; Ling, Groop, Del Guerra, & Lupi, 2009). Calpain-10 has been suggested to influence both insulin secretion and resistance (Baier et al., 2000; Parnaud, Hammar, Rouiller, & Bosco, 2005; Sreenan et al., 2001). The calpain-10 gene might serve as an important T2DM susceptibility gene (Cox, Hayes, Roe, Tsuchiya, & Bell, 2004; Weedon et al., 2003). Calpain-10 gene is located on chromosome 2q37.3, and consists of 15 exons spanning 31 kb. It encodes a 672 amino-acid intracellular protease. Several case–control and association studies indicated that polymorphisms in CAPN10 are associated with the development of T2DM and insulin resistance (Fullerton et al., 2002; Horikawa et al., 2000; Tsuchiya et al., 2006). The susceptibility seems to be attributable to combination of haplotypes of multiple polymorphisms such as SNPs in the intronic regions, -44 (intron 3), -43 (intron 3), -63 (intron 13) and del/ins -19 (intron 6) (Bodhini et al., 2011; Horikawa et al., 2000; Rasmussen et al., 2002).

M. Buraczynska et al. / Journal of Diabetes and Its Complications 27 (2013) 54–58

The aim of the present study was to examine whether the CAPN10 variants were associated with the risk of diabetes and/or its complications in a large, hospital based population of T2DM patients of Polish origin. 2. Materials and methods 2.1. Study subjects The study was designed to analyze the calpain 10 gene polymorphisms in type 2 diabetes (T2DM) patients. To compare the prevalence of polymorphisms in patients with a healthy population, we included a control group of healthy individuals. The study population (cases) consisted of 880 unrelated T2DM patients, consecutively enrolled between 2004 and 2009 from the Departments of Nephrology and Endocrinology of Medical University of Lublin and outpatient diabetes clinic. All subjects were Caucasians of Polish origin. Diabetes was diagnosed according to American Diabetes Association criteria. One or more of the following conditions were met for diagnosis: the presence of classic symptoms of hyperglycemia (polyuria, polydipsia, weight loss), fasting plasma glucose ≥7 mmol/L or random plasma glucose ≥11 mmol/L, the use of insulin or oral hypoglycemic agents. The mean duration of diabetes, estimated from time of the first symptoms attributable to the disease or from time of first detection of glycosuria, was 13.9 years (range 8–31). Glycemic control was evaluated by measuring glycated HbA1c levels by turbidimetric inhibition immunoassay (TINIA) using Tina-quant hemoglobin A1cII (Roche-Hitachi 747). Diabetic nephropathy was diagnosed clinically in the presence of persistent albuminuria ≥300 mg/24 h in at least two consecutive determinations in the absence of hematuria or infection. Diabetic retinopathy was diagnosed by independent ophthalmologists. A complete ophthalmological examination, including corrected visual acuity, fundoscopic examination and fundus photography (three 45° fields per eye) was performed at least every year. Retinopathy was diagnosed according to the Early Treatment Diabetic Retinopathy Study (ETDRS) criteria: the presence of microaneurysms, hemorrhages, cotton wool spots, intraretinal microvascular abnormalities, hard exudates, venous beading and new vessels. Diabetic neuropathy was diagnosed by physical examination. Cardiovascular disease was diagnosed and documented as one or the combination of several pathological states: congestive heart failure, left ventricular hypertrophy, angina pectoris, ischemic heart disease, myocardial infarction, ischemic cerebral stroke, vascular calcifications or atheromatous lesions. Clinical manifestations of CVD were confirmed by appropriate biochemical, radiographic, echocardiographic and vascular diagnostic criteria. There was a substantial overlap between categories. Among diabetic patients 625 individuals (71%) were hypertensive, as defined according to World Health Organization criteria. Hypertensive patients had persistent systolic blood pressure N140 mm Hg and diastolic blood pressure N90 mm Hg and/or were receiving antihypertensive treatment. Healthy control subjects of Polish origin (n=560) were volunteers (mostly blood donors and hospital staff members) with no history of diabetes or cardiovascular disease. Subjects with a positive family history of diabetes or cardiovascular disease in first degree relatives were excluded from the study. A written informed consent was obtained from all subjects in accordance with principles of the Declaration of Helsinki. The protocol of the study was approved by the institutional ethics committee. 2.2. Genotype determination Genomic DNA was extracted from peripheral blood leukocytes (obtained from EDTA anticoagulated blood) using a standard

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technique (Madisen, Hoar, Holroyd, Crisp, & Hodes, 1987). All subjects were genotyped for the calpain-10 SNPs -43, -19 and -63. SNP -43 was genotyped with previously published primers (Zaharna, Abed, & Sharif, 2010). The PCR product was digested with Nde I restriction endonuclease (Fermentas, Vilnius, Lithuania). Digestion products were separated on 3% agarose gel. Allele G (allele 1) was detected as a 254 bp fragment and allele A (allele 2) as 223 bp and 31 bp fragments. For SNP -19 the DNA fragment containing 32 bp insertion/deletion was amplified using previously published primers and conditions (Evans et al., 2001). The PCR products were separated on a 3% agarose gel. Allele 1 (2 repeats of 32 bp sequence) was 155 bp and allele 2 (3 repeats) was 187 bp. The SNP -63 was genotyped by previously published protocol (Evans et al., 2001). PCR product was digested with Hha I restriction endonuclease (Fermentas, Vilnius, Lithuania) and resulting fragments were separated on a 3% agarose gel. The sizes of fragments were 162 and 30 bp for the C allele (allele 1) and 192 bp for the T allele (allele 2). The quality of genotyping was controlled by using blind DNA duplicates for some samples (96 for each polymorphism). In addition, 10 samples were randomly selected for each genotype of three studied SNPs and the PCR products were sequenced in CEQ 8000 Genetic Analysis System (Beckman Coulter, High Wycombe, England). Observed concordance between genotyping assays was 100%. 2.3. Statistical analysis Statistical calculations were performed using SPSS version 11.0 for Windows (SPSS, Inc., Chicago, IL, USA). For baseline characteristics the normally distributed continuous variables are presented as means± SD. The Hardy–Weinberg equilibrium was evaluated with the χ 2 test. Genotype distribution and allele frequencies were compared between groups using a Pearson χ 2 test of independence with 2×2 contingency and z statistics. ANOVA was used to compare average values of biochemical parameters. Haplotype frequencies were estimated using the EH program (Jurg Ott, Rockefeller University, New York). Student's t-test and Mann–Whitney test were used for comparing discrete and continuous variables. For significant allelic and genotyping associations the odds ratios (OR) with corresponding 95% confidence intervals (CI) were calculated. Power calculations were performed with the program of Purcell et al. (Purcell, Cherny, & Sham, 2003) (available at http://pngu.mgh.harvard.edu/~purcell/gpc/). The prevalence of CVD was 63% and the frequency of SNP -43 allele 1 was 0.75. The study had 99.5% power (α=0.05) to detect an association (OR vs CVD- patients 1.89, 95% CI 1.51-2.37). An interaction of the polymorphisms with diabetic complications and various risk factors was examined with multiple logistic regression analysis. The Bonferroni correction was applied for multiple comparisons. Statistical significance was set at pb0.05. 3. Results The demographic and clinical profiles of studied subjects with T2DM and healthy controls are presented in Table 1. Among the 880 patients with T2DM 387 had diabetic nephropathy, 399 had retinopathy and 211 had polyneuropathy. There was a statistically significant difference in age between control group and patients with diabetes (pb0.05). The gender distribution was similar in both diabetic and control groups. Significant differences were observed regarding BMI (pb0.001) and total cholesterol (pb0.001). The genotypes of the calpain 10 SNPs -43, -19 and -63 were determined in 880 patients with type 2 diabetes and 560 healthy individuals. No genotyping errors were found in the total of 378 samples checked. The frequencies of genotypes and alleles for the studied SNPs in T2DM patients and controls are shown in Table 2. The genotype frequencies were consistent with the Hardy-Weinberg

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M. Buraczynska et al. / Journal of Diabetes and Its Complications 27 (2013) 54–58

Table 1 Demographic and clinical profile of studied subjects. Variable

T2DM patients

Controls

N Male/female Age at study (years) Age at diagnosis (years) Diabetes duration (years) Diabetic retinopathy (%) Diabetic nephropathy (%) Polyneuropathy Hypertension (%) HbA1c (%) Fasting plasma glucose (mmol/l) Total cholesterol (mmol/l) HDL cholesterol (mmol/l Triglycerides (mmol/l) BMI (kg/m2) Family history of diabetes

880 449/431 56±24 47±7 13.9±9 399 (45.3) 387 (44) 211 (24) 625 (71) 8.2±2.7 7.92±2.8 5.4±1.22 1.2±0.66 2.3±1.39 28.6±4.6 218 (25)

560 288/272 53±17 NA NA 0 0 0 0 ND 5.67±2.1a 4.6±1.29 ND ND 26.8±4.3 0

p value

Genotypesa NS b0.01

b0.001

b 0.001

T2DM, type 2 diabetes mellitus. Values are presented as mean±SD or numbers (%). NA, not applicable; ND, not determined. aDetermined in 70% of subjects.

equilibrium for all studied polymorphisms : SNP -43 (χ 2 =2.24; p= 0.134), SNP -19 (χ 2 =0.94; p=0.332) and SNP -63 (χ 2 =0.41; p= 0.521). The frequencies in the control group were similar to those reported earlier for Polish population (Malecki et al., 2002). There were no statistically significant differences in the frequency of genotypes/alleles of the calpain 10-19 and −63 polymorphisms between T2DM patients and healthy individuals. There were also no significant differences when we compared patients with any microvascular complications with those without complications after 10 or more years diabetes duration (Table 2). The -43 polymorphism was significantly more frequent in the T2DM patients with coexisting cardiovascular disease than in patients without CVD (p=0.001) (Table 3). The odds ratio for the allele 1 for CVD+ versus CVD- patients was 1.89, 95% CI 1.52–2.35. We also determined the association of -43 SNP with CVD after adjusting for potential risk factors. Variables identified as potentially significant – male gender, age, BMI, dyslipidemia and hypertension were included in further multivariate analysis. The -43 SNP was still significantly associated with the risk of CVD. Adjusted OR for covariates, for allele 1, in multivariate regression analysis was 1.63 (1.16–2.67), p=0.011. The p value range for other variables was 0.26–0.53. For evaluating the effect of studied polymorphisms on development of different microvascular complications, the genotype frequencies were compared separately in T2DM subgroups with diabetic

Table 2 Genotype and allele distribution of calpain 10 SNPs. Genotypes 1/1 SNP -43 T2DM patients Controls T2DM MVC+ T2DM MVCSNP -19 T2DM patients Controls T2DM MVC+ T2DM MVC− SNP -63 T2DM patients Controls T2DM MVC+ T2DM MVC−

493 262 344 77 84 59 56 77 749 466 468 121

2/2

(56) (47) (60) (54)

334 253 214 53

(38) (45) (38) (37)

(9.5) (10.5) (10) (54)

426 261 288 53

(48.5) (46.5) (51) (37)

(85) (83) (83) (85)

128 91 90 21

(14.5) (16.5) (16) (15)

2

0.75 0.69 0.80 0.73

0.25 0.31 0.20 0.27

(42) (43) (39) (9)

0.34 0.34 0.35 0.73

0.66 0.66 0.65 0.27

3 (0.5) 3 (0.5) 6 (1) 0 (0)

0.92 0.91 0.91 0.93

0.08 0.09 0.09 0.07

370 240 220 12

1/2

339 (61) 154 (47)

202 (37) 132 (40)

2/2

1

2

12 (2) 41 (13)

0.80 0.67

0.20 0.33

p=0.001

50 (9) 34 (10.5)

274 (50) 152 (46.5)

229 (41) 141 (43)

0.34 0.34

0.66 0.66

p=0.72

466 (84.5) 283 (86.5)

84 (15) 44 (13.5)

3 (0.5) 0 (0)

0.92 0.93

0.08 0.07

p=0.49

Genotype distributions are shown as numbers (%). ap values at 2 degrees of freedom. OR for SNP -43 allele 1 was 1.89 (95% CI 1.51-2.37), p=0.008 vs. no CVD patients. For T2DM CVD+ n=553; for T2DM no CVD n=327.

nephropathy, retinopathy and neuropathy and without these complications (Table 4). There was an increase in the frequency of 1/1 homozygotes of -43 SNP in diabetic retinopathy patients but the difference did not reach the statistical significance ( p=0.057). No differences were observed in the nephropathy and neuropathy subgroups (pN0.05). The results of CAPN10 haplotype analysis are presented in Table 5. Four haplotypes occurred with the frequency N1%. The distribution of haplotypes was comparable in T2DM patients and controls. In the analysis of haplotype combinations, the 121/121 homozygous combination was more frequent in T2DM patients than in control group (18.4% versus 10.5%, p=0.019). Observed frequencies of haplotype combinations were similar to other studies of Caucasian subjects (Evans et al., 2001; Horikawa et al., 2000; Malecki et al., 2002). 4. Discussion There is a growing evidence of calpain 10 role in the pathogenesis of type 2 diabetes. CAPN10 gene has been associated with T2DM in some studies, but some failed to reproduce the findings (Ezzidi et al., 2010; Horikawa et al., 2000; Iwasaki et al., 2005; Martinez-Gomez et al., 2011; Tsuchiya et al., 2006; Zaharna et al., 2010). One of calpain 10 variants, SNP -43 and at risk haplotype combination 112/121, defined by SNP -43, SNP -19 and SNP -63 polymorphisms, confirmed increased risk of T2DM in some but not all populations (Weedon et al., 2003; Rasmussen et al., 2002; del Bosque-Plata et al., 2004; Iwasaki et al., 2005). We have studied the effect of calpain 10 gene variants SNP -43, SNP -19 and SNP -63 on the risk of T2DM and its macro- and

p value

1

53 (6) 45 (8) 6 (2) 12 (9)

SNP -43 T2DM CVD+ T2DM no CVD SNP -19 T2DM CVD+ T2DM no CVD SNP -63 T2DM CVD+ T2DM no CVD

Alleles

1/1

a

Alleles 1/2

Table 3 Genotype and allele distribution of calpain 10 SNPs in T2DM patients with and without CVD.

0.19 0.11

0.90 0.83

0.86 0.69

MVC, microvascular complications, MVC-, no microvascular complications after diabetes duration ≥10 years. Genotype distributions are shown as numbers (%). a T2DM vs. controls, MVC+ vs. MVC−.

Table 4 Genotype distribution of calpain 10 SNPs in T2DM patients with microvascular complications. SNP -43 Retinopathy (%) Nephropathy (%) Neuropathy (%) SNP -19 Retinopathy (%) Nephropathy (%) Neuropathy (%) SNP -63 Retinopathy (%) Nephropathy (%) Neuropathy (%)

1/1 253 (51) 196 (40) 182 (37) 1/1 36 (43) 38 (45) 38 (45) 1/1 336 (45) 337 (45) 320 (43)

1/2 130 (39) 172 (51) 185 (55) 1/2 196 (46) 202 (47) 197 (46) 1/2 62 (48.5) 49 (38) 67 (52)

2/2 16 (30) 19 (36) 20 (38) 2/2 167 (45) 147 (40) 152 (41) 2/2 1 (33) 1 (33) 0 (0)

a,b p 0.057 0.22 0.12

0.97 0.74 0.85 0.17 0.45 c 0.30

Genotype distributions in patients with microvascular complications are shown as numbers and % of numbers for given genotype in entire T2DM group. aCompared as retinopathy vs. no retinopathy, nephropathy vs. no nephropathy, neuropathy vs. no neuropathy. bp values at 2 degrees of freedom. cFor 1/1 and 1/2 genotypes.

M. Buraczynska et al. / Journal of Diabetes and Its Complications 27 (2013) 54–58 Table 5 Calpain 10 haplotype distribution in T2DM patients and controls. -43, -19, -63

T2DM

Haplotype 121 656 (37.3)a 111 471 (26.8) 112 115 (6.5) 221 518 (29.4) Haplotype combination 111/111 61 (6.9) 111/112 33 (3.7) 111/121 178 (20.3) 111/221 156 (17.8) 112/121 55 (6.2) 112/221 35 (4.0) 121/121 162 (18.4) 121/221 124 (14.1) 221/221 76 (8.6)

Controls

pb

OR (95% CI)b

360 309 103 348

(32.1)a (27.6) (9.2) (31.1)

0.09 0.77 0.23 0.17

1.25 0.95 0.69 0.95

(1.07–1.47) (0.81–1.13) (0.52–0.91) (0.79–1.09)

37 27 106 96 36 35 59 113 51

(6.6) (4.8) (18.9) (17.1) (6.5) (6.3) (10.5) (20.1) (9.2)

0.91 0.43 0.79 0.81 0.69 0.34 0.019 0.09 0.76

1.05 0.77 1.08 1.04 0.97 0.62 1.91 0.65 0.94

(0.69–1.61) (0.46–1.29) (0.83–1.41) (0.79–1.38) (0.63–1.49) (0.38–1.0) (1.39–2.63) (0.49)–0.86 (0.65–1.37)

Haplotypes are coded for alleles (wild type=1, mutant=2) at each locus. aNumber of haplotypes (%). bp values and odds ratio for each haplotype were computed using the other haplotype combinations as a reference. The results for rare haplotypes are not shown.

microvascular complications. These polymorphisms were selected because of their reported associations, either individually or in combinations, with T2DM. No significant association was observed between T2DM and allele frequencies of studied polymorphisms in our patient population. The -43 polymorphism, however, was significantly more frequent in patients with both T2DM and cardiovascular disease (p=0.001) than in patients without CVD. This association remained significant even after adjusting for several classical risk factors, suggesting that calpain 10 SNP -43 can independently alter the CVD risk in patients with T2DM. There were earlier observations regarding the association of calpain 10 gene with subclinical atherosclerosis and carotid intima-media thickness in nondiabetic subjects (Goodarzi et al., 2005; Goodarzi et al., 2009). To our knowledge, ours is the first assessment of calpain 10 gene polymorphisms in diabetic patients with CVD. Calpains influence processes including inflammation, apoptosis, nitric oxide production, leukocyte adherence to vascular endothelium and platelet function (Randriamboavonjy & Fleming, 2010; Stalker, Skvarka, & Scalia, 2003; Yang et al., 2010). This evidence implicates calpains in the progression of atherosclerosis and cardiovascular disease (Libby & Theroux, 2005). The incidence of CVD is markedly increased in patients with diabetes. The product of calpain gene may affect cardiovascular disease either directly or indirectly via diabetes. The knowledge of gene polymorphisms associated with CVD in patients with type 2 diabetes and those without and their relevance to the diabetes-related vascular complications are essential for effective strategies for prevention. It was shown that calpain 10 gene might affect microvascular function (Shore et al., 2002). There was only one previous study investigating the relationship of calpain 10 gene polymorphisms with diabetic microvascular complications. Although the authors observed an association of SNP -44 with susceptibility to T2DM, it was not associated with any microvascular complications. There was also no association between SNP -19 and SNP -63 and microvascular complications of T2DM (Demirci, Yurtcu, Ergun, Yazici, Karasu, & Yetkin, 2008). In our study we evaluated the effect of -43, -19 and -63 SNPs on development of microvascular complications, by comparing the genotype and allele frequencies in T2 DM subgroups with diabetic nephropathy, retinopathy and neuropathy. There was an increase in the frequency of 1/1 homozygotes of -43 SNP in diabetic retinopathy patients with the difference close to the statistical significance (p= 0.057). No differences were observed in the nephropathy and neuropathy subgroups. The results of animal studies shown that calpains play an important role in retinal degeneration through retinal cell death induced by ischemia-reperfusion in vivo and by

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hypoxia in vitro (Azuma, Sakamoto-Mizutani, Nakajima, KanaamiDaibo, Tamada, & Shearer, 2004). This might suggest the involvement of calpains in human retinopathy. It has been demonstrated that haplotypes define the risk of T2DM better than individual polymorphisms. Horikawa et al. reported an association between T2DM and the heterozygous combination of the three locus haplotypes created by SNPs -43, -19 and -63 (112/121) in a Mexican-American population. Similar results were obtained in the European populations (Horikawa et al., 2000). We were not able to confirm the effect of this haplotype combination on susceptibility to T2DM. Malecki et al. (Malecki et al., 2002) found that the homozygous combination of the same loci (-43, -19 and -63) haplotypes (121/ 121) may be associated with an increased risk of T2DM in a Polish population. Our results are in agreement with this finding. The frequency of the 121/121 haplotype combination for 143, -19 and -63 loci was higher in T2DM patients than in controls. However, in our study there was no difference in the frequency of 121/121 haplotype combination between patients with positive family history of T2DM and those without it. The molecular mechanisms of observed associations have yet to be discovered. Although three studied polymorphisms are located in intronic sequences of CAPN10 gene they might affect the expression of the gene or mRNA stability (Baier et al., 2000; Cox et al., 2004; Tsuchiya, Yan, Vogelstein, Kinzler, & Bell, 2003). As most of the association studies, ours has some limitations. It is a retrospective case–control study thus a selection bias cannot be excluded. To limit this bias we included consecutive patients in the study and tried to adjust for known confounding risk factors. All normoglycemic individuals with traits related to type 2 diabetes, like obesity, hypertension or hyperlipidemia, were excluded from the study. It is possible that -43 SNP is in linkage disequilibrium with other unknown functional mutation that contributes to the progression of cardiovascular disease. Since ours was not a community based study, it was difficult to obtain a number of control subjects matching the number of patients. Although the number of control subjects was lower than the number of cases, it was adequate for all statistical comparisons. The strength of our study is that all patients and controls are of the same ethnic origin. Furthermore, all subjects were examined in a standardized manner, with well defined diagnostic criteria and genotyping was performed blind with respect to case– control status. Undoubtedly, future functional and association studies in independent populations are needed to confirm the observed association of -43 SNP with cardiovascular disease in T2DM patients. In conclusion, our data demonstrate that -43 SNP in the calpain 10 gene is significantly associated with the increased risk of CVD in T2DM patients. No association was found between three studied polymorphisms (SNP -43, SNP -19 and SNP -63) and diabetes, compared to healthy controls. The 121/121 haplotype combination seems to increase the risk of T2DM. Thus, the calpain 10 gene appears to be an interesting candidate for a genetic link in pathogenesis of cardiovascular disease and diabetes.

Acknowledgments This study was supported by the grant from National Research Center No. N N402 522940 (MB) and in part by the grant DS 379/11 (MB) from Medical University of Lublin.

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