engleski

Association of TNFa and PTPN22 gene polymorphisms in type I diabetes in Croatian patients

INTRODUCTION Type I diabetes is autoimmune disease characterized by activated T-cell-mediated destruction of the pancreatic &#946; -cells. Polygenic component of the disease has been supported by identification of major susceptibility alleles in HLA class, insulin and CTLA-4 loci. However, single nucleotide polymorphism in TNF-&#945; and PTPN22 genes has been recently identified as susceptibility factor for inflammatory autoimmune disease. Variants of TNF-alpha and PTPN22 genes have been investigated in diabetic patients of other ethnic groups, but there is no existing data for Croatian patients. We used association study to evaluate relationship between type I diabetes and single nucleotide polymorphisms in TNF-&#945; and PTPN22 genes. MATERIAL AND METHODS Subjects The study included 102 patients with type I diabetes. Patients were carefully assessed and categorized as per recent classification by the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, 2001. All patients were recruited from Vuk Vrhovac University Clinic Zagreb, after giving written informed consent for the participation. The control subjects included 154 unrelated, healthy volunteers, obtained from the Croatian Tumor and DNA bank. DNA extraction from venous blood was performed using sodium chloride method. Single nucleotide polymorphisms The genotyping of TNF-&#945; G-238A, G-308A and PTPN22 C1858T variants was determined by PCR-RFLP method. Amplified products were digested overnight with restriction enzymes. NcoI and MspI were used for detection G to A transition in promoter of TNF-&#945; gene (uncut A allele and cut G allele). RsaI enzyme is used for detection C to T nucleotide variants in coding region of PTPN22 gene (uncut T allele and cut C allele). Digested products were separated by electrophoresis on 10% polyacrilamide gel and stained by silver. Statistical analysis The distribution of tested polymorphisms was compared between patients and healthy controls by the X2 test. A p value &#8804; 0.05 was considered significant. RESULTS The frequency of AG/AA genotype and A allele at TNF-&#945; -308 site was significantly higher in patients with type I diabetes than in controls (X2=66.27607, df=2, p<0.001 ; X2=37.08470, df=1, p<0.001, respectively). The frequency of AG/GG genotype and A allele at TNF-&#945; -238 site was not significantly different between these two groups. The frequency of CT/TT genotypes as well as T allele at 1858 position in PTPN22 gene was significantly higher in patients in comparison to healthy volunteers (X2=33.70588, df=2, p<0.001 ; X2=30.61254, df=1, p<0.001, respectively). CONCLUSION These results present evidence of association between TNF-&#945; A-308G and PTPN22 C1858T genetic polymorphisms and type I diabetes in Croatian population.

Type I diabetes; polymorphisms; TNFa

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