Transcriptomic variation of pharmacogenes in multiple human tissues and lymphoblastoid cell lines.

Variation in the expression level and activity of genes involved in drug disposition and action ('pharmacogenes') can affect drug response and toxicity, especially when in tissues of pharmacological importance. Previous studies have relied primarily on microarrays to understand gene expression differences, or have focused on a single tissue or small number of samples. The goal of this study was to use RNA-sequencing (RNA-seq) to determine the expression levels and alternative splicing of 389 Pharmacogenomics Research Network pharmacogenes across four tissues (liver, kidney, heart and adipose) and lymphoblastoid cell lines, which are used widely in pharmacogenomics studies. Analysis of RNA-seq data from 139 different individuals across the 5 tissues (20-45 individuals per tissue type) revealed substantial variation in both expression levels and splicing across samples and tissue types. Comparison with GTEx data yielded a consistent picture. This in-depth exploration also revealed 183 splicing events in pharmacogenes that were previously not annotated. Overall, this study serves as a rich resource for the research community to inform biomarker and drug discovery and use.

A missense mutation in the dystrophin gene in a Duchenne muscular dystrophy patient.

About two thirds of Duchenne muscular dystrophy (DMD) patients have either gene deletions or duplications. The other DMD cases are most likely the result of point mutations that cannot be easily identified by current strategies. Utilizing a heteroduplex technique and direct sequencing of amplified products, we screened our nondeletion/duplication DMD population for point mutations. We now describe what we believe to be the first dystrophin missense mutation in a DMD patient. The mutation results in the substitution of an evolutionarily conserved leucine to arginine in the actin-binding domain. The patient makes a dystrophin protein which is properly localized and is present at a higher level than is observed in DMD patients. This suggests that an intact actin-binding domain is necessary for protein stability and essential for function.

Interaction between lymphocytes and platelets in the synthesis of prostacyclin.

To test the hypothesis that prostacyclin (PGI2) is formed via a biochemical interaction between platelets and lymphocytes, we measured eicosanoids by high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). A distinct 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) peak was noted when [14C]arachidonic acid ([14C]AA) was added to the mixed cell preparations which was increased by pretreating platelets with 1-benzylimidazole (1-BI). Lymphocytes prelabeled with [14C]AA failed to form 6KPGF1 alpha when stimulated with phytohemagglutinin (PHA) or ionophore A23187. When the prelabeled platelets were suspended together with aspirin-treated lymphocytes and stimulated with ionophore, thrombin, or collagen, a 6KPGF1 alpha peak was detected and enhanced by 1-BI. These results were supported by quantifying the 6KPGF1 alpha content in the HPLC-purified fraction by RIA. Adding prostaglandin H2 (PGH2) directly to lymphocytes led to 6KPGF1 alpha production. Platelet aggregation and release were inhibited by lymphocytes in a dose-related manner. We conclude that lymphocytes possess PGI2 synthase activity which is capable of converting platelet-derived PGH2 into PGI2. PGI2 formed is sufficient to inhibit platelet function.

Serum prostacyclin binding defects in thrombotic thrombocytopenic purpura.

To understand the pathophysiologic significance of abnormal serum prostacyclin (PGI2) binding activities in thrombotic thrombocytopenic purpura (TTP), we evaluated the PGI2 binding characteristics in three chronic TTP sera and 19 normal sera. PGI2 binding by serum was rapid and reversible. The binding activity in TTP sera (22.1 +/- SD, 4.4%) was significantly lower than that of normal sera (42.2 +/- 6.2%). Moreover, the antiaggregating activity and 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) content in the gel filtrates representing the binding peak was proportionally lower in a TTP serum than normal serum. Although normal and TTP sera bound [14C]arachidonate with similar activity, and neither bound [3H]6KPGF1 alpha, there was a difference in prostaglandin E1 (PGE1) binding. Binding of [3H]PGE1 was subnormal in two TTP sera (W.J. and T.G.) and normal in the third (H.S.). Normal serum corrected the binding defects of TTP serum. Interestingly, the mixture of two TTP sera (W.J. and H.S.) mutually corrected their PGI2 binding defects. In addition, although in vivo plasma transfusions improved the PGI2 binding activity of W.J. and H.S., there existed a striking difference in the nature of their response. These observations indicate that there is at least two types of PGI2 binding defects in TTP. Our data indicate that TTP is associated with diminished serum binding of PGI2. This defect may reduce the availability of PGI2 to damaged vascular sites and decrease an important modulator of platelet thrombus formation at times of severe vascular insult.

Dystrophin expression in a Duchenne muscular dystrophy patient with a frame shift deletion.

The exon 45 deletion is a common dystrophin gene deletion. Although this is an out-of-frame deletion, which should not allow for protein synthesis, it has been observed in mildly affected patients. We describe a patient with an exon 45 deletion who produced protein, but still had a severe Duchenne muscular dystrophy phenotype. RT-PCR analysis and cDNA sequencing from the muscle biopsy sample revealed that the exon 45 deletion induced exon skipping of exon 44, which resulted in an in-frame deletion and the production of dystrophin. A conformational change in dystrophin induced by the deletion is proposed as being responsible for the severe phenotype in the patient. We feel that the variable clinical phenotype observed in patients with the exon 45 deletion is not due to exon splicing but may be the result of other environmental or genetic factors, or both.

Detection of cytomegalovirus in liver transplant biopsies. A comparison of light microscopy, immunohistochemistry, duplex PCR and nested PCR.

The polymerase chain reaction was used to detect cytomegalovirus (CMV) in 91 formalin-fixed paraffin-embedded needle biopsies from 38 liver transplant patients with allograft dysfunction. Thirty donor liver biopsies served as negative controls. PCR results were compared with light microscopy (LM), immunohistochemical staining (IH) for CMV early and late antigen, and clinical data. Primers to the major immediate early gene (MIE) and the viral DNA polymerase gene were duplex amplified. PCR product was reamplified with a nested primer set for the MIE and confirmed by electrophoretic mobilities and dot blotting. Primers for human beta-hemoglobin were used as internal controls. Seventeen of 38 patients had clinical evidence of cytomegalovirus disease, 12 of these were IH-positive, 14 were LM-positive, 15 were duplex PCR-positive and 17 were nested PCR-positive. In addition, duplex PCR was positive in one patient without other evidence of CMV disease, while nested PCR was positive in 12 such patients. The sensitivity and negative predictive value of nested PCR was 100%--however, the specificities and positive predictive values were only 42.9 and 58.6%, respectively. The control group was completely negative by LM, IH, and duplex PCR, however, 6 of 30 patients were nested PCR-positive. The number of nested-positive, duplex-negative patients without CMV disease was significantly greater in the transplant group versus the control group (12/21 vs. 6/30, P < 0.009). The incidence of IgG seropositivity was also significantly greater in the transplant group versus the controls (29/32 vs. 15/24, P < 0.02). We conclude that nested PCR may be an overly sensitive technique for the detection of clinically relevant CMV disease. A negative nested PCR assay for CMV may, however, help rule-out symptomatic CMV infection in an individual case. Duplex PCR showed little advantage over LM, while IH was confirmatory but did not add any new information in this study.

ARIC hemostasis study--I. Development of a blood collection and processing system suitable for multicenter hemostatic studies.

In order to carry out a multicenter study aimed at understanding the association of hemostatic factors with atherosclerotic vascular disorders for the Atherosclerosis Risk In Communities (ARIC) Study, we compared a blood collection and processing system developed in our laboratory with the state-of-the-art-procedures. The salient features of our system included the use of a new phlebotomy set for venipuncture, the use of Millipore filters for removing platelet residues in the plasma and the use of a mixture of anticoagulants and antiplatelet agents for inhibiting the in vitro activation of platelets, coagulation and fibrinolytic system. The results derived from systematic evaluations indicate that this newly developed system yields the lowest values of plasma beta TG, PF 4 and FPA when compared with the reported values. The technique also gave reliable values of representative hemostatic measurements such as fibrinogen, factor VII, factor VIII, von Willebrand factor, antithrombin-III, protein C, tissue-type plasminogen activator, and serum thromboxane B2. Further experiments revealed that the samples withstood temporary storage at -70 degrees C and overnight "shipping" manipulations without significant changes in the hemostatic values. We conclude that the described blood collection and processing system may be a valuable asset for conducting multicenter cooperative clinical trials and epidemiologic studies involving blood collection by multiple field centers or clinics.

ARIC hemostasis study--II. Organizational plan and feasibility study.

In our previous paper, we reported the development of a blood collection and processing system (BCPS) suitable for the ARIC multicenter hemostasis study. As an additional step of preparation for the ARIC study, we incorporated this BCPS into an organizational plan to increase efficiency and minimize errors. We initially designed organizational trays for blood collection tubes and aliquot tubes and developed a coordinated step-by-step plan for the orderly processing of blood samples. Once the plan was considered workable, we carried out a pilot study to test the feasibility of this integrated organizational plan. Included in the pilot study were 95 healthy subjects randomly selected from 4 ARIC field centers, whose age and gender were comparable to those projected for the ARIC population. We determined the time lapse of filling the first tube as an index of blood flow. The overall mean time-lapse was 23 s (S.D. = 5). There was no significant difference among the field centers. We also determined the entire time lapse required for completing the sample processing. The total processing time was always less than 60 min. By performing the processing of samples in pairs, all the samples from two subjects could be completely processed in 70 min. This greatly increased the efficiency of field center operation. We evaluated the potential in vitro hemostasis activation by measuring plasma beta-thromboglobulin and platelet factor 4 levels. The geometric means of both proteins were comparable to our previously reported results. Fibrinogen, factor VII, factor VIII, von Willebrand factor, antithrombin III, protein C and activated partial thromboplastin time were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

Germline mosaicism at the fragile X locus.

We have identified a fragile X syndrome pedigree where the disorder is associated with a molecular deletion. The deletion was present in the DNA of 2 sons but was absent in the mother's somatic cell (lymphocyte) DNA. The results are consistent with the deletion arising as a postzygotic event in the mother, who therefore is germinally mosaic. This finding has important implications for counseling fragile X families with deletion mutations.

Heteroduplex analysis of the dystrophin gene: application to point mutation and carrier detection.

Approximately one-third of the Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, we identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. We conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing.

Increased D allele frequency of the angiotensin-converting enzyme gene in pulmonary fibrosis.

An insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme has previously been studied extensively in relationship to cardiovascular and renal disease. The deletion/deletion (D/D) genotype is associated with a poor outcome in immunoglobulin (Ig) A nephropathy. However, the association of this genetic marker in cardiovascular and renal disease has generated controversy, with the exception of the rate of progression and therapeutic responsiveness in IgA nephropathy. Many of the same cytokines and polypeptide mediators involved in fibrosis of the cardiovascular and renal systems have been shown to be involved in pulmonary fibrosis. We examined the I/D polymorphism of the angiotensin-converting enzyme in a group of 24 patents with interstitial pneumonia and moderate to severe pulmonary fibrosis defined by radiographic studies, pulmonary function tests, and histologic findings. The incidence of the D allele in this study population was 69.0%, which is approximately 15.0% higher than the incidence in the general population of 54.0%. The incidence of the D/D genotype was 42.0%, which is approximately 11.0% greater than that in the general population (31.0%). The distribution of the D/D, I/D, and insertion/insertion genotypes of these 24 patients was not significantly different from that of historical controls (P =.1; chi(2) test); there were marginally significantly more D alleles among the 48 observed alleles than would be expected (P =.04).

Optimization of DNA extraction from formalin-fixed tissue and its clinical application in Duchenne muscular dystrophy.

The authors report a case of Duchenne muscular dystrophy in which a deletion was determined by multiplex polymerase chain reaction using postmortem tissue from a proband who died in 1985, 2 years before the cloning of the dystrophin gene. Several extraction methods were analyzed to determine optimal conditions for recovery of DNA from fixed tissue. Variables examined were tissue lysis times and temperatures, a simple salting-out procedure for purification of DNA, polymerase chain reaction amplification of crude lysate versus purified DNA, and lysis of different tissues and tissue quantities. Extracted DNA was analyzed spectrophotometrically, electrophoretically, and by its suitability for polymerase chain reaction amplification using exon flanking primers of the dystrophin gene. Lysis at 37 degrees C for less than 24 hours led to the recovery of predominantly low-molecular-weight DNA, which was unsuitable for polymerase chain reaction in this experience. Lysis at 54 degrees C consistently yielded visible, spoolable quantities of high-molecular-weight DNA in as little as 24 hours. Direct amplification of crude, unpurified lysates was unsuccessful. However, purified samples yielded consistent amplification products. The purification step was simplified by substituting a rapid salting-out procedure for organic extractions.

Production of eicosanoids by deendothelialized rabbit aorta: interaction between platelets and vascular wall in the synthesis of prostacyclin.

Production of eicosanoids by deendothelialized aorta in response to continuous infusions of arachidonic acid and platelet suspensions was determined in a rabbit aorta perfusion model. 6-keto-PGF1 alpha production was stimulated by AA infusion in a dose-related manner. Infusion of AA at 4 micrograms/ml/min led to an initial production rate of 0.64 +/- 0.29 ng/min which gradually increased to 0.93 +/- 0.11 ng/min at the 20th min of infusion. When the concentration of AA infusion was increased to 10 micrograms/ml/min, 6-keto-PGF1 alpha production increased to 1.14 +/- 0.86 ng/min initially but declined with time. PGE2 production in response to AA 10 micrograms/min/ml was steady at around 5 ng/min while PGF2 alpha and TXB2 production were only slightly above the control. Perfusion of rabbit washed platelet suspensions at a rate of 3 X 10(8) plt/ml/min raised 6KPGF1 alpha production. The production was further increased when platelets were pretreated with 1-benzylimidazole (5 mM), along with a concurrent reduction in TXB2 release. Pretreatment of platelets with aspirin, on the other hand, abolished the increase in 6KPGF1 alpha production. Our data indicated that the vascular smooth muscle cells can efficiently utilize PGH2 produced by platelets to synthesize PGI2.

Cholesteryl ester transfer protein polymorphisms, statin use, and their impact on cholesterol levels and cardiovascular events.

The association of nonfunctional variants of the cholesteryl ester transfer protein (CETP) with efficacy of statins has been a subject of debate. We evaluated whether three functional CETP variants influence statin efficacy. The effect of CETP genotype on achieved levels of high-density lipoprotein cholesterol (HDLc), low-density lipoprotein cholesterol (LDLc), and total cholesterol during statin treatment was estimated by meta-analysis of the linear regression outcomes of three studies (11,021 individuals). The effect of these single-nucleotide polymorphisms (SNPs) on statin response in protecting against myocardial infarction (MI) was estimated by meta-analysis of statin × SNP interaction terms from logistic regression in five studies (16,570 individuals). The enhancer SNP rs3764261 significantly increased HDLc by 0.02 mmol/l per T allele (P = 6 × 10(-5)) and reduced protection against MI by statins (interaction odds ratio (OR) = 1.19 per T allele; P = 0.04). Focusing on functional CETP variants, we showed that in carriers of the rs3764261 T variant, HDLc increased more during statin treatment, and protection against MI by statins appeared to be reduced as compared with those in noncarriers.

Dopamine transporter DAT and receptor DRD2 variants affect risk of lethal cocaine abuse: a gene-gene-environment interaction.

Epistatic gene-gene interactions could contribute to the heritability of complex multigenic disorders, but few examples have been reported. Here, we focus on the role of aberrant dopaminergic signaling, involving the dopamine transporter DAT, a cocaine target, and the dopamine D2 receptor, which physically interacts with DAT. Splicing polymorphism rs2283265 of DRD2, encoding D2 receptors, were shown to confer risk of cocaine overdose/death (odds ratio ∼3) in subjects and controls from the Miami Dade County Brain Bank.(1) Risk of cocaine-related death attributable to the minor allele of rs2283265 was significantly enhanced to OR=7.5 (P=0.0008) in homozygous carriers of the main 6-repeat allele of DAT rs3836790, a regulatory VNTR in intron8 lacking significant effect itself. In contrast, carriers of the minor 5-repeat DAT allele showed no significant risk (OR=1.1, P=0.84). DAT rs3836790 and DRD2 rs2283265 also interacted by modulating DAT protein activity in the ventral putamen of cocaine abusers. In high-linkage disequilibrium with the VNTR, DAT rs6347 in exon9 yielded similar results. Assessing the impact of DAT alone, a rare DAT haplotype formed by the minor alleles of rs3836790 and rs27072, a regulatory DAT variant in the 3'-UTR, occurred in nearly one-third of the cocaine abusers but was absent in African American controls, apparently conferring strong risk. These results demonstrate gene-gene-drug interaction affecting risk of fatal cocaine intoxication.

Profiling solute carrier transporters in the human blood-brain barrier.

The neuroprotective function of the blood-brain barrier (BBB) presents a major challenge for drug delivery to the central nervous system (CNS). Critical to this function, BBB membrane transporters include the ATP-binding cassette (ABC) transporters, which limit drug penetration across the BBB, and the less-well-studied solute carrier (SLC) transporters. In this work, expression profiling of 359 SLC transporters, comparative expression analysis with kidney and liver, and immunoassays in brain microvessels (BMVs) identified previously unknown transporters at the human BBB.

Pharmacogenomics of the RNA world: structural RNA polymorphisms in drug therapy.

The use of pharmacogenomic biomarkers can enhance treatment outcomes. Regulatory polymorphisms are promising biomarkers that have proven difficult to uncover. They come in two flavors: those that affect transcription (regulatory single-nucleotide polymorphisms (rSNPs)) and those that affect RNA functions such as splicing, turnover, and translation (termed structural RNA SNPs (srSNPs)). This review focuses on the role of srSNPs in drug metabolism, transport, and response. An understanding of the nature and diversity of srSNPs and rSNPs enables clinical scientists to evaluate genetic biomarkers.

Binding of prostacyclin by plasma glycoproteins.

The binding of prostacyclin (PGI2) to plasma proteins and the resulting increase in PGI2 stability was investigated. Using gel filtration to separate bound and free PGI2, we have found that Cohn Fraction VI can bind PGI2, and retard its hydrolysis to 6-keto-PGF1 alpha (6KPGF1 alpha). The biological activity of the bound PGI2 correlated well with the quantity of bound PGI2, measured as 6KPGF1 alpha by RIA. Fraction VI bound a greater percentage of PGI2 than the other eicosanoids tested (i.e., PGI2 greater than TXB2 greater than LTB4 greater than PGE1 greater than PGF2 alpha). The PGI2 binding activity of Fraction VI was lost after neuraminidase treatment. Our data suggest that Fraction VI glycoproteins may play an important role in the binding and stabilization of PGI2 by plasma proteins.

Case of the month: germline mosaicism in carriers of Duchenne muscular dystrophy.

Carrier testing in a Duchenne muscular dystrophy (DMD) family resulted in the identification of a case of germline mosaicism. Using dystrophin cDNA probes, this phenomenon was ascertained by the demonstration of a deletion junction fragment present in the DNA of the affected patient and one sister but absent in the mother's DNA. As a result of this finding carrier risk estimations, based on restriction fragment length polymorphism (RFLP) studies, were significantly altered. The case demonstrates the importance of cDNA deletion carrier testing and the counseling implications of germline mosaicism.