Association of protein tyrosine phosphatase non-receptor type 22 gene functional variant C1858T, HLA-DQ/DR genotypes and autoantibodies with susceptibility to type-1 diabetes mellitus in Kuwaiti Arabs

Abstract

The incidence of type-1 Diabetes Mellitus (T1DM) has increased steadily in Kuwait during recent years and it is now considered amongst the high-incidence countries. An interaction between susceptibility genes, immune system mediators and environmental factors predispose susceptible individuals to T1DM. We have determined the prevalence of protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene functional variant (C1858T; R620W, rs2476601), HLA-DQ and DR alleles and three autoantibodies in Kuwaiti children with T1DM to evaluate their impact on genetic predisposition of the disease. This study included 253 Kuwaiti children with T1DM and 214 ethnically matched controls. The genotypes of PTPN22 gene functional variant C1858T (R620W; rs2476601) were detected by PCR-RFLP method and confirmed by DNA sequencing. HLA-DQ and DR alleles were determined by sequence-specific PCR. Three autoantibodies were detected in the T1DM patients using radio-immunoassays. A significant association was detected between the variant genotype of the PTPN22 gene (C1858T, rs2476601) and T1DM in Kuwaiti Arabs. HLA-DQ2 and DQ8 alleles showed a strong association with T1DM. In T1DM patients which carried the variant TT-genotype of the PTPN22 gene, 93% had at least one DQ2 allele and 60% carried either a DQ2 or a DQ8 allele. Amongst the DR alleles, the DR3-DRB5, DR3-3, DR3-4 and DR4-4 showed a strong association with T1DM. Majority of T1DM patients who carried homozygous variant (TT) genotype of the PTPN22 gene had either DR3-DRB5 or DRB3-DRB4 genotypes. In T1DM patients who co-inherited the high risk HLA DQ, DR alleles with the variant genotype of PTPN22 gene, the majority were positive for three autoantibodies. Our data demonstrate that the variant T-allele of the PTPN22 gene along with HLA-DQ2 and DQ8 alleles constitute significant determinants of genetic predisposition of T1DM in Kuwaiti children.

Fig 1. Detection of PTPN22 gene functional variant [C1858T; rs2476601] genotypes.

PCR amplification of genomic DNA was carried out (details given in Patients and methods) and products of amplification were cleaved with restriction enzyme RsaI. Lane 1, phiX174 HaeIII cut M_r markers; lane 2, products from subjects having TT genotype; lanes 3–5, products from subjects having CT genotype; lane 6, products from subject with CC genotype. The numbers on the side are sizes of the characteristic bands resulting from PCR-RFLP analysis of subjects with different genotypes. The restriction enzyme cleavage products were analyzed on 2% agarose gel and visualized under UV light after staining with ethidium bromide.

Fig 2. Frequency of HLA-DQ genotypes in Kuwaiti T1DM patients.

Fig 3. Frequency of HLA-DR genotypes in Kuwaiti T1DM patients.

Fig 4. Association of HLA-DQ genotypes with genotypes of PTPN22 gene (C1858T, rs2476601) functional variant in Kuwaiti T1DM patients.

Fig 5. Association of HLA-DR genotypes with genotypes of PTPN22 gene (C1858T, rs2476601) functional variant in Kuwaiti T1DM patients.

Fig 6. The correlation of HLA-DQ allele frequency in Kuwaiti T1DM patients with three autoantibodies.

A) Islet-cell autoantibody; B) GADA autoantibody; C) INS autoantibody.

Fig 7. The correlation of HLA-DR allele frequency in Kuwaiti T1DM patients with three autoantibodies.

A) Islet-cell autoantibody; B) GADA autoantibody; C) INS autoantibody.