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.

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.

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.

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.

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.

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.

Neuroprotective effect of hydroxypropyl-ß-cyclodextrin in hypoxia-ischemia.

Neonatal cerebral ischemic injury is a common and debilitating pathology for which there is currently no known purely pharmacological treatments that are effective when delivered immediately after injury. Cyclodextrins are cyclic oligosaccharides that can remove cholesterol from cell membranes and thereby affect receptor function. Cyclodextrins have previously been shown to be neuroprotective in vitro. We showed that hydroxypropyl-ß-cyclodextrin is neuroprotective in rats in vivo when delivered by intraperitoneal injection 30 min following hypoxia-ischemia, when assessed 15 days after surgery. A single dose of 1 g/kg hydroxypropyl-ß-cyclodextrin reduced brain infarction size by 28.57% compared with control (P<0.001). We also report that the same compound reduces neuronal excitability in hippocampal slices and propose that hydroxypropyl-ß-cyclodextrin is neuroprotective by reducing excitotoxicity in the delayed phase of brain damage.

Clinical implications of pharmacogenetic variation on the effects of statins.

The last decade has seen an increase in the trend of HMG-CoA reductase inhibitor (statin) usage in the Western world, which does not come as a surprise noting that the latest American Heart Association heart and stroke statistics indicate an alarming prevalence of 80 million Americans (one in three) with one or more forms of diagnosed cardiovascular disease (CVD). Meta-analysis of several large-scale, randomized clinical trials has demonstrated statins to be efficacious in significantly reducing CVD-associated mortality in both primary and secondary prevention. Despite their proven efficacy, statins have also gained attention with respect to adverse drug reactions (ADRs) of muscle myopathy, derangements in hepatic function and even ADRs classified as psychiatric in nature. The depletion of cholesterol within the myocyte cell wall and/or the depletion of key intermediates within the cholesterol synthesis pathway are hypothesized as possible mechanisms of statin-associated ADRs. However, pharmacogenetic variability may also be a risk factor for ADRs and can include, for example, enzymes, transporters, cell membrane receptors, intracellular receptors or components of ion channels that contribute to the pharmacokinetics or pharmacodynamics of response to a particular drug. The cytochrome P450 (CYP) enzymatic pathways that comprise the polymorphic genes, CYP2D6, CYP3A4 and CYP3A5, and also a hepatic transporter, solute carrier organic anion transporter (SLCO1B1), which is a single nucleotide polymorphism discovered to be associated with statin-induced myopathy through a genome-wide association study, are discussed with respect to their effect on altering the pharmacokinetic profile of statin metabolism. Variants of the Apolipoprotein E (APO-E) gene, polymorphisms in the cholesteryl ester transfer protein (CETP) gene, the HMG-CoA reductase gene and other proteins are discussed with respect to altering the pharmacodynamic profile of statins. Pharmacogenetics and its application in medicine to individualize drug therapy has been previously shown to be clinically and economically beneficial through quality-adjusted life-year assessment. Therefore, polymorphisms affecting the pharmacokinetic and pharmacodynamic profiles of statins, which are widely used in therapy, with their potential application in the personalized prescribing of statin therapy, need further research. In this review, we update the recent literature with respect to genetic polymorphisms that may influence the pharmacokinetics and pharmacodynamics of statin therapy, and consider the relevance of these findings to the efficacy of treatment, prevention of ADRs and what this may mean for patient tolerance and compliance.