High-throughput high-resolution class I HLA genotyping in East Africa

Abstract

HLA, the most genetically diverse loci in the human genome, play a crucial role in host-pathogen interaction by mediating innate and adaptive cellular immune responses. A vast number of infectious diseases affect East Africa, including HIV/AIDS, malaria, and tuberculosis, but the HLA genetic diversity in this region remains incompletely described. This is a major obstacle for the design and evaluation of preventive vaccines. Available HLA typing techniques, that provide the 4-digit level resolution needed to interpret immune responses, lack sufficient throughput for large immunoepidemiological studies. Here we present a novel HLA typing assay bridging the gap between high resolution and high throughput. The assay is based on real-time PCR using sequence-specific primers (SSP) and can genotype carriers of the 49 most common East African class I HLA-A, -B, and -C alleles, at the 4-digit level. Using a validation panel of 175 samples from Kampala, Uganda, previously defined by sequence-based typing, the new assay performed with 100% sensitivity and specificity. The assay was also implemented to define the HLA genetic complexity of a previously uncharacterized Tanzanian population, demonstrating its inclusion in the major East African genetic cluster. The availability of genotyping tools with this capacity will be extremely useful in the identification of correlates of immune protection and the evaluation of candidate vaccine efficacy.

Figure 1. Distribution of Class I HLA-A, -B, and -C allele frequencies in East Africa.

For each locus, cumulative frequencies of reported alleles in Kenyan Luo , Kenyan Nandi , and Ugandan populations are depicted in decreasing frequency order. Solid dots represent the median of the cumulative frequencies among the three populations, and the error bars represent their range. HLA alleles that provide a population coverage of 80–90% and were selected as the target of the current assay (see text for details) are boxed by a dotted line and listed in the insets. Only alleles that have been reported in at least one of the three East African populations were included in the analysis.

Figure 2. Novel sequence-specific primer (SSP) real-time PCR-based genotyping assay for HLA-A, -B, and -C in East African populations.

The layout of the assay is here exemplified for the HLA-A locus, but proceeds similarly for the HLA-B and -C loci. a) After PCR amplification of a genomic region encompassing exons 2 through 3 of HLA-A using locus-specific primers, the amplicon was distributed in 20 separate multiplex SSP real-time PCRs containing sequence-specific primers (colored arrows), variation-insensitive primers (black arrows), and universal fluorescent TaqMan probes (colored stars). Sequence-specific and variation-insensitive primers targeted areas of converse exons. Sequence-specific primers were designed to more efficiently amplify defined targeted alleles (noted next to each reaction). Variation-insensitive primers were used to allow for internal standardization. b) The cross threshold (Ct) values obtained by monitoring amplification with the sequence-specific and internal standardization reagents were then used to assign samples positive or negative reactivities in each reaction (exemplified in the inset by results from reaction HLA-A018). The aggregate reactivity patterns rendered by the array of reactions were used to define the presence or absence of the addressed alleles. See text for details.