Role of Killer-cell Immunoglobulin-like Receptors and Human Leukocyte Antigens in Transmission and Severity of Malaria in Uganda
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Background Malaria is a life-threatening disease caused by Plasmodium falciparum. In 2018, there were an estimated 228 million cases of malaria worldwide and 405,000 malaria deaths. Malaria burden is greatly affected by host immunity, with immune responses to P. falciparum malaria varying between individuals and populations. Genetic diversity in immune system genes like killer-cell immunoglobulin-like receptors (KIR) and human leukocyte antigens (HLA) is likely to play a role in this heterogeneity. Currently, there is limited data about the association between KIR, their major ligands (HLA-C) and risk to malaria. The few studies that have been carried out are limited by small sample sizes, and offer limited genetic information about KIR and HLA genes. As an alternative approach, we used more comprehensive KIR and HLA-C1 and C2 genotyping techniques to gain deeper insights into the genetic diversity of KIR and their HLA-C ligands in humans living in regions with historically varied malaria transmission intensity, and the association of KIR and their HLA-C ligands with malaria severity in Uganda. The specific objectives of this thesis were: 1. To determine genetic diversity of KIR and copy number variation (CNV) in KIR genes in Ugandan populations with historically varied malaria transmission intensity. 2. To determine the prevalence of HLA-C1 and C2 allotypes in Ugandan populations with historically varied malaria transmission intensity. 3. To determine the association of KIR genetic variants and CNV in KIR genes with malaria severity in Ugandan children. 4. To determine the association of HLA-C1 and C2 allotypes with malaria severity in Ugandan children. Methods: We used high-throughput multiplex quantitative real-time PCR to genotype KIR genetic variants and CNV and developed a high-throughput real-time quantitative PCR method to genotype HLA-C1 and C2 allotypes. To address objectives I and 2, we utilized archived samples of 1,344 participants, aged 6 months to 10 years from Ugandan populations with historically high (Nagongera, Tororo District), medium (Walukuba, Jinja District) and low (Kihihi, Kanungu District) malaria transmission intensity. Chi-square and Mid-P exact tests were used to determine differences in the distribution of KIR and HLA-C genetic variants in the three populations. To address objectives 3 and 4, we used conditional logistic regression to determine the associations between KIR genetic variants and their HLA-C ligands and risks of uncomplicated and severe malaria in Ugandan children by utilizing archived samples of a matched case control study including 325 children with severe malaria, 325 children with uncomplicated malaria and 325 children with asymptomatic parasitemia. Results: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6% vs. 13.2%: p=0.006, 57.2% vs. 66.4%: p=0.005, 33.2% vs. 46.6%: p<0.001 and 19.7% vs. 26.7%: p=0.014 respectively) or Jinja (7.6% vs.18.1%: p<0.001, 57.2% vs. 63.8%: p=0.048, 33.2% vs. 43.5%: p=0.002 and 19.7% vs. 30.4%: p<0.001 respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p=0.043. The KIR AA genotype was protective from severe malaria compared to asymptomatic parasitemia (OR 0.66 (0.47 -0.94); p=0.015). HLA-C2 homozygosity was protective from severe malaria compared to asymptomatic parasitemia (OR 0.57 (0.39 - 0.83); p=0.002). A combination of KIR-AA and HLA-C2C2 genotypes protected children from severe malaria compared to uncomplicated malaria (OR 0.46 (0.23 - 0.91); p=0.015) or asymptomatic parasitemia (OR 0.30 (0.16 - 0.54); P<0.001). KIR2DS2 and KIR2DL2 genes increased the risk of severe malaria compared to asymptomatic parasitemia (OR 1.54 (1.10 - 2.15); p=0.008 and OR 1.49 (1.07 - 2.08); p=0.014 respectively).The KIR2DL5 gene increased the risk of uncomplicated and severe malaria compared to asymptomatic parasitemia (OR 1.68 (1.17 - 2.43); p=0.004 and 1.87 (1.33 - 2.63); p<0.001 respectively). Increase in CNV of KIR2DL2 and KIR2DS2 was associated with increased risk to severe malaria compared to asymptomatic parasitemia (p=0.04 and 0.036 respectively). Increase in CNV of KIR2DL1 was associated with protection from severe malaria compared to uncomplicated malaria (p=0.003). Conclusions: The KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes are potentially beneficial in malaria as these genes have been positively selected for in places with historically high malaria transmission intensity. The association of KIR AA and HLA-C2C2 genotypes with protection from severe malaria may be the result of the trade-off for the selection of genetic variants that protect against severe malaria, but increase the risk of other diseases or syndromes. This may explain the increased burden of preeclampsia in this population, which is increased in mothers with KIR AA haplotypes carrying an HLA-C2 foetus. The high-throughput HLA-C genotyping method we have developed will be useful in disease association studies involving large cohorts.