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.

In vivo magnetic resonance spectroscopy in the brain of Cdkl5 null mice reveals a metabolic profile indicative of mitochondrial dysfunctions.

Mutations in the X-linked CDKL5 gene cause CDKL5 deficiency disorder (CDD), a severe neurodevelopmental condition mainly characterized by infantile epileptic encephalopathy, intellectual disability, and autistic features. The molecular mechanisms underlying the clinical symptoms remain largely unknown and the identification of reliable biomarkers in animal models will certainly contribute to increase our comprehension of CDD as well as to assess the efficacy of therapeutic strategies. Here, we used different Magnetic Resonance (MR) methods to disclose structural, functional, or metabolic signatures of Cdkl5 deficiency in the brain of adult mice. We found that loss of Cdkl5 does not cause cerebral atrophy but affects distinct brain areas, particularly the hippocampus. By in vivo proton-MR spectroscopy (MRS), we revealed in the Cdkl5 null brain a metabolic dysregulation indicative of mitochondrial dysfunctions. Accordingly, we unveiled a significant reduction in ATP levels and a decrease in the expression of complex IV of mitochondrial electron transport chain. Conversely, the number of mitochondria appeared preserved. Importantly, we reported a significant defect in the activation of one of the major regulators of cellular energy balance, the adenosine monophosphate-activated protein kinase (AMPK), that might contribute to the observed metabolic impairment and become an interesting therapeutic target for future preclinical trials. In conclusion, MRS revealed in the Cdkl5 null brain the presence of a metabolic dysregulation suggestive of a mitochondrial dysfunction that permitted to foster our comprehension of Cdkl5 deficiency and brought our interest towards targeting mitochondria as therapeutic strategy for CDD.

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.

Exploring genotype-phenotype relationships in the CDKL5 deficiency disorder using an international dataset.

Characterized by early-onset seizures, global developmental delay and severe motor deficits, CDKL5 deficiency disorder is caused by pathogenic variants in the cyclin-dependent kinase-like 5 gene. Previous efforts to investigate genotype-phenotype relationships have been limited due to small numbers of recurrent mutations and small cohort sizes. Using data from the International CDKL5 Disorder Database we examined genotype-phenotype relationships for 13 recurrent CDKL5 variants and the previously analyzed historic variant groupings. We have applied the CDKL5 Developmental Score (CDS) and an adapted version of the CDKL5 Clinical Severity Assessment (CCSA), to grade the severity of phenotype and developmental outcomes for 285 individuals with CDKL5 variants. Comparisons of adapted CCSA and CDS between recurrent variants and variant groups were performed using multiple linear regression adjusting for age and sex. Individuals with the missense variant, p.Arg178Trp, had the highest mean adapted CCSA and lowest mean developmental scores. Other variants producing severe phenotypes included p.Arg559* and p.Arg178Gln. Variants producing milder phenotypes included p.Arg134*, p.Arg550*, and p.Glu55Argfs*20. There are observed differences in phenotype severity and developmental outcomes for individuals with different CDKL5 variants. However, the historic variant groupings did not seem to reflect differences in phenotype severity or developmental outcomes as clearly as analyzed by individual variants.

Novel FARS2 variants in patients with early onset encephalopathy with or without epilepsy associated with long survival.

Mitochondrial translation is essential for the biogenesis of the mitochondrial oxidative phosphorylation system (OXPHOS) that synthesizes the bulk of ATP for the cell. Hypomorphic and loss-of-function variants in either mitochondrial DNA or in nuclear genes that encode mitochondrial translation factors can result in impaired OXPHOS biogenesis and mitochondrial diseases with variable clinical presentations. Compound heterozygous or homozygous missense and frameshift variants in the FARS2 gene, that encodes the mitochondrial phenylalanyl-tRNA synthetase, are commonly linked to either early-onset epileptic mitochondrial encephalopathy or spastic paraplegia. Here, we expand the genetic spectrum of FARS2-linked disease with three patients carrying novel compound heterozygous variants in the FARS2 gene and presenting with spastic tetraparesis, axial hypotonia and myoclonic epilepsy in two cases.