Peter Jagiello

The Wegener's granulomatosis quantitative trait locus on chromosome 6p21.3 as characterised by tagSNP genotyping.

Background: A genomic region on chromosome 6p21.3, including HLA-DPB1, has been linked to Wegener’s granulomatosis (WG). The basis of this association is difficult to evaluate because of the complex haplotype block architecture of this region. Objective: To identify the causative molecular genetic variation(s) using a detailed HapMap based fine-mapping approach. Methods: 282 patients with WG and 380 healthy controls were genotyped for HLA-DPB1 as well as for 35 informative single nucleotide polymorphisms (SNPs) within the respective region. 25 of these SNPs have been selected as tagging SNPs for another 219 associated SNPs. Allele and genotype frequencies were analysed separately by contingency tables and logistic regression. Finally, the coding region of RING1 was directly sequenced in subjects who carried haplotypes that were correlated with contrasting WG risks. Results: The previously reported strong association of WG with the HLA-DPB1*0401 allele was confirmed in an independent WG sample (n = 108, pc = 6.4×10−8). When the complete cohort (n = 282) was considered, the association remained highly significant in ANCA-positive (pc = 1.26×10−22), but not in ANCA-negative patients. An SNP 3′ of HLA-DPB1 yielded the smallest p value and was associated with WG partly independently from the HLA-DPB1 alleles. Another informative SNP in the vicinity of RING1 showed significant WG association that was also partly independent of HLA-DPB1. RING1 sequencing, however, did not show any variation potentially predisposing to WG. Conclusions: The HLA-DPB1/RING1 region is strongly associated with WG in ANCA-positive subjects. Further analyses of potential cis regulatory sequences of candidate genes HLA-DPB1, RING1 and RXRB appear warranted.

ASK1 and MAP2K6 as modifiers of age at onset in Huntington’s disease

Huntington’s disease (HD) is an autosomal dominantly inherited neurodegenerative disease associated with abnormal expansions of a stretch of perfect CAG repeats in the HD gene. The number of repeat units is predictive for the age at onset (AO) of neurological symptoms. Part of the remaining variation in AO is attributed to modifier genes. In this study, genes involved in apoptosis were investigated as candidates for modulating AO in HD. A panel of 304 candidate genes was screened for allelic associations with motor AO via linked micro-satellite markers by pooling the DNAs of HD individuals from opposite ends of the AO distribution. After genotyping promising markers from the pooling experiment individually, markers revealed consolidated evidence for association in a candidate region comprising the genes MAP3K5 (ASK1)/PEX7 at 6q23.3 and in the gene MAP2K6 at 17q24.3. Fine-mapping of these candidate regions in a cohort of 250 Caucasian HD patients using single nucleotide polymorphism (SNP) markers delimitated the precise locations of association. Certain variations in an ASK1–PEX7 haplotype block explain 2.6% of additional variance in AO in our HD cohort. In males, 4.9% additional variance could be attributed to MAP2K6 genotype variations. Altogether, ASK1–PEX7 haplotypes and MAP2K2 genotype variations explain 6.3% additional variance in AO for HD. We hypothesise that sequence variations of ASK1 and MAP2K6 lead to partially sex-specific changes in the levels and/or phosphorylation states of p38 and p38-regulated proteins that might contribute to the observed delaying effects in the AO of HD.

Promoter polymorphism rs3087456 in the MHC class II transactivator gene is not associated with susceptibility for selected autoimmune diseases in German patient groups

We analysed whether the single nucleotide polymorphism (SNP) rs3087456 in the promoter of the MHC class II transactivator (MHC2TA) gene is associated with manifestation of rheumatoid arthritis, multiple sclerosis, narcolepsy and Wegener granulomatosis. The recently reported association in a northern population of the MHC2TA variation with these autoimmune diseases is not evident in the German population.

Screening for candidate gene regions in narcolepsy using a microsatellite based approach and pooled DNA

Narcolepsy is a complex sleep disorder characterized by excessive daytime sleepiness and cataplexy. Mutations in genes of the hypocretin (orexin) neurotransmitter system cause narcoleptic symptoms in animal models. The absence of hypocretin in the cerebrospinal fluid of human patients is hypothesized to originate from destruction of hypocretinergic cells in the hypothalamus, the cause of which remains unknown. Due to strong HLA association autoimmune models of narcolepsy pathogenesis are still mostly favored. Genetic predisposition factors other than HLA are likely to play a role in causing the disorder. We screened three sets of gene regions (n=254) for association with narcolepsy using a microsatellite based approach and pooled DNA: genes related to immunity, particularly apoptosis; genes related to regulation of circadian rhythmicity; genes coding for several factors of neurotransmission. In relation to apoptosis an association was found for the BAG1 gene region. Interestingly, microsatellites representing four genomic regions related to neurotransmission revealed association with narcolepsy: COMT, DRD2, GABBR1, and HTR2A. These results, although exploratory and still to be confirmed in independent samples, support a complex pathogenetic model for narcolepsy, including disturbances of neurotransmission rather than involvement of autoimmunity.

A case-control study of tyrosine phosphatase (PTPN22) confirms the lack of association with Crohn's disease.

In Crohns disease (CD), the whole gastrointestinal tract can be affected by discontinuous and transmural inflammation. The terminal ileum and colon are especially prone to inflammation that comprises granulomata and later intestinal and perianal fistulas. Genome‐wide linkage and epidemiological studies established genetic predisposition factors to CD.

Association study of Wegener granulomatosis and the functionally relevant A645G polymorphism in the bactericidal/permeability increasing protein (BPI) gene.

In antineutrophil cytoplasmatic antibody (ANCA)‐associated vasculitides (AAV), bactericidal/permeability increasing protein (BPI) ANCAs are detected. Recent observations suggest that BPI‐ANCAs can potentially contribute to a proinflammatory setting in the absence of proteinase 3 (PRTN3) ANCAs during the development of a pulmonary relapse by impeding the elimination of Gram‐negative bacteria (GNB). However, it is as yet not clear whether the genetic background contributes to the generation of BPI‐ANCAs in Wegener granulomatosis (WG) or if BPI polymorphisms are associated with WG. In this study we genotyped the functionally relevant single nucleotide polymorphism (SNP) A645 (Glu216Lys) of the BPI gene in 201 WG patients and 608 healthy controls. To investigate whether further SNPs might be associated with WG, we also examined an intragenic microsatellite marker. No significant differences were found between patients and controls. Thus BPI polymorphisms do not appear to contribute to genetic predisposition to WG. Moreover, our data do not suggest a genetic background for the generation of BPI‐ANCAs in WG.

Polymorphisms of the tumor necrosis factor receptors: no association with narcolepsy in German patients

Narcolepsy is a debilitating sleep disorder characterized by daytime sleepiness and cataplexy. The strong association of narcolepsy with the HLA system suggests an autoimmune cause. Tumor necrosis factor is a cytokine involved in both regulation of immune mechanisms and sleep. Several studies were undertaken to determine a contribution of tumor necrosis factor and its receptors to the pathogenesis of narcolepsy. A significant increase in the 196R allele, a functionally relevant polymorphism in the TNFR2 gene, has been found in Japanese patients, indicating altered transduction of tumor necrosis factor signals. Here we explore polymorphisms in both tumor necrosis factor receptor genes as risk factors in a German population sample. Neither the polymorphism in the TNFR1 nor that in the TNFR2 gene was associated with narcolepsy. Our findings contrast to those previously published and thus provide evidence for genetic heterogeneity between different narcolepsy populations.

Transcriptional profiles from patients with dystrophinopathies and limb girdle muscular dystrophies as determined by qRT-PCR

Abstract.Mutations in genes coding for the dystrophin-glycoprotein complex (DGC) cause inherited muscular dystrophies (MD), including Morbus Duchenne (DMD) and M. Becker (BMB) as well as limb-girdle muscular dystrophies (LGMD). New insights into the pathophysiology of the dystrophic muscle, the identification of compensatory mechanisms and additional proteins interacting with dystrophin are essential for developing new treatments. In order to define molecular mechanisms induced by lack of dystrophin and the subsequent counter-regulatory transcriptional response of degenerating muscle fibres, we have investigated the mRNA expression of 19 functionally linked genes in biopsies of patients with MD by means of real time qRT-PCR. Our results define a uniform transcriptional profile of the dystrophic muscle characterized by degeneration and regeneration. Several genes encoding structural proteins appear remarkably highly expressed.

Association study with Wegener granulomatosis of the human phospholipase Cγ2 gene

BackgroundWegener Granulomatosis (WG) is a multifactorial disease of yet unknown aetiology characterized by granulomata of the respiratory tract and systemic necrotizing vasculitis. Analyses of candidate genes revealed several associations, e.g. with α(1)-antitrypsin, proteinase 3 and with the HLA-DPB1 locus. A mutation in the abnormal limb mutant 5 (ALI5) mouse in the region coding for the hydrophobic ridge loop 3 (HRL3) of the phospholipaseCγ2 (PLCγ-2) gene, corresponding to human PLCγ-2 exon 27, leads to acute and chronic inflammation and granulomatosis. For that reason, we screened exons 11, 12 and 13 coding for the hydrophobic ridge loop 1 and 2 (HRL1 and 2, respectively) and exon 27 of the PLCγ-2 protein by single strand conformation polymorphism (SSCP), sequencing and PCR/ restriction fragment length polymorphism (RFLP) analyses. In addition, we screened indirectly for disease association via 4 microsatellites with pooled DNA in the PLCγ-2 gene.ResultsAlthough a few polymorphisms in these distinct exons were observed, significant differences in allele frequencies were not identified between WG patients and respective controls. In addition, the microsatellite analyses did not reveal a significant difference between our patient and control cohort.ConclusionThis report does not reveal any hints for an involvement of the PLCγ-2 gene in the pathogenesis of WG in our case-control study.

An extended association screen in multiple sclerosis using 202 microsatellite markers targeting apoptosis-related genes does not reveal new predisposing factors

Apoptosis, the programmed death of cells, plays a distinct role in the etiopathogenesis of Multiple sclerosis (MS), a common disease of the central nervous system with complex genetic background. Yet, it is not clear whether the impact of apoptosis is due to altered apoptotic behaviour caused by variations of apoptosis-related genes. Instead, apoptosis in MS may also represent a secondary response to cellular stress during acute inflammation in the central nervous system. Here, we screened 202 apoptosis-related genes for association by genotyping 202 microsatellite markers in initially 160 MS patients and 160 controls, both divided in 4 sets of pooled DNA samples, respectively. When applying Bonferroni correction, no significant differences in allele frequencies were detected between MS patients and controls. Nevertheless, we chose 7 markers for retyping in individual DNA samples, thereby eliminating 6 markers from the list of candidates. The remaining candidate, the ERBB3 gene microsatellite, was genotyped in additional 245 MS patients and controls. No association of the ERBB3 marker with the disease was detected in these additional cohorts. In consequence, we did not find further evidence for apoptosis-related genes as predisposition factors in MS.