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A Genome-Wide Linkage Analysis in 181 German Sarcoidosis Families Using Clustered Biallelic Markers
CHEST
Original Research SARCOIDOSIS
A Genome-Wide Linkage Analysis in 181 German Sarcoidosis Families Using Clustered Biallelic Markers Annegret Fischer, PhD; Michael Nothnagel, PhD; Manfred Schürmann, MD; Joachim Müller-Quernheim, MD; Stefan Schreiber, MD; and Sylvia Hofmann, PhD
Background: Sarcoidosis (SA) is a systemic granulomatous inflammatory disorder with complex etiology and strong clustering in families. Genome-wide association studies have been successful in the identification of common risk variants for the disease. To reveal susceptibility variants with low frequencies but strong effects, we performed a genome-wide linkage scan in a large sample of SA families. Methods: We genotyped 528 members of 181 German SA families for 3,882 single nucleotide polymorphism assays from the SNPlex System Human Linkage Mapping Set 4K. Results: Nonparametric linkage analysis revealed one region of suggestive linkage on chromosome 12p13.31 at 20 cM (logarithm of odds [LOD] 5 2.53; local P value 5 .0003) and another linkage peak of nearly suggestive linkage on 9q33.1 at 134 cM (LOD 5 2.12; local P value 5 .0009). The latter has been reported to show suggestive evidence for linkage in a sample of 229 African American SA families previously. Analysis of acute and chronically affected families revealed a subphenotype-specific linkage pattern and an additional, nearly suggestive linkage peak on chromosome 16p13.11 at 38 cM (LOD 5 2.09; local P value 5 .001), which was confined to acute SA. Conclusion: Our results propose that the respective regions might harbor yet-unidentified, possibly subphenotype-specific risk factors for the disease (eg, with immune-related functions like the tumor necrosis factor receptor 1). They should be proved to be important for SA pathogenesis and investigated in detail with an emphasis on rare variants. Subphenotype-specific risk factors might serve for prognosis of the clinical course of the disease. CHEST 2010; 138(1):151–157 Abbreviations: ANXA11 5 annexin A11; BTNL2 5 butyrophilin-like protein 2; GWAS 5 genome-wide association study; GWLS 5 genome-wide linkage study; HLA 5 human leukocyte antigen; LOD 5 logarithm of odds; NPL 5 nonparametric linkage; SA 5 sarcoidosis; SNP 5 single nucleotide polymorphism; STR 5 short tandem repeat, microsatellite marker; TNFR1 5 tumor necrosis factor receptor-1
(SA) (Mendelian Inheritance in Man Sarcoidosis 181000 http://www.ncbi.nlm.nih.gov/omim) is an
inflammatory disease of unknown cause, affecting multiple organs. It is characterized by noncaseating granulomas primarily in the lung and the lymphatic system. Acute and chronic SA can be distinguished as the two main subphenotypes based on the course of the disease. A genetic component to disease predisposition is suggested by numerous reports on the familial occurrence of SA, a recent twin study,1 and several known genetic risk factors, but environmental factors are also believed to play a role in this complex disease.2-5 The first genome-wide linkage study (GWLS) was conducted by Schürmann et al6 with 63 German SA
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families using 225 microsatellite markers (short tandem repeats [STRs]), where the major peak was found on chromosome 6p12 to 6p22, including the human leukocyte antigen (HLA) region. Minor peaks were identified on chromosomes 1p22, 3p21, and 9q33. A GWLS in African American families agreed with the former study in finding linkage to chromosomes 1p11 and 9q34, whereas the most prominent peak was identified on chromosome 5q11.7 Known genetic risk factors for SA are, among others, located in the HLA region (reviewed in Reference 4) including the butyrophilin-like protein 2 (BTNL2),8,9 and the recently identified annexin A11 (ANXA11)10 and C10ORF67 loci,11 which were both discovered by CHEST / 138 / 1 / JULY, 2010
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genome-wide association studies (GWAS). Besides these disease loci, numerous association studies of potential candidate regions suggested additional susceptibility genes, such as the chemokine (C-C motif) receptor 2 and 5 of 3p21.3, the tumor necrosis factor-a, and several other HLA loci (for review see reference 12). However, many of these show conflicting results or await replication. The aim of this study was to identify novel candidate loci for SA susceptibility by conducting an allelesharing-based GWLS. In contrast to a GWAS that has high power to detect common variants with modest effect sizes, a linkage study aims to identify rare variants with a high impact on the phenotype,13 and can, therefore, complement the GWAS approach at this end of the allelic spectrum. The study comprised 181 German SA families, by far extending the study sample of Schürmann and coauthors,6 and made use of a specially designed marker set of approximately 4,000 SNPs. This way, we were able to identify two linkage peaks on chromosomes 9q33.1 and 12p13.31. Materials and Methods Study Sample For this study 528 members of 181 German SA families with two or more affected relatives (Table 1) were contacted actively through the German Sarcoidosis Patients’ Organization, specialized hospitals and practitioners, and by calls for participation in the study that were published via health insurance institutions. All patients included in this study were classified according to disease presentation on the basis of all available information as described before.8 Briefly, acute SA is characterized by sudden complaints and recovery within 2 years and includes Löfgren syndrome, which is characterized by erythema nodosum, bilateral hilar Manuscript received October 23, 2009; revision accepted January 27, 2010. Affiliations: From the Institute of Clinical Molecular Biology (Drs Fischer and Hofmann), the Institute of Medical Informatics and Statistics (Dr Nothnagel), and the Institute of Clinical Molecular Biology and Department of General Internal Medicine, University Hospital Schleswig-Holstein (Dr Schreiber), Christian-Albrechts University, Kiel; the Institute of Human Genetics (Dr Schürmann), University of Lübeck, Lübeck; and the Department of Pneumology (Dr Müller-Quernheim), University of Freiburg, Freiburg, Germany. Funding/Support: The study was supported by the German Ministry of Education and Research (BMBF) through the National Genome Research Network [NGFN 01GS0809]. It received infrastructure support through the DFG excellence cluster “Inflammation at Interfaces.” Correspondence to: Stefan Schreiber, MD, Institute of Clinical Molecular Biology and Department of General Internal Medicine, University Hospital Schleswig-Holstein, Christian-Albrechts University, Schittenhelmstrasse 12, 24105, Kiel, Germany; e-mail: [email protected] © 2010 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/ site/misc/reprints.xhtml). DOI: 10.1378/chest.09-2526
Table 1—Composition of the 181 German Families Included in the Genome-Wide Linkage Experiment Relationship
No. of Families
ASP, no parents available ASP with 2 parents available ASP with 1 parent available First cousin 3 Affected siblings, no parents available 3 Affected siblings with 1 parent available 3 Affected siblings with 2 parents available 4 Affected siblings, no parents available 4 Affected siblings with one parent available Avuncular Other (2-6 affected relatives, complex relatedness) Sum
57 36 31 18 5 2 1 1 1 19 10 181
ASP 5 affected sibling pair.
lymphadenopathy, and polyarthritis.2 Patients who exhibited subtly intensifying early symptoms, followed by enduring disease activity for 2 years or longer, were classified as chronically affected. The remaining patients showed other phenotypes (eg, solely cutaneous SA) or were detected by radiography for other reasons and had no specific complaints. The linkage family set comprised 398 patients. Of these, 151 individuals (38%) had acute SA, including 43 patients with Löfgren syndrome, whereas 196 patients (49%) were reported to suffer from chronic SA. In 40 families (22%) the patients exclusively experienced acute SA; nine of these families had only patients with Löfgren syndrome. In 55 families (30%) patients suffered from chronic disease concordantly. In the remaining 86 families (discordant families, 48%) more than one phenotype occurred. Details about the composition of the three phenotypic subsamples are given in e-Figure 1. Sixty-two of the 181 families were used in a previous GWLS with STR markers.6 No further information on the phenotype (eg, on distinct organ involvement) was available. Informed written consent was obtained from all study participants and the collection protocols were approved by the institutional ethics committees and data protection authorities. Genotyping and Statistical Analysis Samples were genotyped using the SNPlex System Human Linkage Mapping Set 4K (Applied Biosystems; Foster City, CA) with SNPlex technology as described before.14 Genotyping was successful for 3,561 SNPs, and all linkage analyses were performed using MERLIN analysis software (http://www.sph. umich.edu/csg/abecasis/Merlin).15 Details on the marker set, the applied quality criteria, and the linkage analysis are given in the e-Appendix 1.
Results Genome-Wide Linkage Scan We conducted a GWLS in 181 German SA families with 3,882 biallelic markers using the MERLIN analysis software.15 The two most prominent peaks were identified on chromosomes 12p13.31 at 20 cM (LOD 5 2.53; local P value 5 .0003) and 9q33.1 at 134 cM (LOD 5 2.12; local P value 5 .0009; see Figure 1 for complete results). The local P values
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Figure 1. Results of the genome-wide linkage scan in 181 German sarcoidosis families (black line) and in phenotypic subsets of 40 acute (gray line), 55 chronic (light gray line) families (x-axis: genetic distance in cM; y-axis: LOD score). LOD 5 logarithm of odds.
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were nominally significant, and the LOD score for chromosome 12p13.31 was above the threshold for suggestive linkage,16 but simulation-based correction for multiple testing using 1,000 replications showed that both regions did not reach genome-wide significance (P 5 .17 for chromosome 12p13.31). However, suggestive linkage of the chromosome 9q33-34 region had been found in a previous GWLS including 229 African American SA families.7 The STR marker D9S1825 that marks the linkage peak in this GWLS is located about 8.6 Mb centromeric from the linkage peak in our overall German sample (chromosomal position approximately 118.3 Mb). To investigate whether these major signals were exclusively due to one of the two main subphenotypes, namely acute and chronic SA, we separately analyzed those families that were concordantly affected with acute SA (40 families) or with chronic SA (55 families). The 12p13.31 region showed a comparatively strong signal for acute (LOD 5 1.96; local P value 5 .0013), but not for chronic SA. In contrast, chromosome 9q33.1 showed suggestive linkage with chronic SA (LOD 5 2.23 at 132 cM; local P value 5 .0007), whereas no peak for acute SA was detected. Furthermore, a linkage signal specific to acute SA was detected on chromosome 16p13.11 at 38 cM (LOD 5 2.09; local P value 5 .001). (See Fig 1 for complete results.) In addition, we analyzed the Löfgren subset comprising nine families. No linkage was detected in this very small sample (LOD scores: 20.64 to 1.29; data not shown). The results of our GWLS showed nearly suggestive evidence only for a part of the known susceptibility loci for SA. The gene region of ANXA11 on chromosome 10q22, most recently identified as a risk factor for SA,10 was not found to be linked, with an LOD score of only 0.07 obtained from the complete sample of 181 families. The same was true for chromosome 6p21, which showed only very weak evidence for linkage (LOD 5 0.8, P 5 .03), despite the fact that it harbors HLA-DRB1, the locus that is most consistently associated with SA, and the risk gene BTNL2. On the other hand, the region containing C10ORF67 (chromosome 10p12.2), a recently identified risk locus for both SA and Crohn’s disease,11 showed a weak linkage signal to SA with an LOD score of 1.84 (P 5 .002). Comparison of the Performance of SNP and STR Markers in a Subsample of 62 Families Sixty-two of the 181 German SA families included in this study were genotyped for 225 STRs in a previous linkage experiment.6 To evaluate the performance of the clustered SNPs from the SNPlex System Human Linkage Mapping Set 4K in linkage analysis
in contrast to STRs, the results for these 62 families obtained with the two distinct marker types were directly compared. For this purpose, the STR data were reanalyzed using MERLIN.15 Comparison of these results with those obtained from the clustered SNPs confirmed each other in parts, but also showed some divergence in the number of potentially linked loci and the respective nonparametric linkage (NPL) scores (e-Figure 2). Both methods identified the most prominent peak with an NPL score of 2.99 (STR) and 2.86 (SNP) on chromosome 6p22 at approximately 50 cM, which includes the HLA region as reported by Schürmann et al6 previously.
Discussion This study reports on a GWLS in an extended sample of 181 German SA families using clustered biallelic markers. In addition, the analysis was performed in two subsets of families concordantly comprising patients with acute or chronic SA, thereby aiming at the reduction of linkage heterogeneity. Genetic associations in complex diseases obtained in one population can often not be confirmed in other populations. Ethnicity also plays a role in SA epidemiology. For example, African Americans are more commonly affected with the disease and suffer greater morbidity than Americans of European descent.17 Despite these differences, there are genetic loci that confer risk to SA independently of ethnicity, such as the most consistently replicated HLA-DRB1 locus18 or BTNL2.9 Interestingly, the comparison of our results to a previous GWLS in 229 African American families7 revealed one common region of (nearly) suggestive linkage on chromosome 9q33-34 among all major and minor linkage peaks. Additionally, linkage analyses in families with concordant-acute and concordant-chronic patients suggested that this peak might be driven by the chronic subsample. We believe that these results point to an underlying, yet unidentified, risk factor for SA located on chromosome 9q33-34, which plays a role in both African American and German families with a chronic and therefore more severe course of the disease. This is consistent with the observation that SA tends to be of greater severity in patients of African ancestry compared with those of European ancestry.19 We assume that certain risk factors, which play a role in a reasonable number of the investigated families, are located in the two regions of (nearly) suggestive linkage on chromosomes 9 and 12. The result of this GWLS can therefore serve to generate hypotheses on potentially new candidate loci that could be investigated in further family-based as well as casecontrol association studies. Candidates for potential
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risk factors on chromosome 9q33 could be located in the TNF receptor-associated factor 1/complement component 5 region, which is associated with multiple autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus.20,21 The suggestive linkage region on chromosome 12p13.31 harbors a number of genes with potential functional relevance in SA etiology, such as the complement component 3a receptor 1 and tumor necrosis factor receptor 1 (TNFR1) genes, both of which are involved in immune response (reviewed in References 22 and 23). TNFR1, also known as TNFRSF1A, is a promising candidate, as mutations in this locus have recently been shown to be associated with Crohn’s disease,24 which shares clinical and pathophysiologic characteristics with SA (eg, granuloma formation25) and is also genetically related to SA.11 Because there is growing evidence for a common genetic basis of distinct but related complex diseases, the identification of SA susceptibility loci could also improve the understanding of related granulomatous disorders, such as Wegener granulomatosis or Crohn’s disease. The differences observed between acute and chronically affected families support the hypothesis of a subphenotype-specific genetic basis for the different clinical courses of SA as suggested by previous publications.26-28 Therefore, future genetic studies should investigate acute and chronic SA separately to improve statistical power by using a precise, narrowly defined phenotype. As examples for potential candidate genes for acute SA, the chromosome 16p13 region harbors the proapoptotic Cell Death Involved p53-target, which could be relevant for granuloma formation by influencing apoptotic processes,29 and the MHC II transactivator that is associated with a number of complex inflammatory diseases30,31 and controls the transcription of HLA class 2 genes.32 Such potentially subphenotype-specific genetic risk factors may contribute to a model of SA progression and could possibly serve as future prognostic markers for the clinical course of disease. SA is a complex disease with a genetic component, likely comprising numerous factors. Statistical gene mapping either rests on linkage analysis, which is thought to be most suitable for rare genetic variants with large effects, or on indirect association analysis, which is more suitable for common variants with either large or small effects.13 Therefore, genetic risk factors that are found to be associated with a complex disease are often difficult to detect by linkage analysis. For example, we observed no linkage to ANXA11, the most recently identified risk factor for SA on chromosome 10q22.10 Moreover, no linkage was detected to chromosome 6p21, which harbors HLADRB1 and BTNL2 that are most consistently associated with SA. This finding is concordant with the www.chestpubs.org
results of the above-mentioned GWLS in African American SA families, in which no evidence for linkage to the HLA region was found.7 Furthermore, if multiple unlinked variants independently cause the disease phenotype (ie, under linkage heterogeneity), each rare genetic variant can be expected to be private to a limited number of families. In our study, the linkage analysis results obtained from the 62 German families of the previous linkage scan6 differed markedly from those resulting from the extended sample of 181 families. Although this difference could be simply due to chance, linkage heterogeneity in these two samples of similar phenotypic composition is also a likely explanation, in which case it would point to the underlying genetic complexity of SA. The GWLS in 62 German SA families using 3,882 clustered biallelic markers revealed mostly similar results to the corresponding data using 225 STRs derived from a previous study.6 As both approaches investigated the same families with the same individuals genotyped, the amount of extractable genetic information solely depended on marker density.33 The STR marker set exhibited a relatively low density with distances from 6 to 29 cM (average of 19.6 cM), whereas the clustered SNPs were located in clusters with an average spacing of 2 cM between clusters. Thus, in genomic regions with high STR density (eg, chromosome 6p22.3 to 6p21.1 and chromosome 16p12.3 to 16q23.1) nearly equal NPL scores were observed. In contrast, in regions where the spacing of STRs was sparse (eg, on chromosome 2q35) additional peaks were detected most probably because of the much better coverage by the clustered SNPs. This comparison confirms previous findings that STR sets may have less information content than SNP sets because of their sparse spatial density.34-36 In summary, in the linkage study presented here we identified two linkage peaks on chromosomes 12p13.31 and 9q33.1 in a sample of SA families of European ancestry, whereas the latter has previously been reported to be linked to the disease in African American families. Analysis of families that were concordant for acute and for chronic SA, respectively, revealed a subphenotype-specific linkage pattern and an additional linkage peak on chromosome 16p13.11, which was restricted to acute SA. Our results provide a working hypothesis that the regions of suggestive linkage might harbor yet unidentified, subphenotype-specific, probably rare risk variants for the disease (eg, variants affecting the TNFR1 gene locus on chromosome 12p13.31). Our findings might encourage further detailed investigation of the respective regions, and unlike the classic case-control design, with an emphasis on the detection and characterization of rare variants. CHEST / 138 / 1 / JULY, 2010
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Acknowledgments Author contributions: Dr Fischer: contributed to generating and analyzing the data, drafting the manuscript, and approving the final version of the manuscript. Dr Nothnagel: contributed to the statistical analyses and the interpretation of the results, helping write the manuscript, and approving the final version of the manuscript. Dr Schürmann: contributed to recruiting the SA families and patients, being involved in the conception of the study, and approving the final version of the manuscript. Dr Müller-Quernheim: contributed to recruiting the SA families and patients, being involved in the conception of the study, and approving the final version of the manuscript. Dr Schreiber: contributed to initiating and conceptualizing the study, providing infrastructure and general advice, and approving the final version of the manuscript. Dr Hofmann: contributed to steering the project, making substantial contributions to the interpretation of the data, revising the manuscript critically, and approving its final version. Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Other contributions: We thank all patients, families, and physicians for their cooperation. The support of the German Sarcoidosis Patients Association (Deutsche Sarkoidose-Vereinigung e.V.) is gratefully acknowledged. We thank Applied Biosystems for providing the SNPlex System Human Linkage Mapping Set 4K. We thank Catharina von der Lancken, Birthe Fedders, Tanja Wesse, Tanja Henke, Michael Wittig, and Rainer Vogler at the Institute for Clinical Molecular Biology (Kiel, Germany) for expert technical help. Experiments were performed at the Institute for Clinical Molecular Biology, Christian-Albrechts University, Kiel, Germany. Additional information: The e-Appendix and e-Figures can be found in the Online Supplement at http://chestjournal.chestpubs. org/content/138/1/151/suppl/DC1
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10. Hofmann S, Franke A, Fischer A, et al. Genome-wide association study identifies ANXA11 as a new susceptibility locus for sarcoidosis. Nat Genet. 2008;40(9):1103-1106. 11. Franke A, Fischer A, Nothnagel M, et al. Genome-wide association analysis in sarcoidosis and Crohn’s disease unravels a common susceptibility locus on 10p12.2. Gastroenterology. 2008;135(4):1207-1215. 12. Iannuzzi MC. Genetics of sarcoidosis. Semin Respir Crit Care Med. 2007;28(1):15-21. 13. Risch N, Merikangas K. The future of genetic studies of complex human diseases. Science. 1996;273(5281):1516-1517. 14. Hampe J, Franke A, Rosenstiel P, et al. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet. 2007;39(2):207-211. 15. Abecasis GR, Wigginton JE. Handling marker-marker linkage disequilibrium: pedigree analysis with clustered markers. Am J Hum Genet. 2005;77(5):754-767. 16. Lander E, Kruglyak L. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet. 1995;11(3):241-247. 17. Rybicki BA, Major M, Popovich JJ Jr, Maliarik MJ, Iannuzzi MC. Racial differences in sarcoidosis incidence: a 5-year study in a health maintenance organization. Am J Epidemiol. 1997;145(3):234-241. 18. Rossman MD, Thompson B, Frederick M, et al; ACCESS Group. HLA-DRB1*1101: a significant risk factor for sarcoidosis in blacks and whites. Am J Hum Genet. 2003;73(4):720-735. 19. Baughman RP, Teirstein AS, Judson MA, et al; Case Control Etiologic Study of Sarcoidosis (ACCESS) research group. Clinical characteristics of patients in a case control study of sarcoidosis. Am J Respir Crit Care Med. 2001;164(10 pt 1): 1885-1889. 20. Plenge RM, Seielstad M, Padyukov L, et al. TRAF1-C5 as a risk locus for rheumatoid arthritis—a genomewide study. N Engl J Med. 2007;357(12):1199-1209. 21. Kurreeman FA, Goulielmos GN, Alizadeh BZ, et al. The TRAF1-C5 region on chromosome 9q33 is associated with multiple autoimmune diseases. Ann Rheum Dis. 2010;69:696-699. 22. Gasque P. Complement: a unique innate immune sensor for danger signals. Mol Immunol. 2004;41(11):1089-1098. 23. Guicciardi ME, Gores GJ. Life and death by death receptors. FASEB J. 2009;23(6):1625-1637. 24. Waschke KA, Villani A-C, Vermeire S, et al. Tumor necrosis factor receptor gene polymorphisms in Crohn’s disease: association with clinical phenotypes. Am J Gastroenterol. 2005;100(5):1126-1133. 25. Zumla A, James DG. Granulomatous infections: etiology and classification. Clin Infect Dis. 1996;23(1):146-158. 26. Pabst S, Karpushova A, Dìaz-Lacava A, et al. VEGF gene haplotypes are associated with sarcoidosis. Chest. 2010;137(1):156-163. 27. Grunewald J, Eklund A. Löfgren’s syndrome: human leukocyte antigen strongly influences the disease course. Am J Respir Crit Care Med. 2009;179(4):307-312. 28. Fischer A, Valentonyte R, Nebel A, et al. Female-specific association of C-C chemokine receptor 5 gene polymorphisms with Löfgren’s syndrome. J Mol Med. 2008;86(5):553-561. 29. Brown L, Ongusaha PP, Kim H-G, et al. CDIP, a novel proapoptotic gene, regulates TNFalpha-mediated apoptosis in a p53-dependent manner. EMBO J. 2007;26(14):3410-3422. 30. Swanberg M, Lidman O, Padyukov L, et al. MHC2TA is associated with differential MHC molecule expression and susceptibility to rheumatoid arthritis, multiple sclerosis and myocardial infarction. Nat Genet. 2005;37(5):486-494. 31. Martinez A, Perdigones N, Cenit M, et al. Chromosomal region 16p13: further evidence of increased predisposition to immune diseases. Ann Rheum Dis. 2010;69(1):309-311.
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35. John S, Shephard N, Liu G, et al. Whole-genome scan, in a complex disease, using 11,245 single-nucleotide polymorphisms: comparison with microsatellites. Am J Hum Genet. 2004;75(1):54-64. 36. Middleton FA, Pato MT, Gentile KL, et al. Genomewide linkage analysis of bipolar disorder by use of a high-density single-nucleotide-polymorphism (SNP) genotyping assay: a comparison with microsatellite marker assays and finding of significant linkage to chromosome 6q22. Am J Hum Genet. 2004;74(5):886-897.
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Original Research SARCOIDOSIS
A Genome-Wide Linkage Analysis in 181 German Sarcoidosis Families Using Clustered Biallelic Markers Annegret Fischer, PhD; Michael Nothnagel, PhD; Manfred Schürmann, MD; Joachim Müller-Quernheim, MD; Stefan Schreiber, MD; and Sylvia Hofmann, PhD
Background: Sarcoidosis (SA) is a systemic granulomatous inflammatory disorder with complex etiology and strong clustering in families. Genome-wide association studies have been successful in the identification of common risk variants for the disease. To reveal susceptibility variants with low frequencies but strong effects, we performed a genome-wide linkage scan in a large sample of SA families. Methods: We genotyped 528 members of 181 German SA families for 3,882 single nucleotide polymorphism assays from the SNPlex System Human Linkage Mapping Set 4K. Results: Nonparametric linkage analysis revealed one region of suggestive linkage on chromosome 12p13.31 at 20 cM (logarithm of odds [LOD] 5 2.53; local P value 5 .0003) and another linkage peak of nearly suggestive linkage on 9q33.1 at 134 cM (LOD 5 2.12; local P value 5 .0009). The latter has been reported to show suggestive evidence for linkage in a sample of 229 African American SA families previously. Analysis of acute and chronically affected families revealed a subphenotype-specific linkage pattern and an additional, nearly suggestive linkage peak on chromosome 16p13.11 at 38 cM (LOD 5 2.09; local P value 5 .001), which was confined to acute SA. Conclusion: Our results propose that the respective regions might harbor yet-unidentified, possibly subphenotype-specific risk factors for the disease (eg, with immune-related functions like the tumor necrosis factor receptor 1). They should be proved to be important for SA pathogenesis and investigated in detail with an emphasis on rare variants. Subphenotype-specific risk factors might serve for prognosis of the clinical course of the disease. CHEST 2010; 138(1):151–157 Abbreviations: ANXA11 5 annexin A11; BTNL2 5 butyrophilin-like protein 2; GWAS 5 genome-wide association study; GWLS 5 genome-wide linkage study; HLA 5 human leukocyte antigen; LOD 5 logarithm of odds; NPL 5 nonparametric linkage; SA 5 sarcoidosis; SNP 5 single nucleotide polymorphism; STR 5 short tandem repeat, microsatellite marker; TNFR1 5 tumor necrosis factor receptor-1
(SA) (Mendelian Inheritance in Man Sarcoidosis 181000 http://www.ncbi.nlm.nih.gov/omim) is an
inflammatory disease of unknown cause, affecting multiple organs. It is characterized by noncaseating granulomas primarily in the lung and the lymphatic system. Acute and chronic SA can be distinguished as the two main subphenotypes based on the course of the disease. A genetic component to disease predisposition is suggested by numerous reports on the familial occurrence of SA, a recent twin study,1 and several known genetic risk factors, but environmental factors are also believed to play a role in this complex disease.2-5 The first genome-wide linkage study (GWLS) was conducted by Schürmann et al6 with 63 German SA
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families using 225 microsatellite markers (short tandem repeats [STRs]), where the major peak was found on chromosome 6p12 to 6p22, including the human leukocyte antigen (HLA) region. Minor peaks were identified on chromosomes 1p22, 3p21, and 9q33. A GWLS in African American families agreed with the former study in finding linkage to chromosomes 1p11 and 9q34, whereas the most prominent peak was identified on chromosome 5q11.7 Known genetic risk factors for SA are, among others, located in the HLA region (reviewed in Reference 4) including the butyrophilin-like protein 2 (BTNL2),8,9 and the recently identified annexin A11 (ANXA11)10 and C10ORF67 loci,11 which were both discovered by CHEST / 138 / 1 / JULY, 2010
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genome-wide association studies (GWAS). Besides these disease loci, numerous association studies of potential candidate regions suggested additional susceptibility genes, such as the chemokine (C-C motif) receptor 2 and 5 of 3p21.3, the tumor necrosis factor-a, and several other HLA loci (for review see reference 12). However, many of these show conflicting results or await replication. The aim of this study was to identify novel candidate loci for SA susceptibility by conducting an allelesharing-based GWLS. In contrast to a GWAS that has high power to detect common variants with modest effect sizes, a linkage study aims to identify rare variants with a high impact on the phenotype,13 and can, therefore, complement the GWAS approach at this end of the allelic spectrum. The study comprised 181 German SA families, by far extending the study sample of Schürmann and coauthors,6 and made use of a specially designed marker set of approximately 4,000 SNPs. This way, we were able to identify two linkage peaks on chromosomes 9q33.1 and 12p13.31. Materials and Methods Study Sample For this study 528 members of 181 German SA families with two or more affected relatives (Table 1) were contacted actively through the German Sarcoidosis Patients’ Organization, specialized hospitals and practitioners, and by calls for participation in the study that were published via health insurance institutions. All patients included in this study were classified according to disease presentation on the basis of all available information as described before.8 Briefly, acute SA is characterized by sudden complaints and recovery within 2 years and includes Löfgren syndrome, which is characterized by erythema nodosum, bilateral hilar Manuscript received October 23, 2009; revision accepted January 27, 2010. Affiliations: From the Institute of Clinical Molecular Biology (Drs Fischer and Hofmann), the Institute of Medical Informatics and Statistics (Dr Nothnagel), and the Institute of Clinical Molecular Biology and Department of General Internal Medicine, University Hospital Schleswig-Holstein (Dr Schreiber), Christian-Albrechts University, Kiel; the Institute of Human Genetics (Dr Schürmann), University of Lübeck, Lübeck; and the Department of Pneumology (Dr Müller-Quernheim), University of Freiburg, Freiburg, Germany. Funding/Support: The study was supported by the German Ministry of Education and Research (BMBF) through the National Genome Research Network [NGFN 01GS0809]. It received infrastructure support through the DFG excellence cluster “Inflammation at Interfaces.” Correspondence to: Stefan Schreiber, MD, Institute of Clinical Molecular Biology and Department of General Internal Medicine, University Hospital Schleswig-Holstein, Christian-Albrechts University, Schittenhelmstrasse 12, 24105, Kiel, Germany; e-mail: [email protected] © 2010 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/ site/misc/reprints.xhtml). DOI: 10.1378/chest.09-2526
Table 1—Composition of the 181 German Families Included in the Genome-Wide Linkage Experiment Relationship
No. of Families
ASP, no parents available ASP with 2 parents available ASP with 1 parent available First cousin 3 Affected siblings, no parents available 3 Affected siblings with 1 parent available 3 Affected siblings with 2 parents available 4 Affected siblings, no parents available 4 Affected siblings with one parent available Avuncular Other (2-6 affected relatives, complex relatedness) Sum
57 36 31 18 5 2 1 1 1 19 10 181
ASP 5 affected sibling pair.
lymphadenopathy, and polyarthritis.2 Patients who exhibited subtly intensifying early symptoms, followed by enduring disease activity for 2 years or longer, were classified as chronically affected. The remaining patients showed other phenotypes (eg, solely cutaneous SA) or were detected by radiography for other reasons and had no specific complaints. The linkage family set comprised 398 patients. Of these, 151 individuals (38%) had acute SA, including 43 patients with Löfgren syndrome, whereas 196 patients (49%) were reported to suffer from chronic SA. In 40 families (22%) the patients exclusively experienced acute SA; nine of these families had only patients with Löfgren syndrome. In 55 families (30%) patients suffered from chronic disease concordantly. In the remaining 86 families (discordant families, 48%) more than one phenotype occurred. Details about the composition of the three phenotypic subsamples are given in e-Figure 1. Sixty-two of the 181 families were used in a previous GWLS with STR markers.6 No further information on the phenotype (eg, on distinct organ involvement) was available. Informed written consent was obtained from all study participants and the collection protocols were approved by the institutional ethics committees and data protection authorities. Genotyping and Statistical Analysis Samples were genotyped using the SNPlex System Human Linkage Mapping Set 4K (Applied Biosystems; Foster City, CA) with SNPlex technology as described before.14 Genotyping was successful for 3,561 SNPs, and all linkage analyses were performed using MERLIN analysis software (http://www.sph. umich.edu/csg/abecasis/Merlin).15 Details on the marker set, the applied quality criteria, and the linkage analysis are given in the e-Appendix 1.
Results Genome-Wide Linkage Scan We conducted a GWLS in 181 German SA families with 3,882 biallelic markers using the MERLIN analysis software.15 The two most prominent peaks were identified on chromosomes 12p13.31 at 20 cM (LOD 5 2.53; local P value 5 .0003) and 9q33.1 at 134 cM (LOD 5 2.12; local P value 5 .0009; see Figure 1 for complete results). The local P values
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Figure 1. Results of the genome-wide linkage scan in 181 German sarcoidosis families (black line) and in phenotypic subsets of 40 acute (gray line), 55 chronic (light gray line) families (x-axis: genetic distance in cM; y-axis: LOD score). LOD 5 logarithm of odds.
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were nominally significant, and the LOD score for chromosome 12p13.31 was above the threshold for suggestive linkage,16 but simulation-based correction for multiple testing using 1,000 replications showed that both regions did not reach genome-wide significance (P 5 .17 for chromosome 12p13.31). However, suggestive linkage of the chromosome 9q33-34 region had been found in a previous GWLS including 229 African American SA families.7 The STR marker D9S1825 that marks the linkage peak in this GWLS is located about 8.6 Mb centromeric from the linkage peak in our overall German sample (chromosomal position approximately 118.3 Mb). To investigate whether these major signals were exclusively due to one of the two main subphenotypes, namely acute and chronic SA, we separately analyzed those families that were concordantly affected with acute SA (40 families) or with chronic SA (55 families). The 12p13.31 region showed a comparatively strong signal for acute (LOD 5 1.96; local P value 5 .0013), but not for chronic SA. In contrast, chromosome 9q33.1 showed suggestive linkage with chronic SA (LOD 5 2.23 at 132 cM; local P value 5 .0007), whereas no peak for acute SA was detected. Furthermore, a linkage signal specific to acute SA was detected on chromosome 16p13.11 at 38 cM (LOD 5 2.09; local P value 5 .001). (See Fig 1 for complete results.) In addition, we analyzed the Löfgren subset comprising nine families. No linkage was detected in this very small sample (LOD scores: 20.64 to 1.29; data not shown). The results of our GWLS showed nearly suggestive evidence only for a part of the known susceptibility loci for SA. The gene region of ANXA11 on chromosome 10q22, most recently identified as a risk factor for SA,10 was not found to be linked, with an LOD score of only 0.07 obtained from the complete sample of 181 families. The same was true for chromosome 6p21, which showed only very weak evidence for linkage (LOD 5 0.8, P 5 .03), despite the fact that it harbors HLA-DRB1, the locus that is most consistently associated with SA, and the risk gene BTNL2. On the other hand, the region containing C10ORF67 (chromosome 10p12.2), a recently identified risk locus for both SA and Crohn’s disease,11 showed a weak linkage signal to SA with an LOD score of 1.84 (P 5 .002). Comparison of the Performance of SNP and STR Markers in a Subsample of 62 Families Sixty-two of the 181 German SA families included in this study were genotyped for 225 STRs in a previous linkage experiment.6 To evaluate the performance of the clustered SNPs from the SNPlex System Human Linkage Mapping Set 4K in linkage analysis
in contrast to STRs, the results for these 62 families obtained with the two distinct marker types were directly compared. For this purpose, the STR data were reanalyzed using MERLIN.15 Comparison of these results with those obtained from the clustered SNPs confirmed each other in parts, but also showed some divergence in the number of potentially linked loci and the respective nonparametric linkage (NPL) scores (e-Figure 2). Both methods identified the most prominent peak with an NPL score of 2.99 (STR) and 2.86 (SNP) on chromosome 6p22 at approximately 50 cM, which includes the HLA region as reported by Schürmann et al6 previously.
Discussion This study reports on a GWLS in an extended sample of 181 German SA families using clustered biallelic markers. In addition, the analysis was performed in two subsets of families concordantly comprising patients with acute or chronic SA, thereby aiming at the reduction of linkage heterogeneity. Genetic associations in complex diseases obtained in one population can often not be confirmed in other populations. Ethnicity also plays a role in SA epidemiology. For example, African Americans are more commonly affected with the disease and suffer greater morbidity than Americans of European descent.17 Despite these differences, there are genetic loci that confer risk to SA independently of ethnicity, such as the most consistently replicated HLA-DRB1 locus18 or BTNL2.9 Interestingly, the comparison of our results to a previous GWLS in 229 African American families7 revealed one common region of (nearly) suggestive linkage on chromosome 9q33-34 among all major and minor linkage peaks. Additionally, linkage analyses in families with concordant-acute and concordant-chronic patients suggested that this peak might be driven by the chronic subsample. We believe that these results point to an underlying, yet unidentified, risk factor for SA located on chromosome 9q33-34, which plays a role in both African American and German families with a chronic and therefore more severe course of the disease. This is consistent with the observation that SA tends to be of greater severity in patients of African ancestry compared with those of European ancestry.19 We assume that certain risk factors, which play a role in a reasonable number of the investigated families, are located in the two regions of (nearly) suggestive linkage on chromosomes 9 and 12. The result of this GWLS can therefore serve to generate hypotheses on potentially new candidate loci that could be investigated in further family-based as well as casecontrol association studies. Candidates for potential
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risk factors on chromosome 9q33 could be located in the TNF receptor-associated factor 1/complement component 5 region, which is associated with multiple autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus.20,21 The suggestive linkage region on chromosome 12p13.31 harbors a number of genes with potential functional relevance in SA etiology, such as the complement component 3a receptor 1 and tumor necrosis factor receptor 1 (TNFR1) genes, both of which are involved in immune response (reviewed in References 22 and 23). TNFR1, also known as TNFRSF1A, is a promising candidate, as mutations in this locus have recently been shown to be associated with Crohn’s disease,24 which shares clinical and pathophysiologic characteristics with SA (eg, granuloma formation25) and is also genetically related to SA.11 Because there is growing evidence for a common genetic basis of distinct but related complex diseases, the identification of SA susceptibility loci could also improve the understanding of related granulomatous disorders, such as Wegener granulomatosis or Crohn’s disease. The differences observed between acute and chronically affected families support the hypothesis of a subphenotype-specific genetic basis for the different clinical courses of SA as suggested by previous publications.26-28 Therefore, future genetic studies should investigate acute and chronic SA separately to improve statistical power by using a precise, narrowly defined phenotype. As examples for potential candidate genes for acute SA, the chromosome 16p13 region harbors the proapoptotic Cell Death Involved p53-target, which could be relevant for granuloma formation by influencing apoptotic processes,29 and the MHC II transactivator that is associated with a number of complex inflammatory diseases30,31 and controls the transcription of HLA class 2 genes.32 Such potentially subphenotype-specific genetic risk factors may contribute to a model of SA progression and could possibly serve as future prognostic markers for the clinical course of disease. SA is a complex disease with a genetic component, likely comprising numerous factors. Statistical gene mapping either rests on linkage analysis, which is thought to be most suitable for rare genetic variants with large effects, or on indirect association analysis, which is more suitable for common variants with either large or small effects.13 Therefore, genetic risk factors that are found to be associated with a complex disease are often difficult to detect by linkage analysis. For example, we observed no linkage to ANXA11, the most recently identified risk factor for SA on chromosome 10q22.10 Moreover, no linkage was detected to chromosome 6p21, which harbors HLADRB1 and BTNL2 that are most consistently associated with SA. This finding is concordant with the www.chestpubs.org
results of the above-mentioned GWLS in African American SA families, in which no evidence for linkage to the HLA region was found.7 Furthermore, if multiple unlinked variants independently cause the disease phenotype (ie, under linkage heterogeneity), each rare genetic variant can be expected to be private to a limited number of families. In our study, the linkage analysis results obtained from the 62 German families of the previous linkage scan6 differed markedly from those resulting from the extended sample of 181 families. Although this difference could be simply due to chance, linkage heterogeneity in these two samples of similar phenotypic composition is also a likely explanation, in which case it would point to the underlying genetic complexity of SA. The GWLS in 62 German SA families using 3,882 clustered biallelic markers revealed mostly similar results to the corresponding data using 225 STRs derived from a previous study.6 As both approaches investigated the same families with the same individuals genotyped, the amount of extractable genetic information solely depended on marker density.33 The STR marker set exhibited a relatively low density with distances from 6 to 29 cM (average of 19.6 cM), whereas the clustered SNPs were located in clusters with an average spacing of 2 cM between clusters. Thus, in genomic regions with high STR density (eg, chromosome 6p22.3 to 6p21.1 and chromosome 16p12.3 to 16q23.1) nearly equal NPL scores were observed. In contrast, in regions where the spacing of STRs was sparse (eg, on chromosome 2q35) additional peaks were detected most probably because of the much better coverage by the clustered SNPs. This comparison confirms previous findings that STR sets may have less information content than SNP sets because of their sparse spatial density.34-36 In summary, in the linkage study presented here we identified two linkage peaks on chromosomes 12p13.31 and 9q33.1 in a sample of SA families of European ancestry, whereas the latter has previously been reported to be linked to the disease in African American families. Analysis of families that were concordant for acute and for chronic SA, respectively, revealed a subphenotype-specific linkage pattern and an additional linkage peak on chromosome 16p13.11, which was restricted to acute SA. Our results provide a working hypothesis that the regions of suggestive linkage might harbor yet unidentified, subphenotype-specific, probably rare risk variants for the disease (eg, variants affecting the TNFR1 gene locus on chromosome 12p13.31). Our findings might encourage further detailed investigation of the respective regions, and unlike the classic case-control design, with an emphasis on the detection and characterization of rare variants. CHEST / 138 / 1 / JULY, 2010
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Acknowledgments Author contributions: Dr Fischer: contributed to generating and analyzing the data, drafting the manuscript, and approving the final version of the manuscript. Dr Nothnagel: contributed to the statistical analyses and the interpretation of the results, helping write the manuscript, and approving the final version of the manuscript. Dr Schürmann: contributed to recruiting the SA families and patients, being involved in the conception of the study, and approving the final version of the manuscript. Dr Müller-Quernheim: contributed to recruiting the SA families and patients, being involved in the conception of the study, and approving the final version of the manuscript. Dr Schreiber: contributed to initiating and conceptualizing the study, providing infrastructure and general advice, and approving the final version of the manuscript. Dr Hofmann: contributed to steering the project, making substantial contributions to the interpretation of the data, revising the manuscript critically, and approving its final version. Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Other contributions: We thank all patients, families, and physicians for their cooperation. The support of the German Sarcoidosis Patients Association (Deutsche Sarkoidose-Vereinigung e.V.) is gratefully acknowledged. We thank Applied Biosystems for providing the SNPlex System Human Linkage Mapping Set 4K. We thank Catharina von der Lancken, Birthe Fedders, Tanja Wesse, Tanja Henke, Michael Wittig, and Rainer Vogler at the Institute for Clinical Molecular Biology (Kiel, Germany) for expert technical help. Experiments were performed at the Institute for Clinical Molecular Biology, Christian-Albrechts University, Kiel, Germany. Additional information: The e-Appendix and e-Figures can be found in the Online Supplement at http://chestjournal.chestpubs. org/content/138/1/151/suppl/DC1
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