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Calpain 10 gene polymorphisms are related, not to type 2 diabetes, but to increased serum cholesterol in Japanese
Diabetes Research and Clinical Practice 56 (2002) 147– 152 www.elsevier.com/locate/diabres
Calpain 10 gene polymorphisms are related, not to type 2 diabetes, but to increased serum cholesterol in Japanese Makoto Daimon *, Toshihide Oizumi, Tamotsu Saitoh, Wataru Kameda, Hiroshi Yamaguchi, Hiroshi Ohnuma, Masahiko Igarashi, Hideo Manaka, Takeo Kato Third Department of Internal Medicine, Yamagata Uni6ersity School of Medicine, 2 -2 -2 Iida-Nishi, Yamagata 990 -9585, Japan Received 16 March 2001; received in revised form 2 October 2001; accepted 3 December 2001
Abstract A G-to-A (UCSNP-43) polymorphism of the calpain-10 gene was significantly associated with type 2 diabetes (DM) in Mexican–American, and was postulated, together with a T-to-C (UCSNP-44) polymorphism, as a risk factor for DM. We examined the association of these genotypes with DM in Japanese. Eighty-one subjects with DM and 81 non-diabetic subjects (NGT) were recruited. The number of subjects with genotypes UCSNP-43 G/G, G/A and A/A were 76, 5 and 0, respectively, for the DM and NGT groups. The number of subjects with genotypes UCSNP-44 T/T, T/C and C/C were 66, 14 and 1 for the DM group and 64, 17 and 0 for the NGT group. There was no difference between the groups in terms of frequency of any genotype combinations. No association between the genotypes and DM was observed. We next examined the differences between the genotypes or genotype combinations in terms of the traits related to DM, obesity, hypertension and dyslipidemia. No differences were observed between the genotypes UCSNP43 G/G and G/A, between UCSNP-44 T/T and the others, or between the genotype combination UCSNP-43 G/G and UCSNP-44 T/T and the others, except that the individuals with the genotype combination had significantly increased serum cholesterol levels (212.6 934.3 vs. 198.5 9 29.9, P = 0.020). The genotype combination might be a risk factor, not for DM, obesity and hypertension, but for increased serum cholesterol. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calpain-l0; Polymorphism; Association; Cholesterol; Diabetes
1. Introduction * Corresponding author. Tel.: + 81-23-628-5316; fax: + 8123-628-5318. E-mail address: [email protected] (M. Daimon).
A genome-wide search for type 2 diabetes (DM) genes in Mexican –Americans localized a susceptibility gene, non-insulin-dependent diabetes mellitus1 (NIDDM1), to the D2S125-D2S140 region
0168-8227/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 8 2 2 7 ( 0 1 ) 0 0 3 7 2 - 2
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[1]. Recently, the calpain-10 gene (CAPN10) was positionally cloned within the NIDDM1 region [2]. A G-to-A polymorphism (UCSNP-43) in intron 3 of the gene was significantly associated with DM, with evidence for linkage in the NIDDM1 region in Mexican– American sib pairs concordant for the at-risk genotype (G/G) [2]. This polymorphism was shown in a region that affects transcription of the gene [2], which may explain how it can exert a biological function leading to DM. Furthermore, the G/G homozygotes were found to have reduced CAPN10 mRNA expression in skeletal muscle and insulin resistance [3]. A polymorphism, T-to-C (UCSNP44), located 11 bp upstream from UCSNP-43, was also shown to modulate transcriptional activity [2]. Together, the genotypes UCSNP-43 G/G and UCSNP-44 T/T seem to be risk factors. We examined here whether these genotypes are risk factors for DM and DM-related or insulin-resistance-related conditions, such as obesity, hypertension and dyslipidemia, in Japanese.
Table 1 Characteristics of the diabetic and non-diabetic subjects Trait
DM
Number 81 Sex ratio 45/36 (F/M) Age (year) 65.1 910.7 HbA1c (%) 6.7 91.1 BMI (kg/m2) 25.6 9 3.9 Systolic blood 134.8 920.6 pressure (mmHg) Total 207.8 9 31.9 cholesterol (mg/dl) Triglyceride 155.2 9 196.8 (mg/dl) HDLc 53.3916.7 (mg/dl)
NGT
P
81 46/35
0.8751
62.3 9 8.2 5.3 9 0.3 23.8 93.6 125.3916.4
0.0559 B0.0001* 0.0024* 0.0014*
210.1935.6
0.6594
102.7 952.6
0.0215*
58.9 915.7
0.0294*
Values are mean 9S.D. The statistical significance was determined by the Student’s t-test. PB0.05 is indicated by *.
2. Subjects and methods
2.1. Subject Eighty-one subjects with type 2 diabetes (DM) and 81 non-diabetic subjects (NGT) were recruited from the participants for a health care examination. Age and sex ratios were matched between the groups (age, 65.19 10.7 vs. 62.39 8.2 years; sex ratio (female/male), 45/36 vs. 46/35). The details of the clinical characteristics of the subjects were summarized in Table 1. This study was approved by the ethical committee of Yamagata University School of Medicine, and written informed consents were obtained.
2.2. Genotyping Genotyping was done by the PCR-RFLP method. The polymorphisms do not change any restriction enzyme recognition site. Therefore, we made mutagenic primers 43RM and 44FM, which have one mismatch with the original sequence and introduce NdeI and AccII restriction sites for UCSNP-43 and -44, respectively. The restriction enzyme NdeI cuts the amplified genomic DNA fragment of 212 bp derived from the A allele of UCSNP-43, while the restriction enzyme AccII cuts the amplified genomic DNA fragment of 166 bp derived from the C allele for UCSNP-44. Primers 43RM and 44FM were used together with primers 43F and 44R, respectively. The details of the primers are as follows: 43F:5%-GTGCCCAGTGAGCCCTTCCAT-3%, 43RM:5%-AGGCTTAGCCTCACCTTCAAA-3%, 44FM:5%-GCAGGGCGCTCACGCTTGCTG-3%, and 44R:5%-GCATGGCCCCCTCTCTGATTC-3%. PCR reactions were performed in 50 ml volumes using about 100 ng of genomic DNA, 2.5 U Ex-Taq (Takara, kyoto, Japan), 1 mM of each primer, 200 mM each of dATP, dCTP, dTTP and dGTP, and l × buffer supplied by the vendor. Products were amplified in the Gene Amp 2400 (Perkin–Elmer Cetus, USA) for 38 cycles under the following conditions: denaturing at 94 °C for 30 s, annealing at 57 °C for 30 s and extension at 72 °C for 30 s. The final extension step was for 5 min. The amplified genomic DNA fragments were digested for
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Table 2 Frequency of type 2 diabetes and the Calpain-10 gene polymorphisms UCSNP-43
DM NGT
UCSNP-44
UCSNP-43 and -44
G/G
G/A
A/A
T/T
T/C
C/C
G/G and T/T
The others
76 76
5 5
0 0
61 59
19 22
1 0
56 54
25 27
Data are numbers of the subjects having each genotype.
4– 6 h with either NdeI or AccII restriction enzymes (Toyobo, Tokyo, Japan) as recommended by the vendor. The restriction enzyme-digested samples were electrophoresed in a 3% agarose gel with Tris–acetate EDTA (TAE) buffer, and the bands were visualized by ethidium bromide staining under UV light.
2.3. Association study with type 2 diabetes The differences in the frequencies of the genotypes or genotype combinations between the DM and NGT groups were examined. Kai-square analysis was used to determine the statistical significance.
2.4. Relations to the clinical traits The study groups (DM and NGT) were combined to enlarge the sample size, and then the population was regrouped based on the genotypes. The traits related to type 2 diabetes, obesity, hypertension and dyslipidemia were measured, and the differences in the trait’s values between the genotypes or genotype combinations were examined. The traits examined consisted of fasting plasma glucose (FPG), 2 h plasma glucose after 75-g OGTT (2-h PG), blood HbA1c, serum insulin, height, body weight, waist, hip, waist-tohip ratio, body mass index, percent body fat, systolic and diastolic blood pressures, serum total cholesterol, serum triglyceride and serum HDL cholesterol. Percent body fat was assessed by the Tanita body-fat analyzer [4]. The statistical significance of the differences between the groups was determined by the Student’s t-test, with P B 0.05 accepted as statistically significant.
3. Results
3.1. No association with type 2 diabetes The associations of the genotypes with DM were shown in Table 2. No difference was observed between the groups in terms of frequency of the genotypes UCSNP-43 and UCSNP-44. There was also no difference between the groups in terms of frequency of any genotype combinations, including the genotype combination UCSNP-43 G/G and UCSNP-44 T/T, which was hypothesized as the at-risk genotype combination for DM. The genotype combination UCSNP-43 A/A and -44 C/C is opposite to the genotype combination UCSNP-43 G/G and UCSNP-44 T/ T. But, no individuals had the genotype combination. Therefore, we could not examine the association of the genotype combinations from the opposite side. All together, no association of the genotypes with DM was observed in our study population of Japanese.
3.2. Relation to increased serum cholesterol The relations of CAPN10 to the traits related to insulin resistance were then examined. No difference was observed between the genotype UCSNP-43 G/G and G/A, or between UCSNP-44 T/T and the others for any traits examined (data not shown). We next divided our study population based on genotype combination. The individuals with the genotype combination UCSNP-43 G/G and UCSNP-44 T/T were put into group H, and the others were put into groups L. There were 120 and 42 subjects in groups H and L, respectively. There were no differences between the
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groups in terms of age and sex ratio (age, 63.69 9.5 vs. 63.99 9.8 years; sex ratio (female/male), 67/53 vs. 24/18). The traits related to DM, such as FPG, blood HbA1c and serum insulin levels, did not significantly differ between the groups, nor did the traits related to obesity and hypertension (Table 3). Interestingly, the serum total choles-
terol levels were significantly higher in group H than in group L (Table 3). The other traits of dyslipidemia such as the serum triglyceride and HDL cholesterol levels, did not differ significantly (Table 3). The serum cholesterol levels did not differ significantly between the genotypes UCSNP-43 G/G and G/A or between UCSNP-44
Table 3 Genotypic ditributions of trait values for the CAPN10 polymorphisms Trait
Genotypes
Number Sex ratio(F/M) Age (year) Height (cm) Body weight (kg) FPG (mmole) 2 h plasma glucose (mmole) Fasting serum insulin (mU/ml) HbA1c (%) Waist circumference (cm) Hip circumference (cm) Waist to hip ratio Body mass index (kg/m2) Percent body fat (%) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Total cholesterol (mg/dl) Trigliceride (mg/dl) HDL cholesterol (mg/dl)
P
H (43 G/G and 44 T/T)
L (the others)
120 67/53 63.6 9 9.5 152.69 8.3 57.3 9 10.7 111.79 26.8 177.49 92.2 6.039 4.40 5.9791.02 81.9910.1 93.2 97.9 0.87890.066 24.594.0 28.199 8.79 130.49 18.9 76.39 10.7 212.6934.3 137.99 166.3 56.4917.4
42 24/18 63.9 9 9.8 152.4 9 6.7 58.1 9 8.2 110.6 9 29.7 180.0 9 102.0 5.52 9 3.14 6.00 9 1.25 82.2 9 7.1 93.7 9 6.5 0.878 9 0.063 25.0 9 3.3 28.86 9 8.66 129.1 9 20.0 74.3 9 13.9 198.5 929.9 103.6 9 50.8 55.2 913.1
0.884 0.879 0.893 0.629 0.828 0.879 0.498 0.881 0.863 0.700 0.972 0.470 0.668 0.715 0.343 0.020* 0.192 0.696
Values are mean 9S.D. P values compared individuals carrying genotype combination UCSNP-43 G/G and -44 T/T (H) with individuals carrying the other genotype combinations (L). The statistical significance was determined by the studemt’s t-test. PB0.05 is indicated by *. Table 4 Genotypic ditributions of values of serum lipids for UCSNP-43 and -44 Trait
Genotypes UCSNP-43
Number Total cholesterol (mg/dl) Trigliceride (mg/dl) HDL cholesterol (mg/dl)
UCSNP-44
G/G
G/A
P
T/T
T/C and C/C
P
152 210.09 34.0 130.59 150.3 56.69 16.7
10 193.19 26.5 105.3940.8 49.1 9 9.7
0.126 0.604 0.164
130 211.1 934.1 135.4 9 160.3 55.8 917.0
32 200.3 9 31.1 103.0 9 54.1 57.2 9 13.6
0.104 0.262 0.683
Values are mean 9 S.D. P values compared individuals carrying genotypes UCSNP-43 G/G or-44 T/T with individuals carrying the other genotypes. The statistical significance was determined by the Student’s t-test.
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Table 5 Genotypic ditributions of values of serum lipids for the CAPN10 polymorphisms in DM and NGT groups Trait
Number Total cholesterol (mg/dl) Trigliceride (mg/dl) HDL cholesterol (mg/dl)
DM
NGT
H
L
P
H
L
P
61 211.49 33.8 168.99 223.2 52.5 9 17.3
20 196.79 22.8 113.6 9 57.7 55.8 9 14.6
0.074 0.278 0.443
59 213.8 9 35.2 105.7 955.8 60.4 916.7
22 200.2 935.6 94.6 942.9 54.7 9 12.0
0.128 0.400 0.146
Values are mean 9 S.D. P values compared individuals carrying genotype combination UCSNP-43 G/G and -44 T/T (H) with individuals carrying the other genotype combinations (L). The statistical significance was determined by the Student’s t-test.
T/T and the others (Table 4). However, those levels were significantly increased in the individuals with both the genotypes (the genotype combination UCSNP-43 GIG and -44 T/T)(Table 3). Furthermore, the serum cholesterol levels tended to be increased in the individuals with the genotype combination UCSNP43 G/G and UCSNP-44 T/T in both the DM and NGT groups (Table 5). These results indicate that the genotype combination UCSNP-43 G/G and UCSNP-44 T/T might be a risk factor, not for DM, obesity and hypertension, but for increased serum cholesterol.
4. Discussion The heterogeneity of the polymorphisms UCSNP-43 and -44 of CAPN10 was low (6.2 and 25.3%, respectively). Therefore, these polymorphisms can be considered to be associated only when they are major risk factors influencing the conditions examined. Our results, which showed no association between these polymorphisms and DM, indicate that the polymorphisms of CAPN10 are not major risk factors for DM in Japanese, although they may be minor factors. The results mentioned above indicate that CAPN10 does not have a role in the pathogenesis of DM which is significant enough to be determined by this kind of association study. However, this fact does not exclude the possibility of the relation of CAPN10 to DM. Indeed, one of the polymorphisms (UCSNP-43) has been reported to be related to insulin resistance
[3]. Therefore, we next examined the relations of CAPN10 to the traits related to insulin resistance. The traits examined were those related to DM, obesity, hypertension and dyslipidemia. No difference in the traits was observed between any genotypes or between any genotype combinations except for serum cholesterol levels. The serum cholesterol levels were increased in the individuals with the genotype combination UCSNP-43 G/G and -44 T/T. These results indicate that the genotype combination UCSNP-43 G/G and UCSNP-44 T/T might be a risk factor, not for DM, obesity and hypertension, but for increased serum cholesterol. The individuals with the genotype combination UCSNP-43 G/G and UCSNP-44 T/T tended not to be diabetic, obese or hypertensive, but they had higher serum cholesterol levels than the others. This finding indicates that CAPN10 may play an important role in Cholesterol metabolism independently of the pathogenesis of DM, obesity and hypertension. This idea was further strengthened by the fact that the serum cholesterol levels tended to be increased in the individuals with the genotype combination UCSNP-43 G/G and UCSNP-44 T/T in both the DM and NGT groups. Namely, diabetes did not influence the tendency for serum cholesterol levels to increase in the individuals with the genotype combination. The number of the subjects in the DM and NGT groups was not a large, so the difference in the serum cholesterol levels seemed to be determined as significant when the subjects in the two groups were combined.
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M. Daimon et al. / Diabetes Research and Clinical Practice 56 (2002) 147–152
Our study was essentially designed to evaluate the significance of CAPN10 in DM, not in hypercholesteremia. Therefore, the study subjects may not to be suitable to conclude the relation between CAPN10 and increased serum cholesterol levels. Blood glucose levels also might influence the results, since half of the subjects had DM. But, we believe that this possibility is not so high, since the traits related to blood glucose levels (such as FPG, HbA1c and fasting serum insulin) were not different between the groups (H and L). Further examinations with hypercholesteremic population and more subjects are needed before the conclusion. The polymorphisms of CAPN10 seems to be risk factors for increased serum cholesterol levels, which in turn indicates that CAPN10 plays an important role in cholesterol metabolism. Therefore, we strongly recommend that the effort to
clarify the role of CAPN10 should be focus not only on the pathogenesis of type 2 diabetes but also on cholesterol metabolism.
References [1] C.L. Hanis, E. Boerwinkle, R. Chakraborty, et al., A genome-wide search for human non-insulin-dependent (type 2) diabetes genes reveals a major susceptibility locus on chromosome 2, Nat. Genet. 13 (1996) 161 – 166. [2] Y. Horikawa, N. Oda, N.J. Cox, et al., Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus, Nat. Genet. 26 (2000) 163 – 175. [3] L.J. Baier, P.A. Permana, X. Yang, et al., A calpain-10 gene polymorphism is associated with reduced muscle mRNA levels and insulin resistance, J. Clin. Invest. 106 (2000) R69 – R73. [4] S.A. Jebb, T.J. Cole, D. Doman, P.R. Murgatroyd, A.M. Prentice, Evaluation of the novel Tanita body-fat analyser to measure body composition by comparison with a fourcompartment model, Br. J. Nutr. 83 (2000) 115 – 122.
Calpain 10 gene polymorphisms are related, not to type 2 diabetes, but to increased serum cholesterol in Japanese Makoto Daimon *, Toshihide Oizumi, Tamotsu Saitoh, Wataru Kameda, Hiroshi Yamaguchi, Hiroshi Ohnuma, Masahiko Igarashi, Hideo Manaka, Takeo Kato Third Department of Internal Medicine, Yamagata Uni6ersity School of Medicine, 2 -2 -2 Iida-Nishi, Yamagata 990 -9585, Japan Received 16 March 2001; received in revised form 2 October 2001; accepted 3 December 2001
Abstract A G-to-A (UCSNP-43) polymorphism of the calpain-10 gene was significantly associated with type 2 diabetes (DM) in Mexican–American, and was postulated, together with a T-to-C (UCSNP-44) polymorphism, as a risk factor for DM. We examined the association of these genotypes with DM in Japanese. Eighty-one subjects with DM and 81 non-diabetic subjects (NGT) were recruited. The number of subjects with genotypes UCSNP-43 G/G, G/A and A/A were 76, 5 and 0, respectively, for the DM and NGT groups. The number of subjects with genotypes UCSNP-44 T/T, T/C and C/C were 66, 14 and 1 for the DM group and 64, 17 and 0 for the NGT group. There was no difference between the groups in terms of frequency of any genotype combinations. No association between the genotypes and DM was observed. We next examined the differences between the genotypes or genotype combinations in terms of the traits related to DM, obesity, hypertension and dyslipidemia. No differences were observed between the genotypes UCSNP43 G/G and G/A, between UCSNP-44 T/T and the others, or between the genotype combination UCSNP-43 G/G and UCSNP-44 T/T and the others, except that the individuals with the genotype combination had significantly increased serum cholesterol levels (212.6 934.3 vs. 198.5 9 29.9, P = 0.020). The genotype combination might be a risk factor, not for DM, obesity and hypertension, but for increased serum cholesterol. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calpain-l0; Polymorphism; Association; Cholesterol; Diabetes
1. Introduction * Corresponding author. Tel.: + 81-23-628-5316; fax: + 8123-628-5318. E-mail address: [email protected] (M. Daimon).
A genome-wide search for type 2 diabetes (DM) genes in Mexican –Americans localized a susceptibility gene, non-insulin-dependent diabetes mellitus1 (NIDDM1), to the D2S125-D2S140 region
0168-8227/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 8 2 2 7 ( 0 1 ) 0 0 3 7 2 - 2
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[1]. Recently, the calpain-10 gene (CAPN10) was positionally cloned within the NIDDM1 region [2]. A G-to-A polymorphism (UCSNP-43) in intron 3 of the gene was significantly associated with DM, with evidence for linkage in the NIDDM1 region in Mexican– American sib pairs concordant for the at-risk genotype (G/G) [2]. This polymorphism was shown in a region that affects transcription of the gene [2], which may explain how it can exert a biological function leading to DM. Furthermore, the G/G homozygotes were found to have reduced CAPN10 mRNA expression in skeletal muscle and insulin resistance [3]. A polymorphism, T-to-C (UCSNP44), located 11 bp upstream from UCSNP-43, was also shown to modulate transcriptional activity [2]. Together, the genotypes UCSNP-43 G/G and UCSNP-44 T/T seem to be risk factors. We examined here whether these genotypes are risk factors for DM and DM-related or insulin-resistance-related conditions, such as obesity, hypertension and dyslipidemia, in Japanese.
Table 1 Characteristics of the diabetic and non-diabetic subjects Trait
DM
Number 81 Sex ratio 45/36 (F/M) Age (year) 65.1 910.7 HbA1c (%) 6.7 91.1 BMI (kg/m2) 25.6 9 3.9 Systolic blood 134.8 920.6 pressure (mmHg) Total 207.8 9 31.9 cholesterol (mg/dl) Triglyceride 155.2 9 196.8 (mg/dl) HDLc 53.3916.7 (mg/dl)
NGT
P
81 46/35
0.8751
62.3 9 8.2 5.3 9 0.3 23.8 93.6 125.3916.4
0.0559 B0.0001* 0.0024* 0.0014*
210.1935.6
0.6594
102.7 952.6
0.0215*
58.9 915.7
0.0294*
Values are mean 9S.D. The statistical significance was determined by the Student’s t-test. PB0.05 is indicated by *.
2. Subjects and methods
2.1. Subject Eighty-one subjects with type 2 diabetes (DM) and 81 non-diabetic subjects (NGT) were recruited from the participants for a health care examination. Age and sex ratios were matched between the groups (age, 65.19 10.7 vs. 62.39 8.2 years; sex ratio (female/male), 45/36 vs. 46/35). The details of the clinical characteristics of the subjects were summarized in Table 1. This study was approved by the ethical committee of Yamagata University School of Medicine, and written informed consents were obtained.
2.2. Genotyping Genotyping was done by the PCR-RFLP method. The polymorphisms do not change any restriction enzyme recognition site. Therefore, we made mutagenic primers 43RM and 44FM, which have one mismatch with the original sequence and introduce NdeI and AccII restriction sites for UCSNP-43 and -44, respectively. The restriction enzyme NdeI cuts the amplified genomic DNA fragment of 212 bp derived from the A allele of UCSNP-43, while the restriction enzyme AccII cuts the amplified genomic DNA fragment of 166 bp derived from the C allele for UCSNP-44. Primers 43RM and 44FM were used together with primers 43F and 44R, respectively. The details of the primers are as follows: 43F:5%-GTGCCCAGTGAGCCCTTCCAT-3%, 43RM:5%-AGGCTTAGCCTCACCTTCAAA-3%, 44FM:5%-GCAGGGCGCTCACGCTTGCTG-3%, and 44R:5%-GCATGGCCCCCTCTCTGATTC-3%. PCR reactions were performed in 50 ml volumes using about 100 ng of genomic DNA, 2.5 U Ex-Taq (Takara, kyoto, Japan), 1 mM of each primer, 200 mM each of dATP, dCTP, dTTP and dGTP, and l × buffer supplied by the vendor. Products were amplified in the Gene Amp 2400 (Perkin–Elmer Cetus, USA) for 38 cycles under the following conditions: denaturing at 94 °C for 30 s, annealing at 57 °C for 30 s and extension at 72 °C for 30 s. The final extension step was for 5 min. The amplified genomic DNA fragments were digested for
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Table 2 Frequency of type 2 diabetes and the Calpain-10 gene polymorphisms UCSNP-43
DM NGT
UCSNP-44
UCSNP-43 and -44
G/G
G/A
A/A
T/T
T/C
C/C
G/G and T/T
The others
76 76
5 5
0 0
61 59
19 22
1 0
56 54
25 27
Data are numbers of the subjects having each genotype.
4– 6 h with either NdeI or AccII restriction enzymes (Toyobo, Tokyo, Japan) as recommended by the vendor. The restriction enzyme-digested samples were electrophoresed in a 3% agarose gel with Tris–acetate EDTA (TAE) buffer, and the bands were visualized by ethidium bromide staining under UV light.
2.3. Association study with type 2 diabetes The differences in the frequencies of the genotypes or genotype combinations between the DM and NGT groups were examined. Kai-square analysis was used to determine the statistical significance.
2.4. Relations to the clinical traits The study groups (DM and NGT) were combined to enlarge the sample size, and then the population was regrouped based on the genotypes. The traits related to type 2 diabetes, obesity, hypertension and dyslipidemia were measured, and the differences in the trait’s values between the genotypes or genotype combinations were examined. The traits examined consisted of fasting plasma glucose (FPG), 2 h plasma glucose after 75-g OGTT (2-h PG), blood HbA1c, serum insulin, height, body weight, waist, hip, waist-tohip ratio, body mass index, percent body fat, systolic and diastolic blood pressures, serum total cholesterol, serum triglyceride and serum HDL cholesterol. Percent body fat was assessed by the Tanita body-fat analyzer [4]. The statistical significance of the differences between the groups was determined by the Student’s t-test, with P B 0.05 accepted as statistically significant.
3. Results
3.1. No association with type 2 diabetes The associations of the genotypes with DM were shown in Table 2. No difference was observed between the groups in terms of frequency of the genotypes UCSNP-43 and UCSNP-44. There was also no difference between the groups in terms of frequency of any genotype combinations, including the genotype combination UCSNP-43 G/G and UCSNP-44 T/T, which was hypothesized as the at-risk genotype combination for DM. The genotype combination UCSNP-43 A/A and -44 C/C is opposite to the genotype combination UCSNP-43 G/G and UCSNP-44 T/ T. But, no individuals had the genotype combination. Therefore, we could not examine the association of the genotype combinations from the opposite side. All together, no association of the genotypes with DM was observed in our study population of Japanese.
3.2. Relation to increased serum cholesterol The relations of CAPN10 to the traits related to insulin resistance were then examined. No difference was observed between the genotype UCSNP-43 G/G and G/A, or between UCSNP-44 T/T and the others for any traits examined (data not shown). We next divided our study population based on genotype combination. The individuals with the genotype combination UCSNP-43 G/G and UCSNP-44 T/T were put into group H, and the others were put into groups L. There were 120 and 42 subjects in groups H and L, respectively. There were no differences between the
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M. Daimon et al. / Diabetes Research and Clinical Practice 56 (2002) 147–152
groups in terms of age and sex ratio (age, 63.69 9.5 vs. 63.99 9.8 years; sex ratio (female/male), 67/53 vs. 24/18). The traits related to DM, such as FPG, blood HbA1c and serum insulin levels, did not significantly differ between the groups, nor did the traits related to obesity and hypertension (Table 3). Interestingly, the serum total choles-
terol levels were significantly higher in group H than in group L (Table 3). The other traits of dyslipidemia such as the serum triglyceride and HDL cholesterol levels, did not differ significantly (Table 3). The serum cholesterol levels did not differ significantly between the genotypes UCSNP-43 G/G and G/A or between UCSNP-44
Table 3 Genotypic ditributions of trait values for the CAPN10 polymorphisms Trait
Genotypes
Number Sex ratio(F/M) Age (year) Height (cm) Body weight (kg) FPG (mmole) 2 h plasma glucose (mmole) Fasting serum insulin (mU/ml) HbA1c (%) Waist circumference (cm) Hip circumference (cm) Waist to hip ratio Body mass index (kg/m2) Percent body fat (%) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Total cholesterol (mg/dl) Trigliceride (mg/dl) HDL cholesterol (mg/dl)
P
H (43 G/G and 44 T/T)
L (the others)
120 67/53 63.6 9 9.5 152.69 8.3 57.3 9 10.7 111.79 26.8 177.49 92.2 6.039 4.40 5.9791.02 81.9910.1 93.2 97.9 0.87890.066 24.594.0 28.199 8.79 130.49 18.9 76.39 10.7 212.6934.3 137.99 166.3 56.4917.4
42 24/18 63.9 9 9.8 152.4 9 6.7 58.1 9 8.2 110.6 9 29.7 180.0 9 102.0 5.52 9 3.14 6.00 9 1.25 82.2 9 7.1 93.7 9 6.5 0.878 9 0.063 25.0 9 3.3 28.86 9 8.66 129.1 9 20.0 74.3 9 13.9 198.5 929.9 103.6 9 50.8 55.2 913.1
0.884 0.879 0.893 0.629 0.828 0.879 0.498 0.881 0.863 0.700 0.972 0.470 0.668 0.715 0.343 0.020* 0.192 0.696
Values are mean 9S.D. P values compared individuals carrying genotype combination UCSNP-43 G/G and -44 T/T (H) with individuals carrying the other genotype combinations (L). The statistical significance was determined by the studemt’s t-test. PB0.05 is indicated by *. Table 4 Genotypic ditributions of values of serum lipids for UCSNP-43 and -44 Trait
Genotypes UCSNP-43
Number Total cholesterol (mg/dl) Trigliceride (mg/dl) HDL cholesterol (mg/dl)
UCSNP-44
G/G
G/A
P
T/T
T/C and C/C
P
152 210.09 34.0 130.59 150.3 56.69 16.7
10 193.19 26.5 105.3940.8 49.1 9 9.7
0.126 0.604 0.164
130 211.1 934.1 135.4 9 160.3 55.8 917.0
32 200.3 9 31.1 103.0 9 54.1 57.2 9 13.6
0.104 0.262 0.683
Values are mean 9 S.D. P values compared individuals carrying genotypes UCSNP-43 G/G or-44 T/T with individuals carrying the other genotypes. The statistical significance was determined by the Student’s t-test.
M. Daimon et al. / Diabetes Research and Clinical Practice 56 (2002) 147–152
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Table 5 Genotypic ditributions of values of serum lipids for the CAPN10 polymorphisms in DM and NGT groups Trait
Number Total cholesterol (mg/dl) Trigliceride (mg/dl) HDL cholesterol (mg/dl)
DM
NGT
H
L
P
H
L
P
61 211.49 33.8 168.99 223.2 52.5 9 17.3
20 196.79 22.8 113.6 9 57.7 55.8 9 14.6
0.074 0.278 0.443
59 213.8 9 35.2 105.7 955.8 60.4 916.7
22 200.2 935.6 94.6 942.9 54.7 9 12.0
0.128 0.400 0.146
Values are mean 9 S.D. P values compared individuals carrying genotype combination UCSNP-43 G/G and -44 T/T (H) with individuals carrying the other genotype combinations (L). The statistical significance was determined by the Student’s t-test.
T/T and the others (Table 4). However, those levels were significantly increased in the individuals with both the genotypes (the genotype combination UCSNP-43 GIG and -44 T/T)(Table 3). Furthermore, the serum cholesterol levels tended to be increased in the individuals with the genotype combination UCSNP43 G/G and UCSNP-44 T/T in both the DM and NGT groups (Table 5). These results indicate that the genotype combination UCSNP-43 G/G and UCSNP-44 T/T might be a risk factor, not for DM, obesity and hypertension, but for increased serum cholesterol.
4. Discussion The heterogeneity of the polymorphisms UCSNP-43 and -44 of CAPN10 was low (6.2 and 25.3%, respectively). Therefore, these polymorphisms can be considered to be associated only when they are major risk factors influencing the conditions examined. Our results, which showed no association between these polymorphisms and DM, indicate that the polymorphisms of CAPN10 are not major risk factors for DM in Japanese, although they may be minor factors. The results mentioned above indicate that CAPN10 does not have a role in the pathogenesis of DM which is significant enough to be determined by this kind of association study. However, this fact does not exclude the possibility of the relation of CAPN10 to DM. Indeed, one of the polymorphisms (UCSNP-43) has been reported to be related to insulin resistance
[3]. Therefore, we next examined the relations of CAPN10 to the traits related to insulin resistance. The traits examined were those related to DM, obesity, hypertension and dyslipidemia. No difference in the traits was observed between any genotypes or between any genotype combinations except for serum cholesterol levels. The serum cholesterol levels were increased in the individuals with the genotype combination UCSNP-43 G/G and -44 T/T. These results indicate that the genotype combination UCSNP-43 G/G and UCSNP-44 T/T might be a risk factor, not for DM, obesity and hypertension, but for increased serum cholesterol. The individuals with the genotype combination UCSNP-43 G/G and UCSNP-44 T/T tended not to be diabetic, obese or hypertensive, but they had higher serum cholesterol levels than the others. This finding indicates that CAPN10 may play an important role in Cholesterol metabolism independently of the pathogenesis of DM, obesity and hypertension. This idea was further strengthened by the fact that the serum cholesterol levels tended to be increased in the individuals with the genotype combination UCSNP-43 G/G and UCSNP-44 T/T in both the DM and NGT groups. Namely, diabetes did not influence the tendency for serum cholesterol levels to increase in the individuals with the genotype combination. The number of the subjects in the DM and NGT groups was not a large, so the difference in the serum cholesterol levels seemed to be determined as significant when the subjects in the two groups were combined.
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Our study was essentially designed to evaluate the significance of CAPN10 in DM, not in hypercholesteremia. Therefore, the study subjects may not to be suitable to conclude the relation between CAPN10 and increased serum cholesterol levels. Blood glucose levels also might influence the results, since half of the subjects had DM. But, we believe that this possibility is not so high, since the traits related to blood glucose levels (such as FPG, HbA1c and fasting serum insulin) were not different between the groups (H and L). Further examinations with hypercholesteremic population and more subjects are needed before the conclusion. The polymorphisms of CAPN10 seems to be risk factors for increased serum cholesterol levels, which in turn indicates that CAPN10 plays an important role in cholesterol metabolism. Therefore, we strongly recommend that the effort to
clarify the role of CAPN10 should be focus not only on the pathogenesis of type 2 diabetes but also on cholesterol metabolism.
References [1] C.L. Hanis, E. Boerwinkle, R. Chakraborty, et al., A genome-wide search for human non-insulin-dependent (type 2) diabetes genes reveals a major susceptibility locus on chromosome 2, Nat. Genet. 13 (1996) 161 – 166. [2] Y. Horikawa, N. Oda, N.J. Cox, et al., Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus, Nat. Genet. 26 (2000) 163 – 175. [3] L.J. Baier, P.A. Permana, X. Yang, et al., A calpain-10 gene polymorphism is associated with reduced muscle mRNA levels and insulin resistance, J. Clin. Invest. 106 (2000) R69 – R73. [4] S.A. Jebb, T.J. Cole, D. Doman, P.R. Murgatroyd, A.M. Prentice, Evaluation of the novel Tanita body-fat analyser to measure body composition by comparison with a fourcompartment model, Br. J. Nutr. 83 (2000) 115 – 122.