INTRODUCTION

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Sarcoidosis is a systematic granulomatous disorder of unknown etiology. The active form of vitamin D, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] is produced at sites of granulomatous reactions in sarcoidosis (1). This secosteroid hormone has multiple immunomodulatory effects (1), and is also reported to be a promoter of multinucleated giant cell formation (3, 7, 8). 1,25(OH)₂D₃ is considered to be associated with granuloma formation in sarcoidosis (3, 8).

The hormone binds to nuclear vitamin D receptors. Polymorphism of the vitamin D receptor (VDR) gene recognized by the Bsm 1 restriction enzyme has recently been shown to be related to bone mineral density. The polymorphism exists on twelfth chromosome intron 8 and can be detected with endonuclease Bsm 1. There are three genotypes, homozygous for the digestive allele "bb," homozygous of undigestive allele "BB," and heterozygous "Bb" (9). Recent studies revealed that this polymorphism and closely linked variations may have significance for hyperparathyroidism and risk of prostatic carcinoma (11). Our hypothesis was that it might also affect sarcoidosis, and therefore we investigated polymorphism of the VDR gene recognized by Bsm 1 in a series of patients and control subjects.

	METHODS

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Study Population

The 101 patients with sarcoidosis studied (82 females, 19 males) were all inhabitants of central Japan. Sarcoidosis was diagnosed on the basis of the clinical picture and the presence of epithelioid cell granulomas in biopsy specimens from lung, skin, or lymph nodes. They had a mean age of 56.1 ± 15.4 yr (mean ± SD). Regarding the roentgenographic stage of patients at the first visit to our hospital, all but 12 had chest X-ray evidence of sarcoidosis, 73 with stage I, ten with stage II, and six with stage III disease.

As healthy controls, 105 unrelated healthy subjects living in the same area of Japan were selected. They were composed of 60 females and 45 males with a mean age of 45.4 ± 14.7 yr. They did not have any past history of pulmonary disease or any abnormalities on physical examination, chest radiography, electrocardiogram (ECG), urinalysis, and routine laboratory blood testing. None was receiving medication at the time of the evaluation. Informed consent was obtained from all the subjects.

Determination of the VDR Genotype

DNA was extracted from peripheral leukocytes with standard techniques. The polymerase chain reaction (PCR) and digestion of PCR products were performed as described by Morrison and coworkers (9). Specific oligonucleotide primers (5'-CAA CCA AGA CTA CAA GTA CCG CGT CAG TGA-3', and 5'-AAC CAG CGG GAA GAG GTC AAG GG-3') were utilized with PCR to amplify the fragment of the VDR gene including the Bsm 1 restriction site in intron 8. PCR was performed using denaturation at 94° C for 5 min, followed by 35 cycles at 94° C for 45 s, 60° C for 1 min, and 72° C for 1.5 min and a final extension at 72° C for 7 min (DNA Thermal Cycler 2400; Perkin Elmer-Cetus, Norwalk, CT). PCR products were digested with 1.5 U of Bsm 1 (New England Biolabs, Beverly, MA) at 65° C for 2 h, and run on a 2% ethidium bromide-agarose gel. Bsm 1 causes restriction cleavage of the VDR allele denoted b, whereby the patterns BB, Bb, or bb can be distinguished (Figure 1).

View larger version (115K): [in this window] [in a new window]   Figure 1.   Determination of vitamin D receptor genotypes. Panel shows part of representative 2% agarose gel stained with ethidium bromide and photographed under ultraviolet transillumination after PCR amplification and digestion by Bsm 1. The upper band is the B allele and the lower band is the b allele (arrows). The BB type is shown as a single upper band, the Bb type as a double band, and the bb type as a single lower band. The left lane contains markers.

Statistical Analysis

The allele ratios and genotype distributions in sarcoidosis patients and healthy control subjects, roentgenographic stages, organ involvement and disappearance of shadows on chest radiography among the three genotypes were analyzed with the chi-square test. The Fisher exact test was also applied for comparison of small populations with expected values less than 5. A p value < 0.05 was considered significant.

	RESULTS

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Of the 105 healthy control subjects, 1 had the BB genotype (1.0%), 21 the Bb type (20.0%), and 83 bb type (79.0%). The B allele/b allele (B/b) ratio was 0.110/0.890. Of the 101 sarcoidosis patients, 1 was type BB (1.0%), 38 were Bb (37.6%), and 62 were bb (61.4%). The B/b ratio was 0.198/0.802. A significant difference in the genotype distribution between healthy control subjects and sarcoidosis patients (p < 0.05), and a significant increase in the frequency of the B allele (p < 0.05) were observed (Table 1). We examined genotype distributions among the three roentgenographic stages of sarcoidosis patients, but found no significant correlation with the VDR genotype (Table 2). Next, we examined the relationship with organ involvement. Cases of eye, skin, heart, and involvement of three or more organs were examined, but the results indicated no specific association with the genotype (data not shown). We also examined the association of genotype distribution and disappearance of shadow on chest radiography within 3 yr, but no line was found (data not shown).

	DISCUSSION

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In this study, we found a significant difference in the genotype distribution between healthy control subjects and patients with sarcoidosis, the frequency of the B allele in sarcoidosis patients being elevated, although no correlation between genotype and clinical form of the disease was apparent. The genotype distribution established for the healthy subjects in our study was similar to that reported earlier for a larger population of Japanese (14). These results suggest the VDR gene polymorphism is related to risk of sarcoidosis.

1,25(OH)₂D₃ has a modulating effect on the human immune response regarding production of several cytokines and immunoglobulin. Its binding to the receptor inhibits antigen-induced lymphocyte proliferation and immunoglobulin production, and inhibits the production of several lymphokines, including interleukin-2, interferon gamma, and granulocyte- macrophage colony-stimulating factor (1). In addition, 1,25- (OH)₂D₃ acts as a promotor of monocyte/macrophage differentiation (3, 4, 6), and has biological effects on cells of this lineage including stimulation of the mitotic activity of blood monocytes and promotion of multinucleated giant cell formation (3, 7, 8). Recent studies showed that VDR gene polymorphism affects messenger RNA stability, and VDR messenger RNA expression is reduced in bb genotype in comparison with BB genotype individuals (9, 15). Therefore, we suggest that the granulomatous reaction might be suppressed in the bb type as compared with other genotypes because of a decreased potency of 1,25- (OH)₂D₃. In the present series of patients with sarcoidosis, significant increase of the frequency of B allele was observed. The mechanistic details remain unclear, but our results suggest that VDR gene polymorphism might play an important role and that the B allele might be a genetic risk factor for sarcoidosis. However, our sarcoidosis cases were predominantly stage I patients, possibly owing to racial variation as well as early detection on medical checkups in Japan. This limitation should be borne in mind in interpretation of the results. Firm conclusions can only be drawn for stage I patients.

Several studies of VDR gene polymorphism have revealed a higher frequency of the B allele in white individuals than Japanese or other Asian individuals (9, 14, 16, 17). Previous epidemiological studies have suggested a trend for sarcoidosis to be rare in Asians (18, 19). Though interpretation of epidemiological comparisons may be difficult, the findings may be consistent with our hypothesis. To our knowledge, this is the first investigation of VDR gene polymorphism in sarcoidosis. Further investigation of the general association of this source of variation with regard to sarcoidosis and other granulomatous diseases in white individuals and in other Asian groups appears warranted.