A multiplex pharmacogenetics assay using the MinION nanopore sequencing device.

OBJECTIVES: The MinION nanopore sequencing device opens the opportunity to cost-effective and point-of-care DNA sequencing. As a proof of principle, we developed a multiplex assay targeting pharmacogenetic variants related to clopidogrel and warfarin, the two commonly used drugs that show response variability due to genetic polymorphisms. METHODS: Six reference and 78 clinical DNA samples were amplified by PCR to generate 15 amplicons targeting 27 key variants. These products were then barcoded to enable sample multiplexing in one sequencing run. Four variant calling tools (marginCaller, VarScan 2, nanopolish, Clairvoyante) were used to compare genotyping accuracy. RESULTS: In our cohort, 81 out of 84 samples were successfully sequenced and genotyped. Using nanopolish as the variant calling tool achieved accuracy >95% for all except two variants. A known single base deletion (CYP2C9*6) was successfully detected. CONCLUSION: While minor misgenotyping issues exist, this work demonstrates that drug-specific or broad pharmacogenetic screening assays using small PCR amplicons are possible on the MinION sequencing device.

Pharmacogenetics of angiotensin-converting enzyme inhibitor-induced angioedema.

Angioedema is a rare adverse effect of the commonly used angiotensin-converting enzyme inhibitors (ACEi) and is reported to occur with a prevalence of 0.1%-0.7%. Although most ACEi-induced angioedema (ACEi-A) cases are mild, severe cases requiring intensive care and even resulting in death have been reported in the literature. The mechanisms underlying ACEi-A are not yet fully understood, but bradykinin and/or substance P accumulation resulting from inhibition of ACE is believed to play a crucial role. ACEi-A occurs at variable frequencies across different racial groups, suggesting a genetic association with the development of ACEi-A. To date, one genome-wide association study and several candidate gene studies have been published on the association of genetic variation with ACEi-A. Genetic associations reported have been attributed to several distinct mechanisms: (a) genes coding for alternative enzymes responsible for the degradation of bradykinin and/or substance P in the diminution of ACE activity (b) ACE gene function, (c) bradykinin receptor genes, (d) genes implicated in immune and inflammation regulation and (e) genes in the fibrinolytic and coagulation pathway. Despite several plausible genetic associations, there are currently no genetic variants with sufficient effect to be clinically useful. The low incidence of ACEi-A suggests that a combination of genomic approaches with the capability to detect potentially important variants might be required to shed light on the mechanism of this adverse reaction. Additionally, many non-genetic risk factors associated with ACEi-A suggest the potential contribution of epigenetic dysregulation.

Effect of Cannabinoid Receptor Agonists on Isolated Rat Atria.

Cannabinoid CB2 receptor agonists are under investigation for clinical use. At the same time, synthetic cannabinoids have been implicated in a number of deaths. One cause of death is thought to be cardiac arrest subsequent to extreme tachycardia. Central mechanisms are thought to play a role in this, with CB1 but not CB2 receptors thought to mediate central effects. However, the direct effects of cannabinoids on the heart are less well understood. We therefore tested the effects of cannabinoids on isolated rat atria to test whether activation of myocardial CB1 and CB2 receptors could contribute to tachycardia. Although we found a moderate effect that can be attributed to CB1 receptors, we did not find any evidence for chronotropic effects by a CB2 receptor activation. Our results indicate that cannabinoid cardiotoxicity may partially involve CB1 receptors in the myocardium, and that CB2 receptor agonists are unlikely to have significant effects on the heart.

A method for measuring mitochondrial DNA copy number in pediatric populations.

The mitochondrion is a multifunctional organelle that modulates multiple systems critical for homeostasis during pathophysiological stress. Variation in mitochondrial DNA (mtDNA) copy number (mtDNAcn), a key mitochondrial change associated with chronic stress, is an emerging biomarker for disease pathology and progression. mtDNAcn can be quantified from whole blood samples using qPCR to determine the ratio of mtDNA to nuclear DNA. However, the collection of blood samples in pediatric populations, particularly in infants and young children, can be technically challenging, yield much smaller volume samples, and can be distressing for the patients and their caregivers. Therefore, we have validated a mtDNAcn assay utilizing DNA from simple buccal swabs (Isohelix SK-2S) and report here it's performance in specimens from infants (age = <12 months). Utilizing qPCR to amplify ∼200 bp regions from two mitochondrial (ND1, ND6) and two nuclear (BECN1, NEB) genes, we demonstrated absolute (100%) concordance with results from low-pass whole genome sequencing (lpWGS). We believe that this method overcomes key obstacles to measuring mtDNAcn in pediatric populations and creates the possibility for development of clinical assays to measure mitochondrial change during pathophysiological stress.

Trafficking of mitochondrial double-stranded RNA from mitochondria to the cytosol.

In addition to mitochondrial DNA, mitochondrial double-stranded RNA (mtdsRNA) is exported from mitochondria. However, specific channels for RNA transport have not been demonstrated. Here, we begin to characterize channel candidates for mtdsRNA export from the mitochondrial matrix to the cytosol. Down-regulation of SUV3 resulted in the accumulation of mtdsRNAs in the matrix, whereas down-regulation of PNPase resulted in the export of mtdsRNAs to the cytosol. Targeting experiments show that PNPase functions in both the intermembrane space and matrix. Strand-specific sequencing of the double-stranded RNA confirms the mitochondrial origin. Inhibiting or down-regulating outer membrane proteins VDAC1/2 and BAK/BAX or inner membrane proteins PHB1/2 strongly attenuated the export of mtdsRNAs to the cytosol. The cytosolic mtdsRNAs subsequently localized to large granules containing the stress protein TIA-1 and activated the type 1 interferon stress response pathway. Abundant mtdsRNAs were detected in a subset of non-small-cell lung cancer cell lines that were glycolytic, indicating relevance in cancer biology. Thus, we propose that mtdsRNA is a new damage-associated molecular pattern that is exported from mitochondria in a regulated manner.

The pharmacogenetics of CYP2D6 and CYP2C19 in a case series of antidepressant responses.

Pharmacogenetics has potential for optimizing use of psychotropics. CYP2D6 and CYP2C19 are two clinically relevant pharmacogenes in the prescribing of antidepressants. Using cases recruited from the Understanding Drug Reactions Using Genomic Sequencing (UDRUGS) study, we aimed to evaluate the clinical utility of genotyping CYP2D6 and CYP2C19 in antidepressant response. Genomic and clinical data for patients who were prescribed antidepressants for mental health disorders, and experienced adverse reactions (ADRs) or ineffectiveness, were extracted for analysis. Genotype-inferred phenotyping of CYP2D6 and CYP2C19 was carried out as per Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines. A total of 52 patients, predominantly New Zealand Europeans (85%) with a median age (range) of 36 years (15-73), were eligible for analysis. Thirty-one (60%) reported ADRs, 11 (21%) ineffectiveness, and 10 (19%) reported both. There were 19 CYP2C19 NMs, 15 IMs, 16 RMs, one PM and one UM. For CYP2D6, there were 22 NMs, 22 IMs, four PMs, three UMs, and one indeterminate. CPIC assigned a level to each gene-drug pair based on curated genotype-to-phenotype evidence. We analyzed a subgroup of 45 cases, inclusive of response type (ADRs/ineffectiveness). Seventy-nine (N = 37 for CYP2D6, N = 42 for CYP2C19) gene-drug/antidepressant-response pairs with CPIC evidence levels of A, A/B, or B were identified. Pairs were assigned as 'actionable' if the CYP phenotypes potentially contributed to the observed response. We observed actionability in 41% (15/37) of CYP2D6-antidepressant-response pairs and 36% (15/42) of CYP2C19-antidepressant-response pairs. In this cohort, CYP2D6 and CYP2C19 genotypes were actionable for a total of 38% pairs, consisting of 48% in relation to ADRs and 21% in relation to drug ineffectiveness.

Long-Range Polymerase Chain Reaction.

Polymerase chain reaction (PCR) is a laboratory technique used to amplify a targeted region of DNA, demarcated by a set of oligonucleotide primers. Long-range PCR is a form of PCR optimized to facilitate the amplification of large fragments. Using the adapted long-range PCR protocol described in this chapter, we were able to generate PCR products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples. For some of the long PCRs, successful amplification was not possible without the use of PCR enhancers. Thus, we also evaluated the impact of some enhancers on long-range PCR and included the findings as part of this updated chapter.

Epistaxis and other haemorrhagic events associated with the smoking cessation medicine varenicline: a case series from two national pharmacovigilance centres.

PURPOSE: To present a case series of haemorrhagic events associated with varenicline identified from the New Zealand (NZ) and Netherlands national pharmacovigilance centres and propose a possible mechanism for these adverse events. METHODS: Reports of epistaxis and other haemorrhagic events (in all system organ classes excluding gynaecological) associated with varenicline were identified and assessed in both the NZ Intensive Medicines Monitoring Programme (IMMP) and the Netherlands Pharmacovigilance Centre Lareb (Lareb). Additional reports were identified from the World Health Organisation Uppsala Monitoring Centre (WHO-UMC) datasets, and these also underwent causality assessment. RESULTS: A total of 30 reports of haemorrhagic events were identified by the NZ IMMP (16 reports) and Lareb (14 reports). Six cases of epistaxis were identified, and four patients had a positive dechallenge on withdrawal of varenicline, suggesting a causal association. Another five reports of gingival bleeding were identified, with three patients having a positive dechallenge. Another patient who experienced haemoptysis while taking varenicline had a positive dechallenge and a positive rechallenge. In the WHO datasets, a further 49 reports of epistaxis, 39 reports of haemoptysis and 21 reports of thrombocytopenia were identified. A plausible mechanism for haemorrhagic events associated with varenicline may be a result of interaction with the serotonin (5-HT) receptor system and transporter. CONCLUSIONS: This is the first specific investigation of haemorrhagic events associated with varenicline. The results of our assessment of reports identified by two national pharmacovigilance centres suggest that there may be causal relationship between varenicline and these adverse events.

Omeprazole Treatment Failure in Gastroesophageal Reflux Disease and Genetic Variation at the CYP2C Locus.

Omeprazole is extensively used to manage gastroesophageal reflux disease (GERD). It is primarily metabolized by CYP2C19. The CYP2C19*17 (rs12248560) allele and the recently described CYP2C:TG haplotype (rs11188059 and rs2860840) are associated with increased enzymatic activity, and may reduce omeprazole exposure. This observational study aimed to investigate the association between these genetic variants and omeprazole treatment failure in GERD. We recruited predominantly New Zealand European GERD patients who either did not respond to omeprazole or experienced breakthrough heartburn symptoms despite at least 8 weeks of omeprazole (≥40 mg/day). The GerdQ score was used to gauge symptomatic severity. A total of 55 cases were recruited with a median age (range) of 56 years (19-82) and GerdQ score of 11 (5-17). Of these, 19 (34.5%) were CYP2C19*17 heterozygotes and two (3.6%) were CYP2C19*17 homozygotes. A total of 30 (27.3%) CYP2C:TG haplotypes was identified in our cohort, with seven (12.7%) CYP2C:TG homozygotes, and 16 (29%) CYP2C:TG heterozygotes. No significant differences were observed for overall CYP2C19*17 alleles, CYP2C19*17/*17, overall CYP2C:TG haplotypes, and CYP2C:TG heterozygotes (p > 0.05 for all comparisons). Gastroscopy and 24-h esophageal pH/impedance tests demonstrated objective evidence of GERD in a subgroup of 39 (71%) cases, in which the CYP2C:TG/TG was significantly enriched (p = 0.03) when compared with the haplotype frequencies in a predominantly (91%) New Zealand European reference population, but not the CYP2C19*17/*17 (p > 0.99), when compared with the allele frequencies for the non-Finnish European subset of gnomAD. We conclude that omeprazole treatment failure in GERD is associated with CYP2C:TG/TG, but not CYP2C19*17.

Allelic diversity of the pharmacogene CYP2D6 in New Zealand Maori and Pacific peoples.

The enzyme cytochrome P450 2D6 (CYP2D6) metabolises approximately 25% of commonly prescribed drugs, including analgesics, anti-hypertensives, and anti-depressants, among many others. Genetic variation in drug metabolising genes can alter how an individual responds to prescribed drugs, including predisposing to adverse drug reactions. The majority of research on the CYP2D6 gene has been carried out in European and East Asian populations, with many Indigenous and minority populations, such as those from Oceania, greatly underrepresented. However, genetic variation is often population specific and analysis of diverse ethnic groups can reveal differences in alleles that may be of clinical significance. For this reason, we set out to examine the range and frequency of CYP2D6 variants in a sample of 202 Maori and Pacific people living in Aotearoa (New Zealand). We carried out long PCR to isolate the CYP2D6 region before performing nanopore sequencing to identify all variants and alleles in these samples. We identified twelve variants which have previously not been reported in the PharmVar CYP2D6 database, three of which were exonic missense variations. Six of these occurred in single samples and one was found in 19 samples (9.4% of the cohort). The remaining five variants were identified in two samples each. Identified variants formed twelve new CYP2D6 suballeles and four new star alleles, now recorded in the PharmVar database. One striking finding was that CYP2D6*71, an allele of uncertain functional status which has been rarely observed in previous studies, occurs at a relatively high frequency (8.9%) within this cohort. These data will help to ensure that CYP2D6 genetic analysis for pharmacogenetic purposes can be carried out accurately and effectively in this population group.

Pharmacogenetics of Statin-Induced Myotoxicity.

Statins, a class of lipid-lowering medications, have been a keystone treatment in cardiovascular health. However, adverse effects associated with statin use impact patient adherence, leading to statin discontinuation. Statin-induced myotoxicity (SIM) is one of the most common adverse effects, prevalent across all ages, genders, and ethnicities. Although certain demographic cohorts carry a higher risk, the impaired quality of life attributed to SIM is significant. The pathogenesis of SIM remains to be fully elucidated, but it is clear that SIM is multifactorial. These factors include drug-drug interactions, renal or liver dysfunction, and genetics. Genetic-inferred risk for SIM was first reported by a landmark genome-wide association study, which reported a higher risk of SIM with a polymorphism in the SLCO1B1 gene. Since then, research associating genetic factors with SIM has expanded widely and has become one of the foci in the field of pharmacogenomics. This review provides an update on the genetic risk factors associated with SIM.

The Three Ps: Psychiatry, Pharmacy, and Pharmacogenomics, a Brief Report From New Zealand.

We describe a case series of 22 individuals who were referred to our laboratory by a pharmacist based in a mental health hospital, for pharmacogenetic analysis due to severe or unexpected adverse drug reactions (ADRs) to psychiatric medication. The participants were genotyped for common variation in the CYP2D6, CYP2C19, and CYP2C9 genes, using Sanger sequencing. We tested variants in these genes as they have the strongest evidence with respect to altering the pharmacokinetics of commonly prescribed psychiatric medicine. Looking specifically at the subset of 18 European study participants, we observed a comparatively high but non-significant rate of pharmacogenetic variants, compared to allele frequency surveys in unselected population samples. For CYP2D6, we observed an elevated frequency of both poor (17%) and intermediate (33%) metabolizers when compared with previously reported frequencies (6% and 12% respectively). For CYP2C19, we observed an increased frequency of intermediate (33%) and ultra-rapid (17%) metabolizers compared to expected frequencies (21% and 4% respectively). For CYP2C9, the frequency of intermediate metabolizers (22%) was elevated compared to the expected population frequency (11%). While sample size is a major limitation of this brief report, we can conclude that patients with adverse reactions to antidepressant or antipsychotic drugs selected by a specialist mental health pharmacist appear to have a relatively high rate of genetic variants in pharmacogenes known to affect the pharmacokinetics of these drugs. The selective application of such pharmacogenetic tests by clinical pharmacists may be a valuable approach to clarify the basis for adverse or unusual responses to medication, and to guide ongoing prescribing decisions for this group of patients.

Nanopore sequencing of the pharmacogene CYP2D6 allows simultaneous haplotyping and detection of duplications.

Aim: Long read sequencing offers the promise of overcoming some of the challenges in accurate genotyping of complex genes, along with the advantage of straightforward variant phasing. We have established methods for sequencing and haplotyping of the whole CYP2D6 gene using nanopore sequencing. Materials and methods: 32 samples covering various haplotypes including gene duplication were sequenced on the GridION platform. Results: Haplotypes of 52 alleles matched accurately to known star (*) allele subvariants, with the remaining 12 being assigned as new alleles, or new subvariants of known alleles. Duplicated alleles could be detected by analyzing the allelic balance. Conclusion: Nanopore sequencing of CYP2D6 offers a high throughput method for accurate haplotyping, detection of new variants and determination of duplicated alleles.

Common CYP2D6, CYP2C9, and CYP2C19 Gene Variants, Health Anxiety, and Neuroticism Are Not Associated With Self-Reported Antidepressant Side Effects.

Many patients prescribed an antidepressant stop taking it because of side effects. Genetic factors and psychological factors including state or trait anxiety, may explain variation in side effect outcomes. Our aim was to examine the relative contribution of genetic and psychological factors in people with self-reported antidepressant side effects. We undertook a case control study (n = 194) of people who took a selective serotonin reuptake inhibitor (SSRI) or serotonin/noradrenaline reuptake inhibitor (SNRI) in the past 2 years, recruited via social media advertising. Cases had previously not tolerated at least one trial of an SSRI or SNRI, evidenced by stopping the drug or reducing the dose by at least 50% because of a side effect. Control participants had taken an SSRI or SNRI but did not meet case criteria. Variation in the genes CYP2D6, CYP2C19, and CYP2C9 was analyzed by Sanger sequencing on DNA extracted from blood or saliva. Participants completed the Short Health Anxiety Inventory-18, K10, and NEO-FFI-3 personality questionnaire. Participants were 87.1% female. 70.8% had a current K10 score of 22 or more. There was no consistent evidence that cases had higher psychological distress, health anxiety, or neuroticism. There was low correspondence between participants' CYP2D6, CYP2C19, and CYP2C9 phenotypes and their history of antidepressant tolerability. For this cohort of patients a history of not tolerating SSRI or SNRI therapy was not associated with variation in the pharmacogenes we tested, nor was it associated with health anxiety or neuroticism.

Long Fragment Polymerase Chain Reaction.

Polymerase chain reaction (PCR) is an oft-used preparatory technique in amplifying specific DNA regions for downstream analysis. The size of an amplicon was initially limited by errors in nucleotide polymerization and template deterioration during thermal cycling. A variant of PCR, designated long-range PCR, was devised to counter these drawbacks and enable the amplification of large fragments exceeding a few kb. In this chapter we describe a protocol for long-range PCR, which we have adopted to obtain products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples.

Relationship between metabolic phenotypes and genotypes of CYP1A2 and CYP2A6 in the Nigerian population.

BACKGROUND: CYP1A2 and CYP2A6 are polymorphic drug-metabolising enzymes that are also implicated in the activation of procarcinogens in humans. Some of their alleles and haplotypes, often varied in prevalence across populations, are thought to influence activity despite the known contribution of environmental factors. This study assessed the potential influence of some genetic variants of CYP1A2 and CYP2A6 on metabolic phenotypes in Nigerians. METHODS: Genomic DNA was extracted from blood samples of 100 healthy, unrelated subjects for whom CYP1A2 and CYP2A6 phenotypes had previously been determined, alongside an additional 80 other individuals for whom phenotype data were unavailable. The samples were screened for CYP1A2 (*1C,*1D,*1E,*1F, *3,*4,*6,*7) and CYP2A6 (*9,*11,*17) alleles using the Sequenom MassARRAY platform for some alleles and direct Sanger sequencing for others. The genetic data acquired were subsequently analysed for haplotypes and assessed for concordance with phenotypes. RESULTS: All five CYP1A2 haplotypes (CYP1A2*1F, 1J, 1N, 1L, 1W) identified in the Nigerian population were not significantly predictive of metabolic phenotypes. Heterozygous CYP1A2*1J carriers and homozygous CYP1A2*1W carriers showed statistically insignificant decrease in CYP1A2 activity. The CYP2A6*9/*17 genotype was, however, significantly associated with the CYP2A6-poor metabolic phenotype, whereas CYP2A6*9 or CYP2A6*17 alone did not show any such association. CYP2A6*11 was not detected in the population. CONCLUSIONS: Our findings suggest that CYP1A2 alleles or haplotypes were not predictive of metabolic phenotypes in the Nigerian population. Carriers of CYP2A6*9/*17 genotype are likely to be poor metabolisers of CYP2A6 substrates and may experience adverse reactions or poor efficacy while using drugs metabolised mainly by CYP2A6.

A New Zealand platform to enable genetic investigation of adverse drug reactions.

A multitude of factors can affect drug response in individuals. It is now well established that variations in genes, especially those coding for drug metabolising enzymes, can alter the pharmacokinetic and/or pharmacodynamic profile of a drug, impacting on efficacy and often resulting in drug-induced toxicity. The UDRUGS study is an initiative from the Carney Centre for Pharmacogenomics to biobank DNA and store associated clinical data from patients who have suffered rare and/or serious adverse drug reactions (ADRs). The aim is to provide a genetic explanation of drug-induced ADRs using methods ranging from Sanger sequencing to whole exome and whole genome sequencing. Participants for the UDRUGS study are recruited from various sources, mainly via referral through clinicians working in Canterbury District Health Board, but also from district health boards across New Zealand. Participants have also self-referred to us from word-of-mouth communication between participants. We have recruited various ADRs across most drug classes. Where possible, we have conducted genetic analyses in single or a cohort of cases to identify known and novel genetic association(s) to offer an explanation to why the ADR occurred. Any genetic results relevant to the ADR are communicated back to the referring clinician and/or participant. In conclusion, we have developed a programme for studying the genetic basis of severe, rare or unusual ADR cases resulting from pharmacological treatment. Genomic analyses could eventually identify most genetic variants that predispose to ADRs, enabling a priori detection of such variants with high throughput DNA tests.

Impact of New Genomic Technologies on Understanding Adverse Drug Reactions.

It is well established that variations in genes can alter the pharmacokinetic and pharmacodynamic profile of a drug and immunological responses to it. Early advances in pharmacogenetics were made with traditional genetic techniques such as functional cloning of genes using knowledge gained from purified proteins, and candidate gene analysis. Over the past decade, techniques for analysing the human genome have accelerated greatly as knowledge and technological capabilities have grown. These techniques were initially focussed on understanding genetic factors of disease, but increasingly they are helping to clarify the genetic basis of variable drug responses and adverse drug reactions (ADRs). We examine genetic methods that have been applied to the understanding of ADRs, review the current state of knowledge of genetic factors that influence ADR development, and discuss how the application of genome-wide association studies and next-generation sequencing approaches is supporting and extending existing knowledge of pharmacogenetic processes leading to ADRs. Such approaches have identified single genes that are major contributing genetic risk factors for an ADR, (such as flucloxacillin and drug-induced liver disease), making pre-treatment testing a possibility. They have contributed to the identification of multiple genetic determinants of a single ADR, some involving both pharmacologic and immunological processes (such as phenytoin and severe cutaneous adverse reactions). They have indicated that rare genetic variants, often not previously reported, are likely to have more influence on the phenotype than common variants that have been traditionally tested for. The problem of genotype/phenotype discordance affecting the interpretation of pharmacogenetic screening and the future of genome-based testing applied to ADRs are also discussed.

Effects of HMG-CoA reductase inhibitors on learning and memory in the guinea pig.

Statins reduce the risk of death from cardiovascular disease in millions of people worldwide. Recent pharmacovigilance data has suggested that people taking statins have an increased risk of psychiatric adverse events such as amnesia and anxiety. This study aimed to investigate the possibility of statin-induced amnesia through animal models of memory and learning. We conducted extracellular field recordings of synaptic transmission in area CA1 of hippocampal slices to examine the effects of acute cholesterol lowering with lipid lowering drugs. We also assessed the effect of six weeks of simvastatin (2mg/kg/d) and atorvastatin (1mg/kg/d) treatment using the Morris water maze. Long Term Potentiation (LTP) was significantly diminished in the presence of 3µM atorvastatin or simvastatin and by the cholesterol sequestering agent methyl-ß-cyclodextrin (MBCD). The effects were reversed in the MBCD but not the statin treated slices by the addition of cholesterol. In the water maze, statin treatment did not cause any deficits in the first five days of reference memory testing, but statin treated guinea pigs preformed significantly worse than control animals in a working memory test. The deficits observed in our experiments in water maze performance and hippocampal LTP are suggestive of statin induced changes in hippocampal plasticity. The effects on LTP are independent of cholesterol regulation, and occur at concentrations that may be relevant to clinical use. Our results may help to explain some of the behavioural changes reported in some people after beginning statin treatment.