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Chapter 8 Pharmacogenomics and Hematologic Diseases 85

Thiopurines and Somatic Variants in NT5C2 from inappropriate warfarin dosing remain among the most common
reasons for hospitalization due to adverse drug reactions.
To explore molecular mechanisms for drug resistance in childhood Pharmacologically, warfarin is a racemic mixture of R- and
ALL, two study groups recently sequenced the transcriptomes and S-enantiomers that differ in their patterns of metabolism and in
whole exomes of diagnostic, remission, and relapse samples from their potency of pharmacologic effects, with S-warfarin being more
ALL patients. 13,14 In B-cell precursor (BCP)-ALL up to 10% and potent. Warfarin dose requirements can be influenced by both modi-
in T-ALL up to 19% of the relapse samples had mutations in the fiable (e.g., compliance, dietary vitamin K intake, therapeutic level
NT5C2 nucleotidase. The NT5C2 nucleotidase can inactivate the surveillance) and nonmodifiable factors (e.g., age, gender, genetics).
thiopurines MP and TG. Remarkably, six of the identified variants Candidate gene studies initially demonstrated that the CYP2C9
in NT5C2 increased the enzyme activity of the variant proteins (up genotype influences warfarin clearance, and alters oral anticoagulant
to 48-fold), thereby protecting ALL blast cells against thiopurine- dose requirements and bleeding risks. CYP2C9 is the principal
induced apoptosis. Of note, no resistance to other antileukemic CYP2C isoenzyme in the human liver, and it is involved in the
agents was observed when the variant proteins were expressed in oxidative metabolism and inactivation of S-warfarin. 15
cell lines. Maintenance therapy with the antimetabolites MP and The two most common CYP2C9 variants with diminished enzyme
methotrexate is an essential element for successful ALL therapy, activities are CYP2C9*2 (rs1799853) and CYP2C9*3 (rs1057910).
and one can speculate that resistance to MP can give rise to early Approximately 35% of Caucasians have one or two of these variant
relapse of ALL. Indeed, a significant association of activating NT5C2 alleles; the *2 and *3 variants are virtually nonexistent in Africans
variants and early ALL relapse was found. 13,14 Novel strategies are and Asians (95% express the wild-type genotype [i.e., extensive
necessary to overcome this drug resistance phenotype in the subset metabolizers]).
of patients with NT5C2 mutations, and such strategies may help to Compared with the wild-type genotype (CYP2C9*1/*1), patients
further improve outcome in ALL by avoiding inappropriate drug with two nonfunctional variants have a reduction of enzyme activity
levels at the target site (e.g., by using drugs that are not inactivated to approximately 12% for CYP2C9*2/*2 and approximately 5% for
via NT5C2 or by design of small molecules that inhibit NT5C2 CYP2C9*3/*3. Therefore, the required dose of warfarin is lowest in
function). homozygous carriers of the CYP2C9*3 variant (e.g., dose reduction
of about 1.6 mg/day) and intermediate in homozygote carriers of
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Inherited Genome Variants in Cytochrome the CYP2C9*2 variant (e.g., dose reduction of about 1 mg/day).
An important finding was the identification of a novel mechanism
P450 Enzymes underlying warfarin resistance—the discovery of sequence variants
in the warfarin target gene VKORC1, which encodes the vitamin
The cytochrome P450 (CYP) superfamily is a system of phase I K epoxide reductase complex 1. This complex regenerates reduced
enzymes involved in the metabolism of endogenous substances and vitamin K for another cycle of catalysis, which is essential for the
exogenous compounds (e.g., drugs, environmental chemicals). In posttranslational γ-carboxylation of vitamin K–dependent clotting
humans the CYP enzymes are encoded by more than 57 genes, and factors. A common noncoding variant (-1639G>A, rs9923231) was
the majority of these genes are polymorphic. Updated information shown to be significantly associated with warfarin dose requirements.
regarding the nomenclature and properties of the variant alleles with Patients with the -1639 AA genotype require lower initial warfarin
links to the dbSNP database is available at the human CYP allele doses (e.g., dose reduction of up to 3 mg/day) when compared with
website (see Table 8.1). individuals with the -1639 GG variant. As the -1639G>A poly-
On the basis of patient genotype (diplotype) CYP variant alleles, morphism affects a VKORC1 transcription factor binding site, the
individuals are often categorized into one of four major predicted functional effect of the variant is thought to be related to decreased
drug metabolism phenotypes: poor metabolizers (having two loss- VKORC1 transcription, leading to lower protein expression. There
of-function alleles), intermediate metabolizers (being deficient in are major differences in the distribution of VKOCR1 haplotypes
one allele), extensive metabolizers (having two copies of functional among ethnic groups, and this may explain interethnic differences
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alleles), and ultrarapid metabolizers (having three or more functional in coumarin requirement. GWAS in patients treated with warfarin
gene copies due to gene duplications, or two increased-activity alleles, showed two major signals in and around VKORC1 and CYP2C9, and
or one functional allele plus one increased activity allele). identified a much weaker association with CYP4F2. The CYP2F4
Different populations of metabolizers have been linked to differ- enzyme catalyzes vitamin K oxidation, and the V433M variant
ent types of variants in the coding region of CYP genes (i.e., SNPs (rs2108622) was identified to require increased warfarin dosing.
that alter the amino acid encoded, thereby altering protein function Overall, VKORC1 explains approximately 25% of the variance in
or stability); SNPs in intronic regions, which can alter CYP gene coumarin dose requirement, CYP2C9 explains about 15%, and
mRNA expression; CNVs (e.g., gene deletions, gene duplications) CYP4F2 explains about 3%. 15
of CYP genes; or differences in the methylation at CpG islands in In 2010 the FDA updated the label on warfarin, providing
promoter and 5′ regions, which alter expression of CYP genes. VKORC1 and CYP2C9 genotype-specific ranges of doses, and
Many pharmacologically relevant variants in CYP genes have been suggested that VKORC1 and CYP2C9 genotypes be taken into
identified. The focus here is on the variants in CYP2C9, which have consideration when the drug is prescribed. Additionally, dosing
been shown to influence the metabolism of an extensively prescribed algorithms are available online (e.g., from the International Warfarin
medication: warfarin. Pharmacogenetics Consortium [IWPC]), including genetic and non-
genetic information that can help to optimize the warfarin starting
CYP2C9, VKORC1, and Warfarin dose (see CPIC Guidelines: Table 8.2).
In the United States, the oral vitamin K antagonist warfarin is still The results of two large randomized trials, which have prospec-
widely used to prevent thromboembolic events in patients with tively evaluated the benefit of genotype-guided warfarin dosing,
chronic conditions such as atrial fibrillation, and the drug is prescribed have recently been reported. Whereas the European Pharmacoge-
to more than 1 million persons annually. A narrow therapeutic index netics of Anticoagulant Therapy (EU-PACT) trial demonstrated
with a risk for serious hemorrhage and interindividual variability that pharmacogenetic-guided dosing is superior to a fixed-dosing
in response to warfarin necessitate individualization of treatment, regimen for achieving therapeutic INRs, the U.S. Clarification of
which has been based primarily on monitoring prothrombin time Optimal Anticoagulation Through Genetics (COAG) study failed
via international normalized ratio (INR) testing. Compared with the to demonstrate an improvement in PTTR with genotype-guided
therapeutic INR range (i.e., 2–3), INR greater than 4 is associated dosing compared with the algorithm-guided dosing control arm.
with a 25-fold higher risk of bleeding in elderly patients treated Potential reasons for the differences include differences in the algo-
with warfarin, and the percentage time in therapeutic range (PTTR) rithmic strategies and control arms, as well as ethnic heterogeneity. In
is a widely accepted read-out for treatment effect. Complications summary, the results of these trials and recent metaanalyses indicate