Tusar K. Giri

Ability of VKORC1 and CYP2C9 to predict therapeutic warfarin dose during the initial weeks of therapy

Summary.  Background: CYP2C9 and VKORC1 genotypes predict therapeutic warfarin dose at initiation of therapy; however, the predictive ability of genetic information after a week or longer is unknown. Experts have hypothesized that genotype becomes irrelevant once international normalized ratio (INR) values are available because INR response reflects warfarin sensitivity. Methods: We genotyped the participants in the Prevention of Recurrent Venous Thromboembolism (PREVENT) trial, who had idiopathic venous thromboemboli and began low‐intensity warfarin (therapeutic INR 1.5–2.0) using a standard dosing protocol. To develop pharmacogenetic models, we quantified the effect of genotypes, clinical factors, previous doses and INR on therapeutic warfarin dose in the 223 PREVENT participants who were randomized to warfarin and achieved stable therapeutic INRs. Results: A pharmacogenetic model using data from day 0 (before therapy initiation) explained 54% of the variability in therapeutic dose (R2). The R2 increased to 68% at day 7, 75% at day 14, and 77% at day 21, because of increasing contributions from prior doses and INR response. Although CYP2C9 and VKORC1 genotypes were significant independent predictors of therapeutic dose at each weekly interval, the magnitude of their predictive ability diminished over time: partial R2 of genotype was 43% at day 0, 12% at day 7, 4% at day 14, and 1% at day 21. Conclusion: Over the first weeks of warfarin therapy, INR and prior dose become increasingly predictive of therapeutic dose, and genotype becomes less relevant. However, at day 7, genotype remains clinically relevant, accounting for 12% of therapeutic dose variability.

Vascular dermatan sulfate regulates the antithrombotic activity of heparin cofactor II

Heparin cofactor II (HCII)-deficient mice form occlusive thrombi more rapidly than do wild-type mice following injury to the carotid arterial endothelium. Dermatan sulfate (DS) and heparan sulfate (HS) increase the rate of inhibition of thrombin by HCII in vitro, but it is unknown whether vascular glycosaminoglycans play a role in the antithrombotic effect of HCII in vivo. In this study, we found that intravenous injection of either wild-type recombinant HCII or a variant with low affinity for HS (K173H) corrected the abnormally short thrombosis time of HCII-deficient mice, while a variant with low affinity for DS (R189H) had no effect. When HCII was incubated with frozen sections of the mouse carotid artery, it bound specifically to DS in the adventitia. HCII was undetectable in the wall of the uninjured carotid artery, but it became concentrated in the adventitia following endothelial injury. These results support the hypothesis that HCII interacts with DS in the vessel wall after disruption of the endothelium and that this interaction regulates thrombus formation in vivo.

Genetic polymorphism at Val80 (rs700518) of the CYP19A1 gene is associated with aromatase inhibitor associated bone loss in women with ER + breast cancer.

PURPOSE Polymorphisms in the CYP19A1 (aromatase) gene have been reported to influence disease-free survival and the incidence of musculoskeletal complaints in patients taking aromatase inhibitors (AIs) for estrogen receptor positive (ER+) breast cancer. Bone loss and fractures are well-recognized complications from AI therapy. The objective of this study is to determine the influence of polymorphisms in the CYP19A1 gene on bone loss among patients taking aromatase inhibitors for ER+ breast cancer. PATIENTS AND METHODS The subjects consisted of 97 postmenopausal women with ER+ breast cancer who were initiated on third-generation AIs. Bone mineral density (BMD) was measured by dual energy X-ray absorptiometry at baseline and at 6 and 12 months. Twenty-four hour urine N-telopeptide (NTX) was measured by Elisa and serum estradiol was measured by ultrasensitive radioimmunoassay at baseline, and at 6 months. Genotyping was done by Taqman SNP allelic discrimination assay. RESULTS Women with the AA genotype for the rs700518 (G/A at Val(80)) developed significant bone loss at the lumbar spine and the total hip at 12 months relative to patients carrying the G allele (GA/GG); both p = 0.03. There was a borderline greater increase in urinary NTX in those with the AA genotype compared to patients with the G allele, p = 0.05; but no significant difference in changes in estradiol levels among the genotypes. CONCLUSION Patients with the AA genotype for the rs700518 polymorphism in the CYP19A1 gene are at risk for AI-associated bone loss and deserve close follow-up during long-term AI therapy.

The Val432Leu polymorphism of the CYP1B1 gene is associated with differences in estrogen metabolism and bone density.

Polymorphisms of the CYP450 genes that encode for the enzymes that metabolize estrogen are linked to hormone-related cancers. We investigated the impact of two polymorphisms of the CYP1B1 gene previously reported to be associated with hormone-related disorders on estrogen metabolism and bone mineral density (BMD), another hormone-dependent condition, in women from different ethnic backgrounds. Four hundred sixty-eight postmenopausal Caucasian women, 220 from St. Louis, MO, USA (mean age=63.5+/-0.53 years) and 248 from Palermo, Italy (mean age=72.9+/-0.44 years) participated in the study. Measurements of urinary estrogen metabolites by enzyme-linked immunoassay, serum estradiol by ultrasensitive radioimmnunoassay, and serum sex hormone-binding globulin by immunoradiometric assay were performed only in the American women, while BMD by dual energy X-ray absorptiometry and genotyping by pyrosequencing were performed in both American and Italian women. Differences in the levels of metabolites, free estradiol index and BMD were analyzed by analysis of covariance. Analysis among the American participants for the Valine432Leucine polymorphism showed that, compared to women with the Val/Val genotype, women with the Leu allele (Val/Leu and Leu/Leu) had significantly higher log-transformed values of total urinary estrogen metabolite (ng/mg-creatinine) levels (1.23+/-0.04, 1.35+/-0.02, and 1.34+/-0.03; p=0.03), and significantly lower BMD (gm/cm(2)) in the lumbar spine (1.009+/-0.02, 0.955+/-0.01 and 0.931+/-0.02; p=0.03) and the femoral neck (0.748+/-0.02, 0.717+/-0.01 and 0.693+/-001, p=0.03) for the Val/Val, Val/Leu and Leu/Leu genotypes respectively. There were no significant differences in the urinary metabolites and BMD in the different genotypes for the Alanine119Serine polymorphism among the American women. Meanwhile, a separate analysis among the Italian women revealed no significant differences in BMD among the different genotypes for the two polymorphisms investigated. In conclusion, women with the Leu allele for the CYP1B1 Val432polymorphism have increased estrogen catabolism, as indicated by higher urinary estrogen metabolites, compared to those with Val/Val genotype. This may lead to relative hypoestrogenism and lower BMD in the lumbar spine and femoral neck in these women. Our data suggest that through its effect on the rate of estrogen catabolism, the Val432Leu polymorphism of the CYP1B1 gene may represent as a possible genetic risk factor for osteoporosis in American women.

Acquisition of Activation Receptor Ligand by Trogocytosis Renders NK Cells Hyporesponsive

Because NK cells secrete cytotoxic granules and cytokines that can destroy surrounding cells and help shape the subsequent immune response, they must be kept under tight control. Several mechanisms, at different levels, are in place to control NK cell function. In this study, we describe a novel mechanism regulating NK cell function in which NK cells acquire ligands for activating receptors from target cells by trogocytosis, rendering the NK cells hyporesponsive. In this model, murine NK cells acquire m157, the murine CMV–encoded ligand for the Ly49H-activating receptor, from target cells both in vitro and in vivo. Although acquisition of m157 requires cell-to-cell contact, it does not require the expression of the Ly49H receptor by the NK cell. Acquired m157 protein is expressed on the NK cell surface with a glycosylphosphatidylinisotol linkage and interacts with the Ly49H receptor expressed on the NK cell. This interaction results in blocking the Ly49H receptor that prevents the NK cells from recognizing m157-expressing targets and continuous engagement of the Ly49H-activating receptor, which results in the hyporesponsiveness of the Ly49H+ NK cell to stimulation through other activating receptors. Thus, NK cell acquisition of a ligand for an activation receptor by trogocytosis renders them hyporesponsive. This mechanism, by which mature NK cell function can be altered, has important implications in regard to how NK cells respond to tumors in specific microenvironments as well as the use of expanded NK cells in treating various malignancies.

Increased 2-hydroxylation of estrogen is associated with lower body fat and increased lean body mass in postmenopausal women.

Menopause is associated with changes in bone, muscle and fat mass. The importance of postmenopausal estrogen metabolism in bone health has been established. However, its relationship to body composition in postmenopausal women remains undetermined. The objective of this study is to determine the association between estrogen metabolism and body composition in postmenopausal women. This is a cross sectional study of 97 postmenopausal Caucasian women, 49-80 y.o., ≥1 year from the last normal menstrual period or those who have had oophorectomy. Inactive [2-hydroxyestrone (2OHE(1))] and active [16α-hydroxyestrone (16α-OHE(1))] urinary metabolites of estrogen were measured by ELISA. The whole and regional body composition was measured by DXA. We have found that both 2OHE(1), and 2OHE(1)/16α-OHE(1) ratio were negatively correlated with % total fat, and % truncal fat but positively correlated with % total lean mass. Comparing the fat and lean parameters of body composition according to tertiles of 2OHE(1) and 2OHE(1)/16αOHE(1) ratio showed that subjects in the lowest tertiles, had the highest % total fat, and % truncal fat and the lowest % total lean mass. Multiple regression analysis also showed 2OHE(1) and calcium intake as statistically significant predictors of all body composition parameters. In conclusion, in postmenopausal women, an increase in the metabolism of estrogen towards the inactive metabolites is associated with lower body fat and higher lean mass than those with predominance of the metabolism towards the active metabolites.

Pooled sequencing of candidate genes implicates rare variants in the development of asthma following severe RSV bronchiolitis in infancy

Severe infection with respiratory syncytial virus (RSV) during infancy is strongly associated with the development of asthma. To identify genetic variation that contributes to asthma following severe RSV bronchiolitis during infancy, we sequenced the coding exons of 131 asthma candidate genes in 182 European and African American children with severe RSV bronchiolitis in infancy using anonymous pools for variant discovery, and then directly genotyped a set of 190 nonsynonymous variants. Association testing was performed for physician-diagnosed asthma before the 7th birthday (asthma) using genotypes from 6,500 individuals from the Exome Sequencing Project (ESP) as controls to gain statistical power. In addition, among patients with severe RSV bronchiolitis during infancy, we examined genetic associations with asthma, active asthma, persistent wheeze, and bronchial hyperreactivity (methacholine PC20) at age 6 years. We identified four rare nonsynonymous variants that were significantly associated with asthma following severe RSV bronchiolitis, including single variants in ADRB2, FLG and NCAM1 in European Americans (p = 4.6x10-4, 1.9x10-13 and 5.0x10-5, respectively), and NOS1 in African Americans (p = 2.3x10-11). One of the variants was a highly functional nonsynonymous variant in ADRB2 (rs1800888), which was also nominally associated with asthma (p = 0.027) and active asthma (p = 0.013) among European Americans with severe RSV bronchiolitis without including the ESP. Our results suggest that rare nonsynonymous variants contribute to the development of asthma following severe RSV bronchiolitis in infancy, notably in ADRB2. Additional studies are required to explore the role of rare variants in the etiology of asthma and asthma-related traits following severe RSV bronchiolitis.

Vitamin D Levels Are Unrelated to the Severity of Respiratory Syncytial Virus Bronchiolitis Among Hospitalized Infants

BACKGROUND Vitamin D deficiency at birth has been reported as a risk factor for respiratory syncytial virus (RSV) lower respiratory tract infection during the first year of life. Limited data are available on whether an infants vitamin D status is associated with the severity of acute RSV bronchiolitis. METHODS Infants < 1 year of age and hospitalized with their first episode of RSV bronchiolitis were enrolled into the RSV Bronchiolitis in Early Life II cohort. We investigated the relationships between vitamin D status at enrollment and the following indicators of bronchiolitis severity: duration of hospitalization, lowest oxygen saturation measured during hospitalization, and bronchiolitis severity score. RESULTS Among the 145 enrolled infants, the median (quartile 1 [Q1], Q3) serum 25-OH-VitD level was 36.8 (29.8, 42.3) ng/mL, with 14 infants (9.7%) having deficient serum vitamin D levels (25-OH-VitD <20 ng/mL). Vitamin D-deficient infants were younger than infants with 25-OH-VitD ≥ 20 ng/mL (2.8 vs 4.5 months, respectively; P = .04) and were less likely to consume infants formula (42.9% vs 87.0%, respectively; P < .01). The following indicators of acute bronchiolitis severity did not differ between infants who were vitamin D-deficient and nondeficient: duration of hospitalization (P = .53), lowest oxygen saturation (P = .45), and bronchiolitis severity score (P = .97), even after adjusting for age, and for infants formula consumption. CONCLUSIONS Among this cohort of infants that were hospitalized for RSV bronchiolitis, vitamin D status at the time of bronchiolitis was not associated with indicators of acute bronchiolitis severity.