Genotype-guided diagnostic reassessment after exome sequencing in neuromuscular disorders: experiences with a two-step approach.

BACKGROUND AND PURPOSE: Next-generation sequencing has greatly improved the diagnostic success rates for genetic neuromuscular disorders (NMDs). Nevertheless, most patients still remain undiagnosed, and there is a need to maximize the diagnostic yield. METHODS: A retrospective study was conducted on 72 patients with NMDs who underwent exome sequencing (ES), partly followed by genotype-guided diagnostic reassessment and secondary investigations. The diagnostic yields that would have been achieved by appropriately chosen narrow and comprehensive gene panels were also analysed. RESULTS: The initial diagnostic yield of ES was 30.6% (n = 22/72 patients). In an additional 15.3% of patients (n = 11/72) ES results were of unknown clinical significance. After genotype-guided diagnostic reassessment and complementary investigations, the yield was increased to 37.5% (n = 27/72). Compared to ES, targeted gene panels (<25 kilobases) reached a diagnostic yield of 22.2% (n = 16/72), whereas comprehensive gene panels achieved 34.7% (n = 25/72). CONCLUSION: Exome sequencing allows the detection of pathogenic variants missed by (narrowly) targeted gene panel approaches. Diagnostic reassessment after genetic testing further enhances the diagnostic outcomes for NMDs.

Hereditary spastic paraplegia caused by compound heterozygous mutations outside the motor domain of the KIF1A gene.

BACKGROUND AND PURPOSE: Hereditary spastic paraplegia is a clinically and genetically heterogeneous group of rare, inherited disorders causing an upper motor neuron syndrome with (complex) or without (pure) additional neurological symptoms. Mutations in the KIF1A gene have already been associated with recessive and dominant forms of hereditary spastic paraplegia (SPG30) in a few cases. METHODS: All family members included in the study were examined neurologically. Whole-exome sequencing was used in affected individuals to identify the responsible candidate gene. Conventional Sanger sequencing was conducted to validate familial segregation. RESULTS: A family of Macedonian origin with two affected siblings, one with slowly progressive and the other one with a more complex and rapidly progressing hereditary spastic paraplegia is reported. In both affected individuals, two novel pathogenic mutations outside the motor domain of the KIF1A gene were found (NM_001244008.1:c.2909G>A, p.Arg970His and c.1214dup, p.Asn405Lysfs*40) that segregate with the disease within the family establishing the diagnosis of autosomal recessive SPG30. CONCLUSIONS: This report provides the first evidence that mutations outside the motor domain of the gene can cause (recessive) SPG30 and extends the genotype-phenotype association for KIF1A-related diseases.

The c.65-2A>G splice site mutation is associated with a mild phenotype in Danon disease due to the transcription of normal LAMP2 mRNA.

Danon disease (DD) is a rare X-linked multisystem disorder caused by mutations of the LAMP2 gene and characterized by intellectual disability, skeletal myopathy and cardiomyopathy. The survival time is severely reduced. Contrasting with the usual disease course, we report on a family with an exceptionally mild phenotype of DD despite having two potentially damaging LAMP2 mutations. Using RNA-Seq analysis, we showed that a c.65-2A>G splice site mutation results in the tissue-specific production of four different transcripts including the full-length mRNA in muscle tissue but not in leukocytes. We confirmed our results by immunohistochemistry and immunoblotting, showing the detection of LAMP2 protein only in muscle. The second mutation (c.586A>T, p.T196S) has been reported before to have an uncertain clinical significance. In our patients, however, neither of the two mutations seem to have a high enough functional impact to cause a severe phenotype. Overall, our study reveals that alternative splicing is a potential mechanism in DD with underlying splice site mutations of the LAMP2 gene in order to rescue the full-length mRNA. Moreover, our report of a mild phenotype complements the DD spectrum, which is of great importance for a rare disease suspected to be underdiagnosed.

Postictal psychosis in temporal lobe epilepsy: a case-control study.

BACKGROUND AND PURPOSE: To investigate the prevalence of postictal psychosis (PP) in patients with temporal lobe epilepsy (TLE) and to estimate the predictive value of various variables for the development of PP. METHODS: By retrospectively reviewing the charts of all patients evaluated with video-electroencephalogram (EEG)-monitoring at our unit between January 1995 and February 2012, we identified 684 patients with TLE, of which 48 patients had a history of PP. Patients with TLE and PP were compared with 200 controls (patients with TLE without a psychotic history) on demographic, clinical, EEG and magnetic resonance imaging (MRI) variables. RESULTS: The prevalence of PP in our TLE sample was 7.0%. Aggressive behaviour during PP was present in 22.9% of the sample. Univariate analysis revealed that PP was significantly associated with early age at epilepsy onset (P = 0.007), longer duration of epilepsy (P = 0.002), presence of ictal fear (P = 0.005), impaired intellectual function (P = 0.045), and bilateral ictal and interictal epileptiform activity (both P < 0.0001). Using logistic regression analysis, ictal fear [odds ratio (OR) 2.88; P = 0.015] and bilateral interictal EEG activity (OR 6.40; P < 0.0001) were predictive of PP development. No association of PP with MRI pathology or epilepsy-relevant aetiological factors was found. CONCLUSIONS: PP is a frequent and potentially dangerous complication within the course of TLE. Bilateral or widespread functional central nervous system disturbances rather than distinct structural brain alterations or certain predisposing aetiologies of epilepsy appear to be a risk factor for the development of PP. Ictal fear may be a predictive clinical variable of PP in TLE.

The effect of early prednisolone treatment on the generalization rate in ocular myasthenia gravis.

BACKGROUND AND PURPOSE: Several small retrospective studies have observed that patients with a purely ocular manifestation of myasthenia gravis (MG) are significantly less likely to convert to a generalized disease when treated early on with corticosteroids. However, given the limited number of reported patients in the literature these findings still remain controversial. METHODS: In order to increase the number of published cases, we performed a retrospective analysis on 44 patients with newly diagnosed ocular MG who were subsequently either treated with corticosteroids or received no immunosuppressive therapy at all. The generalization rate was assessed at the end of a 2-year follow-up period. RESULTS: Whereas none of 17 treated patients generalized, 11 of 27 (41%) untreated patients developed generalized symptoms. The difference between the groups was significant (P=0.003). CONCLUSIONS: Our results agree well with previous studies on this issue. Taken together, published data indicate risk ratios for generalization of below 0.32 under corticosteroid treatment in comparison to untreated patients.

Analysis of four prevalent filaggrin mutations (R501X, 2282del4, R2447X and S3247X) in Austrian and German patients with atopic dermatitis.

BACKGROUND: Recently, mutations in the filaggrin gene (FLG) have been shown to be a major predisposing factor for atopic dermatitis (AD). OBJECTIVE: In this study, we evaluated the influence of four prevalent mutations (R501X, 2282del4, R2447X and S3247X) in a large cohort of 462 Austrian and German AD patients and in 402 control individuals. RESULTS: We found a strong association of the FLG mutations with AD. Subgroup analysis revealed a significantly higher proportion of patients with an early age of disease onset and significantly higher median serum IgE levels among mutation carriers. Furthermore, we observed an overrepresentation of null alleles in AD patients with concomitant asthma compared with those without this co-morbidity. CONCLUSION: Our data confirm and extend the knowledge of the influence of FLG mutations in AD.

A novel mutation in the MFSD8 gene in late infantile neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCL) are lysosomal storage disorders and constitute the most common group of progressive neurodegenerative diseases in childhood. Most NCLs are inherited in a recessive manner and are clinically characterised by a variable age at onset, epileptic seizures, psychomotor decline, visual impairment and premature death. To date, eight causative genes have been identified to underlie various clinical forms of NCL. We performed a genome-wide linkage analysis followed by sequencing the recently described NCL gene MFSD8 in three affected and three unaffected members of a consanguineous Egyptian family with an autosomal recessively inherited progressive neurodegenerative disorder. The clinical picture of the patients was compatible with a late infantile NCL (LINCL); however, impairment of the visual system was not a cardinal symptom in the respective family. By linkage analysis, we identified two putative loci on chromosome 1p36.11-p35.1 and 4q28.1-q28.2. The latter locus (4q28.1-q28.2) contained the MFSD8 gene, comprising a novel homozygous missense mutation in exon 5 (c.362a>g /p.Tyr121Cys), which segregated with the disease in the three affected sibs. We describe a novel mutation in the previously identified MFSD8 gene in a family with a common phenotype of LINCL, but no clinical report of vision loss. Our results enlarge the mutational and perhaps the nosological spectrum of one of the recently identified subtypes of NCL, called CLN7.

Analysis of the prodynorphin promoter polymorphism in atopic dermatitis and disease-related pruritus.

Pruritus is one of the key symptoms in atopic dermatitis (AD). The prodynorphin polypeptide is a precursor protein of pruritus-modulating opioid peptides. It is encoded by the prodynorphin gene (PDYN). To investigate a possible correlation of PDYN promoter polymorphisms with intensity of pruritus in patients with AD, we genotyped 211 Austrian patients with AD and 197 nonatopic controls. No significant association of the PDYN promoter polymorphism with AD in general was found when patients with AD were compared with controls. The analysis of possible associations with pruritus intensity also showed no relevant difference in the allelic distribution between patients with different pruritus-score values. These data argue against an important role of the PDYN promoter polymorphism in AD in general and in the development of disease-related pruritus, although owing to our small sample size, a weak effect cannot be excluded. Additional studies are needed to further evaluate the influence of PDYN polymorphism in pruritus.

Report of the Task Force on pre-graduate education in Europe of the education committee of the European Federation of Neurological Societies Composition of the task force of the education committee on pre-graduate education.

BACKGROUND AND PURPOSE: The demographical evolution and the technological revolution seen in the last decades, in developed countries, have dramatically changed the practice of Neurology. However, the academic curriculum in many medical schools has not been updated accordingly over many of the European Countries. The Education Committee of the European Federation of Neurological Societies (EFNS) implemented in 2000 a Task Force on pre-graduate education trying to give guidelines to adequate pre-graduate education to the present status. METHODS AND DISCUSSION: Based on the results of two questionnaires, the first sent to the delegates of the EFNS and to the delegates of the European Board of Neurology, and the second answered by the Task Force members themselves, this paper describes the Task Force recommendations aimed to improve Neurology Education in the Medical Schools. These recommendations are also discussed with the analyses of the current bibliography available.

Nuclear calmodulin responds rapidly to calcium influx at the plasmalemma.

We have studied the rate and extent of calcium binding to calmodulin in neuronal cytosol and nucleus during brief calcium influx across the plasmalemma. Rat sensory neurones were whole-cell patch clamped using a pipette containing a fluorescent analogue of calmodulin that reports when it has bound calcium. Cytosolic and nuclear signals were separated using a confocal microscope. Plasmalemmal calcium influx during a one second depolarization that activated L type calcium channels caused large fractions of calmodulin in both the cytosol and nucleus to bind calcium. Thus, contrary to previous predictions, nuclear processes that require the calcium:calmodulin complex will be activated readily by even brief cell stimulation.

Expression of adhesion molecules and histocompatibility antigens at the blood-brain barrier.

The migration of inflammatory cells through the blood-brain barrier in health and disease involves complex interactions between hematogenous cells, endothelial cells, the basement membrane and the perivascular glia limitans. Recent evidence is presented, suggesting that part of these interactions involve antigen independent mechanisms, mediated by cellular adhesion molecules. The accessibility of endothelial adhesion molecules in the intact blood-brain barrier is lower compared to vessels in other organs. This may account for the low traffic of hematogenous cells through the normal blood-brain barrier. However, in inflammatory conditions the expression of endothelial adhesion molecules is upregulated, which may lead to recruitment of inflammatory cells into the lesions. Antigen specific activation of T-cells at the blood-brain barrier apparently takes place mainly at perivascular monocytes and microglia cells. In severe inflammatory lesions, however, astrocytes may be additionally involved in antigen presentation.

Altered expression of voltage-dependent calcium channel alpha(1) subunits in temporal lobe epilepsy with Ammon's horn sclerosis.

Voltage-dependent calcium channels, the initial components in the calcium signalling cascade, are increasingly being recognised as relevant factors in the pathology of epilepsy. To further characterise their role in temporal lobe epilepsy associated with Ammon's horn sclerosis, we investigated the immunohistochemical distribution of five different voltage-dependent calcium channel alpha(1) subunits (alpha(1A), alpha(1B), alpha(1C), alpha(1D), alpha(1E)) in 14 hippocampal specimens of patients with Ammon's horn sclerosis in comparison with eight autopsy control cases. In epilepsy specimens an increased immunoreactivity was observed for alpha(1A), alpha(1B), alpha(1D) and alpha(1E) in the neuropil of the dentate gyrus molecular layer. Dentate gyrus granule cells and residual CA3 pyramidal neurones showed enhanced immunoreactivity for alpha(1A), while labelling of these neurones was decreased for alpha(1C). Astrocytes in Ammon's horn sclerosis specimens were strongly immunoreactive for the alpha(1C) subunit contrasting with an absent astrocytic alpha(1C) labelling in controls. Our results suggest that the expression of calcium channels in neurones and glial cells is dynamically regulated in temporal lobe epilepsy, supporting the relevance of calcium signalling pathways for this disease.

Biphasic effect of calcium on neurite outgrowth in neuroblastoma and cerebellar granule cells.

We have examined the effect of cytosolic calcium concentration ([Ca2+]i) on neurite outgrowth in two neuronal cells, cerebellar granule cells and N1E-115 neuroblastoma cells. The set point [Ca2+]i in unstimulated cells bathed in normal extracellular medium was 37 nM and 108 nM, respectively. When we altered extracellular calcium concentration to cause small excursions of [Ca2+]i either above or below the set point, neurite outgrowth from granule cells declined. Thus granule cells show the bell-shaped dependence of neurite outgrowth on [Ca2+]i characteristic of sensory and other neurones [Dev. Brain Res., 70 (1992) 287-290; The Axon, Oxford University Press, New York, 1994]. In contrast, neurite outgrowth from N1E-115 cells increased monotonically as [Ca2+]i was reduced. This result, which is consistent with results obtained by studying individual growth cones [J. Neurosci., 9 (1989) 4007-4020], implies that these transformed cells are aberrant in having no bell-shaped dependence of neurite outgrowth on [Ca2+]i. In both cell types an increase of [Ca2+]i above the set point reduced neurite outgrowth. However, this decline did not persist as [Ca2+]i was set to increasingly high levels by increasing extracellular calcium. Rather, in both cell types, an increase of extracellular calcium above 6.9 mM produced a second, increasing phase of neurite outgrowth.

Buffering intracellular calcium disrupts motoneuron development in intact zebrafish embryos.

Numerous studies, performed mainly on dissociated cells, have shown that calcium signals have a role during different stages of neuronal development. However, the actions of calcium during neuronal development in vivo remain to be established. The present study has investigated the role of intracellular calcium signals during development of motoneurons in the spinal cord of intact zebrafish embryos. Loading blastomeres of early embryos with either the calcium buffer BAPTA or the calcium reporter dye Calcium Green, was shown to disrupt motoneuron development in the spinal cord of embryos at 24 h postfertilisation. Loading the calcium buffer BAPTA, at an intracellular concentration of 1 mM, into the blastomeres of early embryos did not alter the resting levels of intracellular calcium, but significantly dampened transient rises in intracellular calcium in the cells of later stage embryos. Loading cells with 1 mM BAPTA significantly decreased the number of motoneurons present in the spinal cord at 24 h, indicating that calcium signals are important for normal motoneuron differentiation. Furthermore, in those BAPTA-filled cells that did adopt a motoneuron cell fate, axogenesis was found to be inhibited, suggestive of a role for calcium signalling in neurite initiation. This work provides evidence that calcium signals are necessary at several stages of motoneuron development in vivo.

Immunohistochemical localization of the alpha 1, alpha 2 and alpha 3 subunit of the GABAA receptor in the rat brain.

The immunohistochemical distribution of the alpha 1, alpha 2 and alpha 3 subunit of the gamma-aminobutyric acid-A (GABAA) receptor was investigated in the rat brain using affinity-purified antibodies against unique parts of the amino acid sequence of the respective subunits. The distribution of the 3 subunits differed markedly from each other indicating heterogeneity of the GABAA-receptor composition in different brain regions and at various receptive compartments (dendrites or somata) of neuronal cells.

Inflammation in the nervous system. Basic mechanisms and immunological concepts.

The basic questions in the pathogenesis of inflammation in the nervous system are how inflammatory cells reach the brain, where they recognize their antigen, how the nervous system interacts with local immune regulation in the lesion, and how inflammatory cells induce irreversible tissue damage. These questions have been addressed by studying the pathogenesis of experimental models of encephalomyelitis. The minimal requirement to start brain inflammation is the presence of activated circulating T-cells directed against a brain antigen and of antigen presenting cells in meninges and perivascular spaces of the nervous system. Such a constellation, however, only results in the disease after hypersensitization, i.e. in the presence of very high numbers of circulating autoreactive T-cells. Other local and systemic immunological factors may profoundly lower the threshold for the induction of brain inflammation. They include antigen recognition on cells in the brain parenchyma (microglia, astroglia), local upregulation of MHC antigens and possibly adhesion molecules (by cytokines or as a consequence of brain injury) and the presence of additional humoral immune responses against brain antigens (autoantibodies). Focal production of cytokines by inflammatory cells as well as by resident cells of the brain plays an important role in determining the activity of the inflammatory process and in inducing effector cells and inflammatory mediators, responsible for tissue destruction. Whereas in pure T-cell mediated auto-immune encephalomyelitis these activated effector mechanisms have low selectivity and mainly induce a "bystander" damage of CNS tissue, additional presence of autoantibodies may focus the immune reaction to specific targets, thus inducing, in high sensitivity, very selective tissue destruction. The present experimental data suggest that different immunological pathways may finally lead to quite similar inflammatory demyelinating lesions. Thus, brain lesions in individual multiple sclerosis patients may develop on a quite diverse immunological background.

A splice site variant in the sodium channel gene SCN1A confers risk of febrile seizures.

OBJECTIVE: Our aim was to investigate whether the risk of febrile seizures is influenced by a common functional polymorphism in the sodium channel gene SCN1A. This single nucleotide polymorphism (IVS5N+5 G>A, rs3812718) was shown to modify the proportion of two alternative transcripts of the channel. METHODS: We performed an exploratory case-control association analysis in 90 adult epilepsy patients with childhood febrile seizures vs 486 epilepsy patients without a history of febrile seizures and also vs 701 population controls. In the replication step, we investigated children with febrile seizures without concomitant epilepsy at the time of their inclusion. We compared the genotypes of 55 of those children against population controls and performed a within-family association analysis in an additional 88 child-parent trios with febrile seizures. RESULTS: We observed a significant association of the splice-site interrupting A-allele with febrile seizures (p value in the exploratory step: 0.000017; joint p value of the replication: 0.00069). Our data suggest that the A-allele of this variant confers a threefold genotype relative risk in homozygotes and accounts for a population attributable fraction of up to 50% for the etiology of febrile seizures. CONCLUSIONS: The A-allele of the SCN1A single nucleotide polymorphism IVS5N+5 G>A (rs3812718) represents a common and relevant risk factor for febrile seizures. A limitation of the present study is that patients of the exploratory and replication steps differed in aspects of their phenotype (febrile seizures with and without additional epilepsy).

The stigma of mental illness: anticipation and attitudes among patients with epileptic, dissociative or somatoform pain disorder.

The aim of this study was to survey the attitudes of 101 consecutive in- and out-patients with epileptic, dissociative or somatoform pain disorders (mean age: 43 [+/-11] years; 58% female) from either the Department of Psychiatry or Neurology toward anticipated mental illness stigma. The patients were administered a modified 12-item version of Links Stigma Questionnaire. Nearly 60% of all 101 patients believe that "most people" would not allow a mental patient "to take care of their children", "most young women" would be "reluctant to date a man" who has been treated for a mental illness and "most employers would pass over" the application of a psychiatric patient in favour of another applicant. Fifty five percent of the respondents assume that "most people think less of a person who has been in a mental hospital" and over a half of all patients interviewed assert that the general population thinks that psychiatric patients are "less intelligent, less trustworthy and that their opinion is taken less seriously by others". Gender, age and education had no influence on the overall results. There is a high stigmatisation concerning psychiatry even in patients with epilepsy and somatoform/dissociative symptoms with psychiatric comorbidity. Fear of being stigmatized is more pronounced among somatoform pain patients as compared to patients suffering from epileptic or dissocative disorders, with particular reference to close personal relationships.

Idiopathic generalized epilepsy phenotypes associated with different EFHC1 mutations.

We sequenced 61 patients with various idiopathic generalized epilepsy (IGE) syndromes for mutations in the EFHC1 gene. We detected three novel heterozygous missense mutations (I174V, C259Y, A394S) and one possibly pathogenic variant in the 3' UTR (2014t>c). The mutation I174V was also detected in 1 of 372 screened patients with temporal lobe epilepsy. We conclude that mutations in the EFHC1 gene may underlie different types of epilepsy syndromes.