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Department of General Internal Medicine and Kiel Center of the German National Genotyping Platform
Institute for Medical Informatics and Statistics, Christian-Albrects-University Kiel, University Clinics Schleswig-Holstein Campus Kiel, Kiel
Department of Pneumology, University of Freiburg, Freiburg
Institute of Human Genetics, University of Leeck, Leeck, Germany
ABSTRACT
Prevailing models of sarcoidosis pathogenesis involve the activation of alveolar macrophages, aggregation of CD4+ T lymphocytes, and their accumulation in epithelioid cell granulomas. Increasing evidence suggests that each of these steps is modified by the host genetic constitution. Consequently, candidate susceptibility genes have been selected based on their potential function under this model. The C-C chemokine receptor 2 (CCR2) is involved in Th1 immune activity by recruiting competent cells and possibly by balancing response. CCR2 gene variants have been shown to be associated with sarcoidosis or, more specifically, with Lfgren's syndrome, a distinct form of acute sarcoidosis. We have studied three CCR2 gene polymorphisms (c.190G>A, c.840C>T, and c.4385A>T) in an extended sample of 1,203 patients with sarcoidosis and their relatives. Case-control comparisons and family-based genetic analyses did not support previous findings of an association between CCR2 gene variability and the risk of sarcoidosis. However, they confirmed linkage disequilibrium and showed positive linkage results (p = 0.034) and therefore suggest a susceptibility gene in the surrounding chromosomal region.
Key Words: genetic predisposition to disease genotype Lfgren's syndrome transmission disequilibrium test
Sarcoidosis is a multiorgan inflammatory disease of unknown cause that primarily affects the lung and the lymphatic systems, but can afflict almost every other organ of the body. The disorder is characterized by an exaggerated cellular immune reaction that is believed to be triggered by so far unidentified, but presumably inhaled, exogenous agents (1, 2). The prevailing model of sarcoidosis pathogenesis involves activation of alveolar macrophages, aggregation of CD4+ T lymphocytes, and the accumulation of these cells in epithelioid cell granulomas, which are typical at the sites of inflammation (3, 4).
Clinical presentation of the disorder varies widely, with two main phenotypes: acute and chronic sarcoidosis. Acute sarcoidosis is predominantly associated with a self-limited course of disease and a favorable prognosis. A subset of patients with acute disease present with symptoms of Lfgren's syndrome, which are typically a combination of fever, bilateral hilar lymphadenopathy, erythema nodosum, and arthritis, often of the ankle joints. In contrast, chronic sarcoidosis tends to start with subtle, creeping symptoms and has a considerable risk of proceeding to enduring inflammation and subsequent organ damage (1, 2, 4).
Inherited susceptibility to sarcoidosis is well documented through differing prevalence rates in different populations and through an increased risk of occurrence in close relatives of patients (5eC7). The spectrum of clinical presentation and pattern of organ involvement differs between ethnic groups (8, 9). A complex blend of predisposing and protective gene variants is believed to contribute to an individual's susceptibility to sarcoidosis, as well as to the various presentations and prognoses of the disease. Major candidate gene variants conferring susceptibility or resistance to sarcoidosis have been identified in the HLA gene cluster of chromosome 6 using case-control study designs and genetic linkage analyses (10eC17). However, these gene effects are not sufficient to explain the approximately 20-fold increased manifestation risk in close relatives of patients with sarcoidosis, as observed in European populations (18, 19).
In the search for additional predisposing genes, candidate genes have been selected for analysis because of their potential function in the pathogenesis of sarcoidosis, with a focus on antigen recognition, processing, and elimination. Among these functional candidate genes, the C-C chemokine receptor 2 (CCR2) has been studied in different settings with remarkable results. The CCR2 gene is located on the short arm of chromosome 3 (cytogenetic band 3p21). A nonsynonymous polymorphism in exon 1 (c.190G>A) leads to the substitution of valine by isoleucine (p.V64I) in the transmembrane region of the protein. A decreased risk of sarcoidosis in carriers of the rarer allele, c.190A (p.64I), has been described in Japanese patients (20). This was not replicated in a sample of Czech patients, even though there was a nominal, but not significant, excess of c.190A in control subjects (21). Recently, a set of eight single nucleotide polymorphisms of the CCR2 gene, including c190G>A, has been analyzed in a Dutch sarcoidosis case-control study (22). The superior resolution of the haplotype analysis enabled the demonstration of an association between one specific CCR2 haplotype and an increased risk of Lfgren's syndrome. The fact that the CCR2 gene locus on the short arm of chromosome 3 (3p21) coincides with a linkage peak in a genomewide search for predisposing loci (23) further contributes to the candidate status of CCR2 gene variants in the etiology of sarcoidosis. Here, we contribute novel association and genetic linkage data for CCR2 alleles from an extended sample of patients with sarcoidosis and their families.
METHODS
Patients, Families, and Control Subjects
Patients were actively contacted through the German Sarcoidosis Patients' Organization (Deutsche Sarkoidose-Vereinigung e.V.; www.sarkoidose.de), specialized hospitals, and practitioners, and by calls for participation in the study that were published via health insurance institutions. Parents were included when possible. Patients from families with two or more affected members were interviewed by telephone about their sarcoidosis history and family structure.
All patients completed a questionnaire on the course of disease. Patients' physicians were contacted to confirm the diagnosis and to provide records. The average age at diagnosis was 36.4 years (SD, 10.9 years) and ranged from 12 to 80 years. Sarcoidosis was confirmed by biopsy in 1,001 of 1,203 (83.2%) cases. In the remaining 202 patients, the clinical course, in combination with radiology and laboratory findings, was consistent with the diagnosis of sarcoidosis. The control group comprised 545 healthy blood donors recruited through the Department of Transfusion Medicine at Kiel University Hospital, Kiel, Germany. The control subjects consisted of 268 (49.1%) males and 277 (50.9%) females with an average age at inclusion of 43 years (SD, 12.7 years). All families with two or more patients were of German descent. All other patients and relatives were German residents. All participants gave written, informed consent before inclusion in the study. Study protocols were approved in writing by the institutional ethics and data protection authorities.
Patients were classified according to disease presentation on the basis of all available information (questionnaires completed by patients and physicians, hospital records, and interview information for familial cases). The entire sarcoidosis sample consisted of 1,203 patients and 1,084 close relatives (mainly parents, without sarcoidosis). Three hundred and seventy patients had acute sarcoidosis, with sudden complaints and recovery within 2 years. One hundred and sixty-five of these patients with acute sarcoidosis exhibited the characteristic combination of symptoms of Lfgren's syndrome (bilateral hilar lymphadenopathy, erythema nodosum, and arthropathy). Seven hundred and thirty-nine patients suffered from chronic sarcoidosis. The majority of these exhibited subtly intensifying early symptons followed by enduring disease activity for 2 years or longer. The remaining 94 patients showed other phenotypes (e.g., solely cutaneous sarcoidosis), or were detected serendipitously by radiography for other reasons and had no specific complaints. These individuals and their relatives were genotyped but excluded from further data analysis with the exception of the linkage disequilibrium analysis. An overview of the study population of patients with sarcoidosis is given in Table 1.
CCR2 Genotyping
Three polymorphic sites of the CCR2 gene—c.190G>A (p.V64I, rs1799864), c.840C>T (p.N260N, rs1799865), and c.4385 A>T (rs1034382, in the downstream intergenic sequence)—were genotyped using Taqman (24) and TaqMan-MGB (25) assays. Ready-to-use Assay-on-Demand and Assay-by-Design assays were used for typing c.840C>T and c.4385T>A, respectively (all assays were from Applied Biosystems, Foster City, CA; www.store.appliedbiosystems.com). For the c.190G>A variant, a TaqMan assay was designed manually using the Primer Express 2.0 software (Applied Biosystems). Assay details are given in Table 2.
Data Analysis
Marker genotypes were tested for Hardy-Weinberg equilibrium in the case and control study populations. Allele and genotype frequencies in cases and control subjects were compared using Pearson's 2 test of the SPSS statistical software package (SPSS, Inc., Chicago, IL). Markers in families and trios (single patients with parents) were evaluated for distorted transmission using the transmission disequilibrium test of the Genehunter 2.1 program (26). The same program was used for nonparametric linkage analysis and haplotype reconstruction in families. The allelic correlation coefficient r2 was computed as an index of the pairwise linkage disequilibrium between markers (27).
RESULTS
The entire collection of 2,832 samples (1,203 patients, 1,084 relatives, and 545 control individuals) was genotyped for three single nucleotide polymorphisms in a high-throughput setting. The overall genotype calling rate was 0.98. The observed allele and genotype frequencies of the three CCR2 polymorphisms in the sarcoidosis patient study population (323 patients with acute sarcoidosis and 680 patients with chronic sarcoidosis, after selecting a single patient at random from families with two or more patients of the same type) and control individuals are listed in Table 3. Genotype distributions in cases and control subjects corresponded to the expectations of Hardy-Weinberg equilibrium, with the exception of c.190G>A genotypes in cases (p = 0.018). This mainly resulted from a lack of AA homozygotes (observed 1, expected 6.5). The preferential drop-out of AA genotypes does not appear likely if drop-out genotypes are reconstructed from family data. The random exclusion of one AA homozygote from a family with two patients may have contributed to the low frequency of AA homozygotes but cannot fully explain it. There were no significant (p < 0.1) differences between any sample of patients (Lfgren's syndrome; acute sarcoidosis, including Lfgren's syndrome; chronic sarcoidosis; acute and chronic sarcoidosis) and the control group or between different samples of patients. There was a strong linkage disequilibrium between c.840C>T and c.4385A>T with an r2 value of 0.72 in the control population. In the families, the alleles of the three markers cosegregated with no evident haplotype recombination based on 527 meioses. Three hundred and sixty-two unequivocal haplotype transmissions could be derived from the pedigree and genotype data. Only four of eight possible haplotypes occurred with notable frequencies, and one rare haplotype segregated in one family, but was not transmitted to the patient, who had Lfgren's syndrome. The observed transmission rates of alleles and haplotypes are given in Table 4. There was no significant (p < 0.05) transmission disequilibrium in the study population or in any of the subsets. A suggestive transmission disequilibrium (0.05 < p < 0.1) for two haplotypes was observed in the patients with acute sarcoidosis. One of these distortions was also evident in the subset of patients with Lfgren's syndrome. These haplotypes were divergent with respect to the c.190G>A polymorphism: the c.190A allele (corresponding to p.64I) was overtransmitted and the c.190G allele (corresponding to p.64V) was undertransmitted to patients with sarcoidosis.
Ninety-three families with two or more affected siblings were available for genetic linkage analysis. These showed significant (p = 0.034) concordance of affected sib pairs for at least one parental CCR2 haplotype, as revealed by nonparametric multipoint linkage (NPL) analysis (NPL score, 1.78). There were no significant results when only chronic patients (NPL score, 0.59; p = 0.12) or only acute patients (NPL score, 0.08; p = 0.43) were considered. We could extract 75 independent informative pairs of affected siblings from the set of families (one pair randomly chosen from families with more than two affected siblings). Seventy-three of these 150 patients showed acute sarcoidosis and 77 suffered from chronic sarcoidosis. Twenty-four sib pairs were concordant for the acute sarcoidosis phenotype and 26 sib pairs for chronic sarcoidosis, whereas the remaining 25 pairs of affected siblings exhibited discordant sarcoidosis phenotypes. In 9 of 24 sib pairs concordant for acute sarcoidosis, both patients had Lfgren's syndrome. In three of these nine families, both patients had at least one copy of the c.4385T allele, and in five families, both patients were negative. In two families with Lfgren's syndrome present in one of two patients, the patients were discordant for the c.4385T allele carrier status. In both families, the patients with Lfgren's syndrome were negative for the putative Lfgren's syndrome risk allele and the patient without Lfgren's syndrome was positive.
DISCUSSION
Sarcoidosis is an inflammatory disease with a diverse clinical presentation. The disorder appears to develop gradually, through the interaction of some exogeneous trigger and the immune system of a susceptible host. In fact, the presumed initial stage of the process, bilateral hilar lymphadenopathy, appears to be relatively common, with an adult lifetime risk of 1 in 85 for women and 1 in 105 for men, as derived from a 15-year longitudinal study in Sweden (28). It is not clear which attributes of the patients influence the progression of the disease from the inflammatory reaction to the persistence of specific granulomas in a minority of affected people. However, it has been suggested by previous publications that variants in several genes, especially in the major histocompatibility gene complex, are associated with the development of either acute or chronic sarcoidosis (10eC12, 15, 29eC31).
CCR2 could be another effective gene in fine-tuning the granulomatous immune reaction. The gene product is expressed on the surface of macrophages, monocytes, dendritic cells, and T lymphocytes, and is a receptor of monocyte chemotactic protein 1 and related chemokines. It is involved in inflammatory cell recruitment, and CCR2 knockout mice are unable to initiate immune control of Mycobacterium tuberculosis infection (32). Recently, a dual role of CCR2, influencing subsequent downregulation of the inflammatory reaction, has been discussed (33).
Three previous reports from Japanese (20), Czech (21), and Dutch (22) study populations have communicated the results of CCR2 c.190G>A genotyping in the search for association with sarcoidosis. A significantly reduced allele frequency of 12.5% for the rarer allele (c.190A) was found in 100 patients from Japan as compared with a frequency of 26.2% in 122 healthy control subjects (p = 0.00072; odds ratio [OR], 0.372; 95% confidence interval [CI], 0.209eC0.666) (20). Underrepresentation of the same variant, c.190A, was observed in a study of 65 Czech patients and 80 control subjects, but this did not achieve significance (c.190A frequencies: 6.9% in cases and 11.9% in control subjects, p = 0.17) (21). In the third, more recent, publication, c.190A allele frequencies were similar ( 6%) in 137 Dutch cases (47 patients with Lfgren's syndrome and 90 patients with non-Lfgren's sarcoidosis) and 167 control subjects. When only patients with Lfgren's syndrome were considered, the c.190A allele frequency was only marginally reduced to 5% (22). The observed c.190A allele frequencies of 8% in the 938 German patients with sarcoidosis and 545 control subjects in our study (p = 0.53; OR, 1.09; CI, 0.83eC1.45) do not support the association between this allele and a reduced risk of sarcoidosis, as has been reported for Japanese and Czech patients (20, 21). Rather, our results confirm the findings from the Dutch study populations (22), although the frequency of the rarer allele is slightly higher in our cases and control subjects (8%) when compared with the Dutch samples (5eC6%).
However, Spagnolo and colleagues (22) could demonstrate, by the simultaneous analysis of seven additional polymorphisms across the CCR2 gene, a significantly increased carrier frequency of one haplotype, not bearing the c.190A allele, in Dutch patients with Lfgren's syndrome (74%) when compared with patients without Lfgren's syndrome (carrier frequency, 38%; p < 0,0001; OR, 4.80; CI, 2.2eC10.5) or when compared with control subjects (carrier frequency, 38%; p < 0,0001; OR, 4.81; CI, 2.3eC10.0). Furthermore, this major Lfgren's syndrome risk haplotype was clearly distinct from the eight other haplotypes found in cases and control subjects, bearing the alternate alleles at four of the eight polymorphic sites. Our results confirm the strong linkage disequilibrium among the CCR2 polymorphisms and the single nucleotide polymorphism allele c.4385T is identical with one of the alleles indicative of the Lfgren's syndrome risk haplotype in Dutch patients. Therefore, our results can be compared—with limitations—to the haplotype data of Spagnolo and coworkers (22).
There was a significantly increased c.4385T allele frequency in Dutch patients with Lfgren's syndrome (44%) when compared with patients with non-Lfgren's sarcoidosis (23%; p = 0.008, corrected for multiple testing) and with control subjects (21%; p = 0.0008, corrected for multiple testing) (22). In contrast, we did not observe significantly (p < 0.1) different c.4385T allele frequencies in German control subjects (23%) and cases (24%), irrespective of stratification for chronic (24%) or acute (26%) sarcoidosis or Lfgren's syndrome (27%; p = 0.21; OR, 1.21; CI, 0.90eC1.62 when compared with the healthy control sample). The same was true when genotype frequencies were considered instead of allele frequencies. Seventy-one patients of the Lfgren's syndrome sample (47%) and 214 individuals of the control group (41%) were heterozygote or homozygote carriers of c.4385T (p = 0.20; OR, 1.27; CI, 0.88eC1.82). Consequently, regarding individual CCR2 gene polymorphisms, including c.4385A>T, which tags the Lfgren's syndrome risk haplotype reported by Spagnolo and coworkers (22), we are unable to confirm significant differences between control subjects and cases with sarcoidosis or sarcoidosis subtypes in our German study populations. The sample size in the present study greatly exceeds that in the previous three studies described previously and therefore our inability to detect significant association is unlikely to be caused by lack of power. Our sample has 100% power to detect the allele OR of 2.87 reported by Spagnolo and colleagues (22) for the association between Lfgren's syndrome and c.4385A>T (at = 0.05 and a risk-factor frequency of 0.24 [present data]). Furthermore, our sample has 100% power to detect both the OR of 0.37 reported by Hizawa and coworkers (20) and the OR of 0.52 reported by Petrek and coworkers (21) for carriership of the c.190A allele in patients with sarcoidosis (acute and chronic; at = 0.05 and a risk-factor frequency of 0.17 [present data]).
One reason for conflicting results in the field of association studies could be undetected bias of the control sample. To overcome this problem, family-based methods, such as the transmission disequilibrium test, have been developed that work without a control sample (26). The transmission disequilibrium test counts the number of transmissions of parental gene variants to affected offspring. Deviation from random transmission argues for a predisposing effect of the more frequently transmitted allele. The transmission disequilibrium test can be applied to individual polymorphisms as well as to strings of adjacent polymorphisms, thus analyzing the transmission of haplotypes. In our families, the c.4385T allele segregated exclusively with c.190G (see Table 4), thus confirming strong linkage disequilibrium between both loci as found in the Dutch study populations. The c.4385T allele is included in, and tags, the Lfgren's syndrome risk haplotype communicated by Spagnolo and coworkers (22). In the informative families of our study population, the equivalent haplotype (c.190G eC c.840C eC c.4385T) is in almost perfect transmission equilibrium, being transmitted from heterozygous parents to patients on 111 of 221 occasions. The transmission of the putative risk haplotype to patients with acute sarcoidosis or Lfgren's syndrome was not significantly more frequent than expected, with transmission rates of 37 in 71 (expected 35.5, p = 0.72) and 23 in 42 (expected 21, p = 0.53), respectively.
In summary, we cannot confirm previous reports that suggest a significant impact of CCR2 alleles on the risk, or on the presentation, of sarcoidosis. The scope and the structure of our samples, with the majority of patients being suitable for family-based analyses, facilitated the use of robust statistics. However, an obvious disadvantage of a recruitment strategy that is based on calls for participation is the risk of ascertainment bias, whereby severe and unusual cases may be overrepresented. Furthermore, approximately two of three patients without a family history of sarcoidosis suffered from chronic sarcoidosis, even though this phenotype is clearly less common than this among patients with sarcoidosis in Germany. In the set of families with two or more patients, one of two patients exhibited chronic sarcoidosis. However, considering the markedly wide spectrum of sarcoidosis presentation, any hospital-based study using consecutive patients would be prone to underrepresentation of less severe cases.
In addition to ascertainment bias, stratification for sarcoidosis phenotypes could be hampered by inaccuracies. In our study, this classification is based on various sources, including reports from hospitals, doctors, and patients. Certainly there is a risk of incomplete and misleading information. On the other hand, it appears doubtful that Lfgren's syndrome represents a clear-cut segment of the sarcoidosis patient population. The course of disease and its outcome is specific to, and to some extent predictable in, the majority of patients with Lfgren's syndrome. However, a portion of these cases will develop chronic sarcoidosis, sometimes after an interval of many years (34). Twenty-five of 75 informative pairs of affected siblings in this study exhibited discordant acute or chronic sarcoidosis phenotypes. This argues against acute sarcoidosis being a distinct heritable sarcoidosis phenotype. The examination of nine families with siblings who suffered from Lfgren's syndrome did not support a role for the c.4385T allele in the etiology of Lfgren's syndrome; however, this subset was too small for a meaningful statistical analysis.
The Dutch control subjects of Spagnolo and coworkers (22) and both the German patients and control subjects in the current study exhibit similar CCR2 allele and haplotype frequencies (with c.4385T representing the Lfgren's risk haplotype of Spagnolo and coworkers). The definition of the Lfgren's syndrome phenotype is congruent in both studies. It seems unlikely that the German Lfgren's sample includes a notable proportion of patients with non-Lfgren's sarcoidosis. We cannot exclude that we have missed some patients with Lfgren's syndrome in the acute sarcoidosis sample as a result of incomplete information, but this would not explain the lack of association between our Lfgren's syndrome sample and c.4385T. Perhaps Dutch patients with Lfgren's syndrome are different from German patients and Dutch and German control subjects because of regional differences in the composition of exogenous triggers. The Dutch patients (all from one hospital) presumably originate from a more homogeneous environment than the German patients, who came from all over Germany. In addition, historical trigger variables may be important because some of the German patients suffered and recovered from Lfgren's syndrome more than 20 years ago.
Our results provide little support for the assertion that variants of the CCR2 gene itself contribute significantly to the etiology of sarcoidosis. However, the inclusion of 93 families in our study containing siblings suffering from sarcoidosis permits us to address the more general question of whether the chromosomal segment that carries the CCR2 gene sequence is linked to the risk of sarcoidosis. A positive NPL score of 1.7 (p = 0.034) indicated an excess of haplotype sharing in affected sib pairs, and was the most pronounced result from different types of analyses presented here. This positive linkage result is in concordance with data from a previous genomewide linkage analysis for sarcoidosis (23) that included 10 highly polymorphic microsatellite markers, evenly distributed over the short arm of chromosome 3. The scan was based on 63 of the 93 families of the current set and revealed a linkage peak (NPL score, 2.39; p = 0.009) at the microsatellite marker D3S1766 locus. This is located approximately 5 Mb closer to the centromere of chromosome 3 than CCR2. The lower NPL score of the present study despite a larger number of analyzed families could be explained by the lower information content of the biallelic single nucleotide polymorphisms when compared with multiallelic microsatellite markers. Genetic linkage results decrease with increasing distance between the analyzed marker and the disease gene. Therefore, our linkage result could also mean that the CCR2 polymorphisms under study are located close to, but not exactly at the site of, the real predisposing gene in this chromosomal region.
Conflicting results between CCR2 investigations in sarcoidosis could be explained by linkage disequilibrium between CCR2 gene variants and polymorphisms in a neighboring sarcoidosis susceptibility gene. Linkage disequilibrium within the CCR2 gene has been reported by Spagnolo and colleagues (22) and could be confirmed by our data. Linkage disequilibrium between polymorphic genes within this chromosomal segment has also been documented (35). Containing a number of other reasonable candidate genes, including a cluster of additional chemokine receptors, macrophage-stimulating protein, or MST1, and its receptor, and Toll-like receptor 9, the short arm of chromosome 3 remains a promising target in the search for sarcoidosis susceptibility genes.
Acknowledgments
The authors thank all patients, families, and physicians for their cooperation. The efforts of the German Sarcoidosis Patients' Organization (Deutsche Sarkoidose-Vereinigung e.V.) and the contribution of pulmonary specialist physicians are gratefully acknowledged. The authors thank Melanie Albrecht (Leeck), Tanja Wesse, and Tanja Henke (Kiel) for expert technical help. Carl Manaster (Kiel) is gratefully acknowledged for database and computer support.
REFERENCES
Hunninghake GW, Costabel U, Ando M, Baughman R, Cordier JF, du Bois R, Eklund A, Kitaichi M, Lynch J, Rizzato G, et al. Statement on sarcoidosis: joint statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS board of directors and by the ERS executive committee, February 1999. Am J Respir Crit Care Med 1999;160:736eC755.
Newman LS, Rose CS, Maier LA. Sarcoidosis. N Engl J Med 1997;336:1224eC1234.
Moller DR. Cells and cytokines involved in the pathogenesis of sarcoidosis. Sarcoidosis Vasc Diffuse Lung Dis 1999;16:24eC31.
Ziegenhagen MW, Me筶ler-Quernheim J. The cytokine network in sarcoidosis and its clinical relevance. J Intern Med 2003;253:18eC30.
Rybicki BA, Major M, Popovich J Jr, Maliarik MJ, Iannuzzi MC. Racial differences in sarcoidosis incidence: a 5-year study in a health maintenance organization. Am J Epidemiol 1997;145:234eC241.
Rybicki BA, Iannuzzi MC, Frederick MM, Thompson BW, Rossman MD, Bresnitz EA, Terrin ML, Moller DR, Barnard J, Baughman RP, et al. Familial aggregation of sarcoidosis: a case-control etiologic study of sarcoidosis (ACCESS). Am J Respir Crit Care Med 2001;164:2085eC2091.
McGrath DS, Goh N, Foley PJ, du Bois RM. Sarcoidosis: genes and microbes—soil or seed Sarcoidosis Vasc Diffuse Lung Dis 2001;18:149eC164.
Pietinalho A, Ohmichi M, Hiraga Y, Lofroos AB, Selroos O. The mode of presentation of sarcoidosis in Finland and Hokkaido, Japan: a comparative analysis of 571 Finnish and 686 Japanese patients. Sarcoidosis Vasc Diffuse Lung Dis 1996;13:159eC166.
Baughman RP, Teirstein AS, Judson MA, Rossman MD, Yeager H Jr, Bresnitz EA, DePalo L, Hunninghake G, Iannuzzi MC, Johns CJ, et al. Clinical characteristics of patients in a case control study of sarcoidosis. Am J Respir Crit Care Med 2001;164:1885eC1889.
Foley PJ, McGrath DS, Puscinska E, Petrek M, Kolek V, Drabek J, Lympany PA, Pantelidis P, Welsh KI, Zielinski J, et al. Human leukocyte antigen-DRB1 position 11 residues are a common protective marker for sarcoidosis. Am J Respir Cell Mol Biol 2001;25:272eC277.
Sato H, Grutters JC, Pantelidis P, Mizzon AN, Ahmad T, Van Houte AJ, Lammers JW, Van Den Bosch JM, Welsh KI, Du Bois RM. HLA-DQB10201: a marker for good prognosis in British and Dutch patients with sarcoidosis. Am J Respir Cell Mol Biol 2002;27:406eC412.
Grutters JC, Sato H, Pantelidis P, Lagan AL, McGrath DS, Lammers JW, van den Bosch JM, Wells AU, du Bois RM, Welsh KI. Increased frequency of the uncommon tumor necrosis factor eC857T allele in British and Dutch patients with sarcoidosis. Am J Respir Crit Care Med 2002;165:1119eC1124.
Iannuzzi MC, Maliarik MJ, Poisson LM, Rybicki BA. Sarcoidosis susceptibility and resistance HLA-DQB1 alleles in African Americans. Am J Respir Crit Care Med 2003;167:1225eC1231.
Rossman MD, Thompson B, Frederick M, Maliarik M, Iannuzzi MC, Rybicki BA, Pandey JP, Newman LS, Magira E, Beznik-Cizman B, et al. HLA-DRB11101: a significant risk factor for sarcoidosis in blacks and whites. Am J Hum Genet 2003;73:720eC735.
Grunewald J, Eklund A, Olerup O. Human leukocyte antigen class I alleles and the disease course in sarcoidosis patients. Am J Respir Crit Care Med 2004;169:696eC702.
Martinetti M, Luisetti M, Cuccia M. HLA and sarcoidosis: new pathogenetic insights. Sarcoidosis Vasc Diffuse Lung Dis 2002;19:83eC95.
Schemann M, Lympany PA, Reichel P, Me筶ler-Myhsok B, Wurm K, Schlaak M, Me筶ler-Quernheim J, du Bois RM, Schwinger E. Familial sarcoidosis is linked to the major histocompatibility complex region. Am J Respir Crit Care Med 2000;162:861eC864.
Wirnsberger RM, de Vries J, Wouters EF, Drent M. Clinical presentation of sarcoidosis in The Netherlands: an epidemiological study. Neth J Med 1998;53:53eC60.
McGrath DS, Daniil Z, Foley P, du Bois JL, Lympany PA, Cullinan P, du Bois RM. Epidemiology of familial sarcoidosis in the UK. Thorax 2000;55:751eC754.
Hizawa N, Yamaguchi E, Furuya K, Jinushi E, Ito A, Kawakami Y. The role of the C-C chemokine receptor 2 gene polymorphism V64I (CCR2eC64I) in sarcoidosis in a Japanese population. Am J Respir Crit Care Med 1999;159:2021eC2023.
Petrek M, Drabek J, Kolek V, Zlamal J, Welsh KI, Bunce M, Weigl E, Du Bois R. CC chemokine receptor gene polymorphisms in Czech patients with pulmonary sarcoidosis. Am J Respir Crit Care Med 2000;162:1000eC1003.
Spagnolo P, Renzoni EA, Wells AU, Sato H, Grutters JC, Sestini P, Abdallah A, Gramiccioni E, Ruven HJ, du Bois RM, et al. C-C chemokine receptor 2 and sarcoidosis: association with Lofgren's syndrome. Am J Respir Crit Care Med 2003;168:1162eC1166.
Schemann M, Reichel P, Me筶ler-Myhsok B, Schlaak M, Me筶ler-Quernheim J, Schwinger E. Results from a genome-wide search for predisposing genes in sarcoidosis. Am J Respir Crit Care Med 2001;164:840eC846.
Livak KJ. Allelic discrimination using fluorogenic probes and the 5' nuclease assay. Genet Anal 1999;14:143eC149.
Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, Singer MJ, Walburger DK, Lokhov SG, Gall AA, et al. 3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res 2000;28:655eC661.
Clayton D, Jones H. Transmission/disequilibrium tests for extended marker haplotypes. Am J Hum Genet 1999;65:1161eC1169.
Hill WG, Robertson A. Linkage disequilibrium in finite populations. Theor Appl Genet 1968;38:226eC231.
Hillerdal G, Nou E, Osterman K, Schmekel B. Sarcoidosis: epidemiology and prognosis. A 15-year European study. Am Rev Respir Dis 1984;130:29eC32.
Seitzer U, Swider C, Stuber F, Suchnicki K, Lange A, Richter E, Zabel P, Me筶ler-Quernheim J, Flad HD, Gerdes J. Tumour necrosis factor alpha promoter gene polymorphism in sarcoidosis. Cytokine 1997;9:787eC790.
Maliarik MJ, Rybicki BA, Malvitz E, Sheffer RG, Major M, Popovich J Jr, Iannuzzi MC. Angiotensin-converting enzyme gene polymorphism and risk of sarcoidosis. Am J Respir Crit Care Med 1998;158:1566eC1570.
Grutters JC, Sato H, Welsh KI, du Bois RM. The importance of sarcoidosis genotype to lung phenotype. Am J Respir Cell Mol Biol 2003;29:S59eCS62.
Peters W, Scott HM, Chambers HF, Flynn JL, Charo IF, Ernst JD. Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2001;98:7958eC7963.
Bruhl H, Cihak J, Schneider MA, Plachy J, Rupp T, Wenzel I, Shakarami M, Milz S, Ellwart JW, Stangassinger M, et al. Dual role of CCR2 during initiation and progression of collagen-induced arthritis: evidence for regulatory activity of CCR2+ T cells. J Immunol 2004;172:890eC898.
Mana J, Gomez-Vaquero C, Montero A, Salazar A, Marcoval J, Valverde J, Manresa F, Pujol R. Lofgren's syndrome revisited: a study of 186 patients. Am J Med 1999;107:240eC245.
Smith MW, Dean M, Carrington M, Winkler C, Huttley GA, Lomb DA, Goedert JJ, O'Brien TR, Jacobson LP, Kaslow R, et al. Contrasting genetic influence of CCR2 and CCR5 variants on HIV-1 infection and disease progression. Hemophilia Growth and Development Study (HGDS), Multicenter AIDS Cohort Study (MACS), Multicenter Hemophilia Cohort Study (MHCS), San Francisco City Cohort (SFCC), ALIVE study. Science 1997;277:959eC965.