Association of an HLA-DQ Allele With Clinical Tuberculosis FREE

ABOUT A THIRD of the earth's population is infected with Mycobacterium tuberculosis,^1- 3 the bacteria that causes tuberculosis (TB). Infection with M tuberculosis results in a variety of conditions ranging from asymptomatic infection to progressive pulmonary or extrapulmonary TB and death.⁴ Approximately 1 in 10 of those infected will progress to active disease during their lifetime.^1- 3 Tuberculosis is the leading cause of death due to an infectious disease worldwide, accounting for approximately 3 million deaths annually.^1- 3

Individuals who have impaired cell-mediated immunity due to chemotherapy, steroid use, neoplasia, or the acquired immunodeficiency syndrome (AIDS) have a greatly increased risk of activation of quiescent infection with M tuberculosis .^4- 6 Poverty also has been implicated as a cofactor in disease progression.^7- 8 Certain populations are at risk for increased susceptibility to infection and progressive disease due to M tuberculosis,^9- 13 and in several populations the HLA class II DR2 serotype is associated with clinical TB.^11- 13 Mutations in the interferon-γ receptor gene have been associated with progressive atypical mycobacterial infection¹⁴ and with Calmette-Guérin bacillus (Mycobacterium bovis) infection.¹⁵ Tumor necrosis factor α (TNF-α) appears to play an important role in TB pathogenesis, including granuloma formation and containment of TB infection^16- 17 and impaired TNF-α secretion due to defective signaling through the interferon-γ receptor gene may be involved in disease progression.^14- 15

To determine whether specific HLA class I or class II alleles are associated with clinical TB, we performed a 2-stage study of molecular typing of HLA class I and class II alleles and also tested for the presence of 2 TNF-α alleles in Cambodian patients with clinical TB and in control individuals who did not have a history of TB.

The study subjects were unrelated Cambodian patients recruited from a TB treatment program in eastern rural Cambodia. Two different groups of patients and controls were recruited for the 2 stages of the study, the first group in 1995 and the second group in 1996. The patients were randomly selected from all inpatients or outpatients picking up their monthly supply of TB medicines at Chantrea, Rumduol, or Kompong Rho District Hospitals in Svay Rieng Province.

The diagnosis of clinical TB was made on site on the basis of light microscopy demonstrating the presence of acid-fast bacilli in sputum, pleural fluid, or lymph node drainage. One of us (S.T.) conducted family interviews of the household members of the patients and verified that no patients in the study were related. Control individuals were recruited from patients visiting the same hospitals for minor complaints. Based on detailed clinical history, controls did not have a history of TB or current symptoms consistent with TB. All patients and controls were followed up for 6 months to confirm their diagnosis or control status.

After consent was obtained, blood was drawn from each subject and stored at 4°C for 4 to 10 days. Plasma and peripheral blood mononuclear cells (PBMCs) were prepared according to standard techniques and stored at −70°C. Plasma samples were screened for evidence of antibodies to human immunodeficiency virus (HIV) types 1 and 2 and human T-lymphotropic virus (HTLV) types 1 and 2 by enzyme-linked immunosorbent assay.

The DNA was prepared from PBMCs by a quick isolation method,¹⁸ and allele-specific polymerase chain reaction (PCR) was performed. For HLA class I typing, PCR amplification of exons 2 and 3 of the HLA-A and HLA-B loci was performed. For class II typing, PCR amplification of exon 2 of the HLA-DRB, DQB1, and DQA1 loci was performed.^19- 21 The PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide, and photographed. Dot-blot, prehybridization, and hybridization procedures were carried out according to the manufacturer's instructions (Lifecodes Corp, Stamford, Conn). Class I and class II alleles were identified in the PCR-amplified products by sequence-specific oligonucleotide probe hybridization.^20,22- 25

The TNF-α alleles were determined by restriction fragment length polymorphism (RFLP) analysis using primers designed to incorporate a polymorphic site (the nucleotide A vs G) at position −308 nucleotides (nt) relative to the TNF-α transcription start site. The TNF2 (A at −308 nt) polymorphism creates an Nco I restriction site and can be differentiated by size (107 nt for the TNF1 and 87 nt for the TNF2 allele) from the TNF1 allele by agarose gel electrophoresis.²⁶

The frequencies of independent HLA alleles and TNF-α alleles in patients and controls were determined by direct counting. The statistical significance of the difference in frequency of individual HLA and TNF-α alleles between the 2 groups was calculated by the Fisher exact test with the aid of INSTAT software (GraphPad, San Diego, Calif), and levels of significance were reported as P values along with 95% confidence intervals according to the program used. In the first stage of the study, since each subject was tested for several HLA alleles and 2 TNF-α alleles, and the same data were used to compare the frequency of all detected alleles, significant associations may have arisen by chance due to multiple comparisons. In the second stage, we tested for the presence of a single allele identified in the first stage, and thus, correction of the data for multiple comparisons was not necessary.

The first stage of our study included 78 patients with TB (mean age, 47 years; range, 11-77 years; 24% male; 76 patients with pulmonary TB, 1 with pleural TB, and 1 with scrofula) and 49 control individuals (mean age, 40 years; range, 15-68 years; 53% male). Of these 127 individuals, no patient with TB had antibodies to HIV-1 or HIV-2, and 1 patient with TB was positive for antibodies to HTLV-1.

Among the 156 alleles derived from 78 patients with TB, there were 7 DQB1*0503 alleles, whereas this allele was not found among the 98 alleles from the 49 controls (P=.04) (Table 1). When HLA-DR15 and HLA-DR16 alleles were combined, there was a slight increase in HLA-DR2 alleles (52 of 156) among patients with TB compared with 32 of 98 alleles in controls (Table 1). HLA-B38 was present in 16 of 138 alleles among patients with TB compared with 2 of 96 alleles in controls (P=.005) (data not shown).

An RFLP analysis scoring for the 2 TNF-α promoter variants revealed 11 TNF2 alleles of 156 alleles derived from patients with TB and 8 TNF2 alleles of 96 alleles derived from controls with no difference in TNF2 variant expression in either group. Seventeen of the 19 TNF2 alleles were found in HLA-DR3–positive subjects (data not shown). Thus, as in other populations,²⁶ the TNF2 allele was in linkage disequilibrium with HLA-DR3.

Based on the first stage of our analysis, we chose to further investigate the association of the single class II allele, DQB1*0503, and TB. This second stage included 48 patients with pulmonary TB (mean age, 46 years; range, 25-76 years; 40% male) and 39 controls (mean age, 40 years; range, 19-67 years; 30% male). One control subject tested positive for antibodies to HIV-1 and was excluded from the HLA analysis. Among the 96 alleles derived from 48 patients with TB, there were 9 DQB1*0503 alleles. No DQB1*0503 alleles were detected among the 76 alleles derived from 38 controls (P=.005).

In Cambodia, which has a population of approximately 10 million, it is estimated that up to 40000 new cases of TB and 13000 deaths due to TB occur every year.^27- 28 Tuberculosis is the major cause of morbidity and mortality in Cambodian men aged 18 to 40 years.^27- 29 Although there are no reliable data, it is assumed that the majority of Cambodians harbor M tuberculosis and thus are chronically infected with TB,³⁰ but that only a subset of these individuals progress to active pulmonary or extrapulmonary TB.⁴ In the Cambodian population we studied, poverty (ie, an annual family income of about $180 per year) and poor nutritional status, known cofactors of TB progression, are pervasive and were equivalent between the patient and control groups. In an ongoing pilot study in Svay Rieng, of 450 people randomly screened, 75% were purified protein derivative positive with a skin reaction of greater than 5 mm of induration (S.T. and A.E.G., unpublished data). Although HIV-1 infection and AIDS are rapidly increasing in Cambodia,³¹ we ruled out HIV-1 as contributing to TB progression in this study group.

We did not detect a difference in the presence of the TNF2 allele in patients with TB vs controls consistent with the lack of detectable effect of this polymorphism on TNF-α gene expression.³² Rather, the TNF2 allele appears to serve as a marker in the HLA region for genetic associations with susceptibility to certain inflammatory and infectious diseases.

Previous studies using serologic testing methods reported an association between progressive TB and the HLA-DR2 serotype in populations from India, Indonesia, and Russia.^11- 13 However, serologic methods can result in false assignment of the HLA class II type in up to 25% of samples when compared with more sensitive molecular DNA–based methods.³³ Another study using molecular typing failed to identify a specific allele that was associated with disease progression although it supported the general association between the HLA-DR2 serotype and TB progression in an Indian population.³⁴ We found that when HLA-DR2 alleles were combined (ie, the HLA-DR15 and HLA-DR16 alleles), they were slightly increased in patients with TB vs in controls. However, no specific HLA-DR2 alleles were increased significantly in the patient group even before correction for multiple comparisons (Table 1). We found an association between clinical TB and the HLA-DQB1*0503 allele, and established the significance of this association by its confirmation in a second study sample.

Based on the crystal structure of the class II molecules,³⁵ it has been proposed that peptides bound by the HLA molecules form hydrogen bonds with amino acid residues conserved in most class II alleles. The side chains of the residues of antigenic peptides are accommodated in smaller cavities, called pockets, in the binding site of the HLA molecules.³⁶ These pockets appear to determine the peptide-binding specificity of the different class II molecules.³⁶ Thus, differentiation of HLA alleles is crucial for the interpretation of HLA and disease associations because point mutations in the class II genes are critical for peptide binding and presentation and commonly occur in or near the peptide-binding pockets.³⁵

Our evidence for an association between a specific allele, HLA-DQB1*0503, and progressive clinical TB is particularly intriguing because the DQB1*0503 allele encodes for a change at amino acid position 57 of the β chain (β57), which influences the charge in the putative peptide binding pocket, P9, of the DQ molecule.³⁶ The DQB1*0503 allele, which is part of the DQ1 serologic specificity, encodes the negatively charged aspartic acid at β57 in place of the more common, uncharged, and hydrophobic amino acid valine. The MHC-restricted presentation of peptides by TB-infected macrophages may be affected in patients who express this particular P9 pocket. The negatively charged P9-binding pocket may bind TB antigens less effectively or elicit a diminished immunogenic response.

In our combined samples of 126 patients with TB, 15 (12%) carried the HLA-DQB1*0503 allele. Analysis of the protein sequences of all DQ molecules reveals that only 2 other DQB1 alleles (DQB1*0603 and 0607), both also part of the DQ1 serologic specificity, encode an identical P9 pocket. In the first stage of our study, we found 2 additional patients with TB who carried the HLA-DQB1*0603 allele, but we did not find either the DQB1*0603 or the 0607 allele among any of the controls. Thus, 13.5% of the patients with TB carried the HLA-DQB1*0503 or DQB1*0603 alleles (1 patient was homozygous for HLA-DQB1*0503).

Our findings identify the HLA-DQB1*0503 allele as, to our knowledge, the first gene associated with TB progression. These results provide a clue to the complex process of mycobacterial antigen presentation and containment by the host immune system and support the hypothesis that variability in the human major histocompatibility complex confers relative susceptibility or resistance to infectious disease.