Genetic variation among 82 pharmacogenes: The PGRNseq data from the eMERGE network.

Genetic variation can affect drug response in multiple ways, although it remains unclear how rare genetic variants affect drug response. The electronic Medical Records and Genomics (eMERGE) Network, collaborating with the Pharmacogenomics Research Network, began eMERGE-PGx, a targeted sequencing study to assess genetic variation in 82 pharmacogenes critical for implementation of "precision medicine." The February 2015 eMERGE-PGx data release includes sequence-derived data from ∼5,000 clinical subjects. We present the variant frequency spectrum categorized by variant type, ancestry, and predicted function. We found 95.12% of genes have variants with a scaled Combined Annotation-Dependent Depletion score above 20, and 96.19% of all samples had one or more Clinical Pharmacogenetics Implementation Consortium Level A actionable variants. These data highlight the distribution and scope of genetic variation in relevant pharmacogenes, identifying challenges associated with implementing clinical sequencing for drug treatment at a broader level, underscoring the importance for multifaceted research in the execution of precision medicine.

A genome-wide association study identifies variants in KCNIP4 associated with ACE inhibitor-induced cough.

The most common side effect of angiotensin-converting enzyme inhibitor (ACEi) drugs is cough. We conducted a genome-wide association study (GWAS) of ACEi-induced cough among 7080 subjects of diverse ancestries in the Electronic Medical Records and Genomics (eMERGE) network. Cases were subjects diagnosed with ACEi-induced cough. Controls were subjects with at least 6 months of ACEi use and no cough. A GWAS (1595 cases and 5485 controls) identified associations on chromosome 4 in an intron of KCNIP4. The strongest association was at rs145489027 (minor allele frequency=0.33, odds ratio (OR)=1.3 (95% confidence interval (CI): 1.2-1.4), P=1.0 × 10(-8)). Replication for six single-nucleotide polymorphisms (SNPs) in KCNIP4 was tested in a second eMERGE population (n=926) and in the Genetics of Diabetes Audit and Research in Tayside, Scotland (GoDARTS) cohort (n=4309). Replication was observed at rs7675300 (OR=1.32 (1.01-1.70), P=0.04) in eMERGE and at rs16870989 and rs1495509 (OR=1.15 (1.01-1.30), P=0.03 for both) in GoDARTS. The combined association at rs1495509 was significant (OR=1.23 (1.15-1.32), P=1.9 × 10(-9)). These results indicate that SNPs in KCNIP4 may modulate ACEi-induced cough risk.

Genetic variation in the HLA region is associated with susceptibility to herpes zoster.

Herpes zoster, commonly referred to as shingles, is caused by the varicella zoster virus (VZV). VZV initially manifests as chicken pox, most commonly in childhood, can remain asymptomatically latent in nerve tissues for many years and often re-emerges as shingles. Although reactivation may be related to immune suppression, aging and female sex, most inter-individual variability in re-emergence risk has not been explained to date. We performed a genome-wide association analyses in 22,981 participants (2280 shingles cases) from the electronic Medical Records and Genomics Network. Using Cox survival and logistic regression, we identified a genomic region in the combined and European ancestry groups that has an age of onset effect reaching genome-wide significance (P>1.0 × 10(-8)). This region tags the non-coding gene HCP5 (HLA Complex P5) in the major histocompatibility complex. This gene is an endogenous retrovirus and likely influences viral activity through regulatory functions. Variants in this genetic region are known to be associated with delay in development of AIDS in people infected by HIV. Our study provides further suggestion that this region may have a critical role in viral suppression and could potentially harbor a clinically actionable variant for the shingles vaccine.

Design and anticipated outcomes of the eMERGE-PGx project: a multicenter pilot for preemptive pharmacogenomics in electronic health record systems.

We describe here the design and initial implementation of the eMERGE-PGx project. eMERGE-PGx, a partnership of the Electronic Medical Records and Genomics Network and the Pharmacogenomics Research Network, has three objectives: (i) to deploy PGRNseq, a next-generation sequencing platform assessing sequence variation in 84 proposed pharmacogenes, in nearly 9,000 patients likely to be prescribed drugs of interest in a 1- to 3-year time frame across several clinical sites; (ii) to integrate well-established clinically validated pharmacogenetic genotypes into the electronic health record with associated clinical decision support and to assess process and clinical outcomes of implementation; and (iii) to develop a repository of pharmacogenetic variants of unknown significance linked to a repository of electronic health record-based clinical phenotype data for ongoing pharmacogenomics discovery. We describe site-specific project implementation and anticipated products, including genetic variant and phenotype data repositories, novel variant association studies, clinical decision support modules, clinical and process outcomes, approaches to managing incidental findings, and patient and clinician education methods.

Human PON1, a biomarker of risk of disease and exposure.

Human paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme that exhibits a broad substrate specificity. In addition to protecting against exposure to some organophosphorus (OP) pesticides by hydrolyzing their toxic oxon metabolites, PON1 is important in protecting against vascular disease by metabolizing oxidized lipids. Recently, PON1 has also been shown to play a role in inactivating the quorum sensing factor N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) of Pseudomonas aeruginosa. Native, untagged engineered recombinant human PON1 (rHuPON1) expressed in Escherichia coli and purified by conventional column chromatographic purification is stable, active, and capable of protecting PON1 knockout mice (PON1(-/-)) from exposure to high levels of the OP compound diazoxon. The bacterially derived rHuPON1 can be produced in large quantities and lacks the glycosylation of eukaryotic systems that can produce immunogenic complications when inappropriately glycosylated recombinant proteins are used as therapeutics. Previous studies have shown that the determination of PON1 status, which reveals both PON1(192) functional genotype and serum enzyme activity level, is required for a meaningful evaluation of PON1's role in risk of disease or exposure. We have developed a new two-substrate assay/analysis protocol that provides PON1 status without use of toxic OP substrates, allowing for use of this protocol in non-specialized laboratories. Factors were also determined for inter-converting rates of hydrolysis of different substrates. PON1 status also plays an important role in revealing changes in HDL-associated PON1 activities in male patients with Parkinson disease (PD). Immunolocalization studies of PONs 1, 2 and 3 in nearly all mouse tissues suggest that the functions of PONs 1 and 3 extend beyond the plasma and the HDL particle.

MR imaging of adventitial vasa vasorum in carotid atherosclerosis.

Vasa vasorum in the adventitia of atherosclerotic arteries may play a role in plaque progression. In this investigation, a method for characterizing vasa vasorum in the carotid artery is proposed, in which the perfusion properties of the adventitia are probed via dynamic contrast-enhanced (DCE) MRI. A parametric "vasa vasorum image" is automatically generated that depicts the plasma volume (vp) and transfer constant (K trans). The average K trans within the adventitia is proposed as a quantitative measurement related to the extent of the vasa vasorum. In 25 subjects with lesions meeting the requirements for carotid endarterectomy (CEA) significantly higher adventitial K trans of 0.155 +/- 0.045 min(-1) was observed, compared to 0.122 +/- 0.029 min(-1) in the remaining 20 subjects with moderate disease (P < 0.01). In the 25 subjects with endarterectomy specimens, histological evaluation showed that adventitial K trans was significantly correlated with the amount of neovasculature (R = 0.41; P = 0.04) and macrophages (R = 0.49; P = 0.01) in the excised plaque. In the remaining 20 subjects without histology, elevated adventitial K trans was significantly correlated with the log of C-reactive protein (CRP) levels (R = 0.57; P = 0.01) and was elevated in active smokers compared to nonsmokers (0.141 +/- 0.036 vs. 0.111 +/- 0.017 min(-1); P = 0.02). Because these factors are all associated with higher risk of atherosclerotic complications, these results suggest that adventitial K(trans) may be a marker of risk as well.

Clinical characteristics of prostate cancer in an analysis of linkage to four putative susceptibility loci.

PURPOSE: Hereditary prostate cancer is an etiologically heterogeneous disease with six susceptibility loci mapped to date. We aimed to describe a collection of high-risk prostate cancer families and assess linkage to multiple markers at four loci: HPC1 (1q24-25), PCaP (1q42.2-43), HPCX (Xq27-28), and CAPB (1p36). EXPERIMENTAL DESIGN: Medical record data on 505 affected men in 149 multiply-affected prostate cancer families were reviewed, and correlations of clinical traits within each family were calculated. Logarithm of odds (LOD) score and nonparametric (NPL) linkage analyses were performed; white families were stratified by age of diagnosis, grade and stage of disease, and evidence of linkage to the other loci to increase genetic homogeneity. RESULTS: Age at diagnosis was the most correlated clinical trait within families. A maximum NPL score of 2.61 (P = 0.007) appeared to confirm HPC1 linkage for families that had a prevalence of high-grade or advanced-stage prostate cancer and which were not likely to be linked to PCaP, HPCX, or CAPB. Because the NPL scores improved when families more likely to be linked to the other loci were excluded, HPC1 may act independently of the other loci. The relationship of HPC1 and aggressive disease was strongest in families with median age at diagnosis > or =65 years (NPL, 3.48; P = 0.0008). CONCLUSIONS: The current results suggest that HPC1 linkage may be most common among families with more severe prostate cancer. Stratification by clinical characteristics may be a useful tool in prostate cancer linkage analyses and may increase our understanding of hereditary prostate cancer.

Germline mutations in the p73 gene do not predispose to familial prostate-brain cancer.

BACKGROUND: Analysis of high-risk prostate cancer (PC) families with at least one confirmed case of primary brain cancer (BC) has identified a region of genetic linkage on chromosome 1p36 termed CAPB. The p36 region of chromosome one has been reported to have frequent loss of heterozygosity (LOH) in brain and central nervous system (CNS) tumors and epidemiological studies have shown an increased relative risk of BC and tumors of the CNS in PC families. In 1997 a reported tumor suppressor with high homology to p53, termed p73, was mapped to the p36 region of chromosome one. Here, we examine the p73 gene as a potential candidate for CAPB. METHODS: Ninety-four members from the 12 prostate-brain cancer families in which linkage was originally found were examined. The complete coding region and intron-exon boundaries of the p73 gene were analyzed for germline mutations by Single Stranded Conformational Polymorphism analysis (SSCP) and direct DNA sequencing. RESULTS: Silent nucleotide substitutions only were detected within the coding regions of the gene in affected individuals. Nucleotide changes were detected in introns 1, 6, 8, 9, and 10, but all were located >or=16 base pairs from the splice site, and are thus unlikely to be deleterious mutations. CONCLUSIONS: Germline mutations in the p73 gene are unlikely to be critical for inherited susceptibility to PC in this specified subset of families.

Effects of 5' regulatory-region polymorphisms on paraoxonase-gene (PON1) expression.

Human HDL-associated paraoxonase (PON1) hydrolyzes a number of toxic organophosphorous compounds and reduces oxidation of LDLs and HDLs. These properties of PON1 account for its ability to protect against pesticide poisonings and atherosclerosis. PON1 also hydrolyzes a number of lactone and cyclic-carbonate drugs. Among individuals in a population, PON1 levels vary widely. We previously identified three polymorphisms in the PON1 regulatory region that affect expression levels in cultured human hepatocytes. In this study, we determined the genotypes of three regulatory-region polymorphisms for 376 white individuals and examined their effect on plasma-PON1 levels, determined by rates of phenylacetate hydrolysis. The -108 polymorphism had a significant effect on PON1-activity level, whereas the -162 polymorphism had a lesser effect. The -909 polymorphism, which is in linkage disequilibrium with the other sites, appears to have little or no independent effect on PON1-activity level in vivo. Other studies have found that the L55M polymorphism in the PON1-coding region is associated with differences in both PON1-mRNA and PON1-activity levels. The results presented here indicate that the L55M effect of lowered activity is not due to the amino acid change but is, rather, largely due to linkage disequilibrium with the -108 regulatory-region polymorphism. The codon 55 polymorphism marginally appeared to account for 15.3% of the variance in PON1 activity, but this dropped to 5% after adjustments for the effects of the -108 and Q192R polymorphisms were made. The -108C/T polymorphism accounted for 22.8% of the observed variability in PON1-expression levels, which was much greater than that attributable to the other PON1 polymorphisms. We also identified four sequence differences in the 3' UTR of the PON1 mRNA.

Polymorphisms in the human paraoxonase (PON1) promoter.

Paraoxonase (PON1) is a protein component of high-density lipoprotein (HDL) particles that protects against oxidative damage to both low-density lipoprotein and HDL and detoxifies organophosphorus pesticides and nerve agents. A wide range of expression levels of PON1 among individuals has been observed. We examined the promoter region of PON1 for genetic factors that might affect PON1 activity levels. We conducted a deletion analysis of the PON1 promoter region in transient transfection assays and found that cell-type specific promoter elements for liver and kidney are present in the first 200bp upstream of the coding sequence. Sequence analysis of DNA from a BAC clone and a YAC clone identified five polymorphisms in the first 1000 bases upstream of the coding region at positions -108, -126, -162, -832 and -909. Additionally, the promoter sequences of two individuals expressing high levels of PON1 and two individuals expressing low levels of PON1 were analysed. The two polymorphisms at -126 and -832 had no apparent effect on expression level in the reporter gene assay. The polymorphisms at position -909, -162 (a potential NF-I transcription factor binding site) and -108 (a potential SP1 binding site) each have approximately a two-fold effect on expression level. The expression level effects of the three polymorphisms appear not to be strictly additive and may depend on context effects.

Genetic linkage analysis of prostate cancer families to Xq27-28.

OBJECTIVES: A recent linkage analysis of 360 families at high risk for prostate cancer identified the q27-28 region on chromosome X as the potential location of a gene involved in prostate cancer susceptibility. Here we report on linkage analysis at this putative HPCX locus in an independent set of 186 prostate cancer families participating in the Prostate Cancer Genetic Research Study (PROGRESS). METHODS: DNA samples from these families were genotyped at 8 polymorphic markers spanning 14.3 cM of the HPCX region. RESULTS: Two-point parametric analysis of the total data set resulted in positive lod scores at only two markers, DXS984 and DXS1193, with scores of 0.628 at a recombination fraction (theta) of 0.36 and 0.012 at theta = 0.48, respectively. The stratification of pedigrees according to the assumed mode of transmission increased the evidence of linkage at DXS984 in 81 families with no evidence of male-to-male transmission (lod = 1.062 at theta = 0.28). CONCLUSIONS: Although this analysis did not show statistically significant evidence for the linkage of prostate cancer susceptibility to Xq27-28, the results are consistent with a small percentage of families being linked to this region. The analysis further highlights difficulties in replicating linkage results in an etiologically heterogeneous, complexly inherited disease.

Paraoxonase (PON1) phenotype is a better predictor of vascular disease than is PON1(192) or PON1(55) genotype.

The paraoxonase (PON1) PON1-Q192R and PON1-L55M polymorphisms have been inconsistently associated with vascular disease. Plasma PON1 activity phenotypes vary markedly within genotypes and were, therefore, expected to add to the informativeness of genotype for predicting vascular disease. The case-control sample included 212 age- and race-matched men (mean age 66.4 years). The 106 carotid artery disease (CAAD) cases had >80% carotid stenosis, and the 106 controls had <15%. Two PON1 substrate hydrolysis rates (paraoxon [POase] and diazoxon [DZOase]) were significantly lower in cases than in controls and were significant predictors of CAAD by use of logistic regression (POase, P=0.005; DZOase, P=0.019). DZOase predicted vascular disease independently of lipoprotein profile, high density lipoprotein subfractions, apolipoprotein A-I, and smoking. PON1-192 and PON1-55 genotypes or haplotypes did not predict case-control status unless the activity phenotype was also included as a predictor by use of logistic regression. When phenotype was included as a predictor, PON1-192 and PON1-55 genotypes or combined haplotypes were significant predictors (P<0.05). In conclusion, examining PON1-192 and/or PON1-55 genotypes alone may mistakenly lead to the conclusion that there is no role of PON1 in CAAD. These results support the benefit of a "level crossing" approach that includes intervening phenotypes in the study of complexly inherited disease.

The PON1 gene and detoxication.

It has been assumed since its discovery that serum paraoxonase (PON1) plays a major role in the detoxication of specific organophosphorus compounds. It was also assumed that individuals with low PON1 activity would be more susceptible to paraoxon/parathion poisoning than individuals with higher PON1 activity. Evidence supporting this hypothesis was provided by injection of rabbit PON1 into rodents. Injected PON1 protected against paraoxon toxicity in rats and chlorpyrifos oxon toxicity in mice. The recent availability of PON1 knockout mice has provided an in vivo system with which one can more closely examine the role of PON1 in detoxication. PON1 knockout mice demonstrated dramatically increased sensitivity to chlorpyrifos oxon and diazoxon and moderately increased sensitivity to the respective parent compounds. The PON1 knockout mutation also resulted in the elimination of liver PON1 activity, accounting for the dramatic increase in sensitivity to chlorpyrifos oxon and diazoxon. Totally unexpected was our finding that the PON1 knockout mice were not more sensitive to paraoxon. This was particularly surprising in light of the earlier enzyme injection experiments. Differences in the relative catalytic efficiencies of rabbit vs. mouse PON1 for the specific oxon forms explain these observations. Mouse PON1 has good catalytic efficiency for the hydrolysis of diazoxon and chlorpyrifos oxon, but a poor efficiency for paraoxon hydrolysis relative to rabbit PON1. The human PON1Q192 isoform has a catalytic efficiency similar to that of mice, whereas the human PON1R192 isoform has a much better catalytic efficiency, predicting that individuals expressing high levels of the PONIR192 isoform may have increased resistance to paraoxon toxicity.

Analysis of paraoxonase (PON1) L55M status requires both genotype and phenotype.

Paraoxonase (PON1) is tightly associated with high-density lipoprotein particles and is believed to contribute to the prevention of atherosclerosis by metabolizing oxidized lipids. PON1 also hydrolyses the bioactive oxon forms of organophosphorus pesticides such as parathion, diazinon and chlorpyrifos. Two common polymorphisms have been identified in the coding sequence of human PON1: L55M and R192Q. Several previous studies have found that the presence of the PON1R192 allele raises the risk of cardiovascular disease while others found no correlation. The studies, however, have focused on the genotype of PON1 and not the expression level of the protein. We found that the PON1 expression level in plasma, as determined by the rates of paraoxon and diazoxon hydrolysis, varies widely among individuals and within a genotype. Previous studies found that individuals having Met at PON155 have lower levels of both PON1 mRNA and activity. In this study, we determined the plasma activity levels of PON1 and examined the relationships between PON155 genotype and PON1 level. As with PON1192, we found considerable overlap in activity among the PON155 genotypes. Of the 317 individuals whose PON1 status was determined in this study, 48.9% were PON1Q192 homozygotes. Analysis of the PON1QQ192 population showed that while the average PON1 activity (diazoxon hydrolysis) was 12266 U/L for PON1LL55 and 7777 U/L for PON1MM55, a given PONMM55 individual could have more than twice the activity of a PON1LL55 individual. PON1 status, which includes PON1 level as well as PON1192 genotype, may be a better predictor for cardiovascular disease or organophosphate susceptibility than PON1 genotype alone.

Autosomal dominant thrombocytopenia: incomplete megakaryocyte differentiation and linkage to human chromosome 10.

We studied a large kindred with nonsyndromic autosomal dominant thrombocytopenia to define the phenotype and used genomic linkage analysis to determine the locus of the abnormal gene. Affected family members are characterized by lifelong moderate thrombocytopenia (mean = 42.7 x 10(9)/L) with moderate propensity toward easy bruising and minor bleeding. Megakaryocytes are present in bone marrow with reduced frequency, and there are no apparent abnormalities of myeloid or erythroid cells. This type of inherited thrombocytopenia has no evident association with hematopoietic malignancy or progression to aplastic anemia. In the past, members of this family have failed therapeutic trials of immunosuppression and splenectomy. In our investigation, we found that affected individuals had normal platelet size compared with unaffected family members and modestly increased thrombopoietin levels. Hematopoietic colony assays from bone marrow and peripheral blood demonstrated that megakaryocyte precursors (CFU-Mk) were dramatically increased in both number and size in affected individuals. Bone marrow cells grown in liquid culture with thrombopoietin failed to develop polyploid cells greater than 8N. Also, electron microscopy demonstrated that megakaryocytes from an affected individual had markedly delayed nuclear and cytoplasmic differentiation. Genome-wide linkage analysis established a single locus for the disease gene on the short arm of chromosome 10 with a maximum 2-point lod score of 5.68 (at theta = 0). By recruiting additional family members, the genomic region was narrowed to 17 centimorgans. We conclude that a gene in this locus plays an important role in megakaryocyte endomitosis and terminal maturation.

A genomic scan of families with prostate cancer identifies multiple regions of interest.

A 10-cM genomewide scan of 94 families with hereditary prostate cancer, including 432 affected men, was used to identify regions of putative prostate cancer-susceptibility loci. There was an average of 3.6 affected, genotyped men per family, and an overall mean age at diagnosis of 65.4 years. A total of 50 families were classified as early onset (mean age at diagnosis <66 years), and 44 families were classified as later onset (mean age at diagnosis > or =66 years). When the entire data set is considered, regions of interest (LOD score > or =1.5) were identified on chromosomes 10, 12, and 14, with a dominant model of inheritance. Under a recessive model LOD scores > or =1.5 were found on chromosomes 1, 8, 10, and 16. Stratification by age at diagnosis highlighted a putative susceptibility locus on chromosome 11, among the later-onset families, with a LOD score of 3. 02 (recombination fraction 0) at marker ATA34E08. Overall, this genomic scan suggests that there are multiple prostate cancer loci responsible for the hereditary form of this common and complex disease and that stratification by a variety of factors will be required for identification of all relevant genes.