ABSTRACT.

p: probability uncorrected (significant when p<0.05)

INTRODUCTION.

MATERIALS AND METHODS

REFERENCES

CONCLUSIONS

Kettaneh A, et al. Int J Tuberc Lung Dis; 10: 717-25. 2006.

Goldfield A. Delgado J; Thim S et al. JAMA. 21, 279 (03). 226-28, 1998.

Delgado J, Baena A, Thim S, Goldfeld A. J Immunol; 176: 1090-7. 2006.

Takiff H. In: Tuberculosis 2007. Chapter 6, Palomino, Leão, Ritacco (Editors). 207-262. 2007.

Lombard Z, Dalton DL, Venter PA, Williams RC, Bornman L. Hum Immunol; 67: 643-54. 2006.

ROLE OF HLA VARIABILITY IN TUBERCULOSIS.

Ángel R. Villasmil C, Mercedes Fernández-Mestre, Violeta Ogando and Zulay Layrisse.

Laboratorio de Fisiopatología, Instituto Venezolano de Investigaciones Científicas.

Caracas, Venezuela.

Tuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis

continues to be a problem of public health in developing countries, including

Venezuela. By still unknown reasons, only 30% of the individuals exposed are

infected, only 5% of them develop the illness within the first year of the

infection and 95% remain in phase of latency of which 5-10% develops the

illness in the course of their lives. The susceptibility to TB is multifactorial

and genetic factors of the host play an important role. The aim of this study

was to investigate the variability of HLA-DRB1 and HLA-DQB1 genes in

patients with clinical diagnosis of pulmonary tuberculosis and healthy

individuals exposed to the Koch bacillus. We have examined these

polymorphisms in a case-control study of 194 Venezuelan ethnically-mixed

adults, including 93 patients with smear positive pulmonary TB (mean of age

37,9 years) and 101 unrelated healthy controls (mean age 41,4 years), selected

from the non-medical staff of the José Ignacio Baldó Hospital in Caracas.

Definition of HLA variants was done using Dynal RELI SSO HLA Typing

Kits. Allelic and genotypic frequencies were determined by direct count and

the association was established as odd ratios (OR). Several statistically

significant frequency differences were observed. Increased frequencies of

DRB1*12 and DQB1*0602 were observed in patients. In contrast, increased

frequencies of HLA- DRB1*03 and HLA-DQB1*0306 were found in controls.

The analysis of HLA-DQB1 alleles that encode or not aspartate at position 57

at the β chain (ASP-β57) showed the presence of the phenotypes:

DQB1*0301-0302 (ASP-β57/No ASP-β57), DQB1*0301-0307 (ASP-β57/No

ASP-β57) and DQB1*0202-0501(No ASP-β57/ No ASP-β57) only in patients.

The DRB1*13-DQB1*06 haplotype was observed also increased in the

controls vs. patients (OR: 0, 26; p: 0.031). The results suggest an influence of

HLA polymorphism in the occurrence of TB in our patients.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the most

common mycobacterial disease in the world and remains a leading public

health problem. 90% of people infected with Mtb have latent infection with no

symptoms and an immune response that contains the bacilli. In 10% of

infected individuals, symptoms develop and most commonly manifest as

pulmonary disease, which accounts for 80% of all forms of TB disease.

Multiple genetic factors explain at least why some people resist infection more

successfully than others, such as the genes of the Leucocitary Human Antigen

(HLA) have been previously associated with the susceptibility to the illness,

and different studies have revealed an association between HLA-DRB1 and

HLA-DQB1 with susceptibility to TB pulmonary, affecting the presentation of

processed peptides of phagocytosed pathogens to CD4+ T cells.

The aim of this study was to investigate the variability of HLA-DRB1 and

HLA-DQB1 genes in patients with clinical diagnosis of pulmonary

tuberculosis and healthy individuals exposed to the Koch bacillus.

Patient Population

Whole blood was collected from 194 ethnically mixed Venezuelan

individuals: 93patients with confirmed pulmonary TB who were seen at the

Hospital José Ignacio Baldó, Caracas (mean of age 37,9 years; 60% female

and 40% male) and 101 unrelated healthy controls (mean age 41,4 years;

77,22% female and 22,77% male), were randomly selected from the non-

medical staff of the Hospital.

A local ethics committee approved this study.

HLA Typing

Genomic DNA was extracted from blood samples using a modified salting-

out procedure. HLA typing was carried out by polymerase chain reaction-

sequence-specific oligonucleotide reverse dot blot using the Dynal RELI

SSO HLA- DRB and HLA- DQB test kits.

Statistical Analysis

Frequencies were determined by direct counting. The statistical

significance of allele frequency was estimated by Fisher's exact test and p

values were corrected according to Bonferroni. Odds ratio (OR) with

corresponding 95% confidence intervals (95% CI) were calculated to

measure the strength of associations.

Finally, the DRB1*13-DQB1*06 haplotype was observed also increased in the

controls vs. patients (OR: 0, 26; p: 0.031).

Several statistically significant frequency differences were observed. Increased

frequencies of DRB1*12 and DQB1*0602 were observed in patients. In

contrast, increased frequencies of HLA- DRB1*03 and HLA-DQB1*0306 were

found in controls (Table 1).

The analysis of HLA-DQB1 alleles that encode or not aspartate at position 57 at

the β chain (ASP-β57) showed the presence of the phenotypes: DQB1*0301-0302

(ASP-β57/No ASP-β57), DQB1*0301-0307 (ASP-β57/No ASP-β57) and

DQB1*0202-0501(No ASP-β57/ No ASP-β57) only in patients. Our results did

not confirm the data previously reported by Delgado J (2006) in Camboya

population. (Figure1, Table 2).

RESULTS

Table 1

HLA allelic group showing frequency differences in cases (TB) and controls

Figure 1

The figure illustrates that HLA-DQB alleles encoding an aspartic acid at position

57 (including HLA-DQB1*0503, shown in red) and the HLA-DQB alleles

encoding HLA-DQB 57-non-Asp (Delgado et al. 2006)

The results suggest an influence of HLA polymorphism in the occurrence of TB in

our patients.

To confirm the obtained results would be necessary to increase the size of the

sample and to carry out studies in families.

HLA-DRB1 TB

n=66

Controls

n=78

OR

(IC95%)

p

*03 0,03 0,096 0,27 0,017

*12 0,053 0,006 9,14 0,018

HLA-DQB1 TB

n=81

Controls

n=88

OR

(IC95%)

p

*0306 0,012 0,079 0,14 0,004

*0602 0,043 0,005 8,09 0,02

Table 2 Distribution of HLA-Asp-57 and HLA-no 57 frequencies in cases (TB)

and controls

p: probability uncorrected (significant when p<0.05)

Genotypic

Combinations

DQB1-DQB1

Phenotype

TB

n=66

Controls

n=77

OR (IC95%)

p

*0202-*0501 No Aspβ57- No

Aspβ57

6,1 % (4) 0 11,2 0,04*

*0301-*0302 Aspβ57- No

Aspβ57

7,6% (5) 0 13,8 0,02*

*0301-*0307 Aspβ57-No

Aspβ57

6,1 % (4) 0 11,2 0,04*