INTRODUCTION

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Sarcoidosis is characterized by the presence of epithelioid granulomas in affected organs that may lead to fibrosis and dysfunction (1). Epithelioid cells secrete over 40 different cytokines and other mediators including the angiotensin-converting enzyme (ACE), which is thought to reflect granuloma burden in sarcoidosis (2, 3). Liberman (4), over 20 years ago, first reported elevations of serum ACE activity in sarcoidosis patients and more recently, studies suggest that ACE plays a direct role in sarcoidosis pathophysiology (5).

ACE acts as a dipeptidylcarboxypeptidase cleaving the C terminal dipeptide from angiotensin I to give the octapeptide angiotensin II, a potent vasoconstrictor. Epithelioid cells of sarcoid granulomas contain ACE and ACE synthesis in monocyte cell culture can be modulated by T lymphocytes obtained from sarcoid patients (6). Studies have also demonstrated that ACE inactivates bradykinin and substance P (7, 8) and that angiotensin II is chemotactic for T cells (9). Serum ACE levels in healthy individuals (10) and sarcoidosis patients (11) are influenced by an insertion (I)/deletion (D) ACE gene polymorphism (10). This polymorphism is due to a 287 base pair Alu sequence either present or absent in intron 16 that results in three genotypes: insertion/insertion (II), insertion/deletion (ID), and deletion/deletion (DD). The DD polymorphism is associated with the highest plasma levels, ID with intermediate levels, and II with the lowest levels.

Elevated serum ACE levels are associated with active sarcoidosis (12), but the use of serum ACE as a marker for disease is limited. ACE concentrations at the time of disease presentation have also been examined as a marker for development of chronic sarcoidosis, but a consistent association has not been found (2, 16). The discovery of the I/D ACE gene polymorphism has led to a reexamination of ACE levels as a marker for both disease and disease activity in sarcoidosis. Several studies have shown that controlling for ACE genotype increases the sensitivity of serum ACE levels as a disease marker (11).

At present, the role of the ACE gene in sarcoidosis susceptibility and progression is inconclusive. No studies to date have included African Americans, even though they are more susceptible to both sarcoidosis (19) and chronic disease (20) compared with Caucasians. Because the ACE polymorphism has a direct effect on serum ACE levels, we investigated whether this polymorphism is associated with sarcoidosis susceptibility and clinical course in a well-characterized patient population of Caucasians and African Americans.

	METHODS

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Study Population

Unrelated sarcoidosis cases were recruited from an ongoing sarcoidosis patient registry at Henry Ford Hospital in Detroit, Michigan. Diagnosis was based on a review of clinical and radiographic presentation, as well as histology and special stains and cultures for acid-fast bacillus and fungus. A total of 183 African-American and 60 Caucasian sarcoidosis patients participated in the study. Cases were diagnosed between 1950 and 1996 and had been followed for an average of 10.5 yr. A total of 111 African-American and 48 Caucasian control individuals were randomly selected from hospital employees. A comparison of the case and control groups by race is shown in Table 1. African-American cases were an average of 3 yr younger than Caucasian cases at diagnosis. Furthermore, African Americans were more likely to be female and have their diagnosis confirmed by biopsy. Both control groups were similar in age to the respective case groups at time of diagnosis, but the African-American control group had a higher percentage of females (79%) than the Caucasian control subjects and the two case groups. All subjects were enrolled after giving informed consent, and all procedures conformed to human subject protocols approved by the institutional review board at Henry Ford Hospital.

Phenotyping and Other Patient Characteristics

The clinical course of each sarcoidosis case was determined by reviewing medical records from time of diagnosis to present. Based on clinical features, each subject was 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.

For the purposes of analysis, the mild and moderate categories were combined into one category and compared with severe sarcoidosis cases.

Patients were classified as having familial sarcoidosis if they had a first- or second-degree relative with sarcoidosis. Baseline and follow-up chest radiographic data were available for a subset of African-American subjects. For longitudinal analyses, we compared the chest radiograph at time of disease presentation with the first radiograph taken at least 2, but no more than 4 yr, after initial diagnosis. Radiographic stages were stratified into five levels as previously defined (21): 0a normal chest radiograph; Ithe presence of bilateral hilar adenopathy only; IIadenopathy with pulmonary infiltrates; III pulmonary infiltrates without adenopathy; and IVpulmonary fibrosis with loss of lung volume. Additional clinical data collected included the presence of eye and/or skin involvement.

Genotyping

Blood samples and DNA were prepared as previously described (22). The diallelic ACE polymorphism was genotyped by enzymatic amplification of a segment within intron 16 with the 190 and 490 base pair alleles resolved by 6% polyacrylamide gel electrophoresis. The primers were designed from the published flanking sequence (10) flanking 5'-CTGGAGACCACTCCCATCCTTTCT-3' (sense) and 5'-GATGTGGCCATCACATTCGTCAGAT-3' (antisense). One hundred nanograms of genomic DNA were amplified with 10 picomole of each primer at 94° C for 1 min, 58° C for 1 min, and 72° C for 2 min, for 30 cycles.

To confirm that genotypes with only a D band were true homozygotes rather than mistyped ID heterozygotes due to preferential amplification of the D allele, a second reaction was performed using the sense primer labeled with gamma [³²P] ATP (23). Five microliters of the reaction were separated on a 6% polyacrylamide gel in 9 mM tris-borate buffer, 1 mM EDTA buffer, pH 8.0. The gels were stained with ethidium bromide to display molecular weight markers and autoradiographed to detect alleles.

Statistical Analysis

Differences in genotype frequencies between groups were examined using chi-square tests. Odds ratios (OR) and 95% confidence intervals (CI) were calculated for ID and DD genotypes with the II genotype used as the reference. All case-control comparisons were stratified by race. Adjusted ORs were calculated using logistic regression models.

	RESULTS

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Table 2 shows the distribution of the ACE genotype in African-American and Caucasian cases and control subjects. In African Americans, an increasing gradient of risk with the number of D alleles was found. The DD genotype conferred a threefold increased risk for sarcoidosis (OR = 3.17; 95% CI = 1.51 to 3.17). Overall, we observed a marked difference in the distribution of the ACE genotype between the African-American cases and control subjects (p = 0.005). For Caucasians, the risk associated with the D allele was in the opposite direction compared with African Americans. However, both odds ratios for the ID or DD genotypes had wide confidence intervals, and we found no overall difference in the distribution of the ACE genotype between Caucasian cases and control subjects (p = 0.577). Because of the lack of association between the ACE gene polymorphism and sarcoidosis in Caucasians, and our small sample size in this group, all further analyses were restricted to African Americans.

We next tested whether the association found between the ACE polymorphism and sarcoidosis in African Americans varied with case characteristics. The above analysis was repeated stratifying by sex, age, family history of disease, and disease severity (Table 3). The odds ratios for the ID and DD genotypes were similar between strata, except when stratifying cases by family history. The DD genotype showed a marked increase in risk for cases with a positive family history (OR = 4.83; 95% CI = 1.86 to 12.59) compared with those who had a negative family history (OR = 2.52; 95% CI = 1.12 to 5.66).

Further analyses of the ACE polymorphism in African-American sarcoidosis cases included testing for associations with organ involvement and disease prognosis (Table 4). Of the disease characteristics examined, only skin involvement was associated with ACE genotype (p = 0.044) with a disproportionate number having the ID genotype. For a subset of patients (n = 117), we had radiographic results at the time of initial presentation with disease. We found no association between the ACE polymorphism and radiographic stage either when analyzing each stage separately (p = 0.238) or when condensing cases into advanced (stage II or greater) and less advanced (stage 0 or I) categories (p = 0.399). In the latter analysis, those with the ID and DD genotypes had an increased risk for advanced radiographic stage at disease presentation (OR = 1.90; 95% CI = 0.75 to 4.85).

Of the 117 subjects with radiographic data at disease presentation, most (n = 84) had an additional radiograph 2 to 4 yr later. In the group with follow-up radiographs, 31% had improved, 43% stayed the same, and 26% progressed. In subjects with a stage I or higher radiograph at baseline, 21% had complete radiographic resolution at follow-up. Figure 1 shows overall radiographic change for the three separate ACE genotypic groups. While overall radiographic change was not associated with the ACE polymorphism (p = 0.375), it appeared that the ID and DD groups had similar patterns of radiographic changes that differed from those in the II group. To explore these differences further, we did a reanalysis of these data where the IDs and DDs were collapsed into one group and radiographic progression was the outcome of interest. In this analysis, those with the II genotype had a threefold greater risk for radiographic progression (OR = 2.97; 95% CI = 1.01 to 8.76). Because initial chest radiograph is likely related to whether an individual radiographically progresses during follow-up, we reran the above longitudinal analyses adjusting for initial chest radiograph. Even after adjustment, the pattern shown in Figure 1 persisted (data not shown). Likewise, the risk associated with the II genotype and progression was still elevated (OR = 2.71; 95% CI = 0.86 to 8.51).

View larger version (24K): [in this window] [in a new window]   Figure 1.   Radiographic change 2 to 4 yr after initial presentation and ACE genotype in 84 African-American sarcoidosis cases.

	DISCUSSION

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A growing body of evidence suggests the ACE gene insertion/ deletion polymorphism plays a role in chronic disorders such as cardiovascular (24) and renal (27, 28) disease. These findings and the long-standing clinical use of serum ACE levels as a marker for disease activity in sarcoidosis makes the ACE gene an attractive candidate gene for sarcoidosis. If the level of circulating serum ACE is related to granuloma burden, then the genetic polymorphism that in large part determines ACE concentrations could be related to risk of sarcoidosis. In our study, we found an elevated risk for African Americans with the ACE DD genotype (OR = 3.17) and a significant heterogeneity in risk among the three genotypes (p = 0.005). These findings were not duplicated in our Caucasian sample where no association with the ACE polymorphism was found.

Few studies have investigated the role of the ACE polymorphism in sarcoidosis and none have studied African Americans. Of the two case-controls studies of the ACE gene and sarcoidosis in Japanese populations, one found no increased risk (13) and the other found an increased risk with the ID and DD genotypes (OR = 2.18) only in females (11). In our stratified analyses of African Americans, we found no difference in risk between the sexes. Two other case-control studies in Caucasians, one in Italians (14), and the other in a Western European Caucasian population (12), were both consistent with our lack of positive findings between ACE polymorphism and sarcoidosis in Caucasians. In contrast to these negative studies, the association we found in African Americans is the strongest evidence to date that the ACE gene may play a role in sarcoidosis susceptibility. Based on our Caucasian results and those of others, we might infer that some other genetic or environmental factor unique to African Americans may need to interact with the ACE gene before an increased risk for sarcoidosis is manifest. This inference is bolstered by our stratified analyses that found the risk of the ACE DD genotype was greater when cases were restricted to those with a positive family history. This group of cases would most likely have other genetic risk factors for sarcoidosis.

Another important question is what role, if any, the ACE gene may play in sarcoidosis progression. Although a majority of sarcoidosis cases are acute and self-limiting, in a significant percentage of patients, especially among African Americans, the disease fails to resolve (20). Predicting who goes on to chronic sarcoidosis is difficult. A stage I chest X-ray at presentation usually indicates a good chance (60 to 80%) of remission. However, the worse prognosis for patients at a more advanced radiographic stage may be confounded with timing in the ascertainment of incident cases. The concentration of serum ACE at presentation is generally not a reliable marker of disease progression (2, 16), but recent studies of the ACE polymorphism in sarcoidosis patients have shown that controlling for the ACE genotype increases the sensitivity of serum ACE in detecting clinically active disease (11).

If serum ACE levels correlate with granuloma burden, then the ACE gene may play a role in disease progression. Only one study to date has examined the role of the ACE gene in chronic sarcoidosis. Tomita and coworkers (13) found no association between the ACE polymorphism and rate of chest radiographic resolution 3 and 5 yr after diagnosis in a Japanese population. We examined the role of the ACE gene in sarcoidosis progression in African-American cases. The ACE polymorphism was not associated with severe sarcoidosis, defined as having progressive disease with organ dysfunction and evidence of scarring on chest radiograph, nor was there any association with eye involvement and initial chest radiograph. We did find an association between the ACE polymorphism and skin involvement, but all the increased risk was found in ID genotype where ACE levels are intermediate. This would suggest that the association was more likely spurious than causal in nature.

Using an objective marker of progressive sarcoid, we examined radiographic change over time in a subset of African-American patients. When ACE genotypes were analyzed separately we did not find an association with radiographic change. Grouping the ID and DD genotypes together, we did find an increased risk associated with the II genotype and radiographic progression (OR = 2.90). The small number of cases with the II genotype in our case sample (n = 18), and the select nature of this sample, limits the inference we can make from these results. Nevertheless, further investigation of the role of the ACE gene in disease progression, especially in African Americans, seems warranted from our results.

Allele frequencies for I and D polymorphisms vary greatly among ethnic groups (29, 30), and within subgroups of the African-American population (29, 31). Previously reported allele frequencies among African-American control study populations vary from 36% (32) to 51.4% (31), and 48.6% to 64%, for I and D alleles, respectively. In contrast, reported I/D allele frequencies among both European and American Caucasian sample populations have a smaller range of variation, from 43% (29) to 50% (24) and 57% to 50% for I and D alleles, respectively. This suggests the African-American population may be heterogeneous within the United States, and careful geographic matching of control and patient samples is important.

In our study, neither the Caucasian nor African-American control groups came from the same population from which the cases were drawn in that both comprised convenience samples of hospital employees. The genotypic frequencies in our Caucasian control group were consistent with reported results in other Caucasian populations (10, 33). In a control sample drawn from the same geographic area as our study, the reported genotypic frequencies in African Americans were similar to our results (31). Based on these comparisons, we think that our sampling procedure of control subjects introduced a minimal amount of bias in genotypic frequencies. Cases were drawn from a sarcoidosis clinic that has nearly complete ascertainment of all cases in the Henry Ford Health System. In our case group, we likely oversampled those with severe sarcoidosis because mild and moderate cases diagnosed before the time of our recruitment period were less likely to return to the clinic for follow-up. Because severe sarcoidosis in African Americans was not associated with the ACE polymorphism, it is unlikely an overrepresentation of severe cases would bias our initial risk estimates. The subset of cases in our longitudinal analyses of radiographic progression were also more likely to be those with severe disease. Nevertheless, this group had similar ACE genotypic frequencies as those without longitudinal data.

In summary, we found an association of the ACE gene polymorphism and sarcoidosis susceptibility in African Americans, but not Caucasians. An increased odds ratio when cases were restricted to those with a positive family history suggests other factors that cluster in families may interact with the ACE gene. Although the ACE polymorphism was not associated with either severe disease or overall radiographic change in African-American sarcoidosis patients, cases with the II genotype may be at greater risk for radiographic progression. Our results represent the strongest evidence to date for a role of the ACE gene in sarcoidosis. ACE gene susceptibility may be limited to ethnic groups such as African Americans who show greater preponderance of familial disease (34, 35). Further elucidation of other genetic risk factors in sarcoidosis will likely help determine the role of the ACE gene in the overall disease pathophysiology.