hla-b5701 genotype is a major determinant of drug-induced ......1 hla-b5701 genotype is a major...

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HLA-B*5701 genotype is a major determinant of drug-induced liver injury due to

flucloxacillin

Ann K Daly1, Peter T Donaldson1, Pallav Bhatnagar1, Yufeng Shen2, Itsik Pe'er2, Aris

Floratos2, Mark J Daly3, David B Goldstein4, Sally John5, Matthew R Nelson6, Julia

Graham1, B Kevin Park7, John F Dillon8, William Bernal9, Heather J Cordell1, Munir

Pirmohamed7, Guruprasad P Aithal10,11, Christopher P Day1,11 for the DILIGEN study12 and

International SAE Consortium12

Nature Genetics: doi:10.1038/ng.379

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Supplementary note

DILIGEN study team

Investigators: AK Daly (PI), CP Day, PT Donaldson, C Donaldson (Newcastle University); GP

Aithal (Nottingham Digestive Diseases Centre); M Pirmohamed, BK Park, S Khoo, I Gilmore

(University of Liverpool); W Bernal (Kings College Hospital, London). Research nurses: J

Henderson (Newcastle University); C Davies (Nottingham Digestive Diseases Centre); K Hawkins,

A Hanson, J Evely (University of Liverpool); J Calera (Kings College Hospital). Other contributors

to flucloxacillin case recruitment: N Thompson (Freeman Hospital, Newcastle), R Williams and M

Morgan (Royal Free and University College Medical School), H Hussaini (Truro), EM Phillips

(Hexham), P Mills (Glasgow), M Groom and M Miller (Ninewells Hospital, Dundee), M Patel

(Merthyr Tydfil), H Mitchison (Sunderland), W Griffiths (Addenbrooks Hospital, Cambridge), JG

Kingham (Singleton Hospital, Swansea), D Das (Stepping Hill Hospital, Stockport), J Collier (John

Radcliffe Infirmary, Oxford), A Brind (North Staffordshire), N Fisher (Dudley), J Shearman (South

Warwick), E Elias (Birmingham), A Grant (Leicester Royal Infirmary), S Hellier (Worcester), A

Austin (Derby).

International serious adverse events consortium (SAEC) management team

Arthur L Holden [SAEC], Brian Spear [Abbott], Joe Walker [Daiichi-Sankyo], Dan Burns

[GlaxoSmithKline], Lon Cardon [GlaxoSmithKline], Eric Lai [GlaxoSmithKline], Matt Nelson

[GlaxoSmithKline], Allen Roses [GlaxoSmithKline], Nadine Cohen [Johnson & Johnson],

Quingqin Serena Li [Johnson & Johnson], Joanne Meyer [Novartis], Steve Lewitzky [Novartis],

Sally John [Pfizer], Duncan McHale [Pfizer], Klaus Lindpaintner [Roche], Steven Kovacs [Sanofi-

Aventis], Leonardo Sahelijo [Takeda], Michael Dunn [Wellcome Trust], Maha C Karnoub [Wyeth],

and Michael E Burczynski [Wyeth].


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Supplementary Results

Eigenstrat analysis

To account for the possibility of associations resulting from population stratification, we applied

EIGENSTRAT on the data set. The chi-square statistics from trend test conditioned on the first 10

eigen-vectors were in close correlation with the original trend test (correlation coefficient = 0.91;

Supplementary Fig. 2a), and the signals from chromosome 6 and the top SNPs were still genome-

wide significant (Supplementary Fig. 2b).

Copy Number Variation (CNV) analysis

We exported the Log R Ratio (LRR) and B Allele Frequency (BAF) values of all the Illumina 1M

probes for all samples from Illumina BeadStudio with default parameters. The LRR value is the

normalized signal intensity for each probe. We discarded samples with large LRR standard

deviations or low average LRR values to prevent spurious CNV calls (Supplementary Fig. 4).

Eleven cases and 10 population controls were removed. We used PennCNV1 to call CNVs for each

individual samples based on LRR and BAF values (Supplementary Table 9). We then applied

PLINK v1.04 to calculate the association of CNVs in the remaining 40 cases and 272 controls. Only

one CNV region from chromosome 11 was significantly associated after permutation: 5 cases

carried 3-copy duplications, 2 cases carried 1-copy deletions, while none of the controls carried it

(p-value = 0.02 after genome-wide permutation). The size of the CNVs varied from 103 kb to 214

kb, with a common region of 50 kb (48,756,916 bp to 48,807,363 bp). The CNV region is about

200 kb away from genes OR4A47 and FOLH1. It is flanked by segmental duplications, and the

location was reported to have CNVs by previous studies (Supplementary Fig. 5)2,3.

For the samples removed due to failure to pass quality control, data for the CNVs that are larger

than 100 kb and contain no less than 10 probes is still likely to be correct. Among the 11 cases and


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10 controls removed, there were two cases carrying chromosome 11 duplications similar to those

described above, one case carrying the deletion and one control carrying the duplication. When

combined with the data from samples that had passed quality control, nominal p-values of 1.4x10-6

(permutation p-value 0.00014) for duplication only and 3x10-8 (permutation p-value 2.3x10-5) for

duplications and deletions were obtained.

References

1. Wang, K. et al. PennCNV: an integrated hidden Markov model designed for high-resolution copy number variation detection in whole-genome SNP genotyping data. Genome Res 17, 1665-74 (2007).

2. Iafrate, A.J. et al. Detection of large-scale variation in the human genome. Nat Genet 36, 949-51 (2004).

3. Redon, R. et al. Global variation in copy number in the human genome. Nature 444, 444-54 (2006).


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Supplementary Table 1. Clinical and biochemical variables of DILI patients exposed to flucloxacillin

Sex (F/M) 36/15

Age at onset (yrs) 63.1 ± 12.2

Time to onset (days) 25.4 ± 22.4

Total days on drug 11.3 ± 7.8

Pattern of liver injury

Cholestatic 44 (86.3)

Hepatocellular 4 (7.8)

Mixed 3 (5.9)

ICC scoring

3-5 (possible) 4 (7.8)

6-8 (probable) 18 (35.3)

>8 (highly probable) 29 (56.9)

Peak Bilirubin (µmol/l) 271 ± 240

Peak ALT (U/l) 398 ± 264

Peak ALP (U/l) 644 ± 874

ALT/ALP decreased by ≥

50% above ULN after drug

discontinuation

Yes 46 (90.2)

No 3 (5.9)

Not known 2 (3.9)

Time taken for ALT/ALP to

decrease to ≥ 50% after

discontinuation 74 days ± 62 days

Percentages are shown in parentheses


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Supplementary Table 2. Number of markers removed in GWAS quality control steps

Criteria Threshold Number of markers

removed

Genotyping missingness > 0.05 27,941

Hardy-Weinberg

Equilibrium test

p <= 10-7 in controls 1,034

Minor Allele Frequency < 0.01 200,835 *

Total number of markers

removed

204,231 **

* This includes 136,223 CNV-specific markers, which are all non-polymorphic.

** The total is not the sum of the numbers from three categories due to overlap.


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Supplementary Table 3. rs2395029 genotypes in cases and POPRES controls

G/G G/T T/T P-value OR (95%CI)

Controls (n=282) 252 (89.4) 30 (10.6) 0

Cases (n=51) 8 (15.7) 39 (76.5) 4 (7.8) 8.7 x10-33 45 (19.4-105)

The odds ratio is for carriage of the T variant and assumes a dominant model. Numbers in

parentheses are percentages. The p-value is not corrected for multiple testing.


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Supplementary Table 4. Distribution of HLA-DRB1 alleles in flucloxacillin DILI cases and drug-exposed controls

HLA DRB1 allele

DILI cases (n=51) DILI controls (n=64)

p-value pc OR (95% CI)

Positive for allele

Negative for allele

Positive for allele

Negative for allele

* 01 7 (13.7) 44 (86.3) 13 (20.3) 51 (79.7) 0.459 * 03 16 (31.4) 35 (68.6) 24 (37.5) 40 (62.5) 0.557 * 04 10 (19.6) 41 (80.4) 22 (34.3) 42 (65.7) 0.096 * 07 36 (70.6) 15 (29.4) 16 (25.0) 48 (75.0) 1.61x10-

6 1.9x10-5 7.2 (3.15

- 16.45) * 08 5 (9.8) 46 (90.2) 1 (1.6) 63 (98.4) 0.086 * 09 0 (0.0) 51

(100.0) 2 (3.1) 62 (96.9) 0.502

* 10 0 (0.0) 51 (100.0)

1 (1.6) 63 (98.4) 1

* 11 4 (7.8) 47 (92.2) 4 (6.3) 60 (93.7) 1 * 13 9 (17.6) 42 (82.4) 14 (21.9) 50 (78.1) 0.643 * 14 1 (1.9) 50 (98.1) 4 (6.3) 60 (93.7) 0.38 * 15 5 (9.8) 46 (90.2) 18 (28.1) 46 (71.9) 0.018 0.21 0.27

(0.09 - 0.81)

* 16 1 (1.9) 50 (98.1) 1 (1.6) 63 (98.4) 1

Pc: corrected p-value (correction factor = 12). Numbers in parentheses represent percentages of

individuals positive or negative for the individual alleles. All *07 alleles were found to be *0701 by

high resolution typing.


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Supplementary Table 5. Extended HLA class II haplotypes in DRB1*0701-positive cases and controls

DRB1*0701-positive

haplotypes

Cases

(n=41)

Controls

(n=19) p-value

DRB1*0701-DQB1*02 8 (7.8) 13 (10.2) 0.647

DRB1*0701-DQB1*0303 33 (32.3) 6 (4.7) 1.91 x 10-8

OR for DRB1*0701-DQB1*0303 possession = 9.72 (95%CI 3.88 - 24.36). Numbers in parentheses

represent percentage of all haplotypes among cases or controls. P-values are uncorrected.


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Supplementary Table 6. TNF and HSPAIL genotypes

TNFα rs1799964 CC CT TT p-value Odds ratio (95% CI) Cases (n=48) 11 (22.9) 31 (64.6) 6 (12.5) 5.64x10-8 12.41 (4.55-33.8) Controls (n=61) 1 (1.7) 21 (34.4) 39 (63.9) rs1800629 AA AG GG p-value Odds ratio (95% CI) Cases (n=51) 0 (0.0) 15 (29.4) 36 (70.6) 0.079 0.46 (0.21-0.99) Controls (n=64) 3 (4.7) 27 (42.2) 34 (53.1) rs1800630 AA AC CC p-value Odds ratio (95% CI) Cases (n=50) 0 (0.0) 10 (20.0) 40 (80.0) 0.544 1.42 (0.58-3.49) Controls (n=61) 1 (1.6) 15 (24.6) 45 (73.8) rs361525 AA AG GG p-value Odds ratio (95% CI) Cases (n=51) 5 (9.8) 34 (66.6) 12 (23.5) 4.33x10-13 38.35 (12.5 - 117.4) Controls (n=64) 0 (0.0) 5 (7.8) 59 (92.2) HSPA1L rs2227956 TT TC CC p-value Odds ratio (95% CI) Cases (n=51) 14 (27.4) 29 (56.9) 8 (15.7) 3.33x10-6 6.25 (2.8 - 14.2) Controls (n=64) 45 (70.3) 19 (29.7) 0 (0.0) Odds ratios are for carriage of the minor allele. p values are uncorrected. Numbers in parentheses

are percentages showing the particular genotype.


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Supplementary Table 7. Conditioning analysis on selected MHC gene data

Conditioning

marker

Test marker

B*5701 TNF-α

rs361525

HSPAIL

rs361525

DRB1*0701

HLA-B*5701 2.7 x 10-16 0.242 0.931 0.256

TNF-α

rs361525

0.00058 5.45 x 10-14 0.755 0.19

HSPAIL

rs2227956

6.9 x 10-11 1.42 x 10-8 7.44 x 10-7 7.4 x 10-4

DRB1*0701 4.1 x 10-11 2.3 x 10-8 3.51 x 10-5 2.7 x 10-4

All p values were calculated using a likelihood ratio chi-square test. P values for individual markers

are shown in bold on the diagonal. The p values shown on the off-diagonals correspond to testing

the effect of adding a particular marker (the test marker) to a model that already includes the

conditioning marker. The top row, conditioned on HLA-B*5701, shows no significant effect for

adding additional markers but in the other rows, conditioned on other markers, the model is

improved by adding B*5701 and, in some cases, other markers also.


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Supplementary Table 8. Effect of B*5701 genotype status on disease severity

B*5701 Positive

(n=43)

B*5701 Negative

(n=8) p-value

Sex (F/M) 31/12 5/3 0.059

Age at onset (years) 63.2 ± 12.5 62.6 ± 11.3 1.00

Time to onset from initial drug

intake (in days) 23.4 ± 11.3 35.7 ± 51.2 0.56

Total days on drug 11.1 ± 7.4 12.6 ± 10.5 0.902

Pattern of liver injury

Cholestatic 37 (86.0) 7 (87.5)

Hepatocellular 4 (9.3) -

Mixed 2 (4.7) 1 (12.5)

1.00

ICC scoring

3-5 (possible) 4 (9.3) -

6-8 (probable) 16 (37.2) 2 (25.0)

>8 (highly probable) 23 (53.5) 6 (75.0)

0.267

Peak Bilirubin (mmol/l) 291 ± 250.5 165.4 ± 140.8 0.195

Peak ALT (U/l) 390 ± 244.4 440.6 ± 371.2 0.866

Peak ALP (U/l) 689 ± 943.7 405.4 ± 181.4 0.265


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Supplementary Table 9. Potential additional signals from chromosomes other than 6

Odds Ratio (OR) was estimated based on allelic frequency. The chromosomal positions are based

on NCBI human genome build 36

3 99498527 rs1603605 TREND 0.3942 0.1738 3.12E-06 3.095 OR5H2

3 99508366 rs1472413 TREND 0.3942 0.1738 3.12E-06 3.095 OR5H2

3 99516090 rs7634235 TREND 0.3942 0.1738 3.12E-06 3.095 OR5H2

3 99517216 rs1497546 TREND 0.1346 0.0231 1.94E-07 6.569 OR5H2

3 99522310 rs11928290 TREND 0.3942 0.1738 3.12E-06 3.095 OR5H2

3 99540350 rs12630857 TREND 0.3942 0.1755 3.20E-06 3.057 OR5K4

9 26596060 rs10812425 TREND 0.4423 0.2252 2.20E-06 2.729 C9org82

9 26604847 rs10812428 TREND 0.5673 0.3149 1.28E-06 2.852 C9org82

12 36965842 rs1973293 TREND 0.6562 0.4029 3.31E-06 2.829 ALG10B

12 36978814 rs1825806 TREND 0.625 0.3865 4.71E-06 2.645 ALG10B

12 36987497 rs6582576 TREND 0.625 0.3865 4.71E-06 2.645 ALG10B

12 36994816 rs1843876 TREND 0.625 0.3865 4.71E-06 2.645 ALG10B

12 36996726 rs4882284 TREND 0.625 0.3865 4.71E-06 2.645 ALG10B

12 37003945 rs6582607 TREND 0.6154 0.3741 3.48E-06 2.677 ALG10B

12 37016722 rs10880934 TREND 0.6154 0.3706 2.74E-06 2.718 ALG10B

12 37029775 rs6582630 TREND 0.6346 0.3812 1.49E-06 2.819 ALG10B

12 37078615 rs7968322 TREND 0.6154 0.3723 4.08E-06 2.697 ALG10B

12 37207850 rs7980932 TREND 0.6154 0.3723 4.66E-06 2.697 ALG10B

15 92740512 rs4984390 TREND 0.1731 0.406 4.20E-06 0.3062 MCTP2

OR Genes nearbyChromo

some

Risk allele

frequency in

cases

Risk allele

frequency in

controls

Position SNP name test p-value


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Supplementary Table 10. The statistics of CNV calls from 40 cases and 272 controls A. Copy number Cases (average number of

CNVs) Controls (average number of CNVs)

0 134 (3.4) 914 (3.4) 1 1767 (44) 12193 (45) 3 1081 (27) 5191 (19) 4 10 (0.25) 69 (0.25) B. Cases Controls Total number of CNVs 2992 18637 Average # of CNVs per sample

75 68

Total size of CNVs (kb) 2988 2459 Average total size of CNVs per sample (kb)

41 38


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Supplementary Table 11. rs10937275 genotyping summary rs1093725-positive rs1093725-negative Cases positive for B*5701/rs2395029(n=48)

25 (52.0) 23 (48.0)

Cases negative for B*5701/rs2395029 (n=10)

1 (10.0) 9 (90.0)

POPRES controls (n=282) 50 (17.7) 232 (82.2) Odds ratio for disease development in positive cases 5.04 (95% CI 2.65-9.60); p-value =1.17 x 10-6

Numbers in parentheses represent percentages.


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Supplementary Figure 1 (a)

(b)

Supplementary Figure 1. Principal component analysis of population structure. Panel (a)

shows an overview of the entire POPRES control cohort and flucloxacillin cases. Panel (b) is an

enlarged view of the Northern European cluster.


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Supplementary Figure 2

(a)

(b)

Supplementary Figure 2. EIGENSTRAT analysis. The first 10 eigen-vectors were included in

the analysis. Chi-square statistics were calculated from the trend test conditioned on the 10

components. Panel (a) shows Eigenstrat chi-square plotted against chi-square for trend. Panel (b)

shows Eigenstrat chi-square plotted against chi square quantiles.


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Supplementary Figure 3 (a)

(b)

Supplementary Figure 3. The p-values of SNPs from the MHC region. The p-values of SNPs

from MHC region. X-axis is the position on chromosome 6 (NCBI build 36). Y-axis is –log10 of p-

values from logistic regression. The approximate positions of three genes (HLA-B, HCP5, and

HLA-DQA1) are marked. (a) p-values of case-control association by logistic regression. (b) p-

values of case-control association conditioned on rs2395029. rs2395029 was modeled as an

additional factor in the logistic regression. The figure indicates that no other SNP in MHC region is

genome-wide significantly associated that are independent of rs2395029 alleles.


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Supplementary Figure 4. CNV quality control: removing poor-quality samples. The figure

shows LRR standard deviations (LRR SD) versus LRR mean values for all samples, each

represented by a dot. The gray lines mark the ad hoc cutoff values of LRR mean (-0.018) and LRR

SD (0.19). Samples in the top-left quarter were removed. It is noticeable that cases are more likely

than controls to be removed.


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Supplementary Figure 5. Genomic features around the CNV in chromosome 11. The red and

blue “PLINK CNV” tracks represent the deletions and duplications respectively of the cases.


hla-b*5701 genotype is a major determinant of drug-induced ......1 hla-b*5701 genotype is a major...

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hla-b5701 genotype is a major determinant of drug-induced ......1 hla-b5701 genotype is a major...