DC-SIGN (CD209), pentraxin 3 and vitamin D receptor gene variants associate with pulmonary tuberculosis risk in West Africans

We investigated the role of DC-SIGN (CD209), long pentraxin 3 (PTX3) and vitamin D receptor (VDR) gene single nucleotide polymorphisms (SNPs) in susceptibility to pulmonary tuberculosis (TB) in 321 TB cases and 347 healthy controls from Guinea-Bissau. Five additional, functionally relevant SNPs within toll-like receptors (TLRs) 2, 4 and 9 were typed but found, when polymorphic, not to affect host vulnerability to pulmonary TB. We did not replicate an association between SNPs in the DC-SIGN promoter and TB. However, we found that two polymorphisms, one in DC-SIGN and one in VDR, were associated in a nonadditive model with disease risk when analyzed in combination with ethnicity (P=0.03 for DC-SIGN and P=0.003 for VDR). In addition, PTX3 haplotype frequencies significantly differed in cases compared to controls and a protective effect was found in association with a specific haplotype (OR 0.78, 95% CI 0.63-0.98). Our findings support previous data showing that VDR SNPs modulate the risk for TB in West Africans and suggest that variation within DC-SIGN and PTX3 also affect the disease outcome.     

Impact of natural selection on global patterns of genetic variation and association with clinical phenotypes at genes involved in SARS-CoV-2 infection

Human genomic diversity has been shaped by both ancient and ongoing challenges from viruses. The current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a devastating impact on population health. However, genetic diversity and evolutionary forces impacting host genes related to SARS-CoV-2 infection are not well understood. We investigated global patterns of genetic variation and signatures of natural selection at host genes relevant to SARS-CoV-2 infection (angiotensin converting enzyme 2 [ACE2], transmembrane protease serine 2 [TMPRSS2], dipeptidyl peptidase 4 [DPP4], and lymphocyte antigen 6 complex locus E [LY6E]). We analyzed data from 2,012 ethnically diverse Africans and 15,977 individuals of European and African ancestry with electronic health records and integrated with global data from the 1000 Genomes Project. At ACE2, we identified 41 nonsynonymous variants that were rare in most populations, several of which impact protein function. However, three nonsynonymous variants (rs138390800, rs147311723, and rs145437639) were common among central African hunter-gatherers from Cameroon (minor allele frequency 0.083 to 0.164) and are on haplotypes that exhibit signatures of positive selection. We identify signatures of selection impacting variation at regulatory regions influencing ACE2 expression in multiple African populations. At TMPRSS2, we identified 13 amino acid changes that are adaptive and specific to the human lineage compared with the chimpanzee genome. Genetic variants that are targets of natural selection are associated with clinical phenotypes common in patients with COVID-19. Our study provides insights into global variation at host genes related to SARS-CoV-2 infection, which have been shaped by natural selection in some populations, possibly due to prior viral infections.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronaviruses are enveloped, positive-sense, and single-stranded RNA viruses, many of which are zoonotic pathogens that crossed over into humans. Seven coronavirus species, including SARS-CoV-2, have been discovered that, depending on the virus and host physiological condition, may cause mild or lethal respiratory disease. There is considerable variation in disease prevalence and severity across populations and communities. Importantly, minority populations in the United States appear to have been disproportionally affected by COVID-19 (1, 2). For example, in Chicago, more than 50% of COVID-19 cases and nearly 70% of COVID-19 deaths are in African Americans (who make up 30% of the population of Chicago) (1). While social and economic factors are largely responsible for driving COVID-19 health disparities, investigating genetic diversity at host genes related to SARS-CoV-2 infection could help identify functionally important variation, which may play a role in individual risk for severe COVID-19 infection.In this study, we focused on four key genes playing a role in SARS-CoV-2 infection (3). The ACE2 gene, encoding the angiotensin-converting enzyme-2 protein, was reported to be a main binding site for severe acute respiratory syndrome coronavirus (SARS-CoV) during an outbreak in 2003, and evidence showed stronger binding affinity to SARS-CoV-2, which enters the target cells via ACE2 receptors (3, 4). The ACE2 gene is located on the X chromosome (chrX); its expression level varies among populations (5); and it is ubiquitously expressed in the lung, blood vessels, gut, kidney, testis, and brain, all organs that appear to be affected as part of the COVID-19 clinical spectrum (6). SARS-CoV-2 infects cells through a membrane fusion mechanism, which in the case of SARS-CoV, is known to induce down-regulation of ACE2 (7). Such down-regulation has been shown to cause inefficient counteraction of angiotensin II effects, leading to enhanced pulmonary inflammation and intravascular coagulation (7). Additionally, altered expression of ACE2 has been associated with cardiovascular and cerebrovascular disease, which is highly relevant to COVID-19 as several cardiovascular conditions are associated with severe disease. TMPRSS2, located on the outer membrane of host target cells, binds to and cleaves ACE2, resulting in activation of spike proteins on the viral envelope and facilitating membrane fusion and endocytosis (8). Two additional genes, DPP4 and LY6E, have been shown to play an important role in the entry of SARS-CoV-2 virus into host cells. DPP4 is a known functional receptor for the Middle East respiratory syndrome coronavirus (MERS-CoV), causing a severe respiratory illness with high mortality (9, 10). LY6E encodes a glycosylphosphatidylinositol-anchored cell surface protein, which is a critical antiviral immune effector that controls coronavirus infection and pathogenesis (11). Mice lacking LY6E in hematopoietic cells were susceptible to murine coronavirus infection (11).Previous studies of genetic diversity at ACE2 and TMPRSS2 in global human populations did not include an extensive set of African populations (5, 12–14). No common coding variants (defined here as minor allele frequency [MAF] > 0.05) at ACE2 were identified in any prior population studies. However, few studies included diverse indigenous African populations whose genomes harbor the greatest diversity among humans. This leads to a substantial disparity in the representation of African ancestries in human genetic studies of COVID-19, impeding health equity as the transferability of findings based on non-African ancestries to African populations can be low (15). Including more African populations in studying the genetic diversity of genes involved in SARS-CoV-2 infection is extremely necessary. Additionally, the evolutionary forces underlying global patterns of genetic diversity at host genes related to SARS-CoV-2 infection are not well understood. Using methods to detect natural selection signatures at host genes related to viral infections helps identify putatively functional variants that could play a role in disease risk.We characterized genetic variation and studied natural selection signatures at ACE2, TMPRSS2, DPP4, and LY6E in ethnically diverse human populations by analyzing 2,012 genomes from ethnically diverse Africans (referred to as the “African diversity” dataset), 2,504 genomes from the 1000 Genomes Project (1KG), and whole-exome sequencing of 15,977 individuals of European ancestry (EA) and African ancestry from the Penn Medicine BioBank (PMBB) dataset (SI Appendix, Fig. S1). The African diversity dataset includes populations with diverse subsistence patterns (hunter-gatherers, pastoralists, agriculturalists) and speaking languages belonging to the four major language families in Africa (Khoesan; Niger–Congo, of which Bantu is the largest subfamily; Afroasiatic; and Nilo-Saharan). We identify functionally relevant variation, compare the patterns of variation across global populations, and provide insight into the evolutionary forces underlying these patterns of genetic variation. In addition, we perform an association study using the variants identified from whole-exome sequencing at the four genes and clinical traits derived from electronic health record (EHR) data linked to the subjects enrolled in the PMBB. The EHR data include diseases related to organ dysfunctions associated with severe COVID-19, such as respiratory, cardiovascular, liver, and renal complications. Our study of genetic variation in genes involved in SARS-CoV-2 infection provides data to investigate infection susceptibility within and between populations and indicates that variants in these genes may play a role in comorbidities relevant to COVID-19 severity.     

Additional polymorphisms at marker loci D9S5 and D9S15 generate extended haplotypes in linkage disequilibrium with Friedreich ataxia.

The gene for Friedreich ataxia (FA), a severe recessive neurodegenerative disease, has previously been shown to be tightly linked to the polymorphic markers D9S15 and D9S5 on human chromosome 9. In addition, the observation of linkage disequilibrium suggested that D9S15 is within 1 centimorgan (cM) of the disease locus, FRDA. Although D9S5 did not show recombination with FRDA, its localization was less precise (0-5 cM) due to its lower informativeness. We have now identified additional polymorphisms at both marker loci. Two cosmids spanning 50 kilobases around D9S5 were isolated, and a probe derived from one of them detects an informative three-allele polymorphism. We have found a highly polymorphic microsatellite sequence at D9S15 which is rapidly typed by the DNA polymerase chain reaction. The polymorphism information contents at the D9S5 and D9S15 loci have been increased from 0.14 to 0.60 and from 0.33 to 0.74, respectively. With the additional polymorphisms the lod (log10 odds ratio) score for the D9S15-FRDA linkage is now 48.10 at recombination fraction theta = 0.005 and for D9S5-FRDA, the lod score is 27.87 at theta = 0.00. We have identified a recombinant between D9S15 and FRDA. However, due to the family structure, it will be of limited usefulness for more precise localization of FRDA. The linkage disequilibrium previously observed between D9S15 and FRDA is strengthened by analysis of the haplotypes using the microsatellite polymorphism, while weaker but significant disequilibrium is found between D9S5 and FRDA. Extended haplotypes that encompass D9S5 and D9S15 show a strikingly different distribution between chromosomes that carry the FA mutation and normal chromosomes. This suggests that both marker loci are less than 1 cM from the FRDA gene and that a small number of mutations account for the majority of FA cases in the French population studied. D9S5 and D9S15 are thus excellent start points to isolate the disease gene.     

Report on the 6th African Society of Human Genetics (AfSHG) Meeting,March 12–15, 2009, Yaoundé, Cameroon

The African Society of Human Genetics (AfSHG), founded in 2003 with its inaugural meeting in Accra, Ghana,¹ has the stated missions of (1) disseminating information about human genetics research in Africa, (2) establishing a mentorship network providing educational resources, including the development of appropriate technology transfer, (3) providing advocacy for human genetic research in Africa, and (4) encouraging collaborative research. Despite its young age, the AfSHG has developed a strong cadre of active researchers, both within and outside of Africa, with more than 400 members (from 16 countries across Africa as well as 8 other countries), and has held six successful meetings, five in Africa and one in the United States.     

Global distribution of a novel trinucleotide microsatellite polymorphism (ATA)n in intron 8 of the SLC11A1 gene and susceptibility to pulmonary tuberculosis

We identified a novel trinucleotide (ATA)n repeat polymorphism in intron 8 of SLC11AI, a candidate gene for susceptibility to tuberculosis (TB) infection. We characterized the frequency of this polymorphism in 485 individuals originating from eight globally diverse human populations and compared the distribution of (ATA)n alleles in 146 adults and in 80 cord blood samples from newborns in the Gambian population. Lastly, we tested for association of this microsatellite with pulmonary TB in 318 TB cases and 146 controls in the Gambian population. We found no significant difference in frequency or distribution of alleles in adult and cord blood samples, and we found no significant association between this marker and pulmonary TB.     

G6PD A- Deficiency and Severe Malaria in The Gambia: Heterozygote Advantage and Possible Homozygote Disadvantage

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is frequent in Africa, because it confers resistance to Plasmodium falciparum malaria; however, the nature of the protection and the genotypes associated with it have been controversial. In 1972, Bienzle and others described protection from malaria in West African females heterozygous for G6PD A-. They determined that G6PD A- heterozygotes had lower parasite counts than A- homozygotes, hemizygous males, and normal individuals. However, other studies have reached different conclusions about the protective genotypes. DNA samples from 135 children with severe malaria and 146 children with mild malaria from The Gambia were genotyped for the G6PD A- mutation that is most frequent among Gambians (G6PD 968 T->C); there was a marked deficiency of heterozygotes and an excess of homozygotes with severe malaria, producing a strong deviation from Hardy–Weinberg equilibrium. Our results support the protective effect in G6PD A- heterozygous females and suggest that homozygotes might be more susceptible to severe malaria attacks.Glucose-6-phosphate dehydrogenase (G6PD) deficiency is widespread across Africa, because it confers resistance to Plasmodium falciparum (and possibly, P. vivax) malaria, but despite this evidence and the previous evidence that G6PD is under strong natural selection, the nature of the protection and genotypes associated with it has been controversial.^1–3 In 1972, Bienzle and others⁴ described protection from malaria in West African females heterozygous for G6PD A-; their study was based on 255 boys and 203 girls with P. falciparum malaria recruited from a holoendemic area in southwestern Nigeria. Bienzle and others⁴ determined that G6PD A- heterozygotes had significantly lower parasite counts than A- homozygous females, hemizygous males, and normal individuals. Since the report by Bienzle and others,⁴ other studies have had conflicting results, including a recent conclusion by Guindo and others⁵ based on a Malian sample of children with severe malaria that hemizygous males and possibly, homozygous but not heterozygous females are protected from disease. The argued protection in hemizygous males was supported by pooled odds ratios from meta-analysis of their data⁵ “and data from a previous study”⁶ that “confirmed highly significant protection against severe malaria in hemizygous males but not in heterozygous females.” However, because the “previous study” that Guindo and others⁵ referred to was based on largely problematic genotyping data,^2,6,7 their meta-analysis is also questionable.The most common enzyme-deficient African variant, A-, has 12% of the normal enzymatic activity, and its frequency is as high as 0.24 in Nigerian Yoruba.^8–10 G6PD A- is molecularly characterized by two distinct variants within the gene: one variant is always 376 A->G (underlying G6PD A, which has 85% enzyme activity and is not considered to be a deficient variant); a second deficiency-causing mutation is 202 G->A (376G/202A; most common), 680 G->T (376G/680T), or 968 T->C (376G/968C; Betica Selma).¹¹ Virtually all of the second A- mutations have been found in the presence of 376 A->G, with only one exception reported so far.¹²Although the 202 G->A mutation is largely responsible for G6PD A- deficiency in much of sub-Saharan Africa, in The Gambia (and likely, the whole of Senegal¹³), the major mutation causing A- is 968 T->C, with a frequency of 7%, whereas 202 G->A is only 2–3%; therefore, approximately 10% (7% + 3%) of Gambian males are G6PD A-.¹⁴ An additional mutation, 542 A->T (376G/542T), that causes a severe deficiency with 2% residual activity (G6PD Santamaria) occurs in about 2% of Gambian males.¹⁴ Thus, the overall frequency of G6PD deficiency in The Gambia is approximately 12%.To address the role of enzymatic deficiency, we analyzed G6PD 202 G->A and 968 T->C genotypes in Gambian children with malaria (age range = 6 months to 16 years), because this combination accounts for most of the deficiency in The Gambia.¹⁵ Subjects for our study were enrolled into a health center-based case-control study that has been described elsewhere.¹⁵ Subjects were clinically assessed by a research physician and stringently classified as children with uncomplicated (mild) malaria (N = 146; 63 females and 83 males) and severe malaria (N = 135; 60 females and 75 males) resident in a restricted periurban area. Mild malaria was defined as an episode of fever (temperature > 37.5°C) within the last 48 hours and more than 5,000 parasites/μL detected by slide microscopy. Subjects were, to the best of our knowledge, unrelated. Relatedness is unlikely given that our samples were drawn from different villages in the greater Banjul area (within 40 km of the city) and several ethnic groups. The study was reviewed and approved by the Gambian Government/Medical Research Council (MRC) Joint Ethics Committee. All patients were enrolled after written informed consent was obtained from the parents or guardians.In addition to the above criteria, severe malaria was defined by the presence of one or more of the following criteria: severe anemia (SA; defined as hemoglobin [Hb] < 6 g/dL); severe respiratory distress (SRD; defined as serum lactate > 7 mmol/L); cerebral malaria (CM; defined as a Blantyre coma score ≤ 2 in the absence of hypoglycemia or hypovolemia, with the coma lasting at least 2 hours); severe prostration (SP; defined as inability to sit unsupported [children > 6 months] or inability to suck [children ≤ 6 months]); and absence of any other obvious cause of severe illness¹⁵ (