Abstract
Killer cell immunoglobulin-like receptor (KIR) genes are expressed by natural killer cells and encoded by a family of genes exhibiting considerable haplotypic and allelic variation. HLA-C molecules, the dominant ligands for KIR, are present in all individuals and are discriminated by two KIR epitopes, C1 and C2. We studied the frequencies of KIR genes and HLA-C1 and C2 groups in a large cohort (n = 492) from Kampala, Uganda, East Africa and compared our findings with published data from other populations in sub-Saharan Africa (SSA) and several European populations. We find considerably more KIR diversity and weaker linkage disequilibrium in SSA compared to the European populations and describe several novel KIR genotypes. C1 and C2 frequencies were similar to other SSA populations with a higher frequency of the C2 epitope (54.9 %) compared to Europe (average 39.7 %). Analysis of this large cohort from Uganda in the context of other African populations reveals variations in KIR and HLA-C1 and C2 that are consistent with migrations within Africa and potential selection pressures on these genes. Our results will help understand how KIR/HLA-C interactions contribute to resistance to pathogens and reproductive success.
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Introduction
Killer cell immunoglobulin-like receptor genes (KIR) are expressed by natural killer (NK) cells and function in infection, cancer, and reproduction. KIR bind some human leukocyte antigen (HLA) class I molecules and other unknown ligands, and a balance between activating and inhibitory signals mediated by KIR regulates NK cell functions. Both KIR and HLA genetic regions are highly variable and can be used as tools in anthropological studies (Parham and Moffett 2013) revealing the history of populations, migration, admixture, or pathogen pressure (Hedrick and Thomson 1983; Piazza et al. 1980). African populations have higher levels of genetic diversity (Tishkoff and Verrelli 2003), which has been documented by many studies using mitochondrial DNA and/or Y chromosome markers and neutral markers as well as HLA polymorphisms (Pilkington et al. 2008; Prugnolle et al. 2005; Tishkoff et al. 2009). KIR variability across the African continent is still not well documented despite the burden of infectious disease including HIV/AIDS, malaria, and tuberculosis. In addition, maternal and reproductive health disorders are a major problem in SSA (WHO databases, www.who.int), and KIR and HLA have been associated with these diseases (Chazara et al. 2004). HLA-C alleles were genotyped as either group C1 or C2 allotypes. Genoty** was performed by PCR-SSP using two pairs of primers for both HLA-C allotypes, as previously detailed (Hiby et al. 2008). Extra KIR primers were designed using sequence information from the IPD-KIR database (release 2.4.0) to detect rare alleles of KIR2DS5 and KIR2DL3 (KIR2DS5, 2DS5rev2: TCC AGA GGG TCA CTG GGA and KIR2DL3, 2DL3rev3: AGA CTC TTG GTC CAT TAC CG) (Robinson et al. 2011). Selected samples (based on their unusual KIR gene content) were typed for copy number by multiplexed quantitative PCR for all 16 KIR genes (KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 2DP1, 3DP1, 3DL1, 3DL2, 3DL3, and 3DS1) (Jiang et al. 2012). KIR haplotypes were defined by matrix subtraction of gene copy numbers using previously characterized common and contracted KIR haplotypes using the KIR Haplotype Identifier software (www.bioinformatics.cimr.cam.ac.uk/haplotypes).
Data analysis and statistical methods
KIR gene carrier frequencies (F) and HLA-C1 and C2 group frequencies were calculated by direct counting. KIR gene frequencies were estimated by the formula \( f=1-\sqrt{\left(1-F\right)} \), where F is the KIR gene carrier frequency in the population. Differences between the Ugandan and the UK populations were compared by χ 2. A centered unscaled principal components analysis was conducted as implemented in the adegenet R package (Jombart and Ahmed 2011). A map was generated using GMT using a modified script available online (www.pypop.org/popdata, Owen Solberg) (Wessel and Smith 1991). Pairwise linkage disequilibrium (Wn*) was calculated for the 12 KIR loci (Single et al. 2008). Statistical significance was obtained using Fisher's exact test, as implemented in PyPop (Lancaster et al. 2007). A graphical view of the linkage disequilibrium (LD) from our study data and that of Single et al. (2008) was achieved using Gold (Abecasis and Cookson 2000).
Results and discussion
KIR and HLA-C group carrier frequencies
We first compared the carrier frequencies of the 12 KIR genes in the Ugandan population with frequencies in the UK cohort (Table 1). The Ugandan population showed marked differences in individual KIR gene frequencies; in particular, KIR3DS1 and KIR2DS1, both associated with the KIR telomeric B region, were significantly less frequent. Additionally, comparatively higher frequencies of KIR2DL2, KIR2DL5, and KIR2DS5 and lower frequencies of KIR2DL3 and KIR2DS3 were seen (KIR2DS3 and KIR2DS5 are alleles of the same gene). Increased KIR2DL2 and decreased KIR2DL3 frequency suggests that the centromeric B region is abundant in the Ugandan population. KIR2DS5 can be found on either the centromeric or telomeric B regions in Africans, similarly to KIR2DL5 in both European and African populations, making precise interpretation of centromeric B frequencies difficult (Hou et al. 2010). KIR gene frequencies in the Uganda population are similar to those reported for the South African Xhosa and San population (Middleton et al. 2007) indicating that African populations have lower frequencies of the telomeric B region and higher frequencies of the centromeric B region compared with European populations.
We compared these results with data from a range of African populations where frequencies for KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, and KIR3DS1 have been reported (Table 2). Of note is that the sample size was under 100 in 19 out of 22 studies. All African populations share similar characteristics compared to UK populations with lower KIR2DS1 and KIR3DS1 carrier frequencies; the highest value for KIR2DS1 was 0.28 (Congo Mbuti) and 0.23 for KIR3DS1 (Ethiopian) compared with, respectively, 0.42 and 0.44 for the UK population. The variability of these frequencies between the African populations may be visualized using principal component analysis (Fig. 1). The Ugandan population localizes to an intermediate position, between Central and West African populations and the East African populations. Therefore, in terms of KIR gene frequencies, the Ugandan population can be regarded as intermediate to these populations. The history of human migration in Africa is consistent with this observation because Uganda is an East African country known to have been populated from the Bantu migrations from West and Central Africa (Campbell and Tishkoff 2008). Furthermore, it is in both the northern and southern hemispheres, being situated on the equatorial line.
Previous reports have revealed extensive allelic diversity at individual KIR loci in African populations. To ensure all variants were detected, we used additional new primers that were added when positive results were obtained with only one pair of primers out of the two classically used or when the individual KIR gene content was unusual. Allele “dropout” was most commonly suspected for KIR2DL3, KIR2DS1, and KIR2DS5. This could be seen where the allele occurred in the hemizygous or homozygous state and was detected in 2.4 % of the individuals. For KIR2DS5, this was usually observed on a contracted haplotype. Without using additional primers, published results for other African populations may not have detected these unusual alleles.
Analysis of the KIR2DL1 and KIR2DS1 ligand, HLA-C alleles encoding the C2 allotype, reveals that in general C2 are more frequent than C1 alleles in Africa compared with elsewhere in the world (Fig. 2). The Ugandan population, with a C2 frequency of 54.9 % and a C1 frequency of 45.1 % is similar to the neighboring populations such as Kenyan Luo (C2 = 59 % and C1 = 41 %) or Rwandan (C2 = 55 % and C1 = 45 %) (Cao et al. 2004; Tang et al. 2000). The distribution of the C2 group frequencies shows limited variation across the continent (Fig. 2). Therefore, in contrast to its activating receptor, KIR2DS1, African populations have high frequencies of HLA-C2, with comparatively little variation, in accordance with previous reports (Single et al. 2007). There must be strong selective pressures within Africa, either from pathogens or reproductive success that are maintaining this high level of C2.
KIR genotypes
Genoty** of the entire cohort for the presence/absence of the 12 variable KIR genes allowed the definition of individual KIR genotypes. Fifty-two KIR gene content profiles were observed in the Ugandan population (Fig. 3). Nineteen genotypes had a frequency above 1 %, accounting for 90.0 % of individuals. Half of the individuals were classified as Ganda (both parents speaking the Ganda language, 50.9 %), but the other groups were broadly similar in KIR genotype frequencies (Table S 1). As in other African populations, the most frequent KIR genotype in the Ugandan population is homozygosity for the KIR A haplotype (28.1 %). The reported frequencies for this KIR AA genotype in Africa are variable, from 12 % in a South Africa Xhosa population to 42 % in Senegal (Williams et al. 2004; Yindom et al. 2010).
The remaining 33 genotypes are rare (<1 %), comprising 9.9 % of the total population. These represent unusual genotypes, including contracted or extended KIR haplotypes containing an unusual combination of KIR genes. In order to investigate these haplotypes, which arise from ancestral unequal recombination events, 15 individuals were fully typed for KIR gene copy number. Firstly, different regions of KIR3DL1 (exons 4 and 9) and of KIR3DL2 (exons 4 and 9) were separately typed for copy number to detect the KIR3DL1/3DL2 hybrid gene (Shilling et al. 2002). Nine out of 15 individuals had one or two copies of this telomeric region (tA01-hybd1; individuals 1–6 and 13–15, Fig. 4). Two additional genotypes (numbers 438 and 252/72/83, Fig. 3) have KIR3DL1 present without KIR2DS4, and these individuals are also likely to have the same contracted telomeric region (tA01-hybd1). This contracted haplotype was observed in this Ugandan cohort at an underestimated frequency of 1.7 %, which is lower than the 6.5 % from other African populations (Norman et al. 2009; Norman et al. 2007), because the common telomeric A region (tA01) will hide this deletion, as seen with individual 6. Secondly, nine different individuals (individuals 7–15) have one or two copies of other contracted haplotypes defined by a deletion of the central framework genes, KIR3DP1, KIR2DL4, and KIR3DS1, defined as del6 (Pyo et al. 2010) and previously reported in individuals of African ancestry (Gómez-Lozano et al. 2003; Norman et al. 2002). Using these individuals and the similar KIR genotypes with KIR2DS1 present without KIR3DS1, the frequency of this deletion in Uganda is greater than 7.25 %.
More KIR profiles were found in the Ugandan cohort (52 genotypes) when compared to a similar sized cohort of women from the UK (40 genotypes) (Fig. 5). The two populations only shared 25 genotypes present in 72.6 % of Ugandans and 88.3 % of UK. Similarly to the six other African populations (from the Allele Frequency Net database: Gabon, Ghana, Senegal, South Africa San, and South Africa Xhosa), the Ugandan population displays a higher diversity at the genotype level, and more than a quarter of these genotypes are not observed in Europeans. Forty-eight genotypes are unique to the African populations (including Uganda) and therefore might be African genotypes that have not spread out of Africa to Europe.
Linkage disequilibrium
The LD between KIR genes was calculated for the 12 KIR loci in order to observe the pairwise association of genes in this population (Table 3). The strongest positive and negative LD values were observed between KIR2DL1 and KIR2DP1 (0.88) and KIR2DL2 and KIR2DL3 (−0.35), consistent with a previous report for six African populations (Single et al. 2008). A graphical analysis of Ugandan compared to these African populations and seven European populations is shown (Fig. 6). The positive LD between KIR2DS1, KIR2DS5, and KIR3DS1 observed in Europeans is absent in Africans; in contrast, a positive association is seen between KIR2DS5 and KIR2DS2-KIR2DL2, consistent with the location of KIR2DS5 gene in the centromeric B region where KIR2DS3 is generally located in Europeans. Overall, the LD values observed for the KIR genes are less extreme in Africa.
Conclusions
This Ugandan cohort is the largest African population typed for KIR genes, and when considered with other published data, the remarkable extent of KIR diversity in Africa is clear. The KIR and HLA-C frequencies observed in this population are consistent with other studies of African populations. A number of novel KIR gene profiles are described, including new genotypes and rare patterns. This East African population will be an important reference group for future disease and anthropological studies in African or African-derived populations. Our study has not considered the extensive allelic variation seen in Africans, for example, for KIR3DL1 or KIR2DL1, where the great KIR diversity will become even more apparent.
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Acknowledgments
We thank all the clinical and laboratory staff who acquired the DNA samples: Margaret Sewagaba, Florence Mugema, Dorothy Mugabi, Anastanzia Karungi, and Prossy Namukwaya. We are grateful to all study participants for voluntary consent. We also thank Paul Norman for his helpful comments on the draft manuscript and Jill Hollenbach for her help with the HLA-C frequency map. This work was funded by the Wellcome Trust (090108/Z/09/Z, 085992/Z/08/Z, and a vacation scholarship in 2011), the British Heart Foundation (PG/09/077/27964), the Center for Trophoblast Research at the University of Cambridge, and a Wellcome Trust Uganda PhD Fellowship in Infection and Immunity held by Annettee Nakimuli, funded by a Wellcome Trust Strategic Award, grant number 084344.
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The authors have declared that no competing interests exist.
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Annettee Nakimuli and Olympe Chazara contributed equally to this work.
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Nakimuli, A., Chazara, O., Farrell, L. et al. Killer cell immunoglobulin-like receptor (KIR) genes and their HLA-C ligands in a Ugandan population. Immunogenetics 65, 765–775 (2013). https://doi.org/10.1007/s00251-013-0724-7
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DOI: https://doi.org/10.1007/s00251-013-0724-7