Association of tumor necrosis factor alpha, interferon gamma and interleukin 10 gene polymorphisms with peripheral neuropathy in South Indian patients with type 2 diabetes

Cytokine 47 (2009) 173–177

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Association of tumor necrosis factor alpha, interferon gamma and interleukin 10 gene polymorphisms with peripheral neuropathy in South Indian patients with type 2 diabetes Venkata Karunakar Kolla a,b, G. Madhavi a, B. Pulla Reddy a, B.M.V. Srikanth Babu a, J. Yashovanthi a, Vijaya Lakshmi Valluri b, Jayanthy Ramesh c, Jyothy Akka a,* a

Institute of Genetics & Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad, Andhra Pradesh, India LEPRA Health in Action, Blue Peter Research Center, Cherlapally, Hyderabad, Andhra Pradesh, India c Osmania General Hospital, Afzalgung, Hyderabad, Andhra Pradesh, India b

a r t i c l e

i n f o

Article history: Received 28 November 2008 Received in revised form 28 May 2009 Accepted 13 June 2009

Keywords: Cytokine Diabetic peripheral neuropathy Tumor necrosis factor alpha Interferon gamma Interleukin 10

a b s t r a c t Diabetic peripheral neuropathy (DPN) is a major global health threat and a common complication of diabetes. Peripheral nerve complications due to irregular cytokine production are eminent factors in many inflammatory diseases. The present study focused on gene polymorphisms of pro and anti-inflammatory cytokines that may be responsible for nerve damage in diabetic neuropathy. We examined three common functional SNPs primarily at the positions on genes of tumor necrosis alpha (TNFa) 308G/A, interferon gamma (IFNc) +874A/T and interleukin (IL) 10 1082G/A in order to establish their association with peripheral neuropathy in type 2 diabetes. Results: Genotypic frequencies obtained from TNFa 308G/A gene analysis in DPN group comprised 86.4% of G/A, 10.6% of G/G and 3% of A/A genotype, where as the control group had 94% of G/A, 2% of G/G and 4% of A/A which could not reach the statistical significance with the disease after Bonferroni correction. The IFNc +874 A/T polymorphism in patient group revealed 33.3% of A/A, 47.5% of A/T and 19.2% of T/T genotype. The A/A genotype had attained statistical significance of P = 0.04 (P corrected); OR 2; 95% CI 1.14–3.64 when compared to controls. The IL10 1082 G/A polymorphism in the patient group has showed 62.6% of A/A, 21.2% of G/A, 16.2% of G/G genotype, revealing significant association with G/G genotype (P < 0.01, OR 2.9; 95% CI 1.47–5.84) when compared to controls. Conclusion: Our findings indicate that the tested markers within the IFNc and IL-10 genes, but not the TNFa gene, are significantly associated with peripheral neuropathy in South Indian type 2 diabetic patients. The study shows that the ‘high-producer’ IL-10 1082 G/G genotype and the ‘low-producer’ IFNc +874 A/A genotype may be responsible for the down regulation of immune response leading to inflammation in this setting. Ó 2009 Elsevier Ltd. All rights reserved.

1. Background Diabetic peripheral neuropathy (DPN) is a major global health threat and a common complication of diabetes, characterized by inflammation and degeneration of peripheral nerves. The morbidity caused by peripheral neuropathy is quite profound in India and is the major public health problem in other developing countries. About 60–70% of people affected with diabetes are prone to neuropathy [1]. The frequency distribution for neuropathies is more or less same in type 1 and 2 diabetes [2].

* Corresponding author. Address: Department of Cell and Molecular Biology, Institute of Genetics & Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad 500016, Andhra Pradesh, India. Tel.: +91 040 23403681. E-mail address: [email protected] (J. Akka). 1043-4666/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2009.06.007

Peripheral nerve complications due to irregular cytokine production are one of the eminent factors in many inflammatory diseases [3,4]. Several studies have identified polymorphisms in cytokine gene regulatory regions that correlated to intra-individual variations in cytokine production [5–7]. The differential production of cytokines will eventually amend the downstream signaling processes that could directly or indirectly affect nerve functions which could lead to neurodegeneration [8–10]. TNFa overproduction has demonstrated the pathogenicity in diabetic polyneuropathy [11]. Human TNFa mutant allele A at the promoter 308 position with increased production of TNFa has been studied in many diseases [12–14]. Its overproduction stimulates the synthesis of other pro-inflammatory cytokines such as interferon gamma (IFNc) which has a pivotal role in the induction of immune mediated inflammatory response [15]. SNP located at the +874A/T position from translation start site in the first intron

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of IFNc gene plays a fundamental role in the induction of IFNc production [16]. The T allele correlates with high IFNc expression, whereas the A allele correlates with low expression [17]. The unusual immune responses like surplus production of inflammatory cytokines, TNFa and IFNc would be suppressed by interleukin (IL) 10 via immunosuppressive and anti-inflammatory cytokine action by inhibiting the activation and effector function of T cells, monocytes, and macrophages thereby giving the protection against the deleterious effects [18,19]. At position 1082 from transcriptional start site, the presence of G allele is associated with higher and A allele with lower production of IL-10 has been reported [20]. The present study focused on gene polymorphisms of pro and anti-inflammatory cytokines that may be responsible for nerve damage in type 2 diabetic patients with peripheral neuropathy. We examined three common functional SNPs primarily at the positions on genes of tumor necrosis alpha (TNFa) -308G/A, interferon gamma (IFNc) +874A/T and interleukin (IL) 10 1082G/A in order to establish an association with diabetic peripheral neuropathy. 2. Materials and methods 2.1. Subjects All the subjects recruited for this study are of South Indian origin and from the Department of Endocrinology & Diabetology of Osmania General Hospital and Institute of Genetics & Hospital for Genetic Diseases, Osmania University, Hyderabad, India. They were recruited during the period from March 2007 to March 2009. We have selected 198 type 2 diabetic patients affected with peripheral neuropathy having poor glycemic controls and disease duration of more than 5 years. Glycemic status of subjects as pre and post prandial plasma glucose levels and BMI has been represented in Table 1. These cases comprised of 124 males and 74 females with mean age 55.27 ± 3.92 years and mean duration of diabetes 10.87 ± 3.5 years. 202 healthy subjects without any ailments were selected (118 males and 84 females with a mean age of 52.71 ± 5.9 years) as controls (Table 1). Mean age, disease duration and severity of disease of the patients according to the gender has been depicted in Table 2. 2.1.1. Inclusion criteria Patients with type 2 diabetes with onset of disease >5 years presenting the symptoms of frequent burning pain, electrical or stabbing sensations, parasthesiae, hyperasthesiae and deep aching pain in the feet and lower limbs. All these patients were recruited after thorough clinical examination of neuropathy, which includes examination of the ankle reflex, pin-prick and temperature perception with cold tuning fork at the great toe and absence or decreased vibration perception threshold (VPT) as documented by biosthesiometry. This was confirmed by applying neuropathy score postulated by Young et al [21]. VPT 15–25 volts per micron (V/l) was considered mild, VPT 25–42 V/l was considered moderate and >42 V/l was considered severe neuropathy, and the score <15 V/l was considered normal, and was excluded from this study. Age Table 1 General characteristics of the study group. Characteristics

Diabetic neuropathy (N = 198)

Controls (N = 202)

Sex (male/female) Age (years) Duration of diabetes (years) BMI (kg/m2) Preprandial plasma glucose (mg/dl) Postprandial plasma glucose (mg/dl)

124/74 55.27 ± 3.92 10.87 ± 3.51 24.58 ± 4.17 185.71 ± 21.5 254.52 ± 45.46

118/84 52.71 ± 5.9 Not applicable 22.84 ± 2.9 96.26 ± 1.22 142.1 ± 16.33

matched healthy individuals with VPT less than 15 V/l were included in control category without any neurological symptoms or diabetic problems. All the healthy and age matched individuals were blood donors and patient attendants attending the two Institutions. The subjects recruited were of similar socioeconomic status. The Institutional Ethical Committee approved this study and informed written consent was obtained from all the subjects before their participation in this study. 2.2. Genomic DNA isolation and genotyping of subjects Genomic DNA was isolated, using Qiagen Flexigene extraction kit according to the manufacturer’s recommendations (QIAGEN Pvt. Ltd., Australia), from 300 ll of whole blood. The polymorphisms in TNFa ( 308 G/A), IFNc (+874 T/A) and IL-10 ( 1082 G/A) genes were typed using amplification refractory mutation system polymerase chain reaction methods (ARMS PCR) [22–24]. In brief, genomic DNA of each subject was amplified for SNPs using 0.5 U of Taq Polymerase (Qiagen, Australia) in two different PCRs for each polymorphism; each reaction mixture for ARMS PCR consists of 40 ng of genomic DNA in a total volume of 20 ll, containing 0.8 lM of each primer, 400 lM dNTPs, 10 mM Tris–HCl (pH 9.0), 1.5 mM MgCl2, 50 mM KCl, 0.01% gelatin. Each reaction employed a generic antisense primer and one of the two allele-specific sense primers. For evaluation of the PCR amplification in both reactions one internal control was amplified using a pair of specific primers. The products were analysed on 2% agarose gel stained with ethidium bromide. 2.3. Statistical analysis All calculations were done using the SPSS program (Version 15.0 for Windows 98; SPSS, Chicago, IL). The differences in the distribution of genotypes and allele frequencies were analyzed using the v2 test. Genotype frequencies were checked for deviation from Hardy–Weinberg equilibrium and were not significantly different from those predicted. Odds ratios and 95% confidence interval (95% CI) were calculated to assess the strength of the relationship between the TNFa, IFNc and IL-10 gene polymorphisms with diabetic neuropathy. Power calculations have been performed using online software (http://pngu.mgh.harvard.edu/~purcell/gpc/) with a P value <0.05. Bonferroni correction (P-corrected) was applied for multiple testing of three SNPs tested in patient group.

3. Results In our case-control study we genotyped the three SNPs TNFa 308G/A, IFNc +874A/T and IL-10 1082G/A in 198 diabetic neuropathy patients and 202 healthy controls. The genotype distributions and allele frequencies of three SNPs are shown in Table 3. The distribution of TNFa -308G/A, IFNc +874A/T and IL-10 1082 G/A genotypes was in Hardy–Weinberg equilibrium among controls. 3.1. The

308 G/A polymorphism of the TNFa gene

Genotype frequency obtained from TNFa gene analysis in patients with diabetic peripheral neuropathy revealed that the majority of them were G/A heterozygotes (86.4%) followed by G/G homozygotes (10%) and the least frequent A/A homozygotes (3%). The homozygous G/G group showed a significant P value of 0.02, which disappeared after Bonferroni correction. The control group had genotypic frequency of 94% heterozygotes followed by diminished frequencies of homozygotes (2% G/G and 4% A/A). The allelic frequency in patient group was 53.8% of G and 46.2% of A, where as the control group showed 51% of A allele and 48% of G

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V.K. Kolla et al. / Cytokine 47 (2009) 173–177 Table 2 Mean age, disease duration and severity of disease of the patients according to the gender. Sex

Age in yearsa

Disease duration in yearsa

Male (124) Female (74)

55.8 ± 4.09 54.41 ± 3.47

11.08 ± 3.39 10.52 ± 3.71

a

Severity of the disease Mild

Moderate

Severe

56 (45.2%) 38 (51.4%)

59 (47.6%) 32 (43.2%)

9 (7.3%) 4 (5.4%)

Values are mean ± SD.

Table 3 Allele and genotype distribution of the TNFa, IFNc and IL-10 polymorphic markers in the study groups

TNFa 308 Allele G A Genotype G/G G/A A/A IFNc +874 Allele A T Genotype A/A A/T T/T IL10 1082 Allele G A Genotype G/G G/A A/A

Diabetic neuropathy patients (N = 198)

Controls (N = 202)

No.

%

No.

%

OR (95% CI)

P value

213 183

53.8 46.2

198 206

21 171 6

10.6 86.4 3.0

226 170

P corrected

48.3 51.0

1.2 (0.90–1.61) Reference

NS

4 190 8

2.0 94.0 4.0

7 (1.25-41.84) 1.2 (0.35-4.28) Reference

0.02 NS

NS

57.1 42.9

195 209

48.3 51.7

1.4 (1.06-1.9) Reference

0.012

0.03

66 94 38

33.3 47.5 19.2

40 115 47

19.8 57.0 23.2

2 (1.14-3.64) 1 (0.59-1.67) Reference

0.015 NS

0.04

106 290

26.8 73.2

67 337

16.6 83.7

1.8 (1.28-2.63) Reference

0.0004

0.001

32 42 124

16.2 21.2 62.6

13 41 148

6.4 20.3 73.3

2.9 (1.47-5.84) 1.2 (0.72-2.06) Reference

0.001 NS

0.003

NS, not significant; P value was calculated by v2 test with 2  2 contingency table and considered <0.05 as significant.

allele. The allelic and genotypic frequencies have not shown any statistical significance in relation to the disease (Table 3). 3.2. The +874 A/T polymorphism of the IFNc gene We next compared allelic and genotypic frequencies between patients and controls for IFNc +874 A/T polymorphism. Patient and control groups showed significant distinguishable allelic frequency for A allele (P value of 0.01, OR 1.4; 95% CI 1.06–1.9). Genotype frequency analysis in patients showed 33% of A/A, 47% of A/T and lower frequency, 19% of T/T genotype. The control population had higher genotypic frequency of heterozygotes 57% A/Ts and diminished frequencies of homozygotes (20% A/As and 23.% T/Ts). The A/A genotype in patients had attained statistical significance (P value 0.01; OR 2; 95% CI 1.14–3.64) (Table 3). 3.3. The IL-10

1082 G/A polymorphism

In the third gene there was a significant difference between genotype frequencies of the IL10 1082 variant between diabetic neuropathy and control subjects (P < 0.001). The allelic frequency of IL-10 1082 G was significantly higher among patients with diabetic neuropathy (26.8%) than among control subjects (16.6%) (Corrected P = 0.001, OR 1.8, 95% CI 1.28–2.63). In the control group, the genotype and allele frequencies of IL-10–1082 were 73.3% for A/A genotype, 20.3% for G/A genotype, 6.4% for G/G genotype and 83.7% for A allele and 16.6% for G allele, while in

the patient group the frequency was 62.6% for A/A genotype, 21.2% for G/A genotype, 16.2% for G/G genotype and 73.2% for A allele and 26.8% for G allele (Table 3). Statistically significant association was observed in only homozygous G/G genotype among patients with diabetic neuropathy (P = 0.001, OR 2.9; 95% CI 1.47–5.84).

4. Discussion Cytokines play a major role in the pathophysiology of development of nerve damage in many inflammatory diseases. Several studies have investigated the possible role of cytokine gene polymorphisms for susceptibility or resistance towards nerve damage in leprosy [25,26]. The present study focused on three cytokine gene polymorphisms in type 2 diabetic patients with peripheral neuropathy, aiming to understand the role of these gene polymorphisms in the clinical outcome of the disease. To the best of our knowledge, this is the first report from South India demonstrating the non-association of TNFa and significant association of IFNc, and IL-10 gene polymorphisms with diabetic peripheral neuropathy. TNFa has been found to induce neuronal damage [27,28] and is considered as a major initiator of inflammation. In pre progression stage of diabetic peripheral neuropathy circulating levels of TNFa are elevated in type 2 diabetes [29]. Compared with the -308G allele, the -308A allele of the TNFa gene has been shown to

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increase transcription two fold and therefore, TNFa concentration [30,31]. Homozygous AA genotype of TNFa has been demonstrated as a key marker in many inflammatory diseases [12–14] as it is a high-producer of TNFa and causes disease susceptibility. In our study we did not find any significant association between TNFa 308 gene polymorphism with diabetic peripheral neuropathy and our data shows a higher frequency of heterozygous (G/A) genotype in South Indian population (Table 3). All genotype frequencies are more or less same in the disease population compared to control group. Similarly, it has also been shown to be non-significant in many other inflammatory diseases [32–34]. From SNPnexus database search (http://www.snp-nexus.org/) revealed that several other non South Indian populations have also shown no connection with metabolic and inflammatory diseases. Furthermore the genotypic frequencies also vary in diverse populations. SNP database search for genotype frequency of TNF (rs1800629) showed a higher frequency of G/G genotype (95%), followed by G/A (6%) and negligible frequency of A/A in Asian population, whereas African Americans have more than 75% of G/G, 24% of G/A and 1% of A/A genotype (http://www.ncbi.nlm.nih.gov/sites/ entrez?db=snp). Similar variations in the frequencies of these genotypes have been observed in Taiwanese (G/G 89.8%, G/A 8.5% and A/A 1.6%), Chinese (G/G 85.6% G/A + A/A 14.4%), Turkish (G/G 66%, G/A 28% and A/A 6%), Dutch (G/G 65.2%, G/A 31.8% and A/A 3%) and Italian (G/G 91.3%, G/A 7.6% and A/A 1.1%) populations [34,41–44]. Nonetheless, the frequency of homozygous G/G and A/A shows a similar trend in these populations. The heterogygous G/A frequency seems to be variable (6–32%). Interestingly in our study on South Indian population we have observed a very high frequency of G/A heterozygotes (94%) in both patients and control groups. Thus, it indicates that TNFa 308 gene polymorphism may not be a precise marker for diabetic peripheral neuropathy in type 2 diabetic patients from South India. The TNFa gene may require further investigation on a functional basis, to elucidate the genetic role of the Th1/Th2 cytokine responses in the aetiopathogenesis of DNP. IFNc is a pivotal proinflammatory cytokine that has a role in the induction of immune mediated inflammatory response [15]. Polymorphism located at the +874A/T position plays a fundamental role in the induction of IFNc production [16]. The T allele of IFNc +874A/T provides a binding site for the transcription factor NF-jB (nuclear factor kappa B), which is able to regulate IFNc expression [17]. This protein plays an important role in the transcriptional regulation of IFNc gene, [35] and the +874T and +874A alleles are responsible for high and low IFNc production, respectively [36]. It is possible that low IFNc production will facilitate not as much of immune response against inflammation rendering these individuals more susceptible to the disease as the downstream process would eventually leads to nerve damage. A previous report from North Indian population has shown that the association of IFNc +874 homozygous A/A with cervical cancer [37]. Our observation shows profound increase in the distribution of IFNc +874 homozygous A/A genotype which is significantly associated with diabetic neuropathy (P = 0.02, OR 2.05, CI 1.04– 1.97) demonstrating two fold increased risk and suggests that this could be a risk factor for susceptibility to diabetic peripheral neuropathy in South India. The superfluous production of inflammatory cytokines, TNFa and IFNc would be suppressed by interleukin (IL) 10 [18,19] thus providing protection against the inflammatory reaction. Turner et.al (1997) showed gene polymorphisms of IL-10 at position 1082 from transcriptional start site and the presence of G allele associated with higher and A allele with lower production of IL10. Reports indicate that the high-producer genotype is associated with inflammatory diseases such as rheumatic heart disease [38] and unstable atherosclerotic disease [39]. The association of G

allele has also been reported in inflammatory bowel disease and ulcerative colitis [40]. Similarly, we also found a significant association of IL-10 1082 G/G genotype (P = 0.003) and G allele (P < 0.001) in diabetic peripheral neuropathy cases of South Indian origin. SNP data base search for IL-10 (rs1800896) population diversity showed that the frequencies of A/A is more than 90% and heterozygous G/A is 8% in Asians, whereas as European population has 20% of A/A, >50% of G/A and 28% of G/G indicating that G and A alleles are in nearly equal frequencies, whereas in Asian population, allele ‘A’ is more predominant. Based on the clinical evaluation of diabetic peripheral neuropathy, the ‘high-producer’ IL-10-1082 G/G genotype and the ‘lowproducer’ IFNc +874 A/A genotype may be responsible for the down regulation of immune response leading to inflammation in diabetic peripheral neuropathy patients. Our findings indicate that the tested markers within the IFNc and IL-10 genes, but not the TNFa gene, are significantly associated with peripheral neuropathy in South Indian type 2 diabetic patients. These results appear to encourage further investigation into the role of cytokines in the pathogenesis of diabetic peripheral neuropathy. In conclusion the present study is limited to diabetic neuropathy with type 2 diabetes only, which may comprise axonal and demyelinating forms of neuropathy, as nerve conduction study only differentiates these forms and also the comparison of type 2 diabetes subjects without neuropathy as control group with disease subjects, which would be performed in future research in order to establish the stringent association towards the diabetic peripheral neuropathy. Acknowledgments Authors are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi for providing Senior Research Fellowship to Venkata K. Kolla. We acknowledge Mousumi Majumder, Indian Statistical Institute, Kolkata, for the Statistical evaluation. We thank all the study subjects for their participation and cooperation during the study and thanks to Ramesh Vajroju for helping in sample collection. References [1] Watkins PJ. Natural History of the Diabetic Neuropathies. QJM 1990;77:1209–18. [2] Dyck PJ, Kratz KM, Karnes JL, Litchy WJ, Klein R, Pach JM. The prevalence by staged severity of various types of diabetic neuropathy, retinopathy, and nephropathy in a populationbased cohort: the rochester diabetic neuropathy study. Neurology 1993;43:817–24. [3] Miesse AM, Willey JS, Bateman TA. Potential role of proinflammatory cytokines in nerve damage related bone loss. Biomed Sci Instrum 2004;40:266–71. [4] Manandhar R, Shrestha N, Butlin CR, Roche PW. High levels of inflammatory cytokines are associated with poor clinical response to steroid treatment and recurrent episodes of type 1 reactions in leprosy. Clin Exp Immunol 2002;128:333–8. [5] Hutchinson IV, Turner DM, Sankaran D, Awad MR, Sinnott PJ. Influence of cytokine genotypes on allograft rejection. Transplant Proc 1998;30:862–3. [6] Hutchinson, Pravica V, Hajeer A, Sinnott PJ. Identification of high and low responders to allografts. Rev mmunogent 1999;1:323–33. [7] Sankaran D, Asderakis A, Ashraf S, Roberts IS, Dyer PA, et al. Cytokine gene polymorphisms predict acute graft rejection following renal transplantation. Kidney Int 1999;56:281–8. [8] Nagatsu T, Mogi M, Ichinose H, Togari A. Cytokines in Parkinson’s disease. J Neural Trans Suppl 2000;58:143–51. [9] Hans-Peter MD. Immune-mediated demyelination Hartung. Annal Neurol 2004;33:563–7. [10] Koski, Lee Carol. Mechanisms of schwann cell damage in inflammatory neuropathy. J Infect Dis 1997;176(S2):169–72. [11] Jo Satoh, Yagihashi Soroku, Toyota Takyoshi. The possible role of tumor necrosis factor alpha in diabetic neuropathy. Experimental Diab Res 2003;4:65–71. [12] Santos AR, Almeida AS, Suffys PN. Tumor necrosis factor promoter polymorphism (TNF2) seemed to protect against development of severe forms of leprosy in a pilot study in Brazilian patients. Int J Lepr Other Mycobact Dis 2000;68:325–7.

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