INTRODUCTION

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Clinicians have long noted the clinical and pathologic similarities between chronic beryllium disease (CBD) and sarcoidosis (1). Both have the histologic hallmark of noncaseating granulomas in affected tissues, both predominantly involve the lung but may involve multiple organs, and both can be progressive. The main difference is that the etiologic agent for CBD is known. CBD results from a hypersensitivity response to beryllium, whereas sarcoidosis is thought to arise from a hypersensitivity response to some unknown antigen.

Granuloma formation is thought to begin with antigen presenting cells (APCs) that process and present antigen to T cells. Interaction of APCs and T cells results in T-cell activation, secretion of cytokines, recruitment of inflammatory cells and fibroblasts, macrophage differentiation, and granuloma formation (2, 3). Evidence supporting T-cell recognition of beryllium via human leukocyte antigen (HLA) class II molecules comes from Saltini and colleagues, who observed that antibodies to major histocompatibility complex (MHC) class II molecules partly block the in vitro beryllium-induced proliferative response of CD4⁺ T lymphocytes (4). Further supporting the role of HLA class II molecules in CBD is the observation by Richeldi and colleagues (5) that 32 of 33 occupationally exposed workers with beryllium disease exhibited a glutamate in position 69 in the HLA-DPB1 gene. This substitution was present in only 30% of beryllium-exposed workers who did not have beryllium disease or sensitization. Saltini and associates found that five of every six CBD-affected workers from an Italian beryllium plant were Glu⁶⁹ positive (6), and Stubbs and coworkers reported that 86% of beryllium sensitized cases as opposed to only 48% of beryllium exposed, nondiseased control subjects, were Glu⁶⁹ positive (7). The functional significance of this substitution is unknown, but on the basis of crystallographic studies of HLA-DR, position 69 probably exists in a critical region for antigen binding to the HLA-DP molecule (8).

Ethnic distribution and familial clustering suggest an inherited susceptibility to sarcoidosis. Sarcoidosis affects African- Americans more commonly and severely than Caucasians (2, 9). Familial sarcoidosis has been reported in several studies, with up to 19% of patients reporting a positive family history of the disorder (12). Harrington and associates (14) described 91 families with sarcoidosis and noted that African- Americans had a higher prevalence of a family history of the disease than did Caucasians (19% versus 5.2%, respectively).

It is appealing to speculate that the region HLA-DPB1 plays a direct role in triggering T-cell recognition of beryllium in CBD as well as the unknown antigen in sarcoidosis. The present study investigated the role of the hypervariable region of the HLA-DPB1 gene in African-American sarcoidosis patients and controls.

	METHODS

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Study Population

The study was approved by the Henry Ford Health System Institutional Review Board. The study sample included 68 African-American sarcoidosis patients and 108 African-American controls. Patients were recruited during visits for their sarcoidosis at the Henry Ford Hospital pulmonary clinic. The diagnosis of sarcoidosis was confirmed by biopsy showing noncaseating granulomas with negative special stains and culture for acid-fast bacilli (AFB) and fungus. Seventy percent of the patients were female. Patients ranged in age between 26 and 73 yr, with an average age of 49 (± 11) [SD] yr. Patients had been diagnosed for an average of 13 (± 10) year at the time of study, and had been followed in the clinic for at least 1 yr. At the time of diagnosis, the average age of patients was 36 (± 9) yr old with a range of 19 and 59 years. A total of 108 healthy African-American controls were used as a comparison group. Controls were hospital employees who had no pulmonary disease at the time of participation and had no known relatives with sarcoidosis. The controls were 78% female and had an average age of 39 (± 10) yr old. All patients gave informed consent for enrollment in the study and for blood drawing.

A positive family history was defined as a report of one or more blood relatives with a history of sarcoidosis. Disease in relatives was verified by contacting the affected relative and asking whether he or she had had a biopsy confirming the disease or had a physician tell them that they had sarcoidosis.

Phenotyping

On the basis of medical chart review, patients were phenotyped according to the following scheme:

1. Mild. Incidental abnormal chest X-ray, normal organ function, never treated, remained asymptomatic after 2 yr of observation; or had a history of acute onset of symptoms with spontaneous remission within 2 yrs.
2. Moderate. Symptomatic disease with minimal organ-function abnormalities occurring with or without therapy.
3. Severe. Progressive disease with organ dysfunction and evidence of progressive scarring on chest X-ray.

DNA Isolation and HLA-DPB1 Sequencing

Blood samples were collected and DNA was prepared from whole blood by organic extraction, as previously described (15).

The second exon of HLA-DPB1, which contains the six hypervariable regions, was amplified, sequenced, and translated to determine its primary structure. Sequence-based typing was performed as follows: Group-specific oligonucleotide primers flanking exon 2 were used in amplification and sequencing (5). Reactions were run in an MJ Research thermocycler (MJ Research, Cambridge, MA). Each reaction contained 200 ng DNA, 10 pmol of each primer, 200 µM of each 2'-deoxyadenosine-5'-triphosphate, 2'-deoxycytidine-5'-triphosphate, 2'-deoxyguanosine-5'-triphosphate, and 2'-deoxythymidine-5'-triphosphate, 2.5 mM MgCl₂ in the purchased buffer, and 1 unit Taq DNA polymerase (Promega, Madison, WI) per 25 µl. Samples were denatured for 2 min at 95° C and were then amplified with 30 cycles of 94° C for 75 s, 55° C for 60 s, and 72° C for 180 s, ending with an extension step of 72° C for 10 min. The product was dried under vacuum, reconstituted in distilled H₂O, and quantified for polymerase chain reaction (PCR) cycle sequencing, using the Promega PCR cycle sequencing kit (Promega, Madison, WI). Samples were denatured by addition of 95% formamide and 20 mM ethylenediamine tetraacetic acid (EDTA), and heating to 95° C. Samples were run on 6% polyacrylamide, 8 M urea gels containing 90 mM Tris-borate buffer, pH 8.3, and 2 mM EDTA, followed by autoradiography. Each DNA sample was assayed at least twice to confirm the HLA-DPB1 sequence.

Statistical Analysis

Residues in the hypervariable regions of exon 2 in the HLA-DPB1 gene were analyzed. Initial analyses included the calculation of a likelihood-ratio test statistic (LRT) to test for heterogeneity between cases and controls in the distribution of haplotypes. The degrees of freedom for this test statistic were the number of haplotypes minus one. This test is conservative in that it tends to underestimate the true significance of overall allele or haplotype effects because of the inclusion of many, often rare, categories, which increases the degrees of freedom and hence the value required to reach significance (16). To avoid the problem of false positive results from multiple testing, individual haplotypes and alleles were examined only for those regions that achieved statistical significance (p < 0.05). For sequences that showed significant heterogeneity, odds ratios (OR) and 95% confidence intervals (95% CI) were calculated for the haplotype and/or allele associated with disease. For alleles with a positive association with sarcoidosis, subgroup analysis and differences between strata-specific ORs were tested.

	RESULTS

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Five of the six HLA-DPB1 hypervariable regions were found to be polymorphic in our sample population. We first used the LRT statistic to look for differences in haplotype frequency between sarcoidosis cases and controls (Table 1). Statistically significant differences were found for regions B and C. For these two regions, we next examined individual haplotypes and amino acids. In particular, Val³⁶ (patients: 60.3%; controls: 39.8%; p =  0.021) and Asp⁵⁵ (patients: 52%; controls: 35%; p =  0.033) were found to be increased in patients compared with controls. Notably, Glu⁶⁹ was only slightly more frequent in patients than in controls, and this difference was not statistically significant.

Table 2 shows the degree of association for individual haplotypes and alleles. Haplotypes in regions B and C had more than two variants; the variants found less frequently in cases were collapsed into one category to calculate ORs. Valine at position 36 had the strongest association with sarcoidosis (OR = 2.30), and glutamate at position 56 had the weakest (OR = 1.99). The haplotype ORs differed little from the individual allele ORs, which made it impossible to determine whether a change in only one or both of these residues is associated with increased susceptibility to sarcoidosis.

To determine whether disease severity or family history correlated with HLA-DPB1 polymorphism, we stratified the cases for these factors and recalculated the ORs for the four positions (Table 3). Values for Glu⁶⁹ are presented for comparison. Cases with severe disease had a slightly lower but statistically nonsignificant association at three of the four residues tested. When the cases were divided into those with and those without a family history of sarcoidosis, the nonfamilial group consistently showed a stronger association with the HLA-DP alleles, with ORs generally doubled in magnitude. Despite these differences, small numbers in the patient subgroups precluded showing any statistically significant differences.

	DISCUSSION

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HLA disease association and susceptibility to abnormal immune reactions are believed to reflect single-amino-acid changes in the hypervariable regions of the HLA molecule that are critical to antigen presentation. Sequence variations in hypervariable regions of HLA class II binding pockets underlie binding to specific residues on an antigen, determining the affinity of peptides for that particular antigenic molecule (17). In the HLA-DPB1 gene, there are six hypervariable coding regions A, B, C, D, E, and F (18), which denote residues 8 to 11, 33 to 36, 55 to 57, 65 to 69, 76, and 84 to 87, respectively. Although the three-dimensional structure of HLA-DPB has not yet been defined, the crystal structure of the closely related HLA-DR1 molecule bound to influenza-virus peptide provides insight into the interactions between an antigen and HLA peptides (8). In this structure, regions B to E are directly involved in binding to the viral peptide, whereas region A is thought to be involved in other peptide interactions.

Several weak to moderate associations between the HLA genes and sarcoidosis have been reported (19). The lack of consistent findings in these studies has made defining the role of the HLA in sarcoidosis difficult. As rules for the specificity of binding between antigen and HLA molecules become defined, understanding the structure of the binding pocket may give clues about the nature of the specific antigen bound, and insight into the role of the MHC in sarcoidosis.

The clinical and histologic similarities between sarcoidosis and CBD suggest a common pathogenic mechanism in the two diseases, and a possibly similar genetic susceptibility to disease. A strong association between Glu⁶⁹ and CBD has been reported, and motivated the present study (6, 7). In a population of Caucasian sarcoidosis patients and controls, Lympany and colleagues found no association with a specific HLA-DP allele, but observed that 26 of 41 patients had Glu⁶⁹-positive alleles, whereas 29 of 76 controls were Glu⁶⁹ positive (30). This group observed no association of HLA-DPB1 position 55 with sarcoidosis. Like Lympany and colleagues, we found no statistically significant increase in any one HLA-DPB1 allele in our African-American sample population. Unlike Lympany and colleagues, we observed no association between Glu⁶⁹-positive alleles and sarcoidosis. Rather, we found weak associations with Val³⁶ and Asp⁵⁵.

Our study sample size allowed us to detect with 80% power a 2.5-fold increase in relative risk (RR) for Glu⁶⁹-positive alleles (p = 0.05). Our results are consistent with those of Saltini and coworkers, who found no difference in frequency of the Glu⁶⁹-positive DPB*0401 allele and Glu⁶⁹-negative DPB*-0201 allele in 24 biopsy-proven cases of sarcoidosis as compared with normal subjects (31). Our results suggest that residues other than Glu⁶⁹ in HLA-DPB1 may contribute to susceptibility to sarcoidosis in African-Americans.