Introduction

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Sarcoidosis is thought to be a hypersensitivity response to some unknown antigen. Several environmental risk factors have been suggested, but few have been confirmed. For example, case-control studies suggest higher rates of infection in patients with sarcoidosis for mycobacteria tuberculosis (1, 2), Mycobacterium orale (3), and Epstein-Barr virus (4, 5). Mycobacterial DNA and RNA have been detected in sarcoid tissue using the polymerase chain reaction (PCR) (6), although no consistent isolation of these organisms has been reported. One clearly identified risk factor for sarcoidosis is race. In the United States, African Americans are more commonly and severely affected than the general population (11), and have a higher prevalence of familial sarcoidosis (10, 17). Harrington and coworkers (17) described 91 families with more than one member affected with sarcoidosis and reported familial sarcoidosis to be more common in African Americans (17%) than in Caucasians (6%). Preponderance of sarcoidosis in certain ethnic groups and familial aggregation suggests that genetic susceptibility may play a role in sarcoidosis etiology.

Resistance to mycobacterial infections also appears to be genetically controlled. The notion that hereditary susceptibility to tuberculosis exists is supported by reports of familial clustering in endemic areas, concordance studies in monozygotic and dizygotic twins, and the apparent influence of ethnic factors on the incidence and severity of mycobacterial infection (18). In the United States, African Americans are twice as likely as Caucasians to become infected with Mycobacterium tuberculosis (22, 23). Studies on murine models of susceptibility to mycobacterial infection revealed that a single gene, denoted Lsh/Ity/Bcg, confers susceptibility to intracellular pathogens such as leishmania, salmonella, and mycobacteria (24). Macrophages from homozygous mutant mice show a variety of specific defects in tumoricidal and antimicrobial activity (25). Investigators identified a gene called natural resistance-associated macrophage protein (Nramp1), which encodes a polytopic, macrophage-specific, integral membrane protein whose function appears to involve membrane transport (26). A single, nonconservative mutation resulting in the substitution of aspartate for glycine 169 was found responsible for resistance to infection by these intracellular parasites in mice. Subsequent cloning and characterization of the human NRAMP1 gene indicates 88% sequence identity to the mouse gene (27, 28). In particular, the glycine responsible for resistance in the mouse is conserved in the human sequence. Recent studies in human populations have found an association of NRAMP1 with susceptibility to tuberculosis in a Gambian population (29) and weak linkage in a family study (30).

Functional studies indicate that NRAMP1 is involved in the early stages of macrophage priming and activation. NRAMP1 may have many effects on macrophage function, including upregulation of tumor necrosis factor (TNF) , interleukin (IL)-1, and major histocompatibility complex class II expression, leading to higher levels in resistant murine macrophages (25, 31). The proinflammatory cytokines TNF- and IL-1 have been reported to be important mediators of the granuloma formation. Increased expression of TNF- (32), IL-1 (33, 34), and human leukocyte-associated antigen-DR (35) has been reported in sarcoid alveolar macrophages.

The involvement of NRAMP1 in macrophage activation makes it an attractive candidate gene for sarcoidosis. We performed a case-control association study to test whether four NRAMP1 polymorphisms were found more frequently in African American patients with sarcoidosis or control subjects.

	Materials and Methods

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Study Population

This study was approved by the Henry Ford Health System Institutional Review Board. The study sample consisted of 157 African American patients with sarcoidosis and 111 healthy African American control subjects. 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 bacillus and fungus. Seventy percent of the patients were female. At the time of the study, patients ranged in age between 26 and 73 yr with an average age of 49 ± 11 yr. Patients had been diagnosed for an average of 13 ± 10 yr at the time of study and followed in the clinic for at least 1 yr. The average age of patients at diagnosis was 36 ± 9 yr with a range between 19 and 59 yr. Control subjects were hospital employees who had no pulmonary disease at the time of participation and no known relatives with sarcoidosis. Control subjects were 78% female and had an average age of 39 ± 10 yr. All patients gave informed consent for enrollment in the study and blood drawing.

Phenotyping

The clinical course of each sarcoidosis case was determined by reviewing medical records. Based on clinical features, 140 of the 157 patients sampled were categorized and placed in one of three phenotypic categories:

Mild: incidental abnormal chest radiograph, normal organ function, never treated, remained asymptomatic after 1 to 2 yr of observation, or history of acute onset of symptoms with spontaneous remission within 2 yr.

Moderate: symptomatic disease with minimal organ function abnormalities occurring with or without therapy.

Severe: progressive disease with organ dysfunction and evidence of scarring on chest radiograph.

Genotyping

High molecular weight DNA was isolated from anticoagulated blood by detergent lysis and organic extraction (36). The NRAMP1 polymorphisms typed were a (CA)_n repeat in the immediate 5' region of the gene, denoted 5'(CA)_n; a single nucleotide change in intron 4 (469+14G/C), denoted INT4; a single base substitution at codon 318 changing alanine to valine (A318V); a nonconservative single base substitution at codon 543 changing aspartic acid to asparagine (D543N); and a TGTG deletion in the 3' untranslated region (1729+55del4), denoted 3' UTR.

Three polymorphisms were analyzed by amplifying the region of interest, followed by restriction enzyme digestion. Amplification primers were used as described by Liu and colleagues (37), encompassing sequence variants at A318V, D543N, and 3' UTR. PCR were performed in 40-µl reaction volumes containing 400 ng genomic DNA, 50 mM Tris-HCl (pH 8.3), 2.5 mM MgCl₂, 0.05% Tween-20, 0.05% NP-40, 20 mM each deoxynucleotide triphosphate, 20 pmol each primer, and 2 U of Taq DNA polymerase (Promega, Madison, WI). Thermocycling parameters were as follows: denaturation at 94°C for 3 min, and 30 cycles of 94°C for 1 min, 59°C for 1 min, and 72°C for 2 min. An aliquot of each amplification reaction was diluted 1:3 and digested with 5 to 10 U of Ava I (D543N), Fok I (3' UTR deletion), and Bso fI (A318V) under conditions recommended by the supplier (New England BioLabs, Beverly, MA). Restriction enzyme products were resolved on 12% polyacrylamide gels in 9 mM Tris-borate buffer, 1 mM ethylenediaminetetraacetic acid (EDTA) buffer, pH 8.0, and stained with ethidium bromide.

Microsatellite polymorphisms were analyzed using primers as previously published (37, 38). Amplification reaction conditions were as described previously, with the addition that one primer in each reaction was labeled at the 5' end with [-³²P]adenosine triphosphate (39). Samples were denatured by the addition of 10 µl 95% formatted, 20 mM EDTA and heating to 70°C for 2 min. Five microliters of sample were run on 4 or 6% polyacrylamide, 8 M urea gels containing 90 mM Tris-borate buffer, pH 8.3, 2 mM EDTA for 3 to 4 h at 55 W. Gels were exposed to film for 18 h at 70°C. Allele sizes were determined by running M13 DNA sequencing ladders in each gel for single base pair resolution. Individuals were genotyped according to allele size. Each DNA sample was assayed at least twice per marker to confirm genotype.

Statistical Analysis

For each polymorphism, genotypic frequency differences between case patients and control subjects were examined using a chi-square test with two degrees of freedom. Additional chi-square analyses included a test for frequency differences of haplotype groupings formed by combining selected polymorphisms. Odds ratios (OR) and 95% confidence intervals (CI) were calculated to quantitatively assess the degree of association between these polymorphisms and sarcoidosis.

	Results

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Of the five loci spanning the NRAMP1 gene that were analyzed, four were polymorphic in this population of African Americans. The fifth polymorphism denoted A318V was not polymorphic in this sample population. Allele frequencies for the microsatellite repeat 5'(CA)_n, the single base transversion in intron 4 (INT4), the single base pair substitution D543N, and the 4-bp deletion in the 3' UTR polymorphisms are listed in Table 1. The results comparing allele frequency distribution of the four polymorphic markers are shown in Table 2. We observed a statistically significant difference in allele frequency distribution of 5'(CA)_n alleles between patients and control subjects (P = 0.014). Compared with the most common 120-bp allele, the less common alleles were underrepresented in the sarcoidosis patient population, suggesting that polymorphism at this site is associated with decreased risk for sarcoidosis. The D543N A allele is apparently in linkage disequilibrium with the 3' UTR del allele, thus these results were not independent of each other. The D543N G/3' UTR del haplotype was observed in this African American population, whereas it is not found in Caucasians and Asians (35) (and unpublished data). The D543N G/3' UTR del haplotype has also been observed in a population of West Africans (27). Combined analysis of the 5'(CA)_n and D543N G polymorphisms showed an increased protective effect (Table 3). Combined analysis of the INT4 and 3' UTR variants, which were strongly associated with tuberculosis (27), showed no effect in patients with sarcoidosis.

One hundred forty patients were characterized with regard to clinical course of disease (see MATERIALS AND METHODS) and placed into mild (n = 50), moderate (n = 57), and severe (n = 33) phenotypic categories. We did find that in mild cases the association of the D543N A allele with decrease risk for sarcoidosis was much stronger (OR, 0.20; 95% CI, 0.05-0.81) than that observed in the total sample of cases (OR, 0.63; 95% CI, 0.32-1.22), but the sample size has inadequate power to draw conclusions.

	Discussion

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There are several characteristics of NRAMP1 that make it an attractive candidate susceptibility gene for sarcoidosis. NRAMP1 is expressed exclusively in macrophages and polymorphonuclear leukocytes. Interspecies conservation of intramembranous residues with charged side chains suggests that transport of a charged molecule may be central to the function of NRAMP1. NRAMP1 also has close sequence homology with other transport proteins, including a nitrate transporter in Aspergillus (26). This suggests that NRAMP1 may transport nitrate intracellularly into an acidic compartment to create nitrous oxide, which would aid in antimicrobial activity. Gene transfection experiments in vitro (31, 40) provide support that NRAMP1 functions early in macrophage activation.

The similarities between sarcoidosis and tuberculosis prompted us to investigate NRAMP1 as a candidate susceptibility gene for sarcoidosis. Our observed decreased risk for sarcoidosis contrasts with results obtained in tuberculosis studies. Bellamy and coworkers (29) evaluated the 5'(CA)_n, INT4, D543N, and the 3' UTR deletion polymorphisms in 800 West Africans and reported a strong association between NRAMP1 polymorphisms and increased risk for tuberculosis (29). Segregation analysis on 98 Brazilian families (30) also suggested a minor role (² = 3.84, P < 0.05) for the NRAMP1 locus. Although the exact contribution is unclear, these studies in sarcoidosis and tuberculosis suggest that NRAMP1 plays a role in granulomatous lung inflammation, which warrants further investigation.

The hypothesis that infectious agents such as mycobacteria play a role in sarcoidosis pathogenesis continues to be debated. Recent studies using techniques with increased sensitivity to detect mycobacterial DNA in sarcoidosis specimens have not resolved the question. Popper and associates (9) reported amplification of mycobacterial DNA sequences by the PCR in biopsy specimens from 11 of 35 patients with sarcoidosis, whereas Vokurka and coworkers (41) detected no mycobacterial DNA in any sarcoidosis biopsy specimen using sequence-capture PCR. Using quantitative PCR, Ishige and colleagues (10) estimated the number of mycobacterial and propionibacterial DNA in sarcoidosis biopsies. They observed mycobacterial genomes in 3 of 15 patients with sarcoidosis, and propionibacterial DNA in 12 of 15 patients with sarcoidosis. Investigating genes involved in resistance to these organisms may lead to a greater understanding of the role that infectious agents play in sarcoidosis.

Sarcoidosis is thought to begin with macrophage-antigen processing with subsequent antigen presentation to T cells. The resulting activated T cells initiate a process which includes secretion of cytokines and growth factors, recruitment of inflammatory cells, macrophage differentiation, and granuloma formation. Macrophages in patients with sarcoidosis have unusually high ability to present antigen due to increased expression of human lymphocyte antigens on the cell surface (35). Aberrant expression of antigen presentation molecules and proinflammatory cytokines by sarcoid macrophages may be a result of overactivation by NRAMP1. Differential macrophage activation by various NRAMP1 alleles could affect the clinical course of sarcoidosis. Our results suggest that at least one NRAMP1 polymorphism is associated with decreased risk for sarcoidosis in African Americans, further supporting a potential role for NRAMP1 in susceptibility to sarcoidosis.