Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

Received; October 9, 2011 Published online; January 24, 2012

Accepted; January 12, 2012 doi; 10.2133/dmpk.DMPK-11-NT-120

DMPK, Note

Development of a rapid and inexpensive assay for detecting a surrogate genetic

polymorphism of HLA-B*58:01: a partially predictive but useful biomarker for

allopurinol-related Stevens-Johnson syndrome/toxic epidermal necrolysis in

Japanese.

Keiko Maekawa1), Jun Nishikawa1), Nahoko Kaniwa1), Emiko Sugiyama1), Tomoko

Koizumi1), Kouichi Kurose1), Masahiro Tohkin2) and Yoshiro Saito1, *)

1) Division of Medicinal Safety Science, National Institute of Health Sciences, Tokyo

158-8501, Japan.

2) Department of Medicinal Safety Science, Graduate School of Pharmaceutical

Sciences, Nagoya City University, 467-8603, Japan.

*Correspondence should be addressed to Dr. Yoshiro Saito, Division of Medicinal

Safety Science, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku,

Tokyo 158-8501, Japan.

Running title: PCR-RFLP assay for detecting a surrogate SNP of HLA-B*58:01

Footnotes

This study was supported in part by a Health and Labour Sciences Research Grant

(H23-Chikyukibo-Shitei-001) from the Ministry of Health, Labour and Welfare, by the

Copyright C 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

Japan Health Sciences Foundation (Research on Publicly Essential Drugs and Medical

Devices, KHB1011), and by KAKENHI (23790611) from the Japan Society for the

Promotion of Science (JSPS) in Japan.

Text: 15 pages

Table: 1 page

Supplementary Figure: 2 pages

2
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

Abstract

Allopurinol-induced Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN)

are strongly associated with HLA-B*58:01 in various populations including Japanese.

We demonstrated that several single nucleotide polymorphisms (SNPs) around the HLA

region on chromosome 6 were strongly linked with HLA-B*58:01 in a previous study

using Japanese allopurinol-related SJS/TEN patients. Their very strong linkage

suggests that these SNPs could be used as surrogate biomarkers to find carriers of

HLA-B*58:01 to avoid these serious adverse effects. In the present study, to expedite

the application of this pharmacogenomic information to the proper usage of allopurinol

in the clinical situation, we developed a polymerase chain reaction-restriction fragment

length polymorphism (PCR-RFLP) assay for the genotyping of rs9263726 in the

psoriasis susceptibility 1 candidate 1 (PSORS1C1) gene, which is in absolute linkage

disequilibrium (r2=1, D’=1) with HLA-B*58:01. Developed PCR-RFLP assay using

FokI restriction enzyme was able to detect three different genotypes, GG, GA, and AA

of rs9263726 robustly, and thus enabled to find HLA-B*58:01 carriers. This robust

and inexpensive assay would be useful for pre-screening the subjects with

HLA-B*58:01, a genetically high risk factor for allopurinol-induced SJS/TEN.

Keywords: allopurinol, PCR-RFLP, screening test, Stevens-Johnson syndrome, toxic

epidermal necrolysis.

3
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

Introduction
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe

cutaneous adverse reactions (SCARs).1) SJS and TEN, considered variants of the same

skin disorder, are characterized by the development of limited (in SJS) or widespread

(in TEN) detachment and blistering of the skin epidermis and mucous epithelium, often

with organ involvement.1,2) The incidence of SJS/TEN is very rare, estimated to occur

at about 2 patients per million individuals per year in Caucasians3), but these SCARs

require intensive care due to the high mortality rates (1-5% for SJS and 20-30% for

TEN) and long-term treatments for subsequent complications, especially ocular

pathologies.1) SJS/TEN are idiosyncratic SCARs that have considered, for a long time,

to be difficult to predict, but human lymphocyte antigen (HLA) types have recently

been reported to be associated with the onset of SJS/TEN in a drug-specific manner.1,4)

Allopurinol is a widely-prescribed urate-lowering drug and has known to be the most

common causative drug for SJS/TEN in Japan.4,5) In 2005, Hung et al. reported that an

HLA allele B variant, HLA-B*58:01, is strongly associated with allopurinol-induced

SCARs consisting of SJS, TEN and hypersensitivity syndrome in Han Chinese

population.6) They found that 100% (51/51 patients) of the case patients had this HLA

type, while only 15% (20/135 patients) of the tolerant control had, giving an odds ratio

(OR) of 580.3 (sensitivity = 100%, specificity = 85%). This association was later

confirmed in Thai (SJS/TEN patients, OR = 348.3, sensitivity = 100%, specificity =

87%)7), Korean (SJS/TEN/drug-induced hypersensitivity syndrome patients, OR = 97.8,

sensitivity = 92%, specificity = 89%)8), European (SJS/TEN patients, OR = 80,

sensitivity = 56%)9) and Japanese (SJS/TEN patients, OR = 62.8, sensitivity = 56%)10,

11)
populations. Although the associations have been partial, especially in Europeans

4
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

and Japanese, HLA-B*58:01 is thought to be a useful biomarker for allopurinol-induced

SJS/TEN.

Recent report showed that based on the very strong association of HLA-B*15:02

allele with SJS/TEN in Han Chinese population (sensitivity = 98%, specificity =

96%),12) prospective testing for HLA-B*15:02 and subsequent avoidance of

carbamazepine therapy result in zero occurrence of SJS/TEN in Taiwan.13) Based on

this result and severity of these adverse reactions, pre-screening test is now mandatory

and covered by the National Health Insurance in Taiwan, although its positive predictive

value could be estimated at around 3% using the values of their study. Thus,

examining HLA-B*58:01 prior to allopurinol administration may be also valuable to

avoid allopurinol-induced SJS/TEN. However, testing HLA types is relatively

laborious, time-consuming and expensive. Very recently, we found that several single

nucleotide polymorphisms (SNPs) around the HLA region on chromosome 6 were

strongly linked with HLA-B*58:01 in a group of SJS/TEN patients.11) In general,

single SNP can be easily genotyped and inexpensively compared to HLA type. Thus,

the linked SNPs could be used as alternatives to testing for HLA-B*58:01 when

deciding on the application of drug therapies involving allopurinol. To expedite the

application of this pharmacogenomic information for the proper usage of allopurinol in

clinical settings, we developed a polymerase chain reaction-restriction fragment length

polymorphism (PCR-RFLP) method that can genotype SNPs easily without high skills

and inexpensively.

5
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

Materials and Methods

Patients

Japanese SJS/TEN patients from unrelated families were recruited from July 2006

through April 2010 at participating institutes of the Japan Severe Adverse Reactions

(JSAR) research group.11) In addition, SJS/TEN patients were recruited through a

nationwide blood-sampling network system in Japan for severe drug adverse reactions

operated by the National Institute of Health Sciences under the auspices of the Ministry

of Wealth, Labour and Welfare and the Federation of Pharmaceutical Manufacturers'

Associations of Japan. Genomic DNA was extracted from blood leukocytes as

described previously.10) DNA samples extracted from the cord blood of healthy

Chinese-Americans were purchased from AllCells (Emeryville, CA, USA). The ethics

committees of the National Institute of Health Sciences and each participating institute

of the JSAR research group approved this study. Written informed consent was

obtained from all cases and healthy Chinese-American subjects.

Genotyping of Single Nucleotide Polymorphism by TaqMan assay and HLA types

HLA-B types were determined by the sequencing-based method as reported in a

previous paper.11) Of the several SNPs linked with HLA-B*58:01, we selected

rs9263726 (110G>A, Arg37His) in psoriasis susceptibility 1 candidate 1 (PSORS1C1)

as a surrogate marker for HLA-B*58:01, because this SNP was in absolute linkage

disequilibrium (r2 = 1, D’ = 1) with HLA-B*58:01 and associated with SJS/TEN with an

odds ratio of 61.2 (p = 3.64 x 10-8) in the dominant genotype mode. 11) This variation

was located ca., 215 kb away from the HLA-B gene, detected at minor allele frequency

of 0.006 (12/1982 alleles), which was the same as that of the reported Japanese

frequency of HLA-B*58:01 (0.006),14) and in Hardy-Weinberg equilibrium (p=0.847).11)

6
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

In allopurinol-related SJS/TEN patients, the minor allele frequency of HLA-B*58:01

and rs9263726 was 0.27811). Rs9263726 was genotyped using TaqMan SNP

Genotyping Assays (C_30352071_10, Life Technologies, Carlsbad, CA, USA)

according to the manufacturer’s instruction using 5 ng of genomic DNA from Japanese

SJS/TEN patients or healthy Chinese-Americans. Hardy-Weinberg equilibrium was

analyzed by Fisher’s exact test using SNPAlyze ver. 3.1 software (Dynacom, Chiba,

Japan).

Genotyping of rs9263726 by PCR-RFLP

PCR primers (forward: 5’-AAGCTCCATCCACCCCTGGT-3’ and reverse:

5’-ACACATTGGGTGGGGGACAT-3’) were designed to amplify a PSORS1C1

genomic fragment containing the rs9263726 SNP locus. PCR was performed using

Ex-Taq (0.625 units) (Takara Bio Inc., Shiga, Japan) with a pair of primers (0.2 µM) and

genomic DNA (50 ng). The PCR conditions were 94°C for 5 min, followed by 30

cycles of 94°C for 30 sec, 60°C for 1 min, 72°C for 1.5 min, and a final extension at

72°C for 7 min. Aliquots of PCR products (5 µl) were then digested by the addition of

0.4 units of FokI restriction endonuclease (New England Biolabs, Beverly, MA, U.S.A.)

in the presence of 1 x Buffer 4 (New England Biolabs) at 37°C for 2 h. Restriction

mixtures were incubated at 65°C for 20 min to inactivate FokI, and then electrophoresed

through a 15-25% gradient acrylamide gel (MULTIGEL II Mini, Cosmo Bio Co., Ltd,

Tokyo, Japan). Following electrophoresis, the gels were stained with ethidium

bromide, and DNA was visualized by placing the gel on an UV transilluminator.

7
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

Results and Discussion

First, we compared the results of genotyping rs9263726 in PSORS1C1 with the

PCR-RFLP and TaqMan SNP Genotyping assays (C_30352071_10). DNAs from

Chinese-Americans were used since the frequency of HLA-B*58:01 in this population is

reportedly higher than in the Japanese population.6,10) Preliminary, experiments using

the TaqMan assay showed that the 200 DNA samples from Chinese-Americans

contained 161 homozygotes of the major allele (GG), 36 heterozygotes (GA), and 3

homozygotes of the minor allele (AA) of rs9263726 (data not shown), which

distribution was in Hardy-Weinberg equilibrium (p=0.550). In addition, we confirmed

that the 3 subjects with homozygous AA surely had homozygous HLA-B*58:01 (data

not shown). From the DNAs from Chinese-Americans genotyped by TaqMan assay, 5

samples with GG, 4 with GA, and 2 with AA of rs9263726 were selected to establish the

PCR-RFLP method. In the developed assay, the 260 bp PCR products derived from

the A allele of rs9263726 were digested with Fok I produced two bands (141 bp and 119

bp), while those derived from the G allele remained as the parent single band (260 bp)

(supplementary Fig. 1A). Genotypes of these samples by PCR-RFLP assay were

100% in concordance with those from the TaqMan SNP assay, indicating that this is a

robust method of genotyping rs9263726.

Next, in order to validate this PCR-RFLP assay, the rs9263726 locus was

genotyped for the DNA samples with or without HLA-B*58:01 of 27 Japanese SJS/TEN

patients for whom HLA-B types had been previously determined.10,11) The following

SJS/TEN samples were selected: 5 HLA-B*58:01 heterozygous carriers and 22 other

HLA-B allele carriers. The other HLA types were selected based on an allele frequency

≥ 0.01 in Japanese control populations,14,15) although a HLA-B*44:02 sample (allele

8
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

frequency = 0.01) was not available. As shown in Table 1 and supplementary Fig. 1B,

the 5 patients with heterozygous HLA-B*58:01 were also heterozygotes for rs9263726

(GA), and the remaining 22 patients with the other HLA-B types were major

homozygotes for this SNP (GG). Thus, our developed PCR-RFLP assay can robustly

predict the HLA-B*58:01 status of SJS/TEN patients.

Very recently, Kostenko et al. generated a monoclonal antibody to recombinant

HLA-B*57:01 protein and developed an flow cytometric assay for the detection of

HLA-B57-positive peripheral blood mononuclear cells.16) This antibody can

cross-react with HLA-B58 proteins and thus could be used to pre-screen for

HLA-B*58:01 carriers. However, this assay method cannot discriminate HLA-B*57:01

from B*58:01 and uses blood cells, making it laborious and expensive (i.e., a flow

cytometer is necessary). In contrast, our PCR-RFLP method does not require a high

skill set, and at a low cost without use of specific machines, although a DNA extraction

step is necessary.

Although the testing of rs9263726 or HLA-B*58:01 cannot perfectly predict

allopurinol-induced SJS/TEN, the HLA-B*58:01-positive patients may be better to

avoid the administration of allopurinol, as the HLA-B*15:02-positive patients for

carbamazepine in Taiwan. Because allopuirol is a xantine oxidase inhibitor, febuxostat,

having same pharmacological effect by different structure, might be an alternative drug

for the HLA-B*58:01-positive patients, although further studies are clearly necessary to

prove that SJS/TEN induced by febuxostat is surely not to be associated with

HLA-B*58:01.

In conclusion, we have developed a robust PCR-RFLP genotyping assay for

rs9263726 in PSORS1C1, which is in absolute linkage disequilibrium with

9
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

HLA-B*58:01, a partially predictive but useful biomarker for allopurinol-related

SJS/TEN in Japanese. The genotyping of rs9263726 by this easy and inexpensive

method makes it useful for the prospective screening of patients with HLA-B*58:01 in

the future.

10
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

References
1) Aihara, M.: Pharmacogenetics of cutaneous adverse drug reactions. J. Dermatol.,

38: 246-254 (2011).

2) Auquier-Dunant, A., Mockenhaupt, M., Naldi, L., Correia, O., Schröder, W. and

Roujeau, J.C.; for the SCAR Study Group.: Correlations between clinical patterns

and causes of erythema multiforme majus, Stevens-Johnson syndrome, and toxic

epidermal necrolysis: results of an international prospective study. Arch.

Dermatol., 138: 1019-1024 (2002).

3) Rzany, B., Mockenhaupt, M., Baur, S., Schröder, W., Stocker, U., Mueller, J.,

Holländer, N., Bruppacher, R. and Schöpf, E.: Epidemiology of erythema

exsudativum multiforme majus, Stevens-Johnson syndrome, and toxic epidermal

necrolysis in Germany (1990-1992): structure and results of a population-based

registry. J. Clin. Epidemiol., 49: 769-773 (1996).

4) Tohkin, M., Ishiguro, A., Kaniwa, N., Saito, Y., Kurose, K. and Hasegawa R.:

Prediction of severe adverse drug reactions using pharmacogenetic biomarkers.

Drug Metab. Pharmacokinet., 25: 122-133 (2010).

5) Sudo, C., Azuma, J., Maekawa, K., Kaniwa, N., Sai, K. and Saito, Y.: Current

movements of four serious adverse events induced by medicinal drugs based on

spontaneous reports in Japan. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho

Hokoku, in press (2011).

6) Hung, S.I., Chung, W.H., Liou, L.B., Chu, C.C., Lin, M., Huang, H.P., Lin, Y.L.,

Lan, J.L., Yang, L.C., Hong, H.S., Chen, M.J., Lai, P.C., Wu, M.S., Chu, C.Y.,

Wang, K.H., Chen, C.H., Fann, C.S., Wu, J.Y. and Chen, Y.T.: HLA-B*5801 allele

as a genetic marker for severe cutaneous adverse reactions caused by allopurinol.

11
Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

Proc. Natl. Acad. Sci. U S A., 102: 4134-4139 (2005).

7) Tassaneeyakul, W., Jantararoungtong, T., Chen, P., Lin, P.Y., Tiamkao, S.,

Khunarkornsiri, U., Chucherd, P., Konyoung, P., Vannaprasaht, S., Choonhakarn,

C., Pisuttimarn, P., Sangviroon, A. and Tassaneeyakul, W.: Strong association

between HLA-B*5801 and allopurinol-induced Stevens-Johnson syndrome and

toxic epidermal necrolysis in a Thai population. Pharmacogenet. Genomics, 19:

704-709 (2009).

8) Kang, H.R., Jee, Y.K., Kim, Y.S., Lee, C.H., Jung, J.W., Kim, S.H., Park, H.W.,

Chang, Y.S., Jang, I.J., Cho, S.H., Min, K.U., Kim, S.H. and Lee, K.W.; Adverse

Drug Reaction Research Group in Korea.: Positive and negative associations of

HLA class I alleles with allopurinol-induced SCARs in Koreans. Pharmacogenet.

Genomics, 21: 303-307 (2011).

9) Lonjou, C., Borot, N., Sekula, P., Ledger, N., Thomas, L., Halevy, S., Naldi, L.,

Bouwes-Bavinck, J.N., Sidoroff, A., de Toma, C., Schumacher, M., Roujeau, J.C.,

Hovnanian, A. and Mockenhaupt, M.; for the RegiSCAR study group.: A

European study of HLA-B in Stevens-Johnson syndrome and toxic epidermal

necrolysis related to five high-risk drugs. Pharmacogenet. Genomics, 18: 99-107

(2008).

10) Kaniwa, N., Saito, Y., Aihara, M., Matsunaga, K., Tohkin, M., Kurose, K.,

Sawada, J., Furuya, H., Takahashi, Y., Muramatsu, M., Kinoshita. S,, Abe, M.,

Ikeda, H., Kashiwagi, M., Song, Y., Ueta, M., Sotozono, C., Ikezawa, Z. and

Hasegawa, R.; on behalf of the JSAR research group.: HLA-B locus in Japanese

patients with anti-epileptics and allopurinol-related Stevens-Johnson syndrome

and toxic epidermal necrolysis. Pharmacogenomics, 9: 1617-1622 (2008).

12
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

11) Tohkin, M., Kaniwa, N., Saito, Y., Sugiyama, E., Kurose, K., Nishikawa, J.,

Hasegawa, R., Aihara, M., Matsunaga, Abe, M., Furuya, H., Takahashi, Y., Ikeda,

H., Muramatsu, M., Ueta, M., Sotozono, C., Kinoshita. S., Ikezawa, Z. and the

Japan Pharmacogenomics Data Science Consortium: A whole-genome association

study of major determinants for allopurinol-related Stevens-Johnson syndrome

and toxic epidermal necrolysis in Japanese patients. Pharmacogenomics J., in

press.

12) Hung, S.I., Chung, W.H., Jee, S.H., Chen, W.C., Chang, Y.T., Lee, W.R., Hu, S.L.,

Wu, M.T., Chen, G.S., Wong, T.W., Hsiao, P.F., Chen, W.H., Shih, H.Y., Fang,

W.H., Wei, C.Y., Lou, Y.H., Huang, Y.L., Lin, J.J. and Chen, Y.T.: Genetic

susceptibility to carbamazepine-induced cutaneous adverse drug reactions.

Pharmacogenet. Genomics, 16: 297-306 (2006).

13) Chen, P., Lin, J.J., Lu, C.S., Ong, C.T., Hsieh, P.F., Yang, C.C., Tai, C.T., Wu, S.L.,

Lu, C.H., Hsu, Y.C., Yu, H.Y., Ro, L.S,. Lu, C.T., Chu, C.C., Tsai, J.J., Su, Y.H.,

Lan, S.H., Sung, S.F., Lin, S.Y., Chuang, H.P., Huang, L.C., Chen, Y.J., Tsai, P.J.,

Liao, H.T., Lin, Y.H., Chen, C.H., Chung, W.H., Hung, S.I., Wu, J.Y., Chang, C.F.,

Chen, L., Chen, Y.T. and Shen, C.Y.; for the Taiwan SJS Consortium.:

Carbamazepine-induced toxic effects and HLA-B*1502 screening in Taiwan. N.

Engl. J. Med., 364: 1126-1133 (2011).

14) Tanaka, H., Akaza, T. and Juji, T.: Report of the Japanese Central Bone Marrow

Data Center. Clin. Transpl., 9: 139-144 (1996).

15) Tokunaga, K., Ishikawa, Y., Ogawa, A., Wang, H., Mitsunaga, S., Moriyama, S., Lin,

L., Bannai, M., Watanabe, Y., Kashiwase, K., Tanaka, H., Akaza, T., Tadokoro, K.

and Juji, T.: Sequence-based association analysis of HLA class I and II alleles in

13
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

Japanese supports conservation of common haplotypes. Immunogenetics, 46:

199-205 (1997).

16) Kostenko, L., Kjer-Nielsen, L., Nicholson, I., Hudson, F., Lucas, A., Foley, B., Chen,

K., Lynch, K., Nguyen, J., Wu, A.H., Tait, B.D., Holdsworth, R., Mallal, S.,

Rossjohn, J., Bharadwaj, M. and McCluskey, J.: Rapid screening for the detection

of HLA-B57 and HLA-B58 in prevention of drug hypersensitivity. Tissue

Antigens, 78: 11-20 (2011).

14
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

Figure Legends

Supplementary Figure 1. Establishment of PCR-RFLP assay for rs9263726 and its

validation by samples with or without HLA-B*58:01. A. Establishment of

PCR-RFLP assay for rs9263726 in PSORS1C1 using FokI restriction digestion on

DNA samples from Chinese-Americans. Genotypes of DNA samples were

pre-determined by TaqMan SNP genotyping assay method. The genotypes were GG

(260 bp) in lanes 4, 6, 7, 9 and 10, GA (260 bp, 141 bp and 119 bp) in lanes 1, 2, 8, and

11, and AA (141 bp and 119 bp) in lanes 3 and 5. M, DNA ladder marker (OneSTEP

Ladder 50, NIPPON GENE CO., LTD, Tokyo, Japan). B. Genotyping of rs9263726

in 27 Japanese SJS/TEN patients with or without HLA-B*58:01. Patient ID

corresponds to those in Table 1. M, DNA ladder marker (All Purpose HI-LO DNA

marker, BIONEXUS Inc., Oakland, CA, USA).

15
Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

Table 1 HLA and rs9263726 genotypes in

Japanese SJS/TEN patients.
ID HLA-B* rs9263726
1 58:01 46:01 G/A
2 58:01 51:01 G/A
3 58:01 51:01 G/A
4 58:01 38:02 G/A
5 58:01 07:02 G/A
6 07:02 51:01 G/G
7 13:01 35:01 G/G
8 15:01 40:02 G/G
9 15:11 40:02 G/G
10 15:18 38:02 G/G
11 35:01 40:02 G/G
12 37:01 40:06 G/G
13 39:01 51:01 G/G
14 40:01 40:01 G/G
15 48:01 51:02 G/G
16 40:02 40:06 G/G
17 40:06 52:01 G/G
18 44:03 44:03 G/G
19 46:01 35:01 G/G
20 51:01 35:01 G/G
21 52:01 52:01 G/G
22 54:01 54:01 G/G
23 55:02 51:01 G/G
24 56:01 46:01 G/G
25 59:01 35:01 G/G
26 67:01 39:01 G/G
27 40:03 54:01 G/G
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

Suppl. Fig. 1A

Lane
M 1 2 3 4 5 6 7 8 9 10 11

2000 bp
1500 bp
1000 bp

500 bp
260 bp
350 bp
250 bp
200 bp 141 bp
150 bp 119 bp
100 bp

50 bp
 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

Supple. Fig. 1B

Patient ID Patient ID Patient ID

M 1 2 3 4 5 6 7 8 9 10 M 11 12 13 14 15 16 17 18 19 M 20 21 22 23 24 25 26 27

1000 bp
500 bp

300 bp 260 bp

200 bp
141 bp
100 bp 119 bp

50 bp

With HLA-B*58:01