CA2911709A1 - Retinoic acid receptor gamma (rarg) gene polymorphisms predictive of anthracycline-induced cardiotoxicity (act) - Google Patents

Abstract

Provided are methods for assessing the susceptibility of a subject to the development of cardiotoxicity in response to receiving one or more anthracycline compounds, the method including determining the presence or absence of one or more polymorphisms, wherein the presence or absence of one or more such polymorphisms is indicative of susceptibility to the development of cardiotoxicity.

Description

RETINOIC ACID RECEPTOR GAMMA (RARG) GENE POLYMORPHISMS
PREDICTIVE OF ANTHRACYCLINE-INDUCED CARDIOTOXICITY (ACT) FIELD OF THE INVENTION
This invention relates to the field of genetic markers for adverse drug reactions. More specifically, methods and compositions useful for identifying individuals that may be at risk for an adverse drug reaction.
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application Serial No.
62/077,702 filed on 10 November 2014, entitled "RETINOIC ACID RECEPTOR GAMMA
(RARG) GENE POLYMORPHISMS PREDICTIVE OF ANTHRACYCLINE-INDUCED
CARDIOTOXICITY (ACT)".
BACKGROUND
Adverse drug reactions (ADRs) are a significant cause of illness, hospitalization and death for both children and adults in the Western world (LAZAROU et al. JAMA 1998;
PIRMOHAMED et al. BMJ 2004). Estimates suggest that 15% of hospitalized children experience an ADR. Those that do survive the ADR may be left disabled (MITCHELL et al., 1988 Pediatrics 82:24-9; MARTINEZ-MIR et al., 1999. Br J Clin Pharmacol 47 :681-8).
Many approved drugs used in children are untested in pediatric populations.
While it is known that children metabolize drugs differently than adults, in many cases pediatric dosage forms are not available. This is of particular concern with chemotherapy drugs, which may frequently be supplied as a single-dose package, and in combination with other agents, excipients and the like. Pediatric populations also represent a more varied population, and this increased variability may be due to developmental differences in the normal expression of drug metabolism genes.
Genetic factors are involved in variability in drug response ¨ ranging from 20-95% in some studies. Age, sex, body weight, health, medical history and the like may be accounted for, but patient genotype is largely an unknown factor (EVANS et al. 2003. NEJM
348:538-549; WEINSHILBOUM 2003. NEJM 348:529-537).
Anthracyclines are used as cytotoxic agents in chemotherapeutic protocols in both children and adults, for a variety of neoplasms. Over 70% of childhood cancers are treated with anthracyclines. However, clinical utility of anthracyclines is limited by anthracycline-induced cardiotoxicity (ACT). This manifests as asymptomatic cardiac dysfunction in 57%
of children and as congestive heart failure in 16-20% of children. Some genetic risk factors have been identified, but much of the variability in the susceptibility to ACT
remains unaccounted for, suggesting the existence of additional genetic factors. ACT
may be characterized by reduced ventricular wall thickness and mass, indicative of decreased cardiac muscle and depressed ventricular contractility. Increased and cumulative dose, nature of the particular anthracycline, administration route, age, sex and prior radiation treatment may affect onset and severity of cardiotoxicity. Administration of enalapril, dexrazoxane or antioxidants such as vitamin E, coenzyme Qio, carnitine, or glutathione, for example may be beneficial in preventing or reducing cardiac injury during chemotherapy. Other agents that may be administered to reduce anthracycline cardiotoxicity are described (WOUTERS et al. 2005. Br. J Hematol 131:561-578).
Examples of anthracyclines and anthracycline analogues include daunorubicin, doxorubicin, idarubicin and epirubicin. For example, anthracyclines may be used in the treatment of solid and hematologic cancers, such as breast cancer, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma, sarcoma, renal cancer and liver cancer.
Dose limits have been empirically set in the clinic, above which the cardiotoxicity is deemed to be unacceptable. Subclinical and clinical cardiotoxicity may occur below these doses (JOHNSON 2006. Seminars in Oncology 33:S33-7o) and affect current and subsequent therapeutic regimens. Liposomal anthracycline compositions may demonstrate reduced cardiotoxicity (EWER et al. 2004. Seminars in Oncology 31:161-181).
Proteomic methods have been developed for early detection of drug-induced cardiotoxicity (PETRICOIN et al. 2004. Toxicol Pathol 32:122-30).
Some polymorphisms in NAD(P)H oxidase are associated with anthracycline-induced cardiotoxicity (WOJNOWSKI et al. 2005. Circulation 112:3754-3762). Similarly, polymorphisms have been identified that associated with ACT in pediatric populations (W02012162812 and W02008058394).
Genotype has been shown to alter response to therapeutic interventions.
Genentech's HERCEPTIN was not effective in its overall Phase III trial but was shown to be effective in a genetic subset of subjects with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. Similarly, Novartis' GLEEVECC) is only indicated for the

2 subset of chronic myeloid leukemia subjects who carry a reciprocal translocation between chromosomes 9 and 22.
SUMMARY
This invention is based in part on the identification that the particular nucleotide (allele) or genotype at the site of a given RARG SNP may be associated with an increased likelihood of cardiotoxicity ('risk genotype') or a decreased likelihood of cardiotoxicity ('decreased risk genotype').
This invention is also based in part on the surprising discovery that n2229774 SNP is useful in predicting a subject's risk of cardiotoxicity following anthracycline treatment.
Furthermore, SNPs in linkage disequilibrium (i.e. rs11170479; rs11170481;
rs7334991.74 and rs57789211) are also useful in predicting a subject's risk of cardiotoxicity following anthracycline treatment. Whereby the subjects having a decreased risk genotype are less likely to experience cardiotoxicity and subjects having a risk genotype are more likely to experience cardiotoxicity from the same treatment.
In accordance with one aspect of the invention, methods are provided for selecting human subjects for anthracycline compound administration, the method including:
(a) performing an amplification reaction using a nucleic acid sample from a subject to amplify polymorphic site: n2229774 or a polymorphic site in linkage disequilibrium thereto selected from one or more of the following: rs1117o479; rs111704.84 rs73309171;
and rs57789211;
(b) performing a sequencing reaction using the amplified nucleic acid from (a) to determine whether the subject has a risk genotype selected from the following:
r52229774 A/A; or rs2229774 A/G; or a reduced risk genotype rs2229774 GIG or the corresponding genotype at a polymorphic site in linkage disequilibrium to n2229774 selected from one or more of the following: rs11170479; rs11170481; rs733091.71; and rs57789211;
and (c) identifying the subject as having a risk genotype or a reduced risk genotype.
In accordance with a further aspect of the invention, methods are provided for assisting in the identification of human subjects at risk for cardiotoxicity from anthracycline compound administration, the method including:
(a) performing an amplification reaction using a nucleic acid sample from a subject to amplify polymorphic site m2229774 or a polymorphic site in linkage disequilibrium thereto selected from one or more of the following: rs11170479; rs11170481;
rs73309171; and

3 rs57789211;
(b) performing a sequencing reaction using the amplified nucleic acid from (a) to determine whether the subject has a risk genotype selected from the following:
rs2229774 A/A; or r52229774 A/G; or a reduced risk genotype n2229774 GIG or the corresponding genotype at a polymorphic site in linkage disequilibrium to n2229774 selected from one or more of the following: rs11170479; rs11170481; rs73309171; and rs57789211; and (c) identifying the subject as having a risk genotype or a reduced risk genotype.
In accordance with a further aspect of the invention, methods are provided for treating a neoplastic disease in a human subject in need thereof, the method including:
(a) administering one or more anthracycline compounds to a subject having a reduced risk genotype n2229774 G/G;
(b) administering one or more anthracycline compounds and heart function monitoring or a cardioprotective agent or both to subjects with a risk genotype selected from the following: n2229774 A/A; and m2229774 A/G;
(c) administering one or more anthracycline compounds in conjunction with a non-anthracycline anti-neoplastic compound and heart function monitoring or a cardioprotective agent or both to subjects with a risk genotype selected from the following:
r52229774 A/A; and m2229774 A/G; or (d) administering one or more non-anthracycline compounds to subjects with a risk genotype selected from one or more of the following: n2229774 A/A; and rs2229774 A/G.
In accordance with a further aspect of the invention, methods are provided for diagnosing a predisposition for cardiotoxicity risk in a human subject from anthracycline administration, the method including: a) determining an identity for one or more of the following single nucleotide polymorphisms (SNPs) in a biological sample from the subject:
S2229774 or a polymorphic site in linkage disequilibrium thereto selected from one or more of the following: r51117o479; rs1117o481; rs73309171; and rs57789211; and b) making a cardiotoxicity risk determination based on the prevalence of risk alleles in the subject sample.
In accordance with a further aspect of the invention, uses of an anthracycline compound are provided having a cardiotoxicity risk for the treatment of a subject, wherein the subject treated has a reduced cardiotoxicity risk genotype at polymorphic site:
m2229774 or a polymorphic site in linkage disequilibrium thereto selected from one or more of the following: rs1117o479; rs1117o481; rs733o9171; and rs57789211; for the subject, where the subject is a candidate for anthracycline administration.

4 In accordance with a further aspect of the invention, there is provided a use of one or more anthracycline compounds or one or more non-anthracycline compounds for the treatment of a neoplastic disease in a human subject in need thereof, wherein the treatment depends on the risk genotype as follows: (a) a subject having a reduced risk genotype rs2229774 G/G would be selected for treatment with one or more anthracycline compounds;
(b) a subject having a risk genotype selected from the following: r52229774 A/A; and 1'52229774 A/G would be selected for treatment with one or more anthracycline compounds and heart function monitoring or a cardioprotective agent or both; (c) a subject having a risk genotype selected from the following: r52229774 A/A; and rs2229774 A/G would be selected for treatment with one or more anthracycline compounds in conjunction with a non-anthracycline anti-neoplastic compound and heart function monitoring or a cardioprotective agent or both; or (d) a subject having a risk genotype selected from one or more of the following: n2229774 A/A; and r52229774 A/G would be selected for treatment with one or more non-anthracycline compounds.
In accordance with a further aspect of the invention anthracyclines are provided for use in a method for treating a neoplastic disease in a subject in need there of, the method including:
(a) selecting a subject having a reduced risk of developing cardiotoxicity, wherein cardiotoxicity is based on the identity of a single nucleotide polymorphism (SNP) at polymorphic site: r52229774 or a polymorphic site in linkage disequilibrium thereto selected from one or more of the following: rs1117o479; n1117 481; rs73309171;
and rs57789211; and (b) administering said subject one or more anthracyclines.
The method may further include selecting a treatment regimen based on the subject's cardiotoxicity risk status, as follows: (i) a subject with a reduced risk genotype is administered the anthracycline compound; (ii) a subject with a risk genotype is administered the anthracycline compound and is given heart function monitoring or a cardioprotective agent or both; (iii) a subject with a risk genotype is administered the anthracycline compound in conjunction with a non-anthracycline anti-neoplastic compound and is given heart function monitoring or a cardioprotective agent or both; (iv) a subject with a risk genotype is administered a non-anthracycline anti-neoplastic compound.
The anthracycline may be selected from one or more of the following:
daunorubicin, daunomycin, rubidomycin, doxorubicin, idarubicin, epirubicin, mitoxantrone, carminomycin, esorubicin, quelamycin, aclarubicin, esorubicin, zorubicin, pirarubicin,

5 amrubicin, iododoxorubicin, mitoxantrone and valrubicin.
The non-anthracycline anti-neoplastic compound may be selected from one or more of:
cyclophosphamide, ifosphamide, fluorouracil, paclitaxel, vincristine, cisplatin, streptozocin, and docetaxel.
The anthracycline compound may be doxorubicin. The polymorphic site may be rs2229774 and wherein the risk genotype is rs2229774 A/A or r52229774 A/G and the reduced risk genotype is rs2229774 G/G. The cardioprotective agent may be dexrazoxane. The non-anthracycline anti-neoplastic compound may be selected from one or more of:
cyclophosphamide, ifosphamide, fluorouracil, paclitaxel, vincristine, cisplatin, streptozocin, and docetaxel. The neoplastic disease may be selected from: breast cancer, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma, sarcoma, renal cancer and liver cancer.
The method may further include administering the anthracycline in accordance with the subject's risk of developing cardiotoxicity.
The identity of a single nucleotide polymorphism may be determined by one or more of the following techniques: restriction fragment length analysis; sequencing; micro-sequencing assay; hybridization; invader assay; gene chip hybridization assays;
oligonucleotide ligation assay; ligation rolling circle amplification; 5' nuclease assay; polymerase proofreading methods; allele specific PCR; matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy; ligase chain reaction assay; enzyme-amplified electronic transduction; single base pair extension assay; and reading sequence data.
In accordance with a further aspect of the invention, uses of an anthracycline in the manufacture of a medicament for the treatment of neoplastic disease, wherein the subjects treated may have a reduced cardiotoxicity risk genotype at one or more of the following RARG polymorphic sites: rs2229774; rs1117o479; rs1117o481; rs73309171; and rs57789211.
The anthracycline may be selected from one or more of the following:
anthracycline antibiotics such as daunorubicin (daunomycin, rubidomycin), doxorubicin, idarubicin, epirubicin, mitoxantrone, carminomycin, esorubicin, quelamycin, aclarubicin, esorubicin, zorubicin, pirarubicin, amrubicin, iododoxorubicin, mitoxantrone and valrubicin or other anthracycline compounds described herein.

6 The method may further include obtaining a biological sample or samples from a subject or subjects. The method may further include administering the anthracycline in accordance with the subject's risk of developing cardiotoxicity.
The identity of a single nucleotide polymorphism may be determined by one or more of the following techniques: restriction fragment length analysis; sequencing; micro-sequencing assay; hybridization; invader assay; gene chip hybridization assays;
oligonucleotide ligation assay; ligation rolling circle amplification; 5' nuclease assay; polymerase proofreading methods; allele specific PCR; matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy; ligase chain reaction assay; enzyme-amplified electronic transduction; single base pair extension assay; and reading sequence data.
In accordance with a further aspect of the invention, there are provided two or more oligonucleotides or peptide nucleic acids of about in to about 400 nucleotides that hybridize specifically to a sequence contained in a human target sequence consisting of a subject's cardiotoxicity associated gene sequence, a complementary sequence of the target sequence or RNA equivalent of the target sequence and wherein the oligonucleotides or peptide nucleic acids are operable in determining the presence or absence of two or more polymorphism(s) in the cardiotoxicity associated gene sequence selected from of the following polymorphic site m2229774 or a polymorphic site in linkage disequilibrium thereto.
In accordance with a further aspect of the invention, a kit is provided for determining a genotype at one or more of the following polymorphic sites: rs2229774;
rs11170479;
rs11170481; rs73309171; and rs57789211; in a subject to assess the subject's risk of cardiotoxicity following anthracycline administration, by distinguishing alternate nucleotides at the polymorphic site; or a labeled oligonucleotide having sufficient complementary to the polymorphic site so as to be capable of hybridizing distinctively to said alternate. The kit may further include an oligonucleotide or a set of oligonucleotides operable to amplify a region including the polymorphic site. The kit may further include a polymerization agent. The kit may further include instructions for using the kit to determine genotype.
In accordance with another aspect of the invention, there is provided a commercial package containing, as active pharmaceutical ingredient, use of anthracycline, or a pharmaceutically acceptable salt thereof, together with instructions for its use for the curative or prophylactic treatment of a neoplastic disease in a subject, wherein the subject treated has a reduced

7 risk polymorphism in one or more of the following polymorphic sites:
rs2229774;
rs11170479; rs11170481; rs73309171; and rs57789211.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1: shows a pharmacogenetic association with susceptibility to anthracycline-induced cardiotoxicity is situated within RARG, wherein the association results are shown for genotyped (circles) and imputed (squares) SNPs along with recombination rates for a 122 kb region of chromosome 12q13.13 and wherein each point represents the nominal P-value (left y-axis) for the stage 1 cohort. P-values are from logistic regression analysis using an additive model, adjusted for age, dose, tumour type (ALL, Ewing's sarcoma and rhabdomyosarcoma) and cardiac radiation therapy. SNPs are coloured according to their pairwise correlation (r2) with r52229774 (purple circle) using the tow Genomes CEU
reference population. Overlaid are the recombination rates (right y-axis) for estimating putative recombination hotspots also based upon the Iwo Genomes CEU
population.
FIGURE 2: shows a functional characterization of RARGS427L. (a), Transcriptional activation of luciferase coupled to an optimized retinoic acid response element (RARE) by transiently transfected RarG (WT) or RARGS427L in HEK293T cells. Data are presented as the average s.e.m. in aggregate (n=48) from three separate experiments of sixteen replicates each. Data represent the fold induction in luminescence upon ATRA
treatment compared to untreated samples. ** denotes P value <0.005 using a t-test analysis. Inset, Immunoblot of 2on HEK293T lysate generated 48 hours post-transfection with empty vector (negative), or the indicated construct using anti-DDK 4C5 (top panel) and anti-GAPDH (bottom panel) antibodies. Untagged wild type RARG has an estimated molecular weight of 50.4 kDa. Molecular sizes are indicated on the left. (b), Relative Top2b expression in untransfected or RARG-transfected H9c2 cells in the presence or absence of ATRA. Data are presented as the average s.e.m. in aggregate (n=6) from two separate experiments of three replicates each. ' denotes P value <0.0001 using a one-way ANOVA
analysis. (c), Repression of Top2b expression in H9c2 cells transfected with RarG WT or RARG

compared to untransfected cells. Data, normalized to the relative expression of the transfected RARG construct, are presented as the average + s.e.m. in aggregate (n=12) from two separate experiments of six replicates each. *** denotes P value <o.000i using a t-test analysis.

8 DETAILED DESCRIPTION
1. Definitions In the description that follows, a number of terms are used extensively, the following definitions are provided to facilitate understanding of the various embodiments of the invention.
An "anthracycline compound" or "anthracycline" or "anthracycline derivatives"
or "anthracycline analogues" as used herein is typically an anthraquinone core attached to a carbohydrate moiety and derivative thereof (see for example, FAN et al. J.
Org. Chem.
(2007) 72:2917-2928; Goodman and Gilman's The Pharmacological Basis of Therapeutics 8th edition editors Alfred Goodman Gilman, Theodore Rall, Alan Nies, Palmer Taylor.
Pergamon Press. 1990 pg 1241-1244). For example, include anthracycline antibiotics such as daunorubicin (daunomycin, rubidomycin), doxorubicin, idarubicin, epirubicin, mitoxantrone, carminomycin, esorubicin, quelamycin, aclarubicin, esorubicin, zorubicin, pirarubicin, amrubicin, iododoxorubicin, detorubicin, marcellomycin, rodorubicin, and valrubicin. Alternatively, the anthracycline may be selected from daunorubicin and doxorubicin.
As used herein "anthracycline-induced cardiotoxicity" or "ACT" is defined based on CTCAEv3 (Common Terminology Criteria for Adverse Events ¨ see Cancer Therapy Evaluation Program - Common Terminology Criteria for Adverse Events- Version 3 in edition 2003) as early- or late-onset left ventricular dysfunction measured by echocardiogram (shortening fraction, SF) and/or symptoms requiring intervention. We used a more stringent threshold of SF..26% at any time during or after anthracycline therapy to better differentiate between cardiotoxicity cases and controls. To exclude transient acute cardiotoxicity, echocardiograms obtained <21 days after a dose of anthracyclines were excluded. Control patients were required to have normal echocardiograms (SF3o%) during and after therapy, with a follow-up of >5 years after completion of anthracycline therapy. Doxorubicin equivalents were used to calculate cumulative anthracycline doses (Altman A.J. editor, Children's Oncology Group.
Supportive care of children with cancer: current therapy and guidelines from the Children's Oncology Group. Baltimore: Johns Hopkins University Press; 2004.412 p.p.).
"Genetic material" includes any nucleic acid and can be a deoxyribonucleotide or ribonudeotide polymer in either single or double-stranded form.

9 A nucleotide represented by the symbol M may be either an A or C, a nucleotide represented by the symbol W may be either an T/U or A, a nucleotide represented by the symbol Y may be either an C or T/U, a nucleotide represented by the symbol S
may be either an G or C, while a nucleotide represented by the symbol R may be either an G or A, and a nucleotide represented by the symbol K may be either an G or T/U.
Similarly, a nucleotide represented by the symbol V may be either A or G or C, while a nucleotide represented by the symbol D may be either A or G or T, while a nucleotide represented by the symbol B may be either G or C or T, and a nucleotide represented by the symbol H may be either A or C or T. A nucleotide represented by the symbol N may be an A or G or T or C.
A "polymorphic site" or "polymorphism site" or "polymorphism" or "single nucleotide polymorphism site" (SNP site) or single nucleotide polymorphism" (SNP) as used herein is the locus or position with in a given sequence at which divergence occurs. A
"polymorphism" is the occurrence of two or more forms of a gene or position within a gene (allele), in a population, in such frequencies that the presence of the rarest of the forms cannot be explained by mutation alone. The implication is that polymorphic alleles confer some selective advantage on the host. Polymorphic sites have at least two alleles, each occurring at frequency of greater than 1%, and may be greater than 10% or 20%
of a selected population. Polymorphic sites may be at known positions within a nucleic acid sequence or may be determined to exist. Polymorphisms may occur in both the coding regions and the noncoding regions (for example, promoters, introns or untranslated regions) of genes. Polymorphisms may occur at a single nucleotide site (SNPs) or may involve an insertion or deletion as described herein.
A "risk genotype" as used herein refers to an allelic variant (genotype) at one or more of the following polymorphic sites: rs2229774; rs1117o479; rs1117o481; rs73309171;
and rs57789211; as described herein, as being indicative of an increased likelihood of cardiotoxicity following administration of an anthracycline. The risk genotype may be determined for either the haploid genotype or diploid genotype, provided that at least one copy of a risk allele is present. Risk genotype may be an indication of an increased risk of cardiotoxicity. Subjects having one copy (heterozygotes) or two copies (homozygotes) of the risk allele are considered to have the "risk genotype" even though the degree to which the subjects is at risk cardiotoxicity may increase, depending on whether the subject is a homozygote rather than a heterozygote. Such "risk genotypes" may be selected from the following: rs2229774 A/A; and rs2229774 A/G; or a polymorphic sites in linkage disequilibrium thereto (risk genotype on the reverse strand; T and C on the forward strand).

A "decreased risk genotype" as used herein refers to an allelic variant (genotype) at polymorphic site rs2229774 or a polymorphic site in linkage disequilibrium thereto, for the subject as described herein, as being indicative of a decreased likelihood of cardiotoxicity following administration of an anthracycline. "Decreased risk genotype" or "reduced risk genotypes" may be and a "reduced risk genotype" may be n2229774 GIG; or a polymorphic site in linkage disequilibrium thereto (risk genotype on the reverse strand; T
and C on the forward strand).
A "clade" is a group of haplotypes that are closely related phylogenetically.
For example, if haplotypes are displayed on a phylogenetic (evolutionary) tree a clade includes all haplotypes contained within the same branch.
The pattern of a set of markers along a chromosome is referred to as a "Haplotype".
Accordingly, groups of alleles on the same small chromosomal segment tend to be transmitted together. Haplotypes along a given segment of a chromosome are generally transmitted to progeny together unless there has been a recombination event.
Absence of a recombination event, haplotypes can be treated as alleles at a single highly polymorphic locus for mapping.
As used herein "haplotype" is a set of alleles of closely linked loci on a chromosome that tend to be inherited together. Such allele sets occur in patterns, which are called haplotypes. Accordingly, a specific SNP or other polymorphism allele at one SNP site is often associated with a specific SNP or other polymorphism allele at a nearby second SNP
site or other polymorphism site. When this occurs, the two SNPs or other polymorphisms are said to be in Linkage Disequilibrium (LD) because the two SNPs or other polymorphisms are not just randomly associated (i.e. in linkage equilibrium).
In general, the detection of nucleic acids in a sample depends on the technique of specific nucleic acid hybridization in which the oligonucleotide is annealed under conditions of "high stringency" to nucleic acids in the sample, and the successfully annealed oligonucleotides are subsequently detected (see for example Spiegelman, S., Scientific American, Vol. 210, p. 48 (1964)). Hybridization under high stringency conditions primarily depends on the method used for hybridization, the oligonucleotide length, base composition and position of mismatches (if any). High-stringency hybridization is relied upon for the success of numerous techniques routinely performed by molecular biologists, such as high-stringency PCR, DNA sequencing, single strand conformational polymorphism analysis, and in situ hybridization. In contrast to Northern and Southern hybridizations, these aforementioned techniques are usually performed with relatively short probes (e.g., usually about 16 nucleotides or longer for PCR or sequencing and about 40 nucleotides or longer for in situ hybridization). The high stringency conditions used in these techniques are well known to those skilled in the art of molecular biology, and examples of them can be found, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998.
"Oligonucleotides" as used herein are variable length nucleic acids, which may be useful as probes, primers and in the manufacture of microarrays (arrays) for the detection and/or amplification of specific nucleic acids. Such DNA or RNA strands may be synthesized by the sequential addition (5'-3' or 3'-5') of activated monomers to a growing chain, which may be linked to an insoluble support. Numerous methods are known in the art for synthesizing oligonucleotides for subsequent individual use or as a part of the insoluble support, for example in arrays (BERNFIELD MR. and ROTTMAN FM. J. Biol. Chem. (1967) 242(18):4134-43; SULSTON J. et al. PNAS (1968) 6o(2):409-415; GILLAM S. et al.
Nucleic Acid Res.(1975) 2(5):613-624; SONORA GM. et al. Nucleic Acid Res.(199o) 18(11):3155-9;
LASHKARI DA. et al. Proc Nat Acad Sci (1995) 92(17)7912-5; MCGALL G. et al.
PNAS
(1996) 93(24):13555-6o; ALBERT TJ. et a/. Nucleic Acid Res.(2003) 31(7):e35;
GAO X. et al. Biopolymers (2004) 73(5):579-96; and MOORCROFT MJ. et al. Nucleic Acid Res.(2005) 33(8):e75). In general, oligonucleotides are synthesized through the stepwise addition of activated and protected monomers under a variety of conditions depending on the method being used. Subsequently, specific protecting groups may be removed to allow for further elongation and subsequently and once synthesis is complete all the protecting groups may be removed and the oligonucleotides removed from their solid supports for purification of the complete chains if so desired.
"Peptide nucleic acids" (PNA) as used herein refer to modified nucleic acids in which the sugar phosphate skeleton of a nucleic acid has been converted to an N-(2-aminoethyl)-glycine skeleton. Although the sugar-phosphate skeletons of DNA/RNA are subjected to a negative charge under neutral conditions resulting in electrostatic repulsion between complementary chains, the backbone structure of PNA does not inherently have a charge.
Therefore, there is no electrostatic repulsion. Consequently, PNA has a higher ability to form double strands as compared with conventional nucleic acids, and has a high ability to recognize base sequences. Furthermore, PNAs are generally more robust than nucleic acids. PNAs may also be used in arrays and in other hybridization or other reactions as described above and herein for oligonucleotides.

An "addressable collection" as used herein is a combination of nucleic acid molecules or peptide nucleic acids capable of being detected by, for example, the use of hybridization techniques or by any other means of detection known to those of ordinary skill in the art. A
DNA microarray would be considered an example of an "addressable collection".
In general the term "linkage", as used in population genetics, refers to the co-inheritance of two or more nonallelic genes or sequences due to the close proximity of the loci on the same chromosome, whereby after meiosis they remain associated more often than the 50%
expected for unlinked genes. However, during meiosis, a physical crossing between individual chromatids may result in recombination. "Recombination" generally occurs between large segments of DNA, whereby contiguous stretches of DNA and genes are likely to be moved together in the recombination event (crossover). Conversely, regions of the DNA that are far apart on a given chromosome are more likely to become separated during the process of crossing-over than regions of the DNA that are close together.
Polymorphic molecular markers, like SNPs, are often useful in tracking meiotic recombination events as positional markers on chromosomes.
Furthermore, the preferential occurrence of a disease gene in association with specific alleles of linked markers, such as SNPs or other polymorphisms, is called "Linkage Disequilibrium" (LD). This sort of disequilibrium generally implies that most of the disease chromosomes carry the same mutation and the markers being tested are relatively close to the disease gene(s).
For example, in SNP-based association analysis and LD mapping, SNPs can be useful in association studies for identifying polymorphisms, associated with a pathological condition, such as sepsis. Unlike linkage studies, association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families. In a SNP association study the frequency of a given allele (i.e. SNP allele) is determined in numerous subjects having the condition of interest and in an appropriate control group. Significant associations between particular SNPs or SNP
haplotypes and phenotypic characteristics may then be determined by numerous statistical methods known in the art.
Association analysis can either be direct or LD based. In direct association analysis, potentially causative SNPs may be tested as candidates for the pathogenic sequence. In LD
based SNP association analysis, SNPs may be chosen at random over a large genomic region or even genome wide, to be tested for SNPs in LD with a pathogenic sequence or pathogenic SNP. Alternatively, candidate sequences associated with a condition of interest may be targeted for SNP identification and association analysis. Such candidate sequences usually are implicated in the pathogenesis of the condition of interest. In identifying SNPs associated with cardiotoxicity, candidate sequences may be selected from those already implicated in the pathway of the condition or disease of interest. Once identified, SNPs found in or associated with such sequences, may then be tested for statistical association with an individual's prognosis or susceptibility to the condition or to the side effect of a medication.
For an LD based association analysis, high density SNP maps are useful in positioning random SNPs relative to an unknown pathogenic locus. Furthermore, SNPs tend to occur with great frequency and are often spaced uniformly throughout the genome.
Accordingly, SNPs as compared with other types of polymorphisms are more likely to be found in close proximity to a genetic locus of interest. SNPs are also mutationally more stable than variable number tandem repeats (VNTRs) and short tandem repeats (STRs).
In population genetics linkage disequilibrium refers to the "preferential association of a particular allele, for example, a mutant allele for a disease with a specific allele at a nearby locus more frequently than expected by chance" and implies that alleles at separate loci are inherited as a single unit (Gelehrter, T.D., Collins, F.S. (1990). Principles of Medical Genetics. Baltimore: Williams & Wilkens). Accordingly, the alleles at these loci and the haplotypes constructed from their various combinations serve as useful markers of phenotypic variation due to their ability to mark clinically relevant variability at a particular position (see Akey, J. et al. Eur J Hum Genet (2001) 9:291-300; and Zhang, K.
et al.
(2002). Am J Hum Genet. 71:1386-1394). This viewpoint is further substantiated by Khoury et al. ((1993). Fundamentals of Genetic Epidemiology. New York: Oxford University Press at p. 16o) who state, "[w]henever the marker allele is closely linked to the true susceptibility allele and is in [linkage] disequilibrium with it, one can consider that the marker allele can serve as a proxy for the underlying susceptibility allele."
As used herein "linkage disequilibrium" (LD) is the occurrence in a population of certain combinations of linked alleles in greater proportion than expected from the allele frequencies at the loci. For example, the preferential occurrence of a disease gene in association with specific alleles of linked markers, such as SNPs, or between specific alleles of linked markers, are considered to be in LD. This sort of disequilibrium generally implies that most of the disease chromosomes carry the same mutation and that the markers being tested are relatively close to the disease gene(s). Accordingly, if the genotype of a first locus is in LD with a second locus (or third locus etc.), the determination of the allele at only one locus would necessarily provide the identity of the allele at the other locus.
When evaluating loci for LD those sites within a given population having a high degree of linkage disequilibrium (i.e. an absolute value for r2 0.5) are potentially useful in predicting the identity of an allele of interest (i.e. associated with the condition of interest). A high degree of linkage disequilibrium may be represented by an absolute value for r2 >
o.6.
Alternatively, a high degree of linkage disequilibrium may be represented by an absolute value for r2 0.7 or by an absolute value for r2 ._. 0.8. Additionally, a high degree of linkage disequilibrium may be represented by an absolute value for r2> 0.85 or by an absolute value for r2 .. 0.9 or by an absolute value for r2 0.95. Accordingly, two SNPs that have a high degree of LD may be equally useful in determining the identity of the allele of interest or disease allele. Therefore, we may assume that knowing the identity of the allele at one SNP may be representative of the allele identity at another SNP in LD.
Accordingly, the determination of the genotype of a single locus can provide the identity of the genotype of any locus in LD therewith and the higher the degree of linkage disequilibrium the more likely that two SNPs may be used interchangeably.
LD may be useful for genotype-phenotype association studies. For example, if a specific allele at one SNP site (e.g. "A") is the cause of a specific clinical outcome (e.g. call this clinical outcome "B") in a genetic association study then, by mathematical inference, any SNP (e.g. "C") which is in significant LD with the first SNP, will show some degree of association with the clinical outcome. That is, if A is associated (¨) with B, i.e. A¨B and C¨A then it follows that C¨B. Of course, the SNP that will be most closely associated with the specific clinical outcome, B, is the causal SNP ¨ the genetic variation that is mechanistically responsible for the clinical outcome. Thus, the degree of association between any SNP, C, and clinical outcome will depend on LD between A and C.
Until the mechanism underlying the genetic contribution to a specific clinical outcome is fully understood, LD helps identify potential candidate causal SNPs and also helps identify a range of SNPs that may be clinically useful for prognosis of clinical outcome or of treatment effect. If one SNP within a gene is found to be associated with a specific clinical outcome, then other SNPs in LD will also have some degree of association and therefore some degree of prognostic usefulness.
Polymorphisms in linkage disequilibrium may be identified, for example, using the Haploview program (BARRETT JC. et al. Bioinformatics (2005) 21(2):263-5) and the LD
function in the Genetics Package in R (R Core Development Group, 2005 - R
Development Core Team (www.R-project.org). Linkage Disequilibrium between markers may be defined using r2 whereby all SNPs available on Hapmap.org (phase II) (cohort H), all SNPs genotyped internally using the Illumina Goldengate assay (cohort I) and SNPs may be sequenced using the Sequenom Iplex Platform (cohort S) for genes of interest.
A minimum r2 of 0.5 may be used as the cutoff to identify LD SNPs.
Numerous sites have been identified as polymorphic sites associated cardiotoxicity following anthracycline administration (see FIGURE 1).
TABLE 1. Single Nucleotide Polymorphisms Associated with Anthracycline-Induced Cardiotoxicity SNP SNP Odds AD
Gene Position Alleles ( P - Ratio SNP ID Chr RP
Symbol (BUILD reverse V value (95%CI
35) strand) 6.0(2.9 rs2229774 4.1X10-8 RARG 53605545 12 T/C (A/G) (A) ¨ 12.5) T/C 3.1x10- 5.9 (2.9 rs11170479 53610627 RARG 12 10 - 11.8) G/A 7.5x10- 5.5 (2.8 rs11170481 53611791 RARG 12 10 - 11.0) C/T 3.8x10- 6.1 (2.9 RARG 12 8 ¨ 12.9) C/T 3.8x10- 6.1 (2.9 rs57789211 53609992 RARG 12 8 ¨ 12.9) Adverse Drug Reaction Predictive Variant (ADRPV) Chromosome (Chr) TABLE 2. below shows the flanking sequences for the SNPs described in TABLE 1 providing their rs designations and corresponding SEQ ID NO designations. Each polymorphism is shown within the flanking sequence, and the polymorphism is identified in bold.

TABLE 2. Sequence for Cardiotoxicity-Associated Polymorphisms with SEQ ID NO
designations SNP SEQ
Alleles.ID
Gene Mnor Sybol SNP ID (* GENOMIC SEQUENCE NO: m Allele reverse strand) AGAACCCTGA AATGTTTGAG
GATGACTCCT
rs222974 GCAGCCTGGT CCCCACCCCA
TIC ATGCCTCTAG CGAGGATGAG
RARG (A/G) A GTTCCTGGGG

GTGTG
rs11170479 TIC
GCGCGCGCGC GCGCGCGCGT
RARG GTGGT

TCACT
rs11170481 G/A A
GATTCTGGCA CAGACACACA
RARG GCCAG

ACACA
rs73309171 C/T
ACTTGGAATT GTGCTGAATT
RARG AAAAA

GTATA
rs57789211 C/T
CTTTGTCAGG TAGTCTACTT
RARG CCCAC
It will be appreciated by a person of skill in the art that further linked polymorphic sites and combined polymorphic sites may be determined. A haplotype of the above genes can be created by assessing polymorphisms in normal subjects using a program that has an expectation maximization algorithm (for example PHASE). A constructed haplotype of these genes may be used to find combinations of SNPs that are in LD with the tag SNPs (tSNPs) identified herein. Accordingly, the haplotype of an individual could be determined by genotyping other SNPs or other polymorphisms that are in LD with the tSNPs identified herein. Single polymorphic sites or combined polymorphic sites in LD may also be genotyped for assessing subject risk of cardiotoxicity following anthracycline treatment.
It will be appreciated by a person of skill in the art that the numerical designations of the positions of polymorphisms within a sequence are relative to the specific sequence and the orientation of the strand being read (i.e. forward or reverse). Also the same positions may be assigned different numerical designations depending on the way in which the sequence is numbered and the sequence chosen. Furthermore, sequence variations within the population, such as insertions or deletions, may change the relative position and subsequently the numerical designations of particular nucleotides at and around a polymorphic site. For example, the sequences represented by accession numbers NM 0 3786, Y17151, BC104952, BCo5o37o, AF15400f all comprise ABCC3 nucleotide sequences, but may have some sequence differences and numbering differences between them. Furthermore, one of skill in the art will appreciate that a variety of sequencing, amplification, extension, genotyping or hybridization primers or probes may be designed to specifically identify the polymorphisms described in TABLE 2, and the sequences flanking the various polymorphisms as provided herein are illustrative examples. One of skill in the art will also appreciate that a variety of sequencing, amplification, extension, genotyping or hybridization primers or probes adjacent to, complimentary to, or overlapping with the sequences provided in TABLE 2, may be developed or designed for the identification of the polymorphisms described herein, without going beyond the scope of various embodiments of the invention as described herein.
One example of a partial gene sequence is a human ABCC3 gene sequence illustrated as GenBank accession # NM 003786. The genomic sequence of the human ABCC3 gene (NC 00 017.9 nucleotides 45979561-46185071) further includes 5' and 3' untranslated sequences, introns and the like. Sequence databases with this information, such as GenBank, operated by the National Centre for Biotechnology Information (NCBI) store such information in a retrievable format, and are publicly accessible. A
person of skill in the art will appreciate the various methods and tools that may be used to access such information, in a context suitable to their particular application of aspects described herein.
Polymorphic sites in SEQ ID NO:1-5 are identified by their variant designation (i.e. M, W, Y, S, R, K, V, B, D, H or by "¨" for a deletion, a "+"or for example "G" etc.
for an insertion).
An "rs" prefix designates a SNP in the database is found at the National Center for Biotechnology Information (NCBI) SNP database. The "rs" numbers are the NCBI
rsSNP
ID form.
The Sequences given in TABLE 2 (SEQ ID NO:1-5) above may be useful to a person of skill in the art in the design of primers and probes or other oligonudeotides or PNAs for the identification of polymorphisms as described herein.
An "allele" is defined as any one or more alternative forms of a given gene.
In a diploid cell or organism the members of an allelic pair (i.e. the two alleles of a given gene) occupy corresponding positions (loci) on a pair of homologous chromosomes and if these alleles are genetically identical the cell or organism is said to be "homozygous", but if genetically different the cell or organism is said to be "heterozygous" with respect to the particular gene.
A "gene" is an ordered sequence of nucleotides located in a particular position on a particular chromosome that encodes a specific functional product and may include untranslated and untranscribed sequences in proximity to the coding regions (5' and 3' to the coding sequence). Such non-coding sequences may contain regulatory sequences needed for transcription and translation of the sequence or introns etc. or may as yet to have any function attributed to them beyond the occurrence of the SNP of interest.
A "genotype" is defined as the genetic constitution of an organism, usually in respect to one gene or a few genes or a region of a gene relevant to a particular context (i.e. the genetic loci responsible for a particular phenotype).
A "phenotype" is defined as the observable characters of an organism. In gene association studies, the genetic model at a given locus can change depending on the selection pressures (i.e., the environment), the population studied, or the outcome variable (i.e., the phenotype).
A similar observation would be seen in a gene association study with the hemoblobin, beta gene (HBB) with mortality as the primary outcome variable. A mutation in the HBB gene, which normally produces the beta chain subunit of hemoglobin (B allele), results in an abnormal beta chain called hemoglobin S (S allele; Allison A (1955) Cold Spring Harbor Symp. Quant. Biol. 20:239-255). Hemoglobin S results in abnormal sickle-shaped red blood cells which lead to anemia and other serious complications including death. In the absence of malaria, a gene association study with the HBB gene would suggest a codominant model (survival(BB) > survival (BS) > survival (SS)). However, in the presence of marlaria, a gene association study with the HBB gene would suggest a heterozygote advantage model (survival(BB) < survival(BS) > survival(SS)).
A "single nucleotide polymorphism" (SNP) occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences.
The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations).
A single nucleotide polymorphism usually arises due to substitution of one nucleotide for another at the polymorphic site. A "transition" is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. A "transversion" is the replacement of a purine by a pyrimidine or vice versa. Single nucleotide polymorphisms can also arise from a deletion (represented by "-" or "del") of a nucleotide or an insertion (represented by "+" or "ins" or "I") of a nucleotide relative to a reference allele. Furthermore, a person of skill in the art would appreciate that an insertion or deletion within a given sequence could alter the relative position and therefore the position number of another polymorphism within the sequence. Furthermore, although an insertion or deletion may by some definitions not qualify as a SNP as it may involve the deletion of or insertion of more than a single nucleotide at a given position, as used herein such polymorphisms are also called SNPs as they generally result from an insertion or deletion at a single site within a given sequence.
A "subject", as used herein, refers to a patient or test subject, for example a human patient.
The subject may have been previously diagnosed with a neoplastic disorder, or may be suspected of having a neoplastic disorder and thus may be a candidate for a chemotherapeutic regimen. The subject may be selected as part of a general population (for example a 'control' subject), or may be selected as part of a particular ethnic, gender, age or genetic subgroup of a population, or may be excluded from selection as part of a particular ethnic, gender, age or genetic subgroup of a population. Patients and test subjects, whether control or not, may be generally referred to as a subject.
As used herein, the terms "cancer" or "neoplastic condition" or "neoplastic disorder" or "neoplastic disease" refer to a proliferative disorder caused or characterized by the proliferation of cells which have lost susceptibility to normal growth control. A "cancer" or neoplastic condition" or "neoplastic disorder" or "neoplastic disease" may include tumors and any other proliferative disorders. Cancers of the same tissue type usually originate in the same tissue, and may be divided into different subtypes based on their biological characteristics. Four general categories of cancers are carcinoma (epithelial tissue derived), sarcoma (connective tissue or mesodermal derived), leukemia (blood-forming tissue derived) and lymphoma (lymph tissue derived). Over 200 different types of cancers are known, and every organ and tissue of the body may be affected. Specific examples of cancers that do not limit the definition of cancer may include melanoma, leukemia, astrocytoma, glioblastoma, retinoblastoma, lymphoma, glioma, Hodgkins' lymphoma and chronic lymphocyte leukemia. Examples of organs and tissues that may be affected by various cancers include pancreas, breast, thyroid, ovary, uterus, testis, prostate, thyroid, pituitary gland, adrenal gland, kidney, stomach, esophagus or rectum, head and neck, bone, nervous system, skin, blood, nasopharyngeal tissue, lung, urinary tract, cervix, vagina, exocrine glands and endocrine glands. Alternatively, a cancer may be multicentric or of unknown primary site (CUPS).
As used herein, a "therapeutic regimen" refers to a chemotherapeutic regimen or a radiotherapy regimen, or a combination thereof.
As used herein, a "chemotherapeutic regimen" or "chemotherapy" refers to the use of at least one chemotherapy agent to destroy cancerous cells. There are a myriad of such chemotherapy agents available for treating cancer. Chemotherapy agents may be administered to a subject in a single bolus dose, or may be administered in smaller doses over time. A single chemotherapeutic agent may be used (single-agent therapy) or more than one agent may be used in combination (combination therapy). Chemotherapy may be used alone to treat some types of cancer. Alternatively, chemotherapy may be used in combination with other types of treatment, for example, radiotherapy or alternative therapies (for example immunotherapy) as described herein. Additionally, a chemosensitizer may be administered as a combination therapy with a chemotherapy agent.
As used herein, a "chemotherapeutic agent" or "chemotherapeutic agent" refers to a medicament that may be used to treat cancer, and generally has the ability to kill cancerous cells directly. Examples of chemotherapeutic agents include alkylating agents, antimetabolites, natural products, hormones and antagonists, and miscellaneous agents.
Examples of alternate names are indicated in brackets. Examples of alkylating agents include nitrogen mustards such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine and thiotepa; alkyl sulfonates such as busulfan;
nitrosoureas such as carmustine (BCNU), semustine (methyl-CCNU), lomustine (CCNU) and streptozocin (streptozotocin); DNA synthesis antagonists such as estramustine phosphate;
and triazines such as dacarbazine (DTIC, dimethyl-triazenoimidazolecarboxamide) and temozolomide .
Examples of antimetabolites include folic acid analogs such as methotrexate (amethopterin); pyrimidine analogs such as fluorouracin (5-fluorouracil, 5-FU, 5FU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside) and gemcitabine; purine analogs such as mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG) and pentostatin (2'-deoxycoformycin, deoxycoformycin), cladribine and fludarabine; and topoisomerase inhibitors such as amsacrine.
Examples of natural products include vinca alkaloids such as vinblastine (VLB) and vincristine; taxanes such as paclitaxel and docetaxel (Taxotere); epipodophyllotoxins such as etoposide and teniposide; camptothecins such as topotecan or irinotecan; antibiotics such as dactinomycin (actinomycin D), bleomycin, mitomycin (mitomycin C);
anthracycline antibiotics such as daunorubicin (daunomycin, rubidomycin), doxorubicin, idarubicin, epirubicin; enzymes such as L-asparaginase; and biological response modifiers such as interferon alpha and interleukin 2. Examples of hormones and antagonists include luteinising releasing hormone agonists such as buserelin;
adrenocorticosteroids such as prednisone and related preparations; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol and related preparations; estrogen antagonists such as tamoxifen and anastrozole; androgens such as testosterone propionate and fluoxymesterone and related preparations; androgen antagonists such as flutamide and bicalutamide; and gonadotropin-releasing hormone analogs such as leuprolide. Examples of miscellaneous agents include thalidomide; platinum coordination complexes such as cisplatin (cis-DDP), carboplatin, oxaliplatin, tetraplatin, ormiplatin, iproplatin or satraplatin;
anthracenediones such as mitoxantrone; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH); adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; RXR agonists such as bexarotene; or tyrosine kinase inhibitors such as imatinib. Alternate names and trade-names of these and additional examples of chemotherapeutic agents, and their methods of use including dosing and administration regimens, will be known to an individual versed in the art, and may be found in, for example "The Pharmacological basis of therapeutics", loth edition.
HARDMAN HG., LIMBIRD LE. editors. McGraw-Hill, New York, or in "Clinical Oncology", 3rd edition. Churchill Livingstone/ Elsevier Press, 2004. ABELOFF, MD. editor.
2. General Methods Once a subject is identified as a candidate for anthracycline administration, then genetic sequence information may be obtained from the subject to determine the risk of cardiotoxicity for the subject. Genetic sequence information may be obtained from a subject by any of several methods. For example, a biological sample comprising genetic material with a sequence or sequences of interest, may be obtained from the subject, for example a blood sample, a saliva sample, a hair sample including a follicle, skin scraping, such as a cheek scraping and the like. Or alternatively genetic sequence information may already have been obtained from the subject. For example, a subject may have already provided a biological sample for other purposes or may have even had their genetic sequence determined in whole or in part and stored for future use. Genetic sequence information may be obtained in numerous different ways and may involve the collection of a biological sample that contains genetic material, particularly, genetic material containing the sequence or sequences of interest. Many methods are known in the art for collecting biological samples and extracting genetic material from those samples. Genetic material can be extracted from blood, tissue, hair and other biological material. There are many methods known to isolate DNA and RNA from biological material. Typically, DNA
may be isolated from a biological sample when first the sample is lysed and then the DNA is separated from the lysate according to any one of a variety of multi-step protocols, which can take varying lengths of time. DNA isolation methods may involve the use of phenol (Sambrook, J. et al., "Molecular Cloning", Vol. 2, pp. 9.14-9.23, Cold Spring Harbor Laboratory Press (1989) and Ausubel, Frederick M. et al., "Current Protocols in Molecular Biology", Vol. 1, pp. 2.2.1-2.4.5, John Wiley & Sons, Inc. (1994)). Typically, a biological sample is lysed in a detergent solution and the protein component of the lysate is digested with proteinase for 12-18 hours. Next, the lysate is extracted with phenol to remove most of the cellular components, and the remaining aqueous phase is processed further to isolate DNA. In another method, described in Van Ness et al. (U.S. Pat. # 5,130,423), non-corrosive phenol derivatives are used for the isolation of nucleic acids. The resulting preparation is a mix of RNA and DNA.
Other methods for DNA isolation utilize non-corrosive chaotropic agents. These methods, which are based on the use of guanidine salts, urea and sodium iodide, involve lysis of a biological sample in a chaotropic aqueous solution and subsequent precipitation of the crude DNA fraction with a lower alcohol. The resulting nucleic acid sample may be used `as-is' in further analyses or may be purified further. Additional purification of the precipitated, crude DNA fraction may be achieved by any one of several methods, including, for example, column chromatography (Analects, (1994) Vol 22, No. 4, Pharmacia Biotech), or exposure of the crude DNA to a polyanion-containing protein as described in Koller (U.S. Pat. # 5,128,247).
Yet another method of DNA isolation, which is described by Botwell, D. D. L.
(Anal.
Biochem. (1987) 162:463-465) involves lysing cells in 6M guanidine hydrochloride, precipitating DNA from the lysate at acid pH by adding 2.5 volumes of ethanol, and washing the DNA with ethanol.
Numerous other methods are known in the art to isolate both RNA and DNA, such as the one described by CHOMCZYNSKI (U.S. Pat. # 5,945,515), whereby genetic material can be extracted efficiently in as little as twenty minutes. EVANS and HUGH (U.S.
Pat. #
5,989,431) describe methods for isolating DNA using a hollow membrane filter.

The level of expression of specific nucleic acids such as mRNAs or microRNAs, copy number of a gene, or the degree of heterozygosity for a polymorphism may also be determined once the nucleic acid sample has been obtained. Quantitative and semi-quantitative methods are known in the art, and may be found in, for example AUSUBEL, supra; SAMBROOK, supra or Harrison's Principles of Internal Medicine 15th ed.
BRAUNWALD et al. eds. McGraw-Hill.
Once a subject's genetic material has been obtained from the subject it may then be further be amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR), Polymerase Chain Reaction (PCR), Transcription Mediated Amplification (TMA), Ligase chain reaction (LCR), Nucleic Acid Sequence Based Amplification (NASBA) or other methods known in the art, and then further analyzed to detect or determine the presence or absence of one or more polymorphisms or mutations in the sequence of interest, provided that the genetic material obtained contains the sequence of interest. Particularly, a person may be interested in determining the presence or absence of a polymorphism in a cardiotoxicity associated gene sequence, as described herein.
Detection or determination of a nucleotide identity, or the presence of one or more single nucleotide polymorphism(s) (SNP typing), may be accomplished by any one of a number methods or assays known in the art. Many DNA typing methodologies are useful for use in the detection of SNPs. The majority of SNP genotyping reactions or assays can be assigned to one of four broad groups (sequence-specific hybridization, primer extension, oligonucleotide ligation and invasive cleavage). Furthermore, there are numerous methods for analyzing/detecting the products of each type of reaction (for example, fluorescence, luminescence, mass measurement, electrophoresis, etc.). Furthermore, reactions can occur in solution or on a solid support such as a glass slide, a chip, a bead, etc.
In general, sequence-specific hybridization involves a hybridization probe, which is capable of distinguishing between two DNA targets differing at one nucleotide position by hybridization. Usually probes are designed with the polymorphic base in a central position in the probe sequence, whereby under optimized assay conditions only the perfectly matched probe target hybrids are stable and hybrids with a one base mismatch are unstable. A strategy which couples detection and sequence discrimination is the use of a molecular beacon", whereby the hybridization probe (molecular beacon) has 3' and 5' reporter and quencher molecules and 3' and 5' sequences which are complementary such that absent an adequate binding target for the intervening sequence the probe will form a hairpin loop. The hairpin loop keeps the reporter and quencher in close proximity resulting in quenching of the fluorophor (reporter) which reduces fluorescence emissions.
However, when the molecular beacon hybridizes to the target the fluorophor and the quencher are sufficiently separated to allow fluorescence to be emitted from the fluorophor.
Similarly, primer extension reactions (i.e. mini sequencing, nucleotide-specific extensions, or simple PCR amplification) are useful in sequence discrimination reactions.
For example, in mini sequencing a primer anneals to its target DNA immediately upstream of the SNP
and is extended with a single nucleotide complementary to the polymorphic site. Where the nucleotide is not complementary, no extension occurs.
Oligonucleotide ligation assays require two sequence-specific probes and one common ligation probe per SNP. The common ligation probe hybridizes adjacent to a sequence-specific probe and when there is a perfect match of the appropriate sequence-specific probe, the ligase joins both the sequence-specific and the common probes.
Where there is - not a perfect match the ligase is unable to join the sequence-specific and common probes.
Probes used in hybridization can include double-stranded DNA, single-stranded DNA and RNA oligonucleotides, and peptide nucleic acids. Hybridization methods for the identification of single nucleotide polymorphisms or other mutations involving a few nucleotides are described in the U.S. Pat. 6,270,961; 6,025,136; and 6,872,530. Suitable hybridization probes for use in accordance with the invention include oligonucleotides and PNAs from about 10 to about 400 nucleotides, alternatively from about 20 to about 200 nucleotides, or from about 30 to about loo nucleotides in length.
A unimolecular segment amplification method for amplifying nucleic acids is described in US patent 5854033. A rolling circle replication reporter system may be used for identification of polymorphisms or mutations.
An invasive cleavage method employs an "InvaderTM" (Applied Biosystems) probe and sequence-specific probes to hybridize with the target nucleic acid, usually DNA, with an overlap of one nucleotide. When the sequence specific probe is an exact match to the site of polymorphism, the overlapping probes form a structure that is specifically cleaved by a FLAP endonuclease, Release of the 5' end of the allele-specific probe may be detected by known methods as described. See for example, Lu, M., et al. J. Am. Chem. Soc.
2001, 124, 7924 ¨ 7931; Lyamichev, etal. 1999. Nature Biotech. 17, 292 ¨ 296; Landegren et al. 1998.
Genome Research, 8, 769 ¨ 776; Brookes, 1999. Gene 234, 177 ¨ 186; Chen, et al. 2004. J.
Am. Chem. Soc. 126, 3016-3017; Wang, D.G., etal. Science 1998, 280, 1077 ¨
1082. The TaqMan' assay (Applied Biosystems) exploits the 5' exonuclease activity of the Taq polymerase to displace and cleave an oligonucleotide probe hybridized to the target nucleic acid, usually DNA, generating a fluorescent signal. See, for example U.S.
Patents 4,683,202, 4,683,195, and 4,965,188.
5' exonuclease activity or TaqManTm assay (Applied BiosystemsTM) is based on the 5' nuclease activity of Taq polymerase that displaces and cleaves the oligonucleotide probes hybridized to the target DNA generating a fluorescent signal. It is necessary to have two probes that differ at the polymorphic site wherein one probe is complementary to the 'normal' sequence and the other to the mutation of interest. These probes have different fluorescent dyes attached to the 5' end and a quencher attached to the 3' end when the probes are intact the quencher interacts with the fluorophor by fluorescence resonance energy transfer (FRET) to quench the fluorescence of the probe. During the PCR
annealing step the hybridization probes hybridize to target DNA. In the extension step the 5' fluorescent dye is cleaved by the 5' nuclease activity of Taq polymerase, leading to an increase in fluorescence of the reporter dye. Mismatched probes are displaced without fragmentation. The presence of a mutation in a sample is determined by measuring the signal intensity of the two different dyes.
The Illumina Golden GateTM Assay uses a combined oligonucleotide ligation assay/ allele-specific hybridization approach (SHEN R etal. Mutat Res 2005573:70-82). The first series of steps involve the hybridization of three oligonucleotides to a set of specific target SNPs;
two of these are fluorescently-labelled allele-specific oligonucleotides (AS0s) and the third a locus-specific oligonucleotide (LSO) binding 1-20 bp downstream of the ASOs.
A second series of steps involve the use of a stringent polymerase with high 3' specificity that extends only oligonucleotides specifically matching an allele at a target SNP. The polymerase extends until it reaches the LSO. Locus-specificity is ensured by requiring the hybridization of both the ASO and LSO in order that extension can proceed.
After PCR
amplification with universal primers, these allele-specific oligonucleotide extension products are hybridized to an array which has multiple discretely tagged addresses (in this case 1536 addresses) which match an address embedded in each LSO. Fluorescent signals produced by each hybridization product are detected by a bead array reader from which genotypes at each SNP locus may be ascertained.
It will be appreciated that numerous other methods for sequence discrimination and detection are known in the art and some of which are described in further detail below. It will also be appreciated that reactions such as arrayed primer extension mini sequencing, tag microarrays and sequence-specific extension could be performed on a microarray. One such array based genotyping platform is the microsphere based tag-it high throughput genotyping array (BORTOLIN S. et al. Clinical Chemistry (2004) 50(11): 2028-36). This method amplifies genomic DNA by PCR followed by sequence-specific primer extension with universally tagged genotyping primers. The products are then sorted on a Tag-It array and detected using the Luminex xMAPTm system.
Mutation detection methods may include but are not limited to the following:
Restriction Fragment Length Polymorphism (RFLP) strategy ¨ An RFLP gel-based analysis can be used to indicate the presence or absence of a specific mutation at polymorphic sites within a gene. Briefly, a short segment of DNA (typically several hundred base pairs) is amplified by PCR. Where possible, a specific restriction endonuclease is chosen that cuts the short DNA segment when one polymorphism is present but does not cut the short DNA
segment when the polymorphism is not present, or vice versa. After incubation of the PCR
amplified DNA with this restriction endonuclease, the reaction products are then separated using gel electrophoresis. Thus, when the gel is examined the appearance of two lower molecular weight bands (lower molecular weight molecules travel farther down the gel during electrophoresis) indicates that the DNA sample had a polymorphism was present that permitted cleavage by the specific restriction endonuclease. In contrast, if only one higher molecular weight band is observed (at the molecular weight of the PCR
product) then the initial DNA sample had the polymorphism that could not be cleaved by the chosen restriction endonuclease. Finally, if both the higher molecular weight band and the two lower molecular weight bands are visible then the DNA sample contained both polymorphisms, and therefore the DNA sample, and by extension the subject providing the DNA sample, was heterozygous for this polymorphism;
For example the Maxam-Gilbert technique for sequencing (MAXAM AM. and GILBERT
W.
Proc. Natl. Acad. Sci. USA (1977) 74(4):56o-564) involves the specific chemical cleavage of terminally labelled DNA. In this technique four samples of the same labeled DNA are each subjected to a different chemical reaction to effect preferential cleavage of the DNA
molecule at one or two nucleotides of a specific base identity. The conditions are adjusted to obtain only partial cleavage, DNA fragments are thus generated in each sample whose lengths are dependent upon the position within the DNA base sequence of the nucleotide(s) which are subject to such cleavage. After partial cleavage is performed, each sample contains DNA fragments of different lengths, each of which ends with the same one or two of the four nucleotides. In particular, in one sample each fragment ends with a C, in another sample each fragment ends with a C or a T, in a third sample each ends with a G, and in a fourth sample each ends with an A or a G. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA
sequence can be read from the pattern of radioactive bands. This technique permits the sequencing of at least 100 bases from the point of labeling. Another method is the dideoxy method of sequencing was published by SANGER et al. (Proc. Natl. Acad. Sci.
USA (1977) 74(12):5463-5467). The Sanger method relies on enzymatic activity of a DNA
polymerase to synthesize sequence-dependent fragments of various lengths. The lengths of the fragments are determined by the random incorporation of dideoxynucleotide base-specific terminators. These fragments can then be separated in a gel as in the Maxam-Gilbert procedure, visualized, and the sequence determined. Numerous improvements have been made to refine the above methods and to automate the sequencing procedures.
Similarly, RNA sequencing methods are also known. For example, reverse transcriptase with dideoxynucleotides have been used to sequence encephalomyocarditis virus RNA
(ZIMMERN D. and KAESBERG P. Proc. Natl. Acad. Sci. USA (1978) 75(9):4257-4261).
MILLS DR. and KRAMER FR. (Proc. Natl. Acad. Sci. USA (1979) 76(5):2232-2235) describe the use of QI3 replicase and the nucleotide analog inosine for sequencing RNA in a chain-termination mechanism. Direct chemical methods for sequencing RNA are also known (PEATTIE DA. Proc. Natl. Acad. Sci. USA (1979) 76(4):1760-1764). Other methods include those of Donis-Keller etal. (1977, Nucl. Acids Res. 4:2527-2538), SIMONCSITS A.
etal. (Nature (1977) 269(5631):833-836), AXELROD VD. etal. (Nucl. Acids Res.(1978) 5(10):3549-3563), and KRAMER FR. and MILLS DR. (Proc. Natl. Acad. Sci. USA
(1978) 75(11):5334-5338). Nucleic acid sequences can also be read by stimulating the natural fluoresce of a cleaved nucleotide with a laser while the single nucleotide is contained in a fluorescence enhancing matrix (U.S. Pat. # 5,674,743); In a mini sequencing reaction, a primer that anneals to target DNA adjacent to a SNP is extended by DNA
polymerase with a single nucleotide that is complementary to the polymorphic site. This method is based on the high accuracy of nucleotide incorporation by DNA polymerases. There are different technologies for analyzing the primer extension products. For example, the use of labeled or unlabeled nucleotides, ddNTP combined with dNTP or only ddNTP in the mini sequencing reaction depends on the method chosen for detecting the products.
Probes used in hybridization can include double-stranded DNA, single-stranded DNA and RNA oligonucleotides, and peptide nucleic acids. Hybridization methods for the identification of single nucleotide polymorphisms or other mutations involving a few nucleotides are described in the U.S. Pat. 6,270,961; 6,025,136; and 6,872,530. Suitable hybridization probes for use in accordance with the invention include oligonucleotides and PNAs from about io to about 400 nucleotides, alternatively from about 20 to about 200 nucleotides, or from about 30 to about too nucleotides in length.

A template-directed dye-terminator incorporation with fluorescent polarization-detection (TDI-FP) method is described by FREEMAN BD. et al. (J Mol Diagnostics (2002) 4(4):209-215) for large scale screening.
Oligonucleotide ligation assay (OLA) is based on ligation of probe and detector oligonucleotides annealed to a polymerase chain reaction amplicon strand with detection by an enzyme immunoassay (VILLAHERMOSA ML. J Hum Virol (2001) 4(5):238-48;
ROMPPANEN EL. Scand J Clin Lab Invest (2001) 61(2):123-9; IANNONE MA. etal.
Cytometry (2000) 39(2):131-40).
Ligation-Rolling Circle Amplification (L-RCA) has also been successfully used for genotyping single nucleotide polymorphisms as described in QI X. et al.
Nucleic Acids Res (2001) 29(22):E116.
5' nuclease assay has also been successfully used for genotyping single nucleotide polymorphisms (AYDIN A. etal. Biotechniques (2001) (4):920-2, 924, 926-8.).
Polymerase proofreading methods are used to determine SNPs identities, as described in WO 0181631.
Detection of single base pair DNA mutations by enzyme-amplified electronic transduction is described in PATOLSKY F et al. Nat Biotech. (2001) 19(3):253-257.
Gene chip or microarray technologies are also known for single nucleotide polymorphism discrimination whereby numerous polymorphisms may be tested for simultaneously on a single array (for example: EP 1120646; and GILLES PN. et al. Nat.
Biotechnology (1999) 17(4):365-7o).
Matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy is also useful in the genotyping single nucleotide polymorphisms through the analysis of microsequencing products (HAFF LA. and SMIRNOV IP. Nucleic Acids Res. (1997) 25(18):3749-50; HAFF LA. and SMIRNOV IP. Genome Res. (1997) 7:378-388; SUN X.
et al. Nucleic Acids Res. (2000) 28 e68; BRAUN A. etal. Clin. Chem. (1997) 43:1151-1158;
LITTLE DP. etal. Eur. J. Clin. Chem. Clin. Bioehem. (1997) 35:545-548; FEI Z.
etal.
Nucleic Acids Res. (2000) 26:2827-2828; and BLONDAL T. et al. Nucleic Acids Res.
(2003) 31(24):e155).

Sequence-specific PCR methods have also been successfully used for genotyping single nucleotide polymorphisms (HAWKINS JR. et a/. Hum Mutat (2002) 19(5):543-553).
Alternatively, a Single-Stranded Conformational Polymorphism (SSCP) assay or a Cleavase Fragment Length Polymorphism (CFLP) assay may be used to detect mutations as described herein.
US 7,074,597 describes methods for multiplex genotyping using solid phase capturable dideoxynucleotides and mass spectrometry. Nucleotide identity is detected at a specific site of a nucleic acid sample by contacting DNA-primer complex with labeled dideoxynucleotides (ddNTPs) to generate labeled single base extended (SBE) primer. The identifying ddNTP may be within the SBE primer.
Multiplex analysis of PCR-amplified products may also be used to detect specific SNPs.
Reporting DNA sequences comprising a fluorophore on a 5' end may be used to combine a multiplex PCR amplification reaction with microsphere based hybridization (US
7,083,951). Other multiplex detection methods include BeadArrayTM and similar hybridization-based methods, for example, those described in US Patent Nos.
6,429,027, 6,396,995, 6,355,431.
Microarray or 'gene chips' of oligonucleotides may be used for SNP
discrimination.
Oligonucleotides may be nucleic acids or modified nucleic acids, including PNAs, and may be 'spotted' onto a solid matrix, such as a glass or plastic slide.
Alternatively, oligonucleotides may be synthesized in situ on the slide. See, for example, GAO et al. 2004.
Biopolymers 73:579-596; US 5,445,934; US 5,744,305, US 5,800,992, US
5,796,715.
Alternatively, if a subject's sequence data is already known, then obtaining may involve retrieval of the subjects nucleic acid sequence data (for example from a database), followed by determining or detecting the identity of a nucleic acid or genotype at a polymorphic site by reading the subject's nucleic acid sequence at the one or more polymorphic sites.
Once the identity of a polymorphism(s) is determined or detected an indication may be obtained as to the subject's risk of cardiotoxicity following anthracycline administration.
Methods for predicting a subject's risk of cardiotoxicity following anthracycline administration may be useful in making decisions regarding the administration of anthracycline(s).

TREATMENT
Anthracycline compounds (for example, doxorubicin) may be used to treat a variety of cancers in children and adults. In a given therapeutic regimen, the anthracycline compound may be administered alone or in combination with other chemotherapeutic agents in various doses and compositions, depending on the type of cancer, age of subject, health of subject, body mass, etc. The choice of dose, chemotherapeutic agents or combinations, methods of administration and the like will be known to those skilled in the art. Further, methods of assessing response to treatment and side effects are also known.
For example, heart function in a subject suspected of experiencing cardiotoxicity may be assessed by various methods including medical history, electrocardiogram (ECG) monitoring, endomyocardial biopsy, radionuclide angiography (MUGA scan) or LVEF
monitoring with serial echo or exercise stress testing, or other methods that may be dependent on the age and condition of the subject, as are known in the art.
Early signs of cardiotoxicity may include persistent reduction in the voltage of the QRS
wave, prolongation of the systolic time interval, or reduction of LVEF as determined by echo or MUGA. A reduction of 10% to below the lower limit of normal, 20% at any level, or an absolute LVEF 45% indicates deterioration of cardiac function.
Response to a therapeutic regimen may be monitored. Tumor staging provides a method to assess the size and spread of a tumor in response to a treatment regimen. The TNM tumor staging system uses three components to express the anatomic extent of disease: T is a measure of the local extent of tumor spread (size), N indicates the presence or absence of metastatic spread to regional lymph nodes, and M specifies the presence or absence of metastatic spread to distant sites. The combination of these classifications combine to provide a stage grouping. Clinical TNM (cTNM) defines the tumor based on clinical evidence. Pathologic TNM (pTNM) defines the tumor based on examination of a surgically resected specimen.
Changes in tumor size may be observed by various imaging methods known to physicians or surgeons in the field of oncology therapy and diagnostics. Examples of imaging methods include positron emission tomography (PET) scanning, computed tomography (CT) scanning, PET/CT scanning, magnetic resonance imaging (MRI), chemical shift imaging, radiography, bone-scan, mammography, fiberoptic colonoscopy or ultrasound.
Contrast agents, tracers and other specialized techniques may also be employed to image specific types of cancers, or for particular organs or tissues, and will be known to those skilled in the art. Changes in rate of metastasis may also be observed by the various imaging methods, considering particularly the appearance, or frequency of appearance, of tumors distal to the primary site. Alternatively, the presence of tumor cells in lymph nodes adjacent and distal to the primary tumor site may also be detected and used to monitor metastasis.
A subject may be tested for a cardiotoxicity-associated polymorphism before undergoing a therapeutic regimen involving an anthracycline compound. If a subject's genotype includes a cardiotoxicity-associated polymorphism, this may indicate that the subject is at a risk for cardiotoxicity when an anthracycline compound is administered.
A subject at risk for cardiotoxicity may be administered a therapeutic regimen involving an anthracycline compound and the cardiac function monitored as described. If a decrease in cardiac function is identified, the therapeutic regimen may be altered to decrease the dose of the anthracycline compound, eliminate the dose of the anthracycline compound, or increase the dose of a second chemotherapeutic agent in the therapeutic regimen.
Examples of chemotherapeutic agents that may be used in combination with an anthracycline compound in a therapeutic regimen may include, for example, cyclophosphamide, Ifosphamide, fluorouracil, Paclitaxel, vincristine, cisplatin, streptozocin, docetaxel, and the like.
A subject at risk for cardiotoxicity may also be administered a therapeutic regimen involving an anthracycline compound and the cardiac function monitored as described.
The therapeutic regimen may be supplemented to include a cardioprotective agent.
Examples of cardioprotective agents are known in the art, and may include those described by Wouters et al. 2005. Br. J Hematol 131:561-578). For example, Dexrazoxane is a cardioprotective agent and is approved for use in conjunction with doxorubicin to reduce the incidence and severity of cardiomyopathy associated with doxorubicin administration.
Alternatively, a subject at risk for cardiotoxicity may be administered a therapeutic regimen that does not involve an anthracycline compound and the cardiac function monitored as described.
Alternatively, a subject at risk for cardiotoxicity may be administered a cardioprotective agent in conjunction with a therapeutic regimen.

GENE
Detailed information relating to the sequence, expression patterns, molecular biology, etc of these and related genes in both Homo sapiens and in other model species is known, and may be found at, for example Entrez Gene (NCBI ) and references therein.
Retinoic acid receptor, gamma (RARG) [Homo sapiensRalternative designations RARC;
NR1B3) maps to chromosome 22q13. A representative human RARG sequence may be found in GenBank under accession number NT o29419.11 BG740799. RARG encodes a retinoic acid receptor that belongs to the nuclear hormone receptor family.
Retinoic acid receptors (RARs) act as ligand-dependent transcriptional regulators and when bound to ligands, RARs activate transcription by binding as heterodimers to the retinoic acid response elements (RARE) found in the promoter regions of the target genes.
Unbound RARs repress transcription of their target genes. RARs are involved in various biological processes, including limb bud development, skeletal growth, and matrix homeostasis.
METHODS
Study Design In stage 1, a total of 280 European Canadian patients (32 cases and 248 controls) were used as the discovery cohort and genotyped on the Illumina Infinium Human Omni ExpressTM panel (740K), to perform a GWAS for ACT (stage 1). Markers that reached P <
i.oxio-5 (ref. (Welter, MacArthur et al. 2014)) in the discovery cohort were tested for replication in 96 European Dutch patients (22 cases and 74 controls) in stage 2. RARG
rs2229774 was further tested for association with ACT in non-European populations in stage 3. We also performed a combined analysis for all European patients (stage 1 and stage 2) and an overall association test for rs2229774 using all populations (stages 1-3). In addition, we performed genetic fine mapping analyses of the associated region and functionally characterized n2229774 in vitro.
Genetic ancestry was self-reported and ascertained by principal component analysis (PCA) using the EIGENSTRAT method, with the GWAS Illumina 740K (Canadian patients), Illumina 4.5K (Dutch patients) and Illumina 8K (USA patients) SNP genotype data sets.
Detailed Analysis of Clinical Data This study was approved by the individual ethics committees/institutional review boards of the universities and institutions where patients were enrolled. Written informed consent/assent was obtained from patients/parents/legal guardians in accordance with the Helsinki Declaration as revised in 2008.

Patients' medical records were reviewed prior to genotyping by a clinical pharmacologist, a cardiologist, an oncologist and an ADR surveillance clinician, who reviewed the echocardiogram test results and other clinical information. The demographic, clinical and therapeutic information was extracted from these medical records and included the following data: demographics, disease characteristics, chemotherapy, diagnostic echocardiograms to document baseline and follow-up cardiac function and any cardiac compromise and its severity and any symptoms and/or signs consistent with ACT.
All patients were children 18 years at cancer diagnosis, who received anthracyclines as part of their chemotherapy protocol, had normal cardiac function before anthracycline chemotherapy and were recruited from outpatient clinics and inpatient units.
ACT was monitored by echocardiograms (or comparable cardiac imaging) according to the "The Children's Oncology Group Long-Term Follow-Up Guidelines for Survivors of Childhood, Adolescent, and Young Adult Cancers". The recommended frequency of echocardiograms (or comparable cardiac imaging) is every year, every two years or every 5 years post-treatment depending on the age at treatment, radiation with potential impact to the heart and the cumulative anthracycline dose. Grading of ACT based on this detailed clinical characterization was performed using the Cancer Therapy Evaluation Program, Common Terminology Criteria for Adverse Events version 3 (CTCAEv3) as previously described (Visscher, Ross et al. 2012; Visscher, Ross et al. 2013). Patients with serious ACT were defined as those with grade 2 or higher CTCAE impairment of cardiac function after treatment with anthracycline.
ACT was defined as early- or late-onset left ventricular dysfunction assessed by echocardiogram measurements using shortening fraction [SF]) and/or symptoms (dyspnea, orthopnea, and/or fatigue) and/or signs (edema, hepatomegaly, and/or rales) of cardiac compromise requiring intervention based on the CTCAEv3. Due to variability in echocardiographic measurements a conservative threshold of SF 24% was used to define asymptomatic cases who developed ACT. All cases had grade 2 or higher ACT.
Also, grade 2-4 ACT is the point at which anthracycline chemotherapy protocols recommend clinical intervention such as heart failure treatment, halting or reducing anthracycline doses and switching to alternate treatments. Early-onset chronic ACT was defined as developing less than 1 year after start of treatment(Rodvold, Rushing et al. 1988; Kremer, van Dalen et al.
2001) while late-onset chronic ACT was defined as developing more than 1 year after start of treatment (Lipshultz, Colan et al. 1991; Lipshultz, Lipsitz et al. 1995) since ventricular dysfunction, heart failure, and arrhythmias can occur years or even decades after the discontinuation of anthracycline therapy(Lipshultz, Colan et al. 1991;
Lipshultz, Lipsitz et al. 1995). To exclude transient acute ACT, only echocardiograms obtained 21 days after an anthracycline dose were considered. To exclude cardiotoxicity unrelated to anthracycline chemotherapy patients with no baseline echocardiogram were excluded from the study. Controls had no signs or symptoms of cardiac compromise at study participation (grade o). Due to the delayed onset of ACT in some patients, SF 30% with 5 years of follow-up after the end of anthracycline treatment was used to define controls. The cumulative anthracycline exposure was calculated using the doxorubicin isotoxic equivalent (Altman 2004) and cumulative dose stratification into low-to-moderate 250 mg/m2) and at high (> 250 mg/m2) anthracycline exposure was performed as previously described (Mulrooney, Yeazel et al. 2009; Blanco, Sun et al. 2012; Wang, Liu et al.
2014) where appropriate. Radiation therapy included significant radiation exposure to the heart or surrounding tissue. This included mantle and mediastinal radiation, whole lung radiation, whole or upper abdominal radiation, left sided flank radiation and total body irradiation.
Study Populations Canadian Patient Populations: We genotyped 434 pediatric oncology patients treated with anthracyclines that were recruited from 13 pediatric oncology units from across Canada between February 2005 and April 2011 (Visscher, Ross et al. 2012;
Visscher, Ross et al. 2013). A total of 13 samples failed quality control (details of quality control procedure described below). In 421 remaining patients (average call rate = 99.5%), we performed principal component analysis (PCA) with GWAS IlluminaTM 740K SNP genotype data set to determine the population structure and defined four distinct population clusters comprised of European, African, East Asian and Aboriginal Canadian patients. Patients with genetic ancestry that fell outside of these four population clusters were excluded from further analysis. We excluded an additional 27 patients with CTCAE grade 1 toxicity (shortening fraction: 24% < SF < 30%). A total of 97 patients were excluded and 337 patients were available for further analyses: Europeans (280 patients; 32 cases and 248 controls), Africans (n = ii patients; 2 cases and 9 controls), East Asians (n = 31 patients; 8 cases and 23 controls) and Aboriginal Canadians (n = 15 patients; 4 cases and ii controls).
Dutch Patient Population: We recruited 128 pediatric oncology patients treated with anthracyclines from Emma Children's Hospital/Academic Medical Centre in Amsterdam, the Netherlands, between July 2009 and April 2011 (Visscher, Ross et al. 2012;
Visscher, Ross et al. 2013). We performed PCA using the Illumina 4.5K SNP genotype data set generated for these patients (Visscher et al. 2015, Pharmacogenomies in the press) and excluded 14 patients not genetically matching European ancestry. Based on the detailed clinical assessment of the 114 patients, we excluded another 18 patients with CTCAE grade 1 toxicity (shortening fraction: 24% < SF < 30%). A total of 96 patients (22 cases and 74 controls) were then available for further analysis.
USA Patient Population: We recruited 164 pediatric oncology patients treated with anthracyclines from Lucile Packard Children's Hospital at Stanford (Palo Alto, USA) between December 2008 and October 2010. We performed a detailed clinical assessment of these patients and excluded 10 patients who had missing or abnormal baseline echocardiogram readings, 8 patients with CTCAE grade 1 toxicity (shortening fraction: 24%
< SF < 30%), and 102 patients with insufficient cardiac follow-up data (either SF>30% but less than 5 year follow-up (97 patients), or SF<24% but <21 days from end of treatment (5 patients)). Principal component analysis identified four clusters of the remaining patients aligning with their self-reported ancestries of Hispanic (specifically reported as "White Hispanic"), Asian, African-American, and European (specifically reported as "White Non-Hispanic"). The cluster of 23 Hispanic patients (5 cases and 18 controls) was selected for analysis in stage 3.
Molecular Genetic Methods and GWAS Quality Control Procedure (QC) DNA Extraction and Molecular Genotyping Genotyping experiments were conducted at the Canadian Pharmacogenomics Network for Drug Safety (CPNDS) genotyping core facility, Child & Family Research Institute, The University of British Columbia, Vancouver, BC, Canada. All patients provided a biologic specimen for DNA extraction. De-identified genomic DNA was extracted using the QIAampTM DNA purification system (QiagenTm, Toronto, Ontario, Canada) and quantified by Quant-iT PicoGreenTm assay (InvitrogenTM, Eugene, OR, USA), according to the manufacturer's protocols. Whole Genome Amplification (WGA) was performed prior to genotyping for all samples available at a low concentration (< 50 ng/ I) and/or low volume (< 20 D. The laboratory assistants were blinded to the case-control status of the patients genotyped in the study. To ensure the accuracy of all genotyping results, multiple positive and negative controls and replicate samples were included in all genotyping assays and plates. The concordance of genotype calls between replicate genotyped samples was 100%.
The genome-wide association study (GWAS) was performed using the Illumina Infinium HumanOmniExpressTM assay (740K), according to the manufacturer's instructions (IlluminaTm, San Diego, CA, USA). This assay provides high sample throughput and coverage of common variants. Genotypes were called with the Illumina Genome StudioTm software package and the SNPs were clustered using the IlluminaTM 7401( cluster file. All samples in the study population were used to determine cluster boundaries in order to maximize clustering accuracy. A detailed GWAS quality control procedure was performed for all SNPs and samples prior to analysis. The quality control analysis was performed by date of genotyping and by plate to check for systematic errors in the generated data set. No systematic errors were found. The quality control procedure was performed with the Illumina Genome Studio software package.
The Dutch and Hispanic patients were genotyped for the GWAS candidate variants using TaqManTm SNP genotyping assays (Applied BiosystemsTM, Streetsville, ON, Canada), according to the manufacturer's protocols. The top replicated SNP from the Illumina Infinium HumanOmniExpressTM assay was re-genotyped in loo randomly selected patients from the discovery patient population using the TaqmanTm SNP genotyping assay.
The concordance rate of genotype calls between the two assays was 100%.
Importing GWAS raw data and pre-QC Steps The raw data was initially imported and clustered using the IlluminaTM 740K
cluster file.
Sample statistics were subsequently updated. Next, an iterative genotyping/cluster QC
process was performed using the highest-quality samples in our specific dataset (all samples with initial call rates < 99% were temporarily removed). A sequence of QC filters was then applied to both SNPs and samples as described below.
GWAS Genetic Markers (SNPs) Detail Quality Control Procedure The quality control for genotype data was performed in Illumina Genome StudioTM
software package. We used a combination of thresholds for various quality control metrics with visual inspection of cluster plots for markers at the boundaries of the thresholds.
Poorly-clustering markers (call rates < 95%) were filtered out for re-clustering. Y
chromosome and mtDNA SNPs were excluded. Poorly-clustered SNPs were re-clustered and their new cluster positions evaluated using various quality control metrics. Newly defined cluster positions were either left alone, manually edited, or dropped altogether using the following quality control metrics available in Illumina Genome StudioTM.
1. SNP Call Rates: Re-clustered SNPs with low call rates (< 0.95) were manually edited and the ambiguous ones were excluded.
2. Cluster separation: Re-clustered SNPs with poor cluster separation < 0.3, were visually reviewed and SNPs with cluster separation from the threshold where the clusters were no longer separable by eye were excluded.
3. Mean Normalized Intensity: Low intensity re-clustered markers k 0.25) were removed.

4. Heterozygote clusters shifted too close to a homozygote cluster: SNPs with an AB T Mean < 0.2 or o.8 were visually inspected and appropriate cutoff for exclusion of poor quality markers determined based on how close the heterozygote cluster was to the homozygotes. Poor quality re-clustered markers were then excluded based on the pre-determined cutoff.
5. Heterozygous Excess: X chromosome loci were ignored here. First, re-clustered markers with heterozygous excess 0.16 were selected and further re-clustered amongst themselves. Then all SNPs with heterozygous excess < -0.3 and > 0.2 were excluded.
6. False Homozygotes: Using R dev 13.05 and AA or BB T Dev 0.05 respectively, SNPs with multiple minor-allele homozygote clusters in the R dimension and SNPs with multiple clusters on the T axis called together as homozygotes were excluded. Also, markers where all three clusters were so close to each other that they get called as a single heterozygous cluster were excluded.
7. Reproducibility/Replication Errors: All SNPs with 1 or more errors were evaluated and any remaining markers with > 3 replication errors were excluded.
8. Call Frequency: All remaining SNPs with call frequency < o.95 were excluded.
9. Hardy-Weinberg Equilibrium: Deviation of the genotype distributions from Hardy-Weinberg equilibrium (HWE) was tested in control patients. All SNPs with Fisher's exact test for HWE P-value < i.oxio-4 were excluded.

10. Previously excluded samples were included at this point and the sample statistics generated.
We implemented the same quality-control procedures such as call rates and HWE
from the GWAS, in the replication cohorts. Nine SNPs in the replication studies had call rates of >90%, while one SNP had a call rate <90% and was subsequently excluded from further analysis.
GWAS Sample Quality Control Procedures Quality control for DNA samples was per-formed with SVS/HelixTree 8.l.1TM.
Samples were excluded if they had a call rate below 95%, if the reported and genotypically inferred genders did not match, if the ancestry departs from the expected homogenous genetic ancestry (principal component analysis via EigenstratTM (Visscher, Ross et al.
2009)), and if they were related (identity by descent estimation).
GWAS Association Testing Quality Control X and Y chromosomes and mitochondria SNPs were excluded from the association analyses in keeping with recent GWAS quality control practices. A total of 657,694 SNPs from the GWAS were retained after QC. Cluster plots for all GWAS associated SNPs were visually inspected.
Functional Assays Constructs, cells and reagents: A Myc-DDK-tagged mammalian expression clone of human RARG was purchased from OrigeneTM (Rockville, MD). The rs2229774 SNP was introduced into this expression vector using the Quikchange IITm kit according to the manufacturer's specifications (Agilent Technologies, Mississauga, ON) with mutagenic primers. Transfections were performed with X-tremeGENE 9T1 (RocheTM, Laval, PQ) or EffecteneTM (QiagenTM, Toronto, ON) reagents according to the manufacturer's specifications. HEK293T and H9c2(2-1) (ATCC CRL-1446) cells were purchased from ATCC (CedarlaneTm, Burlington, ON) and routinely cultured in DMEM supplemented with 10% FBS, looU/m1 Penicillin, loong/m1 Streptomycin, with additional o.25 g/m1 Amphotericin B for H9C2 cells. RARG transcriptional regulation was assayed using the Cignal RARE ReporterTM luciferase kit (QiagenTM, Toronto, ON). Luminescence assays were developed using the Dual-Glo Luciferase Assay SystemTM according to the manufacturer's specifications (PromegaTM, Madison, WI) and read on a POLARstar OmegaTM plate reader (BMG LabtechTm). For real-time RT-PCR (qPCR) experiments RNA
was purified from H9c2 cells using the Ambion Purelink RNATM mini kit with PurelinkTM
homogenizers and PurelinkTM on-column DNAse digestion according to the manufacturer's specifications (Life TechnologiesTm, Burlington, ON). cDNA was generated using the Invitrogen Superscript IIITM first strand synthesis kit according to the manufacturer's specifications (Life TechnologiesTm, Burlington, ON). Rat Top2b, rat Hprti and human RARG gene expression was measured using validated Applied Biosystems TaqManTm assays Rn01537914_mi, Rno152784o_ml, and Hso1559234 mi, respectively (Life Technologies, Burlington, ON). qPCR was performed on the PikoReal 96 Real-Time PCRTM
system (Thermo ScientificTM) and relative gene expression was calculated by the AACq method using the instrument software. The anti-DDK 4C5 antibody (catalog #
TA5ooll) was purchased from OrigeneTM (Rockville, MD) and the anti-GAPDH antibody (catalog #
MAB374) was purchased from MilliporeTM (Etobicoke, ON).
RarG transcriptional regulation assay: To assess whether general RARG activity was affected by the rs2229774 variant, we used a reporter construct fused to an optimized RARETM element (Cignal Reporter SystemTM, SABiosciencesTm). The proprietary constituents of the Cignal reporter system impart a technical limitation for the use of a robustly transfectable cell line. Accordingly we used HEK293T cells that have been validated and recommended by the manufacturer. HEK293T cells were reverse co-transfected in a 96-well plate with song (per well) of either empty vector (pcDNA3.1.), RARG WT or RARG5427L expression constructs and the Cignal RARETM reporter Luciferase kit constructs according to the manufacturer's specifications. After 20-24 hours of transfection cells were washed with PBS and the medium was replaced with 750 Cignal AssayTM medium (OptiMemTm supplemented with 1 % charcoal-stripped FBS, o.imM
NEAA, imM sodium pyruvate, looU/m1 Penicillin and 1oop.g/m1 Streptomycin) containing ipM ATRA or DMSO control for 6 hours. Firefly and Renilla luminescence was measured as indicated above. Firefly luciferase/Renilla luciferase ratios (L/R) were calculated for each well and converted to a Relative response ratio (RRR) = [(L/R)sample (L/R)negative control]/[(L/R)positive control ¨ (L/R)negative control] to allow sample comparison between experiments. The fold induction of ATRA-treated versus untreated samples was calculated using the corrected RRR values (subtraction of empty vector RRR values) for RARG WT-and RARGs427L-transfected samples.
Relative gene expression studies in rat cardiomyoblasts: 7.5 x 104 H9c2 cells were seeded into each well of a 6-well dish in DMEM supplemented with 10% FBS.
The following day, cells were transfected with wg RARG WT or RARGs427L expression constructs for 7 hours before replacing with fresh medium. Where required 250nM ATRA
was added to cells 24 hours post-transfection. Cells were grown for 48 hours post-transfection then total RNA was immediately purified and used for cDNA
synthesis. qPCR
reactions were conducted in a 100 reaction volume that consisted of sal 2X
TaqmanTm Universal Master MixTM, 0.50 TaqmanTm probe and 411 cDNA with standard cycling conditions according to the manufacturer's specifications. Relative gene expression was calculated using Hprti as a housekeeping gene. To calculate the fold repression of Top2b expression in RarG-transfected H9c2 cells, relative Top2b expression in untransfected cells was divided by expression levels in transfected cells and normalized to the relative expression of the appropriate RARG construct.
Statistical Methods We performed statistical analyses using SVS/HelixTree 8.1.1 (Golden HelixTM, Bozeman, MT, USA), R 3.1.0 for Statistical ComputingTm, SPSS Version 18.0 (IBMTm, Armonk, NY), Quanto, HaploviewTm (Barrett, Fry et al. 2005), LocusZoomTm(PruimTm, Welch et al.
201.0), Epi InfoTM Version 7.1.3, Comprehensive Meta-Analysis, BEAGLE 4TM
(ref.

(Browning and Browning 2007)), GraphPad Prism S.oaTM and PikoReal version 21TM

software packages. All statistical tests were 2-sided. Baseline quantitative and qualitative variables were analyzed with Wilcoxon-Mann-Whitney U test and Fisher exact test, respectively. Genetic associations were tested by logistic regression with an additive model and adjusted for appropriate clinical covariates unless indicated otherwise, e.g. where logistic regression was not possible. Covariates for logistic regression were derived from each study cohort and represented relevant baseline (clinical and demographic) differences between cases and controls. Covariates for the European Canadian patients (stage 1) analysis included age at start of treatment, cumulative anthracycline dose, tumor type (acute lymphoblastic leukemia, Ewing's sarcoma and rhabdomyosarcoma) and cardiac radiation therapy. Cumulative anthracycline dose was included as a covariate for the European Dutch patient (stage 2) analysis. Covariates for the combined European patient (stage i and 2) and overall combined (stages 1-3) analyses included age at start of treatment, cumulative anthracycline dose, tumor type (acute lymphoblastic leukemia, Ewing's sarcoma and rhabdomyosarcoma) and cardiac radiation therapy. All effect sizes (odds ratios) were calculated for the minor allele for each SNP.
We performed genetic fine mapping analysis by genotype imputation of additional SNPs not present on the GWAS genotyping platform using BEAGLE 4TM with LD and haplotype information from CEU woo Genomes population as the reference population. We imputed 1,005,286 additional variants on Chromosome 12 region containing RARG. The quality control metric (BEAGLETM allelic R2) was calculated for all imputed SNPs. We then examined evidence of additional genetic associations with ACT in this region based on SNPs with imputed BEAGLETM allelic R2 0.5, using logistic regression adjusted for stage 1 covariates. LD analyses (r2 and D') of the imputed variants were conducted using the moo Genomes CEU reference population.
To control for Type I error, we implemented screening thresholds based upon multiple testing correction for all genetic association analyses. A threshold of P<1x10-5, indicative of putative genetic associations (Welter, MacArthur et al. 2014) was implemented for the stage i analysis. For the stage 2 replication analysis in European Dutch patients, a Bonferroni-adjusted threshold of P < 0.006 (replication testing of SNPs from 9 LD blocks) was implemented. Further stage 3 replication in independent non-European populations was tested using a threshold of P < 0.05 (1 replicated SNP). Potentially confounding clinical risk factors were identified at P <0.05.

To prevent spurious (obscured, false positive and false negative) genetic associations, all patients within each study population shared the same genetic ancestry, which was self-reported and ascertained by principal component analysis (PCA). Then for each study cohort, we computed the genomic inflation factor (XGc) to verify the presence of intra-ethnic fine-scale population structures or admixtures or inflation of the test statistics due to population stratification(Devlin and Roeder 1999): Stage i European-Canadian patient discovery cohort (based on 657,694 SNPs) ¨ XGc = 1.021; stage 2 European Dutch patient replication cohort (based on 4516 SNPs) ¨ XGc = 1.014; and stage 3 Non-European patient replication cohort (based upon 7798 mutual SNPs available to all patients) ¨
XGc = 0.941.
We performed meta-analyses of all European patients (stages 1-2) and of all study populations (stages 1-3) using SVS/HelixTree 8.l.1TM and calculated the heterogeneity by Cochran's Q statistics to assess the diversity across the different study populations using Comprehensive Meta Analysis software. The Manhattan plot of ¨log10 P values and the quantile-quantile distribution were generated using SVS/HelixTree 8.l.1TM. The regional association plot for the associated genomic region was created using LocusZoomTM.
Linkage disequilibrium plots were created using HaploviewTM and color coded as follows:
white (D' < 1, LOD < 2); blue (D' = 1, LOD < 2); pink shading (D' < 1, LOD 2);
bright red (D' = 1, LOD 2).
Statistical analyses of the functional data provided in FIGURE 2 shows that all data (FIGURE 2a¨c) were normally distributed when assessed by the D'Agostino &
Pearson omnibus normality test. Variation coefficients were estimated as: FIGURE 2a ¨
RARG
WT (23.77%), RARGs4271- (31.28%); FIGURE 2h ¨ untransfected (9.30%), RARG WT
(9.37%), RARG WT + ATRA (7.33%); FIGURE 2C ¨ RARG WT (16.48%), RARG54271-(22.69%). F tests showed the variances between groups significantly differed in FIGURE
2b and 2c.

EXAMPLES
Example 1: RARG m2229774 is associated with Anthracycline-associated cardiotoxicity (ACT) We recruited well-phenotyped patients treated with anthracyclines for childhood cancer (.18 years at treatment) from 13 pediatric oncology centers across Canada.
Clinical information was used to assess cardiac function, define cases and controls, and determine important baseline differences between these groups. Cases were defined as exhibiting shortening fractions [SF] of 5_24% or signs and symptoms of cardiac compromise requiring intervention based on CTCAEv3, while controls had SF 3o% and no symptoms of cardiac compromise for at least 5 years after treatment(Visscher, Ross et al. 2012;
Visscher, Ross et al. 2013). A stage 1 discovery analysis was performed in Canadian patients of European ancestry (280 patients; 32 cases and 248 controls) as shown in TABLE 3.
Compared with controls, cases were significantly older at the start of treatment, had higher cumulative anthracycline exposure (dose), were less likely to have acute lymphoblastic leukemia (ALL), but more likely to have rhabdomyosarcoma or Ewing's sarcoma, and receive radiotherapy to the heart (RT) (P<o.o5). Age, dose and RT are established risk factors for ACT and after verifying that the associations with ALL, Ewing's sarcoma and rhabdomyosarcoma tumor types were not exclusively explained by the cumulative anthracycline dose (remained associated with ACT after accounting for dose), these six clinical factors were included as covariates for logistic regression analyses. Notably, the limited sample size may have precluded our detection of other significant clinical differences between cases and controls.
Patients were genotyped using an Illumina HumanOmniExpressTm (740K SNP) assay with 657,694 SNPs passing quality control assessment, conferring a Bonferroni-adjusted multiple testing correction threshold of P<7.6xio-8. The GWAS discovery analysis was performed using logistic regression adjusted for age, dose, ALL, Ewing's sarcoma, rhabdomyosarcoma, and RT (as shown in TABLE 4). Analysis of the test statistics (XGc =
1.021), suggested that they were not influenced by cryptic population stratification (as shown in TABLE 4). Eighteen variants tagging nine distinct linkage disequilibrium (LD) blocks (r2>o.9 and D'>0.9 in the l000 Genomes CEU reference dataset) with P<i.oxio-5 were prioritized for further analysis in an independent patient population (as shown in TABLE 4).

TABLE 3: shows the baseline characteristics of the patients of European Ancestry (Discovery and Replication Patient Populations).
Stage 1- Discovery Canadian Patient Stage 2 - Replication Dutch Patient Combined European Patient Population Population Population (n = 376 patients) Patient Characteristicsa (n = 280 patients) (n =
96 patients) Cases Controls Cases Controls Cases Controls (n = 32) (n = 248) P (n = 22) (n = 74) P (n = 54) (n = 322) P
Age at the Start of Treatment 9.0 (2.5 - 14) 4 (2 -7.5) 0.004e 7.5 (5 - 12) 11 (6 - 14) 0.14 8.5 (4 - 14) 5 (2 - 10) 0.007 Age in yrs, median (IQ range) Gender, female/male (13/0 female) 17/15 (53.1) 112/136 (45.2) 0.45 10/12 (45.5) 36/38 (48.6) 0.81 27/27 (50) 148/174 (46) 0.66 Cumulative Anthracycline Exposure 260 (177.5 - 175 (140-407.5 (270- 277.5 (180- 281.5 (200- 200 (150 -Dose') in mg/m2, median 0.011 0.010 <0.0001 365) 295) 480) 364) 450) 300) (Interquartile range) Chemotherapy (Anthracycline 01..) Doxorubicin 25 (78.1) 178 (71.8) 0.53 13 (59.1) 40 (54.1) 0.81 38 (70.4) 218 (67.7) 0.75 ko 1-, Daunorubicin 2 (6.3) 26 (10.5) 0.75 1 (4.5) 7 (9.5) 0.68 3 (5.6) 33 (10.2) 0.45 .4 Doxorubicin plus daunorubicin 2 (6.3) 37 (14.9) 0.28 0 (0) 4 (5.4) 0.57 2 (3.7) 41 (12.7) 0.063 0 ko Doxorubicin plus other 0(0) 1(0.4) 1.0 2(9.1) 7(9.5) 1.0 2(3.7) 8(2.5) 0.64 1..) Daunorubicin plus other 3(9.4) 6 (2.4) 0.071 0 (0) 0(0) 0.57 3(5.6) 6(1.9) 0.13 0 1-, Epirubicin 0 (0) 0 (0) 1.0 5 (22.7) 13 (17.6) 0.55 5 (9.3) 13 (4) 0.16 Ln Epirubicin plus other 0 (0) 0 (0) 1.0 1 (4.5) 2 (2.7) 0.55 1 (1.9) 2 (0.6) 0.37 1-, 1-, Otherb 0(0) 0(0) 1.0 0(0) 1(1.4) 1.0 0(0) 1(0.3) 1.0 1-, Primary Diagnosis (Tumor type), no.

Acute Lymphoblastic Leukemia 5(15.6) 105 (42.3) 0.0035 5(22.7) 13 (17.6) 0.55 10 (18.5) 118 (36.6) 0.0085 Acute Myelogenous Leukemia 3 (9.4) 8 (3.2) 0.12 0 (0) 7 (9.5) 0.35 3 (5.6) 15 (4.7) 0.73 Other Leukemia 0(0) 4 (1.6) 1.0 0 (0) 1(1.4) 1.0 0 (0) 5(1.6) 1.0 Hodgkin's Lymphoma 4 (12.5) 19(7.7) 0.31 1(4.5) 8(10.8) 0.68 5(9.3) 27(8.4) 0.79 Non-Hodgkin's Lymphoma 3(9.4) 23(9.3) 1.0 6(27.3) 16 (21.6) 0.57 9(16.7) 39 (12.1) 0.38 Osteosarcoma 0 (0) 11(4.4) 0.62 0 (0) 9 (12.2) 0.11 0 (0) 20 (6.2) 0.093 Rhabdomyosarcoma 2(6.3) 2 (0.8) 0.066 3(13.6) 3 (4.1) 0.13 5(9.3) 5(1.6) 0.0073 Ewing's sarcoma 5 (15.6) 8 (3.2) 0.0095 4 (18.2) 5 (6.8) 0.20 9 (16.7) 13 (4) 0.0015 Other sarcoma 1(3.1) 3(1.2) 0.39 0(0) 1(1.4) 1.0 1(1.9) 4(1.2) 0.54 Hepatoblastoma 2 (6.3) 11(4.4) 0.65 0 (0) 0 (0) 1.0 2 (3.7) 11(3.4) 1.0 Neuroblastoma 1(3.1) 28 (11.3) 0.22 0(0) 0(0) 1.0 1(1.9) 28(8.7) 0.099 Wilms Tumor 6(18.8) 26 (10.5) 0.23 3(13.6)

11 (14.9) 1.0 9(16.7) 37 (11.5) 0.27 TABLE 3: Continued

12 (37.5) 40 (16.1) 0.0068 6 (27.3) 18 (24.3) 0.78 18 (33.3) 58 (18.0) 0.016 Rariinthpranv invnlvinn thp hPartd Use of cardioprotectants, no. (%) 2(6.3) 7(2.8) 0.27 0(0.0) 2 (2.7) 1.0 2(3.7) 9(2.8) 0.66 Duration of Follow-up in years, 7.5 (2.5- 9 (7- 12) 0.33 22 (19 -25) 17 (14 -22) 0.012 15.5 (7 - 22) 10 (7 - 15) 0.021 a Age, dose and duration of follow-up were analyzed by Wilcoxon-Mann-Whitney U
test. Gender, anthracycline type, tumor type, radiotherapy involving the heart and use of cardioprotectant were analyzed by Fisher exact test. b Cumulative anthracycline dose in doxorubicin isotoxic equivalent doses. C
Other anthracycline type included idarubicin, epirubicin or mitoxantrone. d Includes mantle and mediastinal radiation, whole lung radiation, whole or upper abdominal radiation, left sided flank radiation and total body irradiation. e Bold font indicates statistically significant P-value (P < 0.05) and covariates for logistic regression.
ci =

TABLE 4: shows Genome-Wide Association Study (GWAS) of ACT in Patients of European Ancestry: Pharmacogenomic Discovery and Replication Analyses.
Stage 1 - Discovery Canadian Patient Stage 2 - Replication Dutch Patient Population Population n = 280 (32 cases; 248 controIs)a n = 96 (22 cases; 74 controls)b ChGenomic Min Odds Ratio MAF
MAF Odds Ratio MAP MAF
Variantg Positiong Function P
rd Region or (95%Ce (Cases) (Cont. P (95%CI) (Case (Contro Allel ) s) Is) rs6895189 5 13430225 CTIVND2 INTG C 2.4 x 10-6 6.1 (2.8 - 0.359 0.105 N/Af N/A N/A N/A
I DNAH5 13.3) rs7731918 5 13397992 CTNND2 INTG A 4.0 x 10-6 5.9, (2.7 - 0.355 0.105 0.98 1.0 (0.34 - 0.114 0.115 Cl I pNAH5 12.9) 3.0) o CTNND2 5.812.7) (2.6 -"
rs2081944 5 13405946 INTG A 4.8 x io-6 0.355 0.105 LD g LD LD LD ko 1-, 1-, -.3 o rs10085086 5 13424707 CTNND2 INTG C 3.6 x 10-6 5.9 (2.7 - 0.355 0.103 LD LD LD LD ko I DNAH5 13.0) n.) o rs15736 21 44273858 WDR4 NONS-C A 2.6 x 10-6 4.4 (2.2 - 8.7) 0.703 0.366 0.44 0.76 (0.38 - 0.386 0.431 ol 1.5) 1-, rs6586252 21 44276387 WDR4 INTRON A
2.6 x 10-6 4.4 (2.2 - 8.8) 0.703 0.367 LD LD LD LD

rs8133752 21 44271989 WDR4 INTRON A
3.3 x 10-6 4.4 (2.2 - 8.6) 0.703 0.370 LD LD LD LD

rs4381672 18 22712791 ZNF521 INTRON A 2.9 x 10-6 4.3 (2.2 - 8.3) 0.597 0.342 0.78 0.89 (0.40 - 0.341 0.372 2.0) S4275929 18 22706688 ZNF52/ INTRON C 2.8 x 10-6 4.3 (2.2 - 8.5) 0.594 0.343 LD LD LD LD
rs4519409 18 22722077 ZNF521 INTRON A
5.6 x 10-6 4.8 (2.3 - 0.516 0.313 LD LD LD LD
10.0) rs358224h 4 22860785 GBA3 I INTG A 3.3 x 10-6 4.2 (2.2 - 8.1) 0.500 0.249 0.47 1.4 (0.56 - 0.289 0.217 PPARGC
3.5) rs412218 4 22843458 RGC INTG C 5.7 x 10-6 4 (2.1 - 7.5) 0.531 0.270 LD LD LD LD
PPA
IA

TABLE 4: Continued rs11946006 4 22850202 GBA3 I INTG G 9.1 x 10-6 3.9 (2.1 - 7.4) 0.531 0.282 LD LD LD LD
PPARG
rs7676830i 4 23169854 GBA3 I INTG G
5.5 x 10-6 4.8 (2.3 - 9.8) 0.500 0.262 0.057 2.3 (0.96 - 0.364 0.243 PPARG
5.4) rs2282889 7 21476188 SP4 INTRON A
4.4 x 10-6 0.2 (0.088 - 0.234 0.446 0.75 0.9 (0.44 - 0.364 0.401 0.44) 1.8) rS22297743 12 53605545 RARG NONS-C A 5.0 x 10-6 7 (2.9 -17) 0.297 o.o8i 0.0043 4.1 (1.5 - 0.25 0.061 11.5) o rs9323880 14 93129810 R/N3 INTRON A 6.8 x 10-6 4.2 (2.1 - 8.2) 0.594 0.348 0.17 1.6 (0.82 - 0.50 0.372 3.1) rs7042745 9 27248177 NCR1VA INTRON A
7.5 x 10-6 4.5 (2.2 - 8.9) 0.484 0.222 0.65 0.85 (0.41 - 0.295 0.319 0 1.8) o tv a Covariates for the Logistic regression were age at treatment, cumulative anthracycline exposure, radiotherapy involving the heart and tumour type l0 I-, (acute lymphoblastic leukemia, rhabdomyosarcoma and Ewing's sarcoma).
.4 b Covariate for the Logistic regression was cumulative anthracycline exposure.

l0 c Variants with P < 1.0 x to-bin the discovery GWAS analysis'.
n.) 5' Chr, chromosome.

1-, e Chromosomal positions in the GRCH37.p13.
ol f Not Applicable; call rates for this SNP were < 90% in the Stage 2 cohort.
1-, g LD (r2> 0.9 and D' > 0.9 in the CEU component of HapMap), therefore only 9 of 18 variants were genotyped in the Stage 2 - Replication Dutch patient 1 1-, population.

The call rate for this SNP was 92% in the replication cohort.
' Tags a distinct LD block in this genomic region.
3 Bold font indicates statistically significant SNP after multiple testing correction (discovery P < 1.ox10-5 and replication P < 0.05/9 LD blocks =
o.006).
INTG = INTERGENIC; NUNS-C = NONSYN-CODING; INTRON = INTRON; and Cont. =
Control TABLE 5: shows the association of RARG rs2229774 with Anthracycline-induced Cardiotoxicity in Childhood Cancer Patients.
Biomarker Pharmacogenomic Analyses Adjusted Logistic Regression Genotypic Test (Additive Model) MAF MAF
SNP Gene Function Study Population P
Odds Ratio (95%CI) P
Cases Controls NON-SYN Stage 1 ¨ Discovery GWASa rs2229774 RARG 0.297 0.081 5.0 x 10 7.0 (2.9 ¨ 17.0) 4.1 x 10-' CODING Canadian European Patient Stage 2 ¨ Replicationb P
0.25 0.061 0.0043 4.1 (1.5 ¨ 11.5) 0.0042 o Dutch European Patients N) ko 1¨`
All European Patientsa 0.278 0.076 7.8 x 10-8 5.4 (2.9 ¨ 10.3) 1.2 x 10-9 ¨.1 ko Stage 3 ¨ Replication 0.158 0 N/A`
N/A 1.2x10-4 N) o Non-European Patients Ui I
1¨`
All Populations' European and Non-European 0.247 0.064 5.9 x 10 4.7 (2.7 ¨ 8.3) 4.3x10-11 Patients a Covatiates for the Logistic regression were age at treatment, cumulative anthracycline exposure, radiotherapy involving the heart and incidence of acute lymphoblastic leukemia, rhabdomyosarcoma and Ewing's sarcoma.
b Covari ate for the Logistic regression was cumulative anthracycline exposure.
' Not applicable, rs2229774 absent in controls.

TABLE 6: shows Top GWAS Associations by Cumulative Anthracycline Exposure.
Pharmacogenomic Associations of Anthracycline Low-to-moderate Anthracycline Exposure High Anthracycline Exposure Cardiotoxicity (5. 250 mg/ml (>250 mg/m2) Stage 1: Discovery" Stage 2: Replication"
Stage 1: Discovery Stage 2: Replication"
Variantb Genomic Regions Function n = 184 patients n =
38 patients n = 96 patients n = 58 patients (16 cases; 168 controls) (5 cases; 33 controls) (16 cases; 80 controls) (17 cases; 41 controls) rs7731918 CTNND2 I DNAH5 INTERGENIC 0.00016 0.70 0.0010 0.85 o P
rs15736 WDR4 NONSYN-CODING 0.0011 0.62 0.00029 0.59 o N) rs4381672 ZNF521 INTRON 6.9 x 10-5 0.29 0.021 0.56 ts) 1-`
1-`
-.1 rs358224 GBA3 I PPARGC1A INTERGENIC 0.00020 0.37 0.0065 0.69 0 ts) rs7676830' GBA3 I PPARGC1A INTERGENIC 9.1 x 105 0.0240 0.0076 0.30 "

1-`
(xi rs2282889 SP4 INTRON 0.0037 0.84 0.00041 (0.05)h 1 1-`
1-`
l rs2229774 RARG NONSYN-CODING 0.00041 0.0036 0.0021 0.084 1-`

rs9323880 R1N3 INTRON 5.8 x 107 0.76 0.31 0.15 rs7042745 NCRNA00032 INTRON 0.0011 0.84 0.0011 0.67 Stratification by cumulative anthracycline exposure as previously performed'''.
Variants with P< 1.0 x 10 in the discovery GWAS analysis'.
P-values are for logistic regression (additive model) with adjustment for covariates.
Covariates for the Logistic regression were age at treatment, cumulative anthracycline exposure, radiotherapy involving the heart and acute lymphoblastic leukemia tumor type.
Covariate for the Logistic regression was cumulative anthracycline exposure.
Covariates for the Logistic regression were age at treatment, cumulative anthracycline exposure, radiotherapy involving the heart and tumor type (acute lymphoblastic leukemia, ha bdomyosarcoma and Ewing's sarcoma).
Tags a distinct LD block in this genomic region.
Regression failed due to absence of variant in cases in this stratified dose, genotypic testing P-value shown.

In stage 2, we genotyped one GWAS candidate variant per LD block in an independent Dutch population of childhood cancer patients of European ancestry (96 patients; 22 cases and 74 controls). In this cohort cases and controls were similarly matched with the exception that cases had higher cumulative anthracycline exposure (TABLE 3).
Genetic associations were tested using logistic regression with adjustment for cumulative anthracycline dose. Of the 9 candidate variants tested, only rs2229774, a nonsynonymous coding variant (p.Ser427Leu) in RARG (Retinoic Acid Receptor Gamma), was replicated (P=o.0o42, OR=4.1 (1.5-11.5); FIGURE 1 and TABLE 5).
Given that the Stage 1 and 2 cohorts differed in some patient characteristics, e.g.
anthracycline dose, the replication analysis merits circumspect interpretation. To address this we performed additional genetic association analyses by logistic regression with the prioritized GWAS variants in subsets of cases and controls stratified by low-to-moderate ( .25o mg/m2) and high (>250 mg/m2) anthracycline exposure (TABLE 6). RARG
rs2229774 was associated with ACT in stages 1, 2 and the combined analysis at both low-to-moderate (P=4.1x10-4, P=o.0036, and P=9.8x10-6 , respectively) and high anthracycline doses (P=o.0021, P=o.o84, and P=8.7xio-4, respectively). By contrast, none of the other top GWAS candidate SNPs were significantly associated with ACT in the replication cohort, even at low-to-moderate anthracycline exposure. Logistic regression analyses in the Stage 1 cohort indicated that RARG rs2229774 was similarly associated with early-onset chronic ACT (n=16 cases; P=2.8x10-4; OR=7.3 (2.3-22.9)) and late-onset chronic ACT
(n=r6 cases;
P=5.oxio-3; OR=5.8 (1.7-19.4)).
In stage 3, we examined the association of RARG rs2229774 with ACT in a third cohort of non-European patients representative of different ancestries (80 patients; 19 cases and 61 controls; TABLE 7). Due to the absence of RARG rs2229774 in controls, logistic regression with adjustment for population stratification and other clinical covariates could not be performed in this cohort (TABLE 8). Instead the association of RARG 1.52229774 with ACT in this cohort was analyzed by genotypic test. RARG 1.52229774 was highly associated with ACT (P=1.2xio-4; TABLE 1) in the stage 3 cohort, and in each of the four non-European populations separately (African, Aboriginal Canadians, Hispanic, and East Asian;
TABLE 8). Notably, genotypic association testing reached genome-wide significance in the discovery and combined cohorts (P<5.oxio-8; TABLE 1).

TABLE 7: shows assessment of Baseline Characteristics in Non-European Patient Populations.
Patient Aboriginal Canadians -Hispanic USA -Stanford African - CPNDS
East Asian - CPNDS
Characteristicsa CPNDS
(n = 23 patients) (n = 11 patients) (n = 31 patients) (n = 15 patients) Cases Controls Cases Controls Cases Controls Cases Controls (n= 5) (n = 18) P (n = 2) (n = 9) P (n = 8) (n = 23) P (n = 4) (n = 11) P
Age at the Start of Treatment 14.0 (12.5- ., 6.o (2.5 - 3.5 (1.5 -5.5 (3 - 12) 0.01.9e 4.5 (4 - 5) 4.o (1.5 - 7.0) 0.73 3.5 (0.5 - 8) 0.32 , 4 (2.5 - 7) 0.49 Age in yrs, median 17.5) 9.5) 5.5) (Interquartile Gender, female/male (% 1/4 (20.0) 7/11 (38.9) 0.62 0/2 (o) 6/3 (66.7) 1.0 5/3 (62.5) 11/12 (47.8) 0.69 (loo.o) 6/5 (54.5) 0.23 Cumulative Anthracycline 200 (141- 162.5 (150 - 319.5 (240 -24o (114 -_ 300.5 (270.0 - 290 (162.5 , 25o (137.5 150 (135 -Exposure Doseb in o.8o o.5o 0.40 0.66 o 245) 300) 399) 382.5) 362.5) - 360) - 330) 245) N.) mg/m2, median ko 1-, (Interquartile 1-, =4 Anthracycline typec, ko Doxorubicin 2 (40) 13 (72.2) 0.30 2 (100) 6 (66.7) 1.0 5 (62.5) 14 (60.9) 1.0 2 (50) 9 (81.8) 0.52 N.) Daunorubicin 1(20) 2(11.1) 0.54 o(o) 2(22.2) 1.0 1(12.5) 1(4.3) 0.46 2(50) 1(9.1) 0.15 1-, ol Doxorubicin plus 2 (40) 3 (16.7) 0.29 o (o) 1 (11.1) 1.0 o (o) 5 (21.7) 0.29 o (o) 1 (9.1) 1.0 I
1-, Daunorubicin o (o) o (o) 1.0 o (o) o (o) 1.0 2(25) 2(8.7) 0.27 o(o) o(o) 1.0 1-, 1-, Doxorubicin plus daunorubicin plus o (o) o (o) 1.0 o (o) o (o) 1.0 o (o) 1 (4.3) 1.0 o (o) o (o) 1.0 others Primary Diagnosis, Acute 2 (40) 13 (72.2) 0.30 1 (5o) 3 (33.3) 1.0 o (o) 8 (34.8) 0.07 2 (50) 5 (45.5) 1.0 Acute 1 (20) 2 (11.1) 0.54 0 (o) o (o) 1.0 2 (25) 2 (8.7) 0.27 1 (25) o (o) 0.27 Other Leukemia 2 (40) 3 (16.7) 0.29 o (o) o (o) Lo 1 (12.5) o (o) 0.26 0 (0) 0 (0) 1.0 Hodgkin's o (o) o (o) 1.0 o (o) 1 (11.1) 1.0 o (o) o (o) 1.0 o (o) 1 (9.1) 1.0 Non-Hodgkin's o (o) o (o) 1.0 o (o) 1 (11.1) 1.0 2 (25) 3 (13) 0.58 o (o) 1 (9.1) 1.0 Osteosarcoma o (o) o (o) 1.0 o (o) 1 (11.1) 1.0 o (o) 3 (13) 0.55 o (o) 1 (9.1) 1.0 Rhabdomyosarco o (o) o (o) 1.0 1 (5o) o (o) o.18 o (o) 1 (4.3) 1.0 o (o) o (o) 1.0 Ewing's sarcoma o (o) o (o) 1.0 o (o) o (o) to o (o) o (o) Lo o (o) 1 (9.1) 1.0 Hepatoblastoma o (o) o (o) 1.0 o (o) o (o) 1.0 2 (25) 2 (8.7) 0.27 0 (0) 1 (9.1) 1.0 TABLE 7: Continued Neuroblastoma o(o) o (o) 1.0 o (o) o (o) 1.0 1 (12.5) 3 (13) 1.0 o (o) 1 (9.1) 1.0 Wilms Tumor o (o) o (o) 1.0 o (o) 3 (33.3) t.o o (0) 1 (4.3) 1.0 1 (25) o (0) 0.27 Radiotherapy involving heartd, no. (%) Data not available for all patients o (0) 2 (22.2) 1.0 0 (0) 0 (0) 1.0 1 (25.0) 0 (0) 0.27 Use of cardioprotectants, no. (%) Data not available for all patients o (o) o (o) 1.0 o (0) 2 (8.7) 1.0 0 (0) 0 (0) 1.0 Duration of Follow-up in years median (range) 4 (3 -4) 6 (5 - 7) 10.5 (9 -12) 8 (6.5 ¨ 10.5) 0.33 8.5 (4 - 14) 7 (6.5 - 8) 0.84 7 (2 - 19) 7 (6.5 -lo) 0.85 a Age, dose and follow-up were analyzed by Wilcoxon-Mann-Whitney U test.
Gender, anthracycline type, tumor type, radiotherapy involving the heart, and use of cardioprotectant were analyzed by Fisher exact test. b Cumulative anthracycline dose in doxorubicin isotoxic equivalent doses. c o Other anthracycline type included idarubicin, epirubicin or mitoxantrone.
d Radiotherapy involving the heart include: mantle and mediastinal radiation, whole lung radiation, whole or upper abdominal radiation, left sided 0 1..) flank radiation and total body irradiation ko 1-, e Bold indicates statistically significant P-value (P < o.o5).
.4 ko iv 1-, (xi 1-, 1-, 1-, TABLE 8: shows association of RARG rs2229774 with ACT in Non-European Populations.
Africans Hispanics East Asians Aboriginal Canadians Combined n = 11 patients n = 23 patients n = 31 patients n = 15 patients n = 80 patients (2 cases; 9 controls) (5 cases;18 controls) (8 cases; 23 controls) (4 cases; 11 controls) (19 cases; 61 controls) MAFa'b Expected 11.0% 5.0% 0%
Unreported N/A
Range 6.0% - 16.0% 3.0% - 8.0% 0%
Unreported N/A
Genetic Association Observed MAF
25.0% vs. 0 /0 20.0% vs. 0% 6.3% vs. 0%
25.0% vs. 0% 15.8% vs. 0%
(Cases vs. Controls) Pc 0.026 0.052 0.085 0.012 1.2x10-4 MAF are from http://www.1000genomes.org.
(xi b Abbreviations: MAP, minor allele frequency; N/A, not applicable.
P-values are for genotypic association tests.

TABLE 9: shows the fine mapping of genetic association signals in the RARG
gene region.
Biomarker Pharmacogenomic Analyses Adjusted Logistic regression (additive model)b LD with MAF MAF
Odds Ratio Conditional Analysis SNP rs-ID` Position Type Source rs2229774 on rs2229774 Cases Controls (95%CI) rs11170481 53611791 Intronic Imputed 1.00(0.84) 0.313 0.081 1.7x10-6 7.0 (3.0 ¨ 16.6) 0.10 rs73309171 53606565 Intronic Imputed 1.00 (0.55) 0.281 0.071 4.1x10-6 7.5 (3.0¨ 18.4) 0.42 rs57789211 53609992 Intronic Imputed 1.00 (0.84) 0.281 0.071 4.1x10-6 7.5 (3.0¨ 18.4) 0.42 rs2229774 53605545 Nonsyn Genotyped 1.00(1.00) 0.297 0.081 5.0x10-6 7.0 (2.9 ¨ 17.0) a1,005,286 additional variants on Chr12 imputed into stage 1 cohort using the CEU component of the 1000 Genomes populations as a reference.
bCovariates for logistic regression analysis (additive model) were age at treatment, cumulative anthracycline exposure, radiotherapy involving the heart and incidence of acute lymphoblastic leukemia, rhabdomyosarcoma, Ewing's sarcoma and rs2229774 where indicated. n.) 'Association analyses for imputed SNPs were restricted to those with BEAGLE
allelic R2 > 0.5.
n.) dChromosomal positions in the GRCH37.p13.

(xi 'Calculated using the CEU component of 1000 Genomes reference population.

TABLE 10: shows stage 1 Discovery Analysis - Results for Previous ACT-associated Regions.
MAF
MAF
IQ, Marker Chra Positionb Gene Function P-Odds Ratio Minor Referen (248 valueb (95%C1) Allele µ''` ce Cases) Controls) rs17583889 2 138746039 HNMT INTRON 0.008 2.4 (1.3 -4.5) A 0.344 0.175 5,6 rs8187710 10 101611294 0.021 4.3 (1.4 -

13.8) A 0.078 0.046 7,8 rs2868177 7 75589903 POR INTRON 0.016 2.1 (1.1 -4.0) G 0.438 0.312 9 rs13240755 7 75606109 POR INTRON 0.033 2.0 (1.0 -3.7) G 0.453 0.349 9 rs4732513 7 75607608 POR INTRON 0.041 1.9 (1.0 -3.6) G 0.466 0.348 9 NONSYN-rs2232228 16 69143577 HAS3 0.18 0.67 (0.36 -1.2) G 0.375 0.427 2 0 CODING

rs3743527 16 16235681 UTR 0.24 0.65 (0.30 -1.4) A 0.172 0.204 10 "

ko 1-, 1-, rs13058338 22 37632770 RA C2 INTRON 0.28 0.68 (0.34 - 1.4) T 0.188 0.245 7,8 --.1 l 0 rs10836235 11 34460704 CAT INTRON 0.46 0.70 (0.26 -1.9) A 0.109 0.118 11 1-, SYN-rs1695 11 67352689 GSTP1 0.46 1.3 (0.68 -2.4) G 0.344 0.349 12-14 1 CODING
1-, 1-, SYN-3,13,15,1 rs1056892 21 37518706 CBR3 0.64 0.85 (0.42 -1.7) A 0.344 0.351 1-, CODING

rs246221 16 16138322 0.68 1.1 (0.60 -2.2) G 0.281 0.274 10 NONSYN-rs1799945 6 26091179 HFE 0.68 0.84 (0.37 -1.9) G 0.125 0.151 8,17 CODING
SYN-rs4673 16 88713236 CYBA 0.81 1.1 (0.59 -2.0) A 0.371 0.356 7,18 CODING
'Abbreviations: Chr, chromosome; MAF, minor allele frequency.
b Chromosomal positions in the GRCH37.p13.
b P-values and odds ratios (95%C1) are for logistic regression analysis (additive model) with adjustment for age at treatment, cumulative anthracycline exposure, radiotherapy involving the heart and incidence of acute lymphoblastic leukemia, rhabdomyosarcoma and Ewing's sarcoma.

The stage 3 cohort, comprised of four distinct populations, was assessed for population stratification using the genomic inflation factor and principal component analysis. PCs i¨lo did not significantly differ between cases and controls (P>0.14) and the Xcc of 0.941 suggested no confounding effect of population stratification on the genetic association of rs2229774 with ACT in this cohort. In line with the reported high incidence of ACT in studies conducted in India (Agarwala, Kumar et al. 2000) and Pakistan(Shaikh, Saleem et al. 2013), and in African-American patients in the USA (Krischer, Epstein et al. 1997; Hasan, Dinh et al. 2004), RARG
p52229774 is more frequent in South Asian (22%) and African populations (11%) compared to European (6%) and Hispanic (5%) populations. By contrast, rs2229774 is very rare in East Asian (0%) populations (woo Genomes). It will be interesting to correlate the frequency of n2229774 with the incidence of ACT in children of different ancestries as data on multiethnic studies become available for comparison.
A meta-analysis of all study populations (456 patients; 73 cases and 383 controls) using logistic regression adjusted for age, dose, ALL, RT, Ewing's sarcoma and rhabdomyosarcoma showed that RARG rs2229774 was significantly associated with ACT (P-5.9x10-8, OR=4.7 (2.7-8.3)), which surpassed the Bonferroni-adjusted multiple testing correction threshold, with no significant heterogeneity across the GWAS and two independent replication analyses (Pheterogeneity=0.402). Overall, rs2229774-carriers had significantly increased odds of developing ACT compared to non-carriers (OR=5.2 (3.0-9.0); P=5.9x10-10).
Example 2: Functional validation of the RARGs427L variant To fine-map the rs2229774 association with ACT, we imputed variants on chromosome 12 using the stage 1 cohort and the woo Genomes CEU reference population, and tested their association with ACT by logistic regression (FIGURE 1). The linkage disequilibrium (D') based upon the moo Genomes CEU population for this region was similarly demonstrated that the associated haplotype is localized to RARG (data not shown). Of the 1,005,286 imputed SNPs, three were associated with ACT at P<I.0x10-5 (rsiii70481, rs73309171 and rs57789211;
TABLE 9). These were all located in introns of RARG, highly correlated with each other (D' r2-0=54), and in high linkage disequilibrium (LD) with rs2229774 (D'=1.0o, r20.55). Since the ACT associations of the imputed variants were similar to rs2229774, and logistic regression with conditioning on n2229774 abolished these associations (TABLE 9), the non-synonymous variant r52229774 was prioritized for functional characterization to gain biological insight into its association with ACT.

Retinoic acid receptors bind to DNA regulatory sequences termed retinoic acid response elements (RARE) and transcriptionally co-regulate downstream gene expression in response to their agonist all-trans retinoic acid (ATRA)(Chambon 1996). Notably, RARG can both activate and repress transcription in response to ATRA(Tang, Chen et al. 2011). To explore the functional properties of rs2229774 (i.e. RARGs4271-), we first examined its transactivation of RARG regulatory elements and then identified a putative role in the dysregulation of a critical gene involved in the development of ACT (see Online Methods). In HEK293T cells expressing RARGs4271-, the ATRA-inducible transcriptional activation of a RARE-coupled luciferase reporter was significantly reduced compared to wild type RARG-expressing cells (FIGURE
2a). Immunoblot analysis verified that both wild type and variant RARG
proteins were detected at similar levels in HEK293T cell lysate (FIGURE 2a inset). The significant 17%
decrease in RARG activity conferred by the rs2229774 variant may be an underestimate since endogenous retinoic acid receptors were present in this assay. These results suggested that dysregulation of an RARG-regulated gene might underlie the association of rs2229774 with ACT.
Anthracyclines are mechanistically and genetically linked with Topoisomerase II beta (Top2b).
Anthracyclines exert their anticancer activity by binding and inhibiting Topoisomerase II
(Minotti, Menna et al. 2004). In addition, Top2b is necessary for the development of ACT in a murine model (Zhang, Liu et al. 2012), while in a rat cardiomyoblast (H9c2) cell line, Top2b levels are decreased by the ACT cardioprotectant, dexrazoxane (Lyu, Kerrigan et al. 2007).
RARG expression has been reported as "particularly high" in the heart (Nuclear Receptor Signaling Atlas) and is highly induced in murine cardiac cells following cardiac injury (Bilbija, Haugen et al. 2012). Since RARG has been shown to bind to the Top2b promoter (Delacroix, Moutier et al. 2010) it was one potential candidate of RARGs4271-dysregulation. We found that Top2b expression in H9c2 cells was significantly decreased when human RARG was added, and this effect was further exacerbated with the addition of ATRA (FIGURE 2b).
By contrast, the RARGs427L variant did not repress Top2b expression as effectively as wild type RARG
(FIGURE 2c). Taken together these results demonstrate that RARG represses Top2b expression and that rat cardiomyoblasts carrying rs2229774 express higher basal levels of Top2b, consistent with an increased susceptibility to ACT.
We have identified a novel genetic biomarker for ACT, RARG rs2229774, using a three-stage genetic association study combined with biological functional analyses. This discovery, despite originating from a relatively small number of patients, is likely due to its large effect size. In general, sample size limitation remains a major challenge in pediatric cancer pharmacogenomic studies (McLeod 2013; Wheeler, Maitland et al. 2013), where the need for a homogenous study population and well defined clinical phenotypes results in reduced numbers of study patients, particular the number of affected individuals.
However, clinically relevant genetic markers of severe ADRs such as ACT, abacavir-induced hypersensitivity reaction, and carbamazepine-induced Stevens-Johnson syndrome, are expected to have large effect sizes and therefore, the number of patients, particularly the number of affected individuals, required to uncover these genetic associations from large-scale genome screens, has been shown to be in the range of 10-100 patients(Nelson, Bacanu et al.
2009) - similar to the number in this study.
Our study was 8o% powered to detect genome-wide associations of P.5.5 x io-8 with per-allele 012.4.5 and MAFo.io. There are likely additional genetic contributions to ACT
including previously reported associations (Wojnowski, Kulle et al. 2005; Blanco, Leisenring et al. 2008;
Rajic, Aplenc et al. 2009; Rossi, Rasi et al. 2009; Windsor, Strauss et al.
2011; Blanco, Sun et al. 2012; Cascales, Sanchez-Vega etal. 2012; Lubieniecka, Liu etal. 2012;
Semsei, Erdelyi etal.
2012; Visscher, Ross et al. 2012; Volkan-Salanci, Aksoy et al. 2012; Armenian, Ding et al.
2013; Cascales, Pastor-Quirante et al. 2013; Lipshultz, Lipsitz et al. 2013;
Lubieniecka, Graham et al. 2013; Sachidanandam, Gayle et al. 2013; Visscher, Ross et al.
2013; Vivenza, Feola et al. 2013; Wang, Liu et al. 2014) that were not uncovered in the current GWAS, potentially owing to the strict ACT case definition and resulting small patient numbers used in this study. For example, ABCC2/MRP2, HNMT and POR, although not reaching genome-wide significance in this study, exhibited strong trends of association (P<o.o5;
TABLE io) in the same direction of effect as previously reported(Wojnowski, Kulle et al. 2005;
Visscher, Ross et al. 2012; Armenian, Ding et al. 2013; Lubieniecka, Graham et al. 2013;
Sachidanandam, Gayle et al. 2013). This underscores the complementary nature of GWAS and candidate gene association studies in identifying variants associated with pharmacogenomic traits.
In agreement with in silico predictions, our in vitro studies demonstrate that rs2229774 causes a relatively tolerated amino acid substitution that results in a moderate, but significant, reduction in RARE transcriptional activation. We established a genetic interaction between RARG and Top2b, where expression of the latter is significantly repressed by RARG in the presence of ATRA. Notably, the treatment of acute promyelocytic leukemia with high cumulative doses of anthracycline and ATRA has resulted in a significantly lower incidence of ACT (Ortega, Madero et al. 2005; Pellicori, Calicchia et al. 2012). We further showed that rs2229774 causes derepression of Top2b in cardiomyoblasts, directly linking this variant with ACT. These data are consistent with a model where rs2229774 carriers have higher basal levels of TOP2B in cardiomyocytes, conferring increased susceptibility to cardiotoxicity when treated with anthracyclines. Nevertheless, despite rs2229774 encoding a non-synonymous variant, it is plausible that the rs2229774 haplotype confers increased susceptibility to ACT
through a regulatory mechanism. To our knowledge a comprehensive analysis of RARG-regulated gene expression in cardiomyocytes has not been reported and it is possible that additional genes in cardiomyocytes may contribute to the development of ACT when dysregulated in RARG
1.52229774 carriers. For example, ATRA-regulated gene expression in cardiomyocytes is important for cardiac development and the progression of cardiomyocyte hypertrophy (Palm-Leis, Singh et at. 2004; Arima, Shiotsugu et al. 2005; Simandi, Balint et at.
2010), though the specific contribution of RARG to these processes is unknown.
The identification of RARG rs2229774, genetically and functionally linked to ACT, provides a clinical tool that may be used to predict genetic risk and improve ACT risk stratification. ACT is a clinically significant ADR, and carriers of 1.52229774 have 5-fold increased odds of ACT. This novel finding merits further exploration of the role of RARG in ACT and of the clinical utility of RARG 1.52229774 predictive testing to inform ACT risk assessment.
Agarwala, S., R. Kumar, et al. (2000). "High incidence of adriamycin cardiotoxicity in children even at low cumulative doses: role of radionuclide cardiac angiography." J
Pediatr Surg 35(12): 1786-1789.
Altman, A. J. (2004). Supportive care of children with cancer: current therapy and guidelines from the Children's Oncology Group. Baltimore, John Hopkins University Press.
Arima, K., J. Shiotsugu, et al. (2005). "Global analysis of RAR-responsive genes in the Xenopus neurula using cDNA microarrays." Dev Dyn 232(2): 414-431.
Armenian, S. H., Y. Ding, et al. (2013). "Genetic susceptibility to anthracycline-related congestive heart failure in survivors of haematopoietic cell transplantation."
Br J
Haematol 163(2): 205-213.
Barrett, J. C., B. Fry, et at. (2005). "Haploview: analysis and visualization of LD and haplotype maps." Bioinformatics 21(2): 263-265.
Bilbija, D., F. Haugen, et al. (2012). "Retinoic acid signalling is activated in the postischemic heart and may influence remodelling." PLoS One 7(9): e44740.
Blanco, J. G., W. M. Leisenring, et al. (2008). "Genetic polymorphisms in the carbonyl reductase 3 gene CBR3 and the NAD(P)H:quinone oxidoreductase 1 gene NQ01 in patients who developed anthracycline-related congestive heart failure after childhood cancer." Cancer 112(12): 2789-2795.
Blanco, J. G., C. L. Sun, et al. (2012). "Anthracycline-related cardiomyopathy after childhood cancer: role of polymorphisms in carbonyl reductase genes--a report from the Children's Oncology Group." J Clin Oncol 30(13): 1415-1421.
Browning, S. R. and B. L. Browning (2007). "Rapid and accurate haplotype phasing and missing-data inference for whole-genome association studies by use of localized haplotype clustering." Am J Hum Genet 81(5): 1084-1097.
Cascales, A., F. Pastor-Quirante, etal. (2013). "Association of anthracycline-related cardiac histological lesions with NADPH oxidase functional polymorphisms." Oncologist 18(4):
446-453.
Cascales, A., B. Sanchez-Vega, et al. (2012). "Clinical and genetic determinants of anthracycline-induced cardiac iron accumulation." Int J Cardiol 154(3): 282-286.
Chambon, P. (1996). "A decade of molecular biology of retinoic acid receptors." FASEB J
10(9): 940-954.
Delacroix, L., E. Moutier, et al. (2010). "Cell-specific interaction of retinoic acid receptors with target genes in mouse embryonic fibroblasts and embryonic stem cells." Mol Cell Biol 30(1): 231-244.
Devlin, B. and K. Roeder (1999). "Genomic control for association studies."
Biometrics 55(4):
997-1004.
Hasan, S., K. Dinh, et al. (2004). "Doxorubicin cardiotoxicity in African Americans." J Natl Med Assoc 96(2): 196-199.
Kremer, L. C., E. C. van Dalen, et al. (2001). "Anthracycline-induced clinical heart failure in a cohort of 607 children: long-term follow-up study." J Clin Oncol 19(1): 191-196.
Krischer, J. P., S. Epstein, etal. (1997). "Clinical cardiotoxicity following anthracycline treatment for childhood cancer: the Pediatric Oncology Group experience." J
Clin Oncol 15(4): 1544-1552.
Lipshultz, S. E., S. D. Colan, et al. (1991). "Late cardiac effects of doxorubicin therapy for acute lymphoblastic leukemia in childhood." N Engl J Med 324(12): 808-815.
Lipshultz, S. E., S. R. Lipsitz, et al. (2013). "Impact of hemochromatosis gene mutations on cardiac status in doxorubicin-treated survivors of childhood high-risk leukemia."
Cancer 119(19): 3555-3562.
Lipshultz, S. E., S. R. Lipsitz, et al. (1995). "Female sex and drug dose as risk factors for late cardiotoxic effects of doxorubicin therapy for childhood cancer." N Engl J Med 332(26): 1738-1743.
Lubieniecka, J. M., J. Graham, etal. (2013). "A discovery study of daunorubicin induced cardiotoxicity in a sample of acute myeloid leukemia patients prioritizes P450 oxidoreductase polymorphisms as a potential risk factor." Front Genet 4: 231.
Lubieniecka, J. M., J. Liu, et al. (2012). "Single-nucleotide polymorphisms in aldo-keto and carbonyl reductase genes are not associated with acute cardiotoxicity after daunorubicin chemotherapy." Cancer Epidemiol Biomarkers Prey 21(11): 2118-2120.
Lyu, Y. L., J. E. Kerrigan, et al. (2007). "Topoisomerase IIbeta mediated DNA
double-strand breaks: implications in doxorubicin cardiotoxicity and prevention by dexrazoxane."
Cancer Res 67(18): 8839-8846.
McLeod, H. L. (2013). "Cancer pharmacogenomics: early promise, but concerted effort needed." Science 339(6127): 1563-1566.
Minotti, G., P. Menna, et al. (2004). "Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity." Pharmacol Rev 56(2):
185-229.
Mulrooney, D. A., M. W. Yeazel, et al. (2009). "Cardiac outcomes in a cohort of adult survivors of childhood and adolescent cancer: retrospective analysis of the Childhood Cancer Survivor Study cohort." BMJ 339: b4606.

Nelson, M. R., S. A. Bacanu, et al. (2009). "Genome-wide approaches to identify pharmacogenetic contributions to adverse drug reactions." Pharmacogenomics J
9(1):
23-33.
Ortega, J. J., L. Madero, et al. (2005). "Treatment with all-trans retinoic acid and anthracycline monochemotherapy for children with acute promyelocytic leukemia:
a multicenter study by the PETHEMA Group." J Clin Oncol 23(30): 7632-7640.
Palm-Leis, A., U. S. Singh, et al. (2004). "Mitogen-activated protein kinases and mitogen-activated protein kinase phosphatases mediate the inhibitory effects of all-trans retinoic acid on the hypertrophic growth of cardiomyocytes." J Biol Chem 279(52):
54905-54917.
Pellicori, P., A. Calicchia, et al. (2012). "Subclinical anthracycline cardiotoxicity in patients with acute promyelocytic leukemia in long-term remission after the AIDA
protocol."
Congest Heart Fail 18(4): 217-221.
Pruim, R. J., R. P. Welch, et al. (2010). "LocusZoom: regional visualization of genome-wide association scan results." Bioinformatics 26(18): 2336-2337.
Rajic, V., R. Aplenc, et al. (2009). "Influence of the polymorphism in candidate genes on late cardiac damage in patients treated due to acute leukemia in childhood." Leuk Lymphoma 50(10): 1693-1698.
Rodvold, K. A., D. A. Rushing, et al. (1988). "Doxorubicin clearance in the obese." J Clin Oncol 6(8): 1321-1327.
Rossi, D., S. Rasi, etal. (2009). "Analysis of the host pharmacogenetic background for prediction of outcome and toxicity in diffuse large B-cell lymphoma treated with R-CHOP21." Leukemia 23(6): 1118-1126.
Sachidanandam, K., A. A. Gayle, et al. (2013). "Unexpected doxorubicin-mediated cardiotoxicity in sisters: possible role of polymorphisms in histamine n-methyl transferase." J Oncol Pharm Pract 19(3): 269-272.
Semsei, A. F., D. J. Erdelyi, et al. (2012). "ABCC1 polymorphisms in anthracycline-induced cardiotoxicity in childhood acute lymphoblastic leukaemia." Cell Biol Int 36(1): 79-86.
Shaikh, A. S., A. F. Saleem, et al. (2013). "Anthracycline-induced cardiotoxicity: prospective cohort study from Pakistan." BMJ Open 3(11): e003663.
Simandi, Z., B. L. Balint, etal. (2010). "Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies." FEBS Lett 584(14): 3123-3130.
Tang, Q., Y. Chen, etal. (2011). "A comprehensive view of nuclear receptor cancer cistromes."
Cancer research 71(22): 6940-6947.
Visscher, H., C. J. Ross, et al. (2009). "Application of principal component analysis to pharmacogenomic studies in Canada." Pharmacogenomics J 9(6): 362-372.
Visscher, H., C. J. Ross, et al. (2012). "Pharmacogenomic prediction of anthracycline-induced cardiotoxicity in children." J Clin Oncol 30(13): 1422-1428.
Visscher, H., C. J. Ross, et al. (2013). "Validation of variants in SLC28A3 and UGT1A6 as genetic markers predictive of anthracycline-induced cardiotoxicity in children." Pediatr Blood Cancer 60(8): 1375-1381.
Vivenza, D., M. Feola, etal. (2013). "Role of the renin-angiotensin-aldosterone system and the glutathione S-transferase Mu, Pi and Theta gene polymorphisms in cardiotoxicity after anthracycline chemotherapy for breast carcinoma." Int J Biol Markers 28(4):
e336-347.
Volkan-Salanci, B., H. Aksoy, et al. (2012). "The relationship between changes in functional cardiac parameters following anthracycline therapy and carbonyl reductase 3 and glutathione S transferase Pi polymorphisms." J Chemother 24(5): 285-291.

Wang, X., W. Liu, et al. (2014). "Hyaluronan synthase 3 variant and anthracycline-related cardiomyopathy: a report from the children's oncology group." J Clin Oncol 32(7): 647-653.
Welter, D., J. MacArthur, et al. (2014). "The NHGRI GWAS Catalog, a curated resource of SNP-trait associations." Nucleic Acids Res 42(Database issue): Mom-1006.
Wheeler, H. E., M. L. Maitland, et al. (2013). "Cancer pharmacogenomics:
strategies and challenges." Nat Rev Genet 14(1): 23-34.
Windsor, R. E., S. J. Strauss, et al. (2011). "Germline genetic polymorphisms may influence chemotherapy response and disease outcome in osteosarcoma: A Pilot Study."
Cancer 118(7): 1856.
Wojnowski, L., B. Kulle, et al. (2005). "NAD(P)H oxidase and multidrug resistance protein genetic polymorphisms are associated with doxorubicin-induced cardiotoxicity."

Circulation 112(24): 3754-3762.
Zhang, S., X. Liu, et al. (2012). "Identification of the molecular basis of doxorubicin-induced cardiotoxicity." Nat Med 18(11): 1639-1642.

Claims (30)

WHAT IS CLAIMED IS:

1. A method of selecting human subjects for anthracycline compound administration, the method comprising:
(a) performing an amplification reaction using a nucleic acid sample from a subject to amplify polymorphic site: rs2229774 or a polymorphic site in linkage disequilibrium thereto selected from one or more of the following: rs11170479; rs11170481;
rs73309171; and rs57789211;
(b) performing a sequencing reaction using the amplified nucleic acid from (a) to determine whether the subject has a risk genotype selected from the following:

rs2229774 A/A; or rs2229774 A/G; or a reduced risk genotype rs2229774 G/G or the corresponding genotype at a polymorphic site in linkage disequilibrium to rs2229774 selected from one or more of the following: rs11170479; rs11170481;
rs73309171; and rs57789211; and (c) identifying the subject as having a risk genotype or a reduced risk genotype.

2. The method of claim 1, wherein the anthracycline compound is doxorubicin.

3- The method of claim 1 or 2, wherein the polymorphic site is rs2229774 and the risk genotype is rs2229774 A/A or 2229774 A/G and the reduced risk genotype rs2229774 G/G.

4- The method of claim 1, 2 or 3, further comprising selecting a treatment regimen based on the subject's cardiotoxicity risk status, as follows:
(i) a subject with a reduced risk genotype is administered the anthracycline compound;
(ii) a subject with a risk genotype is administered the anthracycline compound and is given heart function monitoring or a cardioprotective agent or both;
(iii) a subject with a risk genotype is administered the anthracycline compound in conjunction with a non-anthracycline anti-neoplastic compound and is given heart function monitoring or a cardioprotective agent or both;
(iv) a subject with a risk genotype is administered a non-anthracycline anti-neoplastic compound.

5- The method of claim 1, wherein the anthracycline is selected from one or more of the following: daunorubicin, daunomycin, rubidomycin, doxorubicin, idarubicin, epirubicin, mitoxantrone, carminomycin, esorubicin, quelamycin, aclarubicin, esorubicin, zorubicin, pirarubicin, amrubicin, iododoxorubicin, mitoxantrone and valrubicin.

6. The method of claim 4, wherein the cardioprotective agent is dexrazoxane.

7- The method of claim 4, wherein the non-anthracycline anti-neoplastic compound is selected from one or more of: cyclophosphamide, ifosphamide, fluorouracil, paclitaxel, vincristine, cisplatin, streptozocin, and docetaxel.

8. A method for assisting in the identification of human subjects at risk for cardiotoxicity from anthracycline compound administration, the method comprising:
(a) performing an amplification reaction using a nucleic acid sample from a subject to amplify polymorphic site r52229774 or a polymorphic site in linkage disequilibrium thereto selected from one or more of the following: rs11170479; rs11170481;
rs73309171; and rs57789211;
(b) performing a sequencing reaction using the amplified nucleic acid from (a) to determine whether the subject has a risk genotype selected from the following:

rs2229774 A/A; or rs2229774 A/G; Or a reduced risk genotype r52229774 G/G or the corresponding genotype at a polymorphic site in linkage disequilibrium to rs222974 selected from one or more of the following: rs11170479; rs11170481;
rs73309171; and rs57789211; and (c) identifying the subject as having a risk genotype or a reduced risk genotype.

9. The method of claim 8, wherein the anthracycline compound is doxorubicin.

10. The method of claim 8 or 9, wherein the polymorphic site is rs2229774 and wherein the risk genotype is rs2229774 A/A or rs2229774 A/G and the reduced risk genotype is rs2229774 G/G.

11. The method of claim 8, further comprising selecting a treatment regimen based on the subject's cardiotoxicity risk status, as follows:
(i) a subject with a reduced risk genotype is administered the anthracycline compound;
(ii) a subject with a risk genotype is administered the anthracycline compound and is given heart function monitoring or a cardioprotective agent or both;
(iii) a subject with a risk genotype is administered the anthracycline compound in conjunction with a non-anthracycline anti-neoplastic compound and is given heart function monitoring or a cardioprotective agent or both;
(iv) a subject with a risk genotype is administered a non-anthracycline anti-neoplastic compound.

12. The method of any one of claims 8-11, wherein the anthracycline is selected from one or more of the following: daunorubicin, daunomycin, rubidomycin, doxorubicin, idarubicin, epirubicin, mitoxantrone, carminomycin, esorubicin, quelamycin, aclarubicin, esorubicin, zorubicin, pirarubicin, amrubicin, iododoxorubicin, mitoxantrone and valrubicin.

13. The method of claim 11, wherein the cardioprotective agent is dexrazoxane.

14. The method of claim 11, wherein the non-anthracycline anti-neoplastic compound is selected from one or more of: cyclophosphamide, ifosphamide, fluorouracil, paclitaxel, vincristine, cisplatin, streptozocin, and docetaxel.

15. Use of one or more anthracycline compounds or one or more non-anthracycline compounds for the treatment of a neoplastic disease in a human subject in need thereof, wherein the treatment depends on the risk genotype as follows:
(a) a subject having a reduced risk genotype rs2229774 G/G would be selected for treatment with one or more anthracycline compounds;
(b) a subject having a risk genotype selected from the following: rs2229774 A/A;
and rs2229774 A/G would be selected for treatment with one or more anthracycline compounds and heart function monitoring or a cardioprotective agent or both;
(c) a subject having a risk genotype selected from the following: rs2229774 A/A;
and rs2229774 A/G would be selected for treatment with one or more anthracycline compounds in conjunction with a non-anthracycline anti-neoplastic compound and heart function monitoring or a cardioprotective agent or both; or (d) a subject having a risk genotype selected from one or more of the following:
rs2229774 A/A; and rs2229774 A/G would be selected for treatment with one or more non-anthracycline compounds.

16. The use of claim 15, wherein the anthracycline compound is doxorubicin.

17. The use of claim 15 or 16, wherein the polymorphic site is rs17863783 and wherein the risk genotype is rs2229774 A/A or rs2229774 A/G and the reduced risk genotype is rs2229774 G/G.

18. The use of claim 15, 16 or 17, wherein the anthracycline is selected from one or more of the following: daunorubicin, daunomycin, rubidomycin, doxorubicin, idarubicin, epirubicin, mitoxantrone, carminomycin, esorubicin, quelamycin, aclarubicin, esorubicin, zorubicin, pirarubicin, amrubicin, iododoxorubicin, mitoxantrone and valrubicin.

19. The use of any one of claims 15-18, wherein the cardioprotective agent is dexrazoxane.

20. The use of any one of claims 15-19, wherein the non-anthracycline anti-neoplastic compound is selected from one or more of: cyclophosphamide, ifosphamide, fluorouracil, paclitaxel, vincristine, cisplatin, streptozocin, and docetaxel.

21. The use of any one of claims 15-20, wherein the neoplastic disease is selected from:
breast cancer, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma, sarcoma, renal cancer and liver cancer.

22. A method for diagnosing a predisposition for cardiotoxicity risk in a human subject from anthracycline administration, the method comprising: a) determining an identity for one or more of the following single nucleotide polymorphisms (SNPs) in a biological sample from the subject: rs2229774 or a polymorphic site in linkage disequilibrium thereto selected from one or more of the following: rs11170479; rs11170481;
rs73309171; and rs57789211; and b) making a cardiotoxicity risk determination based on the prevalence of risk alleles in the subject sample.

23. The method of claim 22, wherein the anthracycline is selected from one or more of the following: anthracycline antibiotics such as daunorubicin (daunomycin, rubidomycin), doxorubicin, idarubicin, epirubicin, mitoxantrone, carminomycin, esorubicin, quelamycin, aclarubicin, esorubicin, zorubicin, pirarubicin, amrubicin, iododoxorubicin, detorubicin, marcellomycin, rodorubicin, and valrubicin.

24. The method of claim 22 or 23, wherein the method further comprises administering the anthracycline in accordance with the subject's risk of developing cardiotoxicity.

25. The method of claim 22, 23 or 24, wherein the subject has a cardiotoxicity risk genotype r52229774 A/A or n2229774 A/G or wherein the subject has the reduced risk genotype n2229774 G/G.

26. The method of any one of claims 22-25, wherein the identity of a single nucleotide polymorphism is determined by one or more of the following techniques:
(a) restriction fragment length analysis;
(b) sequencing;
(c) micro-sequencing assay;
(d) hybridization;
(e) invader assay;
(f) gene chip hybridization assays;
(g) oligonucleotide ligation assay;
(h) ligation rolling circle amplification;
(i) 5' nuclease assay;
(j) polymerase proofreading methods;
(k) allele specific PCR;

(l) matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy;
(m)ligase chain reaction assay;
(n) enzyme-amplified electronic transduction;
(o) single base pair extension assay; and (p) reading sequence data.

27. A use of an anthracycline compound having a cardiotoxicity risk for the treatment of a subject, wherein the subject treated has a reduced cardiotoxicity risk genotype at polymorphic site: rs2229774 or a polymorphic site in linkage disequilibrium thereto selected from one or more of the following: rs11170479; rs11170481;
rs73309171; and rs57789211; for the subject, where the subject is a candidate for anthracycline administration.

28. The use of claim 27, wherein the subject has a reduced cardiotoxicity risk genotype rs2229774 G/G.

29. Anthracyclines for use in a method of treating a neoplastic disease in a subject in need there of, the method comprising:
(a) selecting a subject having a reduced risk of developing cardiotoxicity, wherein cardiotoxicity is based on the identity of a single nucleotide polymorphism (SNP) at polymorphic site: rs2229774 or a polymorphic site in linkage disequilibrium thereto selected from one or more of the following: rs11170479; rs11170481;
rs73309171; and rs57789211; and (b) administering said subject one or more anthracyclines.

30. The anthracycline of claim 29, wherein the anthracycline is selected from one or more of the following: anthracycline antibiotics, daunorubicin, daunomycin, rubidomycin, doxorubicin, idarubicin, epirubicin, mitoxantrone, carminomycin, esorubicin, quelamycin, aclarubicin, esorubicin, zorubicin, pirarubicin, amrubicin, iododoxorubicin, detorubicin, marcellomycin, rodorubicin, and valrubicin.