Leveraging Human Plasma-Derived Small Extracellular Vesicles as Liquid Biopsy to Study the Induction of Cytochrome P450 3A4 by Modafinil.

Preparations of plasma-derived small extracellular vesicles (sEVs) were deployed as liquid biopsy to study cytochrome P450 (CYP) 3A4 (CYP3A4) induction following modafinil 400 mg once daily × 14 days (young healthy volunteers, N = 10 subjects). Induction was confirmed using the 4ß-hydroxycholesterol-to-cholesterol (4ßHC/C) ratio, a plasma CYP3A4/5 biomarker, with a mean 2.1-fold increase (Day 15 vs. Day 1; 90% confidence interval (CI) = 1.8-2.3; P value = 0.0004). Proteomic analysis revealed the induction (mean Day 15 vs. Day 1 fold-increase (90% CI)) of both liver (1.3 (1.1-1.5), P value = 0.014) and nonliver (1.9 (1.6-2.2), P value = 0.04) sEV CYP3A4 protein expression. In CYP3A5 nonexpresser subjects, the baseline (pre-dose) 4ßHC/C plasma ratio was more highly correlated with liver sEVs (r = 0.937, P value = 0.001) than nonliver sEVs (r = 0.619, P value = 0.101) CYP3A4 protein expression. When CYP3A5 expressers (CYP3A5*1/*3) were included, the correlation with liver sEVs (r = 0.761, P value = 0.011) and nonliver sEVs (r = 0.391, P value = 0.264) CYP3A4 protein was weaker. Although modafinil-induced changes in plasma 4ßHC/C ratio did not correlate with sEVs CYP3A4 protein expression, the individual subject sEVs proteomic data were used successfully to predict victim drug (midazolam, triazolam, dextromethorphan, 17α-ethinylestradiol, and abemaciclib) area under the plasma concentration-time curve (AUC) ratios (AUCRs) following modafinil. Based on the AUCR values, modafinil was classified as a weak to moderate CYP3A4 inducer (vs. rifampicin). For the first time, it was possible to deploy plasma-derived sEVs to study CYP3A4 induction beyond rifampicin, a more potent CYP3A4 inducer.

Evaluation of the Association of Polymorphisms With Palbociclib-Induced Neutropenia: Pharmacogenetic Analysis of PALOMA-2/-3.

BACKGROUND: The most frequently reported treatment-related adverse event in clinical trials with the cyclin-dependent kinase 4/6 (CDK4/6) inhibitor palbociclib is neutropenia. Allelic variants in ABCB1 and ERCC1 might be associated with early occurrence (i.e., end of week 2 treatment) of grade 3/4 neutropenia. Pharmacogenetic analyses were performed to uncover associations between single nucleotide polymorphisms (SNPs) in these genes, patient baseline characteristics, and early occurrence of grade 3/4 neutropenia. MATERIALS AND METHODS: ABCB1 (rs1045642, rs1128503) and ERCC1 (rs3212986, rs11615) were analyzed in germline DNA from palbociclib-treated patients from PALOMA-2 (n = 584) and PALOMA-3 (n = 442). SNP, race, and cycle 1 day 15 (C1D15) absolute neutrophil count (ANC) data were available for 652 patients. Univariate and multivariable analyses evaluated associations between SNPs, patient baseline characteristics, and early occurrence of grade 3/4 neutropenia. Analyses were stratified by Asian (n = 122) and non-Asian (n = 530) ethnicity. Median progression-free survival (mPFS) was estimated using the Kaplan-Meier method. The effect of genetic variants on palbociclib pharmacokinetics was analyzed. RESULTS: ABCB1 and ERCC1_rs11615 SNP frequencies differed between Asian and non-Asian patients. Multivariable analysis showed that low baseline ANC was a strong independent risk factor for C1D15 grade 3/4 neutropenia regardless of race (Asians: odds ratio [OR], 6.033, 95% confidence interval [CI], 2.615-13.922, p < .0001; Non-Asians: OR, 6.884, 95% CI, 4.138-11.451, p < .0001). ABCB1_rs1128503 (C/C vs. T/T: OR, 0.57, 95% CI, 0.311-1.047, p = .070) and ERCC1_rs11615 (A/A vs. G/G: OR, 1.75, 95% CI, 0.901-3.397, p = .098) were potential independent risk factors for C1D15 grade 3/4 neutropenia in non-Asian patients. Palbociclib mPFS was consistent across genetic variants; exposure was not associated with ABCB1 genotype. CONCLUSION: This is the first comprehensive assessment of pharmacogenetic data in relationship to exposure to a CDK4/6 inhibitor. Pharmacogenetic testing may inform about potentially increased likelihood of patients developing severe neutropenia (NCT01740427, NCT01942135). IMPLICATIONS FOR PRACTICE: Palbociclib plus endocrine therapy improves hormone receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer outcomes, but is commonly associated with neutropenia. Genetic variants in ABCB1 may influence palbociclib exposure, and in ERCC1 are associated with chemotherapy-induced severe neutropenia. Here, the associations of single nucleotide polymorphisms in these genes and baseline characteristics with neutropenia were assessed. Low baseline absolute neutrophil count was a strong risk factor (p < .0001) for grade 3/4 neutropenia. There was a trend indicating that ABCB1_rs1128503 and ERCC1_rs11615 were potential risk factors (p < .10) for grade 3/4 neutropenia in non-Asian patients. Pharmacogenetic testing could inform clinicians about the likelihood of severe neutropenia with palbociclib.

Meta-Analysis of Noncompartmental Pharmacokinetic Parameters of Ertugliflozin to Evaluate Dose Proportionality and UGT1A9 Polymorphism Effect on Exposure.

Ertugliflozin, a sodium-glucose cotransporter 2 inhibitor, is primarily metabolized via glucuronidation by the uridine 5'-diphospho-glucuronosyltransferase (UGT) isoform UGT1A9. This noncompartmental meta-analysis of ertugliflozin pharmacokinetics evaluated the relationship between ertugliflozin exposure and dose, and the effect of UGT1A9 genotype on ertugliflozin exposure. Pharmacokinetic data from 25 phase 1 studies were pooled. Structural models for dose proportionality described the relationship between ertugliflozin area under the plasma concentration-time curve (AUC) or maximum observed plasma concentration (Cmax ) and dose. A structural model for the UGT1A9 genotype described the relationship between ertugliflozin AUC and dose, with genotype information on 3 UGT1A9 polymorphisms (UGT1A9-2152, UGT1A9*3, UGT1A9*1b) evaluated as covariates from the full model. Ertugliflozin AUC and Cmax increased in a dose-proportional manner over the dose range of 0.5-300 mg, and population-predicted AUC and Cmax values for the 5- and 15-mg ertugliflozin tablets administered in the fasted state demonstrated good agreement with the observed data. The largest change in ertugliflozin AUC was in subjects carrying the UGT1A9*3 heterozygous variant, with population-predicted AUC (90% confidence interval) values of 485 ng·h/mL (458 to 510 ng·h/mL) and 1560 ng·h/mL (1480 to 1630 ng·h/mL) for ertugliflozin 5 and 15 mg, respectively, compared with 436 ng·h/mL (418 to 455 ng·h/mL) and 1410 ng·h/mL (1350 to 1480 ng·h/mL), respectively, in wild-type subjects. Overall, the mean effects of the selected UGT1A9 variants on ertugliflozin AUC were within ±10% of the wild type. UGT1A9 genotype did not have any clinically meaningful effects on ertugliflozin exposure in healthy subjects. No ertugliflozin dose adjustment would be required in patients with the UGT1A9 variants assessed in this study.

Exploring the Use of Serum-Derived Small Extracellular Vesicles as Liquid Biopsy to Study the Induction of Hepatic Cytochromes P450 and Organic Anion Transporting Polypeptides.

Liver-derived small extracellular vesicles (sEVs), prepared from small sets of banked serum samples using a novel two-step protocol, were deployed as liquid biopsy to study the induction of cytochromes P450 (CYP3A4, CYP3A5, and CYP2D6) and organic anion transporting polypeptides (OATP1B1 and OATP1B3) during pregnancy (nonpregnant (T0), first, second, and third (T3) trimester women; N = 3 each) and after administration of rifampicin (RIF) to healthy male subjects. Proteomic analysis revealed induction (mean fold-increase, 90% confidence interval) of sEV CYP3A4 after RIF 300 mg × 7 days (3.5, 95% CI = 2.5-4.5, N = 4, P = 0.029) and 600 mg × 14 days (3.7, 95% CI = 2.1-6.0, N = 5, P = 0.018) consistent with the mean oral midazolam area under the plasma concentration time curve (AUC) ratio in the same subjects (0.28, 95% CI = 0.22-0.34, P < 0.0001; and 0.17, 95% CI = 0.13-0.22, P < 0.0001). Compared with CYP3A4, liver sEV CYP3A5 protein (subjects genotyped CYP3A5*1/*3) was weakly induced (≤ 1.5-fold). It was also possible to measure liver sEV-catalyzed dextromethorphan (DEX) O-demethylation to dextrorphan (DXO), correlated with sEV CYP2D6 expression (r = 0.917, P = 0.0001; N = 10) and 3-hour plasma DXO-to-DEX concentration ratio (r = 0.843, P = 0.002, N = 10), and show that CYP2D6 was not induced by RIF. Nonparametric analysis of liver sEV revealed significantly higher CYP3A4 (3.2-fold, P = 0.003) and CYP2D6 (3.7-fold, P = 0.03) protein expression in T3 vs. T0 women. In contrast, expression of both OATPs in liver sEV was unaltered by RIF administration and pregnancy.

Identification of Appropriate Endogenous Biomarker for Risk Assessment of Multidrug and Toxin Extrusion Protein-Mediated Drug-Drug Interactions in Healthy Volunteers.

Endogenous biomarkers are emerging to advance clinical drug-drug interaction (DDI) risk assessment in drug development. Twelve healthy subjects received a multidrug and toxin exclusion protein (MATE) inhibitor (pyrimethamine, 10, 25, and 75 mg) in a crossover fashion to identify an appropriate endogenous biomarker to assess MATE1/2-K-mediated DDI in the kidneys. Metformin (500 mg) was also given as reference probe drug for MATE1/2-K. In addition to the previously reported endogenous biomarker candidates (creatinine and N1 -methylnicotinamide (1-NMN)), N1 -methyladenosine (m1 A) was included as novel biomarkers. 1-NMN and m1 A presented as superior MATE1/2-K biomarkers since changes in their renal clearance (CLr ) along with pyrimethamine dose were well-correlated with metformin CLr changes. The CLr of creatinine was reduced by pyrimethamine, however, its changes poorly correlated with metformin CLr changes. Nonlinear regression analysis (CLr vs. mean total concentration of pyrimethamine in plasma) yielded an estimate of the inhibition constant (Ki ) of pyrimethamine and the fraction of the clearance pathway sensitive to pyrimethamine. The in vivo Ki value thus obtained was further converted to unbound Ki using plasma unbound fraction of pyrimethamine, which was comparable to the in vitro Ki for MATE1 (1-NMN) and MATE2-K (1-NMN and m1 A). It is concluded that 1-NMN and m1 A CLr can be leveraged as quantitative MATE1/2-K biomarkers for DDI risk assessment in healthy volunteers.

Dose-Dependent Inhibition of OATP1B by Rifampicin in Healthy Volunteers: Comprehensive Evaluation of Candidate Biomarkers and OATP1B Probe Drugs.

To address the most appropriate endogenous biomarker for drug-drug interaction risk assessment, eight healthy subjects received an organic anion transporting polypeptide 1B (OATP1B) inhibitor (rifampicin, 150, 300, and 600 mg), and a probe drug cocktail (atorvastatin, pitavastatin, rosuvastatin, and valsartan). In addition to coproporphyrin I, a widely studied OATP1B biomarker, we identified at least 4 out of 28 compounds (direct bilirubin, glycochenodeoxycholate-3-glucuronide, glycochenodeoxycholate-3-sulfate, and hexadecanedioate) that presented good sensitivity and dynamic range in terms of the rifampicin dose-dependent change in area under the plasma concentration-time curve ratio (AUCR). Their suitability as OATP1B biomarkers was also supported by the good correlation of AUC0-24h between the endogenous compounds and the probe drugs, and by nonlinear regression analysis (AUCR-1 vs. rifampicin plasma Cmax (maximum total concentration in plasma)) to yield an estimate of the inhibition constant of rifampicin. These endogenous substrates can complement existing OATP1B-mediated drug-drug interaction risk assessment approaches based on agency guidelines in early clinical trials.

Identification of the caffeine to trimethyluric acid ratio as a dietary biomarker to characterise variability in cytochrome P450 3A activity.

PURPOSE: Cytochrome P450 (CYP) 3A plays an important role in the metabolism of many clinically used drugs and exhibits substantial between-subject variability (BSV) in activity. Current methods to assess variability in CYP3A activity have limitations and there remains a need for a minimally invasive clinically translatable strategy to define CYP3A activity. The purpose of this study was to evaluate the potential for a caffeine metabolic ratio to describe variability in CYP3A activity. METHODS: The metabolic ratio 1,3,7-trimethyluric acid (TMU) to caffeine was evaluated as a biomarker to describe variability in CYP3A activity in a cohort (n = 28) of healthy 21 to 35-year-old males. Midazolam, caffeine, and TMU concentrations were assessed at baseline and following dosing of rifampicin (300 mg daily) for 7 days. RESULTS: At baseline, correlation coefficients for the relationship between apparent oral midazolam clearance (CL/F) with caffeine/TMU ratio measured at 3, 4, and 6 h post dose were 0.82, 0.79, and 0.65, respectively. The strength of correlations was retained post rifampicin dosing; 0.72, 0.87, and 0.82 for the ratios at 3, 4, and 6 h, respectively. Weaker correlations were observed between the change in midazolam CL/F and change in caffeine/TMU ratio post/pre-rifampicin dosing. CONCLUSION: BSV in CYP3A activity was well described by caffeine/TMU ratios pre- and post-induction. The caffeine/TMU ratio may be a convenient tool to assess BSV in CYP3A activity, but assessment of caffeine/TMU ratio alone is unlikely to account for all sources of variability in CYP3A activity.

Plasma extracellular nanovesicle (exosome)-derived biomarkers for drug metabolism pathways: a novel approach to characterize variability in drug exposure.

AIMS: Demonstrate the presence of cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) proteins and mRNAs in isolated human plasma exosomes and evaluate the capacity for exosome-derived biomarkers to characterize variability in CYP3A4 activity. METHODS: The presence of CYP and UGT protein and mRNA in exosomes isolated from human plasma and HepaRG cell culture medium was determined by mass spectrometry and reverse transcription-polymerase chain reaction, respectively. The concordance between exosome-derived CYP3A4 biomarkers and midazolam apparent oral clearance (CL/F) was evaluated in a small proof-of-concept study involving six genotyped (CYP3A4 *1/*1 and CYP3A5 *3/*3) Caucasian males. RESULTS: Exosomes isolated from human plasma contained peptides and mRNA originating from CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2 J2, 3A4 and 3A5, UGT 1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10 and 2B15, and NADPH-cytochrome P450 reductase. Mean (95% confidence interval) exosome-derived CYP3A4 protein expression pre- and post-rifampicin dosing was 0.24 (0.2-0.28) and 0.42 (0.21-0.65) ng ml-1 exosome concentrate. Mean (95% confidence interval) exosome CYP3A4 mRNA expression pre- and post-rifampicin dosing was 6.0 (1.1-32.7) and 48.3 (11.3-104) × 10-11 2-ΔΔCt , respectively. R2 values for correlations of exosome-derived CYP3A4 protein expression, CYP3A4 mRNA expression, and ex vivo CYP3A4 activity with midazolam CL/F were 0.905, 0.787 and 0.832, respectively. CONCLUSIONS: Consistent strong concordance was observed between exosome-derived CYP3A4 biomarkers and midazolam CL/F. The significance of these results is that CYP3A4 is the drug-metabolizing enzyme of greatest clinical importance and variability in CYP3A4 activity is poorly described by existing precision dosing strategies.

No Clinical Impact of CYP3A5 Gene Polymorphisms on the Pharmacokinetics and/or Efficacy of Maraviroc in Healthy Volunteers and HIV-1-Infected Subjects.

Maraviroc is a C-C chemokine receptor type-5 antagonist approved for the treatment of HIV-1. Previous studies show that cytochrome P450 3A5 (CYP3A5) plays a role in maraviroc metabolism. CYP3A5 is subject to a genetic polymorphism. The presence of 2 functional alleles (CYP3A5*1/*1) confers the extensive metabolism phenotype, which is rare in whites but common in blacks. The effect of CYP3A5 genotype on maraviroc and/or metabolite pharmacokinetics was evaluated in 2 clinical studies: a post hoc analysis from a phase 2b/3 study (NCT00098293) conducted in 494 HIV-1-infected subjects (study 1) in which the impact on maraviroc efficacy in 303 subjects was also assessed, and a study conducted in 47 healthy volunteers (study 2). In study 2 (NCT02625207), extensive metabolizers had 26% to 37% lower mean area under the concentration-time curve compared with poor metabolizers (no CYP3A5*1 alleles). This effect diminished to 17% in the presence of potent CYP3A inhibition. The effect of CYP3A5 genotype was greatest in the formation of the metabolite (1S,2S)-2-hydroxymaraviroc. In study 1, the CYP3A5*1/*1 genotype unexpectedly had higher maraviroc area under the curve predictions (20%) compared with those with no CYP3A5*1 alleles. The reason for this disparity remains unclear. The proportions of subjects with viral loads <50 and <400 copies/mL for maraviroc were comparable among all 3 CYP3A5 genotypes. In both studies maraviroc exposures were in the range of near-maximal viral inhibition in the majority of subjects. These results demonstrate that although CYP3A5 contributes to the metabolism of maraviroc, CYP3A5 genotype does not affect the clinical response to maraviroc in combination treatment of HIV-1 infection at approved doses.

Functional confirmation that the R1488* variant in SCN9A results in complete loss-of-function of Nav1.7.

BACKGROUND: Individuals with an extremely rare inherited condition, termed Congenital Insensitivity to Pain (CIP), do not feel pain in response to noxious stimuli. Variants in SCN9A, encoding the transmembrane voltage-gated sodium channel Nav1.7, have previously been reported in subjects with CIP accompanied by anosmia, which are typically transmitted in a recessive pattern. Functional characterisations of some of these SCN9A mutations show that they result in complete loss-of-function of Nav1.7. METHODS: In a consanguineous family we performed whole exome sequencing of three members who have a diagnosis of CIP and one unaffected family member. The functional effects of the segregating variant in SCN9A were determined using patch clamp electrophysiology in human embryonic kidney (HEK) 293 cells transfected with the variant. RESULTS: We found that each CIP subject was homozygous for a putatively nonsense variant, R1488*, in SCN9A. This variant was reported elsewhere in a subject with CIP, though the functional effect was not determined. Using electrophysiology, we confirm that this variant results in a complete loss-of-function of Nav1.7. CONCLUSIONS: We confirm through electrophysiological analysis that this R1488* variant in SCN9A results in complete loss-of-function of Nav1.7, which is consistent with reports on other variants in this gene in subjects with CIP.

Assessment of inter-racial variability in CYP3A4 activity and inducibility among healthy adult males of Caucasian and South Asian ancestries.

PURPOSE: Cytochrome P450 (CYP) 3A4 is responsible for the metabolism of more than 30% of clinically used drugs. Inherent between subject variability in clearance of CYP3A4 substrates is substantial; by way of example, midazolam clearance varies by > 10-fold between individuals before considering the impact of extrinsic factors. Relatively little is known about inter-racial variability in the activity of this enzyme. METHODS: This study assessed inter-racial variability in midazolam exposure in a cohort (n = 30) of CYP3A genotyped, age-matched healthy males of Caucasian and South Asian ancestries. Midazolam exposure was assessed at baseline, following 7 days of rifampicin and following 3 days of clarithromycin. RESULTS: The geometric mean baseline midazolam area under the plasma concentration curve (AUC0-6) in Caucasians (1057 µg/L/min) was 27% greater than South Asians (768 µg/L/min). Similarly, the post-induction midazolam AUC0-6 in Caucasians (308 µg/L/min) was 50% greater than South Asians (154 µg/L/min), while the post-inhibition midazolam AUC0-6 in Caucasians (1834 µg/L/min) was 41% greater than South Asians (1079 µg/L/min). The difference in baseline AUC0-6 between Caucasians and South Asians was statistically significant (p ≤ 0.05), and a trend toward significance (p = 0.067) was observed for the post-induction AUC0-6 ratio, in both unadjusted and genotype adjusted analyses. CONCLUSIONS: Significantly higher midazolam clearance was observed in healthy age-matched males of South Asian compared to Caucasian ancestry that was not explained by differences in the frequency of CYP3A genotypes.

A role for PACE4 in osteoarthritis pain: evidence from human genetic association and null mutant phenotype.

OBJECTIVES: The aim of this study was to assess if genetic variation in the PACE4 (paired amino acid converting enzyme 4) gene Pcsk6 influences the risk for symptomatic knee osteoarthritis (OA). METHODS: Ten PCSK6 single nucleotide polymorphisms were tested for association in a discovery cohort of radiographic knee OA (n=156 asymptomatic and 600 symptomatic cases). Meta-analysis of the minor allele at rs900414 was performed in three additional independent cohorts (total n=674 asymptomatic and 2068 symptomatic). Pcsk6 knockout mice and wild-type C57BL/6 mice were compared in a battery of algesiometric assays, including hypersensitivity in response to intraplantar substance P, pain behaviours in response to intrathecal substance P and pain behaviour in the abdominal constriction test. RESULTS: In the discovery cohort of radiographic knee OA, an intronic single nucleotide polymorphism at rs900414 was significantly associated with symptomatic OA. Replication in three additional cohorts confirmed that the minor allele at rs900414 was consistently increased among asymptomatic compared to symptomatic radiographic knee OA cases in all four cohorts. A fixed-effects meta-analysis yielded an OR=1.35 (95% CI 1.17 to 1.56; p=4.3×10(-5) and no significant between-study heterogeneity). Studies in mice revealed that Pcsk6 knockout mice were significantly protected against pain in a battery of algesiometric assays. CONCLUSIONS: These results suggest that a variant in PCSK6 is strongly associated with protection against pain in knee OA, offering some insight as to why, in the presence of the same structural damage, some individuals develop chronic pain and others are protected. Studies in Pcsk6 null mutant mice further implicate PACE4 in pain.

Comprehensive whole-genome and candidate gene analysis for response to statin therapy in the Treating to New Targets (TNT) cohort.

BACKGROUND: Statins are effective at lowering low-density lipoprotein cholesterol and reducing risk of cardiovascular disease, but variability in response is not well understood. To address this, 5745 individuals from the Treating to New Targets (TNT) trial were genotyped in a combination of a whole-genome and candidate gene approach to identify associations with response to atorvastatin treatment. METHODS AND RESULTS: A total of 291 988 single-nucleotide polymorphisms (SNPs) from 1984 individuals were analyzed for association with statin response, followed by genotyping top hits in 3761 additional individuals. None was significant at the whole-genome level in either the initial or follow-up test sets for association with low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, or triglyceride response. In addition to the whole-genome platform, 23 candidate genes previously associated with statin response were analyzed in these 5745 individuals. Three SNPs in apoE were most highly associated with low-density lipoprotein cholesterol response, followed by 1 in PCSK9 with a similar effect size. At the candidate gene level, SNPs in HMGCR were also significant though the effect was less than with those in apoE and PCSK9. rs7412/apoE had the most significant association (P=6x10(-30)), and its high significance in the whole-genome study (P=4x10(-9)) confirmed the suitability of this population for detecting effects. Age and gender were found to influence low-density lipoprotein cholesterol response to a similar extent as the most pronounced genetic effects. CONCLUSIONS: Among SNPs tested with an allele frequency of at least 5%, only SNPs in apoE are found to influence statin response significantly. Less frequent variants in PCSK9 and smaller effect sizes in SNPs in HMGCR were also revealed.

Frequency and function of CETP variants among individuals of Asian ancestry.

Genetic variation in CETP (cholesteryl ester transfer protein) has been clearly associated with HDL cholesterol levels but its association with cardiovascular disease and related phenotypes has been more controversial, possibly due to variability of polymorphisms and their frequencies across different ethnic populations. To see if there are undetected polymorphisms affecting protein sequence in individuals of Asian ancestry and to determine the functionality of such variants, all exons and adjacent intronic segments were resequenced in 96 individuals and the observed variants cloned and analyzed. Two novel SNPs, including one coding change, S332 to Y332, were identified. Y332 and all other reported variants in Asians were cloned for study in vitro. Secretion efficiency was determined by Western blotting of protein from cell lysates and media. Cholesteryl ester transfer activity was measured in vitro by following the extent of transfer of fluorescently labeled substrate. Y332, Q296 and G442 are all secreted less well than wild type protein but retain significant transfer activity. P151 is not secreted and no transfer activity was detected. These protein variants should all contribute to higher HDL cholesterol in individuals carrying them. Additionally, a splicing variation that causes a protein truncation and non-functional CETP that has been reported predominantly in Asians was also found in two individuals of European ancestry and was on the same haplotype background in the two populations, suggesting a common origin of this null variant. This improved understanding of CETP variation in Asians will allow a more effective comparison of studies across populations.

Association between neprilysin polymorphisms and sporadic Alzheimer's disease.

The deposition of amyloid beta peptide (Abeta) in the form of plaques in the brain is a hallmark of Alzheimer's disease (AD). Neprilysin is the major Abeta-degradating enzyme and reduction in neprilysin activity could contribute to Alzheimer's by increasing the steady-state level of Abeta. To provide further evidence for the role of neprilysin in AD we genotyped 22 polymorphisms, 21 SNPs and the GT repeat in the promoter region, across the neprilysin gene in 298 Caucasian sporadic Alzheimer's patients and 298 age-matched controls. Several SNPs showed genotypic and allelic association to AD. SNP rs1836915, in linkage disequilibrium block 2, showed the greatest extent of genotypic association with AD (p=0.0076). We were unable to replicate any of the SNPs that were previously reported as putatively associated with AD. However, these novel findings add to the weight of evidence supporting the involvement of neprilysin in the aetiology of Alzheimer's disease.

High-density genotyping and functional SNP localization in the CETP gene.

The cholesteryl ester transfer protein gene (CETP) has been the subject of hundreds of genetic analyses that typically focus on a small number of polymorphisms within a single ethnic group. Furthermore, the extent of DNA beyond the transcribed sequence from which single nucleotide polymorphisms (SNPs) may influence CETP expression has not been well defined. To better understand the role of natural variation in modulating CETP and high density lipoprotein-cholesterol (HDL-C) levels, dense genotyping of CETP and regions up to 15 kb on either side of the gene was carried out on >2,000 individuals. A complex, nonlinear set of linkage disequilibrium bins was found, with many bins interspersed along the DNA sequence and spread over large regions of the gene. Bins assigned based on large numbers of individuals matched the small subset of SNPs that had been assigned to bins previously with a small number of individuals. Associations of known functional SNPs with HDL-C were found, but there were suggestions that there are additional functional SNPs not characterized previously. Narrowing of the set of likely functional SNPs was accomplished by comparing associations observed in different ethnic groups. The promoter SNP most highly associated with HDL-C that is likely to be functional, position -4,502, alters a consensus transcription factor binding site.

Significant support for DAO as a schizophrenia susceptibility locus: examination of five genes putatively associated with schizophrenia.

BACKGROUND: Schizophrenia is a complex psychiatric disorder with a strong genetic component. Past linkage studies have implicated several chromosomal regions in the etiology of schizophrenia. Within these regions, several genes have been identified via candidate gene association studies as strong schizophrenia susceptibility loci, including DAO, DAOA, DISC1, DTNBP1, and RGS4. METHODS: The present study attempted to replicate these association findings by analyzing a total of 120 markers across these genes in 311 schizophrenia subjects, 140 schizoaffective subjects, and 291 control subjects. RESULTS: Our study found no association for DAOA and DTNBP1 with schizophrenia. Although no association was seen with DAOA and DTNBP1, several other markers in the other genes resulted in significant association with schizophrenia (p < .05). However, after a conservative Bonferroni correction for multiple testing, only one marker, rs3918346, within DAO remained significant (odds ratio = 1.71, confidence interval = 1.32-2.22, p = 4 x 10(-5)). This significant association was concordant with previous DAO genetic findings. CONCLUSIONS: Our results significantly support DAO as a susceptibility locus for schizophrenia and offer some support for the implication of both RGS4 and DISC1 in the etiology of schizophrenia. However, we see no evidence to support either DAOA or DTNBP1 as schizophrenia disease loci.

Association of torsades de pointes with novel and known single nucleotide polymorphisms in long QT syndrome genes.

BACKGROUND: Reduction of drug-induced adverse events may be achievable through a better understanding of the underlying causes of such events. Identifying phenotypes and genotypes that allow event prediction would provide greater safety margins for new therapeutics. Torsades de pointes (TdP) is one such life-threatening adverse event and can arise from excessive lengthening of the QT interval. This study was designed to better understand the role of genetics in the development of TdP and to determine whether genotypes can be used to predict susceptibility and thus reduce adverse events. METHODS: Seven known familial long QT syndrome genes were scanned for sequence variations in 34 patients with TdP. This group of patients is the largest such cohort ever assembled for this type of analysis. The allele frequencies for novel and known polymorphisms in these patients were compared with those in healthy control subjects. RESULTS: Six novel mutations--4 in ANK2, 1 in KCNQ1, and 1 in SCN5A--were found in the patients with TdP. Two mutations were also found in 595 healthy control subjects, whereas the others were unique to patients with TdP. Two common single nucleotide polymorphisms may be associated with the risk of TdP. The entire ANK2 gene had not been screened in a population this large previously. CONCLUSIONS: Genotypes alone could not be used to completely predict susceptibility to TdP, even when used with phenotypes. The best model using genotypic and phenotypic variables was unable to predict all events. It is unclear what other risk genes or environmental effects might be necessary to predict such cases.

Novel variants in human and monkey CETP.

Variation in CETP has been shown to play an important role in HDL-C levels and cardiovascular disease. To better characterize this variation, the promoter and exonic DNA for CETP was resequenced in 189 individuals with extreme HDL-C or age. Two novel amino acid variants were found in humans (V-12D and Y361C) and an additional variant (R137W) not previously studied in vitro were expressed. D-12 was not secreted and had no detectable activity in cells. C361 and W137 retained near normal amounts of cholesteryl ester transfer activity when purified but were less well secreted than wild type. Torcetrapib, a CETP inhibitor in clinical development with atorvastatin, was found to have a uniform effect on inhibition of wild type CETP versus W137 or C361. In addition, the level of variation in other species was assessed by resequencing DNA from nine cynomolgus monkeys. Numerous intronic and silent SNPs were found as well as two variable amino acids. The amino acid altering SNPs were genotyped in 29 monkeys and not found to be significantly associated with HDL-C levels. Three SNPs found in monkeys were identical to three found in humans with these SNPs all occurring at CpG sites.

Cholesteryl ester transfer protein variants have differential stability but uniform inhibition by torcetrapib.

Cholesteryl ester transfer protein (CETP) is an important modulator of high density lipoprotein cholesterol in humans and thus considered to be a therapeutic target for preventing cardiovascular disease. The gene encoding CETP has been shown to be highly variable, with multiple single nucleotide polymorphisms responsible for altering both its transcription and sequence. Examining nine missense variants of CETP, we found some had significant associations with CETP mass and high density lipoprotein cholesterol levels. Two variants, Pro-373 and Gln-451, appear to be more stable in vivo, an observation mirrored by partial proteolysis studies performed in vitro. Because these naturally occurring variant proteins are potentially present in clinical populations that will be treated with CETP inhibitors, all commonly occurring haplotypes were tested to determine whether the proteins they encode could be inhibited by torcetrapib, a compound currently in clinical trials in combination with atorvastatin. Torcetrapib behaved similarly with all variants, with no significant differences in inhibition.