Use of GWAS and exome sequencing to identify genes relevant to drug-induced liver injury

Ann K. DalyInstitute of Cellular MedicineNewcastle University

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

Drug-induced toxicities associated with 28 drugs withdrawn from the US market 1976 to 2005

Wilke et al. Nature Reviews Drug Discovery 2007; 6, 904-916

Range of drugs give rise to idiosyncratic DILI but due to value of drugs and rarity of problem are still used widely

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

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

UK-wide study on drug-induced liver injury

Worldwide study on DILI genetics-Ann Daly and Guru Aithal-co-chairs

study

Genome-wide association study (GWAS) on flucloxacillin DILI

51 UK cases (DILIGEN study) and population control group (n=282)

Illumina IM chipNo previous genetic studies on this form of

DILIGWAS design allowed for an open study

Manhattan plot for flucloxacillin GWAS

MHC regionStrongest signal with SNP in HCP5 gene which tags HLA-B*57:01 Daly et al., Nature Genetics 2009; 41:816-822.

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

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

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

Amoxicillin-clavulanate DILI GWAS

MHC zoom-in, conditioned on the top SNP from class II (rs9274407)

rs2523822 (A*02:01)

MHC zoom-in

rs9274407rs3135388 (DRB1*15:01)

201 cases- UK DILIGEN (n=77), US DILIN (n=56), Spanish DILI (n=49), EUDRAGENE (n=19); 532 matched controls

MHC region

(Lucena et al., Gastroenterology 2011)

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 (3.67-24.5), p=2.56 x 10-7

Evidence for genetic interaction (p=0.0015)

HLA associations with DILI

Daly, Annual Review of Pharmacology & Toxicology 2012; 52:21-35

FlucloxacillinAbacavir

Clavulanic acid Lumiracoxib

LapatanibXimelagatran

DRB1*07:01-DQA1*02:01

DRB1*15:01

B*57:01

Chemical structures and HLA toxicity associations

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

Abacavir-B*57:01 interaction

From Illing et al., Nature 2012

Norcross et al., 2012; AIDS 26: F21-F29

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?

Very recent findings suggest possible direct effect of flucloxacillin in some B*57:01-positive healthy volunteers

o No covalent adduct

Need to incubate with flucloxacillin for 24 to 48 hours to see cell proliferation

o Covalent adduct formedo Hapten mechanism likely

CD8-positive T cell clones from B*57:01-positive flucloxacillin DILI cases: response to various penicillins

Monshi et al., Hepatology 2013

Wuillemin et al., J Immunol, 2013

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

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

All drug DILI GWAS results

• 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"

All

WithoutFluclox/amoxicillin-clavulanateAll with HLA taken out

Urban et al, 2013 Pharmacogenetics and Genomics

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

Lumiracoxib Diclofenac

HLA-DRB1*15:01 No significant HLA class II association

DILI and related NSAIDs

• Both drugs metabolized by broadly similar pathways

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; 132:272-281.

Majority of these cases plus additional cases from UK/US included in "all drug" GWAS

GWAS on diclofenac DILI cases (n=30)

GWAS QQ ADME gene QQ

Urban et al, 2013 Pharmacogenetics and Genomics

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

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

Controls for exome sequencing/exome chip studies Exome sequencing

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)

Amoxicillin-clavulanate: best results for sequencing plus exome chip with combined data also shown

SGSM3: Small G Protein Signaling Modulator 3

CHR BPSEQ_MAF_

ASEQ_MAF_

U SEQ_PSEQ_O

REC_MAF_

A EC_MAF_U EC_PEC_O

R GENE FUNCT PPHENEVS_MA

F P OR

19 57868483 0 0.021 2.0E-02 0 0.005 0.027 1.4E-02 0.2 ZNF304 missense_variant benign 0.030 3.7E-06 0.1

22 40803845 0.013 0.002 6.3E-02 5.8 0.005 0.000 1.4E-02 19.1 SGSM3 splice_region_variantpossibly_damagin

g 0.000 1.3E-05 27.0

7 107599806 0.114 0.075 6.3E-02 1.6 0.107 0.062 6.6E-04 1.8 LAMB1 missense_variant probably_damaging 0.068 4.0E-05 1.8

15 80137560 0.136 0.081 1.6E-02 1.8 0.135 0.084 5.5E-04 1.7 MTHFS missense_variant benign 0.089 5.1E-05 1.7

14 57092112 0.030 0.012 7.3E-02 2.5 0.024 0.011 1.6E-02 2.3 C14orf101 missense_variant benign 0.006 1.2E-04 3.1

10 55566406 0.360 0.287 3.2E-02 1.4 0.366 0.290 3.0E-03 1.4 PCDH15 missense_variant NA 0.295 1.2E-04 1.4

15 27890491 0.411 0.493 2.8E-02 0.7 0.424 0.498 9.2E-03 1.3 NA NA NA 0.491 1.7E-04 0.7

2 190331180 0.008 0 4.2E-02 NA 0.002 0.000 5.0E-02 NA WDR75 missense_variant possibly_damaging 0.000 1.8E-04 80.8

15 77329417 0.008 0 4.2E-02 NA 0.005 0.000 7.2E-03 38.3 PSTPIP1 missense_variant possibly_damaging 0.000 1.9E-04 21.3

5 96124330 0.415 0.356 9.6E-02 1.3 0.432 0.349 1.7E-04 1.5 ERAP1 missense_variant benign 0.357 1.9E-04 1.4

6 121544432 0.004 0 2.1E-01 NA 0.005 0.000 2.5E-03 NA C6orf170 missense_variant benign 0.000 1.9E-04 78.7

5 96129512 0.267 0.226 2.0E-01 1.2 0.293 0.217 8.6E-05 1.6 ERAP1 synonymous_variant NA 0.220 2.0E-04 1.4

15 42054450 0.008 0 4.2E-02 NA 0.005 0.000 1.4E-02 19.1 MGA missense_variant benign 0.000 2.0E-04 21.1

8 8748032 0.013 0 8.6E-03 NA 0.012 0.002 2.5E-03 6.4 MFHAS1 missense_variant possibly_damaging 0.003 2.8E-04 5.4

5 82491674 0.072 0.034 1.6E-02 2.2 0.073 0.043 1.3E-03 1.9 XRCC4 missense_variant possibly_damaging 0.041 3.0E-04 1.8

2 95947085 0.174 0.223 1.1E-01 0.7 0.176 0.226 6.2E-03 0.7 PROM2 missense_variant benign 0.245 3.1E-04 0.7

2 73786188 0.008 0 4.2E-02 NA 0.005 0.001 3.3E-02 9.6 ALMS1 missense_variant possibly_damaging 0.000 3.2E-04 17.6

17 34893326 0.021 0.068 4.6E-03 0.3 0.029 0.061 1.8E-02 0.5 MYO19 upstream_gene_variant NA 0.053 3.3E-04 0.4

17 72768191 0.008 0.002 1.9E-01 3.9 0.005 0.000 2.5E-03 NA SLC9A3R1 downstream_gene_variant NA 0.001 4.5E-04 15.4

16 77465249 0.004 0 2.1E-01 NA 0.010 0.001 2.2E-03 9.6 ADAMTS18 missense_variant benign 0.001 4.5E-04 9.6

SNP CHR BP MAF_A MAF_U P OR GENE FUNCTION POLYPHENexm-IND5-149355072 5 149374880 0.505 0.398 2.3E-06 1.6 NA NA NAexm813093 10 18439900 0.015 0.001 5.4E-06 23.3 CACNB2 missense_variant benignexm1621613 22 50687883 0.010 0.000 6.1E-06 NA MAPK12 downstream_gene_variant NAexm2267521 13 51133655 0.320 0.445 6.8E-06 0.6 NA NA NAexm748945 9 35618065 0.015 0.001 1.1E-05 19.3 CD72 missense_variant benign

exm1074032 13 86368449 0.029 0.006 2.2E-05 5.1 SLITRK6 missense_variant possibly_damagingexm198643 2 68622914 0.193 0.283 2.9E-05 0.6 PLEK missense_variant benignexm177347 2 25141529 0.010 0.000 2.9E-05 76.9 ADCY3 missense_variant possibly_damaging

exm1026884 12 94965488 0.012 0.001 3.2E-05 24.1 TMCC3 missense_variant probably_damagingexm2270519 6 128709493 0.129 0.077 4.1E-05 1.9 PTPRK intron_variant NAexm941422 11 74883577 0.156 0.097 5.2E-05 1.8 SLCO2B1 missense_variant benign

exm2269450 3 53282188 0.317 0.417 6.4E-05 0.6 TKT intron_variant NAexm-IND19-62843150 19 58151339 0.498 0.398 6.4E-05 1.5 NA NA NA

exm1564814 21 31587859 0.090 0.049 9.6E-05 2.0D21S2091

E upstream_gene_variant NAexm848765 10 101667814 0.068 0.029 1.2E-04 2.3 DNMBP missense_variant benignexm681525 8 10469846 0.007 0.000 1.2E-04 NA RP1L1 missense_variant probably_damaging

exm1389227 18 56400644 0.007 0.000 1.2E-04 NA MALT1 missense_variant probably_damaging

Amoxicillin-clavulanate-exome chip only

CACNB2 not well covered in exome sequencing

Flucloxacillin – Exome chip

• 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

SNP CHR BP P OR F-A F-U A1 GENE FUNCTION (PPH)exm518448 6 17602870 7.26E-26 20.77 0.2571 0.02458 A NA NA (NA)exm1161045 15 50785016 1.46E-23 11.49 0.5071 0.1445 G USP8 missense (probably-damaging)exm-rs1497546 3 98034526 5.36E-06 4.326 0.1071 0.02752 A NA NA (NA)exm2255202 2 241516094 8.85E-06 4.441 0.09286 0.02424 C RNPEPL1 coding-synonymous (unknown)exm-rs4984390 15 94939508 1.77E-05 0.3766 0.1786 0.3613 G NA NA (NA)exm-rs6582630 12 38743508 6.09E-05 2.091 0.6286 0.4595 G NA NA (NA)exm305107 3 43602803 6.71E-05 0.4259 0.2214 0.4029 C ANO10 missense (benign)exm781146 9 127618776 8.72E-05 19.75 0.02143 0.003151 A WDR38 missense (benign)exm1329395 17 42927723 9.23E-05 3.943 0.08571 0.025 A EFTUD2,HIGD missense (possibly-damaging)exm1223806 16 20966362 9.65E-05 2.657 0.1786 0.07941 C DNAH3 missense (probably-damaging)exm1223776 16 20959918 0.0001076 3.084 0.1214 0.05063 A DNAH3 missense (probably-damaging)exm359484 3 151166058 0.0001385 3.397 0.09286 0.03592 A IGSF10 missense (benign)exm359277 3 151161112 0.0001416 3.39 0.09286 0.03613 G IGSF10 missense (benign)exm1588866 22 21989142 0.0001429 4.222 0.07143 0.02248 A CCDC116 missense (probably-damaging)exm1588898 22 21990823 0.0001429 4.222 0.07143 0.02248 A CCDC116 missense (benign)exm1588914 22 21991120 0.0001429 4.222 0.07143 0.02248 A CCDC116 missense (benign)exm952418 11 104879658 0.000163 3.219 0.1071 0.03765 A CASP5 missense (possibly-damaging)

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

Successfully adjudicated new cases in iDILIC to date

Cases mainly from Europe GWAS-exome chip in progress on 747 of these cases Agreed collaboration with DILIN may improve power

Number of cases

Others

Valproate

Sartans

Proton pump inhibitors

Carbamazepine

Co-trimoxazole

Estrogen containing drugs

Anti-TNF agents

Quinolones

Nitrofurantoin

Other NSAIDs

Diclofenac

Flucloxacillin

0 50 100 150 200 250

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

Acknowledgements

NewcastleChris DayPete DonaldsonJulian LeathartJulia PatchRuth WakePallav BhatnagarHeather CordellTom ChamberlainSally Coulthard

Guru Aithal (Nottingham)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)

Supported by the UK Department of Health Pharmacogenetics Initiative and International Serious Adverse Events Consortium

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

Spraggs C F et al. JCO 2011;29:667-673

Human diclofenac toxicity: possible relationship to metabolism

Biliary excretion

MRP2

Covalent adducts

Diclofenac

4'-OH-diclofenac

Diclofenacacylglucuronide

CYP2C9

UGT2B7

Liver injury

Other oxidative metabolitesVariousCYPs

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