Unravelling Molecular Genetic Causes and Disease Mechanisms in Landau Kleffner Syndrome

The cost of epilepsy to an individual lies not just in the burden of having recurrent seizures but also in the condition’s neurodevelopmental, cognitive, psychological and social co-morbidities. Presently, our understanding of the pathophysiological mechanisms underlying epilepsy and its neurocognitive co-morbidities remains severely limited, translating to our current lack of targeted treatment options. This PhD study aims to better understand the pathophysiological mechanisms underlying epilepsy and its neurocognitive co-morbidities through the clinical and molecular genetic study of a cohort of patients with Landau Kleffner syndrome (LKS), an epilepsy syndrome characterised by seizures, and neurodevelopmental regression in the form of loss of speech and language skills. Patients were recruited from a database of children referred for LKS at Great Ormond Street Hospital’s Developmental Epilepsy Clinic. Clinical data was extracted through case note review. As mutations in GRIN2A, a gene encoding the N2A subunit of the Nmethyl-D-Aspartate (NMDA) receptor have previously been described in 8-20% of individuals with LKS and related disorders, recruited individuals were screened for GRIN2A mutations via Sanger Sequencing and multiplex-ligation probe amplification. Functional investigations exploring gene/protein expression, protein localisation and channel function were carried out on missense GRIN2A mutations identified. Individuals who screened negative for GRIN2A variants underwent whole exome sequencing or whole genome sequencing to identify novel genes associated with LKS. This study has drawn conclusions that LKS is a neurodevelopmental disorder and clinical features influencing prognosis include age at onset of regression, non-verbal intelligence, and the presence of motor difficulties. GRIN2A mutations are likely to lead to LKS through overall NMDA receptor loss of function effects. Nonetheless, LKS may be a complex disorder with multi-factorial or oligogenic aetiology. Lastly, the long term potentiation pathway, important for learning and memory mechanisms, features strongly in the pathogenesis of LKS

TBC1D24 Mutations in a Sibship with Multifocal Polymyoclonus

Background: Advances in molecular genetic technologies have improved our understanding of genetic causes of rare neurological disorders with features of myoclonus. Case Report: A family with two affected siblings, presenting with multifocal polymyoclonus and neurodevelopmental delay, was recruited for whole-exome sequencing following unyielding diagnostic neurometabolic investigations. Compound heterozygous mutations in TBC1D24, a gene previously associated with various epilepsy phenotypes and hearing loss, were identified in both siblings. The mutations included a missense change c.457G>A (p.Glu157Lys), and a novel frameshift mutation c.545del (p.Thr182Serfs*6). Discussion: We propose that TBC1D24-related diseases should be in the differential diagnosis for children with polymyoclonus

<i>TBC1D24</i> Mutations in a Sibship with Multifocal Polymyoclonus

<p><strong>Background:</strong>&nbsp;Advances in molecular genetic technologies have improved our understanding of genetic causes of rare neurological disorders with features of myoclonus.</p><p><strong>Case Report:</strong>&nbsp;A family with two affected siblings, presenting with multifocal polymyoclonus and neurodevelopmental delay, was recruited for whole-exome sequencing following unyielding diagnostic neurometabolic investigations. Compound heterozygous mutations in&nbsp;<em>TBC1D24</em>, a gene previously associated with various epilepsy phenotypes and hearing loss, were identified in both siblings. The mutations included a missense change c.457G&gt;A (p.Glu157Lys), and a novel frameshift mutation c.545del (p.Thr182Serfs*6).</p><p><strong>Discussion:</strong>&nbsp;We propose that&nbsp;<em>TBC1D24-</em>related diseases should be in the differential diagnosis for children with polymyoclonus.</p><p>&nbsp;</p

<i>TBC1D24</i> Mutations in a Sibship with Multifocal Polymyoclonus

<p><strong>Background:</strong>&nbsp;Advances in molecular genetic technologies have improved our understanding of genetic causes of rare neurological disorders with features of myoclonus.</p><p><strong>Case Report:</strong>&nbsp;A family with two affected siblings, presenting with multifocal polymyoclonus and neurodevelopmental delay, was recruited for whole-exome sequencing following unyielding diagnostic neurometabolic investigations. Compound heterozygous mutations in&nbsp;<em>TBC1D24</em>, a gene previously associated with various epilepsy phenotypes and hearing loss, were identified in both siblings. The mutations included a missense change c.457G&gt;A (p.Glu157Lys), and a novel frameshift mutation c.545del (p.Thr182Serfs*6).</p><p><strong>Discussion:</strong>&nbsp;We propose that&nbsp;<em>TBC1D24-</em>related diseases should be in the differential diagnosis for children with polymyoclonus.</p><p>&nbsp;</p

Clinical and molecular characterisation of KCNT1-related severe early onset epilepsy

Objective: To characterise the phenotypic spectrum, molecular genetic findings and functional consequences of pathogenic variants in early onset KCNT1-epilepsy. Methods: We identified a cohort of 31 patients with epilepsy of infancy with migrating focal seizures (EIMFS) and screened for variants in KCNT1 using direct Sanger sequencing, a multiple gene next generation sequencing panel and whole exome sequencing. Additional patients with non-EIMFS early onset epilepsy in whom we identified KCNT1 variants on local diagnostic multiple gene panel testing were also included. Where possible, we performed homology modelling to predict putative effects of variants on protein structure and function. We undertook electrophysiological assessment of mutant KCNT1 channels in a Xenopus oocyte model system. Results: We identified pathogenic variants in KCNT1 in 12 patients, four of which are novel. Most variants occurred de novo. Ten had a clinical diagnosis of EIMFS and the other two presented with early onset severe nocturnal frontal lobe seizures. Three patients had a trial of quinidine with good clinical response in one. Computational modelling analysis implicates abnormal pore function (F346L) and impaired tetramer formation (F502V) as putative disease mechanisms. All evaluated KCNT1 variants resulted in marked gain-of-function, with significantly increased channel amplitude and variable blockade by quinidine. Conclusions: Gain-of-function KCNT1 pathogenic variants cause a spectrum of severe focal epilepsies with onset in early infancy. Currently, genotype-phenotype correlations are unclear, though clinical outcome is poor for the majority of cases. Further elucidation of disease mechanisms may facilitate the development of targeted treatments, much needed for this pharmacoresistant genetic epilepsy

Mutations in the histone methyltransferase gene KMT2B cause complex early-onset dystonia.

Histone lysine methylation, mediated by mixed-lineage leukemia (MLL) proteins, is now known to be critical in the regulation of gene expression, genomic stability, cell cycle and nuclear architecture. Despite MLL proteins being postulated as essential for normal development, little is known about the specific functions of the different MLL lysine methyltransferases. Here we report heterozygous variants in the gene KMT2B (also known as MLL4) in 27 unrelated individuals with a complex progressive childhood-onset dystonia, often associated with a typical facial appearance and characteristic brain magnetic resonance imaging findings. Over time, the majority of affected individuals developed prominent cervical, cranial and laryngeal dystonia. Marked clinical benefit, including the restoration of independent ambulation in some cases, was observed following deep brain stimulation (DBS). These findings highlight a clinically recognizable and potentially treatable form of genetic dystonia, demonstrating the crucial role of KMT2B in the physiological control of voluntary movement.Funding for the project was provided by the Wellcome Trust for UK10K (WT091310) and DDD Study. The DDD study presents independent research commissioned by the Health Innovation Challenge Fund [grant number HICF-1009-003] - see www.ddduk.org/access.html for full acknowledgement. This work was supported in part by the Intramural Research Program of the National Human Genome Research Institute and the Common Fund, NIH Office of the Director. This work was supported in part by the German Ministry of Research and Education (grant nos. 01GS08160 and 01GS08167; German Mental Retardation Network) as part of the National Genome Research Network to A.R. and D.W. and by the Deutsche Forschungsgemeinschaft (AB393/2-2) to A.R. Brain expression data was provided by the UK Human Brain Expression Consortium (UKBEC), which comprises John A. Hardy, Mina Ryten, Michael Weale, Daniah Trabzuni, Adaikalavan Ramasamy, Colin Smith and Robert Walker, affiliated with UCL Institute of Neurology (J.H., M.R., D.T.), King’s College London (M.R., M.W., A.R.) and the University of Edinburgh (C.S., R.W.)

Elucidation of the effect of ionic liquid pretreatment on rice husk via structural analyses

<p>Abstract</p> <p>Background</p> <p>In the present study, three ionic liquids, namely 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), and 1-ethyl-3-methylimidazolium diethyl phosphate ([EMIM]DEP), were used to partially dissolve rice husk, after which the cellulose were regenerated by the addition of water. The aim of the investigation is to examine the implications of the ionic liquid pretreatments on rice husk composition and structure.</p> <p>Results</p> <p>From the attenuated total reflectance Fourier transform-infrared (ATR FT-IR) spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM) results, the regenerated cellulose were more amorphous, less crystalline, and possessed higher structural disruption compared with untreated rice husk. The major component of regenerated cellulose from [BMIM]Cl and [EMIM]DEP pretreatments was cellulose-rich material, while cellulose regenerated from [EMIM]OAc was a matrix of cellulose and lignin. Cellulose regenerated from ionic pretreatments could be saccharified via enzymatic hydrolysis, and resulted in relatively high reducing sugars yields, whereas enzymatic hydrolysis of untreated rice husk did not yield reducing sugars. Rice husk residues generated from the ionic liquid pretreatments had similar chemical composition and amorphousity to that of untreated rice husk, but with varying extent of surface disruption and swelling.</p> <p>Conclusions</p> <p>The structural architecture of the regenerated cellulose and rice husk residues showed that they could be used for subsequent fermentation or derivation of cellulosic compounds. Therefore, ionic liquid pretreatment is an alternative in the pretreatment of lignocellulosic biomass in addition to the conventional chemical pretreatments.</p

Evaluation of fractionation and delignification efficiencies of deep eutectic solvents on oil palm empty fruit bunch

Deep eutectic solvent (DES) has been introduced as a new generation green solvent for lignocellulosic biomass pretreatment in recent years. In the current study, six DES varying from acidic to basic nature were synthesized and applied on oil palm empty fruit bunch (EFB) in a single step fractionation and delignification process. The effect of the properties of DES on its fractionation performance was assessed. Mass balance performed on the pretreatment process reveals that pH of DES has a significant impact on biopolymer fractionation efficiency. The dissolution trend of acidic and basic DES resembles those in the conventional acid and alkaline pretreatments. The pretreatment energy requirement for the DES used ranged from 2087 to 2451 J/g. The acidic choline chloride:lactic acid DES succeeded in achieving 100% hemicellulose extraction, 88% delignification and 50% lignin pellet extraction from EFB. Thus, it is feasible for acidic DES to be considered in a biorefinery scheme for biomass fractionation

Effect of functional groups in acid constituent of deep eutectic solvent for extraction of reactive lignin

In this study, acidic deep eutectic solvents (DES) synthesized from various organic carboxylic acid hydrogen bond donors were applied to lignocellulosic oil palm empty fruit bunch (EFB) pretreatment. The influence of functional group types on acid and their molar ratios with hydrogen bond acceptor on lignin extraction were evaluated. The result showed presence of hydroxyl group and short alkyl chain enhanced biomass fractionation and lignin extraction. Choline chloride:lactic acid (CC-LA) with the ratio of 1:15 and choline chloride:formic acid (CC-FA) with 1:2 ratio extracted more than 60 wt% of lignin. CC-LA DES-extracted lignin (DEEL) exhibited comparable reactivity with technical and commercial lignin based on its phenolic hydroxyl content (3.33–3.72 mmol/glignin). Also, the DES-pretreated EFB comprised of enriched glucan content after biopolymer fractionation. Both DES-pretreated EFB and DEEL can be potential feedstock for subsequent conversion processes. This study presented DES as an effective and facile pretreatment method for reactive lignin extraction. © 201

Transformation of Starchy Lignocellulosic Biomass to Ethanol using Ragi Tapai Synergized with Microwave Irradiation Pretreatment

Ethanol production strategy was studied using multiple strain microbes from microwave irradiation (MI) pretreated sago waste. Sago waste (SW) was MI-pretreated for reducing sugars production using 2 heating media (water and sulfuric acid) under pretreatment conditions including MI power, pretreatment duration, and solid loading. When water was used, the pretreatment parameters were optimized using Box-Behnken Design (BBD). However, gelatinized starch and charring of SW led to an insignificant quadratic model. To mitigate the gelatinization problem while determining the best MI pretreatment conditions, water was substituted by sulfuric acid using single factor method. The highest reducing sugar yield of 261.5 mg/g SW was achieved at 7.5% solid loading, 6 min pretreatment duration, and 300 W MI power. The effectiveness of the pretreatment was ascertained by field-emission scanning electron microscopy (FESEM) and chemical-composition analysis. When fermenting MI-pretreated SW using ragi tapai, simultaneous saccharification of starch and ethanol production was evidenced from the sugar/ethanol profile. A resulted yield of 7.24 g ethanol/100 g SW confirmed the fermentability of MI-pretreated SW. The ethanol production was well fitted into the modified Gompertz model