Genetics: Correction

Correction. In the article "Specific HLA-DQB and HLA-DRBI alleles confer susceptibility to pemphigus vulgaris" byStephen J. Scharf, Adam Freidmann, Lawrence Steinman,Chaim Brautbar, and Henry A. Erlich, which appeared in

Proc. Natl. Acad. Sci. USA 86 (1989) 10023

number 16, August 1989, of Proc. Natl. Acad. Sci. USA (86,6215-6219), the authors request that the following error benoted. Fig. 1 on page 6216 contains an incorrect alignment andshould be replaced by the corrected Fig. 1 reproduced here.

10 20 30 40 50 60 70 80 90PROTOTYPE: RFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQKRMVDTYCRHNYGVGESFTVQRR

DRw6: - YSTS HN N F I-DE DRw6a (DRw13)DRw6: - YSTS IN-F A i- - RR-E- DRw6B (DRw14)DRw6: -YSTS E W-HN N R DRw6C (DRw14)

FIG. 1. Amino acid sequences encoded by DRB1 alleles from the DRw6 haplotype. Amino acid sequences were translated from the nucleotidesequences and are given in the standard one-letter amino acid code. The amino acid sequence of the DR4 Dw4 prototype is shown; the otherDRB allele-derived sequences are aligned with it. Lines indicate sequence identity, and letters indicate polymorphic amino acids. The sequencefor the DRBJ 6B and DRBI 6C alleles were obtained by PCR cloning and are shorter than the sequence of the DRBJ 6A allele, which was derivedfrom a genomic clone (13). The DRBI 6C allele was derived from the DRw6' homozygous typing cell (HTC) "Amala." The 6B allele was derivedfrom three PV patients and from the Dw9, DRw6 HTC lines ABO and EK/OH.

Dow

nloa

ded

by g

uest

on

July

21,

202

1 D

ownl

oade

d by

gue

st o

n Ju

ly 2

1, 2

021

Dow

nloa

ded

by g

uest

on

July

21,

202

1 D

ownl

oade

d by

gue

st o

n Ju

ly 2

1, 2

021

Dow

nloa

ded

by g

uest

on

July

21,

202

1 D

ownl

oade

d by

gue

st o

n Ju

ly 2

1, 2

021

Dow

nloa

ded

by g

uest

on

July

21,

202

1

Proc. Nati. Acad. Sci. USAVol. 86, pp. 6215-6219, August 1989Genetics

Specific HLA-DQB and HLA-DRBJ alleles confer susceptibility topemphigus vulgaris

(polymerase chain reaction/autoimmunity/genetic predisposition/oligonucleotide probes/allele-specific DNA amplification)

STEPHEN J. SCHARF*, ADAM FREIDMANNt, LAWRENCE STEINMANt, CHAIM BRAUTBAR§,AND HENRY A. ERLICH**Department of Human Genetics, Cetus Corporation, 1400 Fifty-Third Street, Emeryville, CA 94608; tThe Hebrew University of Jerusalem, The Institute ofLife Sciences, Department of Genetics, Jerusalem, Israel; tDepartment of Neurology, Pediatrics and Genetics, Stanford University, Stanford, CA 94305; and§Hadassah Medical School, The Tissue Typing Unit and Department of Clinical Microbiology, and the Lautenberg Center for General and TumorImmunology, Jerusalem, Israel

Communicated by Leonard A. Herzenberg, May 4, 1989 (received for review January 6, 1989)

ABSTRACT The autoimmune dermatologic disease pem-phigus vulgaris (PV) is associated with the serotypes HLA-DR4and HLA-DRw6. Based on nucleotide sequence and oligonu-cleotide probe analysis of enzymatically amplified DNA encod-ing HLA-DR P chain (HLA-DRB) and HLA-DQ (3 chain(HLA-DQB; henceforth HLA is omitted from designations), weshowed previously that the DR4 susceptibility was associatedwith the DwlO DRBI allele [encoding the mixed lymphocyteculture (MLC)-defined DwlO specificity]. The DRw6 suscep-tibility similarly was shown to be associated with a rare DQBallele (DQBI.3), which differed from another nonsusceptibleallele by only a valine-to-aspartic acid substitution at position57. Given the linkage disequilibrium that characterizes HLAhaplotypes, it is difficult to assign disease susceptibility to aspecific locus rather than to a closely linked gene(s) on the samehaplotype. To address this problem, we have analyzed all of thepolymorphic loci of the class II HLA region (DRB1, DRB3,DQA, DQB, and DPB) on the DRw6 haplotypes in patients andcontrols. In 22 PV patients, 4 different DRw6 haplotypes werefound that encode the same DQ (3 chain (DQBI.3) but con-tained silent nucleotide differences at the DQB locus as well ascoding sequence differences in the DQA and DRB loci. Theseresults, obtained by using a method for allele-specific polymer-ase chain reaction amplification, strongly support the hypoth-esis that the allele DQBI.3 confers susceptibility. This DQBallele is correlated with the MLC-defined Dw9 specificity andis associated with two different DRIll alleles (the common"6A" associated with DRw13 and the rare "6B" associatedwith DRw14). Since 86% (19 of 22) ofDRw6+ patients containthe DQBI.3 allele (vs. 3% of controls), whereas 64% (14 of 22)contain the DRBI allele 6B (vs. 6% of the controls), weconclude that most of the DRw6 susceptibility to PV can beaccounted for by the DQ .8 chain.

Pemphigus vulgaris (PV) is an autoimmune dermatologicdisease mediated by autoantibodies to an epidermal mem-brane protein (1). In population studies, PV is stronglyassociated with the serotypes HLA-DR4 and HLA-DRw6 (2,3) with only 5% of the patients possessing neither marker.The association of two different serologically defined haplo-types with a specific disease could mean either that theyshare an allele or epitope that confers susceptibility or,alternatively, that they contain different sequences respon-sible for the disease association.By using the polymerase chain reaction (PCR) technique

(4-7), nucleotide sequence analysis, and hybridization of theenzymatically amplified DNA with sequence-specific oligo-nucleotide (SSO) probes (8), the distribution of the alleles

encoding the DR ,( chain (DRB), the DQ /3chain (DQB), andthe DQ a chain (DQA) in a panel of PV patients andHLA-matched controls was determined. Analysis of PCR-amplified DNA from a panel of DR4' PV patients andcontrols with a set ofDRBJ SSO probes revealed that nearlyall (>95%) DR4+ PV patients carried the Dw10 DRBI allele,while the frequency of the oligonucleotide-defined DwJODRBE allele in the HLA-matched control group was 5% (U.S.controls) (9) and 60% (Israeli Jewish controls) (10). In addi-tion, sequence and SSO analysis of the DQB allele carried bythe DR4+ PV and control groups showed that 92% of bothpatients and controls contained the DQB3.2 allele (10). Thus,the observed distribution of DRBI and DQB alleles amongDR4+ PV patients and controls suggested that the Dw10DRB1 allele was responsible for the observed associationwith DR4. The Dw1O DRBJ allele differs from the otherDRBJ alleles of the DR4 haplotype in the third hypervariableregion in containing the "IDE" (Ile-67, Asp-70, and Glu-71)epitope which is, therefore, implicated in the susceptibility ofDR4' individuals to PV (10-12). Although the same IDEepitope is found in the third hypervariable region of themost-common DRw6 DRBI allele, 6A (Fig. 1), this allele isnot associated with PV. Instead, a rare DQB allele, DQB1.3,was found in 11 of 11 Israeli DRw6+ PV patients and in 1 of13 ethnically matched controls. This allele differed fromanother common DQB allele (DQBJ.1) by only a valine-to-aspartic acid substitution at position 57 of the DQ 1 chainand is associated with the Dw9 subtype of DRw6. Someindividuals in both the patient (n = 3) and control (n = 1)population failed to hybridize to either of the two probes(CRX02 and CRX03) used to distinguish the sequencesaround codon 57 (10). We speculated that these DQB allelesmight represent uncharacterized sequence polymorphisms(10).Although the DQBJ.3 allele is very strongly associated

with PV (1), it is difficult to assign susceptibility to a specificlocus rather than to other linked genes because of the stronglinkage disequilibrium characteristic of most HLA haplo-types. In order to assess the role of the DQBJ.3 allele in PVsusceptibility, we have analyzed the distribution of the otherpolymorphic class II (-chain alleles in patients and HLA-matched controls. In this study, we have used in vitroamplified DNA, dot-blot analysis with oligonucleotideprobes, and DNA sequencing to determine the DQ (-chaingene sequence of those DRw6+ individuals that failed to typewith our previous panel of SSO probes for the DQB allelicsubtypes. We also have examined the DRBI and DRB3alleles of DRw6+ PV patients and controls and the DPB

Abbreviations: PV, pemphigus vulgaris; PCR, polymerase chainreaction; SSO, sequence-specific oligonucleotide; HTC, homozy-gous typing cell.

6215

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 -solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 86 (1989)

10 20 30 40 50 60 70 80 90

PROTOTYPE: RFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQKRAAVDTYCRHNYGVGESFTVQRR

DRw6: -YSTS HN- N F I-DE DRw6A (DRw13)DRw6: - YSTS 11H F A H RR- E DRw6B (DRw14)DRw6: -YSTS E - N N R DRw6C (DRw14)

FIG. 1. Amino acid sequences encoded by DRB1 alleles from the DRw6 haplotype. Amino acid sequences were translated from the nucleotidesequences and are given in the standard one-letter amino acid code. The amino acid sequence of the DR4 Dw4 prototype is shown; the otherDRB allele-derived sequences are aligned with it. Lines indicate sequence identity, and letters indicate polymorphic amino acids. The sequence

for the DRBI 6B and DRBI 6C alleles were obtained by PCR cloning and are shorter than the sequence of the DRBI 6A allele, which was derivedfrom a genomic clone (13). The DRBI 6C allele was derived from the DRw6+ homozygous typing cell (HTC) "Amala." The 6B allele was derivedfrom three PV patients and from the Dw9, DRw6 HTC lines ABO and EK/OH.

alleles in DR4+ PV patients and controls. This analysisallowed the potential contribution of these other class II,B-chain sequences to be evaluated.

MATERIALS AND METHODSPCR Amplification. Genomic DNA from PV patients and

control subjects was prepared from whole blood (3), and 1 ,ugwas amplified by the PCR technique by using either Esche-richia coli DNA polymerase I Klenow fragment or Thermusaquaticus (Taq) DNA polymerase (7). The DRB genes wereamplified by using the PCR primers GH46 and GH50 (11) at1 uM, the DQA genes were amplified by using the PCRprimers GH26 and GH27 (6), and the DQB genes wereamplified by using the PCR primers GH28 and GH29 (11) at1 ,uM. In the DR4/DRw6 heterozygotes, the DRw6 DRBJalleles were selectively amplified by allele-specific amplifi-cation so that probes used to type DRw6 alleles would notcross-hybridize to nucleotide sequences shared on the DR4haplotypes.

Allele-Specific Amplification. The PCR primer CRX11 (5'-TCTAGAAGTACTCTACGTCT-3') used in conjunctionwith the DRB primer GH50 provides selective amplificationof the DRBI allele of the DR3, DR5, DRw6, and DRw8haplotypes. CRX11/GH50 (1 ,uM) was used in place of the 1,uM GH46/GH50 primers in a Taq DNA polymerase DRBPCR mix (10) with the following PCR conditions: denatur-ation at 94°C for 15 sec, hybridizing of primers at 40°C for 30sec, and enzymatic extension by Taq DNA polymerase at70°C for 30 sec. Reactions were run for 35 cycles. Thespecificity of the reaction is shown in Fig. 2. DRB3, DRB4alleles and DRBI alleles from other haplotypes were notamplified (data not shown). DPB was amplified by usingDB01 and DBO3 as primer oligonucleotides (14).SSO Analysis of Amplified DNA Samples. Amplified DNA

was analyzed by filter dot-blot hybridization with SSO probesas described (8, 10; Table 1). The DRw6 DRBI alleles wereselectively amplified by allele-specific amplification so that

350 40 450 500r r ---

DR43 * * * 4ASO DR0I PCR PrimersGeneral Probe * * *General DRO3 PCR Primers

A B A B A B A B

A - DRw6 HTCB - DR4 HTC

FIG. 2. Allele-specific amplification. DRB sequences from theDR4 HTC LS40 and the DRw6 HTC HHK were amplified as de-scribed (ref. 10; and Materials and Methods). The lower row ofsamples was amplified with generalDRB primers GH46/GH50 (9, 10),which amplify all DRB loci and alleles. The upper row of samples wasamplified with the DRw6 DRBI-specific primers CRX11/GH50. Theprimers were hybridized to the template at the temperatures shown inthe figure. The reactions were analyzed by dot-blot hybridization asdescribed (10) and probed with the generalDRB oligonucleotide probeGH22, which hybridizes to all DRB alleles (Table 1). The generalprimers amplify both the DR4 and DRw6 HTC DNA, whereas theDRw6-specific primers amplify only the DRw6 DNA.

probes used to type DRw6 DRBI alleles would not cross-hybridize to nucleotide sequences shared on the DR4 hap-lotypes. The DRBI allele 6A was typed by hybridization withthe probe GH78 (10, 11; Table 1). The DRBI allele 6B was

typed by hybridization of the probes CRX23 and CRX36(Table 1). CRX23 and CRX36 hybridize to the nucleotidesequences encoding amino acids of the DRw6 DRBI secondexon at the following positions: CRX23 from position 56through 61 ("A-H" in Fig. 1) and CRX36 from position 70through 75 ("RR" in Fig. 1). The DRw52 serotypes weredetermined by the pattern of hybridization of the probesCRX21, CRX22, and GH54. CRX21 + GH54 corresponds tothe DRw52a allele; CRX22 alone corresponds to the DRw52ballele, and CRX22 + GH54 corresponds to the DRw52callele. CRX21 and CRX22 hybridize to the nucleotide se-

quences encoding amino acids of the DRB3 second exon atpositions 11 though 14; GH54 hybridizes to positions 57through 61 (not shown). The filters were prehybridized in 10ml of hybridization solution for 15 min at the temperaturesgiven in the legend to Table 1. After prehybridization, 0.2pmol of SSO probe was added to the solution and hybridizedas described in Table 1. After the appropriate washes for eachprobe, the filters were placed under Kodak XAR-5 film witha single intensifying screen (DuPont Cronex Lightning Plus)at -70°C for autoradiography.Sequence Determination of DQB, DQA, DRBI, DRB3, and

DPB Alleles. Of the PCR reaction product, 1/32 was clonedin phage M13mplO (6), and DNA from purified phage clones

Table 1. Sequence of SSO probesCodon

Probe Specificity Sequence numbers

DRB-specific probesCRX21 DRB3 AGACTTACGCAGCTC 9-13

("LR-S")CRX22 DRB3 GAGCTGCTTAAGTCT 9-13

("LL-S")CRX23 DRw6B DRBI CCTGCTGCGGAGCACTG 56-61

("A-H")CRX36 DRw6B DRBI CCCGCCTCCGCTCCA 70-75

("RR")GH22 General DRB CTTCAATGGGACGGAG 17-22GH54 DRB3 ("S") GCTGTTCCAGGACTC 59-63

DQB-specific probesCRX19 DQBI.2 AGGGGCGGCCTAGCGCCGA 53-59CRX20 DQBI.3.2 TCGGCATCAGGCCGCCCCT 53-59

All blot hybridizations were for 16 hr at 420C (unless otherwiseindicated) in 0.1% polyvinylpyrrolidone/0.1% Ficoll/0.1% bovineserum albumin/0.5% NaDodSO4 containing SSPE at the concentra-tions indicated (1 x SSPE is 0.18 M NaCI/10 mM sodium phosphate,pH 7.4/1 mM EDTA). All wash solutions contained 0.1% NaDod-SO4. CRX21, CRX22, and GH54 were hybridized in 2x SSPE.CRX21 was washed for 10 min in 0.5x SSPE at 42°C. CRX22 andGH54 were washed for 10 min in 0.2x SSPE at 50°C and 42°C,respectively. CRX23 and CRX36 were hybridized in 5x SSPE andwashed for 10 min at 50°C in 0.2x SSPE and 1 x SSPE, respectively.CRX19 and CRX20 were hybridized in 2x SSPE at 50°C and washedfor S min at 50C in 0.Sx SSPE.

6216 Genetics: Scharf et al.

Proc. Natl. Acad. Sci. USA 86 (1989) 6217Genetics: Scharf et al.

P rototypeDQB1.1:

DQB1 .2:

DQB1 .3:

DQB1 .6:

DQB1 .7:

10 20 30 40 50 60 70 80 90DFVYQFKGMCYFTNGTERVRLVTRYIYNREEYARFDSDVGVYRAVTPLGPPAAEYWNSQKEVLERTRAELDTVCRHNYQLELRTTLQRR

L C H V Q R-V GA-SVR EVAY-GI

GH69 CRX02

: I V Q R-S GA-SV-R

GH69 CRX19

: V QRD GA-SV-R

GH69 CRX03, CRX20

H Q .1DWRC

GH80 CRX03

HI 0 F-V EVGY-GI-

GH80 CRX02

DQB1.8: Q R-V

CRX02

FIG. 3. Amino acid sequences encoded by DQB alleles from DRw6, DQw1 haplotypes. Amino acid sequences were translated from thenucleotide sequences and are given in the standard one-letter amino acid code. The amino acid sequence of the DQB3.2 prototype is shown;the otherDQB allele-derived amino acid sequences are aligned with it. Lines indicate sequence identity, and letters indicate polymorphic aminoacids. The DNA sequences for the alleles DQBJ.2, DQBJ.3, DQBJ.6, and DQBJ.8 were obtained by PCR cloning and are shorter than theprototype and DQB1.1 and DQB1.7 sequences, which were obtained from cDNA clones. The six DQBJ SSOs hybridize to the nucleotidesequences encoding the amino acid residues underscored in each alignment. The DQB1.8 sequence is previously unreported and was derivedfrom two different Chinese individuals (R. Griffith, private communication).

was sequenced by the dideoxy chain-termination method(15). The DQA gene segment was amplified and sequenceddirectly (16). Sequences and suggested nomenclature for theDQB and DQA alleles are reported in Horn et al. (17).

RESULTSDQB Alielic Variation. Nucleotide sequence analysis of

PCR-amplified DQB DNA has identified thus far six differentDQB alleles on DRw6, DQwl haplotypes (Fig. 3). Thesedifferent sequences have been identified from a variety (n =60) of cells from patients and controls. The most frequentallele (=50%o) on control DRw6 haplotypes is the DQBI.6allele. The DQB sequence analysis of DNA from threeDRw6' PV patients and one control that had failed to typewith a set of SSO probes revealed, in two PV patients,¶ asilent base substitution (GAC -- GAT) in codon 57 encodingaspartic acid. This substitution altered its hybridization pat-tern with the SSO probes CRX02 and CRX03 but did notchange its amino acid coding potential. The GAC codon isfound in most DQB alleles with Asp-57, however, the GATcodon is found in the DQBJ.5 (DR2, Dw12) allele. Thus, theDQB1.3 allele can now be subdivided on the basis of a silentnucleotide polymorphism, into DQBI .3.1 andDQBJ .3.2 (Fig.4). This observation is consistent with the notion that theDQBJ.3 allele is directly involved with susceptibility to PVand is not simply a marker for some "disease" haplotype. Inanother PV patient carrying the DRw6 haplotype (DR4/DRw6) who also failed to type with the SSO probes CRX02and CRX03, another DQB allele was found that codes for aserine at codon 57 (Fig. 3). This sequence (DQBI.2) haspreviously been found only on the rare DR2, AZH haplotype(13, 18) but is present, in this patient, on the DRw6 haplotype(Fig. 3). In this patient, susceptibility to PV may be conferredby the DR4, DwlO haplotype and not by this unusual DRw6haplotype.

DRB and DPB Variation. The DRBI alleles from severalDRw6' PV patients were sequenced by using an allele-specific amplification primer (see Materials andMethods andthe legend to Fig. 2). This amplification strategy significantlysimplifies the subsequent analysis by both nucleotide se-quence determination and SSO probe hybridization.The sequence analysis of DRw6' patients revealed the

presence of two different DRBI alleles: 6A and 6B (Fig. 1)associated with the serological splits ofDRw6. The 6A allele,associated with DRw13, is the most common DRBI allele,while the 6B allele, associated with DRw14, is rare but hasbeen recently reported (19). We have designed an oligonu-cleotide (CRX23) to detect the "A-H" sequence encoded bycodons 57-61 and an oligonucleotide (CRX36) to identify thepolymorphic sequences around codons 70. The distributionof DRBI alleles on DRw6, DQBJ.3 haplotypes is shown inTable 2 and Fig. 4. The observation that most (19 of 22 or86%) DRw6+ patients contain the DQBI.3 allele (vs. 3% ofcontrols) while 64% contain the DRBI allele 6B (vs. 6% ofcontrols) and 36% contain the 6A allele suggests that most ofthe DRw6 susceptibility to PV can be accounted for by theDQBI.3 chain. That is, DQB1.3 haplotypes confer riskwhether they contain 6A or 6B. The increase in DRBI allele6B among PV patients may be due to linkage disequilibriumwith DQBI.3. Alternatively, this allele also could contributeto PV susceptibility. This distinction can be made only byanalyzing DQB1.3+ controls. However, the incidence of theDQBI.3 allele among DRw6+ controls is very low (only 1 of38 random controls). This one rare DQBI.3 haplotype con-tains the DRBI allele 6A. Clearly, any conclusion about thepotential contribution of the DRBI allele 6B to PV suscep-tibility requires the analysis of more DQBI.3 haplotypes inrandom controls.The DRB3 sequences in DRw6+ PV patients and their

matched controls were also analyzed by PCR amplificationand SSO probe typing. Sixteen of 21 (76%) of the DRw6+patients carried the DRB3 allele 52c, while 10 of 15 controlDRw6+ haplotypes carried the 52c allele (Table 2). Thus, the52c allele is slightly increased among patients, but since itsfrequency is 66% in controls, this locus does not appear to beinvolved in predisposition to PV.

IThe third PV patient has a conventional DQ31.3 nucleotide se-quence and, on repeated SSO testing, yielded the expected probetyping result.

Proc. Natl. Acad. Sci. USA 86 (1989)

IlArgMTCysTyrPhe ThrAsnGlyThrGluArgValArgGlyVal ThrArgHisIl a1 GGATCCGCATGTGCTACTTCACCAACGGGACGGAGCGCGTGCGGGGTGTGACCAGACACA D

.__________________________________------------------------- D)QB 1.3.1OQB 1.3.2

FIG. 4. Nucleotide sequenceTyrAsnArgGluGluTyrVa.LArgPheAspSerAspValGlyValTyrArgAlaValThr of the DQBJ.3 alleles. The nucle-

61 TCTATAACCGAGAGGAGTACGTGCGCTTCGACAGCGACGTGGGGGTGTATCGGGCGGTGA DQB 1.3.1 otide sequence of the DQBJ.3.1-______________________________________________------------- DQB 1.3.2 allele is shown, and the DQBI.3-2

allele is shown aligned with it.ProGlnGlyArgProAspAlaGluTyrTrpAsnSerGlnLysGluVallLsuGluArgAla Dashes indicate sequence iden-121 CGCCGCAGGGGCGGCCTGACGCCGAGTACTGGAACAGCCAGAAGGAAGTCCTGGAGAGGG DQB 1 3 1 . q-T-DQB 1 3:2 tity, and letters indicate polymor--------------------.T---------------------------------------- DQB 1.3.2 tiy an letr iniaeplmr

phic nucleotides. The amino acidArgAlaSerVaLAspArgVal sequences inferred from the nucle-

181 GGCCCGGGCCCTCGGTGGACAGAGTGTGCAGACACAkCTACCTGCAG DQB 1.3.1 otide sequences are shown in ital------------------------------------------------ DQB 1.3.2 ics above the DQBJ.3.1 allele.

Lastly, the allelic variation at theDPB locus allele in all PVpatients (DR4 and DRw6) and their HLA-matched controlswas studied by using the newly developed DPB SSO probetyping system (ref. 14; unpublished data). Table 2 shows theresult of this typing. In summary, 20 of 27 Israeli controls

Table 2. SSO probe analysis of HLA class II subtypesHLA class II haplotypes

DRw6 haplotypes

Patient no.Israeli patients

117118119120121122123125128129130132137

Austrian patients106107108109110111112114115

Austrian controls116117118

Israeli controls122123124125128129130131132133134135

DR DQw DQA DQBI DRBI DRB3 DPB

4, 64, 64, 64, 64, 64, 64, 64, 65, 65, 65, 66, -5, 6

4, 64, 64, 64, 64, 65, 65, 66, -6, 7

4, 65, 66, 7

1,31,31,31,31,31,31,31,31,31,31,31,31,3

1.2 1.2NT 1.3.11.1 1.3.11.1 1.3.11.1 1.3.11.1 1.3.11.1 1.3.11.1 1.3.11.1 1.3.11.1 1.3.11.1 1.3.11.1 1.3.11.1 1.3.1

1,3 1.3 1.3.21, 3 1.1 1.3.11, 3 NT 1.71,3 1.3 1.3.21,3 1.1 1.3.21, 3 1.1 1.3.11,3 1.3 1.71 1.1 1.3.11,2 1.1 1.3.2

6B6B*6A6B6B6B6B6B6B6B6B6A6B

6A6B6A6A6B6A6A6A6B

52c 4.1, 152c 4.2, -52b NT52c 4.1, -52c 4.1, 2.152c 4.1, -52c 4.1, 952c 4.1, -S2c NT52c NT52c 4.1,352c 4.1,252b NT

52c 2.1, -52c NTNT 4.1, -52c 2.1,-52c 2.1,-52b NT52b NT52c 2.1, 1052c NT

1, 3 1.2 1.7 6A 52c 1, 31, 3 NT 1.7 6A 52c 2.11, 2 NT 1.6 6A 52c NT

4,6 1,34,6 1,34,6 1,34,6 1,35,6 1,35,6 1,35,6 1,3HTC 6 1HTC 6 1HTC 6 16,- 16, 7 1,2

1.1 1.3.11.3 1.61.3 1.61.3 NT1.2 1.71.3 1.61.2 1.11.3 1.61.3 1.61.2 1.11.1 1.11.3 1.6

6A 52b 4.1, 56A 52c 4.2, -6A 52c 4.1,-6A 52c NT6A 52c 4.1, 2.16B 52b 4.1, 2.16A 52b 4.1, 2.16A 52b 4.1,-6A 52a NT6A 52c 2.1,-6A 52c NT6A 52c 4.1, 1

were found to carry the DPB4.1 allele, while 21 of 27 IsraeliPV patients carried this DPB4.1 allele. No specificDPB allelewas increased in the patient population. In the Israeli popu-lation, DPB4.1 is very frequent and appears to be in stronglinkage disequilibrium with DR4. In the Austrian PV popu-lation, by contrast, the DPB2.1 allele was found to be presentin three of four Austrian PV patients as well as in one of twocontrols. Only one individual in the Austrian population (aPV patient) had the DPB4.1 allele. In general, these findingssuggest that the DPB gene on DR4 and DRw6 haplotypes isnot involved in susceptibility to PV.

DISCUSSIONSpecific HLA class II alleles that are strongly associated withPV (i.e., Dw10 DRBJ on DR4 haplotypes and DQB1.3 onDRw6 haplotypes) could be markers for extended "disease"haplotypes or they could directly confer susceptibility. Theanalysis of allelic sequence variation at all the polymorphicclass II loci revealed that there were four different DRw6haplotypes encoding the DQB1.3 chain (Fig. 5). This heter-ogeneity of DRw6, DQB1.3 haplotypes made it possible toask whether a specific allele contributes to PV predispositionby examining whether this allele is more highly associatedwith PV than is a given haplotype. To address these issues,we have analyzed the distribution of class II alleles in PVpatients and in HLA-matched controls.The sequence analysis ofDNA from three PV patients and

one control that failed to hybridize with the DQB SSO probes(CRX01 and CRX02) revealed two variant sequences. Twopatients carry a silent base substitution (GAC -- GAT) incodon 57 coding for aspartic acid (Fig. 4). The finding that,in PV patients, two different DNA-defined DQB alleles(DQBI.3.1 and -1.3.2) encode the same DQ B chain suggeststhat the DQB1.3 protein is not simply a marker for a diseasehaplotype. This result, together with our previous observa-tion that the DQB1.3 allele is very strongly associated withPV (10) lends further support to the notion that the DQX3-chain itself is involved in susceptibility to PV. With the

DOQ

1.

2.

3.

4.

DQ(X DRPI

..3.I A_1.3.1 1.1 6A

1.3.1 1.1 6B

1.3.2 1.1 6B

1.32 1.3 6A

FIG. 5. Heterogeneity of DRw6, DQBI.3 (Dw9) haplotypes.

NT, sample not typed.*This sample amplified weakly with the DRw6-specific PCR primerCRX11, though it typed positively with SSO probes used to assignthe DRw6B allele.

6218 Genetics: Scharf et al.

Proc. Natl. Acad. Sci. USA 86 (1989) 6219

inclusion of these newly characterizedDQB alleles in DRw6'patients, the frequency of the DQBJ.3 marker in IsraeliDRw6+ patients is 92% (12 of 13) and in Austrian DRw6+patients is 78% (7 of 9). In all but one exceptional case (pa-tient 112), the other HLA haplotype is a susceptible DR4,Dw10 haplotype. One PV patient (117) (DR4/Dw6) was foundto carry aDQB allele with serine at position 57 (DQBJ.2) (Fig.2), previously found only on the rare DR2, AZH haplotype(13, 18).The analysis of DQB, DQA, and DRBI alleles in DRw6'

PV patients revealed the existence of a variety of haplotypesencoding the DQB1.3 chain (Fig. 5). Two different DRBIalleles were identified on these haplotypes: (i) the common6A allele, associated with DRw13, and (it) the rare 6B allele,associated with DRw14 (Fig. 1). The observed heterogeneityof DQB1.3-encoding haplotypes is consistent with a directcontribution ofthe DQB1.3 chain toPV susceptibility and notwith the notion of an extended disease haplotype "marked"by the DQBJ.3 allele.The frequency of the DRBJ allele 6B is increased among

DRw6+ patients (64%; 14 of 22) relative to matched controls(6%; 1 of 17), consistent with our previous finding that the 6Aallele was decreased among DRw6+ patients (10). This in-crease could be due simply to linkage disequilibrium between6B and the DQBI.3 allele and not necessarily reflect anyinvolvement of this DRB1 allele in PV susceptibility. Thisissue can be resolved only by comparing the distribution ofDRBI alleles on DQBJ.3 haplotypes between patient andcontrol populations. However, in control DRw6+ individu-als, the DQB1.3 allele is very rare. We have found only onesuch individual (122) whose DQBJ.3 haplotype contains the6A allele (Table 2). However, a panel of four DRw6, Dw9HTCs contain the DQBI.3 allele, the DQAJ.I allele, and theDRBI 6B allele (Table 3). Most (14 of 22) patient DQB1.3haplotypes contain the 6B allele. It is possible that theDQBJ.3, DRBI 6B haplotype confers more risk than does theDQBI.3, DRBI 6A haplotype, but this can be evaluated onlyby analyzing many random control DQB1.3 haplotypes. Thehypothesis that different risks are associated with specificcombinations ofDQB and DRBI alleles is consistent with ourpreviously reported observations on the DR4 associationwith insulin-dependent diabetes mellitus (IDDM) (13, 20).For IDDM, a combination ofthe DQB3.2 allele and the DRBIallele Dw4 or Dw10 is associated with increased risk.The distribution ofDRB3 alleles was similar amongDRw6+

PV patients and controls. DPB allelic variation in patientsand controls also failed to show any strong association withPV. Interestingly, in the Israeli population, the DPB4.1 allelewas found in the majority ofindividuals tested, whereas in theAustrian population, DP2.1 was found in about 50%o ofindividuals (Table 2). These findings may reflect differentpatterns of linkage disequilibrium or of allele frequencies inthe different ethnic groups. In general, these results argueagainst a putative extended disease haplotype and, thus, lendstrong support for a direct role of the Dw10 DRBI allele (inDR4+ PV patients) and of the DQBI.3 allele (in DRw6+ PVpatients) in disease susceptibility.

Table 3. SSO analysis of DRw6 Dw9 HTCsHLA class II haplotypes

HTC Dw DQw DQA DPB DRBI DRB2ABO 9 1 1.1 1.3 6B 52bl.3EK/OH 9 1 1.1 1.3 6B 52cl.3TEM 9 1 1.1 1.3 6B 52bl.3KOSE 9,19 1 1.1,1.2 1.3,1.7 6B 52c or

52b, 52c

The most conclusive demonstration linking DR and DQmolecules to PV and establishing their functional role willultimately come from immunologic studies. In animal modelsof autoimmunity, multiple discrete autoimmunogenic peptidefragments of self-protein are presented to the immune systemby distinct class II major histocompatibility complex mole-cules. Thus, for myelin basic protein, which triggers paralysisin the murine model. autoimmune disease, experimental aller-gic encephalomyelitis, multiple discrete encephalitogenic T-cell epitopes have been demonstrated (21). Some epitopes bindto class II molecules homologous to DR, while others bind toclass II molecules homologous to DQ. Such studies have beenextended for the first time to human autoimmune disease.Thus, in humans with myastheniagravis, myasthenics who areDR3' mount T-cell and antibody responses to peptide-(257-269) of the a chain of the acetylcholine. receptor, whileDR5' individuals respond to peptide-(15-212) (22). When thepeptide sequence of the epidermal membrane protein (1)responsible forPV is known, such studies could be performedon PV patients. Only then will the functional associations ofHLA class II molecules with disease be understood.

In summary, the data presented here support the hypoth-esis that two different class II genes, DRBJ Dw10 (DR4), andDQBJ.3 (DRW6), directly contribute to the predisposition toPV. Susceptibility conferred by the DR4 haplotype and theDRB1 Dw10 allele may involve recognition of a differentepitope or utilize a different T-cell receptor than does the riskconferred by the DRw6 haplotype and the DQBJ.3 allele.

1. Razzaque, A. (1983) Dermatology 1, 1-158.2. Brautbar, C., Moscovitz, M., Livshits, T., Haim, S., Hacham-Zadeh, S.,

Cohen, H. A., Sharon, R. & Nelken,- D. (1985) Tissue Antigens 16,238-243.

3. Szafer, F., Brautbar, C., Tzfoni, E., Frankel, G., Sherman, L., Cohen,I., Hacham-Zadeh, S., Aberer, W., Tappeiner, G., Holubar, K., Stein-man, L. & Friedmann, A. (1987) Proc. Nati. Acad. Sci. USA 84,6542-6545.

4. Mullis, K. B. & Faloona, F. (1987) Methods Enzymol. 155, 335-350.5. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich,

H. A. & Arnheim, A. (1985) Science 230, 1350-1354.6. Scharf, S. J., Horn, G. T. & Erlich, H. A. (1986) Science 233, 1076-1078.7. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R.,

Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science 239, 487-491.8. Saiki, R. K., Bugawan, T. L., Horn, G. T., Mullis, K. B. & Erlich,

H. A. (1986) Nature (London) 324, 163-166.9. Hansen, J. A., Beatty, P. G., Anasett, C., Martin, P. J., Mickelson, E.

& Thomas, E. D. (1987) Br. Med. Bull. 43, 203-207.10. Scharf, S. J., Friedmann, A., Brautbar, C., Szafer, F., Steinman, L.,

Horn, G., Gyllensten, U. & Erlich, H. (1988) Proc. Natl. Acad. Sci. USA85, 3504-3508.

11. Scharf, S. J., Long, C. M. & Erlich, H. A. (1988) Hum. Immunol. 22,61-69.

12. Sinha, A. A., Brautbar, C., Szafer, F., Friedmann, A., Tzfoni, E., Todd,J. A., Bell, J. I. & McDevitt, H. 0. (1988) Science 239, 1026-1029.

13. Erlich, H. A., Horn, G., Scharf, S. & Bugawan, T. (1989) in MolecularBiology ofHLA Class Il Antigens, ed. Silver, J. (CRC, Boca Raton, FL),in press.

14. Bugawan, T. L., Horn, G. T., Long, C. M., Mickelson, E., Hansen,J. A., Ferrara, G. B., Angelini, G. & Erlich, H. A. (1988) J. Immunol.141, 4024-4030.

15. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci.USA 74, 5463-5467.

16. Gyllensten, U. & Erlich, H. A. (1988) Proc. Natl. Acad. Sci. USA 85,7562-7566.

17. Horn, G. T., Bugawan, T. L., Long, C. M. & Erlich, H. A. (1988) Proc.Natl. Acad. Sci. USA 85, 6012-6016.

18. Lee, B. S. M., Bell, J. I., Rust, N. A. & McDevitt, H. 0. (1987) Immu-nogenetics 26, 85-91.

19. Gorski, J. (1989) Hum. Immunol. 24, 145-149.20. Sheehy, M. J., Scharf, S., Rowe, J. R., Neme de Gimenez, M. H.,

Meske, L. M., Erlich, H. A. & Nepom, B. S. (1989) J. Clin. Invest. 83,830-835.

21. Zamvil, S., Mitchell, D., Rothbard, J. & Steinman, L. (1988) J. Exp. Med.168, 1181-1186.

22. Brocke, S., Brautbar, C., Steinman, L., Abramsky, O., Rothbard, J.,Neumann, D., Fuchs, S. & Mozes, E. (1988) J. Clin. Invest. 82,1894-1900.

Genetics: Scharf et al.