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- Subjects and methods
Sarcoidosis is a multi-factorial systemic disease of granulomatous inflammation. Current concepts of the aetiology include interactions of unknown environmental triggers with an inherited susceptibility. Toll-like receptors (TLRs) are main components of innate immunity and therefore TLR genes are candidate susceptibility genes in sarcoidosis. Ten members of the human TLR gene family have been identified and mapped to seven chromosomal segments. The aim of this study was to investigate all known TLR gene loci for genetic linkage with sarcoidosis and to follow positive signals with different methods. We analysed linkage of TLR gene loci to sarcoidosis by use of closely flanking microsatellite markers in 83 families with 180 affected siblings. We found significant linkage between sarcoidosis and markers of the TLR4 gene locus on chromosome 9q (non-parametric linkage score 2·63, P = 0·0043). No linkage was found for the remaining TLR gene loci. We subsequently genotyped 1203 sarcoidosis patients from 997 families, 1084 relatives and 537 control subjects for four single nucleotide polymorphisms of TLR4, including Asp299Gly and Thr399Ile. This genotype data set was studied by case–control comparisons and transmission disequilibrium tests, but showed no significant results. In summary, TLR4 − w ith significant genetic linkage results − appears to be the most promising member of the TLR gene family for further investigation in sarcoidosis. However, our results do not confirm the TLR4 polymorphisms Asp299Gly and Thr399Ile as susceptibility markers. Our results rather point to another as yet unidentified variant within or close to TLR4 that might confer susceptibility to sarcoidosis.
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- Subjects and methods
Sarcoidosis is a multi-organ inflammatory disease with exaggerated cellular immune activity that leads to formation of non-caseating granulomas in affected organs. The aetiology of sarcoidosis is unknown and presumably complex. The lung and adjacent lymph nodes appear to be the first and primary side of immune reaction in the majority of patients [1,2]. This has led to the hypothesis that an inhaled ‘sarcoidosis antigen’ triggers the process, and a wide variety of organic and inorganic agents have been suspected to cause sarcoidosis. Major candidates are Gram-negative microbes such as mycobacteria and propriobacteria [3,4], but as different substances such as photocopier dust or crystalline silica have also been discussed [5,6]. An inherited susceptibility is assumed to contribute to the risk of sarcoidosis. This risk varies with ethnicity and it is increased greatly in close relatives of patients .
Hypothetical candidate susceptibility genes in sarcoidosis presumably play a role in the concerted molecular and cellular interactions of antigen recognition and immune response. Among the first molecules potentially involved in this process are the Toll-like receptors (TLRs), a family of related transmembrane molecules that are expressed on the surface of a variety of antigen-detecting cells. Different TLRs are activated specifically by different pathogen-associated molecular patterns, such as TLR-4 by cell membrane lipopolysaccharides (LPS) of Gram-negative bacteria, TLR-5 by flagillin and TLR-9 by unmethylated cytosine–phosphate–guanosine (CpG)-rich DNA . The downstream signalling pathway includes activation of nuclear factor (NF)-kappa B that has been demonstrated in the pathogenesis of sarcoidosis . Two of the TLR genes, TLR4 and TLR9, reside at chromosomal positions that have shown linkage peaks in a previous whole genome scan for predisposing gene loci . Taken together, the TLR gene family appears to be a promising target in the search for sarcoidosis susceptibility genes. TLR2 and TLR4 gene variations have been studied in the search for susceptibility to sarcoidosis and showed conflicting results [11–14].
The human TLR genes, TLR1–TLR10, have been mapped to seven different positions on human chromosomes. TLR1, TLR6 and TLR10 are clustered at one location on the short arm of chromosome 4, and TLR7 and TLR8 on the X chromosome (http://www.ncbi.nlm.nih.gov/Omim). We have studied these seven chromosomal loci and their flanking regions with highly polymorphic DNA markers, so-called microsatellites, for linkage with sarcoidosis in a panel of 83 families with two or more affected siblings. Basically, above-average sharing of microsatellite alleles in the set of affected siblings was an indicator of linkage between the chromosomal locus under study and the risk of sarcoidosis. This approach allows for effective scanning of candidate susceptibility gene regions prior to fine-mapping efforts and the search for disease-associated gene mutations.
Subjects and methods
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- Subjects and methods
The study population consisted of two sets of participants: (i) 83 families with two or more siblings with sarcoidosis investigated for the linkage study (‘sib-pair families’) and (ii) an extended collection of 1203 sarcoidosis patients from 997 families, 1084 relatives and 537 controls for the population- and family-based association experiments. In the linkage study sample, 63 of the sib-pair families have been studied previously with a set of more widely spaced markers in a whole genome linkage experiment . Parents of the sib-pair families were included whenever possible. In families with only one or no parents available, unaffected siblings were included to facilitate parental genotype reconstruction. Altogether, the sib-pair families comprised 302 individuals, including 180 affected siblings, 104 females and 76 males. The average age at diagnosis was 36·7 years [standard deviation (s.d.) 10·9 years] in these patients.
Most of the patients and families were recruited by calls for participation forwarded by the German patients' organization Deutsche Sarkoidose-Vereinigung or by specialized hospitals. All patients were interviewed by telephone or questionnaire, and their doctors were contacted to confirm the diagnosis and to provide records. The diagnosis was confirmed by biopsy in 120 of 180 patients from the sib-pair families, and the clinical course of the disease in combination with radiology and laboratory findings was consistent with the diagnosis of sarcoidosis in the remaining patients.
Seventy of 180 patients from the sib-pair families had acute sarcoidosis, with sudden complaints and recovery within 2 years. Eighty-three patients suffered from chronic sarcoidosis, mainly with a stealthy beginning, and with enduring disease activity for 2 years or longer. Ten patients had acute sarcoidosis in the beginning but experienced a relapse more than 2 years later. The remaining 17 patients showed other phenotypes, e.g. sole cutaneous sarcoidosis, or were detected incidentally by radiography for other reasons and had no specific complaints.
The sib-pair families were genotyped for 28 highly polymorphic microsatellite markers that flanked the chromosomal loci of the TLR genes. Markers were chosen from either a commercially available genome-wide linkage marker set (Research Genetics Inc., Huntsville, AL, USA) or from the public National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/mapview). Details of the TLR loci and marker positions are listed in Table 1. Marker and TLR gene positions are given in Megabase (Mb) on the chromosomal sequence. Recombination fractions used in linkage analysis were derived from genetic linkage map positions (in centiMorgan, cM) whenever available. If no genetic map position was available or if two markers had the same genetic map position, recombination fractions were derived from the distance on the DNA sequence. Nine of the 28 markers (D1S549; D3S2409; D4S408; D4S1625; D4S1627; D4S1629; D4S2632; D9S934; DXS9902) have been studied in 63 families of the present sib-pair families sample on the occasion of a previous whole genome linkage analysis . Microsatellites of the previous whole genome set have been included into the present study to estimate the effect contributed by the additional sib-pair families. Genomic DNA of the participants was prepared and genotypes were determined as described previously . In brief, microsatellite alleles were amplified through a polymerase chain reaction (PCR) amplification technique with M13 labelled primers, and fluorescent PCR products were separated according to size on an automated sequence analysis apparatus .
Table 1. Characteristics and non-parametric linkage (NPL) analysis results of 28 microsatellite markers that flank seven Toll-like receptor (TLR) gene loci.
|TLR gene||Marker/locus||Cytogenetic position||Sequence position (Mb)||Number of observed alleles||NPL score||P-value|
|TLR1, 6, 10||D4S2632*|| ||35·4||14||0·67||0·25|
|TLR1, 6, 10||4p14||38·5|| || || |
|TLR2||4q32||154·8|| || || |
|TLR3||4q35||187·2|| || || |
|TLR4||9q33||119·5|| || || |
|TLR5||1q41||221·4|| || || |
|TLR7, 8||DXS9895|| ||7·4||6||−0·94||0·83|
|TLR7, 8||Xp22||12·8|| || || |
|TLR9||3p21||52·2|| || || |
As an internal control, segregation of microsatellite and Single nucleotide polymorphism (SNP) alleles were checked for Mendelian inheritance by use of Pedcheck . genehunterversion 2·0  was used to calculate non-parametric linkage (NPL) probabilities and to perform transmission disequilibrium tests (TDTs) in the sib-pair families.
The sib-pair families are included in a much larger sarcoidosis study population that has been compiled for high throughput genotyping procedures and family-based statistical procedures. The entire collection comprises 1203 patients and 1084 relatives from 997 families. The complete composition of this population has been described previously  and is shown in detail in Table 2. All patients completed a questionnaire on the course of disease, and patients from families with more than one patient were also interviewed by telephone. Patients' physicians were contacted to confirm the diagnosis and to provide records. The average age at diagnosis was 36·4 years (s.d. 10·9 years) and ranged from 12 to 80 years. Sarcoidosis was confirmed by biopsy in 1001 of 1203 (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.
Table 2. Details of the study population of sarcoidosis patients (acute sarcoidosis: sudden complaints and recovery within 2 years; Löfgren's syndrome: acute sarcoidosis with bilateral hilar lymphadenopathy, erythema nodosum and arthropathy; chronic sarcoidosis: stealthy beginning, enduring disease activity for 2 years or longer).
|Family structure||Number of families||Number of patients||Male patients||Female patients||Löfgren's syndrome||Acute sarcoidosis*||Chronic sarcoidosis||Other form of sarcoidosis|
|Single patient, without parents||309||309||108||201||36||83||205||21|
|Single patient, with one parent||124||124||60||64||10||28||91||5|
|Single patient, with both parents||385||385||159||226||55||104||257||24|
|Two patients, siblings||75||150||63||87||28||62||68||20|
|Two patients, parent and offspring||57||114||47||67||18||46||54||14|
|Two patients, 2nd- or 3rd-degree relatives||24||48||28||20||6||17||25||6|
|More than two patients||23||73||38||35||12||30||39||4|
Further genotyping of the entire collection of DNA was performed at the only locus with significant linkage results, TLR4. One intragenic SNP c.-4036A > G (rs2737191) located upstream of the TLR4 transcription initiation site, and one intronic SNP c.94-837G > A (rs1927911) were genotyped according to the protocol of the supplier of the assays (Assays-on-Demand from Applied Biosystems; http://www.appliedbiosystems.com).
Two downstream coding polymorphisms Asp299Gly (rs4986790) and Thr399Ile (rs4986791) [19,20] were genotyped by use of the TaqMan™ technique as described previously . Primer and probes of the Asp299Gly assay were: forward primer 5′-TGACCATTGAAGAATTCCGATTAGCA; reverse primer 5′-ACACTCACCAGGGAAAATGAAGAA; VIC probe 5′-TACCTCGATGATATTATT; FAM probe 5′-CCTCGATGGTATTATT. The Thr399Ile assay contained the following: forward primer 5′-ACCAAGCCTTGAGTTTCTAGATCTCA; reverse primer 5′-GGTAATAACACCATTGAAGCTCAGATC; VIC probe 5′-TGATTTTGGGACA ATCA; FAM probe 5′-TGATTTTGGGACAACCA. Genotypes of SNPs were checked for conformance with Hardy–Weinberg equilibrium. Allele and genotype frequencies in cases and controls were compared using Pearson's χ2 test with 1 and 2 degrees of freedom respectively. In addition, TDTs for single markers and four-marker haplotypes were performed in sarcoidosis families and trios (single patients with parents) by using genehunter version 2·1 .
For case–control comparisons of SNP allele and genotype frequencies, genotype information was extracted from the entire collection. One patient per family carrying the phenotype under scrutiny, i.e. Löfgren's syndrome (characterized by acute sarcoidosis with bilateral hilar lymphadenopathy, erythema nodosum and arthropathy), acute sarcoidosis including Löfgren's syndrome, chronic sarcoidosis, and either acute or chronic sarcoidosis, was selected randomly from the sample of sarcoidosis families. This resulted in groups of 152, 323, 680 and 938 unrelated patients with Löfgren's syndrome, acute sarcoidosis (including Löfgren's syndrome) chronic sarcoidosis, and either acute or chronic sarcoidosis respectively. Statistical power of the samples was calculated by use of the program ps (Power and Sample Size Calculations) version 2·1·30 .
The control group comprised 537 healthy blood donors recruited through the Department of Transfusion Medicine at Kiel University Hospital, Kiel Germany. The controls consisted of 263 (49%) males and 274 (51%) females with an average age at inclusion of 40·9 years (s.d. 12·8 years). All patients, parents and controls were German by residence. All participants gave written informed consent for participation in the study. Study protocols were approved in writing by the institutional ethics and data protection authorities.
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Toll-like receptors constitute a family of evolutionarily conserved transmembrane proteins that function as front-line pathogen recognition receptors of the innate immune defence system. They act as homodimers or heterodimers in the recognition of specific molecular determinants that represent characteristic components of infectious pathogens. These ligands include bacterial cell-surface LPS and lipoproteins, bacterial flagellin and fungal hyphae, double-stranded RNA of viruses and the unmethylated CpG islands of bacterial and viral DNA . Some of these agents have been considered to be possibly involved in the aetiology of sarcoidosis, and a wide variety of other agents have also been accused [3–7]. Because of the wide spectrum of ligands of the different TLRs we decided to check whether any member of the TLR gene family is linked to the risk of sarcoidosis. Such a finding could help to focus on a distinct class of pathogens in the search for the cause of sarcoidosis.
So far, TLR4 and more recently TLR2 gene variations have been studied in sarcoidosis. An association between two intragenic non-synonymous TLR4 point mutations, Asp299Gly and Thr399Ile and the risk to develop chronic sarcoidosis has been found in a study of 141 German patients and 141 controls . However, this finding could not be replicated in two case–control association studies of 156 Dutch and 100 Greek patients [12,13]. It has been discussed that an accidentally low frequency of the presumed risk allele in the German control group contributed to the conflicting results . A SNP at position -16934 of the promoter region of TLR2 showed significant association with chronic sarcoidosis in patients from the Netherlands. However, this result could not be confirmed in a validation cohort of the same study .
We have applied a two-stage strategy including three types of raw data interpretation − NPL analysis, TDT and case–control association study − to test whether the TLR genes contribute to the increased inherited risk of sarcoidosis. NPL analysis and TDT do not require an independent matched control group, and do therefore not raise the issue of population stratification. Genetic linkage analysis of affected relatives is a most straightforward procedure to check extended segments of chromosomes for susceptibility gene candidates. TDT can be applied on the same genotype data set to test if a specific allele or haplotype within the shared chromosomal region is transmitted preferentially from parents to affected offspring, thus proving an association between the genetic marker and the risk of disease without the need of a control group.
The risk of sarcoidosis is increased in first-degree relatives of patients. According to different reports from European populations the sibling recurrence risk is approximately one in 100, or increased 20-fold compared with the prevalence in the general population . Pedigrees with more than two affected members, as often seen in monogenic diseases with Mendelian inheritance, are rare in sarcoidosis, and complex multi-case families are unique exceptions . These observations indicate that the effect of any single sarcoidosis susceptibility gene is presumably small, and that extended study groups are needed to demonstrate such genes, especially if the risk allele frequency is low as in the case of TLR4 Asp299Gly. We have analysed extended groups of cases and controls for four TLR4 polymorphisms − including two more common polymorphisms that ensured better coverage of the TLR4 gene region and had a minor allele frequency of 0·25 or greater − to overcome the problem of insufficient power to detect association. Haplotypes were used only if they could be derived from family genotype information.
Microsatellite analysis showed significant results only at the TLR4 gene locus both in NPL analysis and TDT. The inclusion of microsatellite marker D9S934 from the whole genome marker set  into the present study made comparison possible of both linkage analyses. While the NPL score at D9S934 in the previous study of 63 families was 1·82 (P = 0·034), it increased to 2·41 (P = 0·008) with the inclusion of 20 additional sib-pair families. This increase of significance with additional families underlines the relevance of this locus in the search for sarcoidosis susceptibility genes. In the only other genome-wide linkage scan in sarcoidosis, based on African American families, a peak on the long arm of chromosome 9 was one of the two coinciding peaks of both whole genome studies . This peak was labelled in the African American data set mainly by the microsatellite D9S1825. However, as this marker is located approximately 7·4 Mb telomeric to TLR4 and D9S1864, no further conclusions can be drawn without closer and overlapping linkage information from the African American families sample.
The TLR4 gene was chosen for subsequent SNP analysis in a high throughput genotyping setting, but revealed no significant result in the following case–control comparisons. This outcome supported the assumption derived from Dutch and Greek studies that no association exists between TLR4 Asp299Gly and the risk to develop sarcoidosis or to proceed to chronic sarcoidosis. The minor allele frequency of TLR4 Asp299Gly in our German control group (0·056) is close to the frequency in the control group from the Netherlands (0·063) . It differs grossly from the frequency found in the German control group communicated by Pabst et al. (0·028) . This appears to support the assumption that an accidentally low allele frequency in the control group of Pabst et al. contributed to significant differences between the chronic sarcoidosis group and controls in their study. Recently, a study about the worldwide distribution of TLR4 Asp299Gly and Thr399Ile has been published that included 632 and 209 healthy individuals from Germany and the Netherlands, respectively . The minor Asp299Gly allele frequency was 0·057 in the German group and 0·072 in the Dutch group. One of the largest study groups to analyse the Asp299Gly polymorphism has been recruited at five different places in western Germany . It included 3000 individuals and has been designed as a prospective community-based study that represents the local German population. In this large sample was the minor Asp299Gly allele frequency 0·04 and thus closer to the frequency in the control group of Pabst et al.
In summary, we are left with conflicting results: linkage studies point to the TLR-4 locus on chromosome 9, but case–control association studies largely exclude the functional polymorphism Asp299Gly and three others from having a meaning in sarcoidosis. However, our TDT results of TLR4 SNP haplotypes showed that the only haplotype with the presumed risk allele of Asp299Gly was transferred more often than expected by chance from heterozygous parents to offspring who developed sarcoidosis. As a consequence of the low frequency of the TLR4 Asp299Gly polymorphism, only 69 parents of the study population were heterozygotes and contributed information to the test. On 42 of 69 occasions the presumed risk allele was transferred, on 27 occasions the other allele was transferred (P = 0·07). In the absence of evidence that TLR4 Asp299Gly is associated directly with the risk of sarcoidosis, the distorted transmission could be explained by an extended haplotype that carries the real risk allele and is labelled by TLR4 Gly299. This hypothetical susceptibility allele could be located outside the coding sequence of TLR4, possibly affecting regulatory elements of the gene. Interestingly, a comparable finding was reported by Oostenbrug et al. , who searched for association between TLR4 polymorphisms and inflammatory bowel disease (IBD) after conflicting reports on association between Asp299Gly and the risk of IBD have been published. IBD and sarcoidosis have in common a complex multi-organ inflammation and the formation of granulomas. Oostenbrug et al. genotyped 781 IBD cases and 315 controls from the Netherlands for Asp299Gly and Thr399Ile and for four flanking microsatellite markers, including D9S1864. No association was found between both SNPs and the risk of IBD, but significant microsatellite haplotype sharing was observed in the patients' group. This effect was due mainly to the contribution of D9S1864 to the risk haplotypes, which otherwise carried the common alleles of Asp299Gly and Thr399Ile. The authors proposed that IBD susceptibility is associated with other variants of TLR4. Given the similarity of our results, this proposed susceptibility could be linked to a common inflammatory feature of IBD and sarcoidosis.
Alternatively, another gene in the vicinity of TLR4 could be responsible for the NPL peak and positive TDT result of our study. Genes from this chromosomal region on the long arm of chromosome 9 have been studied for association with sarcoidosis before. In fact, one of the first genetic studies in sarcoidosis focused on the ABO blood groups and found increased risk of sarcoidosis associated with blood group A in 518 sarcoidosis patients . However, the ABO gene locus resides approximately 15 Mb towards the telomere of chromosome 9q and is therefore most probably too far away to account for the linkage peak at the TLR4 locus (all positions and distances according to NCBI Map Viewer, build 36·2). By contrast, the ORM1 gene that codes for the acute phase protein, orosomucoid, is located closer to the linkage peak region of this study, at a distance of approximately 3·4 Mb. Association of the allele ORM*1 with sarcoidosis has been communicated from a study of Swedish patients . At least two other reasonable functional candidate genes are located close to peak at the TLR4 gene locus, TNFSF15 (3·5 Mb from TLR4) and tumour necrosis factor receptor-associated factor 1 (TRAF1) (3·2 Mb from TLR4). TNFSF15 is a member of the tumour necrosis factor super-family. Haplotypes of this gene have shown a highly significant association with Crohn's disease in Japanese and European study populations . TRAF1 is involved in the regulation of NF-kappa B activity, which plays a crucial role in sarcoidosis [9,30].
From the study of all seven TLR gene loci presented here, we conclude that the TLR4 gene and its chromosomal vicinity appear to be a promising target region for further efforts in the search for sarcoidosis susceptibility genes.