De novo and biallelic DEAF1 variants cause a phenotypic spectrum.

PURPOSE: To investigate the effect of different DEAF1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and on DEAF1 activity in vitro. METHODS: We assembled a cohort of 23 patients with de novo and biallelic DEAF1 variants, described the genotype-phenotype correlation, and investigated the differential effect of de novo and recessive variants on transcription assays using DEAF1 and Eif4g3 promoter luciferase constructs. RESULTS: The proportion of the most prevalent phenotypic features, including intellectual disability, speech delay, motor delay, autism, sleep disturbances, and a high pain threshold, were not significantly different in patients with biallelic and pathogenic de novo DEAF1 variants. However, microcephaly was exclusively observed in patients with recessive variants (p < 0.0001). CONCLUSION: We propose that different variants in the DEAF1 gene result in a phenotypic spectrum centered around neurodevelopmental delay. While a pathogenic de novo dominant variant would also incapacitate the product of the wild-type allele and result in a dominant-negative effect, a combination of two recessive variants would result in a partial loss of function. Because the clinical picture can be nonspecific, detailed phenotype information, segregation, and functional analysis are fundamental to determine the pathogenicity of novel variants and to improve the care of these patients.

Targeted next generation sequencing as a diagnostic tool in epileptic disorders.

PURPOSE: Epilepsies have a highly heterogeneous background with a strong genetic contribution. The variety of unspecific and overlapping syndromic and nonsyndromic phenotypes often hampers a clear clinical diagnosis and prevents straightforward genetic testing. Knowing the genetic basis of a patient's epilepsy can be valuable not only for diagnosis but also for guiding treatment and estimating recurrence risks. METHODS: To overcome these diagnostic restrictions, we composed a panel of genes for Next Generation Sequencing containing the most relevant epilepsy genes and covering the most relevant epilepsy phenotypes known so far. With this method, 265 genes were analyzed per patient in a single step. We evaluated this panel on a pilot cohort of 33 index patients with concise epilepsy phenotypes or with a severe but unspecific seizure disorder covering both sporadic and familial cases. KEY FINDINGS: We identified presumed disease-causing mutations in 16 of 33 patients comprising sequence alterations in frequently as well as in less commonly affected genes. The detected aberrations encompassed known and unknown point mutations (SCN1A p.R222X, p. E289V, p.379R, p.R393H; SCN2A p.V208E; STXBP1 p.R122X; KCNJ10 p.L68P, p.I129V; KCTD7 p.L108M; KCNQ3 p.P574S; ARHGEF9 p.R290H; SMS p.F58L; TPP1 p.Q278R, p.Q422H; MFSD8 p.T294K), a putative splice site mutation (SCN1A c.693A> p.T/P231P) and small deletions (SCN1A p.F1330Lfs3X [1 bp]; MFSD8 p.A138Dfs10X [7 bp]). All mutations have been confirmed by conventional Sanger sequencing and, where possible, validated by parental testing and segregation analysis. In three patients with either Dravet syndrome or myoclonic epilepsy, we detected SCN1A mutations (p.R222X, p.P231P, p.R393H), even though other laboratories had previously excluded aberrations of this gene by Sanger sequencing or high-resolution melting analysis. SIGNIFICANCE: We have developed a fast and cost-efficient diagnostic screening method to analyze the genetic basis of epilepsies. We were able to detect mutations in patients with clear and with unspecific epilepsy phenotypes, to uncover the genetic basis of many so far unresolved cases with epilepsy including mutation detection in cases in which previous conventional methods yielded falsely negative results. Our approach thus proved to be a powerful diagnostic tool that may contribute to collecting information on both common and unknown epileptic disorders and in delineating associated phenotypes of less frequently mutated genes.

Isolation and molecular characterization of brain microvascular endothelial cells from human brain tumors.

Brain tumor formation and growth is accompanied by the proliferation and infiltration of blood capillaries. The phenotypes of endothelial cells that make up capillaries are known to differ not only in the tissues in which endothelial cells are located but also as a result of the microenvironment to which they are exposed. For this reason, primary cultures of brain endothelial cells were isolated from human brain tumors removed by surgery and compared with cells from normal tissue. The primary confluent monolayers that grew out of isolated capillary fragments consisted of closely associated, elongated, fusiform-shaped cells. But brain tumor-derived endothelial cells in culture exhibited significantly less expression of endothelial-specific Factor VIII-related antigen compared with cells isolated from normal tissue. Cultured cells that exhibited binding of Ulex europaeus lectin were shown to take up Dil-Ac-Ldl and formed continuous monolayers that were joined together by tight junctions. The cells also exhibited characteristics of the cells of the brain microvasculature in vitro as seen by the presence of large numbers of mitochondria and few pinocytotic vesicles and by the absence of Weibel-Palade bodies within the cells. The expression of vascular cell adhesion molecule-1, E-Selectin, and the tight junction associated protein ZO-1 but not intercellular adhesion molecule-1 was demonstrated by immunohistological staining or reverse transcriptase-polymerase chain reaction methodologies. Comparative studies of these endothelial cells with endothelial cells from normal tissue will be useful for determining and understanding how the blood-brain barrier differs and functions in tumor and healthy tissues and may lead to strategies for brain tumor therapeutic approaches.

On the application of cannabis in paediatrics and epileptology.

An initial report on the therapeutic application of delta 9-THC (THC) (Dronabinol, Marinol) in 8 children resp. adolescents suffering from the following conditions, is given: neurodegenerative disease, mitochondriopathy, posthypoxic state, epilepsy, posttraumatic reaction. THC effected reduced spasticity, improved dystonia, increased initiative (with low dose), increased interest in the surroundings, and anticonvulsive action. The doses ranged from 0.04 to 0.12 mg/kg body weight a day. The medication was given as an oily solution orally in 7 patients, via percutaneous gastroenterostomy tube in one patient. At higher doses disinhibition and increased restlessness were observed. In several cases treatment was discontinued and in none of them discontinuing resulted in any problems. The possibility that THC-induced effects on ion channels and transmitters may explain its therapeutic activity seen in epileptic patients is discussed.

Etiology and site of temporal lobe epilepsy influence postictal cytokine release.

Inflammatory mechanisms are involved in the pathogenesis of epilepsy. Vice versa, immune functions are regulated by the brain. We measured postictal changes in serum levels of the immuno-modulating cytokines IL-1beta, IL-6 and TNFalpha in patients with well-defined temporal lobe epilepsy (TLE) and determined modifying factors. Serum levels of IL-1beta, IL-6 and TNFalpha were quantified by ELISA at baseline as well as immediately, 1h and 24h after a complex partial (CPS) or secondary generalized tonic-clonic seizure (GTCS) during video-EEG monitoring in 25 patients suffering from temporal epilepsy. IL-6 increased by 51% immediately after the seizure (p<0.01) and remained elevated for 24h. This increase lacked in patients with hippocampal sclerosis (HS; n=16, mean increase 28%, p>0.5, vs. 112%, p<0.01 in patients without HS). IL-6 levels were higher after right-sided seizures as compared to left-sided seizures 24h after the seizure (8.7pg/mL vs. 3.4pg/mL, p<0.05). In patients taking valproate (VPA, n=9), the levels of IL-1beta were higher as compared to patients not treated with VPA. The results suggest a relationship between the cytokine system and characteristics of TLE such as side and pathology.

NK and CD4+ T cell changes in blood after seizures in temporal lobe epilepsy.

UNLABELLED: Immunological phenomena may affect the course of focal epilepsy. We analyzed prospectively the pre- and postictal distribution of leukocyte subsets in epileptic patients. METHODS: Twenty-two patients (age 36.6+/-10.8 years, 50% men) with temporal lobe epilepsy were included. Distribution of leukocyte subsets and serum levels of epinephrine were measured in peripheral blood immediately and 24 h after seizures and compared to baseline values. RESULTS: In the immediate postictal state (10+/-6 min), we observed a significant relative increase of total leukocytes (42%, p=0.0004), neutrophil leukocytes (55%, p=0.0007), total lymphocytes (45%, p=0.0019), natural killer (NK) cells (104%, p=0.0017), and epinephrine (454%, p=0.0014). CD4(+) T cells decreased by 13% (p=0.0113). These postictal changes remained significant considering only complex partial seizures (n=17). The alterations were more pronounced in patients with hippocampal sclerosis. Treatment with valproic acid (VPA) was accompanied by a greater postictal decrease of CD4(+) T cells (25% compared to 5% in patients without VPA, p=0.041) while treatment with levetiracetam (LEV) correlated with a low postictal increase of NK-like T cells (4% versus 41%, p=0.016). Twenty-four hours after the seizures the alterations had resolved. CONCLUSION: Profound postictal changes in the immune cell composition of the peripheral blood may have been mediated by epinephrine release. The greater immune response in patients with mesial temporal lobe epilepsy due to hippocampal sclerosis may reflect a close relationship between mesial temporal structures and the sympathetic nerve system. VPA and LEV may have an impact on seizure induced immunological changes.