MATERIALS AND METHODS

Subjects

The 155 patients (118 female and 37 male) with RA studied were all inhabitants of central Japan. The diagnosis of RA was based on the criteria of the American college of Rheumatology. They had a mean (SD) age of 59.6 (12.4) years (table 1). To evaluate the correlation between clinical characteristics and polymorphism, age at onset and presence or absence of pulmonary fibrosis, osteoarthritis, and joint replacement were also investigated. The patients had a mean (SD) age at onset of 49.8 (12.5) years. Of the 155 patients, 17 (11%) had pulmonary fibrosis as detected by chest radiography or computed tomography findings.

One hundred and ten unrelated healthy subjects (68 female and 42 male) living in the same area of Japan were selected as controls. They were all volunteers and joined sequentially. Their mean (SD) age was 44.5 (18.9) years (table 1). They had no history of articular disease or any abnormalities on physical examination, chest radiography, electrocardiography, urinary analysis, or routine laboratory blood testing. They were not receiving medication at the time of the evaluation. Informed consent was obtained from all the patients and healthy controls.

Determination of TGFβ1 T869C genotypes

DNA was extracted from peripheral leucocytes using standard techniques. Genotypes were determined by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) based on the method previously described by Wood et al.⁵ Specific oligonucleotide primers 5'-TTCCCTCGAGGC CCTCCTA-3' and 5'-GCCGCAGCTTGGACAGGATC-3' were used in the PCR to amplify a fragment of the TGFβ gene, with denaturation at 96°C for 10 minutes, followed by 35 cycles at 96°C for 75 seconds, 62°C for 75 seconds, 73°C for 75 seconds, and a final extension at 73°C for five minutes (DNA Thermal Cycler 2400, Perkin Elmer-Cetus, Norwalk, CT, USA). Aliquots of the PCR products were analysed on 2% agarose gels stained with ethidium bromide before digestion to control for correct amplification of the 294 bp fragments. MspA1I (New England Biolabs, Hitchin, UK) digestion of the 294 bp fragments at 37°C for 180 minutes resulted in fragments of the T allele of 161, 67, 40, and 26 bp, and the C allele of 149, 67, 40, 26, and 12 bp. The samples were then analysed by electrophoresis on a 4% agarose gel stained with ethidium bromide and the genotypes were determined. There were three genotypes, homozygous for the alleles TT, CC, and heterozygous CT.

Statistical analysis

Genotypic distributions in the patients and healthy control subjects were analysed with Fisher's exact test. Bonferroni's test was used for evaluation of the age at onset. A p value<0.05 was considered significant.

RESULTS

Genotypic distribution of TGFβ1 T869C

Of the 110 healthy subjects, 33 (30%) were CC type, 53 (48%) were CT type, and 24 (22%) were TT type. Of the 155 patients with RA, 29 (19%) were CC type, 92 (59%) were CT, and 34 (22%) were TT type. A significantly higher proportion of patients with RA carrying the T allele (grouped CT heterozygous or TT homozygous) was found compared with those carrying the CC homozygous allele (p=0.039) (table 1), although this difference in genotypic distribution between the controls and patients was not significant when we compared all three genotypes.

Genotypes and clinical characteristics of patients with RA

For CC, CT, and TT genotypes, the mean (SD) ages at onset of patients with RA were 48.2 (14.3), 50.3 (12.6), and 49.9 (11.1), respectively. No significant differences in age of onset between genotypes was found. Of the 155 patients, 17 (11%) had pulmonary fibrosis. The genotypic distribution of these 17 patients was four CC type, seven CT type, and six TT type. No difference in genotypic distribution was found between the patients with or without pulmonary fibrosis. We also investigated patients with osteoarthritis and genotype and those with joint replacement and genotype. No associations were found (data not shown).

DISCUSSION

In this study, significantly more patients carried the T allele (CT or TT type) than the CC type. The genotype distribution found for the healthy subjects in our study was similar to that reported earlier in a larger population of Japanese.⁹ From these results, we speculate that this polymorphism is related to the risk of developing RA.

Previous studies have shown that TGFβ1 is produced in large amounts in synovial fluid and is strongly expressed in the joints of patients with RA. Synovial cells have also been reported to be able to produce TGFβ1 in vitro and in vivo.^6,7 TGFβ1 is related to immunosuppression and anti-inflammatory reactions, and is considered to be associated with remission of arthritis. Furthermore, recent investigations have reported that TGFβ1 gene therapy in an animal model showed improvement of arthritis of the junction. Thus, TGFβ1 is considered to be a modulator of reduced inflammation in RA.^7,8

In several previous studies the T allele of T869C polymorphism has been reported to be associated with reduced production of TGFβ1 proteins.^9,10 These findings were supported by a study of another polymorphism, C-509T, which is linked to T869C polymorphism.¹¹ Therefore, we speculate that the T alleles of T869C polymorphism might be associated with relatively low production of TGFβ1, and may correlate with the rate of progression of the disease, as reduced TGFβ1 may result in increased inflammation in RA. On the other hand, Awad et al reported that the T allele of T869C was linked to higher production of TGFβ1.¹² The association between TGFβ1 T869C polymorphisms and their protein concentrations remains unclear and we have no data of our own to show TGFβ1 concentrations by genotype. Thus, care must be taken in interpretation of our results. However, we speculate that our data and those of several other investigations are in agreement in indicating lower production of TGFβ1 in patients with the T allele of T869C.

TGFβ1 is also considered to be a regulator of fibrosing processes, and TGFβ1 polymorphism was associated with the risk of lung fibrosis in pulmonary transplant recipients, or deteriorated lung function in cystic fibrosis.^12,13 In our patient group there were no significant correlations between TGFβ1 genotypes and the presence or absence of pulmonary fibrosis. However, as there were only 17 patients with RA and pulmonary fibrosis, we speculate that the small population size may have an effect on comparisons. Further study is necessary to evaluate the association between pulmonary fibrosis in RA and TGFβ1 polymorphisms.

Although RA is an inflammatory disease of unknown cause, several investigators have suggested that there may be several genetic risk factors that affect the susceptibility for this disease.¹⁴ The details of the mechanisms remain unclear, but our results suggest that in TGFβ1 gene polymorphism the T allele might be one of the genetic risk factors for RA. Limitations of this study were its small size, and the fact that it only considered Japanese patients, as racial differences may be important. Furthermore, recent investigations showed that there are other polymorphic sites of the TGFβ1 gene and TGFβ type III receptor gene.^5,15 Further investigation of the association between the TGFβ related genetic variation and RA seems warranted.