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Ann K. Daly Institute of Cellular Medicine Newcastle University

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1 Ann K. Daly Institute of Cellular Medicine Newcastle University
Use of GWAS and exome sequencing to identify genes relevant to drug-induced liver injury Ann K. Daly Institute of Cellular Medicine Newcastle University

2 Idiosyncratic adverse drug reations
Serious health problem which is expensive for society Common cause of death Loss of new drugs late in development or soon after licensing Typical incidence 1 in 10,000 to 100,000 patients exposed Local drug concentration may contribute but concentration-independent effects important Contribution from immune system in many but not all cases Examples Drug-induced liver injury Hypersensitivity reactions and skin rash Cardiotoxicity Muscle toxicity 2 2

3 Drug-induced toxicities associated with 28 drugs withdrawn from the US market 1976 to 2005
Range of drugs give rise to idiosyncratic DILI but due to value of drugs and rarity of problem are still used widely Wilke et al. Nature Reviews Drug Discovery 2007; 6,

4 Drug-induced liver injury (DILI)
Rare but serious idiosyncratic toxicity Several phenotypes Cholestatic Effects on bile secretion Hepatocellular Inflammatory disease Aims Develop strategies to prevent DILI reactions by genotyping patients prior to treatment Develop screening approaches for use during drug development 4

5 UK-wide study on drug-induced liver injury
Aim: Find genes predisposing to DILI Drugs Anti-TB medication or Flucloxacillin or Amoxicillin-clavulanate Multicentre collaboration involving Newcastle, Liverpool, Nottingham and London UK-wide recruitment of cases Retrospective and prospective 5 5

6 Table 1. Case adjudication results
study Worldwide study on DILI genetics-Ann Daly and Guru Aithal-co-chairs Genotyping methods Confirmed cases of flucloxacillin DILI were genotyped for HLA-B*57:01 by genotyping for rs in HCP5. The assay involved amplification of a 250bp fragment by PCR followed by digestion with BstNI and resolution of restriction digest patterns on an 10% polyacrylamide gel. Positive cases were confirmed by direct genotyping using SSP-PCR for HLA-B*57:01 using an AllSet+™ Gold SSP B17 High Res Kit (Invitrogen). Results Recruitment and Adjudication A total of 1333 cases have been recruited to date. As summarised in Table 1, 856 (64%) have passed adjudication. Table 1. Case adjudication results The ethnicity of cases is summarized in Table 2 which shows that the vast majority of study participants are of white European ethnicity. Table 2. Case ethnicity Samples for genome-wide association study From the 856 samples that have passed adjudication, 747 have been selected for inclusion in a genome-wide association study (GWAS) on the basis of the number of cases available for particular drugs. The GWAS is being performed in collaboration with the US DILIN network and will include ethnically-matched controls from selected worldwide control sets including the POPRES cohort used in previous studies. Rare coding variants will also be genotyped (exome chip). The drugs represented among the iDILIC cases are shown in Figure 2. Fig 2: Number of cases relating to particular drugs included in the genome-wide association study. The other drugs category comprises imatinib, coumarin anticoagulants, methotrexate, sulphasalazine, ticlopidine, montelukast, enalapril, cyproterone, cyclophosphamide, ebrotidine, fenofibrate, sevoflurane, dronedarone, gabapentin, phenytoin, lamotrigine, interferon and retinoids. The anti-TB drug category includes various combinations of isoniazid, rifampicin, pyrazinamide and ethambutol.

7 Genome-wide association study (GWAS) on flucloxacillin DILI
51 UK cases (DILIGEN study) and population control group (n=282) Illumina IM chip No previous genetic studies on this form of DILI GWAS design allowed for an open study 7

8 Manhattan plot for flucloxacillin GWAS
MHC region Strongest signal with SNP in HCP5 gene which tags HLA-B*57:01 Daly et al., Nature Genetics 2009; 41: 8

9 Flucloxacillin DILI and B*57:01
Association with HLA B*57:01 similar to that for abacavir hypersensitivity 83% of 150 cases now studied are B*57:01-positive No indication that other forms of DILI show this particular association Sensitivity of B*57:01 genotyping lower as predictor of flucloxacillin DILI than abacavir hypersensitivity Other genetic and non-genetic risk factors may contribute 9 9 9

10 HLA genes-common thread in serious adverse drug reactions
HLA class I genes expressed on most cells Proteins present antigens (particularly peptides) to CD8-positive cytotoxic T cells A, B and C genes HLA class II genes expressed on antigen presenting cells Proteins present antigens to T cells-mainly CD4-positive T helper cells DR, DQ, DP genes 10

11 Amoxicillin-clavulanate-induced liver injury
Important cause of DILI worldwide Several small independent candidate gene studies showed role for HLA class II DRB1*15:01 allele in susceptibility GWAS involving international collaboration performed to identify additional risk factors 11

12 Amoxicillin-clavulanate DILI GWAS
201 cases- UK DILIGEN (n=77), US DILIN (n=56), Spanish DILI (n=49), EUDRAGENE (n=19); 532 matched controls MHC region MHC zoom-in rs rs (DRB1*15:01) MHC zoom-in, conditioned on the top SNP from class II (rs ) rs (A*02:01) (Lucena et al., Gastroenterology 2011) 12

13 HLA typing and amoxicillin-clavulanate DILI
Detailed HLA typing performed on 177 cases and 219 controls DRB1*15:01 and A*02:01 alleles associated independently with risk of DILI (OR approx. 2.5 for each) Additional risk also seen from individual A*02:01 and DRB1*15:01 alleles (not on same haplotype) If positive for both OR= 9.47 ( ), p=2.56 x 10-7 Evidence for genetic interaction (p=0.0015) 13

14 HLA associations with DILI
Daly, Annual Review of Pharmacology & Toxicology 2012; 52:21-35 14 14

15 Chemical structures and HLA toxicity associations
DRB1*07:01-DQA1*02:01 Lapatanib Ximelagatran DRB1*15:01 Clavulanic acid Lumiracoxib B*57:01 Abacavir Flucloxacillin 15 15

16 HLA and related gene associations with DILI-underlying mechanism
Specific HLA protein interacts with drug or metabolite inappropriately Triggers T-cell response Local cellular damage 16 16

17 Abacavir-B*57:01 interaction
From Illing et al., Nature 2012

18 Effect of different drugs on peptide binding in B
Effect of different drugs on peptide binding in B*57:01-positive cell line No apparent direct effect by flucloxacillin unlike abacavir Different mechanism for antigen presentation to abacavir? Norcross et al., 2012; AIDS 26: F21-F29

19 CD8-positive T cell clones from B
CD8-positive T cell clones from B*57:01-positive flucloxacillin DILI cases: response to various penicillins Need to incubate with flucloxacillin for 24 to 48 hours to see cell proliferation Covalent adduct formed Hapten mechanism likely Monshi et al., Hepatology 2013 Very recent findings suggest possible direct effect of flucloxacillin in some B*57:01-positive healthy volunteers No covalent adduct Wuillemin et al., J Immunol, 2013

20 Is metabolism of flucloxacillin relevant to DILI reactions?
Flucloxacillin is a PXR ligand so may induce its own metabolism? Induction will also affect bile acid synthesis and transporter levels Our attempts to demonstrate that CYP3A4 or other CYPs can generate 5'-hydroxymethyl metabolite unsuccessful Human liver microsomes with 1 mM flucloxacillin Overlay of 5'-hydroxymethylflucloxacillin standard

21 Studies on pooled DILI cases
DILI due to any prescribed drug Cases from UK (Diligen), Spain (Spanish DILI) and US (DILIN) Total number of cases =783 21

22 All drug DILI GWAS results
Without Fluclox/amoxicillin-clavulanate All with HLA taken out 783 cases, 3,001 population controls After correcting for known HLA risk alleles for flucloxacillin- and amoxicillin-clavulanate-DILI, no deviation from expected p-value distribution No SNPs in HLA region approaching genome-wide significance for "other drugs" Urban et al, 2013 Pharmacogenetics and Genomics 22

23 Diclofenac-induced liver injury
Rare toxicity associated with diclofenac use Serious hepatotoxicity 11 per 100,000 patients Believed to be due to metabolic idiosyncracy ?Specific reactive metabolite which binds covalently to proteins

24 DILI and related NSAIDs
Lumiracoxib Diclofenac No significant HLA class II association HLA-DRB1*15:01 Both drugs metabolized by broadly similar pathways

25 Candidate gene studies on genetic susceptibility to diclofenac-related DILI
UGT2B7*2 and ABCC2 C-24T previously identified as significant risk factors in small (n=24) candidate gene study Daly et al. Gastroenterology 2007; 132: Majority of these cases plus additional cases from UK/US included in "all drug" GWAS

26 GWAS on diclofenac DILI cases (n=30)
GWAS QQ ADME gene QQ Urban et al, 2013 Pharmacogenetics and Genomics

27 GWAS hits on diclofenac DILI cases and data on ADME genes as candidates
PPAR-gamma association did not replicate in 26 additional cases ADME associations in agreement with our earlier UGT2B7 finding but PXR an interesting additional candidate

28 Exome sequencing and exome chip studies on DILI
Exome sequencing performed on 125 amoxicillin-clavulanate DILI cases 66 from UK and 59 from Spain All from previous GWAS Exome chip studies 112 further amoxicillin-clavulanate DILI cases from iDILIC study and 93 from US DILIN network for replication of exome sequencing 73 UK flucloxacillin DILI cases

29 Controls for exome sequencing/exome chip studies
353 NIMH (white US) plus 102 from Spain (EGA) Exome chip 3900 controls from several cohorts Combined studies 4300 controls from European sequencing project (EVS)

30 splice_region_variant
Amoxicillin-clavulanate: best results for sequencing plus exome chip with combined data also shown CHR BP SEQ_MAF_A SEQ_MAF_U SEQ_P SEQ_OR EC_MAF_A EC_MAF_U EC_P EC_OR GENE FUNCT PPHEN EVS_MAF P OR 19 0.021 2.0E-02 0.005 0.027 1.4E-02 0.2 ZNF304 missense_variant benign 0.030 3.7E-06 0.1 22 0.013 0.002 6.3E-02 5.8 0.000 19.1 SGSM3 splice_region_variant possibly_damaging 1.3E-05 27.0 7 0.114 0.075 1.6 0.107 0.062 6.6E-04 1.8 LAMB1 probably_damaging 0.068 4.0E-05 15 0.136 0.081 1.6E-02 0.135 0.084 5.5E-04 1.7 MTHFS 0.089 5.1E-05 14 0.012 7.3E-02 2.5 0.024 0.011 2.3 C14orf101 0.006 1.2E-04 3.1 10 0.360 0.287 3.2E-02 1.4 0.366 0.290 3.0E-03 PCDH15 NA 0.295 0.411 0.493 2.8E-02 0.7 0.424 0.498 9.2E-03 1.3 0.491 1.7E-04 2 0.008 4.2E-02 5.0E-02 WDR75 1.8E-04 80.8 7.2E-03 38.3 PSTPIP1 1.9E-04 21.3 5 0.415 0.356 9.6E-02 0.432 0.349 1.5 ERAP1 0.357 6 0.004 2.1E-01 2.5E-03 C6orf170 78.7 0.267 0.226 2.0E-01 1.2 0.293 0.217 8.6E-05 synonymous_variant 0.220 2.0E-04 MGA 21.1 8 8.6E-03 6.4 MFHAS1 0.003 2.8E-04 5.4 0.072 0.034 2.2 0.073 0.043 1.3E-03 1.9 XRCC4 0.041 3.0E-04 0.174 0.223 1.1E-01 0.176 6.2E-03 PROM2 0.245 3.1E-04 0.001 3.3E-02 9.6 ALMS1 3.2E-04 17.6 17 4.6E-03 0.3 0.029 0.061 1.8E-02 0.5 MYO19 upstream_gene_variant 0.053 3.3E-04 0.4 1.9E-01 3.9 SLC9A3R1 downstream_gene_variant 4.5E-04 15.4 16 0.010 2.2E-03 ADAMTS18 SGSM3: Small G Protein Signaling Modulator 3 30

31 Amoxicillin-clavulanate-exome chip only
SNP CHR BP MAF_A MAF_U P OR GENE FUNCTION POLYPHEN exm-IND 5 0.505 0.398 2.3E-06 1.6 NA exm813093 10 0.015 0.001 5.4E-06 23.3 CACNB2 missense_variant benign exm 22 0.010 0.000 6.1E-06 MAPK12 downstream_gene_variant exm 13 0.320 0.445 6.8E-06 0.6 exm748945 9 1.1E-05 19.3 CD72 exm 0.029 0.006 2.2E-05 5.1 SLITRK6 possibly_damaging exm198643 2 0.193 0.283 2.9E-05 PLEK exm177347 76.9 ADCY3 exm 12 0.012 3.2E-05 24.1 TMCC3 probably_damaging exm 6 0.129 0.077 4.1E-05 1.9 PTPRK intron_variant exm941422 11 0.156 0.097 5.2E-05 1.8 SLCO2B1 exm 3 0.317 0.417 6.4E-05 TKT exm-IND 19 0.498 1.5 exm 21 0.090 0.049 9.6E-05 2.0 D21S2091E upstream_gene_variant exm848765 0.068 1.2E-04 2.3 DNMBP exm681525 8 0.007 RP1L1 exm 18 MALT1 CACNB2 not well covered in exome sequencing

32 Flucloxacillin – Exome chip
SNP CHR BP P OR F-A F-U A1 GENE FUNCTION (PPH) exm518448 6 7.26E-26 20.77 0.2571 A NA NA (NA) exm 15 1.46E-23 11.49 0.5071 0.1445 G USP8 missense (probably-damaging) exm-rs 3 5.36E-06 4.326 0.1071 exm 2 8.85E-06 4.441 C RNPEPL1 coding-synonymous (unknown) exm-rs 1.77E-05 0.3766 0.1786 0.3613 exm-rs 12 6.09E-05 2.091 0.6286 0.4595 exm305107 6.71E-05 0.4259 0.2214 0.4029 ANO10 missense (benign) exm781146 9 8.72E-05 19.75 WDR38 exm 17 9.23E-05 3.943 0.025 EFTUD2,HIGD missense (possibly-damaging) exm 16 9.65E-05 2.657 DNAH3 exm 3.084 0.1214 exm359484 3.397 IGSF10 exm359277 3.39 exm 22 4.222 CCDC116 exm exm exm952418 11 3.219 CASP5 USP8: ubiquitin specific protease 8 Chromosome 6 includes processed pseudogene with strong homology so finding may due to LD with B*57:01 CASP5-caspase 5 Replication studies on this looking positive

33 Sequencing and exome chip studies on DILI: progress so far and where next?
Studies show little evidence that rare coding variants are consistently contributing to risk of DILI due to amoxicillin-clavulanate and flucloxacillin Findings fairly similar to those for other complex diseases Moving towards whole genome sequencing is challenging but may be valuable Further GWAS involving new sample collection with improved power may identify novel associations

34 Successfully adjudicated new cases in iDILIC to date
Number of cases Cases mainly from Europe GWAS-exome chip in progress on 747 of these cases Agreed collaboration with DILIN may improve power

35 Summary Genomics has increased understanding of individual risk for idiosyncratic drug-induced liver injury HLA associations Strong association for flucloxacillin toxicity with HLA-B*57:01 Underlying mechanism could involve inappropriate T cell response Probably does not involve metabolism HLA genotype not predictor of DILI with all hepatotoxic drugs Increasing evidence that genetically-determined metabolism to reactive intermediate is an important step in some idiosyncratic DILI reactions Diclofenac Attempts to identify additional risk factors by exome sequencing largely unsuccessful to date Larger sample collections for individual drugs and genome sequencing may provide more complete understanding and better application to drug development process 35

36 Acknowledgements Guru Aithal (Nottingham) Newcastle
Munir Pirmohamed (Liverpool) Kevin Park (Liverpool) Dean Naisbitt (Liverpool) Yufeng Shen (Columbia) Aris Floratos (Columbia) Mark Daly (Harvard) Jackie Goldstein (Harvard) Matt Nelson (GSK) Paul Watkins (North Carolina) Tom Urban (Duke) Maribel Lucena (Malaga) Raul Andrade (Malaga) Mariam Molokhia (London) Newcastle Chris Day Pete Donaldson Julian Leathart Julia Patch Ruth Wake Pallav Bhatnagar Heather Cordell Tom Chamberlain Sally Coulthard Supported by the UK Department of Health Pharmacogenetics Initiative and International Serious Adverse Events Consortium 36 36 36

37 37

38

39 Cumulative incidence of ALT elevations in lapatinib-plus-letrozole treatment group for HLA-DQA1*02:01 carrier and noncarrier subsets, compared with letrozole-only treatment group Cumulative incidence of ALT elevations to greater than 3 × upper limit of normal in lapatinib-plus-letrozole treatment group for HLA-DQA1*02:01 carrier and noncarrier subsets, compared with letrozole-only treatment group in the confirmatory study. Spraggs C F et al. JCO 2011;29:

40 Human diclofenac toxicity: possible relationship to metabolism
Liver injury Covalent adducts Other oxidative metabolites Various CYPs Diclofenac acylglucuronide UGT2B7 Diclofenac CYP2C9 MRP2 4'-OH-diclofenac Biliary excretion

41 Abacavir hypersensitivity-success story for pharmacogenomic screening
Abacavir is a reverse-transcriptase inhibitor AIDS treatment Approx. 5% of patients develop hypersensitivity reactions which resolve on discontinuation Rechallenge results in more severe reaction Up to 100% of proven hypersensitivity cases have one or two HLA B*57:01 alleles Not all patients with this genotype will show detectable reaction 41


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