562 Letters to the Editor J ALLERGY CLIN IMMUNOL

MARCH 2004

Letters to the editorHLA association in aspirin-intolerant asthma:DPB1*0301 as a strong marker in a Koreanpopulation

To the Editor:Aspirin (ASA)-intolerant asthma (AIA) affecting 10%

to 20% of adults with asthma is characterized by ASAhypersensitivity, bronchial asthma, and chronic rhinosi-nusitis with nasal polyposis.1 Although the pathogenicmechanism is not fully understood, a possible involve-ment of immunologic mechanisms in association withHLA alleles, particularly DPB1*0401 and DPB1*0301,was suggested in a white population with AIA.2,3 How-

ever, these data have focused on whites and are requiredto be confirmed in other ethnic populations. In this study,to evaluate HLA associations in AIA development in aKorean population, we defined HLA-DRB1, DQB1, andDPB1 alleles and haplotypes by high-resolution geno-typings in a larger number of patients with AIA com-pared with patients with ASA-tolerant asthma (ATA) andnormal control subjects. Seventy-six patients with AIA(34 men; mean age, 45.2 years), 73 with ATA (21 men;mean age, 46.5 years), and 91 control subjects (74 men;mean age, 30.7 years) were enrolled. Twenty-ninepatients with AIA (38.2%) and 38 with ATA (30.5%) hadatopy. Fifty-nine patients with AIA (77.6%) and 38 withATA (53.4%) had chronic rhinosinusitis (P < .01). Twen-ty-eight (68.3%) among 41 patients with AIA undergoingrhinoscopy had nasal polyps. AIA versus ATA was clas-sified on the basis of lysine-ASA bronchoprovocationtest results.4 All subjects gave informed consent, whichwas approved by the ethics committee of Ajou Universi-ty Hospital, Suwon, Korea.

Genomic DNA from the study subjects was extractedwith the use of an Extraction Kit (Intron Biotechnol,Seoul, Korea). HLA-DQB1 and HLA-DPB1 high-resolu-tion genotypings were obtained from amplified gene prod-ucts with the use of locus-specific primers5 and directsequencing with the ABI 3100 Genetic analyzer (Applied

Biosystems, Foster City, Calif). In the case of DRB1, thepolymorphic exon 2 was amplified from each sample,using 8 sets of allele group–specific primers.5 The alleleand haplotype frequencies were calculated with an E-Malgorithm,6 with the use of SAS/Genetics software (SASInstitute Inc; Cary, NC), and the frequencies between twogroups were compared by means of allele and haplotypeprocedures of the software, which are based on χ2 statis-tics. P values were corrected for multiple comparisons bymultiplying by 10, as 9 common alleles/haplotypes and allothers considered as one were tested. The clinical charac-teristics between the two groups were compared by meansof χ2 and Student t tests. Significance level was <.05.

Twenty-nine DRB1 alleles, 16 DQB1 alleles, and 21DPB1 alleles were noted in this study. The alleles in theDRB1 locus were in Hardy-Weinberg equilibrium,whereas the alleles in DQB1 and DPB1 loci were not (P< .05) when tested by χ2 test. Table I summarizes theallele frequencies showing significant differencesbetween two groups. The frequency of HLA-DPB1*0301 was 13.8% in patients with AIA, comparedwith 4.1% in patients with ATA (odds ratio [OR] = 5.2,95% CI = 1.8 to 14.7, P = .004) and 2.2% in control sub-jects (OR = 8.3, 95% CI = 2.7 to 25.5, P < .0001). Theseresults remained significant after P values were correct-ed for multiple comparisons (Pc = .04, Pc < .001). The

TABLE I. Allele frequencies of HLA-DRB1, DQB1, and DPB1 in study subjects

P value (OR)

Allele AIA (%) n = 76 ATA (%) n = 73 NC (%) n = 91 AIA vs ATA AIA vs NC

DRB1 *0101 3 (2.0) 8 (5.5) 12 (6.6) .042 (0.3)*0301 9 (5.9) 2 (1.4) 4 (2.2) .037 (9.7)*0901 28 (18.4) 14 (9.6) 19 (9.4) .028 (2.3) .037 (2.2)

DQB1 *0501 5 (3.3) 10 (6.8) 16 (8.8) .039 (0.4)DPB1 *0301 21 (13.8) 6 (4.1) 4 (2.2) .004 (5.2) <.001 (8.3)

TABLE II. Haplotype analysis of HLA-DRB1, DQB1, and DPB1

Haplotype frequencies (%) P value

Haplotype* AIA (n = 76) ATA (n = 73) NC (n = 91) AIA vs ATA AIA vs NC

DRB1-DQB1-DPB11. 0901-0303-0501 12.0 3.8 7.4 .006 .102. 0901-0303-0201 4.9 2.2 0.0 .23 .0063. 1501-0602-0201 4.6 2.7 2.5 .59 .194. 0406-0302-0201 4.5 2.5 2.0 .27 .185. 0301-02-0301 3.3 0.0 0.6 .03 .05

DRB1-DQB11. 0901-0303 17.8 9.6 10.4 .04 .05

DRB1-DPB11. 0901-0501 11.6 4.1 7.3 .01 .242. 0901-0201 5.2 2.6 0.0 .26 .0023. 0301-0301 3.9 0.0 0.5 .02 .02

DQB1-DPB11. 0303-0501 12.8 4.6 7.2 .01 .032. 0303-0201 6.2 2.4 0.5 .09 .0043. 02-0301 5.0 0.0 0.5 .004 .008

*Five frequent 3-locus haplotypes in A1A group are listed in frequency order.

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564 Letters to the Editor J ALLERGY CLIN IMMUNOL

MARCH 2004

Letters to the editor

frequencies of DRB1*0101, DRB1*0301, DRB1*0901,and DQB1*0501 were significantly increased ordecreased in patients with AIA than in patients with ATAor control subjects; however, P values were not signifi-cant after correction for multiple comparisons. Whenclinical features of patients with AIA were comparedaccording to DPB1*0301 genotype, the patients withDPB1*0301 (n = 21) tended to have lower FEV1 levels(76.4 ± 18.9% vs 83.1% ± 23.3%, P = .14), higher ratioof women (71.4% vs 49.1%, P = .08), and higher preva-lence of rhinosinusitis and/or nasal polyps (90.5% vs72.7%, P = .13) than those without it (n = 55).

For HLA DRB1-DQB1-DPB1 loci, 31 haplotypeswere observed with >1% frequencies. Table II shows themost common haplotypes in the AIA group. The fre-quency of DRB1*0901-DQB1*0303-DPB1*0501 hap-lotype was significantly higher in patients with AIA(12%) than in patients with ATA (3.8%, P = .006, Pc =.06). The frequency of DRB1*0901-DQB1*0303-DPB1*0201 haplotype was much higher in patients withAIA (4.9%) than in control subjects (0%, P = .006, Pc =.06). In 2-locus haplotype analysis, DRB1*0901-DPB1*0501 and DQB1*0303-DPB1*0501 haplotypesshowed a significant association with AIA (AIA vs ATA,P = .01, Pc = .1). DRB1*0901-DPB1*0201 andDQB1*0303-DPB1*0201 haplotypes also showed a sig-nificant association with AIA (AIA vs NC, P < .0005,Pc < .05). The frequency of DQB1*02-DPB1*0301 hap-lotype was significantly higher in patients with AIA thanin those with ATA (P = .004, Pc = .04) and control sub-jects (P= .008, Pc = .08).

This is the first study to demonstrate a strong associa-tion between DPB1*0301 allele and AIA development inan Asian population, which is comparable to a Polishstudy,3 and to perform haplotype analysis in patients withAIA. In addition, clinical manifestations of patients withAIA with DPB1*0301 are more identical to typical man-ifestation of AIA described previously.1 Consistency ofDPB1*0301 as a positive factor in two different ethnicpopulations suggested that genes controlling AIA suscep-tibility are closely related to the HLA-DPB1 locus. On thebasis of these findings and the previous study demon-strating higher incidences of circulating autoantibodies inpatients with AIA,7 it can be speculated that autoimmuni-ty is one pathogenesis of AIA development. Furthermore,the HLA gene region contains a series of disparate genessuch as TNF-α, complement 4, and so forth.8 The poly-morphisms of TNF gene have been reported to be associ-ated with variation in the expression of TNF-α and thepresence of asthma.9 However, polymorphisms of manyother genes in the HLA gene region have not been inves-tigated for a role in asthma. Further studies will be need-ed to investigate other markers and genes in the context oflinkage disequilibrium with the DPB1*0301 allele.

The HLA haplotype analysis in this study demonstrat-ed that two of the DRB1*0901-associated haplotypesappeared to be significant in AIA susceptibility, althoughthe P values did not remain significant after multiplecomparisons (Pc = .06). However, further studies with a

larger sample size will be needed to elucidate a border-line association of these haplotypes with AIA. The fre-quency of DQB1*02-DPB1*0301 haplotype in AIA waslower than that of DPB1*0301, although the haplotypeshowed significant association with AIA, which wouldbe more evidence that the single allele of DPB1*0301 isan independent risk factor for AIA development. In con-clusion, we suggest that DPB1*0301 may be involved inAIA development. A possible involvement ofDRB1*0901-associated haplotypes was also suggested.

Jeong Hee Choi, MDa

Kyung Wha Lee, PhDb

Heung Bum Oh, MD, PhDc

Kwan Jeh Lee, PhDd

Yu Jin Suh, MDa

Choon Sik Park, MD, PhDe

Hae-Sim Park, MD, PhDa

aDepartment of Allergy and RheumatologyAjou University Hospital

Suwon, KoreabHallym Institute for Genome Application

Hallym UniversityAnyang, Korea

cDepartment of Laboratory MedicineUniversity of Ulsan College of Medicine

dDepartment of StatisticsDongguk University

Seoul, KoreaeDivision of Allergy and Respiratory Medicine

Soonchunhyang University HospitalBucheon, Korea

This study was supported by a grant of the Korean Health 21 R& D project (01-PJ10-PG6-01GN14-0007). Drs Choi and Lee con-tributed equally to this work. Reprint requests: Dr Hae-Sim Park,Ajou University, Suwon, Korea.

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3. Dekker JW, Nizankowska E, Schmitz-Schumann M, Pile K, Bochenek G,Dyczek A, et al. Aspirin-induced asthma and HLA-DRB1 and HLA-DPB1 genotypes. Clin Exp Allergy 1997;27:574-7.

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5. Tilanus MGJ, Hansen JA, Hurley CK. IHWG Technical Manual. Seattle:International Histocompatibility Working Group; 2000.

6. Long CJ, Williams RC, Urbanek M. An E-M algorithm and testing strat-egy for multiple-locus haplotype. Am J Hum Genet 1995;56:799-810.

7. Szczeklik A, Nizanlowska E, Serafin A, Dyczek A, Duplaga M, Musial J.Autoimmune phenomena in bronchial asthma with special reference toaspirin intolerance. Am J Respir Crit Care Med 1995;152:1753-6.

8. Acton RT. The major histocompatibility complex. In: Rich RR, FleisherTA, Shearer WT, Kotzin BL, Schroeder HW Jr, editors. Clinicalimmunology. 2nd edition. London: Mosby; 2001. p. 6.1-6.13.

9. Li Kam Wa TC, Mansur AH, Britton J, Williams G, Pavord I, RichardsK, et al. Association between -308 tumour necrosis factor promoter poly-morphism and bronchial hyperreactivity in asthma. Clin Exp Allergy1999;29:1024-8.

doi:10.1016/j.jaci.2003.12.012