Using a genomewide approach, we have identified an association between genetic variants in the HLA class II region harboring HLA-DQA1, HLA-DQB1, and HLA-DRB1 and podoconiosis, a neglected tropical disease affecting an estimated 4 million people worldwide. The most strongly associated SNPs were validated in a set of families by means of family-based association testing, and HLA typing showed that specific HLA alleles and haplotypes were significantly associated with differential risk of disease. We estimated that the HLA SNPs associated with podoconiosis in this study explained 15.6% of the genetic variance in podoconiosis, conferring an increase in risk by a factor of 2 to 3.
The association of HLA class II suggests that podoconiosis is a T-cell–mediated inflammatory disease. The initial trigger for T-cell activation is recognition of an antigenic peptide bound to an HLA molecule on antigen-presenting cells.16
Although HLA class II molecules are central to the presentation of exogenous (i.e., foreign and usually pathogen-derived) antigens, they have also been implicated in diseases triggered by minerals, such as silicosis and berylliosis. In berylliosis, which is associated with HLA-DPGlu69, possible mechanisms of pathogenesis include modification of a self peptide by beryllium, allowing for presentation of the self peptide by HLA class II molecules or direct binding of beryllium to, and alteration of, the HLA-peptide binding pocket.17-21
In podoconiosis, the DRB1*0701, DQA1*0201,
alleles may have a functional role in antigen presentation to T cells, leading to induction of the immune response and development of disease in response to a currently undefined soil antigen or mineral.
HLA genetic data from African populations are scarce. De Bakker and colleagues included samples from the Nigerian Yoruba population in their construction of a high-resolution HLA and SNP haplotype map.22
This involved typing the classical HLA genes and more than 7500 SNPs that were analyzed together to identify informative tag SNPs that captured the variation in the HLA region. Of the HLA SNPs we found to be associated with podoconiosis, only rs106335 has been identified as an informative tag SNP in the Yoruba. The absence of convergence of more than 1 SNP between the two studies may not be surprising, given the wide diversity of African populations in terms of allele frequencies, local linkage-disequilibrium patterns, and extensive population substructure.23,24
To demonstrate this, we compared the allele frequencies and haplotype structure for 8 SNPs within 20 kb of the most strongly associated marker from our study (rs17612858) among two HapMap (release 3.2) populations from Africa: Yoruba from Ibadan, Nigeria, and Maasai from Kinyawa, Kenya. Six of these 8 SNPs were heterozygous; 5 of these 6 had minor allele frequencies that differed significantly between the two populations, as well as different haplotype structures (Table 8 and Fig. 4 in the Supplementary Appendix
). This finding indicates that a single group in Africa cannot be taken as a proxy for predicting HLA alleles of all other populations on the continent, including the Ethiopian Wolaita population.
We selected barefoot, older participants as “supercontrols” — people who had been sufficiently exposed to the environmental risk factor to have podoconiosis develop yet it did not. These controls were included under the assumption that they would possess fewer genetic susceptibility variants than the case patients. Although the use of such controls may introduce bias with respect to other factors, it is most important in a case–control study that the control group is disease-free.25
This approach has been used successfully in other studies,22,26
and was helpful during this discovery (stage 1) genomewide association study to identify the risk variants for podoconiosis. The possibility that variants involved in survival against other diseases may have been overrepresented in the controls is small, because the findings of the family-based association testing corroborated those of the genomewide association study and because HLA has no known effect on longevity.27
Identification of additional genetic risk factors reaching genomewide significance may have been limited by the small sample and the lower information content of the markers included in the SNP array used for our study population of African ancestry, as compared with populations of European ancestry.28,29
Replication of our findings in a larger sample of patients with podoconiosis and controls with the use of population-specific, denser SNP arrays is needed to inform future research directions.