No association between the −1031 polymorphism in the TNF-α promoter region and type 1 diabetes

No Association Between the ⴚ1031 Polymorphism in the TNF-␣ Promoter Region and Type 1 Diabetes Hadia Shbaklo, Sami T. Azar, Henry Terwedow, Georges Halaby, Roy P. Naja, and Pierre A. Zalloua ABSTRACT: Tumor necrosis factor alpha (TNF-␣) is an important immunomodulator and is believed to be involved in the development or progression of type 1 diabetes. In the following study, we evaluated TNF-␣ promoter polymorphisms at positions ⫺863 and ⫺1031 and their association with type 1 diabetes in a group of 210 diabetic patients from Lebanon. Our results show that in our population, the C allele is predominant at position ⫺863, whereas the A allele is very rare (2%). At position ⫺1031, however, the C and T allele distribution was similar in both the patient (17.8% vs 82.2%, respectively) and the control (21.4% vs 79.6%) groups. No association of TNF-␣ genotype at position 1031 with type 1 diabetes was found as demonstrated by the family-based associaABBREVIATIONS T1D type 1 diabetes TNF-␣ tumor necrosis factor alpha

INTRODUCTION Tumor necrosis factor alpha (TNF-␣) is a potent immunomodulator and plays an important role in inflammatory processes [1, 2]. It is primarily synthesized by monocytes and is believed to produce cytotoxic actions on pancreatic ␤-cells, mainly through potentiation of interleukin-1 and interferon-␥ effects [3– 6]. This consequence on insulin secretion has prompted a wide range of studies on the role of TNF-␣ in type 1 diabetes (T1D). Despite conflicting reports on whether higher or lower

From the Genetics Research Laboratory, Chronic Care Center, Beirut, Lebanon (H. S., R. P. N., P. A. Z.); Division of Endocrinology, American University of Beirut, Beirut, Lebanon (S. T. A); Program for Population Genetics, Harvard School of Public Health, Boston, MA, USA (H. T., P. A. Z); and the Department of Endocrinology, Hoˆtel-Dieu de France, Beirut, Lebanon (G. H.). Address reprint requests to: Pierre A. Zalloua, PhD, Genetics Research Laboratory, Chronic Care Center, Hazmieh, Lebanon. Tel: (⫹961) 5-455101; Fax: (⫹961) 5-952856; E-mail: [email protected]. Received October 17, 2002; revised February 18, 2003; accepted February 19, 2003. Human Immunology 64, 633– 638 (2003) © American Society for Histocompatibility and Immunogenetics, 2003 Published by Elsevier Inc.

tion test and the transmission disequilibrium test. However, when patient genotypes were compared, the recessive CC genotype was only found in type 1 diabetic males but not in type 1 diabetic females. This observation, however, requires further investigation in a larger sample before conclusive association to gender is suggested. In conclusion, our results demonstrate that no association between TNF-␣ polymorphism and type 1 diabetes seems to exist in our population. Human Immunology 64, 633– 638 (2003). © American Society for Histocompatibility and Immunogenetics, 2003. Published by Elsevier Inc. KEYWORDS: type 1 diabetes; tumor necrosis factoralpha, polymorphism, HLA

HLA

human leukocyte antigen

levels of TNF-␣ are involved in the development of T1D [7–11], its role in the pathogenesis of the disease and in other autoimmune diseases is routinely reported [12– 14]. Furthermore, because of the localization of the TNF-␣ gene on chromosome 6 within the major histocompatibility class II region, and because of the association of some human leukocyte antigen (HLA) class II alleles with T1D [15–17], a relation between TNF-␣ genetic variability and T1D is widely suggested. Moreover, polymorphisms within the TNF-␣ promoter region seem to modulate the risk to T1D, mostly in individuals carrying the HLA DQA1*0501-DQB1*0201/ DQA1* 0301-DQB1*0302 genotypes [18, 19]. During the last 5 years, researchers have focused on the polymorphism of the human TNF-␣ promoter region, mostly at positions ⫺238 [20 –22], ⫺308 [20 –23], ⫺857 [21, 24], ⫺863 [21, 24] and ⫺1031 [21] and their possible association with several diseases, including Crohn’s disease [14], human T-cell lymphotropic virus type-1 uve0198-8859/03/$–see front matter doi:10.1016/S1098-8859(03)00053-3

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itis [13], juvenile rheumatoid arthritis [12], and T1D [25]. In this study, we evaluated TNF-␣ promoter polymorphism at positions ⫺863 and ⫺1031 in a group of 210 Lebanese T1D patients and its role as a susceptibility factor for this disease. These polymorphisms were chosen to determine whether, as in the Japanese [25], they are associated with T1D in our population.

H. Shbaklo et al.

TABLE 1 Age of onset, age and HbA1c mean values of T1D patients by gender

Female (100) Males (110) Total

Age of onset ⫾ SD

Age ⫾ SD

HbA1c ⫾ SD

9.42 ⫾ 4.61 8.60 ⫾ 4.67 8.99 ⫾ 4.54

15.03 ⫾ 6.01 13.52 ⫾ 5.55 14.23 ⫾ 5.81

8.67 ⫾ 1.69 8.72 ⫾ 1.58 8.69 ⫾ 1.63

Abbreviations: T1D ⫽ type 1 diabetes; SD ⫽ Standard deviation.

MATERIALS AND METHODS Subjects Two hundred and ten patients (from 196 families) enrolled at the Chronic Care Center who developed T1D between January 1993 and July 2001 were the subject of the present study. The subjects were followed at 1- or 2-month intervals. In addition to a standardized medical history questionnaire, a routine medical evaluation, an HbA1c measurement and antiglutamic acid decarboxylase antibody titers (anti-GAD) determination were made. The HbA1c value reported in this study represents the average value of the last three measurements recorded during the period of the study. The ethics committees of the Chronic Care Center and the Harvard School of Public Health approved the study protocol and an informed written consent was obtained from the participating subjects and their parents. The diagnosis of T1D was made on the basis of ketoacidosis or ketosis with severe symptoms of acute onset at presentation and continuous dependence on insulin within 6 months of diagnosis [26]. Individuals who were suspected to have maturity-onset diabetes of the young or Wolfram syndrome were excluded from the study. All patients were younger than 26 years of age at presentation. Seventy-six percent of the patients with onset of T1D at 5 years or less tested positive for antiGAD. Ninety-six healthy individuals (males and females) were chosen as controls and were randomly selected from the Lebanese population. All control individuals were older than 25 years of age, as recommended by the DiaMond protocol [27] and had passed through the high-risk period (0 –15 years of age), during which time T1D is most likely to develop. HLA Genotyping and TNF-␣ Promoter Polymorphism Sample Preparation and DNA Extraction. Five ml of blood were collected from each patient and his or her father and mother in EDTA-containing tubes. DNA was extracted with a QIAamp Blood Kit (Quiagen, Hilden, Germany) according to the blood and body fluid protocol recommended by the manufacturer. The DNA was eluted in Tris-EDTA buffer (20 mM Tris, 2 mM EDTA) and stored at ⫺20°C until use.

Polymerase Chain Reaction (PCR) Amplification and Analysis. The DNA samples were amplified with HLADQB1*0201, HLA-DQB1*0302, HLA-DQA1*0501, and HLA-DQA1*0301 sequence specific primers [28, 29]. Each sample was also amplified with a primer pair, used as internal positive amplification controls, which detects a conserved region of the DRB1 gene. Genotyping of the ⫺863C/A and ⫺1031T/C polymorphisms was evaluated using sequence specific primers followed by digestion with the restriction enzymes TaiI and BbsI (New England Biolabs, Beverly, MA, USA), respectively [30]. Digestions with the appropriate restriction enzymes were done as recommended by the manufacturer. PCR products were detected by agarose gel electrophoresis. For the ⫺863C/A polymorphism, a single band of 126 bp or 101 bp indicates a CC or AA genotype, respectively, whereas two bands of 126 and 101 bp are indicative of a heterozygous genotype. As for the ⫺1031T/C polymorphism, a 250 bp band or a 194 bp band indicates a TT or CC genotype, respectively, whereas two bands of 250 bp and 194 bp represent a heterozygous phenotype. Statistical Analysis Differences between the groups were analyzed by the Student’s t-test or the chi-square test statistic. A p value of ⬍ 0.05 was considered significant. RESULTS Patients Two hundred and ten patients ages 14.23 ⫾ 5.81 participated in the study. The mean age of onset was 8.99 ⫾ 4.54 years: 9.42 ⫾ 4.61 years for females and 8.60 ⫾ 4.67 years for males. The HbA1c mean value calculated from the average of the last three observations for each patient was 8.67 ⫾ 1.69% for females, 8.72 ⫾ 1.58% for males, and 8.69 ⫾ 1.63% for females and males combined. These results are presented in Table 1, and statistical analysis shows that gender does not influence the age of onset nor the HbA1c value. HLA Typing HLA genotyping shows a predominance of the DQB1*0201 and *0302 alleles and the DQA1 *0501

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TABLE 2 HLA DQB1 and DQA1 genotypes in T1D patients and controls

TABLE 4 TNF-␣ genotypes in T1D patients and controls by gender

Patients n (%)

Controls n (%)

Chi square, p value

DQB1*0201 ⫹ 153 (72.9%) DQB1*0201 ⫺ 57 (27.1%)

29 (30.2%) 67 (69.8%)

49.72, p ⬍ 0.0001

DQA1*0501 ⫹ 156 (74.3%) DQA1*0501 ⫺ 54 (25.7%)

41 (42.7%) 55 (57.3%)

28.65, p ⬍ 0.0001

TC

DQB1*0302 ⫹ 66 (31.4%) DQB1*0302 ⫺ 144 (68.6%)

18 (18.8%) 78 (81.2%)

5.32, p ⫽ 0.0211

TT

DQA1*0301 ⫹ 103 (49.0%) DQA1*0301 ⫺ 107 (51.0%)

4 (4.2%) 92 (93.8%)

HLA

58.36, p ⬍ 0.0001

Abbreviations: HLA ⫽ human leukocyte antigen; T1D ⫽ type 1 diabetes.

and *0301 alleles among T1D patients compared with the controls (Table 2). Table 3 shows the most common of these haplotypes in both patients and controls. TNF-␣ Promoter Polymorphism in T1D Patients and Controls TNF-␣ ⫺863C/A polymorphism was evaluated in all 210 patients and their parents. Our findings show that 99% of patients had the CC genotype, whereas only 1% had the CA and none had the AA genotype. The A allele frequency was also rare (⬍2%) among the parents (500 individuals from the 196 families were tested and only 11 of the 1000 chromosomes carried the A allele) and the controls (results not shown). Because of the low frequency of heterogeneity of this polymorphic site, no further analysis was performed. The distribution of the TNF-␣ ⫺1031T/C polymorphism is presented in Table 4. Comparison between the patients and controls showed no significant differences for all three genotypes CC, TC, and TT (␹2 ⫽ 2.13, p ⫽ 0.344). Family-based association testing (FBAT [31, TABLE 3 Most common HLA haplotypes among T1D patients and controls HLA haplotypes DQA1* DQB1* DQA1* DQB1* 0301 0302 0501 0201 ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹

⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺

⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺

Patients

Controls

19 (9%) 28 (13.3%) 16 (7.6%) 15 (7.1%) 56 (26.7%) 10 (4.8%) 9 (4.3%) 5 (2.4%) 12 (5.7%)

0 0 1 (1.0%) 0 11 (11.5%) 22 (22.3%) 12 (12.5%) 26 (27.1%) 0

Abbreviations: HLA ⫽ human leukocyte antigen; T1D ⫽ type 1 diabetes.

CC

Patients n ⫽ 210

Controls n ⫽ 96

Females Males Total Females Males Total Females Males Total

0* (0%) 6** (5.45%) 6 (2.85%) 39 (39%) 31 (28.18%) 70 (33.3%) 61 (61%) 73 (66.36%) 134 (63.85%)

4 (8%) 2 (4.34%) 6 (6.25%) 12 (21%) 17 (36.96%) 29 (30.2%) 34 (68%) 27 (58.70%) 61 (63.55%)

* p ⬍ 0.05 compared with female controls. ** p ⬍ 0.05 compared with female patients. Abbreviations: TNF ⫽ tumor necrosis factor; T1D ⫽ type 1 diabetes.

32]) and transmission disequilibrium test (TDT [33]) also showed that there is no association between TNF-␣ ⫺1031T/C polymorphism and T1D (␹2 ⫽ 3.6, p ⬎ 0.05). For the TDT, only parents with a heterozygote genotype for the TNF-␣ ⫺1031 promoter region polymorphism were considered (20 families). In multiplex families (in which more than one child was diabetic), only the first patient was included in the analysis. The absence of the CC genotype among female patients was noted as being significantly (p ⬍ 0.05) different from female controls (8% CC) and from male patients (5.45% CC). However, when C versus T allele frequencies were compared among the patients (17.8% vs. 82.2%, respectively) and controls (21.4% vs. 79.6%), no differences were observed even when the groups were divided according to gender. HLA Haplotypes and TNF-␣ Promoter Polymorphism Sixteen different HLA haplotypes were identified in patients with TT genotype, compared with nine HLA haplotypes in controls with TT genotypes. Three of the most common haplotypes among the patients were not found in the controls. Patient and control groups having the CC TNF-␣ genotype had four haplotypes each. Only one haplotype was common to both groups: TNF-␣ promoter polymorphism, age of onset, and HbA1c values. Table 5 shows that only 6 of the 210 patients presented the CC genotype, and they are all males. The mean HbA1c value for these patients is 10.58 ⫾ 2.25% and is significantly higher than that of male or female patients having the TC (p ⫽ 0.018) or TT (p ⫽ 0.002) genotypes. As for the age of onset, no difference was observed among the three TNF-␣ ⫺1031 CC, TC, and TT geno-

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TABLE 5 Age of onset and HbA1c mean values of T1D patients by TNF-␣ genotype and gender

CC TC TT

Females Males Total Females Males Total Females Males Total

n

Age of onset ⫾ SD

HbA1c ⫾ SD

0 6a 6 39 31 70 61 73 134

– 9.18 ⫾ 3.30 9.18 ⫾ 3.30 9.69 ⫾ 5.10 8.72 ⫾ 3.60 9.26 ⫾ 4.49 9.24 ⫾ 4.30 8.50 ⫾ 4.90 8.84 ⫾ 4.63

– 10.58 ⫾ 2.25 10.58 ⫾ 2.25b 8.76 ⫾ 1.78 8.83 ⫾ 1.62 8.79 ⫾ 1.70 8.61 ⫾ 1.64 8.52 ⫾ 1.42 8.55 ⫾ 1.52

Pearson chi-square p ⬍ 0.05. Student’s t-test p ⫽ 0.018 compared with total TC and p ⫽ 0.002 compared with total TT. Abbreviations: T1D ⫽ type 1 diabetes; TNF ⫽ tumor necrosis factor.

a

b

types, even when the three groups were compared with each other according to gender (see Table 5). DISCUSSION Polymorphisms within the TNF-␣ promoter and their frequencies were reported by several groups during the past 6 years [20 –23], and a large number of studies have associated TNF-␣ promoter polymorphism with certain autoimmune diseases, among which is T1D [18, 25] and type 2 diabetes [34]. The most linked polymorphic location with T1D within the TNF-␣ promoter region seems to be the ⫺863 C/A and ⫺1031 T/C sites, which are in linkage disequilibrium with each other and with the HLA locus in many populations [21, 25]. In this study, we first investigated the – 863 polymorphic site, which seems to lead to a reduced TNF-␣ level production [30], and found that 98 –99% of individuals tested, whether patients or controls, had the CC genotype. Because the A allele frequency was very low (1–2%) compared with that reported for Caucasians and Japanese (14%; 17% and 21.1%, respectively) [21, 24, 30], it was not investigated any further. Furthermore, the analysis of the ⫺1031 promoter polymorphism of TNF-␣ showed that T and C allele frequencies in our T1D patients (82.2% T and 17.8% C) and in the control group (79.6% T and 21.4% C) were similar to each other and to those reported elsewhere in the literature for Caucasians (80% T and 20% C) [30]. Family-based association testing and TDT showed no association of this polymorphism with T1D. A different finding was described in a recent study done on the ⫺1031 TNF-␣ promoter polymorphism and its role in T1D by Hamaguchi et al. ([25], who reported an asso-

ciation between T1D and the TNF-␣ ⫺863A/-1031C alleles in the Japanese population. However, despite the lack of association, our results showed a gender difference in the CC genotype of the ⫺1031 TNF-␣ promoter site among patients. This is the first time that such a difference has been reported. It is worth noting that T1D did not show gender preference in regard to disease occurrence, age of onset, HbA1c, or HLA genotyping for DQB1*0201 and DQB1*0302 [35]. We detected a positive association between HLA class II haplotypes and T1D, confirming the results of previous studies [15–17]. Sixteen haplotypes were found among our T1D patients, whereas only nine were observed among the controls. This is almost certainly the result of the greater number of patients in the present study. None of the nine haplotypes common to the two population groups occurred at the same frequency. Repeating the analysis with TNF-␣ in the haplotype did not make a difference to the significance of the HLA association, which is consistent with the observation that TNF-␣ is not involved in T1D in our population. In the present study, we also found significantly higher HbA1c values among patients with the CC genotype at ⫺1031 TNF-␣ promoter region. However, further investigation showed that four of the six patients had a duration of diabetes of less than 2 years, a phase during which most T1D patients exhibit poor glycemic control. This finding does not completely rule out a possible association, however, between ⫺1031 TNF-␣ CC genotype and glycemic control, especially because previous studies report finding that high glucose concentrations increase TNF-␣ production [36], and a positive correlation exists between TNF-␣ and glucose levels [37]. A larger number of patients should be investigated, and their HbA1c and serum TNF-␣ levels measured, before reaching conclusions on the functional effects of the ⫺1031 TNF-␣ promoter polymorphism. In conclusion, we report that in our population, the TNF-␣ ⫺863 promoter polymorphism is rare and that the ⫺1031 polymorphism is not associated with T1D. The finding that the CC genotype at position ⫺1031 was restricted to males among the patients needs to be more extensively investigated in a larger sample. The levels of TNF-␣ should be also assessed and compared in relation to the ⫺1031 TNF-␣ genotype, gender, and HbA1c levels. ACKNOWLEDGMENTS

We thank our nurses, our social worker, as well as our laboratory assistants for their skillful help and input in carrying out this study. Part of the work was funded by a grant from the Lebanese National Council for Scientific Research.

TNF-␣ Promoter Polymorphism and Diabetes

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