Genomic characterization of HLA class I and class II genes in ethnically diverse sub-Saharan African populations: A report on novel HLA alleles.

HLA allelic variation has been well studied and documented in many parts of the world. However, African populations have been relatively under-represented in studies of HLA variation. We have characterized HLA variation from 489 individuals belonging to 13 ethnically diverse populations from rural communities from the African countries of Botswana, Cameroon, Ethiopia, and Tanzania, known to practice traditional subsistence lifestyles using next generation sequencing (Illumina) and long-reads from Oxford Nanopore Technologies. We identified 342 distinct alleles among the 11 HLA targeted genes: HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1, and -DPB1, with 140 of those alleles containing novel sequences that were submitted to the IPD-IMGT/HLA database. Sixteen of the 140 alleles contained novel content within the exonic regions of the genes, while 110 alleles contained novel intronic variants. Four alleles were found to be recombinants of already described HLA alleles and 10 alleles extended the sequence content of already described alleles. All 140 alleles include complete allelic sequence from the 5' UTR to the 3' UTR that are inclusive of all exons and introns. This report characterizes the HLA allelic variation from these individuals and describes the novel allelic variation present within these specific African populations.

The genetic and evolutionary basis of gene expression variation in East Africans.

BACKGROUND: Mapping of quantitative trait loci (QTL) associated with molecular phenotypes is a powerful approach for identifying the genes and molecular mechanisms underlying human traits and diseases, though most studies have focused on individuals of European descent. While important progress has been made to study a greater diversity of human populations, many groups remain unstudied, particularly among indigenous populations within Africa. To better understand the genetics of gene regulation in East Africans, we perform expression and splicing QTL mapping in whole blood from a cohort of 162 diverse Africans from Ethiopia and Tanzania. We assess replication of these QTLs in cohorts of predominantly European ancestry and identify candidate genes under selection in human populations. RESULTS: We find the gene regulatory architecture of African and non-African populations is broadly shared, though there is a considerable amount of variation at individual loci across populations. Comparing our analyses to an equivalently sized cohort of European Americans, we find that QTL mapping in Africans improves the detection of expression QTLs and fine-mapping of causal variation. Integrating our QTL scans with signatures of natural selection, we find several genes related to immunity and metabolism that are highly differentiated between Africans and non-Africans, as well as a gene associated with pigmentation. CONCLUSION: Extending QTL mapping studies beyond European ancestry, particularly to diverse indigenous populations, is vital for a complete understanding of the genetic architecture of human traits and can reveal novel functional variation underlying human traits and disease.

Signatures of Convergent Evolution and Natural Selection at the Alcohol Dehydrogenase Gene Region are Correlated with Agriculture in Ethnically Diverse Africans.

The alcohol dehydrogenase (ADH) family of genes encodes enzymes that catalyze the metabolism of ethanol into acetaldehyde. Nucleotide variation in ADH genes can affect the catalytic properties of these enzymes and is associated with a variety of traits, including alcoholism and cancer. Some ADH variants, including the ADH1B*48His (rs1229984) mutation in the ADH1B gene, reduce the risk of alcoholism and are under positive selection in multiple human populations. The advent of Neolithic agriculture and associated increase in fermented foods and beverages is hypothesized to have been a selective force acting on such variants. However, this hypothesis has not been tested in populations outside of Asia. Here, we use genome-wide selection scans to show that the ADH gene region is enriched for variants showing strong signals of positive selection in multiple Afroasiatic-speaking, agriculturalist populations from Ethiopia, and that this signal is unique among sub-Saharan Africans. We also observe strong selection signals at putatively functional variants in nearby lipid metabolism genes, which may influence evolutionary dynamics at the ADH region. Finally, we show that haplotypes carrying these selected variants were introduced into Northeast Africa from a West-Eurasian source within the last ∼2,000 years and experienced positive selection following admixture. These selection signals are not evident in nearby, genetically similar populations that practice hunting/gathering or pastoralist subsistence lifestyles, supporting the hypothesis that the emergence of agriculture shapes patterns of selection at ADH genes. Together, these results enhance our understanding of how adaptations to diverse environments and diets have influenced the African genomic landscape.

Redondovirus Diversity and Evolution on Global, Individual, and Molecular Scales.

Redondoviridae is a newly established family of circular Rep-encoding single-stranded (CRESS) DNA viruses found in the human ororespiratory tract. Redondoviruses were previously found in ∼15% of respiratory specimens from U.S. urban subjects; levels were elevated in individuals with periodontitis or critical illness. Here, we report higher redondovirus prevalence in saliva samples: four rural African populations showed 61 to 82% prevalence, and an urban U.S. population showed 32% prevalence. Longitudinal, limiting-dilution single-genome sequencing revealed diverse strains of both redondovirus species (Brisavirus and Vientovirus) in single individuals, persistence over time, and evidence of intergenomic recombination. Computational analysis of viral genomes identified a recombination hot spot associated with a conserved potential DNA stem-loop structure. To assess the possible role of this site in recombination, we carried out in vitro studies which showed that this potential stem-loop was cleaved by the virus-encoded Rep protein. In addition, in reconstructed reactions, a Rep-DNA covalent intermediate was shown to mediate DNA strand transfer at this site. Thus, redondoviruses are highly prevalent in humans, found in individuals on multiple continents, heterogeneous even within individuals and encode a Rep protein implicated in facilitating recombination. IMPORTANCERedondoviridae is a recently established family of DNA viruses predominantly found in the human respiratory tract and associated with multiple clinical conditions. In this study, we found high redondovirus prevalence in saliva from urban North American individuals and nonindustrialized African populations in Botswana, Cameroon, Ethiopia, and Tanzania. Individuals on both continents harbored both known redondovirus species. Global prevalence of both species suggests that redondoviruses have long been associated with humans but have remained undetected until recently due to their divergent genomes. By sequencing single redondovirus genomes in longitudinally sampled humans, we found that redondoviruses persisted over time within subjects and likely evolve by recombination. The Rep protein encoded by redondoviruses catalyzes multiple reactions in vitro, consistent with a role in mediating DNA replication and recombination. In summary, we identify high redondovirus prevalence in humans across multiple continents, longitudinal heterogeneity and persistence, and potential mechanisms of redondovirus evolution by recombination.

Genomic evidence for shared common ancestry of East African hunting-gathering populations and insights into local adaptation.

Anatomically modern humans arose in Africa ∼300,000 years ago, but the demographic and adaptive histories of African populations are not well-characterized. Here, we have generated a genome-wide dataset from 840 Africans, residing in western, eastern, southern, and northern Africa, belonging to 50 ethnicities, and speaking languages belonging to four language families. In addition to agriculturalists and pastoralists, our study includes 16 populations that practice, or until recently have practiced, a hunting-gathering (HG) lifestyle. We observe that genetic structure in Africa is broadly correlated not only with geography, but to a lesser extent, with linguistic affiliation and subsistence strategy. Four East African HG (EHG) populations that are geographically distant from each other show evidence of common ancestry: the Hadza and Sandawe in Tanzania, who speak languages with clicks classified as Khoisan; the Dahalo in Kenya, whose language has remnant clicks; and the Sabue in Ethiopia, who speak an unclassified language. Additionally, we observed common ancestry between central African rainforest HGs and southern African San, the latter of whom speak languages with clicks classified as Khoisan. With the exception of the EHG, central African rainforest HGs, and San, other HG groups in Africa appear genetically similar to neighboring agriculturalist or pastoralist populations. We additionally demonstrate that infectious disease, immune response, and diet have played important roles in the adaptive landscape of African history. However, while the broad biological processes involved in recent human adaptation in Africa are often consistent across populations, the specific loci affected by selective pressures more often vary across populations.

Correction: Evaluation of Direct Colorimetric MTT Assay for Rapid Detection of Rifampicin and Isoniazid Resistance in Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0169188.].

Evaluation of Direct Colorimetric MTT Assay for Rapid Detection of Rifampicin and Isoniazid Resistance in Mycobacterium tuberculosis.

With the spread of multidrug-resistant tuberculosis (MDR-TB) strains there is an increasing need for new accurate and cost-effective methods for a rapid diagnostic and drug susceptibility testing (DST), particularly in low-income countries where tuberculosis is hyperendemic. A colorimetric assay using 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) has been suggested as a promising method for DST, especially to rifampicin. In this study, we standardized and evaluated the MTT assay for a rapid direct detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains from sputum specimens using Lowenstein-Jensen (LJ) culture medium as a gold standard. The MTT assay sensitivity, specificity, positive and negative predictive values for rifampicin were 100%, 86%, 100%, 99%, respectively. For isoniazid, the MTT assay had a 100% sensitivity, specificity, positive and negative predictive values. Interestingly, the MTT assay gave interpretable results within two weeks for 94% of the samples compared to 7-14 weeks for LJ media. Overall, an excellent agreement was observed between MTT assay and LJ proportion method (Kappa, 0.91 for rifampicin and 1.00 for isoniazid). In conclusion, the direct colorimetric MTT assay simultaneously detects susceptible and resistant strains of M. tuberculosis within three weeks. It significantly shortens the time required to obtain a DST result and could be a reliable alternative method for rapid detection of drug-resistant TB strains in high-TB-burden resource-limited settings.

Limited evidence for adaptive evolution and functional effect of allelic variation at rs702424 in the promoter of the TAS2R16 bitter taste receptor gene in Africa.

Bitter taste perception, mediated by receptors encoded by the TAS2R loci, has important roles in human health and nutrition. Prior studies have demonstrated that nonsynonymous variation at site 516 in the coding exon of TAS2R16, a bitter taste receptor gene on chromosome 7, has been subject to positive selection and is strongly correlated with differences in sensitivity to salicin, a bitter anti-inflammatory compound, in human populations. However, a recent study suggested that the derived G-allele at rs702424 in the TAS2R16 promoter has also been the target of recent selection and may have an additional effect on the levels of salicin bitter taste perception. Here, we examined alleles at rs702424 for signatures of selection using Extended Haplotype Homozygosity (EHH) and FST statistics in diverse populations from West Central, Central and East Africa. We also performed a genotype-phenotype analysis of salicin sensitivity in a subset of 135 individuals from East Africa. Based on our data, we did not find evidence for positive selection at rs702424 in African populations, suggesting that nucleotide position 516 is likely the site under selection at TAS2R16. Moreover, we did not detect a significant association between rs702424 alleles and salicin bitter taste recognition, implying that this site does not contribute to salicin phenotypic variance. Overall, this study of African diversity provides further information regarding the genetic architecture and evolutionary history of a biologically-relevant trait in humans.

Chronic meningitis in immunocompromised adult Ethiopians visiting Tikur Anbessa Teaching Hospital and Ye'huleshet Clinic from 2003-2004.

BACKGROUND: Chronic meningitis is inflammation of the meninges where signs and symptoms develop and last for at least four weeks without alleviation. Little is known about the current etiology and incidence of the disease in adults living in developing countries. OBJECTIVE: The objective of this study was to elucidate the most common etiologies of chronic meningitis in adult Ethiopian patients and give an aid in the empiric therapy. METHODOLOGY: A total of 53 adult patients (median age 32 years) having chronic meningitis and who were admitted at Tikur Anbessa Teaching Hospital and Ye'huleshet Clinic, Addis Ababa, Ethiopia were recruited between 2003 and 2004. Of the 53 patients, bacteriological, molecular and immunological investigations were done for 52 of the study participants to detect Cryptococcus neoformans, Mycobacterium tuberculosis, Toxoplasma gondii, Brucella and Neisseria meningitides infections. RESULTS: Forty eight of the participants were HIV positive and 15% (8/52) of the CSF were positive with Cryptococcal latex antigen detection test; in addition, M. tuberculosis DNA was detected using PCR from CSF ofpatients infew of the patients. Multiple infections were observed in studyparticipants with < 0.1 to 1 CD4 to CD8 ratio. CONCLUSION: Chronic meningitis mostly occurred in HIV infected patients, where most of the infections were attributed to Cryptococcus neoformans whereas M. tuberculosis appeared secondary.

Origin and differential selection of allelic variation at TAS2R16 associated with salicin bitter taste sensitivity in Africa.

Bitter taste perception influences human nutrition and health, and the genetic variation underlying this trait may play a role in disease susceptibility. To better understand the genetic architecture and patterns of phenotypic variability of bitter taste perception, we sequenced a 996 bp region, encompassing the coding exon of TAS2R16, a bitter taste receptor gene, in 595 individuals from 74 African populations and in 94 non-Africans from 11 populations. We also performed genotype-phenotype association analyses of threshold levels of sensitivity to salicin, a bitter anti-inflammatory compound, in 296 individuals from Central and East Africa. In addition, we characterized TAS2R16 mutants in vitro to investigate the effects of polymorphic loci identified at this locus on receptor function. Here, we report striking signatures of positive selection, including significant Fay and Wu's H statistics predominantly in East Africa, indicating strong local adaptation and greater genetic structure among African populations than expected under neutrality. Furthermore, we observed a "star-like" phylogeny for haplotypes with the derived allele at polymorphic site 516 associated with increased bitter taste perception that is consistent with a model of selection for "high-sensitivity" variation. In contrast, haplotypes carrying the "low-sensitivity" ancestral allele at site 516 showed evidence of strong purifying selection. We also demonstrated, for the first time, the functional effect of nonsynonymous variation at site 516 on salicin phenotypic variance in vivo in diverse Africans and showed that most other nonsynonymous substitutions have weak or no effect on cell surface expression in vitro, suggesting that one main polymorphism at TAS2R16 influences salicin recognition. Additionally, we detected geographic differences in levels of bitter taste perception in Africa not previously reported and infer an East African origin for high salicin sensitivity in human populations.

Genetic adaptation to high altitude in the Ethiopian highlands.

BACKGROUND: Genomic analysis of high-altitude populations residing in the Andes and Tibet has revealed several candidate loci for involvement in high-altitude adaptation, a subset of which have also been shown to be associated with hemoglobin levels, including EPAS1, EGLN1, and PPARA, which play a role in the HIF-1 pathway. Here, we have extended this work to high- and low-altitude populations living in Ethiopia, for which we have measured hemoglobin levels. We genotyped the Illumina 1M SNP array and employed several genome-wide scans for selection and targeted association with hemoglobin levels to identify genes that play a role in adaptation to high altitude. RESULTS: We have identified a set of candidate genes for positive selection in our high-altitude population sample, demonstrated significantly different hemoglobin levels between high- and low-altitude Ethiopians and have identified a subset of candidate genes for selection, several of which also show suggestive associations with hemoglobin levels. CONCLUSIONS: We highlight several candidate genes for involvement in high-altitude adaptation in Ethiopia, including CBARA1, VAV3, ARNT2 and THRB. Although most of these genes have not been identified in previous studies of high-altitude Tibetan or Andean population samples, two of these genes (THRB and ARNT2) play a role in the HIF-1 pathway, a pathway implicated in previous work reported in Tibetan and Andean studies. These combined results suggest that adaptation to high altitude arose independently due to convergent evolution in high-altitude Amhara populations in Ethiopia.

Primary drug resistance in newly diagnosed smear positive tuberculosis patients in Addis Ababa, Ethiopia.

BACKGROUND: Drug resistance monitoring is an important aspect of tuberculosis (TB) control. OBJECTIVE: To determine the sensitivity of mycobacterial isolates from newly diagnosed pulmonary TB patients to first line anti-TB drugs in Addis Ababa. METHODS: A survey on primary anti-tuberculosis drug resistance was conducted on smear positive pulmonary TB patients visiting 19 health centers and 3 hospitals in Addis Ababa from September 2004 to December 2005. Sputum was digested and decontaminated using Petroff's method and inoculated on Lowenstein-Jensen (LJ) media. Mycobacterial isolates were tested for sensitivity to isoniazid, rifampicin, ethambutol and streptomycin on Middlebrook 7H10 media using the standard indirect proportion method. RESULTS: Among the M. tuberculosis strains isolated from 173 patients, 21.4% were resistant to at least one drug. Single drug resistance to streptomycin was observed in 16.2%, to isoniazid in 13.3%, to rifampicin in 1.2% and to ethambutol in 3.5% of the isolates. The prevalence of resistance to at least one drug was 15.7% and 23.7% among patients with and without HIV co-infection, respectively (p > 0.05). Only one patient (0.6%) had a multidrug resistant (MDR) strain. However, the prevalence of resistance to more than one drug was 10.4%. CONCLUSION: Although the prevalence of MDR was low in this study, the precursors of MDR are accumulating in Addis Ababa.