Similarity of Phenotype in Three Male Patients With the c.320A>G Variant in ALG13: Possible Genotype-Phenotype Correlation.

BACKGROUND: Congenital disorders of glycosylation (CDG) are a group of neurometabolic diseases that result from genetic defects in the glycosylation of proteins and/or lipids. Multiple pathogenic genes contribute to the varying reported phenotypes of individuals with CDG-1 syndromes, most of which are inherited as autosomal recessive traits, although X-linked inheritance has also been reported. Pathogenic variants in the asparagine-linked glycosylation 13 homolog (ALG13) gene have been implicated in the aetiology of developmental and epileptic encephalopathy (DEE) 36 (OMIM:*300776, DEE36). The NM_001099922.3:c.320A>G; p.(Asn107Ser) variant is the most frequently described pathogenic variant in ALG13, with 59 females and 2 males with this variant reported to date. METHODS: We report on a male with a de novo, hemizygous variant in ALG13: c.320A>G; p.(Asn107Ser), whose phenotype resembles that of two previously reported males with the same variant. RESULTS: All three males have a de novo mutation, infantile spasms, DEE, drug-resistant epilepsy, intellectual disability, dysmorphic findings, recurrent infections, skeletal anomalies, brain abnormalities and a movement disorder: a phenotype not consistently reported in males with other pathogenic variants in ALG13. CONCLUSION: The similarity of phenotype in the three males with the c.320A>G variant in ALG13, suggests a possible genotype-phenotype correlation.

Novel CDKL5 targets identified in human iPSC-derived neurons.

CDKL5 Deficiency Disorder (CDD) is a debilitating epileptic encephalopathy disorder affecting young children with no effective treatments. CDD is caused by pathogenic variants in Cyclin-Dependent Kinase-Like 5 (CDKL5), a protein kinase that regulates key phosphorylation events in neurons. For therapeutic intervention, it is essential to understand molecular pathways and phosphorylation targets of CDKL5. Using an unbiased phosphoproteomic approach we identified novel targets of CDKL5, including GTF2I, PPP1R35, GATAD2A and ZNF219 in human iPSC-derived neuronal cells. The phosphoserine residue in the target proteins lies in the CDKL5 consensus motif. We validated direct phosphorylation of GTF2I and PPP1R35 by CDKL5 using complementary approaches. GTF2I controls axon guidance, cell cycle and neurodevelopment by regulating expression of neuronal genes. PPP1R35 is critical for centriole elongation and cilia morphology, processes that are impaired in CDD. PPP1R35 interacts with CEP131, a known CDKL5 phospho-target. GATAD2A and ZNF219 belong to the Nucleosome Remodelling Deacetylase (NuRD) complex, which regulates neuronal activity-dependent genes and synaptic connectivity. In-depth knowledge of molecular pathways regulated by CDKL5 will allow a better understanding of druggable disease pathways to fast-track therapeutic development.

Developmental epileptic encephalopathy caused by homozygosity of a c.172+1G>C variant in the WWOX gene.

BACKGROUND: Variations in the WWOX gene have been identified as the leading cause of several central nervous system disorders. However, most previous reports have focused on the description of clinical phenotype, neglecting functional verification. Herein, we presented a case of a patient with developmental epileptic encephalopathy (DEE) caused by WWOX gene variation. CASE PRESENTATION: Our patient was a 13-month-old girl with abnormal facial features, including facial hypotonia, arched eyebrows, a broad nose, and a depressed nasal bridge. She also had sparse and yellow hair, a low anterior hairline, and a short neck. Before the age of 8 months, she was suffering from mild seizures. Her developmental delay gradually worsened, and she suffered infantile spasms. After treatment with vigabatrin, seizures subsided. WWOX gene homozygous variation c.172+1G>C was identified using whole exome sequencing. Further minigene assay confirmed that the variation site affected splicing, causing protein truncation and affecting its function. CONCLUSION: Clinical phenotype and minigene results suggest that WWOX gene homozygous variation c.172+1G>C can cause severe DEE. We also concluded that vigabatrin can effectively treat seizures.

Identification of a novel splice-site WWOX variant with paternal uniparental isodisomy in a patient with infantile epileptic encephalopathy.

WOREE syndrome is an early infantile epileptic encephalopathy characterized by drug-resistant seizures and severe psychomotor developmental delays. We report a case of a WWOX splice-site mutation with uniparental isodisomy. A 1-year and 7-month-old girl presented with nystagmus and epileptic seizures from early infancy, with no fixation or pursuit of vision. Physical examination revealed small deformities, such as swelling of both cheeks, folded fingers, rocking feet, and scoliosis. Brain imaging revealed slight hypoplasia of the cerebrum. Electroencephalogram showed focal paroxysmal discharges during the interictal phase of seizures. Vitamin B6 and zonisamide were administered for early infantile epileptic encephalopathy; however, the seizures were not relieved. Despite altering the type and dosage of antiepileptic drugs and ACTH therapy, the seizures were intractable. Whole-exome analysis revealed the homozygosity of WWOX(NM_016373.4):c.516+1G>A. The WWOX mRNA sequencing using peripheral blood RNA confirmed that exon 5 was homozygously deleted. Based on these results, the patient was diagnosed with WOREE syndrome at 5 months. The WWOX variant found in this study is novel and has never been reported before. WOREE syndrome being extremely rare, further case series and analyses of its pathophysiology are warranted.

Novel Homozygous Variants of SLC13A5 Expand the Functional Heterogeneity of a Homogeneous Syndrome of Early Infantile Epileptic Encephalopathy.

BACKGROUND: Early infantile epileptic encephalopathy 25 (EIEE25) is a distinct type of neonatal epileptic encephalopathy caused by autosomal recessive mutations in the SLC13A5 gene. SLC13A5 encodes a transmembrane sodium/citrate cotransporter required for regulating citrate entry into cells. METHODS: Four families with recessively inherited epileptic encephalopathy were sequenced by clinically accredited laboratories using commercially available epilepsy gene panels. Patients were examined by a neurologist and were clinically diagnosed with infantile epileptic encephalopathy. RESULTS: We present four families with global developmental delay, intellectual disability, and defective tooth development with four novel homozygous mutations in SLC13A5. The neurological examination showed spastic quadriplegia with increased deep tendon reflexes. Brain magnetic resonance imaging showed nonspecific signal abnormality of the bilateral hemispheric white matter. Despite similar clinical features, the conditions were based on different molecular mechanisms acting on SLC13A5 (abnormal splicing, large-scale deletions, and tandem-residue insertion). CONCLUSIONS: Our results extend the landscape of autosomal recessive inherited homozygous mutations in SLC13A5 that cause a distinctive syndrome of severe neonatal epileptic encephalopathy. Our observations confirm the homogeneity of epileptic encephalopathy and dental abnormalities as a distinct clinical marker for EIEE25 despite the heterogeneous functional and mutational background.

Aicardi syndrome in a Nigerian female child: A case report and literature review of a rare neuro-developmental disorder from North-Western Nigeria.

Aicardi syndrome is a very rare neurodevelopmental disorder, inherited as an X-linked dominant condition with a triad of infantile spasm, partial or complete agenesis of the corpus callosum, and chorio-retinal "lacunae." We report a case of a female infant with the classical triad of Aicardi syndrome. A female infant presented to the Paediatric Neurology Clinic of the Federal Medical Centre Birnin-Kebbi, North-western Nigeria, at the age of two months with complaints of recurrent afebrile convulsions typical for infantile spasms. The patient was delivered at term with normal Apgar scores and anthropometry. Examination revealed an infant with no dysmorphic features and normal systemic examination. Magnetic Resonance Imaging (MRI) of the brain however, showed complete agenesis of the corpus callosum and dilatation of the posterior horn of the lateral and third ventricles. Fundoscopy showed multiple yellowish spots along the vascular arcades in the right eye. The left eye had a one-disc diameter lacuna in the superior nasal quadrant adjacent to the optic disc with multiple yellowish spots. A diagnosis of Aicardi syndrome was made. The child was placed on oral phenobarbital and followed up. At the age of 18 months, the child can only sit without support, hold an object in each hand, smile socially, and babble. The frequency of the seizures had also reduced from >100 episodes per day to 2-3 episodes per day, but the child had developed right-sided spastic hemiparesis. The patient was commenced on physiotherapy and the anti-epileptic drugs were maintained. We recommend clinicians consider Aicardi syndrome in the differential diagnosis of any child presenting with infantile spasms.

Focal signs in infantile spasms.

BACKGROUND: Infantile spasms belong to the group of epileptic encephalopathies that typically occur in early infancy and are often associated with severe developmental delay. Little is known about whether focal features are part of the syndrome and thus occur independently of etiology, or whether focal features always indicate a cerebral lesion. METHODS: In our study we included all patients with infantile spasms documented by prolonged video-electroencephalogram (EEG) monitoring between 7/2003 and 11/2020 and analysed symptoms such as tonic posturing, clonic movements, deviation of the eyes and unilateral deviation of the mouth. These symptoms were classified as lateralizing or non-lateralizing and the correlation to the presence of a lesion was investigated. RESULTS: Eighteen patients (9 w/9 m) were included in the study. Lateralizing tonic posturing was found in 66.6% of the patients. Deviation of the eyes to one side and unilateral deviation of the mouth were detected in 61.1% and 11.1% of patients, respectively. Taking into account all symptoms (tonic posturing, clonic movements, deviation of the eyes, unilateral deviation of the mouth), focal signs were observed in a total of 94.4%, with only half of the total patient population having a cerebral lesion. CONCLUSION: In our study, lateralizing symptoms in infantile spasms occurred independently of the presence of a lesion. In contrast, focal symptoms in older children or adults usually correlate with the presence and localization of a lesion. A possible hypothesis could be that the brain is still maturing in infancy.

Exome first approach to reduce diagnostic costs and time - retrospective analysis of 111 individuals with rare neurodevelopmental disorders.

This single-center study aims to determine the time, diagnostic procedure, and cost saving potential of early exome sequencing in a cohort of 111 individuals with genetically confirmed neurodevelopmental disorders. We retrospectively collected data regarding diagnostic time points and procedures from the individuals' medical histories and developed criteria for classifying diagnostic procedures in terms of requirement, followed by a cost allocation. All genetic variants were re-evaluated according to ACMG recommendations and considering the individuals' phenotype. Individuals who developed first symptoms of their underlying genetic disorder when Next Generation Sequencing (NGS) diagnostics were already available received a diagnosis significantly faster than individuals with first symptoms before this cutoff. The largest amount of potentially dispensable diagnostics was found in genetic, metabolic, and cranial magnetic resonance imaging examinations. Out of 407 performed genetic examinations, 296 (72.7%) were classified as potentially dispensable. The same applied to 36 (27.9%) of 129 cranial magnetic resonance imaging and 111 (31.8%) of 349 metabolic examinations. Dispensable genetic examinations accounted 302,947.07€ (90.2%) of the total 335,837.49€ in potentially savable costs in this cohort. The remaining 32,890.42€ (9.8%) are related to non-required metabolic and cranial magnetic resonance imaging diagnostics. On average, the total potentially savable costs in our study amount to €3,025.56 per individual. Cost savings by first tier exome sequencing lie primarily in genetic, metabolic, and cMRI testing in this German cohort, underscoring the utility of performing exome sequencing at the beginning of the diagnostic pathway and the potential for saving diagnostic costs and time.

CDKL5 kinase controls transcription-coupled responses to DNA damage.

Mutations in the gene encoding the CDKL5 kinase are among the most common genetic causes of childhood epilepsy and can also give rise to the severe neurodevelopmental condition CDD (CDKL5 deficiency disorder). Despite its importance for human health, the phosphorylation targets and cellular roles of CDKL5 are poorly understood, especially in the cell nucleus. Here, we report that CDKL5 is recruited to sites of DNA damage in actively transcribed regions of the nucleus. A quantitative phosphoproteomic screen for nuclear CDKL5 substrates reveals a network of transcriptional regulators including Elongin A (ELOA), phosphorylated on a specific CDKL5 consensus motif. Recruitment of CDKL5 and ELOA to damaged DNA, and subsequent phosphorylation of ELOA, requires both active transcription and the synthesis of poly(ADP-ribose) (PAR), to which CDKL5 can bind. Critically, CDKL5 kinase activity is essential for the transcriptional silencing of genes induced by DNA double-strand breaks. Thus, CDKL5 is a DNA damage-sensing, PAR-controlled transcriptional modulator, a finding with implications for understanding the molecular basis of CDKL5-related diseases.

Pathogenic DNM1L Variant (1085G>A) Linked to Infantile Progressive Neurological Disorder: Evidence of Maternal Transmission by Germline Mosaicism and Influence of a Contemporary in cis Variant (1535T>C).

Mitochondria are dynamic organelles undergoing continuous fusion and fission with Drp1, encoded by the DNM1L gene, required for mitochondrial fragmentation. DNM1L dominant pathogenic variants lead to progressive neurological disorders with early exitus. Herein we report on the case of a boy affected by epileptic encephalopathy carrying two heterozygous variants (in cis) of the DNM1L gene: a pathogenic variant (PV) c.1085G>A (p.Gly362Asp) accompanied with a variant of unknown significance (VUS) c.1535T>C (p.Ile512Thr). Amplicon sequencing of the mother's DNA revealed the presence of the PV and VUS in 5% of cells, with the remaining cells presenting only VUS. Functional investigations performed on the patient and his mother's cells unveiled altered mitochondrial respiratory chain activities, network architecture and Ca2+ homeostasis as compared with healthy unrelated subjects' samples. Modelling Drp1 harbouring the two variants, separately or in combination, resulted in structural changes as compared with Wt protein. Considering the clinical history of the mother, PV transmission by a maternal germline mosaicism mechanism is proposed. Altered Drp1 function leads to changes in the mitochondrial structure and bioenergetics as well as in Ca2+ homeostasis. The novel VUS might be a modifier that synergistically worsens the phenotype when associated with the PV.

Biallelic mutations in ELFN1 gene associated with developmental and epileptic encephalopathy and joint laxity.

ELFN1, a transmembrane leucine rich repeat protein, is involved in signal transduction in both neural cells and ROD ON-bipolar synaptogenesis. We present three siblings with developmental and epileptic encephalopathy and co-morbidities due to ELFN1 gene mutation; this is the first report in literature defining the human phenotype of ELFN1 gene mutation. Clinical, electrophysiological, and radiological findings along with comprehensive genetic studies of the patients and their family members are presented. Developmental and epileptic encephalopathy, autistic features, pyramidal signs, joint laxity, and dysmorphic features are the characteristic findings of this new clinical entity, involving mainly nervous system and possibly connective tissue. Whole exome sequence analysis followed by Sanger sequencing in all family members revealed disease-causing 8 bp frameshift mutation depicted as NM_001128636.2: c.42_49delGGCCGCCA; p. (Ala15Profs*241) in ELFN1. The variant, located in the signal peptide domain in the ELFN1 gene, was found to be homozygous in three patients, and heterozygous in the parents and three healthy siblings. Segregation analysis in family members together with pathogenicity assessment tools strongly supported the damaging effect of the frameshift variant on the function of the ELFN1 protein. Mutations in ELFN1 gene may be considered in patients with neonatal and infantile-onset epileptic encephalopathy before the full clinical picture is apparent.

Description of a novel patient with the TRPM3 recurrent p.Val837Met variant.

De novo heterozygous missense mutations in TRPM3 have been shown to cause developmental and epileptic encephalopathies (DEE). It is a very rare condition, as only 9 patients have been described to date. We report here a novel patient carrying the recurrent p.Val837Met variant and presenting new clinical features, such as trigonocephaly, expanding the phenotypical spectrum of the disease.

X-linked neonatal-onset epileptic encephalopathy associated with a gain-of-function variant p.R660T in GRIA3.

The X-linked GRIA3 gene encodes the GLUA3 subunit of AMPA-type glutamate receptors. Pathogenic variants in this gene were previously reported in neurodevelopmental diseases, mostly in male patients but rarely in females. Here we report a de novo pathogenic missense variant in GRIA3 (c.1979G>C; p. R660T) identified in a 1-year-old female patient with severe epilepsy and global developmental delay. When exogenously expressed in human embryonic kidney (HEK) cells, GLUA3_R660T showed slower desensitization and deactivation kinetics compared to wildtype (wt) GLUA3 receptors. Substantial non-desensitized currents were observed with the mutant but not for wt GLUA3 with prolonged exposure to glutamate. When co-expressed with GLUA2, the decay kinetics were similarly slowed in GLUA2/A3_R660T with non-desensitized steady state currents. In cultured cerebellar granule neurons, miniature excitatory postsynaptic currents (mEPSCs) were significantly slower in R660T transfected cells than those expressing wt GLUA3. When overexpressed in hippocampal CA1 neurons by in utero electroporation, the evoked EPSCs and mEPSCs were slower in neurons expressing R660T mutant compared to those expressing wt GLUA3. Therefore our study provides functional evidence that a gain of function (GoF) variant in GRIA3 may cause epileptic encephalopathy and global developmental delay in a female subject by enhancing synaptic transmission.

Confirming the contribution and genetic spectrum of de novo mutation in infantile spasms: Evidence from a Chinese cohort.

OBJECTIVE: We determined the yield, genetic spectrum, and actual origin of de novo mutations (DNMs) for infantile spasms (ISs) in a Chinese cohort. The efficacy of levetiracetam (LEV) for STXBP1-related ISs was explored also. METHODS: Targeted sequencing of 153 epilepsy-related candidate genes was applied to 289 Chinese patients with undiagnosed ISs. Trio-based amplicon deep sequencing was used for all DNMs to distinguish somatic/mosaic mutations from germline ones. RESULTS: Total of 26 DNMs were identified from 289 recruited Chinese patients with undiagnosed ISs. Among them, 24 DNMs were interpreted as pathogenic mutations based on American College of Medical Genetics and Genomics guidelines, contributing to 8.3% (24/289) of diagnosis yield in the Chinese IS cohort. CDKL5 and STXBP1 are the top genes with recurrent DNMs, accounting for 3.1% (9/289) of yield. Further deep resequencing for the trio members showed that 22.7% (5/22) of DNMs are actually somatic in the proband or a parent. These somatic carriers presented milder seizure attacks than those with true germline DNMs. After treatment with LEV for half a year, three patients with DNM in STXBP1 showed improved clinical symptoms, including seizure-free and normal electroencephalogram, except for a patient with a second DNM in DIAPH3. SIGNIFICANCE: Our study confirmed the contribution and genetic spectrum of DNMs in Chinese IS patients. Somatic mutation account for a quarter of DNMs in IS cases. Treatment with LEV improved the prognosis of STXBP1-related ISs.

A description of novel variants and review of phenotypic spectrum in UBA5-related early epileptic encephalopathy.

Early infantile epileptic encephalopathy-44 (EIEE44, MIM: 617132) is a previously described condition resulting from biallelic variants in UBA5, a gene involved in a ubiquitin-like post-translational modification system called UFMylation. Here we report five children from four families with biallelic pathogenic variants in UBA5 All five children presented with global developmental delay, epilepsy, axial hypotonia, appendicular hypertonia, and a movement disorder, including dystonia in four. Affected individuals in all four families have compound heterozygous pathogenic variants in UBA5 All have the recurrent mild c.1111G > A (p.Ala371Thr) variant in trans with a second UBA5 variant. One patient has the previously described c.562C > T (p. Arg188*) variant, two other unrelated patients have a novel missense variant, c.907T > C (p.Cys303Arg), and the two siblings have a novel missense variant, c.761T > C (p.Leu254Pro). Functional analyses demonstrate that both the p.Cys303Arg variant and the p.Leu254Pro variants result in a significant decrease in protein function. We also review the phenotypes and genotypes of all 15 previously reported families with biallelic UBA5 variants, of which two families have presented with distinct phenotypes, and we describe evidence for some limited genotype-phenotype correlation. The overlap of motor and developmental phenotypes noted in our cohort and literature review adds to the increasing understanding of genetic syndromes with movement disorders-epilepsy.

Brain morphological abnormalities in children with cyclin-dependent kinase-like 5 deficiency disorder.

BACKGROUND: To quantitatively evaluate the brain MRI morphological abnormalities in patients with cyclin-dependent kinase-like 5 deficiency disorder (CDD) on a group level and longitudinally. METHODS: We performed surface-based MRI analysis on high-resolution T1-weighted images on three CDD patients scanned at age of three years, and compared with 12 age- and gender-matched healthy controls. We further examined the longitudinal morphological changes in one patient with a follow-up of 5 years. RESULTS: CDD patients presented significant reductions in total intracranial volume, total gray matter (GM) volume and subcortical GM volume compared to controls. For subcortical regions, significant GM volume reductions were seen in the brain stem, bilateral thalamus, bilateral hippocampus, bilateral cerebellum and left amygdala. Although GM volume of cortical mantle did not show statistical differences overall, significant reduction was detected in bilateral parietal, left occipital and right temporal lobes. Cortical thickness exhibited significant decreases in bilateral occipital, parietal and temporal lobes, while surface area did not show any significant differences. Longitudinal follow-up in one patient revealed a monotonic downward trend of relative volume in the majority of brain regions. The relative surface area appeared to gain age-related growth, whereas the relative cortical thickness exhibited a striking progressive decline over time. CONCLUSIONS: Quantitative morphology analysis in children with CDD showed global volume loss in the cortex and more notably in the subcortical gray matter, with a progressive trend along with the disease course. Cortical thickness is a more sensitive measure to disclose cortical atrophy and disease progression than surface area.

Tuber Locations Associated with Infantile Spasms Map to a Common Brain Network.

OBJECTIVE: Approximately 50% of patients with tuberous sclerosis complex develop infantile spasms, a sudden onset epilepsy syndrome associated with poor neurological outcomes. An increased burden of tubers confers an elevated risk of infantile spasms, but it remains unknown whether some tuber locations confer higher risk than others. Here, we test whether tuber location and connectivity are associated with infantile spasms. METHODS: We segmented tubers from 123 children with (n = 74) and without (n = 49) infantile spasms from a prospective observational cohort. We used voxelwise lesion symptom mapping to test for an association between spasms and tuber location. We then used lesion network mapping to test for an association between spasms and connectivity with tuber locations. Finally, we tested the discriminability of identified associations with logistic regression and cross-validation as well as statistical mediation. RESULTS: Tuber locations associated with infantile spasms were heterogenous, and no single location was significantly associated with spasms. However, >95% of tuber locations associated with spasms were functionally connected to the globi pallidi and cerebellar vermis. These connections were specific compared to tubers in patients without spasms. Logistic regression found that globus pallidus connectivity was a stronger predictor of spasms (odds ratio [OR] = 1.96, 95% confidence interval [CI] = 1.10-3.50, p = 0.02) than tuber burden (OR = 1.65, 95% CI = 0.90-3.04, p = 0.11), with a mean receiver operating characteristic area under the curve of 0.73 (±0.1) during repeated cross-validation. INTERPRETATION: Connectivity between tuber locations and the bilateral globi pallidi is associated with infantile spasms. Our findings lend insight into spasm pathophysiology and may identify patients at risk. ANN NEUROL 2021;89:726-739.

Adenosine-to-inosine RNA editing in neurological development and disease.

Adenosine-to-inosine (A-to-I) editing is one of the most prevalent post-transcriptional RNA modifications in metazoan. This reaction is catalysed by enzymes called adenosine deaminases acting on RNA (ADARs). RNA editing is involved in the regulation of protein function and gene expression. The numerous A-to-I editing sites have been identified in both coding and non-coding RNA transcripts. These editing sites are also found in various genes expressed in the central nervous system (CNS) and play an important role in neurological development and brain function. Aberrant regulation of RNA editing has been associated with the pathogenesis of neurological and psychiatric disorders, suggesting the physiological significance of RNA editing in the CNS. In this review, we discuss the current knowledge of editing on neurological disease and development.