The role of vitamin D in the aetiology of type 1 diabetes mellitus in the South African black population
Background: Type 1 diabetes (T1D) is a condition associated with the autoimmune mediated destruction of the pancreatic β-cells. Individuals who develop T1D often express autoantibodies (AAbs) which target β-cell epitopes (such as the 65kDa isoform of glutamic acid decarboxylase (GAD65), protein tyrosine phosphatase related islet antigen 2 (IA-2) and Zinc transporter 8 (ZnT8)). Recently, vitamin D3 has been shown to play an immunomodulatory role in T1D. Vitamin D3 is formed by two successive hydroxylation steps in the liver and kidney by the enzymes encoded by the CYP2R1 and CYP27B1 genes, respectively. Vitamin D3 exerts its effect through the vitamin D receptor (VDR), a ligand activated transcription factor. Activation of the VDR, through binding to vitamin D3 mediates the suppression of pro-inflammatory cytokines which inhibit proliferation of Th1 cells. Th1 cells are responsible for the death of the βcells. Four VDR single nucleotide polymorphisms (SNPs; BsmI [rs1544410], FokI [rs2228570], ApaI [rs7975232], TaqI [rs731236]) have been shown to be associated with the development of T1D however, these findings are not consistent. In addition, polymorphisms in the CYP2R1 (rs10741657) and CYP27B1 (rs10877012) genes have been shown to be associated with susceptibility to T1D. To our knowledge there is no data looking at these associations in the black South African population. This study, therefore, aimed to determine the prevalence of polymorphisms in the CYP and VDR genes and relate the allelic frequencies to serum levels of vitamin D3 and T1D disease status in the South African black population. In addition, we aimed to determine GAD65, IA-2 and ZnT8 AAb positivity within the black South African population. Methods: Clinically diagnosed black T1D patients (n=186) and non-diabetic black control participants (n=153) were recruited. All participants were genotyped for the four VDR SNPs and two CYP SNPs using PCR-RFLP. Vitamin D3 was measured by HPLC using the ClinRep High Performance Liquid Chromatography. GAD65, IA-2 and ZnT8 AAbs were measured by ELISA and glucose was measured on the Advia Chemistry System. Results: In our cohort the mean age at diagnosis was 20.7 ± 8.4 years with a median duration of 7 [2; 11] years. The VDR and CYP gene SNPs were not associated with T1D in the South African black population. Similarly, low levels of vitamin D3 were not associated with the disease. Multiple regression analysis showed that vitamin D3 levels were 5.34 nmol/L lower in individuals carrying the FokI CC genotype compared to individuals with CT/TT genotypes (p = 0.021). In addition, an increasing number of VDR risk alleles were associated with lower vitamin D3 levels (0 risk alleles: 66.53 ± 23.00 nmol/L vs. 8 risk alleles: 47.30 ± 14.87 nmol/L; p = 0.006). In our T1D patients 55.6% were AAb positive (GAD65 = 51%, IA-2 = 12.9% and ZnT8 = 17.6%). IA-2 and ZnT8 AAb positivity was associated with a significantly younger age at diagnosis (16.40 ± 5.53 vs. 21.62 ± 8.56 years; p < 0.001 and 17.00 ± 6.49 vs. 21.48 ± 8.50 years; p = 0.006 respectively) and shorter duration of disease (p = 0.032 and p = 0.008, respectively). GAD65 positive individuals had a 23-fold higher risk of developing T1D (p < 0.001). Conclusion: We found no association between the VDR and CYP gene polymorphisms in the black South African population. However, we did find that the FokI CC genotype was associated with lower levels of vitamin D3. Whilst AAbs are markers of T1D, AAb negativity is not sufficient to exclude a T1D diagnosis in our cohort. AAb positive patients were more likely to develop T1D at an earlier age than those who were AAb negative. The South African black population has an average age at diagnosis a decade later than that seen in white populations. In addition, the frequency of AAbs was lower than that observed in white populations. These findings suggest South African black T1D patients have a unique disease aetiology.
A dissertation submitted to the faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science in Medicine. Johannesburg, 2018