Investigation of sex-specific effects of apolipoprotein E on severity of EAE and MS.

BACKGROUND: Despite pleiotropic immunomodulatory effects of apolipoprotein E (apoE) in vitro, its effects on the clinical course of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) are still controversial. As sex hormones modify immunomodulatory apoE functions, they may explain contentious findings. This study aimed to investigate sex-specific effects of apoE on disease course of EAE and MS. METHODS: MOG(35-55) induced EAE in female and male apoE-deficient mice was assessed clinically and histopathologically. apoE expression was investigated by qPCR. The association of the MS severity score (MSSS) and APOE rs429358 and rs7412 was assessed across 3237 MS patients using linear regression analyses. RESULTS: EAE disease course was slightly attenuated in male apoE-deficient (apoE (-/-) ) mice compared to wildtype mice (cumulative median score: apoE (-/-) = 2 [IQR 0.0-4.5]; wildtype = 4 [IQR 1.0-5.0]; n = 10 each group, p = 0.0002). In contrast, EAE was more severe in female apoE (-/-) mice compared to wildtype mice (cumulative median score: apoE (-/-) = 3 [IQR 2.0-4.5]; wildtype = 3 [IQR 0.0-4.0]; n = 10, p = 0.003). In wildtype animals, apoE expression during the chronic EAE phase was increased in both females and males (in comparison to naïve animals; p < 0.001). However, in MS, we did not observe a significant association between MSSS and rs429358 or rs7412, neither in the overall analyses nor upon stratification for sex. CONCLUSIONS: apoE exerts moderate sex-specific effects on EAE severity. However, the results in the apoE knock-out model are not comparable to effects of polymorphic variants in the human APOE gene, thus pinpointing the challenge of translating findings from the EAE model to the human disease.

Association of TNFAIP3 and TNFRSF1A variation with multiple sclerosis in a German case-control cohort.

Variations in two genes of the tumour necrosis factor (TNF) alpha pathway have been implicated in the pathogenesis of autoimmune diseases: polymorphisms in the TNFRSF1A gene, encoding TNF receptor 1, showed significant association with MS in genomewide association scans, and variation in or near the TNFAIP3 gene, coding for a negative regulator of NFkB, was associated with MS, systemic lupus erythematosus, diabetes and rheumatoid arthritis. This study aimed at investigating association of MS with variation in the TNFRSF1A gene as well as in the TNFAIP3 gene region in an independent German case-control cohort. Four hundred and ninety-seven unrelated patients with MS and 878 healthy controls were genotyped with restriction enzyme digestion or TaqMan assays for three polymorphisms in the TNFRSF1A gene and seven in the region of the TNFAIP3 gene. Allele, genotype and haplotype frequencies were compared between cases and controls by chi-square testing. We found significant association of rs10499194, located in the intergenic region upstream of TNFAIP3, with MS (pc = 3.4 × 10(-4) ). Further, the intronic SNP rs1800693 in TNFRSF1A showed moderate association (pc = 0.033) with MS. In conclusion, evidence is accumulating that polymorphisms in both TNFAIP3 and TNFRSF1A genes play a role in MS pathogenesis. Additional studies are warranted to further elucidate the role of TNF pathway variation for MS development.

Ring chromosome 22 and neurofibromatosis type II: proof of two-hit model for the loss of the NF2 gene in the development of meningioma.

Carriers of a ring chromosome 22 are mentally retarded and show variable facial dysmorphism. They may also present with features of neurofibromatosis type II (NF2) such as vestibular schwannomas and multiple meningiomas. In these cases, tumourigenesis has been suspected to be caused by the loss of both alleles of the NF2 gene, a tumour suppressor localized in 22q12.2. Here, we describe an 18-year-old patient with constitutional ring chromosome 22 and mental retardation who developed rapid-onset spastic paraparesis at the age of 15 years. The causative spinal meningioma at the level of T3, which compressed the spinal cord, was surgically removed, and the patient regained ambulation. Array comparative genomic hybridization (array CGH) and multiplex ligation-dependent probe amplification (MLPA) analyses in blood revealed a terminal deletion in 22q13.32, not comprising the NF2 gene. In tumour tissue, loss of the whole ring chromosome 22 including one NF2 gene due to mitotic instability constituted the likely first hit, while a point mutation in the other allele of the NF2 gene (c.784C>T, p.R262X) was shown as second hit. We review all cases from the literature and suggest clinical guidelines for surveillance of patients with ring chromosome 22.

New cases of adult-onset Sandhoff disease with a cerebellar or lower motor neuron phenotype.

Sandhoff disease is a lipid-storage disorder caused by a defect in ganglioside metabolism. It is caused by a lack of functional N-acetyl-beta-d-glucosaminidase A and B due to mutations in the HEXB gene. Typical, early-onset Sandhoff disease presents before 9 months of age with progressive psychomotor retardation and early death. A late-onset form of Sandhoff disease is rare, and its symptoms are heterogeneous. As drug trials that aim to intervene in the disease mechanism are emerging, the recognition and identification of Sandhoff disease patients-particularly those with atypical phenotypes-are becoming more important. The authors describe six new late-onset Sandhoff cases demonstrating cerebellar ataxia or lower motor neuron (LMN) involvement combined with, mostly subclinical, neuropathy. Two different mutations were found: IVS 12-26 G/A and c.1514G-->A. In patients with either progressive cerebellar ataxia or LMN disease in the setting of a possibly recessive disorder, Sandhoff disease should be suspected, even when the onset age is over 45 years.

Novel association of the CD226 (DNAM-1) Gly307Ser polymorphism in Wegener's granulomatosis and confirmation for multiple sclerosis in German patients.

Recently, there has been increasing evidence that a non-synonymous exchange (Gly307Ser) in the gene for CD226 is linked to several autoimmune diseases including type 1 diabetes, multiple sclerosis (MS), rheumatoid arthritis and Grave's disease. Here we present evidence that this polymorphism also predisposes to Wegener's granulomatosis (WG), an autoimmune condition belonging to the group of ANCA (antineutrophil cytoplasmic autoantibody)-associated vasculitides. We found a significant association of the 307Ser allele in separate panels of 520 Northern German (P=0.016, odds ratio (OR)=1.20) and 122 Southern German (P=0.020, OR=1.37) WG cases compared with 1226 healthy controls. The importance of this single-nucleotide polymorphism in the etiopathology of ANCA-associated vasculitides is supported by similar effect sizes that we found in British WG cases (n=105) and German patients with Churg-Strauss syndrome (n=119), which, however, miss significance level because of the relatively small cohorts available for these rare disorders. Finally, we confirm the association with MS in a cohort of 422 German patients (P=0.011, OR=1.23).

Variation in the IL7RA and IL2RA genes in German multiple sclerosis patients.

Variation in the genes encoding the interleukin (IL) 7 and IL2 receptor alpha chains (IL7RA, IL2RA) was recently found associated with multiple sclerosis (MS). We evaluated the role of these two genes in a large German MS case-control cohort. Five single nucleotide polymorphisms (SNPs) in IL7RA and four in IL2RA were genotyped in 1319 MS patients and 908 controls by means of restriction enzyme digestion or TaqMan assays and subsequently evaluated for association with MS. IL7RA expression was measured via quantitative real time PCR in 24 subjects. We replicated the association of exon 6 variation (rs6897932) in IL7RA with MS. Yet, this association was only found in patients with primary progressive (pp) or secondary progressive (sp) disease course (p=0.0004). Expression analysis did not show differences in IL7RA expression depending on genotypes at this locus, while reduced expression of the soluble receptor was observed in patients with pp and sp MS irrespective of genotype. In the IL2RA gene, significant associations of SNPs in introns 3 and 7 with MS subtypes were obvious. Together these results confirm involvement of polymorphisms in the IL7RA and IL2RA genes in MS pathogenesis and suggest that IL7RA variation may primarily affect chronic disease courses.

Variation in genes of the epidermal differentiation complex in German atopic dermatitis patients.

The filaggrin (FLG) gene is one of the most widely replicated susceptibility genes for atopic dermatitis (AD) so far. Yet, FLG mutations cannot fully account for the original linkage peak on chromosome 1q21, a region comprising the so-called epidermal differentiation complex (EDC). Since the EDC contains numerous genes relevant for epidermal differentiation, we sought to evaluate variation in other genes located in this region in a German AD case-control cohort. Thirty-two single nucleotide polymorphisms (SNPs) in 21 genes across the EDC were genotyped in 402 unrelated AD patients and 325 non-atopic controls by means of restriction enzyme digestion or TaqMan assays. Allele and genotype frequencies were tested for differences between patients and controls by logistic regression. Haplotype frequencies were evaluated using the famhap software. Except for the already known association with FLG, we did not identify any additional significant associations of EDC genes with AD. Thus, in this German cohort, there is no evidence that additional genes in the EDC region apart from FLG contribute substantially to AD pathogenesis.

[The genetics of chronic obstructive pulmonary disease]. / Zur Genetik der chronisch-obstruktiven Lungenerkrankung.

COPD is a heterogenous disease caused by the interaction of genetic susceptibility and environmental influences. The best example to support this is tobacco smoke. Although cigarette smoking is the most important aetiological factor, only up to half of chronic smokers develop significant COPD. There are three main themes within the pathogenesis of COPD: 1) imbalance between proteases and anti-proteases, 2) oxidative stress, 3) inflammation. Disparity between levels of exogeneous oxidants, e. g., tobacco smoke, and endogeneous antioxidants leads to oxidative stress which, in turn, causes an inflammatory response involving pro-inflammatory mediators. The activated inflammatory cells release further proteases and oxidants, leading to chronic inflammation and irreversible destruction of connective tissue in the lung. Individual genetic variations influence these processes in many ways. This article summarises the results of recent candidate gene studies for COPD.

Cervical cord area is associated with infratentorial grey and white matter volume predominantly in relapsing-remitting multiple sclerosis: A study using semi-automated cord volumetry and voxel-based morphometry.

BACKGROUND AND PURPOSE: Atrophy of the brain and the upper cervical cord, which both have major impact on the severity of clinical symptoms in multiple sclerosis (MS), may be interrelated by neuraxonal degeneration. Aiming to identify possible spatially remote effects of neuraxonal brain damage on spinal cord atrophy, we studied regional and global brain volumes and the upper cervical cord area (UCCA) in a large group of MS patients and a healthy control group. METHODS: In a group of 132 MS patients (71 relapsing-remitting MS; 61 secondary progressive MS; median [range] of EDSS: 5 [0-7], respectively 6 [2-8.5] and mean±standard deviation of age/disease duration: 37±11 years/6.7±6.3 years; respectively: 49±8 years/14.5±8.0 years) and 45 healthy subjects UCCA, regional and global brain volumes, and brain lesion load were assessed. Associations between MRI results and clinical parameters in the entire cohort and differentiated according to MS-subtype were investigated using t-tests, partial correlation analyses, voxel-based morphometry and statistical parametric mapping. RESULTS: Exclusively in RRMS, a significant positive correlation of UCCA with cerebellar cortical grey matter (GM) in the vermis and with regional white matter volume in the entire brainstem, corresponding to the corticospinal tracts, was detected. Although SPMS patients were considerably more affected by disability and decrease of UCCA (RRMS:75.2±10.4 mm(2); SPMS: 66.0±11.8 mm(2),controls: 84.5±8.7mm(2)), brain grey matter (RRMS:585.8±53.6 ml; SPMS: 528.2±61.5 ml, controls: 608.7±48.1 ml) and total brain volume (RRMS:1162.9±41.8 ml; SPMS: 1117.9±51.2 ml, controls: 1194.1±19.5 ml) than RRMS patients, significant positive associations in this group were found only between UCCA and a cluster of white matter in the medulla, but not in grey matter. CONCLUSION: Cervical cord and brain atrophy were present in both, RRMS and even more severe in SPMS. Still, spatial associations between cervical cord area and remote cerebellar and brainstem volume, possibly driven by neuraxonal degeneration, were detected mostly in RRMS patients with predominantly short disease durations. Future longitudinal studies may elucidate the interplay between affection of spinal cord and infratentorial structures in MS, and contribute to the understanding of the conversion processes from relapsing-remitting to secondary progressive MS.

Genetic dissection of marfan syndrome and related connective tissue disorders: an update 2012.

Marfan syndrome (MFS) is an autosomal dominant disorder of the connective tissue characterized by early development of thoracic aortic aneurysms/dissections together with symptoms of the ocular and skeletal systems. While most patients/families with a classic phenotypic expression of MFS harbour mutations in the gene encoding fibrillin-1 (FBN1), genetic studies of the recent years revealed that the clinical features, as well as the mutated genes, show a high degree of overlap between MFS and other connective tissue diseases (e.g. Loeys-Dietz syndrome, Ehlers-Danlos syndrome, familial thoracic aneurysms and dissections and others). We summarize herein the current knowledge about the wide spectrum of differential diagnoses and their genetic background as well as novel therapeutic approaches in order to provide appropriate counselling and clinical follow-up for the patients.

On the role of the epidermal differentiation complex in ichthyosis vulgaris, atopic dermatitis and psoriasis.

Undisturbed epidermal differentiation is crucial for an intact skin barrier function. The epidermal differentiation complex (EDC) is a cluster of genes on chromosome 1q21 encoding proteins that fulfil important functions in terminal differentiation in the human epidermis, including filaggrin, loricrin, S100 proteins and others. Recently, evidence emerged that variation within EDC genes plays an important role in the pathogenesis of three common skin disorders, ichthyosis vulgaris, atopic dermatitis (AD) and psoriasis. Two loss-of-function mutations in the filaggrin (FLG) gene, R501X and 2282del4, were identified as causative for ichthyosis vulgaris in 15 affected European families, and the mode of inheritance was found to be semidominant. As ichthyosis vulgaris and AD often occur concomitantly in affected individuals, these two mutations were subsequently investigated in AD patients and found to be strongly associated with the disease. Following this first report, seven replication studies have been performed that all confirm an association of these two mutations with AD (or AD subtypes) in several European cohorts. Additionally, two unique loss-of-function mutations in the FLG gene were identified in Japanese ichthyosis vulgaris families and found to be associated with AD in a Japanese cohort. Thus, the FLG mutations are among the most consistently replicated associations for AD. Additionally, linkage analysis has suggested that variation within the EDC might also predispose for psoriasis but the exact susceptibility variation(s) have not yet been elucidated. Taken together, these findings convincingly demonstrate the important role of barrier dysfunction in various common skin disorders.

Asthma genetics 2006: the long and winding road to gene discovery.

Asthma and atopy are complex phenotypes that are influenced by both genetic and environmental factors. A review of nearly 500 papers on disease association studies identified 25 genes that have been associated with an asthma or atopy phenotype in six or more populations. An additional 54 genes have been associated in 2-5 populations. Here, we discuss the methods that have been used to identify susceptibility genes for common diseases and overview the status of asthma genetic research. Finally, current challenges and future directions are discussed.

Association screen for atopic dermatitis candidate gene regions using microsatellite markers in pooled DNA samples.

Atopic dermatitis (AD) is a chronic inflammatory skin disease affecting up to 16% of children in developed countries. A complex genetic background for AD has been suggested, with genetic as well as environmental factors influencing disease susceptibility. Among other factors, dysregulation in both the innate and the adaptive immune system has been proposed to play a role in AD pathophysiology. We present here an extended association screen for AD using microsatellite markers in 154 genes related to innate and adaptive immunity in pooled DNA samples from 150 German children with AD and 100 controls. After Bonferroni correction, no marker revealed a significant association with AD. Yet, markers representing the nuclear factor kappa B (NFKB)1 and chemokine receptor (CCR)4 genes showed differences in allelic distributions between cases and controls for both pooled DNA analysis and individual genotyping and were thus further investigated. Evaluation of additional single nucleotide polymorphisms (SNP) in the NFKB1 and CCR4 genes revealed no association of individual SNPs with AD. In contrast, haplotype analyses showed a significantly different haplotype distribution between patients and controls for CCR4 (P < 0.001). Furthermore, when SNP-SNP interaction effects were analysed for these two genes, we found significant evidence for epistatic interactions between SNPs within each of the two genes but no evidence for a gene-gene interaction, suggesting that variation in or near both the CCR4 and the NFKB1 genes might individually contribute to AD pathogenesis.

Association of interleukin-8 receptor alpha polymorphisms with chronic obstructive pulmonary disease and asthma.

Chronic obstructive pulmonary disease (COPD) and asthma are common complex diseases characterized by airflow obstruction and inflammatory processes in the small airways. lnterleukin 8 (IL-8) is a potent proinflammatory cytokine which interacts with the IL-8 receptor alpha (IL8RA, CXCR1) and beta (IL8RB, CXCR2), leading to activation and migration of leukocytes. In order to evaluate the role of the IL8RA gene in the pathogenesis of COPD and asthma, we screened the coding region of IL8RA for mutations by means of single-strand conformation polymorphism analysis in 50 COPD patients and identified three exchanges (M31R, S276T and R335C). These three polymorphisms were subsequently genotyped in 182 adult patients with COPD, 68 adult patients and 130 children with asthma as well as 454 healthy controls. The frequencies of the IL8RA 31R and 335C alleles were significantly increased in patients with COPD and in children with asthma compared to healthy controls (P=0.0073 and 0.023, respectively). Thus, these polymorphisms may play a role in the pathogenesis of COPD and asthma.

Effect of recombinant human DNase on alpha1-proteinase inhibitor function: an experimental approach to the combined clinical use of rhDNase and alpha1-PI in CF patients.

Chronic inflammation in cystic fibrosis (CF) airways leads to high concentrations of deoxyribonucleic acid (DNA) and neutrophil elastase (NE). Both play a major role in CF lung pathophysiology and are aims of new therapeutic approaches: rhDNase degrades highly viscosic DNA and alpha1-proteinase inhibitor (alpha1-PI) inhibits NE activity and thereby pulmonary inflammation and hypersecretion. Given the reports on increased sputum NE concentrations upon rhDNase inhalation, there is a rationale for a combined rhDNase/alpha1-PI treatment. With the question of whether a combined therapy is feasible, we first investigated in vitro whether incubation of CF sputum with rhDNase changes proteolytic and secretagogue activity of sputum supernatants and its inhibition by alpha1-PI. Next, we studied whether incubation of alpha1-PI with rhDNase impairs the inhibitory effect of alpha1-PI on proteolytic activity of NE and the inhibitory effect of alpha1-PI on NE-induced secretion from a human mucoepidermoid cell line. Incubation of CF sputum with rhDNase led to a twofold increase in sputum NE activity. Correspondingly, the inhibitory effect of alpha1-PI on sputum NE activity and on secretion induced by these sputum samples was significantly reduced by rhDNase. Preincubation of alpha1-PI with rhDNase significantly reduced the inhibitory effect of alpha1-PI on purified NE activity and on NE-induced secretion. However, this effect was limited to alpha1-PI concentrations lower than those achievable after inhalation. Therefore, impairment of alpha1-PI function by rhDNase is not likely to be relevant in vivo, provided that a sufficient dosage of alpha1-PI is inhaled.

RSV bronchiolitis and risk of wheeze and allergic sensitisation in the first year of life.

Severe respiratory syncytial virus (RSV) infection has been hypothesised to be a risk factor for the development of allergy and asthma, but epidemiological studies in older children have been inconclusive. The current study hypothesises that the effect of RSV bronchiolitis might be most prominent during the first year after bronchiolitis. Forty-two infants had experienced RSV bronchiolitis severe enough to cause hospitalisation. For each child with RSV infection, two controls were acquired from a birth cohort and matched for date of birth and sex. All the children were followed prospectively and underwent a follow-up examination at a mean age of 1 yr, which included physical examination, and serum immunoglobulin (Ig) E tests for common food and inhaled allergens. Risk factors for the development of recurrent wheezing and IgE antibodies were analysed for the whole group of 126 children. A positive test for IgE antibodies was noted in 14 of 42 (33%) RSV children and in 2 of 84 (2.3%) children in the control group. RSV bronchiolitis was the most important risk factor for allergic sensitisation. Likewise, 13 children (15.5%) of the RSV group and three (3.6%) children of the control group suffered from recurrent wheezing, and RSV bronchiolitis posed a considerable risk for recurrent wheezing. Severe respiratory syncytial virus bronchiolitis during the first year of life is an important risk factor for the development of recurrent wheezing and sensitisation to common allergens during the subsequent year.

Respiratory syncytial virus decreases the capacity of myeloid dendritic cells to induce interferon-gamma in naïve T cells.

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants under 6 months of age. Since an RSV infection does not necessarily prevent a reinfection, we asked whether RSV might subvert an effective immune response by interfering with the function of dendritic cells (DCs). Immature DCs cultured from cord blood stem cells and infected with RSV reduced the rate of interferon-gamma (IFN-gamma) production in co-cultured autologous naïve T cells stimulated with the superantigen TSST-1. Maturation of DCs in response to poly(IC) but not to CD40 ligand did overcome the inhibitory effect of RSV. Further experiments demonstrated that induction of apoptosis, a selective increase in CD86 expression and lack of release of pro-inflammatory cytokines were associated with inhibition of IFN-gamma generation. In addition, RSV replication seemed to be essential for modulation of IFN-gamma production because a virus preparation inactivated by UV irradiation had no effect. Hence, one reason for multiple reinfections by RSV might be the subversion of antiviral immune responses by interference of RSV with DC function.

Severe respiratory syncytial virus infections and reduced interferon-gamma generation in vitro.

To study the consequences of the interaction of respiratory syncytial virus (RSV) with dendritic cells in vitro, we established a model of the primary immune response using dendritic cells, autologous naive T cells and the superantigen toxic shock syndrome toxin 1 (TSST 1). About 10% of the naive T cells express the T cell receptor chain Vbeta2. These cells were stimulated by TSST 1 and could be analysed by flow cytometry. Cultures infected with RSV produced significantly less interferon-gamma compared to uninfected cultures. In a first set of experiments we evaluated whether this culture model using isolated CD4(+) CD45RA(+) T cells, in fact, reflects the primary immune response. In a prospective study, cells were isolated from 13 children at birth, at 1 year of age and at 4 years of age. RSV reduced interferon-gamma production at all the age groups analysed and the results were stable over time within a given individual. In a second set of experiments, we asked whether clinical differences in the course of RSV infection are due to variations in the cellular immune response. At the age of 1 year (5-9 months after the RSV epidemic) dendritic cells and naive T cells were obtained from 27 children with a history of bronchiolitis, from 15 children with a benign course of RSV infection and from 26 controls without RSV infection. The frequency of interferon-gamma-producing cells in RSV infected cultures was significantly lower (P < 0.001) in cultures from children with a history of RSV bronchiolitis compared to children with mild RSV infection. Cultures from children without infection displayed a wide range of results. Overall, interferon-gamma generation in this group was still lower (P < 0.05) than in the group with mild RSV infection. Because we have ruled out that memory cells play a role in the experiments performed, the most likely explanation for our results is that a high generation of interferon-gamma in the primary immune response protects from severe RSV mediated disease.