- Email: [email protected]
Association of PTPN22 C1858T and CTLA-4 A49G polymorphisms with type 1 diabetes in Croatians
diabetes research and clinical practice 86 (2009) e54–e57
Contents lists available at ScienceDirect
Diabetes Research and Clinical Practice journal homepage: www.elsevier.com/locate/diabres
Brief report
Association of PTPN22 C1858T and CTLA-4 A49G polymorphisms with type 1 diabetes in Croatians Marina Korolija a, Ivana Pavlic´ Renar b,c, Mirko Hadzˇija a, Edita Pape Medvidovic´ c, Pajica Pavkovic´ c, Mladen Jokic´ a, Marijana Popovic´ Hadzˇija a,* a
Ruper Bosˇkovic´ Institute, Division of Molecular Medicine, Zagreb, Croatia University Hospital Center, Department of Endocrinology, Zagreb, Croatia c Vuk Vrhovac University Clinic, Zagreb, Croatia b
article info
abstract
Article history:
In this case–control study the association between the PTPN22 1858T and CTLA-4 49G gene
Received 20 June 2009
variants and T1D in Croatian population was examined. We found that distribution of
Accepted 10 September 2009
PTPN22 C1858T and CTLA-4 A49G genotypes between T1D patient (n = 102) and control
Published on line 7 October 2009
(n = 193) groups differ significantly ( p < 0.0001 and p = 0.012, respectively). Moreover, although the risk alleles of both SNPs are distributed more frequently in patients, the
Keywords:
significant difference is observed only for PTPN22 1858T allele ( p < 0.0001). This is therefore
PTPN22
the first evidence that analyzed gene variants contribute to T1D pathogenesis in Croatian
CTLA-4
population.
Polymorphism
# 2009 Elsevier Ireland Ltd. All rights reserved.
Type 1 diabetes
The most relevant non-HLA genes identified as susceptible for type 1 diabetes (T1D) are those connected with the T-cellmediated immune response. T-cells activity level and their effectors functions are determined by intracellular signaling pathways and related genes, like PTPN22 and CTLA-4, both of which prevent spontaneous activation of auto-reactive cells and development of autoimmunity [1,2]. However, polymorphic variants of these genes, due to a single nucleotide polymorphism (SNP), might augment autoimmunity, thus expanding the repertoire of genetic components that could influence T1D development. Association of PTPN22 C1858T or CTLA-4 A49G gene variants with T1D has been examined in more than one ethnic group [3– 7]. To the best of our knowledge, such reports for the Croatian population do not exist. The present study was undertaken to
ascertain the possible risk of PTPN22 C1858T and CTLA-4 A49G polymorphisms on T1D in Croatian patients. One hundred and two Croatian patients with T1D (38 males and 64 females; ratio 1:1.7) were enrolled in this study which included diabetic patients treated and categorized as Type I at the University Clinic Vuk Vrhovac, Zagreb, during 2006/2007. The clinical parameters of patients, shown as median (interquartile range), were 37 (31.5–44) for patients’ age, 23.5 (17–29.5) for age at the disease onset and 11.5 (5–17) for diabetes duration. Glycosylated hemoglobin was measured at the time of the study for the assessment of glycaemic control, and the mean value was 7.6 1.3%. All the patients required insulin for glycaemic control (the average dose was 50.0 22.3 units). The study was approved by the Ethics Committee of the University Clinic.
* Corresponding author at: Division of Molecular Medicine, Ruper Bosˇkovic´ Institute, Laboratory for Molecular Endocrinology and Transplantation, Bijenicˇka 54, 10 000 Zagreb, Croatia. Tel.: +385 1 4561064; fax: +385 1 4561010. E-mail address: [email protected] (M.P. Hadzˇija). 0168-8227/$ – see front matter # 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2009.09.012
e55
diabetes research and clinical practice 86 (2009) e54–e57
The control group consisted of 193 unrelated healthy subjects (103 males and 90 females; ratio 1:0.9). Controls as well as patients were Caucasian of Croatian nationality. Control subjects were screened by a questionnaire to ensure the absence of any diagnostic evidence of autoimmune diseases or family history of T1D. Median age for controls was 70 (interquartile range 58–78). Informed consent was obtained from patients and controls prior to blood sampling. Peripheral blood samples were collected in 3 mL volumes by venous puncture and genomic DNA was extracted by a salting out procedure, as described previously [8]. Two appropriate fragments of PTPN22 and CTLA-4 genes were amplified in PCR using two sets of primers:
PTPN22 F 50 -ACTGATAATGTTGCTTCAACGG-30 ; PTPN22 R 50 -TCACCAGCTTCCTCAACCAC-30 ; CTLA-4 F 50 -GCTCTACTTCCTGAAGACCT-30 ; CTLA-4 R 50 -AGTCTCACTCACCTTTGCAG-30 . PCR mixture contained 100 ng DNA, 10 pmoles of each primer, 50 mM of dNTPs, 1 PCR buffer with MgCl2, 1 U Hot Start Taq DNA polymerase (Eppendorf, Germany) and QH2O up to 25 mL. PCR reaction was carried out in the Mastercycler personal (Eppendorf) machine. Annealing temperatures, optimized in the pilot experiment, were 60 8C (for PTPN22) and 58 8C (for CTLA-4). Samples were initially denatured for 2 min at 94 8C followed by 30 cycles of 30 s at 94 8C, 30 s at 60 8C or 58 8C and 90 s at 70 8C. The product was checked on 2% agarose gel containing ethidium bromide. Amplified products of PTPN22 were digested with RsaI (Fermentas, Germany) overnight at 37 8C. The C allele creates restriction site producing two fragments (172 bp and 46 bp), while T allele does not (218 bp). Amplified products of CTLA-4 were digested with BbvI (Fermentas, Germany) overnight at 65 8C. The A allele does not create restriction site (162 bp), while G allele creates restriction site producing two fragments (88 bp and 74 bp). The SNPs in both genes were genotyped by electrophoresis on 12% polyacrilamide gel (PAGE) containing 1 Tris–borate– EDTA (TBE) buffer. DNA fragments were stained with SYBR Gold dye (Crucell, Lieden, Netherlands) and visualized under UV light. Observed genotypes at PTPN22 and CTLA-4 loci in patient and control groups are tested for Hardy–Weinberg equilibrium
using HW calculator ( p-value less than 0.05 was considered as not consistent with HWE). The differences in the frequency of various genotypes and alleles between patients and healthy controls were evaluated using x2 test. p-Value less than 0.05 (two-tailed test) was considered as significant. Odds ratio (OR) at 95% confidence intervals (CI) was also determined to describe the strength of association. As the test of homogeneity of variances was satisfied, the one-way analysis of variance (ANOVA) was applied to compare means of age at T1D diagnosis among individuals with different PTPN22 or CTLA-4 genotypes. LSD test was further used to detect if the means between genotype groups are different from each other. Statistical analysis was performed using SPSS13 software. The frequencies of the genotypes assessed at PTPN22 C1858T and CTLA-4 A49G polymorphic positions in both patient and control groups were in Hardy–Weinberg equilibrium (at PTPN22 0.09 vs. 0.52; at CTLA-4 0.07 vs. 0.6). A significant main effect was observed for PTPN22 (F(2,286) = 15.469, p < 0.0001, effect size f = 0.329) and CTLA-4 (F(2,286) = 4.147, p = 0.017, effect size f = 0.169). Distribution of PTPN22 C1858T genotypes between patients with T1D and healthy controls differ significantly (Table 1). The frequency of risk allele T (tryptophan containing allele) was also significantly higher in the patient than the control group (Table 1), and the importance of T allele for diabetes risk was indicated by high odds ratio (3.06). These results indicate connection between PTPN22 1858T allele and T1D in Croatian population, like it has also been reported for North American, Sardinian [3], Finnish [9], German [10], Danish [11] and Estonian [12] populations. The distribution of CTLA-4 genotypes amongst Croatian T1D patients and healthy controls differ significantly (Table 2), as has also been reported in Japanese [13], Estonian [14], and Iranian [7] case–control studies. It has to be pointed out that in our population CTLA-4 G/G homozygous individuals are overrepresented in patient group, compared to controls, with 2.7 OR at 95% CI in patients (Table 2). It is in agreement with the finding that in G/G homozygous individuals risk for T1D development increases more than twofold, as is reported in meta-analysis of 12 case–control studies that have examined the association of T1D with CTLA-4 49G polymorphism in European populations [15]. In our study, the frequency of the risk allele G was 6.8% higher in T1D than in healthy subjects, but it did not reach significant difference (Table 2). The higher frequency of G allele in patients than in control subjects is also
Table 1 – Distribution of the PTPN22 C1858T polymorphism in patients with T1D and healthy controls. Patient n (%)
Control n (%)
x2
p
Genotypes C/C C/T T/T
47 (46.1) 51 (50) 4 (3.9)
149 (77.1) 43 (22.4) 1 (0.5)
30.1
<0.0001*
1.0 (Ref)a 3.73 (2.21–6.29) –
Alleles C T
145 (71.1) 59 (28.9)
341 (88.3) 45 (11.7)
27.1
<0.0001*
1.0 (Ref)a 3.06 (1.98–4.7)
a *
The most frequent genotype or allele was used as a reference for OR calculations. Significantly different.
OR at 95% CI in patients
e56
diabetes research and clinical practice 86 (2009) e54–e57
Table 2 – Distribution of the CTLA-4 A49G polymorphism in patients with T1D and healthy controls.
Genotypes A/A A/G G/G Alleles A G a *
Patient n (%)
Control n (%)
x2
p
48 (47.0) 36 (35.3) 18 (17.7)
96 (49.7) 84 (43.6) 13 (6.7)
8.7
0.012*
1.0 (Ref)a 0.86 (0.51–1.44) 2.7 (1.25–6.12)
132 (64.7) 72 (35.3)
276 (71.5) 110 (28.5)
2.89
0.089
1.0 (Ref)a 1.36 (0.95–1.96)
OR at 95% CI in patients
The most frequent genotype or allele was used as a reference for OR calculations. Significantly different.
Table 3 – Dependence of age at T1D onset on the presence of risk alleles at the PTPN22 or CTLA-4 locus.
Acknowledgements
Genotypes
The authors thank Ms. Marina Marsˇ for the excellent technical assistance. This work was supported by the Croatian Ministry for Science, Education and Sports, Grant Nos. 098-09824642460 and 098-0982464-2508.
N
Mean age at diagnosis (years)
p
PTPN22 CC CT+TT
47 55
24.1 22.4
0.304
CTLA-4 AA AG+GG
48 54
24.8 21.4
0.046*
references
T—risk allele of PTPN22 and G—risk allele of CTLA-4. Significantly different compared to non-risk genotype.
*
a feature of most European populations (studies summarized in 15) with range of frequency from 2.9% to even 54.3%. The accumulated data from numerous studies demonstrate that CTLA-4 A49G SNP is genetic determinant of T1D, although some discrepancy between findings exists [6,16]. It may be attributed to a variable degree of association between an autoimmunity predisposing allele of the same gene and the disease among various populations. It has previously been shown that genetic susceptibility factors can affect the age at T1D onset, like a well known example of the high-risk DR3/DR4 genotype that is much more prevalent in individuals who have T1D onset at an early age [17]. In this study, we tested if risk genotypes of PTPN22 or CTLA-4 genes have an effect on age at T1D onset. We found that patients with CTLA-4 risk genotypes get diabetes earlier compared to patients with non-risk genotypes (F(1,101) = 4.08, p < 0.05) (Table 3). Although displaying tendency, results for PTPN22 risk genotypes did not reach statistical significance (F(1,101) = 1.067, p > 0.05) (Table 3). In conclusion, our data strongly suggest that polymorphisms PTPN22 C1858T and, to a lower extent CTLA-4 A49G, correlate with T1D incidence in the Croatian population. Preliminary results also indicate an effect of these polymorphisms on age at onset. However, more comprehensive study is needed to evaluate the impact of analyzed SNPs in T1D pathogenesis and to test the idea that combined data obtained from genotyping more than one susceptible gene variant allows identification of people at high-risk of developing T1D.
Conflict of interest There are no conflicts of interest.
[1] N. Bottini, T. Vang, F. Cucca, T. Mustelin, Role of PTPN22 in type 1 diabetes and other autoimmune disease, Semin. Immunol. 18 (2006) 207–213. [2] B.T. Fife, J.A. Bluestone, Control of peripheral T-cell tolerance and autoimmunity via the CTLA-4 and PD-1 pathways, Immunol. Rev. 224 (2008) 166–182. [3] N. Bottini, L. Musumeci, A. Alonso, S. Rahmouni, K. Nika, M. Rostamkhani, et al., A functional variant of lymphoid tyrosine phosphates is associated with type 1 diabetes, Nat. Genet. 36 (2004) 337–338. [4] W. Zheng, J.X. She, Genetic association between a lymphoid tyrosine phosphatase (PTPN22) and type 1 diabetes, Diabetes 54 (2005) 906–908. [5] D. Smyth, J.D. Cooper, J.E. Collins, J.M. Heward, J.A. Franklyn, J.M.M. Howson, et al., Replication of an association between the lymphoid tyrosine phosphates locus (LYP/PTPN22) with type 1 diabetes, and evidence for its role as a general autoimmunity locus, Diabetes 53 (2004) 3020–3023. [6] V. Baniasadi, N. Narain, R. Goswami, S.N. Das, Promoter region-318C/T and -1661 A/G CTLA-4 single nucleotide polymorphisms and type 1 diabetes in North Indians, Tissue Antigens 67 (2006) 383–389. [7] Z. Mojtahedi, G.R. Omrani, M. Doroudchi, A. Ghaderi, CTLA4+49 A/G polymorphism is associated with predisposition to type 1 diabetes in Iranians, Diab. Res. Clin. Pract. 68 (2005) 111–116. [8] S.A. Miller, D.D. Dykes, H.F. Polesky, A simple salting out procedure for extracting DNA from human nucleated cells, Nucleic Acids Res. 16 (1988) 1215. [9] R. Hermann, K. Lipponen, M. Kiviniemi, T. Kakko, R. Veijola, O. Simell, et al., Lymphoid tyrosine phosphates (LYP/ PTPN22) Arg620Trp variant regulates insulin autoimmunity and progression to type 1 diabetes, Diabetologia 49 (2006) 1198–1208. [10] H. Kahles, E. Ramos-Lopez, B. Lange, O. Zwermann, M. Reincke, K. Badenhoop, Sex-specific association of PTPN22 1858T with type 1 diabetes but not with Hashimoto’s thyroiditis or Addison’s disease in the German population, Eur. J. Endocrinol. 153 (2005) 895–899.
diabetes research and clinical practice 86 (2009) e54–e57
[11] C. Nielsen, D. Hansen, S. Husby, S.T. Lillevang, Sex-specific association of the human PTPN22 1858T-allele with type 1 diabetes, Int. J. Immunogenet. 34 (2007) 469–473. [12] K. Douroudis, E. Prans, K. Haller, V. Nemvalts, T. Rajasalu, V. Tillmann, et al., Protein tyrosine phospatase nonreceptor type 22 gene variants at position 1858 are associated with type 1 and type 2 diabetes in Estonian population, Tissue Antigens 72 (2008) 425–430. [13] A. Ide, E. Kawasaki, N. Abiru, S. Fuyan, M. Kobayashi, T. Fukushima, et al., Association between IL-18 gene promoter polymorphisms and CTLA-4 gene 49A/G polymorphism in Japanese patients with type 1 diabetes, J. Autoimmun. 22 (2004) 73–78. [14] K. Haller, K. Kisand, H. Pisarev, L. Salur, T. Laisk, V. Nemvalts, et al., Insulin gene VNTR, CTLA-4+49A/G and
e57
HLA-DQB1 alleles distinguish latent autoimmune diabetes in adults from type 1 diabetes and from type 2 diabetes group, Tissue Antigens 69 (2007) 121–127. [15] K.F. Kavvoura, P.A. Ioannidis, CTLA-4 gene polymorphisms and susceptibility to type 1 diabetes mellitus: a huge review and meta-analysis, Am. J. Epidemiol. 162 (2005) 3–16. [16] S. Genc, K. Genc, O. Sercan, S. Kizildag, Z. Gulay, T. Atay, et al., Analysis of cytotoxic T lymphocyte antigen-4 (CTLA4) exon 1 polymorphisms in patients with type 1 diabetes mellitus in a Turkish population, J. Pediatr. Endocr. Met. 17 (2004) 731–735. [17] S. Caillant-Zucman, H.J. Garchon, J. Timsit, C. Assan, I. Boitard, P. Dijalah-Saiah, et al., Age-dependent HLA genetic heterogeneity of type 1 insulin-dependent diabetes mellitus, J. Clin. Invest. 90 (1992) 2242–2250.
Contents lists available at ScienceDirect
Diabetes Research and Clinical Practice journal homepage: www.elsevier.com/locate/diabres
Brief report
Association of PTPN22 C1858T and CTLA-4 A49G polymorphisms with type 1 diabetes in Croatians Marina Korolija a, Ivana Pavlic´ Renar b,c, Mirko Hadzˇija a, Edita Pape Medvidovic´ c, Pajica Pavkovic´ c, Mladen Jokic´ a, Marijana Popovic´ Hadzˇija a,* a
Ruper Bosˇkovic´ Institute, Division of Molecular Medicine, Zagreb, Croatia University Hospital Center, Department of Endocrinology, Zagreb, Croatia c Vuk Vrhovac University Clinic, Zagreb, Croatia b
article info
abstract
Article history:
In this case–control study the association between the PTPN22 1858T and CTLA-4 49G gene
Received 20 June 2009
variants and T1D in Croatian population was examined. We found that distribution of
Accepted 10 September 2009
PTPN22 C1858T and CTLA-4 A49G genotypes between T1D patient (n = 102) and control
Published on line 7 October 2009
(n = 193) groups differ significantly ( p < 0.0001 and p = 0.012, respectively). Moreover, although the risk alleles of both SNPs are distributed more frequently in patients, the
Keywords:
significant difference is observed only for PTPN22 1858T allele ( p < 0.0001). This is therefore
PTPN22
the first evidence that analyzed gene variants contribute to T1D pathogenesis in Croatian
CTLA-4
population.
Polymorphism
# 2009 Elsevier Ireland Ltd. All rights reserved.
Type 1 diabetes
The most relevant non-HLA genes identified as susceptible for type 1 diabetes (T1D) are those connected with the T-cellmediated immune response. T-cells activity level and their effectors functions are determined by intracellular signaling pathways and related genes, like PTPN22 and CTLA-4, both of which prevent spontaneous activation of auto-reactive cells and development of autoimmunity [1,2]. However, polymorphic variants of these genes, due to a single nucleotide polymorphism (SNP), might augment autoimmunity, thus expanding the repertoire of genetic components that could influence T1D development. Association of PTPN22 C1858T or CTLA-4 A49G gene variants with T1D has been examined in more than one ethnic group [3– 7]. To the best of our knowledge, such reports for the Croatian population do not exist. The present study was undertaken to
ascertain the possible risk of PTPN22 C1858T and CTLA-4 A49G polymorphisms on T1D in Croatian patients. One hundred and two Croatian patients with T1D (38 males and 64 females; ratio 1:1.7) were enrolled in this study which included diabetic patients treated and categorized as Type I at the University Clinic Vuk Vrhovac, Zagreb, during 2006/2007. The clinical parameters of patients, shown as median (interquartile range), were 37 (31.5–44) for patients’ age, 23.5 (17–29.5) for age at the disease onset and 11.5 (5–17) for diabetes duration. Glycosylated hemoglobin was measured at the time of the study for the assessment of glycaemic control, and the mean value was 7.6 1.3%. All the patients required insulin for glycaemic control (the average dose was 50.0 22.3 units). The study was approved by the Ethics Committee of the University Clinic.
* Corresponding author at: Division of Molecular Medicine, Ruper Bosˇkovic´ Institute, Laboratory for Molecular Endocrinology and Transplantation, Bijenicˇka 54, 10 000 Zagreb, Croatia. Tel.: +385 1 4561064; fax: +385 1 4561010. E-mail address: [email protected] (M.P. Hadzˇija). 0168-8227/$ – see front matter # 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2009.09.012
e55
diabetes research and clinical practice 86 (2009) e54–e57
The control group consisted of 193 unrelated healthy subjects (103 males and 90 females; ratio 1:0.9). Controls as well as patients were Caucasian of Croatian nationality. Control subjects were screened by a questionnaire to ensure the absence of any diagnostic evidence of autoimmune diseases or family history of T1D. Median age for controls was 70 (interquartile range 58–78). Informed consent was obtained from patients and controls prior to blood sampling. Peripheral blood samples were collected in 3 mL volumes by venous puncture and genomic DNA was extracted by a salting out procedure, as described previously [8]. Two appropriate fragments of PTPN22 and CTLA-4 genes were amplified in PCR using two sets of primers:
PTPN22 F 50 -ACTGATAATGTTGCTTCAACGG-30 ; PTPN22 R 50 -TCACCAGCTTCCTCAACCAC-30 ; CTLA-4 F 50 -GCTCTACTTCCTGAAGACCT-30 ; CTLA-4 R 50 -AGTCTCACTCACCTTTGCAG-30 . PCR mixture contained 100 ng DNA, 10 pmoles of each primer, 50 mM of dNTPs, 1 PCR buffer with MgCl2, 1 U Hot Start Taq DNA polymerase (Eppendorf, Germany) and QH2O up to 25 mL. PCR reaction was carried out in the Mastercycler personal (Eppendorf) machine. Annealing temperatures, optimized in the pilot experiment, were 60 8C (for PTPN22) and 58 8C (for CTLA-4). Samples were initially denatured for 2 min at 94 8C followed by 30 cycles of 30 s at 94 8C, 30 s at 60 8C or 58 8C and 90 s at 70 8C. The product was checked on 2% agarose gel containing ethidium bromide. Amplified products of PTPN22 were digested with RsaI (Fermentas, Germany) overnight at 37 8C. The C allele creates restriction site producing two fragments (172 bp and 46 bp), while T allele does not (218 bp). Amplified products of CTLA-4 were digested with BbvI (Fermentas, Germany) overnight at 65 8C. The A allele does not create restriction site (162 bp), while G allele creates restriction site producing two fragments (88 bp and 74 bp). The SNPs in both genes were genotyped by electrophoresis on 12% polyacrilamide gel (PAGE) containing 1 Tris–borate– EDTA (TBE) buffer. DNA fragments were stained with SYBR Gold dye (Crucell, Lieden, Netherlands) and visualized under UV light. Observed genotypes at PTPN22 and CTLA-4 loci in patient and control groups are tested for Hardy–Weinberg equilibrium
using HW calculator ( p-value less than 0.05 was considered as not consistent with HWE). The differences in the frequency of various genotypes and alleles between patients and healthy controls were evaluated using x2 test. p-Value less than 0.05 (two-tailed test) was considered as significant. Odds ratio (OR) at 95% confidence intervals (CI) was also determined to describe the strength of association. As the test of homogeneity of variances was satisfied, the one-way analysis of variance (ANOVA) was applied to compare means of age at T1D diagnosis among individuals with different PTPN22 or CTLA-4 genotypes. LSD test was further used to detect if the means between genotype groups are different from each other. Statistical analysis was performed using SPSS13 software. The frequencies of the genotypes assessed at PTPN22 C1858T and CTLA-4 A49G polymorphic positions in both patient and control groups were in Hardy–Weinberg equilibrium (at PTPN22 0.09 vs. 0.52; at CTLA-4 0.07 vs. 0.6). A significant main effect was observed for PTPN22 (F(2,286) = 15.469, p < 0.0001, effect size f = 0.329) and CTLA-4 (F(2,286) = 4.147, p = 0.017, effect size f = 0.169). Distribution of PTPN22 C1858T genotypes between patients with T1D and healthy controls differ significantly (Table 1). The frequency of risk allele T (tryptophan containing allele) was also significantly higher in the patient than the control group (Table 1), and the importance of T allele for diabetes risk was indicated by high odds ratio (3.06). These results indicate connection between PTPN22 1858T allele and T1D in Croatian population, like it has also been reported for North American, Sardinian [3], Finnish [9], German [10], Danish [11] and Estonian [12] populations. The distribution of CTLA-4 genotypes amongst Croatian T1D patients and healthy controls differ significantly (Table 2), as has also been reported in Japanese [13], Estonian [14], and Iranian [7] case–control studies. It has to be pointed out that in our population CTLA-4 G/G homozygous individuals are overrepresented in patient group, compared to controls, with 2.7 OR at 95% CI in patients (Table 2). It is in agreement with the finding that in G/G homozygous individuals risk for T1D development increases more than twofold, as is reported in meta-analysis of 12 case–control studies that have examined the association of T1D with CTLA-4 49G polymorphism in European populations [15]. In our study, the frequency of the risk allele G was 6.8% higher in T1D than in healthy subjects, but it did not reach significant difference (Table 2). The higher frequency of G allele in patients than in control subjects is also
Table 1 – Distribution of the PTPN22 C1858T polymorphism in patients with T1D and healthy controls. Patient n (%)
Control n (%)
x2
p
Genotypes C/C C/T T/T
47 (46.1) 51 (50) 4 (3.9)
149 (77.1) 43 (22.4) 1 (0.5)
30.1
<0.0001*
1.0 (Ref)a 3.73 (2.21–6.29) –
Alleles C T
145 (71.1) 59 (28.9)
341 (88.3) 45 (11.7)
27.1
<0.0001*
1.0 (Ref)a 3.06 (1.98–4.7)
a *
The most frequent genotype or allele was used as a reference for OR calculations. Significantly different.
OR at 95% CI in patients
e56
diabetes research and clinical practice 86 (2009) e54–e57
Table 2 – Distribution of the CTLA-4 A49G polymorphism in patients with T1D and healthy controls.
Genotypes A/A A/G G/G Alleles A G a *
Patient n (%)
Control n (%)
x2
p
48 (47.0) 36 (35.3) 18 (17.7)
96 (49.7) 84 (43.6) 13 (6.7)
8.7
0.012*
1.0 (Ref)a 0.86 (0.51–1.44) 2.7 (1.25–6.12)
132 (64.7) 72 (35.3)
276 (71.5) 110 (28.5)
2.89
0.089
1.0 (Ref)a 1.36 (0.95–1.96)
OR at 95% CI in patients
The most frequent genotype or allele was used as a reference for OR calculations. Significantly different.
Table 3 – Dependence of age at T1D onset on the presence of risk alleles at the PTPN22 or CTLA-4 locus.
Acknowledgements
Genotypes
The authors thank Ms. Marina Marsˇ for the excellent technical assistance. This work was supported by the Croatian Ministry for Science, Education and Sports, Grant Nos. 098-09824642460 and 098-0982464-2508.
N
Mean age at diagnosis (years)
p
PTPN22 CC CT+TT
47 55
24.1 22.4
0.304
CTLA-4 AA AG+GG
48 54
24.8 21.4
0.046*
references
T—risk allele of PTPN22 and G—risk allele of CTLA-4. Significantly different compared to non-risk genotype.
*
a feature of most European populations (studies summarized in 15) with range of frequency from 2.9% to even 54.3%. The accumulated data from numerous studies demonstrate that CTLA-4 A49G SNP is genetic determinant of T1D, although some discrepancy between findings exists [6,16]. It may be attributed to a variable degree of association between an autoimmunity predisposing allele of the same gene and the disease among various populations. It has previously been shown that genetic susceptibility factors can affect the age at T1D onset, like a well known example of the high-risk DR3/DR4 genotype that is much more prevalent in individuals who have T1D onset at an early age [17]. In this study, we tested if risk genotypes of PTPN22 or CTLA-4 genes have an effect on age at T1D onset. We found that patients with CTLA-4 risk genotypes get diabetes earlier compared to patients with non-risk genotypes (F(1,101) = 4.08, p < 0.05) (Table 3). Although displaying tendency, results for PTPN22 risk genotypes did not reach statistical significance (F(1,101) = 1.067, p > 0.05) (Table 3). In conclusion, our data strongly suggest that polymorphisms PTPN22 C1858T and, to a lower extent CTLA-4 A49G, correlate with T1D incidence in the Croatian population. Preliminary results also indicate an effect of these polymorphisms on age at onset. However, more comprehensive study is needed to evaluate the impact of analyzed SNPs in T1D pathogenesis and to test the idea that combined data obtained from genotyping more than one susceptible gene variant allows identification of people at high-risk of developing T1D.
Conflict of interest There are no conflicts of interest.
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