Hepatocyte gene expression and DNA methylation as ancestry-dependent mechanisms in African Americans.

African Americans (AAs) are an admixed population with widely varying proportion of West African ancestry (WAA). Here we report the correlation of WAA to gene expression and DNA methylation in AA-derived hepatocytes, a cell type important in disease and drug response. We perform mediation analysis to test whether methylation is a mediator of the effect of ancestry on expression. GTEx samples and a second cohort are used as validation. One hundred and thirty-one genes are associated with WAA (FDR < 0.10), 28 of which replicate and represent 220 GWAS phenotypes. Among PharmGKB pharmacogenes, VDR, PTGIS, ALDH1A1, CYP2C19, and P2RY1 nominally associate with WAA (p < 0.05). We find 1037 WAA-associated, differentially methylated regions (FDR < 0.05), with hypomethylated genes enriched in drug-response pathways. In conclusion, WAA contributes to variability in hepatocyte expression and DNA methylation with identified genes previously implicated for diseases disproportionately affecting AAs, including cardiovascular (PTGIS, PLAT) and renal (APOL1) disease, and drug response (CYP2C19).

Genome-wide association study identifies pharmacogenomic loci linked with specific antihypertensive drug treatment and new-onset diabetes.

We conducted a discovery genome-wide association study with expression quantitative trait loci (eQTL) annotation of new-onset diabetes (NOD) among European Americans, who were exposed to a calcium channel blocker-based strategy (CCB strategy) or a ß-blocker-based strategy (ß-blocker strategy) in the INternational VErapamil SR Trandolapril STudy. Replication of the top signal from the SNP*treatment interaction analysis was attempted in Hispanic and African Americans, and a joint meta-analysis was performed (total 334 NOD cases and 806 matched controls). PLEKHH2 rs11124945 at 2p21 interacted with antihypertensive exposure for NOD (meta-analysis P=5.3 × 10-8). rs11124945 G allele carriers had lower odds for NOD when exposed to the ß-blocker strategy compared with the CCB strategy (Odds ratio OR=0.38(0.24-0.60), P=4.0 × 10-5), whereas A/A homozygotes exposed to the ß-blocker strategy had increased odds for NOD compared with the CCB strategy (OR=2.02(1.39-2.92), P=2.0 × 10-4). eQTL annotation of the 2p21 locus provides functional support for regulating gene expression.

Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for Pharmacogenetics-Guided Warfarin Dosing: 2017 Update.

This document is an update to the 2011 Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for CYP2C9 and VKORC1 genotypes and warfarin dosing. Evidence from the published literature is presented for CYP2C9, VKORC1, CYP4F2, and rs12777823 genotype-guided warfarin dosing to achieve a target international normalized ratio of 2-3 when clinical genotype results are available. In addition, this updated guideline incorporates recommendations for adult and pediatric patients that are specific to continental ancestry.

Integrated analysis of genetic variation and gene expression reveals novel variant for increased warfarin dose requirement in African Americans.

Essentials Genetic variants controlling gene regulation have not been explored in pharmacogenomics. We tested liver expression quantitative trait loci for association with warfarin dose response. A novel predictor for increased warfarin dose response in African Americans was identified. Precision medicine must take into account population-specific variation in gene regulation. SUMMARY: Background Warfarin is commonly used to control and prevent thromboembolic disorders. However, because of warfarin's complex dose-requirement relationship, safe and effective use is challenging. Pharmacogenomics-guided warfarin dosing algorithms that include the well-established VKORC1 and CYP2C9 polymorphisms explain only a small proportion of inter-individual variability in African Americans (AAs). Objectives We aimed to assess whether transcriptomic analyses could be used to identify regulatory variants associated with warfarin dose response in AAs. Patients/Methods We identified a total of 56 expression quantitative trait loci (eQTLs) for CYP2C9, VKORC1 and CALU derived from human livers and evaluated their association with warfarin dose response in two independent AA warfarin patient cohorts. Results We found that rs4889606, a strong cis-eQTL for VKORC1 (log10 Bayes Factor = 12.02), is significantly associated with increased warfarin daily dose requirement (ß = 1.1; 95% confidence interval [CI] 0.46 to 1.8) in the discovery cohort (n = 305) and in the replication cohort (ß = 1.04; 95% CI 0.33 -1.7; n = 141) after conditioning on relevant covariates and the VKORC1 -1639G>A (rs9923231) variant. Inclusion of rs4889606 genotypes, along with CYP2C9 alleles, rs9923231 genotypes and clinical variables, explained 31% of the inter-patient variability in warfarin dose requirement. We demonstrate different linkage disequilibrium patterns in the region encompassing rs4889606 and rs9923231 between AAs and European Americans, which may explain the increased dose requirement found in AAs. Conclusion Our approach of interrogating eQTLs identified in liver has revealed a novel predictor of warfarin dose response in AAs. Our work highlights the utility of leveraging information from regulatory variants mapped in the liver to uncover novel variants associated with drug response and the importance of population-specific research.

Population differences in S-warfarin pharmacokinetics among African Americans, Asians and whites: their influence on pharmacogenetic dosing algorithms.

Using population pharmacokinetic analysis (PPK), we attempted to identify predictors of S-warfarin clearance (CL(S)) and to clarify population differences in S-warfarin pharmacokinetics among a cohort of 378 African American, Asian and white patients. Significant predictors of CL(S) included clinical (age, body weight and sex) and genotypic (CYP2C9*2,*3 and *8) factors, as well as African American ethnicity, the median CL(S) being 30% lower in the latter than in Asians and whites (170 versus 243 and 250 ml h-1, P<0.01). The plasma S-warfarin (Cp(S)) time courses following the genotype-based dosing algorithms simulated using the PPK estimates showed African Americans with CYP2C9*1/*1 and any of the VKORC1 genotypes would have an average Cp(S) at steady state 1.5-1.8 times higher than in Asians and whites. These results indicate warfarin dosing algorithms should be evaluated in each respective ethnic population. Further study of a large African American cohort will be necessary to confirm the present findings.

De Novo delayed onset hypothermia secondary to therapeutic doses of risperidone in bipolar affective disorder.

The commonly reported side effects related to risperidone include dizziness, nausea, weight gain, sleep disturbances, and sexual dysfunction. A rather rare and very much less documented side effect of risperidone is hypothermia: traditionally defined as a drop in core body temperature below 35°C (95°F). We report a case of a 75-year-old woman who had been treated for bipolar affective disorder for nearly 3 years with risperidone went on to develop hypothermia which was reversed with the withdrawal of the offending drug. This case is unique as it reported a rare but potentially serious side effect occurring after a prolonged administration of risperidone contrary to the previous reports in which hypothermia occurred only a few hours or days after the administration of risperidone and occurred in a patient who was diagnosed as having bipolar affective disorder as opposed to schizophrenia, the most common psychiatric disorder associated with previously reported hypothermia. The authors would like to emphasize the importance of this idiosyncratic potentially life-threatening adverse effect of risperidone-induced hypothermia to all clinicians, which occurs regardless of the duration of drug intake, in order to help them identify the condition early and treat it effectively.

Ethnicity-specific pharmacogenetics: the case of warfarin in African Americans.

Using a derivation cohort (N=349), we developed the first warfarin dosing algorithm that includes recently discovered polymorphisms in VKORC1 and CYP2C9 associated with warfarin dose requirement in African Americans (AAs). We tested our novel algorithm in an independent cohort of 129 AAs and compared the dose prediction to the International Warfarin Pharmacogenetics Consortium (IWPC) dosing algorithms. Our algorithm explains more of the phenotypic variation (R(2)=0.27) than the IWPC pharmacogenomics (R(2)=0.15) or clinical (R(2)=0.16) algorithms. Among high-dose patients, our algorithm predicted a higher proportion of patients within 20% of stable warfarin dose (45% vs 29% and 2% in the IWPC pharmacogenomics and clinical algorithms, respectively). In contrast to our novel algorithm, a significant inverse correlation between predicted dose and percent West African ancestry was observed for the IWPC pharmacogenomics algorithm among patients requiring ⩾60 mg per week (ß=-2.04, P=0.02).

The missing association: sequencing-based discovery of novel SNPs in VKORC1 and CYP2C9 that affect warfarin dose in African Americans.

It is well recognized that the genetic variants VKORC1-1639, CYP2C9*2, and CYP2C9*3 contribute to warfarin dose response. This has led to warfarin dosing algorithms that include these polymorphisms and explains between 47% and 56% of variability in dose in Caucasians. However, these polymorphisms explain significantly less of the variance in dose among African Americans. In order to identify novel variations that affect warfarin dose in African Americans, we used a targeted resequencing strategy that examined evolutionarily conserved sequences and regions of putative transcriptional binding. Through ethnicity-specific warfarin dose model building in 330 African Americans, we identified two novel genetic associations with higher warfarin dose, namely, VKORC1-8191 (rs61162043, P = 0.0041) and 18786 in CYP2C9 (rs7089580, P = 0.035). These novel finds are independent of the previous associations with these genes. Our regression model, encompassing both genetic and clinical variables, explained 40% of the variability in warfarin dose in African-American subjects, significantly more than any model thus far.

Prediction of CYP3A4 enzyme activity using haplotype tag SNPs in African Americans.

The CYP3A locus encodes hepatic enzymes that metabolize many clinically used drugs. However, there is marked interindividual variability in enzyme expression and clearance of drugs metabolized by these enzymes. We utilized comparative genomics and computational prediction of transcriptional factor binding sites to evaluate regions within CYP3A that were most likely to contribute to this variation. We then used a haplotype tagging single-nucleotide polymorphisms (htSNPs) approach to evaluate the entire locus with the fewest number of maximally informative SNPs. We investigated the association between these htSNPs and in vivo CYP3A enzyme activity using a single-point IV midazolam clearance assay. We found associations between the midazolam phenotype and age, diagnosis of hypertension and one htSNP (141689) located upstream of CYP3A4. 141689 lies near the xenobiotic responsive enhancer module (XREM) regulatory region of CYP3A4. Cell-based studies show increased transcriptional activation with the minor allele at 141689, in agreement with the in vivo association study findings. This study marks the first systematic evaluation of coding and noncoding variation that may contribute to CYP3A phenotypic variability.

Feasibility of canine oral rabies vaccination in Sri Lanka--a preliminary report.

OBJECTIVE: To test the feasibility of introducing oral rabies vaccine (vaccinia recombinant rabies glycoprotein) to improve domestic dog immunisation coverage in a selected area. DESIGN: A prospective field trial. SETTING: Panadura, an area relatively isolated by waterways, making it a suitable field laboratory. METHODS: A routine parenteral vaccination program was carried out. A house to house survey identified the residual non-vaccinated dogs in the selected area. Oral vaccine was offered to the non-vaccinated domestic dogs. RESULTS: Of 4322 dogs in the households, 1242 dogs (28.7%) were eligible for oral vaccine. 659 (53%) were considered to have accepted the oral vaccine with release of the vaccine in the oral cavity. CONCLUSION: It is feasible to use oral rabies vaccine to enhance immunisation coverage.

Endometrial-myometrial interface: relationship to adenomyosis and changes in pregnancy.

The endometrial-myometrial interface (EMI) is an important region of the human uterus, which has attracted little research attention. This mucosal-muscular interface has characteristic features when compared with other similar interfaces in the human body. It lacks an intervening tissue layer and as a result, the endometrium sits directly on the myometrium rendering it vulnerable to invasion by the endometrium. Both endometrium and myometrium are sensitive to sex steroids, and their structure and function depend to a large extent on the sex hormonal milieu. Endometrium, which forms one border of the EMI, is a complex tissue consisting of several polarized microenvironments. At a cellular level, sex steroids interact with local mediators secreted by a variety of cell types and are important in maintaining the complex structure and function of the endometrium. Basal endometrium contains prominent aggregates of leukocytes that may be important in controlling local cell growth and function. Myometrium also has a distinct zonal anatomy. The recently described junctional zone differs structurally and functionally from the outer myometrium, although these functions are not yet clearly understood. Embryologically, it originates from müllerian ducts together with endometrium, whereas the outer myometrium has a non-müllerian origin. During early pregnancy, the EMI is disturbed by invading trophoblast. Alterations of myometrial intercellular matrix proteins together with expression of appropriate receptors by the trophoblast seem to regulate this unique interaction. The EMI also is disrupted in adenomyosis. The sequence of events taking place at the EMI during development of this pathology is still debated.

Determination of sodium and chloride ions in teicoplanin lyophilized vials by nonsuppressed, single-column ion chromatography.

Two nonsuppressed, single-column ion chromatographic procedures have been developed and validated for the determination of the total sodium and chloride ion content in lyophilized vials containing teicoplanin. The assays served to determine the amount of sodium chloride added to the vials and to estimate the amount of sodium associated with the teicoplanin molecule.

Respective contribution of intracellular calcium release and extracellular calcium influx for interleukin-2 synthesis in activated T-cell hybrids.

Triggering of the T-cell receptor (TcR)alpha beta/CD3 receptor complex with anti-allotypic antibodies or concanavalin A (Con A) induced a rapid release of intracellular calcium in a murine T-cell hybridoma model system. Internal calcium release preceded the influx of extracellular calcium, as judged by comparative analysis of time-dependent changes in Quin 2 fluorescence following T-cell activation in the presence and absence of extracellular calcium. The magnitude of intracellular calcium release and extracellular calcium influx depended on the degree of receptor-occupancy and cross-linking. Correlations between the concentration of stimulating ligand, cytosolic calcium increase and IL-2 synthesis indicated a positive but non-linear relationship. Our data suggest that TcR cross-linking may provide a third T-cell activation signal which, in conjunction with protein kinase C activation and cytosolic calcium elevation, together form a signal triad responsible for interleukin-2 (IL-2) synthesis.

Pretransplant crossmatch status and immunoglobulin production in renal transplant recipients.

The factors responsible for B cell regulation in stable renal transplant patients are unknown. To examine these control mechanisms, the B cell responses to 4 mitogens (pokeweed, wheat germ agglutinin, lipopolysaccharide, and SAC-1), which are known to stimulate B cells by different mechanisms, were measured in two groups of stable long-term (greater than 1 year post-transplant) renal transplant recipients. The first group were patients who had been transplanted under conditions of a negative crossmatch on all available pretransplant sera (negative crossmatch group) and the second group were patients who might have a unique regulation of B cell function in that they had been successfully transplanted under the conditions of a negative crossmatch using sera at the time of transplant but with pretransplant sera that gave a positive donor-specific T cell crossmatch (positive crossmatch group). Stable transplant patients were found to have significantly impaired responses to all four mitogens tested. Furthermore there were no differences in the responses of the negative crossmatch patients as compared with the positive crossmatch patients. The lack of response to mitogens was not due to a lack of proliferation of cells or to a loss of viability in culture. The number of cells in culture was the same in negative crossmatch patients and controls but was significantly less than controls in positive crossmatch patients (P less than 0.001). These findings suggest an intrinsic B cell defect in all stable transplant patients, and that the degree of the impaired B cell responses is the same in positive and negative crossmatch patients. In addition in positive crossmatch patients, there is a decrease in the percentage B cells that resulted after separation on a Ficoll-Paque gradient.

Effects of adult thymectomy on the contact sensitivity skin reaction and the unresponsiveness caused by feeding contact sensitizing agents.

Unresponsiveness was produced in mice by three feeds of high doses of oxazolone or picryl chloride. The mice were then sensitized and contact sensitivity assessed by ear swelling. Adult thymectomy prevented the unresponsiveness caused by feeding. In some but not all experiments adult thymectomy also increased the response to a single skin paint. This effect of thymectomy was apparent at 1 week and reached a plateau at 2-3 weeks. The effect of adult thymectomy, on unresponsiveness caused by feeding and its ability to increase the contact sensitivity skin reaction, was reversed by treatment with crude thymosin fraction V.

The production of contact sensitivity by the injection into the footpads of recipients of the lymph node cells from mice 1 day after painting the skin with contact sensitizing agent: requirement for matching at the major histocompatibility complex between donor and recipient mice.

Donor mice were painted on the skin of the abdomen with the contact sensitizing agent, oxazolone. One day later 2-5 x 10(6) cells from the regional lymph nodes were injected into the footpads of recipient mice. Contact sensitivity was detected 6 days later by challenging the ears of the recipients and measuring the increase of thickness at 24 h. Good contact sensitivity was obtained when CBA cells were injected into CBA mice and BALB/c cells injected into BALB/c mice; the injection of BALB/c (H-2d) cells into CBA (H-2k) mice and vice versa failed to give rise to contact sensitivity. Hybrid F1 cells gave intermediate responses. The contact sensitivity caused by the injection of small numbers of lymph node cells into the footpad is interpreted as a mode of active immunization and the present results show that this only occurs when there is genetic matching at the major histocompatibility complex between the donor and the recipient mouse.

Contact sensitivity and the DNA response in mice to high and low doses of oxazolone: low dose unresponsiveness following painting and feeding and its prevention by pretreatment with cyclophosphamide.

Cyclophosphamide was used to assess the role of suppressor cells in the contact sensitivity reaction. A single painting with 300 microgram and 30 microgram oxazolone produced poor contact sensitivity reactions (ear swelling). Cyclophosphamide (200 mg/kg) 2 days before painting increased the response to the lower doses but had less effect on the response to 3 mg oxazolone. A single feed with 10 mg oxazolone caused strong contact sensitivity while lower doses (10-1000 microgram) caused poor responses. Cyclophosphamide increased the response to the lower doses but not to the highest dose of oxazolone. These results suggested that the poor response to painting and feeding lower doses of oxazolone was due to a suppressor system which was sensitive to cyclophosphamide. A different result was obtained when contact sensitivity was measured by arrival of radioactively labelled cells. Cyclophosphamide had the greatest effect on cell arrival when high doses were fed. This indicates that ear swelling and cell arrival measure separate aspects of the contact sensitivity response. The lower doses of oxazolone, which caused little contact sensitivity, reduced the response to a standard immunizing dose. This low dose unresponsiveness occurred after either painting or feeding (Chase-Sulzberger phenomenon). It did not occur in mice treated with cyclophosphamide before the first exposure to oxazolone. This suggested that the low dose unresponsiveness was due to suppressor cells. The response to oxazolone was also assessed by DNA synthesis in the regional lymph nodes. A small dose of oxazolone (30 microgram) caused a peak of DNA synthesis on day four while a high dose (3 mg) caused a peak on day three. Pretreatment with cyclophosphamide depressed the response to 30 microgram although it increased contact sensitivity. The secondary response was smaller than the primary on days 3, 4 and 5 after immunization but larger on day two. The depression but not the increase was prevented by cyclophosphamide and was probably due to a suppressor system.

Induction of contact sensitivity. Selective induction of delayed hypersensitivity by the injection of cells from draining lymph nodes into the footpads of normal recipients.

Cells taken from the draining lymph nodes of mice 1 day after painting with picryl chloride can induce contact hypersensitivity when injected into the footpads of normal recipients. Previous studies have presented evidence to show that this is an immunizing process rather than a transfer of sensitized cells. This study shows that footpad injection of 5 times 10(6) 1-day draining lymph node cells induces a similar degree of hypersensitivity to the original skin painting with picryl chloride but no antibody, as judged by anti-TNP splenic PFC and serum antibody, in contrast to the moderate antibody response found after skin painting. The apparent inability to induce a response did, however, correlate with the finding that the draining lymph nodes of mice painted with picryl chloride had few PFC. Some effect of the cells on antibody was noted in that after mice were challenged on the ear to produce contact sensitivity reactions they produced an antibody response larger than that of mice not injected with cells. This was not a large phenomenon but was unusual because 2-5 times 10(6) cells was more effective than either 10(6) or 5 times 10(6) cells, even though 5 times 10(6) cells produced the largest contact sensitivity reactions. The augmentation was antigen specific and the same dose-response effect could be obtained with irradiated cells (2000 rad). It is suggested that the ability of these cells to induce large contact sensitivity reactions without antibody indicates that they may have an important role in the immunogenicity of contact sensitizing agents, which can induce large delayed-type hypersensitivity reactions accompanied only by moderate antibody responses.