Dominant VPS16 Pathogenic Variants: Not Only Isolated Dystonia.

BACKGROUND: VPS16 pathogenic variants have been recently associated with inherited dystonia. Most patients affected by dominant VPS16-related disease display early-onset isolated dystonia with prominent oromandibular, bulbar, cervical, and upper limb involvement, followed by slowly progressive generalization. CASES: We describe six newly reported dystonic patients carrying VPS16 mutations displaying unusual phenotypic features in addition to dystonia, such as myoclonus, choreoathetosis, pharyngospasm and freezing of gait. Response to bilateral Globus Pallidus Internus Deep Brain Stimulation (GPi-DBS) is reported in three of them, associated with significant improvement of dystonia but only minor effect on other hyperkinetic movements. Moreover, five novel pathogenic/likely pathogenic variants are described. CONCLUSIONS: This case collection expands the genetic and clinical spectrum of VPS16-related disease, prompting movement disorder specialists to suspect mutations of this gene not only in patients with isolated dystonia.

Fatigue and depression predict health-related quality of life in patients with pediatric-onset multiple sclerosis.

BACKGROUND: Fatigue, depression and loss in health-related quality of life (HRQoL) have been reported to occur in a substantial amount of patients with pediatric-onset MS (POMS). This study aims to evaluate depression, fatigue and HRQoL and its relationship in a cohort of patients with POMS and matched healthy controls (HCs). METHODS: In a multicenter cross-sectional study, Beck Depression Inventory II, Depressionstest für Kinder, the Pediatric Quality of Life Inventory (PedsQL™) 4.0 Generic Core Scale and the PedsQL™ Multidimensional Fatique Scale were performed. RESULTS: In a cohort of 106 patients with POMS and 210 matched HCs, patients were significantly more often depressed (21.7% vs. 11.4%, p = 0.014) experienced greater fatigue (40.6% vs. 17.3%, p < 0.001) and a greater loss of HRQoL (43.4% vs. 15%, p < 0.001) than controls. Depression predicted 51.8% of variance of fatigue. Fatigue was also predicted by female gender. Loss of HRQoL was predicted by EDSS, depression and fatigue. Depression and fatigue together explained 67.7% of variance of HRQoL. CONCLUSION: Patients with POMS are at a significant increased risk for depression, fatigue and loss of HRQoL. Furthermore, fatigue and depression significantly predict reduced HRQoL in POMS, suggesting that testing for these symptoms and early therapy is of utmost importance in all patients with POMS.

Clinical and magnetic resonance imaging features of children, adolescents, and adults with a clinically isolated syndrome.

BACKGROUND: The diagnosis of multiple sclerosis (MS) both in children and adults is based on clinical and magnetic resonance imaging (MRI) features according to the McDonald criteria. Little is known about differences in the presentation between pre-pubertal children, adolescents, and adult patients at disease onset. OBJECTIVE: To compare (1) the clinical, cerebrospinal fluid (CSF), and MRI characteristics, and (2) the diagnostic performance of the 2010 McDonald criteria between pre-pubertal, adolescent, and adult patients with a clinically isolated syndrome (CIS). METHODS: We performed a retrospective analysis of the initial brain and spinal cord MRI scans from 11 pre-pubertal children, 46 adolescents, and 56 adults with a CIS. Furthermore, clinical, CSF characteristics, and the performance of the 2010 McDonald criteria were compared. RESULTS: The first inter-attack interval tended to increase with age. With respect to MRI presentation, significantly fewer pre-pubertal children presented with juxtacortical and callosal lesions. We found no significant differences in the fulfillment of the 2010 McDonald criteria between the groups. CONCLUSION: In this retrospective series, subtle differences between children, adolescents, and adults with a CIS were noted. Larger samples are required in order to establish distinct features of the different age groups.

Natalizumab therapy for highly active pediatric multiple sclerosis.

IMPORTANCE: Given the high frequency of failure of first-line therapies, there is an urgent need for second-line treatment strategies for pediatric patients with multiple sclerosis (MS). OBJECTIVE: To report the use of natalizumab in pediatric MS. Natalizumab, a humanized monoclonal antibody targeting α4 integrin, is effective against active relapsing-remitting MS in adults. DESIGN: Retrospective study. SETTING: Eleven centers for neurology and pediatric neurology in Germany and Austria. PARTICIPANTS: A total of 20 pediatric patients with MS who started treatment with natalizumab prior to 18 years of age. These patients underwent magnetic resonance imaging as clinically indicated, despite the fact that 19 of these 20 patients were undergoing first-line disease-modifying therapy. The mean (SD) age at initiation of natalizumab therapy was 16.7 (1.1) years, and the mean (SD) pretreatment period was 18 (10) months. INTERVENTION: Natalizumab, 300 mg every 4 weeks. MAIN OUTCOME MEASURES: Annualized relapse rates, Expanded Disability Status Scale scores, number of new T2/fluid-attenuated inversion recovery lesions and contrast-enhancing lesions on magnetic resonance imaging, number of adverse events, the prevalence of neutralizing antibodies against natalizumab, and serum JC virus-antibody status. RESULTS Treatment with natalizumab was associated with reductions in mean annualized relapse rates (3.7 without treatment vs 0.4 with treatment; P < .001), median Expanded Disability Status Scale scores (2 without treatment vs 1 with treatment; P < .02), and mean number of new T2/fluid-attenuated inversion recovery lesions per year (7.8 without treatment vs 0.5 with treatment; P < .001). Two patients developed high-titer neutralizing antibodies against natalizumab and had to stop therapy. Adverse events included headaches, asthenia, infections, and hypersensitivity. Abnormal laboratory results were found for 8 patients. JC virus antibodies were found in 5 of 13 patients. After the discontinuation of natalizumab therapy, relapse activity occurred in 6 of 8 patients within 6 months. CONCLUSIONS AND RELEVANCE: Our data indicate that natalizumab may be safe and effective against MS in pediatric patients with breakthrough disease.

Neuropsychological aspects of childhood multiple sclerosis: an overview.

While cognitive impairment, major depression, and fatigue have been well documented in adult patients with multiple sclerosis (MS), there is still little information regarding MS-associated cognitive disabilities in infants and adolescents who represent 3 to 5% of all MS cases. Recent studies show that cognitive decline related to MS profoundly interferes with academic success and psychosocial adjustment. Neuropsychological dysfunction affects quality of life more significantly than mere Expanded Disability Status Scale is able to reflect. We herein give an overview of the knowledge available to date. Affective and emotional disturbances together with other comorbidities interfering with cognition are also reviewed. Finally, possible suggestions and future directions for the assessment of cognitive capabilities in children with MS are envisioned.

Axonemal localization of the dynein component DNAH5 is not altered in secondary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a heterogeneous genetic disorder characterized by recurrent airway infections and situs inversus in half of affected individuals. Diagnosis currently relies on demonstration of abnormal ciliary ultrastructure or altered ciliary beat. Alterations encountered in secondary ciliary dyskinesia (SCD) caused by inflammation often complicate the diagnostic workup. We have recently shown that in respiratory epithelial cells from PCD patients with outer dynein arm defects the dynein protein DNAH5 is mislocalized and either completely or partially absent from the ciliary axoneme. In this study, we addressed the question whether SCD might affect axonemal DNAH5 localization in respiratory cells. To induce SCD in vitro, we treated primary human respiratory epithelial cell cultures with interleukin-13 (IL-13). Ciliary function and ultrastructure were assessed by high-speed videomicroscopy and transmission electron microscopy, respectively. For in vivo localization of DNAH5, we performed nasal brushing biopsies in patients with evidence of SCD. Expression of DNAH5 was analyzed by immunofluorescence microscopy. IL-13-treated cells showed evidence of SCD. Ciliary beat frequency was significantly reduced and ultrastructural analyses showed axonemal disorganization compared with control cells. High-resolution immunofluorescence studies of respiratory epithelial cells with SCD identified in vitro and in vivo normal axonemal DNAH5 localization. DNAH5 localization is not altered by SCD, indicating a high potential for immunofluorescence analysis as a novel diagnostic tool in PCD.

Primary ciliary dyskinesia: clinical presentation, diagnosis and genetics.

Primary ciliary dyskinesia (PCD) is a phenotypically and genetically heterogeneous disorder with an autosomal-recessive inheritance pattern. Only rarely other modes of inheritance such as X-linked transmission are observed. The disease phenotype is caused by defects of respiratory cilia, sperm tails and the cilia of the embryonic node. The lack of mucociliary clearance contributes to recurrent respiratory tract infections, that might progress to permanent lung damage (bronchiectasis). The goal of therapy is prevention of bronchiectasis. Male infertility due to sperm tail dysmotility is another frequent finding in PCD. Half of affected individuals have situs inversus (Kartagener's syndrome) due to randomization of left/right body asymmetry. Currently three genes (DNAI1, DNAH5, DNAH11) that encode for dynein proteins have been linked to recessive PCD. Mutations in RPGR located on the X chromosome have been identified in males with retinitis pigmentosa and PCD. As a screening test nasal nitric oxide (NO) measurement is widely used. Establishment of diagnosis currently relies on electron microscopy, direct evaluation of ciliary beat by light microscopy, and/or the novel method of high-resolution immunofluorecent analysis of respiratory cilia.

A cysteinyl leukotriene 2 receptor variant is associated with atopy in the population of Tristan da Cunha.

The clinical heterogeneity of asthma suggests that the contribution of genetic variability in candidate gene loci to well-defined phenotypes, such as atopy, may be examined to identify appropriate genetic risk factors for asthma. The gene encoding the cysteinyl leukotriene 2 (CysLT2) receptor has been implicated in atopy since it is localized to a region of chromosome 13q14 that has been linked to atopy in several populations and the cysteinyl leukotrienes are known to activate eosinophils and mast cells in atopy. Accordingly, we analysed the contribution of CysLT2 receptor gene variation to atopy in the inhabitants of Tristan da Cunha, a population characterized by both a founder effect and a 47% prevalence of atopy. Single-stranded conformational polymorphism analysis revealed four variants. Among these, the M201V [corrected] variant was activated with four-fold less potency by leukotriene D4 (LTD4) in a calcium flux assay. The CysLT2 receptor partial agonist, BAY u9773, also showed four-fold lower potency on the M201V [corrected] variant. The M201V [corrected] mutation is located within the extracellular region of the fifth transmembrane spanning domain of CysLT2 receptor, a position that may alter ligand binding and effector signalling. The novel M201V [corrected] CysLT2 receptor variant was associated with atopy (21%) on Tristan da Cunha compared with those who were non-atopic (7%) (Fisher's exact test, P=0.0016) in a manner that was independent of asthma (two-way ANOVA, P=0.0015). This represents the first association of a coding mutation in the CysLT2 receptor gene, located on chromosome 13q14, with the atopic phenotype found in the Tristan da Cunha population.

Monocyte chemoattractant protein-4 core promoter genetic variants: influence on YY-1 affinity and plasma levels.

Monocyte chemoattractant protein-4 (MCP-4) is a CC chemokine implicated in the recruitment of eosinophils, monocytes, and T-lymphocytes in diseases of mucosal inflammation, including asthma. We tested the hypothesis that there is a genetic basis for differences in MCP-4 expression among individuals by evaluating the effects of core promoter variants on MCP-4 expression. We identified two single-nucleotide T-to-C polymorphisms in the MCP-4 core promoter that occur 896 and 887 base pairs preceding the transcription initiation site. The -887 variant alters a consensus binding motif for the transcription factor YY-1. Electrophoretic mobility shift assay demonstrated that YY-1 containing nuclear extracts from tumor necrosis factor-alpha-stimulated peripheral blood mononuclear cells had greater avidity for the wild-type (YY-1 motif intact) sequence than for the variant sequence. Increasing doses of a YY-1 expression vector induced significantly greater reporter activity from MCP-4 core promoter expression constructs of the wild-type compared with the variant sequence in transient transfection experiments. The external validity of these observations was demonstrated by measuring plasma levels of MCP-4 from individuals with the alternative forms of the gene. Individuals bearing haplotypic variants of the MCP-4 core promoter that avidly bind the transcription factor YY-1 had higher plasma levels of MCP-4 than did individuals with variants with lower binding avidity (490, 360, and 360 pg/ml; P < 0.01). Our findings suggest that the MCP-4 core promoter YY-1 binding motif is functional, modulates the transcriptional regulation of the MCP-4 gene, and that part of the variance in the systemic expression of MCP-4 is determined by core promoter genetic variants.

Association of a missense mutation in the NOS3 gene with exhaled nitric oxide levels.

There is evidence that genetic factors affect nitric oxide formation and that sequence variants in the nitric oxide synthase genes contribute to the observed variance of nitric oxide levels in exhaled air (fraction of expired nitric oxide, FENO) in subjects with asthma. We identified a strong association between a known functional NOS3 missense sequence variant in the endothelial nitric oxide gene (G894T) and FENO level in a cohort of subjects with asthma. Age- and sex-adjusted FENO levels were lowest in asthmatic subjects with the TT genotype (geometric mean FENO [95% CI] = 7.17 [4.48 to 11.48] ppb) and were significantly higher in those with either the GT genotype (geometric mean FENO [95% CI] = 17.11 [13.80 to 21.23] ppb) or the GG genotype (geometric mean FENO [95% CI] = 12.06 [9.91 to 14.67] ppb) (F2,59 = 5.97, p = 0.004). The G894T DNA variant explained 16.3% of the residual variance in FENO levels. Our results demonstrate that the endothelial nitric oxide synthase, a nitric oxide synthase constitutively expressed in epithelial cells, plays an important role in determining measured levels of exhaled nitric oxide, a marker of the asthmatic condition.

A randomized controlled trial on the effect of montelukast on sputum eosinophil cationic protein in children with corticosteroid-dependent asthma.

Previous adult studies demonstrated the clinical efficacy of an additional treatment with leukotriene receptor antagonists on steroid-dependent asthma, but there is little knowledge about anti-inflammatory add-on effects within the lung. In this study, we hypothesized that steroid-treated children exhibit a decrease in bronchial inflammation in induced sputum under additional treatment with montelukast. Twenty-five asthmatic children aged 6 to 14 y, who had been taking inhaled corticosteroids (400-800 microg/d budesonide) regularly for at least 12 wk, were randomized to receive additional treatment with either montelukast (5 mg orally, once daily) or placebo over a 4-wk period. As primary efficacy variable, eosinophil cationic protein (ECP) in induced sputum as direct measurement of bronchial inflammation was assessed before and after treatment. To assure a baseline level of inflammation, an ECP concentration above 100 microg/L was required. Sputum eosinophil count, concentration of exhaled nitric oxide, urinary excretion of eosinophil protein X, and quality-of-life items were considered as secondary outcome variables. After treatment with montelukast, ECP in sputum was significantly reduced (montelukast: median -975 microg/L [5 to 95% confidence interval: -4295 to 583 microg/L]; placebo: 561 microg/L [-1335 to 3320 microg/L]; p < 0.01) and the quality-of-life score had significantly improved (p < 0.05) compared with placebo. Partly explained by low baseline levels, no statistically significant change in concentration of exhaled nitric oxide (p > 0.05), urinary excretion of eosinophil protein X (p > 0.05), or eosinophil count (p > 0.05) was found. In conclusion, add-on treatment with montelukast can suppress sputum ECP in children with steroid-dependent asthma, while at the same time an improvement in quality of life items occurs.

Effects of sex and of gene variants in constitutive nitric oxide synthases on exhaled nitric oxide.

Genetic factors may contribute to the variability of exhaled nitric oxide in healthy individuals. We studied exhaled nitric oxide and genetic variants in both neuronal and endothelial nitric oxide synthases in 105 healthy nonsmoking and smoking subjects. Genomic DNA was screened for a repeat polymorphism in intron 20 of the neuronal nitric oxide synthase gene and for the 894G/T mutation of the endothelial nitric oxide synthase gene. Exhaled nitric oxide was significantly higher in males than females among both nonsmokers (p < 0.0001) and smokers (p = 0.003). No association was found between exhaled nitric oxide and the endothelial nitric oxide synthase gene variant. However, healthy nonsmoking females with greater numbers of repeats (i.e., both alleles with 12 or more repeats) in neuronal nitric oxide synthase had significantly lower nitric oxide levels than did females with fewer numbers of repeats (i.e., at least one allele with fewer than 12 repeats) (13.6 +/- 1.6 versus 19.4 +/- 1.6 ppb, p = 0.02). No association was found between exhaled nitric oxide and neuronal nitric oxide synthase genotype in males. These data suggest that variants in the neuronal nitric oxide synthase gene contribute to the variability of airway nitric oxide concentrations in healthy females.

Endothelial nitric oxide synthase variants in cystic fibrosis lung disease.

Variants in the genes encoding for the nitric oxide synthases may act as disease modifier loci in cystic fibrosis, affecting both an individual's nitric oxide level and pulmonary function. In this study, the 894G/T variant in exon 7 of the endothelial nitric oxide synthase gene was related to exhaled nitric oxide and pulmonary function in 70 cystic fibrosis patients who were aged 14.8 +/- 6.9 years (mean +/- SD), with a FEV1 of 69.4 +/- 24.8% predicted. Although there was no association between endothelial nitric oxide synthase genotypes and exhaled nitric oxide in males, nitric oxide levels were significantly higher in female cystic fibrosis patients with an 894T mutant allele, compared with female patients homozygous for the 894G wild-type allele (7.0 +/- 4.4 versus 3.6 +/- 1.9 parts per billion, p = 0.02). Furthermore, in female patients, colonization of airways with Pseudomonas aeruginosa was significantly (p < 0.05) less frequent when carrying an 894T mutant allele as compared with wild type. These data suggest that the 894T variant in the endothelial nitric oxide synthase gene is associated with increased airway nitric oxide formation in female cystic fibrosis patients, possibly affecting colonization of airways with P. aeruginosa.

IFN regulatory factor-1 regulates IFN-gamma-dependent cathepsin S expression.

Cathepsin S is a cysteine protease with potent endoproteolytic activity and a broad pH profile. Cathepsin S activity is essential for complete processing of the MHC class II-associated invariant chain within B cells and dendritic cells, and may also be important in extracellular matrix degradation in atherosclerosis and emphysema. Unique among cysteine proteases, cathepsin S activity is up-regulated by IFN-gamma. Given its importance, we sought to elucidate the pathway by which IFN-gamma increases cathepsin S expression. Our data demonstrate that the cathepsin S promoter contains an IFN-stimulated response element (ISRE) that is critical for IFN-gamma-induced gene transcription in a cell line derived from type II alveolar epithelial (A549) cells. IFN response factor (IRF)-2 derived from A549 nuclear extracts associates with the ISRE oligonucleotide in gel shift assays, but is quickly replaced by IRF-1 following stimulation with IFN-gamma. The time course of IRF-1/ISRE complex formation correlates with increased levels of IRF-1 protein and cathepsin S mRNA. Overexpression of IRF-1, but not IRF-2, markedly augments cathepsin S promoter activity in A549 cells. Furthermore, overexpression of IRF-1 increases endogenous cathepsin S mRNA levels in 293T epithelial cells. Finally, freshly isolated bone marrow cells from IRF-1(-/-) mice fail to up-regulate cathepsin S activity in response to IFN-gamma. Thus, IRF-1 is the critical transcriptional mediator of IFN-gamma-dependent cathepsin S activation. These data elucidate a new pathway by which IRF-1 may affect MHC class II processing and presentation.

Cloning and functional analysis of the mouse 5-lipoxygenase promoter.

5-lipoxygenase (ALOX5), an enzyme essential for the formation of all leukotrienes, is highly regulated at multiple levels, including gene transcription. The human ALOX5 promoter sequence has been cloned and is well characterized. Several important cis-acting elements have been identified including a G+C-rich sequence approximately 145-179 base pairs (bp) upstream from the ATG start codon. This region contains consensus-binding sites for the transcription factor serum protein 1, a zinc-finger transcription factor (SP1) and early growth-response protein 1, a zinc-finger transcription factor (EGR-1) and is unique in that functionally significant polymorphisms alter these sequences. To further understand the significance of these polymorphisms and other regulatory sequences in the promoter we cloned approximately 2,000 bp of the mouse promoter sequence from a 129/SvJ BAC library for direct comparison with the human gene. Like the human promoter, the mouse Alox5 promoter lacks a TATA box and has multiple start sites. The first 292 bp immediately upstream of the translational start site function as a core promoter that is capable of mediating high basal transcription in RAW cells but not 3T3 cells. There are vast differences in the distribution of consensus cis elements between human and mouse genes; however, three areas of strong homology exist and they contain consensus-binding sites for the SP1, GATA, GGAGA, and ETS family of transcription factors. We show that Sp1/Sp3 is essential for constitutive promoter-reporter activity.

Circadian variation of exhaled nitric oxide and urinary eosinophil protein X in asthmatic and healthy children.

Asthmatic symptoms and the frequency of admissions to hospital because of acute asthma tend to increase in the early morning hours, and it is therefore possible that airway inflammation increases during the night. To elucidate the hypothetical circadian variation of airway inflammation, we measured concentrations of exhaled nitric oxide (FeNo), urinary eosinophil protein X excretion (EPX), and forced expiratory volume in the first second (FEV1) in 20 asthmatic and 6 nonatopic nonasthmatic children every 3 h during a 21-h period. Compared with control subjects, asthmatic subjects had higher FeNo (median, 22.7 versus 10.3 ppb, p = 0.016) and lower FEV1 % predicted (median, 91.0 versus 101.9%, p = 0.045), but did not differ significantly in EPX (median, 153.8 versus 148.7 microg/mmol creatinine, p = 0.83) at 7 AM. However, differences in gender and age do not allow direct comparisons between asthmatic and control children. FeNo and EPX demonstrated a cosinelike circadian rhythm (log FeNo, p = 0.0001; log EPX, p = 0.0001) with lowest levels at 7 PM and highest at 7 AM. This was also the case for FEV1 % (p = 0.01). No difference in the amplitude of circadian rhythm was observed between asthmatic and healthy control children for log FeNo (p = 0.35), log EPX (p = 0.57), and FEV1 % (p = 0.17). A stratified analysis showed a significant circadian rhythm in the control group for log FeNo (p = 0.014) and log EPX (p = 0.0001). Our results therefore suggest a circadian rhythm of inflammatory markers, which peaks in the early morning. Rhythmicity of EPX excretion and FeNo in healthy children suggests a physiologic mechanism; however, pathologic effects during the night might occur under conditions of asthma-specific inflammation.