Different Selected Mechanisms Attenuated the Inhibitory Interaction of KIR2DL1 with C2+ HLA-C in Two Indigenous Human Populations in Southern Africa.

The functions of human NK cells in defense against pathogens and placental development during reproduction are modulated by interactions of killer cell Ig-like receptors (KIRs) with HLA-A, -B and -C class I ligands. Both receptors and ligands are highly polymorphic and exhibit extensive differences between human populations. Indigenous to southern Africa are the KhoeSan, the most ancient group of modern human populations, who have highest genomic diversity worldwide. We studied two KhoeSan populations, the Nama pastoralists and the ≠Khomani San hunter-gatherers. Comprehensive next-generation sequence analysis of HLA-A, -B, and -C and all KIR genes identified 248 different KIR and 137 HLA class I, which assort into ∼200 haplotypes for each gene family. All 74 Nama and 78 ≠Khomani San studied have different genotypes. Numerous novel KIR alleles were identified, including three arising by intergenic recombination. On average, KhoeSan individuals have seven to eight pairs of interacting KIR and HLA class I ligands, the highest diversity and divergence of polymorphic NK cell receptors and ligands observed to date. In this context of high genetic diversity, both the Nama and the ≠Khomani San have an unusually conserved, centromeric KIR haplotype that has arisen to high frequency and is different in the two KhoeSan populations. Distinguishing these haplotypes are independent mutations in KIR2DL1, which both prevent KIR2DL1 from functioning as an inhibitory receptor for C2+ HLA-C. The relatively high frequency of C2+ HLA-C in the Nama and the ≠Khomani San appears to have led to natural selection against strong inhibitory C2-specific KIR.

An Unexpectedly Complex Architecture for Skin Pigmentation in Africans.

Approximately 15 genes have been directly associated with skin pigmentation variation in humans, leading to its characterization as a relatively simple trait. However, by assembling a global survey of quantitative skin pigmentation phenotypes, we demonstrate that pigmentation is more complex than previously assumed, with genetic architecture varying by latitude. We investigate polygenicity in the KhoeSan populations indigenous to southern Africa who have considerably lighter skin than equatorial Africans. We demonstrate that skin pigmentation is highly heritable, but known pigmentation loci explain only a small fraction of the variance. Rather, baseline skin pigmentation is a complex, polygenic trait in the KhoeSan. Despite this, we identify canonical and non-canonical skin pigmentation loci, including near SLC24A5, TYRP1, SMARCA2/VLDLR, and SNX13, using a genome-wide association approach complemented by targeted resequencing. By considering diverse, under-studied African populations, we show how the architecture of skin pigmentation can vary across humans subject to different local evolutionary pressures.

Structural and functional effects of nucleotide variation on the human TB drug metabolizing enzyme arylamine N-acetyltransferase 1.

The human arylamine N-acetyltransferase 1 (NAT1) enzyme plays a vital role in determining the duration of action of amine-containing drugs such as para-aminobenzoic acid (PABA) by influencing the balance between detoxification and metabolic activation of these drugs. Recently, four novel single nucleotide polymorphisms (SNPs) were identified within a South African mixed ancestry population. Modeling the effects of these SNPs within the structural protein was done to assess possible structure and function changes in the enzyme. The use of molecular dynamics simulations and stability predictions indicated less thermodynamically stable protein structures containing E264K and V231G, while the N245I change showed a stabilizing effect. Coincidently the N245I change displayed a similar free energy landscape profile to the known R64W amino acid substitution (slow acetylator), while the R242M displayed a similar profile to the published variant, I263V (proposed fast acetylator), and the wild type protein structure. Similarly, principal component analysis indicated that two amino acid substitutions (E264K and V231G) occupied less conformational clusters of folded states as compared to the WT and were found to be destabilizing (may affect protein function). However, two of the four novel SNPs that result in amino acid changes: (V231G and N245I) were predicted by both SIFT and POLYPHEN-2 algorithms to affect NAT1 protein function, while two other SNPs that result in R242M and E264K substitutions showed contradictory results based on SIFT and POLYPHEN-2 analysis. In conclusion, the structural methods were able to verify that two non-synonymous substitutions (E264K and V231G) can destabilize the protein structure, and are in agreement with mCSM predictions, and should therefore be experimentally tested for NAT1 activity. These findings could inform a strategy of incorporating genotypic data (i.e., functional SNP alleles) with phenotypic information (slow or fast acetylator) to better prescribe effective treatment using drugs metabolized by NAT1.

N-acetyltransferase genotypes and the pharmacokinetics and tolerability of para-aminosalicylic acid in patients with drug-resistant pulmonary tuberculosis.

The aim of this study was to examine the relationships between N-acetyltransferase genotypes, pharmacokinetics, and tolerability of granular slow-release para-aminosalicylic acid (GSR-PAS) in tuberculosis patients. The study was a randomized, two-period, open-label, crossover design wherein each patient received 4 g GSR-PAS twice daily or 8 g once daily alternately. The PAS concentration-time profiles were modeled by a one-compartment disposition model with three transit compartments in series to describe its absorption. Patients' NAT1 and NAT2 genotypes were determined by sequencing and restriction enzyme analysis, respectively. The number of daily vomits was modeled by a Poisson probability mass function. Comparisons of other tolerability measures by regimens, gender, and genotypes were evaluated by a linear mixed-effects model. The covariate effects associated with efavirenz, gender, and NAT1*3, NAT1*14, and NAT2*5 alleles corresponded to 25, 37, -17, -48, and -27% changes, respectively, in oral clearance of PAS. The NAT1*10 allele did not influence drug clearance. The time above the MIC of 1 mg/liter was significantly different between the two regimens but not influenced by the NAT1 or NAT2 genotypes. The occurrence and intensity of intolerance differed little between regimens. Four grams of GSR-PAS twice daily but not 8 g once daily ensured concentrations exceeding the MIC (1 mg/liter) throughout the dosing interval; PAS intolerance was not related to maximum PAS concentrations over the doses studied and was not more frequent after once-daily dosing. We confirm that the slow phenotype conferred by the NAT1*14 and NAT1*3 alleles resulted in higher PAS exposure but found no evidence of increased activity of the NAT1*10 allele.

Acquired drug resistance during inadequate therapy in a young child with tuberculosis.

Drug resistance in children with tuberculosis is usually primary (transmitted); however, resistance acquisition during treatment is possible. We describe a child with tuberculosis who acquired drug resistance while receiving directly observed but inadequate first-line therapy and the programmatic and clinical factors that may have contributed to resistance acquisition.

Isoniazid pharmacokinetics, pharmacodynamics, and dosing in South African infants.

AIMS: There are limited data on isoniazid (INH) pharmacokinetics in infants and young children and, therefore, uncertainty on appropriate dosing. METHODS: Pharmacokinetic data were obtained from perinatally HIV-exposed South African infants aged 3-24 months receiving INH 10-20 mg·kg·d orally for Mycobacterium tuberculosis prophylaxis. INH pharmacokinetic parameters were characterized using a population pharmacokinetic approach. Dosing simulations were performed to evaluate weight-based INH doses in children based on N-acetyltransferase 2 enzyme (NAT2) genotype, age, maximum concentrations (Cmax) ≥3 mg/L, and area under the curve (AUC0-24) ≥10.52 mg·h/L. RESULTS: In 151 infants (53% female, 48% HIV positive) receiving a mean INH dose of 14.5 mg·kg·d, mean (±SD) Cmax at 3, 6, and 23 months of age were 10.0 (3.5), 8.6 (2.6), and 9.3 (3.8) mg/L, respectively, mean (±SD) AUC0-24 were 53.6 (26.8), 42 (19.9), and 44 (30.7) mg·h/L, respectively, and mean (±SD) half-lives were 2.1 (0.7), 1.9 (0.6), and 1.8 (0.9) hours, respectively. A trimodal apparent oral clearance of INH as a function of the NAT2 genotype was apparent as early as 3 months. INH was well tolerated. At an average INH dose of 14.5 mg·kg·d, 99% of infants aged 3-24 months have an INH Cmax ≥3 mg/L, and 98% have an INH AUC0-24 ≥10.52 mg·h/L. CONCLUSIONS: INH at an average dose of 14.5 mg/kg once daily was well tolerated in infants and achieved INH Cmax values ≥3 mg/L and AUC0-24 values ≥10.52 mg·h/L.

The pharmacogenetics of NAT2 enzyme maturation in perinatally HIV exposed infants receiving isoniazid.

The roles of the NAT2 genotype and enzyme maturation on isoniazid pharmacokinetics were investigated in South African infants with perinatal HIV exposure enrolled in a randomized, double-blind, controlled trial of isoniazid for prevention of tuberculosis disease and latent infection. Plasma concentration-time measurements of isoniazid from 151 infants (starting at 3-4 months of age) receiving isoniazid 10 to 20 mg/kg/d orally during the course of the 24-month study were incorporated in a population analysis along with NAT2 genotype, body weight, age, and sex. The results showed a different NAT2 enzyme maturation profile for each of the 3 acetylation groups, with the 70-kg body weight-normalized typical apparent clearance for the fast and intermediate acetylators increasing from 14.25 L/h and 10.88 L/h at 3 months of age to 22.84 L/h and 15.58 L/h at 24 months of age, respectively, with no significant change in the apparent clearance of the slow group during this period. A hypothesis is proposed to explain the genotype-dependent enzyme maturation processes for the NAT2 enzyme.

Isoniazid plasma concentrations in a cohort of South African children with tuberculosis: implications for international pediatric dosing guidelines.

BACKGROUND: In most countries with a high burden of tuberculosis, children with tuberculosis are prescribed isoniazid at dosages of 4-6 mg/kg/day, as recommended by international authorities. METHODS: We studied isoniazid concentrations in 56 hospitalized children (median age, 3.22 years; interquartile range [IQR], 1.58-5.38 years) who received isoniazid daily (median dosage, 5.01 mg/kg/day; range, 2.94-15.58 mg/kg/day) as part of antituberculosis treatment. At 1 and 4 months after initiation of treatment, isoniazid concentrations were measured in plasma samples at 0.75, 1.5, 3, 4, and 6 h after a treatment dose, to describe pharmacokinetic measures by using noncompartmental analysis. The effects of dose in milogram per kilogram, acetylator genotype, age, sex, and clinical diagnosis of kwashiorkor and human immunodeficiency virus (HIV) infection on isoniazid concentrations were evaluated. RESULTS: Median peak concentrations of isoniazid in children prescribed a dose of 4-6 mg/kg were 58% lower than those in children prescribed a dose of 8-10 mg/kg (2.39 mg/L [IQR, 1.59-3.40] vs. 5.71 mg/L [IQR, 4.74-7.62]). Peak concentrations were <3 mg/L in 70% of children prescribed a dose of 4-6 mg/kg. In contrast, children prescribed a dose of 8-12 mg/kg achieved peak concentrations approximating those in adults treated with 300 mg of isoniazid daily. Intermediate or fast acetylator genotype independently predicted a 38% (95% confidence interval [CI], 21%-51%) reduction in peak concentrations, compared with the slow-acetylator genotype. Each 1-mg/kg increase in the dose and each year increase in age were associated with increases in peak concentrations of 21% (95% CI, 16%-25%) and 6% (95% CI, 3%-10%), respectively. CONCLUSIONS: Younger children require higher doses of isoniazid per kilogram of body weight to achieve isoniazid concentrations similar to those in adults. A daily isoniazid dose of 8-12 mg/kg should be recommended.

NAT2 polymorphisms and their influence on the pharmacology and toxicity of isoniazid in TB patients.

Tuberculosis is a global pandemic that threatens to overwhelm healthcare budgets in many developing countries. Despite the availability of adequate effective treatment, many patients default on treatment, experience adverse side effects from antibiotics or fail to respond rapidly and recover. Isoniazid, one of the most important first-line tuberculosis drugs, is acetylated in the liver to a variable degree in different individuals giving rise to fast, intermediate and slow acetylator phenotypes. We present the view that the acetylation status of individuals plays an important contributory role in the tuberculosis pandemic. It is important to study the acetylation alleles, and to understand isoniazid metabolism and the manner in which it could affect patient compliance, isoniazid-toxicity and the emergence of drug-resistant strains of mycobacteria.

Allele frequencies for glutathione S-transferase and N-acetyltransferase 2 differ in African population groups and may be associated with oesophageal cancer or tuberculosis incidence.

Glutathione S-transferase (GST) and arylamine N-acetyltransferase 2 (NAT2) metabolise many environmental and chemotherapeutic agents, which influence susceptibility to disease. Polymorphisms in these enzymes result in different host phenotypes and contribute to different disease profiles or responses to toxic or chemotherapeutic agents, depending on their frequency in different populations. GST and NAT2 polymorphisms were investigated in different population groups, including African populations, and a range of allelic frequencies have been observed. The GSTM1 null genotype frequency, reported in this paper in two South African ethnic groups, is the lowest reported (0.19-0.21). In contrast, these same groups have a high GSTT1 null frequency (0.41-0.54), which is considerably higher than in African-Americans, or other Africans. The GSTT1 null frequency is comparable to the Chinese, a population with a very high oesophageal cancer incidence, similar to that in the African group. The frequency of the GSTPi Val105 variant in the South African Xhosas was also high (0.53), differing significantly from the low frequency in other Africans. These variants could therefore be associated with high cancer susceptibility. In addition, the high proportion of NAT2 "fast" alleles may partially explain the high tuberculosis prevalence in South Africans, due to reduced isoniazid efficacy in the presence of rapid acetylation.

NAT2 slow acetylator function as a risk indicator for age-related cataract formation.

OBJECTIVES: To show that the slow arylamine N-acetyltransferase type 2 (NAT2) catalysed acetylator function is associated with the development of age-related cataracts. METHODS: Both the acetylator phenotype and genotype of 139 patients with age-related cataracts were determined, and the distribution of the acetylator subtypes in the case population was compared with the distribution in the general (control) population. The genotype was determined by restriction-enzyme analysis of DNA, and the phenotype was determined using the elimination characteristics of isoniazid as discriminant. RESULTS: The frequency of alleles coding for slow acetylator characteristics was higher in the patients than in the controls, and the difference was significant (P = 0.013). CONCLUSIONS: Slow acetylators are at higher risk of developing age-related cataracts than fast acetylators and we suggest that exogenous factors, which can be detoxified by acetylation, are aetiological agents for cataract formation. Identification of and avoidance of such (environmental) agents should reduce the incidence of age-related cataracts.