PHARMACOGENOMICS

Nerea Albert Colomer

Advanced Genetics MasterGenomics

• INTRODUCTION

• DESCRIBING A NEW TEST

• EXAMPLES

• ADVANTAGES AND DISADVANTAGES

• Pharmacogenetics:

Studying an individual's genetic make up in order to predict responses to a drug and guide prescription.

• Pharmacogenomics:

Analyzing entire genomes, across groups of individuals, to identify the genetic factors influencing response to a drug.

“Personalized medicine”

Pharmacogenetics ≠ Pharmacogenomics

Pharmacogenetic ≠ Pharmacogenomics ???

Describing a new test

© 2001 Macmillan Magazines LtdNATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | JANUARY 2002 | 41

R E V I EW S

matrix and cell-to-cell communications that establishre-entrant circuits, to list just a few of the possibilities.Hence, basic physiological considerations, driven byclinical research and molecular and cellular studies,including (where appropriate) model systems, such asmouse, yeast, or Caenorhabditis elegans, can all con-tribute to the definition of these biological contexts andso have a role in defining candidate sets. Indeed, theresponse of yeast to exogenous stimuli has been suffi-ciently well characterized that a ‘compendium’ of virtu-ally all responses has been described53 and used toidentify pathways modulated by drugs with mecha-nisms that were previously poorly understood. It wouldbe desirable to be able to create a similar compendiumof all possible responses of human cells to exogenousstimuli, but this vision remains futuristic.

Identification of polymorphismsThe next step in this algorithm is the identification ofDNA variants in candidate genes or gene sets. Thehuman genome probably includes 3–10 × 106 singlenucleotide polymorphisms (SNPs)54 and, althoughconsiderable progress has been made in identifyingthese and assigning them to specific genes, the sheernumbers raise considerable questions as to how any sensecan be made of statistical analyses of these numerousvariants in relatively small (even thousands) numbers ofpatients (BOX 1). One approach is to confine the analysisto polymorphisms that alter primary amino-acidsequences, known as NON-SYNONYMOUS CODING-REGION POLY-

MORPHISMS. As technologies advance, polymorphisms inknown promoter regions, or in or near intron–exonboundaries, can readily be included in such a ‘first-pass’analysis. However, these strategies make assumptionsabout our understanding of transcriptional and transla-tional control mechanisms that are not well supported;the formal possibility exists that SYNONYMOUS CODING-

REGION POLYMORPHISMS or distant intronic sequences mightmodulate the efficiency of transcription. The recognitionthat many SNPs are actually in LINKAGE DISEQUILIBRIUM, andthat the number of haplotypes in the human genome isprobably far less than the millions of SNPs, might pro-vide a new tool with which to approach the problem ofgenomic variation as a contributor to disease and drugresponse55,56. Rather than requiring analysis at each ofthe millions of polymorphic sites, it might be possible toperform such analyses with thousands, or, at most, tensof thousands, of haplotypes.

Once a list of candidate DNA variants is generatedthat might modulate the phenotype of interest, patientsand controls need to be genotyped. The main problemshere are choice of platform, the costs, and the issues ofpolymorphism versus haplotype that were discussedabove. Furthermore, the biostatistical challenges in asso-ciating variant DNA sequences with pre-defined drugresponses are considerable (BOX 1). Finally, after apolymorphism or a set of polymorphisms that predictdrug response is identified, it should be subjected to aprospective test in a new population to establish that thepredictive value is reproducible, and cost–benefit issuesshould be analysed. In addition, the polymorphism

be expected to modulate the phenotype. Both candi-date-gene and genome-wide approaches have beenproposed. Genome-wide scans have been useful inPOSITIONAL-CLONING STRATEGIES to find genes in Mendeliandisorders, but the approach is much more difficult toadapt to pharmacogenetics, as kindreds are not generallyavailable. Nevertheless, with advances in technology andin statistical approaches, it may be possible to identify acircumscribed set of polymorphisms, perhaps repre-senting most of the HAPLOTYPES in the human genome,which could be used in genome-wide scans for importantdrug-response end points in large numbers of well-characterized patients and controls. The main advantageof such an approach is that genes or pathways that areunidentified at present may be implicated in mediatingdrug responses. Until such advances are made, however,it seems likely that pharmacogenetics will rely on a can-didate-gene approach. Some candidate genes are rela-tively obvious. For example, association of an adversedrug response with elevated plasma concentrationswould naturally focus attention on genes with productsthat mediate drug disposition — often a relatively cir-cumscribed and tractable set. For pharmacodynamicissues, the most obvious candidate genes are those thatencode the drug targets. However, as discussed above,genes with products that modulate the biological con-text in which the drug–target interactions occur shouldalso be considered as candidates. Returning to theexample of variable responses to anti-arrhythmic drugs,the list of candidates includes not only genes encodingion channels, but also those encoding function-modify-ing subunits, elements of intracellular signalling path-ways that modulate ion channel function, factors thatcontrol intracellular calcium (a key mediator ofarrhythmias), and modulators of the extracellular

POSITIONAL CLONING

An experimental technique toidentify genes that contribute toa phenotype by first identifyingthe chromosomal locus(position). Positional cloningmakes no assumptions as tounderlying physiology, and socan identify genes withrelationships to a specifiedphenotype that had notpreviously been suspected.

HAPLOTYPE

The arrangement of individualalleles on a chromosome.

NON-SYNONYMOUS CODING-

REGION POLYMORPHISM

A DNA polymorphism in thecoding region of a gene thatresults in a change in theencoded amino acid.

SYNONYMOUS CODING-REGION

POLYMORPHISM

A DNA polymorphism in thecoding region of a gene that doesnot result in a change in theencoded amino acid.

LINKAGE DISEQUILIBRIUM

The association between a pairof allelic variants that occursmore often than by chance.

Define the drug response (phenotype) of interest and accumulate DNA from patients with the defined phenotype

Identify candidate gene(s)• Physiology, often through rare monogenic diseases• Genome-wide (unbiased) scan

Identify polymorphisms in candidate genes

Is there evidence that the phenotype is genetically determined?• Epidemiology• Family studies

Relate the identified polymorphisms to the phenotype• Clinical studies • Among patients with/without the phenotype • Across ethnic groups• In vitro studies• Genetic epidemiology

Refine the phenotype Prospective study

Figure 3 | An algorithm for evaluating the role of genetic factors in drug actions. This flowchart, which is discussed further in the text, highlights the interactive and collaborative nature of theresearch that is required to accomplish the goal of defining the role of genetic factors in variabledrug actions.

Roden DM, George Jr AL. The genetic basis of variability in drug responses. Nat Rev Drug Discov [Internet]. 2002 Jan;1(1):37–44.

Pharmacogenomic test should be accomplish:

1. Analytical validity, refers to the reproducibility of a given test in the laboratory.

2. Clinical validity, refers to the ability of a test to act as a robust predictor of a clinical parameter.

3. Clinical utility, assesses the ability of a test to reliably predict a change in treatment required by the result of the genetic test.

Advantages / Disadvantages

Describing a new test

CYP2D6

CYP2D6 gene

CYP2D6 enzyme

Drug metabolism

CYP450

CYP enzyme

CYP enzyme

CYP enzyme

Monooxygenases

⇈ Polymorphic

PM

EM

UM

CYP2D6

• PM (poor metabolizer) = lower or not function• IM (intermediate metabolizer) = metabolize at a rate between PM and EM• EM (extensive metabolizer) = normal function• UM (ultra-rapid metabolizer) = greater than normal function

ALLELE CYP2D PHENOTYPE

CYP2D6*1 Normal EM

CYP2D6*2 Increase UM

CYP2D6*3 None PM

CYP2D6*4 None PM

CYP2D6*5 None PM

CYP2D6*9 Drecrease PM

CYP2D6*10 Decrease PM

CYP2D6*17 Decrease PM

CYP2D6 in pregnancyNARCOTICS

• PM phenotype ➔ 7% of Caucasian population:

⇊ ⇊ capacity to convert codeine to morphine and do not obtain adequate pain relief from codeine.

• UM phenotype ➔ 2–3% of Caucasian population:

Codeine is rapidly converted to morphine, potentially leading to toxicity.

with clinical doses of codeine have been reported.18–20 Insome of these individuals, the presence of a variant thatreduces the activity of UDP-glucuronosyltransferase-2B7(UGT2B7), the enzyme responsible for inactivation of mor-phine, may have also contributed to the toxic concentrationsof morphine. After the report of an infant death associated

with the CYP2D6 UM genotype of a breastfeeding mothertaking codeine for post-cesarean pain relief,21 the U.S. FDAissued a Public Health Advisory cautioning women on the useof narcotic analgesics during breastfeeding.22 Additionally,the Clinical Pharmacogenomics Implementation Consortium(CPIC) has issued guidelines on the use of codeine withrespect to CYP2D6 genotype.17 While hydrocodone and oxy-codone undergo similar metabolic activation via CYP2D6,there are not adequate data regarding the consequences ofPM or UM phenotype for use of these agents.

Antihypertensives

Commonly used to treat hypertension in pregnancy, metoprololis metabolized primarily by CYP2D6. A meta-analysis of studiesin non-pregnant individuals recently identified a 15-fold differ-ence in apparent oral clearance of metoprolol between ultra-rapid and poor metabolizers for CYP2D6.23 In addition to thepharmacogenetic variation of CYP2D6, the enzyme's increasedactivity in pregnancy15 may necessitate increased doses ofmetoprolol compared to those used in non-pregnant women.Similar to metoprolol, labetalol's half-life is decreased

during pregnancy.24 A recent study found that gestationalage and lean body weight were significantly associated withoral clearance.25 Labetalol is cleared predominantly by glu-curonidation through UGT1A1 and UGT2B7.26 The docu-mented increase in UGT1A1 expression during pregnancyhas been attributed to the induction of UGT1A1 by

Table 1 – Pharmacogenomic information in the FDA label of drugs commonly administered to pregnant women.

Drug name Gene Pharmacogenomic information in FDA label

Tramadol CYP2D6 Concentrations in PMs were 20% higher than in EMsCodeine CYP2D6 Respiratory depression and death have occurred in UM children and in breast-fed infants whose

mothers are UMsHydralazine NAT1-2 Mean absolute bioavailability varies from 10% to 26% with higher percentages in PMs; EMs have lower

exposureMetoprolol CYP2D6 EMs who concomitantly take CYP2D6 inhibitors and PMs have increased concentrations, decreasing

metoprolol's cardioselectivityGlyburide G6PD Hemolytic anemia linked to G6PD deficiencyEsomeprazole and

omeprazoleCYP2C19 Induction of CYP3A4 by St. John's wort led to a 37.9% decrease in omeprazole AUC in PMs and a 43.9%

decrease of AUC in EMsLansoprazole CYP2C19 Concomitant administration with tacrolimus may increase whole blood levels of tacrolimus,

especially in transplant patients who are IMs or PMs. Coadministration to EMs taking clopidogrelreduced the AUC of clopidogrel's active metabolite by 14%.

Pantoprazole CYP2C19 PMs have elimination half-life of 3.5–10 h; in EMs, 71% of the dose is excreted in urine and 18%through biliary excretion

Metoclopramide CYB5R1-4 Patients with NADH-cytochrome b5 reductase deficiency are at an increased risk of developingmethemoglobinemia and/or sulfhemoglobinemia when metoclopramide is administered. Inpatients with G6PD deficiency who experience metoclopramide-induced methemoglobinemia,methylene blue treatment is not recommended.

G6PD

Nitrofurantoin G6PD Hemolytic anemia linked to G6PD deficiencyCitalopram CYP2C19 Cmax and AUC increased by 68% and 107% in PMs. Highest recommended dose in PMs is 20 mg/d due

to risk of QT prolongationFluoxetine CYP2D6 PMs have higher concentrations of S-fluoxetine, and lower concentrations of S-norfluoxetine, at a

steady state. There is no effect of CYP2D6 metabolism status on pharmacodynamics of fluoxetine.Paroxetine CYP2D6 In EMs, concomitant administration of paroxetine increased the AUC and Cmax of atomoxetine.

CYP2D6, cytochrome P450 2D6; NAT1-2, N-acetyltransferase 1 and 2; G6PD, glucose-6-phosphate dehydrogenase; CYP2C19, cytochrome P4502C19; CYB5R1-4, cytochrome b5 reductase 1-4; PM, poor metabolizer; EM, extensive metabolizer; UM, ultrarapid metabolizer; AUC, area underthe plasma concentration-time curve; Cmax, maximum plasma concentration.

Table 2 – Common CYP2D6 alleles, functional effect, andfrequency in African Americans and Caucasians.

Allele ActivityaAllele frequency, mean (range)%b

African Americans Caucasians

*1 Normal 40 (30–83) 54 (28–83)*2 Normal 14 (4–29) 27 (10–40)*3 Non-functional 0.3 (0–0.6) 1 (0–3)*4 Non-functional 6.2 (4–8) 18 (10–33)*5 Non-functional 6 (2–9) 3 (0–7)*6 Non-functional 0.2 (0–1) 1 (0–3)*9 Reduced 0.5 (0–1) 2 (0–5)*10 Reduced 4 (3-8) 3 (0.4–15)*17 Reduced 18 (13–26) 0.3 (0–1.1)*36 Non-functional 0.6 (0–1) 0*41 Reduced 9 (2–15) 9 (4–14)*1 ! N Increased 0.4 (0–1.2) 0.8 (0–4)*2 ! N Increased 1.6 (0.1–2) 1.3 (0–6)*4 ! N Non-functional 2 (0.3–4) 0.3 (0–1)

a Crews et al.17b ⟨http://www.pharmgkb.org/download.action?filename=CYP2D6_allele_frequency_table_R2.xlsx⟩, updated August 2013.

S E M I N A R S I N P E R I N A T O L O G Y 3 8 ( 2 0 1 4 ) 5 3 4 – 5 4 0536

Quinney SK, Patil AS, Flockhart DA. Is personalized medicine achievable in obstetrics? Semin Perinatol [Internet]. 2014 Oct 1 [cited 2014 Dec 1];38(8):534–40.

DPD

DPYD gene

DPD enzyme

Drug metabolism

Pyrimidine

⇈ Polymorphic DPD deficiency

DPD

MUTATION ALLELE PHENOTYPE

C.1905+1G>A DPYD*2A DPD deficiency

C. 1679T>A DPYD*13 DPD deficiency

C.2846A>T - DPD deficiency

C.1129-5923C>G - DPD deficiency

DPYD in cancer

5-FU

• DPD deficiency ➔ 3- 5% Caucasian population:

⇊ ⇊ capacity to metabolize 5-FU and its produg capecitabine, consequently develop toxicity at therapeutical doses.

Advantages

• Increase drug response

• Decrease adverse events

• Improve the adherence

• Reduce treatment costs

• Cost - Benefits

• Reproducibility

• Invasivity

Disadvantages

1. American T, Pharmacogenomics T. Pharmacogenetics and pharmacogenomics. 2001;345–7.

2. Quinney SK, Patil AS, Flockhart DA. Is personalized medicine achievable in obstetrics? Semin Perinatol [Internet]. 2014 Oct 1 [cited 2014 Dec 1];38(8):534–40. Available from: http://www.sciencedirect.com/science/article/pii/S0146000514001050

3. Roden DM, George Jr AL. The genetic basis of variability in drug responses. Nat Rev Drug Discov [Internet]. 2002 Jan;1(1):37–44. Available from: http://dx.doi.org/10.1038/nrd705

4. van Staveren MC, Guchelaar HJ, van Kuilenburg a BP, Gelderblom H, Maring JG. Evaluation of predictive tests for screening for dihydropyrimidine dehydrogenase deficiency. Pharmacogenomics J [Internet]. Nature Publishing Group; 2013 Oct [cited 2014 Dec 1];13(5):389–95. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23856855