Annegret Fischer

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

Genome-Wide Association Analysis in Sarcoidosis and Crohn's Disease Unravels a Common Susceptibility Locus on 10p12.2

BACKGROUND & AIMS Crohns disease (CD) and sarcoidosis (SA) are chronic inflammatory barrier diseases that share several clinical and immunological features, including the occurrence of granulomas. METHODS A 100k genome-wide association study with 83,360 single-nucleotide polymorphisms (SNPs) was performed on 382 CD patients, 398 SA patients, and 394 control individuals. The 24 SNPs that were most strongly associated in the combined CD/SA phenotype were selected for verification in an independent sample of 1,317 patients (660 CD and 657 SA) and 1,091 controls. RESULTS The most significant association (Bonferroni corrected P = .036) was obtained at SNP rs1398024 on chromosome 10p12.2, with an odds ratio (OR) for both diseases of 0.81 (95% confidence interval [CI], 0.69-0.96) for carriership of the rarer allele A. The P value in the overall combined sample was 4.24 x 10(-6). During further follow-up, a moderate association (OR, 0.83; 95% CI, 0.72-0.96; P = .015) was observed between rs1398024 and ulcerative colitis (1,080 patients vs 1,091 controls), the second main subphenotype of inflammatory bowel disease in addition to CD. Extensive fine mapping of the 10p12.2 locus points to yet unidentified variants in the C10ORF67 gene region as the most likely underlying risk factors. CONCLUSION Our study demonstrates that the combined analysis of different, albeit clinically related, phenotypes can lead to the identification of common susceptibility loci.

Genetics of Sarcoidosis

Sarcoidosis is a multigenic and multifactorial disease. Predisposing genes have been identified and fast progress in molecular technologies including systematic genome-wide association studies and large-scale resequencing will aid the discovery of further risk loci and variants. The exploration of the molecular epidemiology of genetic variants in the pathogenesis of sarcoidosis will allow an assessment of their prognostic usefulness. To this end, different granulomatous disorders of known and unknown etiology should be investigated jointly by genetic, immunobiological, and proteomic approaches. The definition of individual genetic risk profiles in sarcoidosis and other chronic inflammatory disorders seems achievable and a useful route for clinical translation.

A genome-wide association study reveals evidence of association with sarcoidosis at 6p12.1

Sarcoidosis is a complex systemic inflammatory disease of unknown aetiology that is influenced by a variety of genetic and environmental factors. To identify further susceptibility loci for sarcoidosis, a genome-wide association study (GWAS) was conducted in 381 patients and 392 control individuals based on Affymetrix 100k GeneChip data. The top 25 single-nucleotide polymorphisms (SNPs) were selected for validation in an independent study panel (1,582 patients versus 1,783 controls). Variant rs10484410 on chromosome 6p12.1 was significantly associated, with a Bonferroni-corrected p-value of 2.90×10−2 in the validation sample and a nominal p-value of 2.64×10−4 in the GWAS. Extensive fine mapping of the novel locus narrowed down the signal to a region comprising the genes BAG2, C6orf65, KIAA1586, ZNF451 and RAB23. Verification of the sarcoidosis-associated nonsynonymous SNP rs1040461 in a further independent case–control sample and quantitative mRNA expression studies point to the RAB23 gene as the most likely risk factor. RAB23 is proposed to be involved in antibacterial defence processes and regulation of the sonic hedgehog signalling pathway. The identified association of the 6p12.1 locus with sarcoidosis implicates this locus as a further susceptibility factor and RAB23 as a potential signalling component that may open up new perspectives in the pathophysiology of sarcoidosis.

Identification of Immune-Relevant Factors Conferring Sarcoidosis Genetic Risk

RATIONALE Genetic variation plays a significant role in the etiology of sarcoidosis. However, only a small fraction of its heritability has been explained so far. OBJECTIVES To define further genetic risk loci for sarcoidosis, we used the Immunochip for a candidate gene association study of immune-associated loci. METHODS Altogether the study population comprised over 19,000 individuals. In a two-stage design, 1,726 German sarcoidosis cases and 5,482 control subjects were genotyped for 128,705 single-nucleotide polymorphisms using the Illumina Immunochip for the screening step. The remaining 3,955 cases, 7,514 control subjects, and 684 parents of affected offspring were used for validation and replication of 44 candidate and two established risk single-nucleotide polymorphisms. MEASUREMENTS AND MAIN RESULTS Four novel susceptibility loci were identified with genome-wide significance in the European case-control populations, located on chromosomes 12q24.12 (rs653178; ATXN2/SH2B3), 5q33.3 (rs4921492; IL12B), 4q24 (rs223498; MANBA/NFKB1), and 2q33.2 (rs6748088; FAM117B). We further defined three independent association signals in the HLA region with genome-wide significance, peaking in the BTNL2 promoter region (rs5007259), at HLA-B (rs4143332/HLA-B*0801) and at HLA-DPB1 (rs9277542), and found another novel independent signal near IL23R (rs12069782) on chromosome 1p31.3. CONCLUSIONS Functional predictions and protein network analyses suggest a prominent role of the drug-targetable IL23/Th17 signaling pathway in the genetic etiology of sarcoidosis. Our findings reveal a substantial genetic overlap of sarcoidosis with diverse immune-mediated inflammatory disorders, which could be of relevance for the clinical application of modern therapeutics.

Study of Toll-like receptor gene loci in sarcoidosis

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.

Expression of putative virulence factors by clinical isolates of Klebsiella planticola

A total of 92 clinical isolates of Klebsiella planticola from man was examined with respect to the production of haemagglutinins and siderophores, serum resistance and distribution of capsular types. For comparison, a group of 207 clinical isolates of K. pneumoniae was also studied. The percentages of K. planticola strains able to express mannose-sensitive haemagglutination, indicating type 1 fimbriae (83%) and mannose-resistant and Klebsiella-like agglutination, indicating type 3 fimbriae (69%), as well as to produce the siderophores enterobactin (100%) and aerobactin (2.2%) were almost identical to those of the K. pneumoniae strains. Similarly, the proportion of serum-resistant strains (30%) was comparable to that of K. pneumoniae (25%). The capsule types most often detected in K. planticola were K14 (13%), K2 (9%) and K70 (9%). The incidence of K2, which is the predominant capsular type in K. pneumoniae, was similar in both species. These findings show that K. planticola, which is being detected with increasing frequency in clinical specimens from man, has the ability to express similar putative virulence factors to K. pneumoniae, suggesting that they may have similar pathogenicity.

Genetics of sarcoidosis.

Sarcoidosis is a multifactorial and polygenic disorder. Recently, several novel predisposing genes have been identified by genome-wide association studies, and fast progress in molecular technologies such as systematic and large-scale resequencing will aid the discovery of further risk loci and variants. In this article, the current knowledge of its genetics will be presented, including known and candidate risk variants and loci, with a focus on loci in the human leukocyte antigen region. Some of these factors are shared with other, clinically distinct diseases. This may lead to the development of new hypotheses on pathomechanisms, which associate sarcoidosis with other granulomatous disorders but also with diseases with significantly different phenotypes. In the near future system, biology approaches will help unravel the differing and common features of these disorders and allow the development of new therapeutic strategies and tools to predict the course and response to treatment of individual patients.

A Novel Sarcoidosis Risk Locus for Europeans on Chromosome 11q13.1

RATIONALE Sarcoidosis is a complex inflammatory disease with a heterogeneous clinical picture. Among others, an acute and chronic clinical course can be distinguished, for which specific genetic risk factors are known. OBJECTIVES To identify additional risk loci for sarcoidosis and its acute and chronic subforms, we analyzed imputed data from a genome-wide association scan for these phenotypes. METHODS After quality control, the genome-wide association scan comprised nearly 1.3 million imputed single-nucleotide polymorphisms based on an Affymetrix 6.0 Gene Chip dataset of 564 German sarcoidosis cases, including 176 acute and 354 chronic cases and 1,575 control subjects. MEASUREMENTS AND MAIN RESULTS We identified chromosome 11q13.1 (rs479777) as a novel locus influencing susceptibility to sarcoidosis with genome-wide significance. The marker was significantly associated in three distinct German case-control populations and in an additional German family sample with odds ratios ranging from 0.67 to 0.77. This finding was further replicated in two independent European case-control populations from the Czech Republic (odds ratio, 0.75) and from Sweden (odds ratio, 0.79). In a meta-analysis of the included European case-control samples the marker yielded a P value of 2.68 × 10(-18). The locus was previously reported to be associated with Crohn disease, psoriasis, alopecia areata, and leprosy. For sarcoidosis, fine-mapping and expression analysis suggest KCNK4, PRDX5, PCLB3, and most promising CCDC88B as candidates for the underlying risk gene in the associated region. CONCLUSIONS This study provides striking evidence for association of chromosome 11q13.1 with sarcoidosis in Europeans, and thus identified a further genetic risk locus shared by sarcoidosis, Crohn disease and psoriasis.

Association of inflammatory bowel disease risk loci with sarcoidosis, and its acute and chronic subphenotypes

Sarcoidosis is a complex granulomatous inflammatory disorder that shares several clinical and pathogenic features with inflammatory bowel disease (IBD). Postulating a common genetic basis of inflammatory diseases, we tested 106 single-nucleotide polymorphisms (SNPs) that are known or have been suggested to be associated with IBD for a potential association with sarcoidosis and its acute and chronic subphenotypes. We genotyped 1,996 German sarcoidosis patients, comprising 648 acutely and 1,161 chronically affected individuals, 2,622 control subjects, and 342 German trios with affected offspring using SNPlex™ technology. The nonsynonymous SNP rs11209026 (Arg381Gln) in the interleukin (IL)-23 receptor (IL23R) gene was associated with chronic sarcoidosis (OR 0.63; p = 5.58×10−5), which was supported by the result of a transmission disequilibrium test analysis in the independent family sample (OR 0.50; p = 0.031). Marker rs12035082 located at chromosome 1q24.3 was found to be associated with the acute subphenotype (OR 1.36; p = 6.80×10−7) and rs916977 (HERC2 locus; OR 1.30; p = 4.49×10−5) was associated with sarcoidosis. Our results highlight the potential importance of the IL-23 signalling pathway for the development of chronic sarcoidosis. The finding links sarcoidosis pathogenesis to other inflammatory conditions and may contribute to new hypotheses on disease mechanisms.