To the Editor:
Pulmonary fibrosis is a well recognized extraarticular manifestation of rheumatoid arthritis (RA)1. Several studies have shown an association between the HLA-DRB1 gene and extraarticular manifestations of RA2,3. We analyzed the effect of the HLA-DRB1 gene and cytokine gene polymorphisms on the association of pulmonary fibrosis in Japanese patients with RA.
We examined 131 adult patients with RA, who fulfilled the American Rheumatism Association 1987 revised criteria for RA4. Clinical characteristics were (mean ± SD) age at study 62.4 ± 13.0 years and disease duration 11.2 ± 4.7 years. Pulmonary fibrosis was diagnosed by chest radiograph and chest high-resolution computed tomography (HRCT). Patients with dry cough or exertional dyspnea or suspicious findings for interstitial changes in chest radiography were further investigated with HRCT at suspended end-inspiratory effort without intravenous contrast material. The abnormalities used to screen for pulmonary fibrosis were ground-glass attenuation and honeycombing defined as areas of cystic spaces with thickened walls. Patients with RA with other autoimmune diseases or those with other pulmonary complications, such as drug (methotrexate)-induced interstitial pneumonia diagnosed according to the criteria of Kremer, et al5 or airway diseases, were excluded. Table 1 presents the characteristics of the patients with RA. Pulmonary fibrosis was diagnosed in 44 patients among these 131 patients with RA. The remaining 87 patients showed no radiographic evidence of pulmonary fibrosis.
Biallelic polymorphism within the transforming growth factor-ß (TGF-ß) gene in codon 10 was determined by a polymerase chain reaction-scanning spectral polarimeter (PCR-SSP) technique employing commercial primers (One Lambda Inc., Canoga Park, CA, USA). Interleukin 4 (IL-4) promoter (−590) and intron 3 polymorphisms were studied by PCR-restriction fragment-length polymorphism (PCR-RFLP) or PCR methods as described6. Typing for HLA-DRB1 was performed by PCR and sequence-specific oligonucleotide probe hybridization method using the Genoscience HLA-DRB1 kit (G&G Science, Fukushima, Japan). Genotype frequencies were compared in cases and controls by 2 × 2 contingency tables, and 2-tailed probabilities were calculated using the Fisher exact test. Adjustment for multiple comparisons was made after second-stage typing using the Bonferroni method. Corrected p value was calculated by multiplying the p value by the number of alleles found in Japanese patients (n = 26).
The genotypes at TGF-ß codon 10, IL-4 promoter (−590), and intron 3 polymorphisms were in Hardy-Weinberg equilibrium in both healthy subjects and patients with RA (data not shown). The genotype distributions of TGF-ß polymorphisms at codon 10, IL-4 promoter (−590), and intron 3 polymorphisms were not significantly different among patients with RA with pulmonary fibrosis and those without pulmonary fibrosis (Table 2). As shown in Table 3A, the allele frequencies of HLA-DRB1 shared epitope (SE) were significantly increased in patients with RA compared to healthy controls (48.1% vs 26.2%, respectively; p < 0.0001). We also compared the HLA-DRB1 genotypes between patients with RA with and without pulmonary fibrosis (Table 3B). There were no significant differences in the frequencies of SE between RA patients with and without pulmonary fibrosis (45.5% vs 49.4%). Further, the HLA-DR4 (*04) status had no significant effect on the association of pulmonary fibrosis in patients with RA (patients with pulmonary fibrosis 42.0% vs patients without pulmonary fibrosis 47.1%). On the other hand, the allele frequencies of HLA-DRB1*1502 were significantly associated with pulmonary fibrosis in patients with RA.
Despite advances in molecular genetic techniques, there have been few genetic studies of RA-related pulmonary fibrosis to date. Several studies have demonstrated an association between HLA-DRB1 SE and extraarticular manifestations or disease severity of RA2,7,8. We analyzed the genetic background of RA patients with pulmonary fibrosis compared to those without pulmonary fibrosis and healthy controls. However, the frequencies of the HLA-DR1 SE in patients with RA with pulmonary fibrosis were not significantly different compared to those without pulmonary fibrosis. Thus, the SE was not implicated in the association with pulmonary fibrosis in Japanese patients with RA. These findings suggest that the importance of the SE may be variable for different manifestations and other genetic factors, or environmental factors may have more influence on RA-related pulmonary fibrosis. By contrast, significantly increased frequencies of DRB1*1502 were observed in patients with RA with pulmonary fibrosis compared with those without pulmonary fibrosis, and healthy subjects. Genetic associations between pulmonary fibrosis and the genes encoding TGF-ß and IL-4 have been reported9,10. However, we did not find any significant association between these genetic polymorphisms and RA-related pulmonary fibrosis.
Our data indicate that HLA-DRB1 SE is not associated with an increased risk for RA-related pulmonary fibrosis. However, we found that the presence of HLA-DRB1*1502 is associated with pulmonary fibrosis in Japanese patients with RA. To firmly establish the relationship between the HLA-DRB1 genotype and pulmonary fibrosis in RA patients, further large-scale studies are required including individuals of other ethnicities.
REFERENCES
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- 4.
- 5.
- 6.
- 7.
- 8.
- 9.
- 10.