Antenatal Uterotonics as a Risk Factor for Intrapartum Stillbirth and First-day Death in Haryana, India: A Nested Case-control Study.

BACKGROUND: Use of uterotonics like oxytocin to induce or augment labor has been shown to reduce placental perfusion and oxygen supply to the fetus, and studies indicate that it may increase the risk of stillbirth and neonatal asphyxia. Antenatal use of uterotonics, even without the required fetal monitoring and prompt access to cesarean section, is widespread, yet no study has adequately estimated the risk of intrapartum stillbirth and early neonatal deaths ascribed to such use. We conducted a case-control study to estimate this risk. METHODS: We conducted a population-based case-control study nested in a cluster-randomized trial. From 2008 to 2010, we followed pregnant women in rural Haryana, India, monthly until delivery. We visited all live-born infants on day 29 to ascertain whether they were alive. We conducted verbal autopsies for stillbirths and neonatal deaths. Cases (n = 2,076) were the intrapartum stillbirths and day-1 deaths (early deaths), and controls (n = 532) were live-born babies who died between day 8 and 28 (late deaths). RESULTS: Antenatal administration of uterotonics preceded 74% of early and 62% of late deaths, translating to an adjusted odds ratio (95% confidence interval [CI]) for early deaths of 1.7 (95% CI = 1.4, 2.1), and a population attributable risk of 31% (95% CI = 22%, 38%). CONCLUSIONS: Antenatal administration of uterotonics was associated with a substantially increased risk of intrapartum stillbirth and day-1 death. See video abstract: http://links.lww.com/EDE/B707.

Association between pro-inflammatory alleles and allergic phenotypes in Xhosa adolescents.

BACKGROUND: Significant differences exist in the prevalence, spectrum, and severity of allergic diseases between developing and developed countries and between subpopulations within single countries. These discrepancies likely result from a complex interaction between genetic and environmental factors. However, the precise nature of the contribution of ethnicity to genetic differences in the predisposition to allergic disease is not yet fully understood. In particular, there is a paucity of literature regarding the genetic determinants of allergic disease in people of black African origin with little or no genetic admixture. OBJECTIVE: We aimed to analyze associations between 27 single nucleotide polymorphisms (SNPs) and allergy phenotypes in the local Xhosa population. METHODS: A convenience sample of 213 Xhosa teenagers was enrolled at a local high school. Phenotypic data were collected in the form of a symptom questionnaire, skin prick tests for common food and aeroallergens, total serum IgE, and IgE to Ascaris lumbricoides. In addition, genotyping was performed to establish the prevalence of putative pro-inflammatory alleles. RESULTS: We demonstrated several significant associations between polymorphisms and allergy phenotypes. In particular, 2 polymorphisms in the IL-10 gene (IL10 -592A>C and IL10 -1082A>G) and 1 in the IL-4 gene (IL4 -589C>T) showed multiple associations with allergic sensitization and asthma phenotypes. Other polymorphisms, across a multitude of genes with discrepant functions, showed less consistent associations. CONCLUSION: This study represents an important first step in genotype/phenotype association in this population. Further research is required to confirm or refute our findings.

Hypothalamic-pituitary-adrenal axis variants and childhood trauma influence anxiety sensitivity in South African adolescents.

Anxiety sensitivity (AS) is characterised by the fear of anxiety-related symptoms and is a risk factor for the development of anxiety-related disorders. We examined whether genetic variation in three stress response genes, CRHR1, NR3C1, and FKBP5, interact with childhood trauma (CT) to predict AS in South African adolescents. Xhosa (n = 634) and Coloured (n = 317) students completed self-report measures of AS and CT, and a total of eighteen polymorphisms within CRHR1, NR3C1, and FKBP5 were genotyped. Differences in AS based on genetic variation and CT were analysed within population and gender groups using multiple linear regression. Associations were found between AS and FKBP5 rs9296158 (p = 0.025) and rs737054 (p = 0.045) in Coloured males. Analysis of gene x CT interactions indicated that NR3C1 rs190488 CC-genotype, NR3C1 rs10482605 G-allele addition, and FKBP5 rs3800373 C-allele addition protect against AS with increasing CT in Xhosa females (p = 0.009), Xhosa males (p = 0.036) and Coloured males (p = 0.049), respectively. We identified two different protective single nucleotide polymorphism (SNP) combinations in a four-SNP CRHR1 haplotype in Coloured males. An analysis of the interaction between CT and a six-SNP FKBP5 haplotype in Coloured males revealed both protective and risk allelic combinations. Our results provide evidence for the influence of both genetic variation in CRHR1, NR3C1 and FKBP5, as well as CT x SNP interactions, on AS in South African adolescents. This study reinforces the importance of examining the influence of gene-environment (G X E) interactions within gender and population groups.

Acquisition of Rifampin Resistance in Pulmonary Tuberculosis.

Mycobacterium tuberculosis strains with spontaneous mutations conferring resistance to rifampin (RIF) are exceedingly rare, and fixed drug combinations typically prevent augmentation of resistance to single drugs. Fourteen newly diagnosed tuberculosis patients were treated with RIF alone for 14 days, and bacterial loads, including mutation frequencies, were determined. A statistical model estimated that 1% of the remaining viable mycobacteria could be RIF resistant after 30 days of monotherapy. This indicates that temporal and spatial windows of RIF monotherapy due to uneven drug distribution within lung lesions could contribute to the acquisition of resistance to RIF.

Time Trends in Sputum Mycobacterial Load and Two-Day Bactericidal Activity of Isoniazid-Containing Antituberculosis Therapies.

Recent early bactericidal activity (EBA) studies of isoniazid-based antituberculosis therapies have shown a lower EBA over the first two treatment days than in earlier years. To quantify this trend and evaluate factors contributing to it, we extracted individual data from 18 studies with a total of 182 participants using isoniazid-containing therapies between 1992 and 2015 at a single site and laboratory in Cape Town, South Africa. We recalculated EBA as the daily fall in CFU per milliliter sputum up to day 2 of therapy (EBA0-2) for individual patients and treatment groups and used mixed-effects linear models to investigate the correlation between pretreatment CFU, EBA0-2, and year of study. We found that mean pretreatment CFU and year of study accounted for 46% and 47%, respectively, of the variation in mean EBA0-2 Mean pretreatment CFU differed between the periods 1992 to 2001 and 2007 to 2015 by 0.92 log10 CFU (95% confidence interval [CI], 0.57 to 1.28; P < 0.0001). On average, pretreatment CFU dropped by 0.053 log10 CFU (95% CI, 0.029 to 0.076; P = 0.0004) and EBA0-2 by 0.012 log10 CFU (95% CI, 0.006 to 0.018; P = 0.001) per year. The EBA0-2 of isoniazid-based antituberculosis therapy is strongly correlated with baseline mycobacterial load and shows a declining trend over the past 2 decades.

Association between serotonin transporter gene polymorphisms and increased suicidal risk among HIV positive patients in Uganda.

BACKGROUND: Persons living with HIV/AIDS (PLWHA) are at an increased risk of suicide. Increased suicidal risk is a predictor of future attempted and completed suicides and has been associated with poor quality of life and poor adherence with antiretroviral therapy. Clinical risk factors have low predictive value for suicide, hence the interest in potential neurobiological correlates and specific heritable markers of suicide vulnerability. The serotonin transporter gene has previously been implicated in the aetiology of increased suicidal risk in non-HIV infected study populations and its variations may provide a platform for identifying genetic risk for suicidality among PLWHA. The present cross-sectional study aimed at identifying two common genetic variants of the serotonin transporter gene and their association with increased suicidal risk among human immunodeficiency virus (HIV)-positive adults in Uganda. RESULTS: The prevalence of increased suicidal risk (defined as moderate to high risk suicidality on the suicidality module of the Mini Neuropsychiatric Interview (M.I.N.I) was 3.3% (95% CI, 2.0-5.3). The 5-HTTLPR was found to be associated with increased suicidal risk before Bonferroni correction (p-value = 0.0174). A protective effect on increased suicidal risk was found for the 5-HTTLPR/rs25531 S A allele (p-value = 0.0046)- which directs reduced expression of the serotonin transporter gene (5-HTT). CONCLUSION: The S A allele at the 5-HTTLPR/rs25531 locus is associated with increased suicidal risk among Ugandan PLWHA. Further studies are needed to validate this finding in Ugandan and other sub-Saharan samples.

The identification of novel genetic variants associated with antipsychotic treatment response outcomes in first-episode schizophrenia patients.

BACKGROUND: Although antipsychotics are integral to the treatment of schizophrenia, drug efficacy varies between patients. Although it has been shown that antipsychotic treatment response outcomes are heritable, our understanding of the genetic factors that are involved remains incomplete. Therefore, this study aims to use an unbiased scan of the genome to identify the genetic variants contributing toward antipsychotic treatment response outcomes. MATERIALS AND METHODS: This study utilized whole-exome sequencing of patients on extreme ends of the treatment response spectrum (n=11) in combination with results from previous antipsychotic studies to design a panel of variants that were genotyped in two well-characterized first-episode schizophrenia cohorts (n=103 and 87). Association analyses were carried out to determine whether these variants were significantly associated with antipsychotic treatment response outcomes. RESULTS: Association analyses in the discovery cohort identified two nonsynonymous variants that were significantly associated with antipsychotic treatment response outcomes (P<2.7 × 10(-5)), which were also significantly associated with the corresponding treatment response outcome in an independent replication cohort. Computational approaches showed that both of these nonsynonymous variants--rs13025959 in MYO7B (E1647D) and rs10380 in MTRR (H622Y)--were predicted to impair the functioning of their corresponding protein products. CONCLUSION: The use of whole-exome sequencing in a subset of patients from a well-characterized cohort of first-episode schizophrenia patients, for whom longitudinal depot treatment response data were available, allowed for (i) the removal of confounding factors related to treatment progression and compliance and (ii) the identification of two genetic variants that have not been associated previously with antipsychotic treatment response outcomes and whose results were applicable across different classes of antipsychotics. Although the genes that are affected by these variants are involved in pathways that have been related previously to antipsychotic treatment outcomes, the identification of these novel genes will play an important role in improving our understanding of the specific variants involved in antipsychotic treatment response outcomes.

Impact of MammaPrint on Clinical Decision-Making in South African Patients with Early-Stage Breast Cancer.

The aim of the study was to evaluate the impact of MammaPrint on treatment decision-making in patients with breast cancer. Clinicopathologic information of all breast cancer patients referred for MammaPrint testing in South Africa was collected from 2007 until 2014. A total of 107 patients (109 tumors) with estrogen receptor/progesterone receptor positive and human epidermal growth factor receptor-2 negative tumors were selected with tumors ≥10 mm, or when 1-3 nodes were involved without extra-nodal extension. None of the clinical indicators correlated significantly with the MammaPrint risk classification, which changed the decision for adjuvant chemotherapy in 52% of patients. Of 60 patients who were clinically high risk, 62% had a low-risk MammaPrint result and of the 47 clinically low -risk patients 40% had a high-risk MammaPrint result. This study indicates that MammaPrint could reduce the need for adjuvant chemotherapy by 17% using the selection criteria stipulated. The significant impact on treatment decisions confirmed the clinical utility of MammaPrint independent of standard clinicopathologic risk factors as supported by long-term clinical outcome studies.

Genomic medicine and risk prediction across the disease spectrum.

Genomic medicine is based on the knowledge that virtually every medical condition, disease susceptibility or response to treatment is caused, regulated or influenced by genes. Genetic testing may therefore add value across the disease spectrum, ranging from single-gene disorders with a Mendelian inheritance pattern to complex multi-factorial diseases. The critical factors for genomic risk prediction are to determine: (1) where the genomic footprint of a particular susceptibility or dysfunction resides within this continuum, and (2) to what extent the genetic determinants are modified by environmental exposures. Regarding the small subset of highly penetrant monogenic disorders, a positive family history and early disease onset are mostly sufficient to determine the appropriateness of genetic testing in the index case and to inform pre-symptomatic diagnosis in at-risk family members. In more prevalent polygenic non-communicable diseases (NCDs), the use of appropriate eligibility criteria is required to ensure a balance between benefit and risk. An additional screening step may therefore be necessary to identify individuals most likely to benefit from genetic testing. This need provided the stimulus for the development of a pathology-supported genetic testing (PSGT) service as a new model for the translational implementation of genomic medicine in clinical practice. PSGT is linked to the establishment of a research database proven to be an invaluable resource for the validation of novel and previously described gene-disease associations replicated in the South African population for a broad range of NCDs associated with increased cardio-metabolic risk. The clinical importance of inquiry concerning family history in determining eligibility for personalized genotyping was supported beyond its current limited role in diagnosing or screening for monogenic subtypes of NCDs. With the recent introduction of advanced microarray-based breast cancer subtyping, genetic testing has extended beyond the genome of the host to also include tumor gene expression profiling for chemotherapy selection. The decreasing cost of next generation sequencing over recent years, together with improvement of both laboratory and computational protocols, enables the mapping of rare genetic disorders and discovery of shared genetic risk factors as novel therapeutic targets across diagnostic boundaries. This article reviews the challenges, successes, increasing inter-disciplinary integration and evolving strategies for extending PSGT towards exome and whole genome sequencing (WGS) within a dynamic framework. Specific points of overlap are highlighted between the application of PSGT and exome or WGS, as the next logical step in genetically uncharacterized patients for whom a particular disease pattern and/or therapeutic failure are not adequately accounted for during the PSGT pre-screen. Discrepancies between different next generation sequencing platforms and low concordance among variant-calling pipelines caution against offering exome or WGS as a stand-alone diagnostic approach. The public reference human genome sequence (hg19) contains minor alleles at more than 1 million loci and variant calling using an advanced major allele reference genome sequence is crucial to ensure data integrity. Understanding that genomic risk prediction is not deterministic but rather probabilistic provides the opportunity for disease prevention and targeted treatment in a way that is unique to each individual patient.

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.

Associations between human leukocyte antigen class I variants and the Mycobacterium tuberculosis subtypes causing disease.

BACKGROUND: The development of active tuberculosis disease has been shown to be multifactorial. Interactions between host and bacterial genotype may influence disease outcome, with some studies indicating the adaptation of M. tuberculosis strains to specific human populations. Here we investigate the role of the human leukocyte antigen (HLA) class I genes in this biological process. METHODS: Three hundred patients with tuberculosis from South Africa were typed for their HLA class I alleles by direct sequencing. Mycobacterium tuberculosis genotype classification was done by IS6110 restriction fragment length polymorphism genotyping and spoligotyping. RESULTS: We showed that Beijing strain occurred more frequently in individuals with multiple disease episodes (P < .001) with the HLA-B27 allele lowering the odds of having an additional episode (odds ratio, 0.21; P = .006). Associations were also identified for specific HLA types and disease caused by the Beijing, LAM, LCC, and Quebec strains. HLA types were also associated with disease caused by strains from the Euro-American or East Asian lineages, and the frequencies of these alleles in their sympatric human populations identified potential coevolutionary events between host and pathogen. CONCLUSIONS: This is the first report of the association of human HLA types and M. tuberculosis strain genotype, highlighting that both host and pathogen genetics need to be taken into consideration when studying tuberculosis disease development.

Using multi-way admixture mapping to elucidate TB susceptibility in the South African Coloured population.

BACKGROUND: The admixed South African Coloured population is ideally suited to the discovery of tuberculosis susceptibility genetic variants and their probable ethnic origins, but previous attempts at finding such variants using genome-wide admixture mapping were hampered by the inaccuracy of local ancestry inference. In this study, we infer local ancestry using the novel algorithm implemented in RFMix, with the emphasis on identifying regions of excess San or Bantu ancestry, which we hypothesize may harbour TB susceptibility genes. RESULTS: Using simulated data, we demonstrate reasonable accuracy of local ancestry inference by RFMix, with a tendency towards miss-calling San ancestry as Bantu. Regions with either excess San ancestry or excess African (San or Bantu) ancestry are less likely to be affected by this bias, and we therefore proceeded to identify such regions, found in cases but not in controls (642 cases and 91 controls). A number of promising regions were found (overall p-values of 7.19×10-5 for San ancestry and <2.00×10-16 for African ancestry), including chromosomes 15q15 and 17q22, which are close to genomic regions previously implicated in TB. Promising immune-related susceptibility genes such as the GADD45A, OSM and B7-H5 genes are also harboured in the identified regions. CONCLUSION: Admixture mapping is feasible in the South African Coloured population and a number of novel TB susceptibility genomic regions were uncovered.

Mitochondrial impairment observed in fibroblasts from South African Parkinson's disease patients with parkin mutations.

Parkinson's disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients' fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD pathogenesis will have important implications for the design of new and more effective therapies.

Significance of dietary folate intake, homocysteine levels and MTHFR 677 C>T genotyping in South African patients diagnosed with depression: test development for clinical application.

Low folate intake in the presence of the functional MTHFR 677 C > T (rs1801133) polymorphism is an important cause of elevated homocysteine levels previously implicated in major depressive disorder (MDD) and many other chronic diseases. In this study the clinical relevance and inter-relationship of these aspects were evaluated in 86 South African patients diagnosed with MDD and 97 population-matched controls participating in a chronic diseases screening program. A questionnaire-based clinical and nutrition assessment was performed, homocysteine levels determined, and all study participants genotyped for MTHFR 677 C > T (rs1801133) using allele-specific TaqMan technology. The folate score was found to be significantly lower in the patient group compared to controls (p = 0.003) and correlated with increased body mass index (BMI), particularly in females with MDD (p = 0.009). BMI was significantly higher in the MDD patients compared with controls after adjustment for age and sex (p = 0.015), but this association was no longer significant after further adjustment for the level of folate intake in the diet. In MDD patients but not controls, the minor T-allele of MTHFR 677 C > T was associated with increased BMI (p = 0.032), which in turn correlated significantly with increased homocysteine levels. The significant association between BMI and homocysteine levels was observed in both the MDD patient (p = 0.049) and control (p = 0.018) study groups. The significantly higher homocysteine levels observed in MDD patients compared to controls after adjustment for age and sex (p = 0.030), therefore appears to be mediated by the effects of MTHFR 677 C > T and low folate intake on BMI. Detection of the low-penetrance MTHFR 677 C > T mutation reinforces the importance of folate intake above the recommended daily dose to prevent or restore dysfunction of the methylation pathway.

The fat mass and obesity-associated FTO rs9939609 polymorphism is associated with elevated homocysteine levels in patients with multiple sclerosis screened for vascular risk factors.

UNLABELLED: The previously reported link between homocysteine and obesity, both identified as established risk factors for multiple sclerosis (MS), has not previously been studied in relation to the fat mass and obesity-associated (FTO) gene. AIM: To investigate the mechanism underlying homocysteine accumulation in MS patients. A total of 114 patients and 195 population-matched controls were analysed for the FTO rs9939609 polymorphism. Homocysteine levels were measured in a subgroup of 60 patients and 87 controls screened for multiple vascular risk factors. After adjustment for potential confounders, the risk-associated FTO rs9939609 A-allele was associated with raised homocysteine levels (p = 0.003) in patients diagnosed with MS, but not in controls. Homocysteine levels correlated positively with body mass index (BMI) (p = 0.045) and total cholesterol levels (p = 0.048). Both homocysteine (p = 0.011) and BMI (p = 0.017) were significantly reduced with higher intake of folate in the diet. Higher BMI also correlated with increased intake of saturated/trans fat (p < 0.01) and low physical activity (p < 0.006). Daily intake of at least five fruits and vegetables had a favourable lowering effect on the Expanded Disability Status Scale (EDSS) (p = 0.035), while smoking increased MS disability (p < 0.001). This study has shown for the first time that having a diagnosis of MS moderates the effect of the FTO rs9939609 polymorphism on homocysteine levels. This is consistent with the role of FTO in demethylation and epigenetic changes. Identification of FTO rs9939609 reinforces the importance of adequate fruit, vegetable and folate and restriction of saturated/trans fat intake in the diet.

Variants within the COMP and THBS2 genes are not associated with Achilles tendinopathy in a case-control study of South African and Australian populations.

Cartilage oligomeric matrix protein is a structural protein of the extracellular matrix, while thrombospondin-2 is a matricellular protein involved in cell-matrix interactions. Recent studies have shown that genetic variation is a significant risk factor for Achilles tendinopathy, and the genes encoding cartilage oligomeric matrix protein (COMP) and thrombospondin-2 (THBS2) were identified as good candidate genes for association with Achilles tendinopathy. This study aimed to test the association of sequence variants within these candidate genes with the risk of Achilles tendinopathy in participants from South Africa (SA) and Australia (AUS). Three-hundred and forty (133 SA; 207 AUS) control participants with no history of Achilles tendinopathy and 178 (94 SA; 84 AUS) participants clinically diagnosed with Achilles tendinopathy were genotyped for five single nucleotide polymorphisms within the COMP and THBS2 genes in this case-control study. There was no difference in genotype distributions between control and tendinopathy groups for either the THBS2 variants rs9505888, rs6422747 and rs9283850, or the COMP variants rs730079 and rs28494505 in the SA and AUS populations. As the selection of COMP and THBS2 as candidate genes was hypothesis driven, based on biological function, the possibility that other variants within these genes are associated with Achilles tendinopathy cannot be excluded.

Case/control studies with follow-up: Constructing the source population to estimate effects of risk factors on development, disease, and survival.

If individuals in a case/control study are subsequently observed as a cohort of cases and a cohort of controls, weighted regression analyses can be used to estimate the association between the exposures initially recorded and events occurring during the follow-up of the 2 cohorts. Such analyses can be conceptualized as being undertaken on a reconstructed source population from which cases and controls stem. To simulate this population, the cohort of cases is added to the cohort of controls expanded with the reciprocal of the case disease incidence odds (the sampling weight) to include all individuals in the source population who did not develop the case disease. We use a simulated dataset to illustrate how weighted generalized linear model regression can be used to estimate the association between an exposure captured during the case/control study component and an outcome that occurs during follow-up.