Association of an HLA-DQ Allele with Clinical Tuberculosis

Brief Report Association of an HLA-DQ Allele With Clinical Tuberculosis

Anne E. Goldfeld, MD; Julio C. Delgado, MD; Sok Thim; M. Viviana Bozon, MD; Adele M. Uglialoro; David Turbay, MD; Carol Cohen; Edmond J. Yunis, MD

Context.—Although tuberculosis (TB) is the leading worldwide cause of death clinical TB, we performed a 2-stage study due to an infectious disease, the extent to which progressive clinical disease is as- of molecular typing of HLA class I and sociated with genetic host factors remains undefined. class II alleles and also tested for the pres- α Objective.—To determine the distribution of HLA antigens and the frequency of ence of 2 TNF- alleles in Cambodian pa- 2 alleles of the tumor necrosis factor ␣ (TNF-␣) gene in unrelated individuals with tients with clinical TB and in control individuals who did not have a history of TB. clinical TB (cases) compared with individuals with no history of clinical TB (controls) in a population with a high prevalence of TB exposure. Methods Design.—A 2-stage, case-control molecular typing study conducted in 1995-1996. The study subjects were unrelated Cam- Setting.—Three district hospitals in Svay Rieng Province in rural Cambodia. bodian patients recruited from a TB treat- Patients.—A total of 78 patients with clinical TB and 49 controls were included ment program in eastern rural Cambodia. in the first stage and 48 patients with TB and 39 controls from the same area and Two different groups of patients and con- socioeconomic status were included in the second stage. trols were recruited for the 2 stages of the Main Outcome Measures.—Presence of HLA class I and class II alleles deter- study, the first group in 1995 and the sec- mined by sequence-specific oligonucleotide probe hybridization and presence of 2 ond group in 1996. The patients were ran- TNF-␣ alleles determined by restriction fragment length polymorphism analysis. domly selected from all inpatients or out- Results.—In the first stage, 7 DQB1*0503 alleles were detected among 156 al- patients picking up their monthly supply of TB medicines at Chantrea, Rumduol, or leles derived from patients with TB, whereas no DQB1*0503 alleles were found among the 98 alleles derived from controls (P=.04). There was no detectable dif- Kompong Rho District Hospitals in Svay Rieng Province. ference in the distribution of the 2 TNF-␣ alleles in patients with TB compared with The diagnosis of clinical TB was made controls. In the second stage, we tested for the presence of a single variable, the on site on the basis of light microscopy DQB1*0503 allele, and found 9 DQB1*0503 alleles among 96 alleles derived from demonstrating the presence of acid-fast patients with TB and no DQB1*0503 alleles among 78 alleles in controls (P=.005). bacilli in sputum, pleural fluid, or lymph Conclusions.—The HLA-DQB1*0503 allele is significantly associated with sus- node drainage. One of us (S.T.) conducted ceptibility to TB in Cambodian patients and, to our knowledge, is the first identified family interviews of the household mem- gene associated with development of clinical TB. bers of the patients and verified that no JAMA. 1998;279:226-228 patients in the study were related. Con- trol individuals were recruited from pa- ABOUT A THIRD of the earth’s popula- quired immunodeficiency syndrome tients visiting the same hospitals for mi- tion is infected with Mycobacterium tu- (AIDS) have a greatly increased risk of nor complaints. Based on detailed clinical berculosis,1-3 the bacteria that causes activation of quiescent infection with M history, controls did not have a history of tuberculosis (TB). Infection with M tuber- tuberculosis .4-6 Poverty also has been im- TB or current symptoms consistent with culosis results in a variety of conditions plicated as a cofactor in disease progres- TB. All patients and controls were fol- ranging from asymptomatic infection to sion.7,8 Certain populations are at risk for lowed up for 6 months to confirm their progressive pulmonary or extrapulmo- increased susceptibility to infection and diagnosis or control status. nary TB and death.4 Approximately 1 in progressive disease due to M tuberculo- After consent was obtained, blood was 10 of those infected will progress to ac- sis,9-13 and in several populations the HLA drawnfromeachsubjectandstoredat4°C tive disease during their lifetime.1-3 Tu- class II DR2 serotype is associated with for 4 to 10 days. Plasma and peripheral berculosis is the leading cause of death due clinical TB.11-13 Mutations in the inter- blood mononuclear cells (PBMCs) were to an infectious disease worldwide, ac- feron-␥ receptor gene have been associ- prepared according to standard tech- counting for approximately 3 million ated with progressive atypical mycobac- niques and stored at −70°C. Plasma deaths annually.1-3 terial infection14 and with Calmette- sampleswerescreenedforevidenceofan- Individuals who have impaired cell- Gue´rin bacillus (Mycobacterium bovis) tibodies to human immunodeficiency vi- mediated immunity due to chemo- infection.15 Tumor necrosis factor ␣ (TNF- rus (HIV) types 1 and 2 and human T- therapy, steroid use, neoplasia, or the ac- ␣) appears to play an important role in lymphotropic virus (HTLV) types 1 and 2 TB pathogenesis, including granuloma by enzyme-linked immunosorbent assay. From the Divisions of Adult Oncology (Dr Goldfeld and formation and containment of TB infec- The DNA was prepared from PBMCs Ms Uglialoro) and Immunogenetics (Drs Delgado, Bo- tion16,17 and impaired TNF-␣ secretion by a quick isolation method,18 and allele- zon, Turbay, and Yunis) and the Blood Bank (Ms Cohen), Dana-Farber Cancer Institute, Boston, Mass, and the due to defective signaling through the in- specific polymerase chain reaction (PCR) Cambodian Health Committee, Phnom Penh (Mr Sok). terferon-␥ receptor gene may be in- was performed. For HLA class I typing, Reprints: Anne E. Goldfeld, MD, Division of Adult volved in disease progression.14,15 PCR amplification of exons 2 and 3 of the Oncology, Dana-Farber Cancer Institute, 44 Binney St, Boston, MA 02115 (e-mail: anne_goldfeld@ To determine whether specific HLA HLA-A and HLA-B loci was performed. macmailgw.dfci.harvard.edu). class I or class II alleles are associated with For class II typing, PCR amplification of

Downloaded From: https://jamanetwork.com/ on 10/01/2021 exon 2 of the HLA-DRB, DQB1, and DQA1 (Table). HLA-B38 was present in 16 of 138 Frequencies of Major Histocompatibility Complex loci was performed.19-21 The PCR prod- alleles among patients with TB com- (MHC) Class II Antigens in Patients With Tubercu- ucts were separated by agarose gel elec- pared with 2 of 96 alleles in controls losis (TB) and Control Individuals From Cambodia* trophoresis, stained with ethidium bro- (P=.005) (data not shown). TB, Controls, mide, and photographed. Dot-blot, An RFLP analysis scoring for the 2 No. (%) No. (%) P prehybridization, and hybridization pro- TNF-α promoter variants revealed 11 Allele (n=156†) (n=98†) Value cedures were carried out according to the TNF2 alleles of 156 alleles derived from DRB1 1501 12 (7.7) 7 (7.1) .99 manufacturer’s instructions (Lifecodes patients with TB and 8 TNF2 alleles of 1502 26 (16.7) 18 (18.4) .74 Corp, Stamford, Conn). Class I and class 96 alleles derived from controls with no II alleles were identified in the PCR- difference in TNF2 variant expression 1602 14 (9.0) 7 (7.1) .65 amplified products by sequence-specific oli- in either group. Seventeen of the 19 0301 14 (9.0) 11 (11.2) .67 gonucleotide probe hybridization.20,22-25 TNF2 alleles were found in HLA-DR3– 0403 1 (0.6) 0 .99 TheTNF-αalleles were determined by positivesubjects(datanotshown).Thus, 0404 1 (0.6) 0 .99 restriction fragment length polymor- as in other populations,26 the TNF2 al- 0405 5 (3.2) 7 (7.1) .22 phism (RFLP) analysis using primers de- lele was in linkage disequilibrium with 0406 1 (0.6) 1 (1) .16 signed to incorporate a polymorphic site HLA-DR3. 1101 4 (2.6) 2 (2) .99 (the nucleotide A vs G) at position −308 Based on the first stage of our analy- 1106 0 2 (2) .15 nucleotides (nt) relative to the TNF-α sis, we chose to further investigate the as- 1201 2 (1.3) 0 .52 transcription start site. The TNF2 (A at sociation of the single class II allele, 1202 23 (14.7) 17 (17.3) .60 −308 nt) polymorphism creates an NcoI DQB1*0503, and TB. This second stage in- 1301 2 (1.3) 0 .52 restriction site and can be differentiated cluded 48 patients with pulmonary TB 1302 1 (0.6) 1 (1) .16 by size (107 nt for the TNF1 and 87 nt for (mean age, 46 years; range, 25-76 years; 1312 2 (1.3) 0 .52 the TNF2 allele) from the TNF1 allele by 40% male) and 39 controls (mean age, 40 1401 7 (4.5) 5 (5.1) .99 26 agarose gel electrophoresis. years; range, 19-67 years; 30% male). One 1404 5 (3.2) 0 .16 The frequencies of independent HLA control subject tested positive for anti- 1405 1 (0.6) 0 .99 α alleles and TNF- alleles in patients bodies to HIV-1 and was excluded from 1407 3 (1.9) 1 (1) .99 and controls were determined by direct the HLA analysis. Among the 96 alleles 0701 12 (7.7) 9 (9.2) .81 counting. The statistical significance of derived from 48 patients with TB, there 0802 3 (1.9) 2 (2) .99 the difference in frequency of individual were 9 DQB1*0503 alleles. No DQB1*0503 0803 3 (1.9) 0 .29 HLA and TNF-α alleles between the 2 alleles were detected among the 76 alle- groups was calculated by the Fisher les derived from 38 controls (P=.005). 0901 11 (7.1) 7 (7.1) .99 exact test with the aid of INSTAT soft- 1001 3 (1.9) 1 (1) .99 Comment DRB3 ware (GraphPad, San Diego, Calif), and 0101 3 (1.9) 0 .29 levels of significance were reported as P In Cambodia, which has a population of 02 37 (23.7) 20 (20.4) .64 values along with 95% confidence inter- approximately 10 million, it is estimated 0301 25 (16.0) 17 (17.3) .86 vals according to the program used. In that up to 40 000 new cases of TB and DRB4 the first stage of the study, since each 13 000 deaths due to TB occur every 01 30 (19.2) 21 (21.4) .75 27,28 subject was tested for several HLA al- year. Tuberculosis is the major cause DQA1 leles and 2 TNF-α alleles, and the same of morbidity and mortality in Cambodian 0101 28 (17.9) 18 (18.4) .52 data were used to compare the fre- men aged 18 to 40 years.27-29 Although 0102 28 (17.9) 19 (19.4) .87 quency of all detected alleles, significant there are no reliable data, it is assumed 0103 4 (2.6) 0 .30 associations may have arisen by chance that the majority of Cambodians harbor 0104 15 (9.6) 7 (7.1) .65 due to multiple comparisons. In the sec- M tuberculosis and thus are chronically 0201 11 (7.1) 8 (8.2) .81 30 ond stage, we tested for the presence of infected with TB, but that only a subset 03 20 (12.8) 16 (16.3) .46 asinglealleleidentifiedinthefirststage, oftheseindividualsprogresstoactivepul- 0401 4 (2.6) 3 (3.1) .99 4 and thus, correction of the data for mul- monary or extrapulmonary TB. In the 0501 23 (14.7) 16 (16.3) .60 tiple comparisons was not necessary. Cambodian population we studied, pov- 0601 23 (14.7) 11 (11.3) .46 erty (ie, an annual family income of about DQB1 Results $180peryear)andpoornutritionalstatus, 0501 23 (14.7) 16 (16.3) .72 The first stage of our study included 78 known cofactors of TB progression, are 0502 33 (21.2) 22 (22.4) .87 patients with TB (mean age, 47 years; pervasive and were equivalent between 0503 7 (4.5) 0 .04 range, 11-77 years; 24% male; 76 patients the patient and control groups. In an on- 0601 10 (6.4) 4 (4.1) .57 with pulmonary TB, 1 with pleural TB, going pilot study in Svay Rieng, of 450 0603 2 (1.3) 0 .52 and 1 with scrofula) and 49 control indi- people randomly screened, 75% were pu- 0605 1 (0.6) 1 (1) .16 viduals (mean age, 40 years; range, 15-68 rified protein derivative positive with a 0201 14 (9.0) 11 (11.2) .67 years; 53% male). Of these 127 individu- skin reaction of greater than 5 mm of in- 0202 9 (5.8) 9 (9.2) .32 als, no patient with TB had antibodies to duration (S.T. and A.E.G., unpublished 0301 30 (19.2) 18 (18.4) .99 HIV-1 or HIV-2, and 1 patient with TB data). Although HIV-1 infection and 0302 6 (3.8) 4 (4.1) .99 was positive for antibodies to HTLV-1. AIDS are rapidly increasing in Cambo- 0303 12 (7.7) 5 (5.1) .60 Among the 156 alleles derived from 78 dia,31 we ruled out HIV-1 as contributing patients with TB, there were 7 DQB1*0503 to TB progression in this study group. 04 9 (5.8) 8 (8.2) .45 DRB5 alleles, whereas this allele was not found We did not detect a difference in the 0101 24 (15.4) 16 (16.3) .86 among the 98 alleles from the 49 controls presence of the TNF2 allele in patients 0102 24 (15.4) 12 (12.2) .58 (P=.04) (Table). When HLA-DR15 and with TB vs controls consistent with the 02 3 (1.9) 3 (3.1) .68 HLA-DR16 alleles were combined, there lack of detectable effect of this polymor- was a slight increase in HLA-DR2 alleles phism on TNF-α gene expression.32 *MHC class II antigens were determined using sequence-speciﬁc oligonucleotide hybridization as de- (52 of 156) among patients with TB com- Rather, the TNF2 allele appears to serve scribed in “Methods.” pared with 32 of 98 alleles in controls as a marker in the HLA region for genetic †Total alleles for each group.

Downloaded From: https://jamanetwork.com/ on 10/01/2021 associations with susceptibility to certain Inourcombinedsamplesof126patients Pospelov LE. Tuberculosis in patients with various inflammatory and infectious diseases. with TB, 15 (12%) carried the HLA- HLA phenotypes. Tubercle. 1990;71:187-192. 14. Newport MJ, Huxley CM, Huston S, et al. A mu- Previous studies using serologic test- DQB1*0503 allele. Analysis of the protein tation in the interferon-␥-receptor gene and suscep- ing methods reported an association be- sequencesofallDQmoleculesrevealsthat tibility to mycobacterial infection. N Engl J Med. tween progressive TB and the HLA-DR2 only 2 other DQB1 alleles (DQB1*0603 1996;335:1941-1949. 15. Jouanguy E, Altare F, Lamhamedi S, et al. Inter- serotype in populations from India, Indo- and 0607), both also part of the DQ1 sero- feron-␥-receptor deficiency in an infant with fatal nesia, and Russia.11-13 However, serologic logic specificity, encode an identical P9 bacille Calmette-Gue´rin infection. N Engl J Med. methods can result in false assignment of pocket. In the first stage of our study, we 1996;335:1956-1961. 16. Flynn JL, Goldstein MM, Chan J, et al. Tumor the HLA class II type in up to 25% of found 2 additional patients with TB who necrosisfactor-␣isrequiredintheprotectiveimmune samples when compared with more sen- carried the HLA-DQB1*0603 allele, but response against Mycobacterium tuberculosis in sitive molecular DNA–based methods.33 we did not find either the DQB1*0603 or mice. Immunity. 1995;2:561-572. the 0607 allele among any of the controls. 17. Rook GAW. Mycobacteria, cytokines and antibi- Another study using molecular typing otics. Pathol Biol. 1990;38:276-280. failed to identify a specific allele that was Thus, 13.5% of the patients with TB car- 18. Miller SA, Dykes DD, Polesky HF. A simple salt- associated with disease progression al- riedtheHLA-DQB1*0503orDQB1*0603 ing out procedure for extracting DNA from human though it supported the general associa- alleles (1 patient was homozygous for nucleated cells. Nucleic Acid Res. 1988;16:1215. 19. CerebN,MayeP,LeeS,KongY,YangSY.Locus tionbetweentheHLA-DR2serotypeand HLA-DQB1*0503). specific amplification of HLA class I genes from ge- TB progression in an Indian population.34 Our findings identify the HLA- nomic DNA. Tissue Antigens. 1995;45:1-11. We found that when HLA-DR2 alleles DQB1*0503 allele as, to our knowledge, 20. Bozon MV, Delgado JC, Turbay D, et al. Com- parison of HLA-A antigen typing by serology with were combined (ie, the HLA-DR15 and the first gene associated with TB pro- two polymerase chain reaction based DNA typing HLA-DR16alleles),theywereslightlyin- gression. These results provide a clue to methods. Tissue Antigens. 1996;47:512-518. creased in patients with TB vs in controls. the complex process of mycobacterial 21. Yunis JJ, Delgado MB, Lee-Lewandroski E, Yu- antigen presentation and containment nis EJ, Bing DH. Rapid identification of HLA- However, no specific HLA-DR2 alleles DRw53-positive samples by a generic DRB-PCR am- wereincreasedsignificantlyinthepatient by the host immune system and support plification without further analysis. Tissue Antigens. group even before correction for multiple the hypothesis that variability in the hu- 1992;40:41-44. comparisons (Table). We found an asso- man major histocompatibility complex 22. Kimura A, Sasazuki T. Eleventh International Histocompatibilty Workshop reference protocol for ciation between clinical TB and the HLA- confers relative susceptibility or resis- the HLA DNA-typing technique. In: Tsuji K, Aizawa DQB1*0503 allele, and established the tance to infectious disease. M, Sasazuki T, eds. HLA 1991: Proceedings of the significance of this association by its con- Eleventh International Histocompatibility Work- This work was supported by grants from the Na- shop and Conference. New York, NY: Oxford Uni- firmation in a second study sample. tional Institutes of Health (MH 40279, HL29583) versity Press Inc; 1992:397-419. Based on the crystal structure of the and from the Milton Fund of Harvard University. 23. Salazar M, Yunis JJ, Delgado MB, Bing D, Yunis class II molecules,35 it has been proposed We are grateful to Sharon Alosco and Marcela EJ. HLA-DQB1 allele typing by a new PCR-RFLP Salazar of the Northeastern Region American Red method. Tissue Antigens. 1992;40:116-123. that peptides bound by the HLA mol- Cross for assistance in HLA class II typing. We thank 24. YunisJJ,SalazarM,DelgadoMB,AlperCA,Bing ecules form hydrogen bonds with amino Barbara Barrett, MA, for assistance with the perfor- DH, Yunis EJ. HLA-DQA1, DQB1 and DPB alleles acid residues conserved in most class II mance of serologic tests and Abbott Laboratories for on HLA-DQ2- and DQ9-carrying extended haplo- alleles. The side chains of the residues of donating the ELISA kits. We thank Patricia Pe- types. Tissue Antigens. 1993;41:37-41. savento, PhD, Ellis McKenzie, William Marshall, MD, 25. Bodmer JG, Marsh SGE, Albert ED, et al. No- antigenic peptides are accommodated in and Nadeem Mirza, MD, for their help in preparing menclatures for factors of the HLA system, 1995. Tis- smaller cavities, called pockets, in the blood samples and Joel Charny for help in transport- sue Antigens. 1995:46:1-18. binding site of the HLA molecules.36 ing the samples. We thank Brian Heidel and Steven 26. Wilson AG, Vries N, Pociot F, di Giovine FS, van Miles, MD, for their support and encouragement. der Putte LBA, Duff GW. An allelic polymorphism These pockets appear to determine the within the human tumor necrosis factor ␣ promoter peptide-binding specificity of the differ- References region is strongly associated with HLA A1, B8, and ent class II molecules.36 Thus, differentia- DR3 alleles. J Exp Med. 1993;177:557-560. 1. World Health Organization (WHO) Report on the 27. Congressional Hunger Caucus Round Table tion of HLA alleles is crucial for the inter- TB Epidemic. Geneva, Switzerland: WHO; 1994. Hearing on Tuberculosis Emergency (statement of pretationofHLAanddiseaseassociations 2. Porter JDH, McAdam KPWJ. The re-emergence Anne E. Goldfeld, MD, February 10, 1994). because point mutations in the class II of tuberculosis. Annu Rev Public Health. 1994;15: 28. Rith DN. Tuberculosis in Cambodia. 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