Functional Characteristics of the Nav1.1 p.Arg1596Cys Mutation Associated with Varying Severity of Epilepsy Phenotypes.

Mutations of the SCN1A gene, which encodes the voltage-dependent Na+ channel's α subunit, are associated with diverse epileptic syndromes ranging in severity, even intra-family, from febrile seizures to epileptic encephalopathy. The underlying cause of this variability is unknown, suggesting the involvement of additional factors. The aim of our study was to describe the properties of mutated channels and investigate genetic causes for clinical syndromes' variability in the family of five SCN1A gene p.Arg1596Cys mutation carriers. The analysis of additional genetic factors influencing SCN1A-associated phenotypes was conducted through exome sequencing (WES). To assess the impact of mutations, we used patch clamp analysis of mutated channels expressed in HEK cells and in vivo neural excitability studies (NESs). In cells expressing the mutant channel, sodium currents were reduced. NESs indicated increased excitability of peripheral motor neurons in mutation carriers. WES showed the absence of non-SCA1 pathogenic variants that could be causative of disease in the family. Variants of uncertain significance in three genes, as potential modifiers of the most severe phenotype, were identified. The p.Arg1596Cys substitution inhibits channel function, affecting steady-state inactivation kinetics. Its clinical manifestations involve not only epileptic symptoms but also increased excitability of peripheral motor fibers. The role of Nav1.1 in excitatory neurons cannot be ruled out as a significant factor of the clinical phenotype.

Genetic Risk Factors for Neurological Disorders in Children with Adverse Events Following Immunization: A Descriptive Study of a Polish Case Series.

Studies conducted on large populations show a lack of connection between vaccination and serious neurological symptoms. However, there are isolated cases that indicate such a relationship. These reports on adverse effects following immunization (AEFI) reduce social confidence in vaccination; however, their background may be rare genetic defects. The aim of the presented study was to examine if neurological AEFI in children may be associated with variants in genes related to neurodevelopment. To identify such possible associations, a descriptive study of the Polish case series was conducted. We performed next-generation sequencing in patients who, up to 4 weeks of injection of any vaccine, manifested neurological AEFI. We included 23 previously normally developing children with first seizures that occurred after vaccination. We identified pathogenic/likely pathogenic variants in genes engaged in neurodevelopment in nine patients and variants of uncertain significance in another nine patients. The mutated genes belonged to the group of genes related to epilepsy syndromes/epileptic encephalopathy. We showed that AEFI might have a genetic background. We hypothesized that in some AEFI patients, the vaccine might only trigger neurological symptoms that would have been manifested anyway as a result of a pathogenic variant in a gene engaged in neurodevelopment.

Bullous diseases caused by KRT1 gene mutations: from epidermolytic hyperkeratosis to a novel variant of epidermolysis bullosa simplex.

INTRODUCTION: Mutations in the KRT1 gene encoding keratin 1 cause epidermolytic hyperkeratosis characterized by blistering in the neonatal period followed by ichthyotic hyperkeratosis in childhood and adolescent life. We observed a spectrum of clinical manifestations of blistering disorders caused by different mutations in the same KRT1 gene. AIM: To analyse the phenotypic spectrum of blistering disorders caused by the KRT1 mutations. MATERIAL AND METHODS: Four patients with an epidermal barrier defect manifesting as blistering with the KRT1 mutations were included to the study. The clinical course of the disease was analysed, histology, immunofluorescence and electron microscopic examinations were performed. RESULTS: An adult patient with severe ichthyosis with p.Asn188Lys mutation in exon 1 of KRT1 who occasionally develops blisters in adolescence represents epidermolytic hyperkeratosis, a newborn child who died 4 days after birth due to disruption of the epidermal barrier (extensive blister and erosions) with mutation p.Ser193Pro in the KTR1 gene and two adult sisters harbouring heterozygous mutation c.591+1A>G in the KRT1 gene who present superficial blisters induced by mild trauma from the birth up to adolescent life without ichthyosis suggesting the diagnosis of epidermolysis bullosa simplex. Histopathology in all adult patients showed cytoplasm disruption in keratinocytes of the stratum spinosum with keratohyalin granule-like structures and, on the ultrastructural level, the presence of keratin clumping confirming the pathology of keratin intermediate filaments. CONCLUSIONS: This study extends the knowledge of the clinical spectrum for the KRT1 gene mutations. This is the first description of familial dominant epidermolysis bullosa simplex linked to the KRT1 mutation.

SCN3A-Related Neurodevelopmental Disorder: A Spectrum of Epilepsy and Brain Malformation.

OBJECTIVE: Pathogenic variants in SCN3A, encoding the voltage-gated sodium channel subunit Nav1.3, cause severe childhood onset epilepsy and malformation of cortical development. Here, we define the spectrum of clinical, genetic, and neuroimaging features of SCN3A-related neurodevelopmental disorder. METHODS: Patients were ascertained via an international collaborative network. We compared sodium channels containing wild-type versus variant Nav1.3 subunits coexpressed with ß1 and ß2 subunits using whole-cell voltage clamp electrophysiological recordings in a heterologous mammalian system (HEK-293T cells). RESULTS: Of 22 patients with pathogenic SCN3A variants, most had treatment-resistant epilepsy beginning in the first year of life (16/21, 76%; median onset, 2 weeks), with severe or profound developmental delay (15/20, 75%). Many, but not all (15/19, 79%), exhibited malformations of cortical development. Pathogenic variants clustered in transmembrane segments 4 to 6 of domains II to IV. Most pathogenic missense variants tested (10/11, 91%) displayed gain of channel function, with increased persistent current and/or a leftward shift in the voltage dependence of activation, and all variants associated with malformation of cortical development exhibited gain of channel function. One variant (p.Ile1468Arg) exhibited mixed effects, with gain and partial loss of function. Two variants demonstrated loss of channel function. INTERPRETATION: Our study defines SCN3A-related neurodevelopmental disorder along a spectrum of severity, but typically including epilepsy and severe or profound developmental delay/intellectual disability. Malformations of cortical development are a characteristic feature of this unusual channelopathy syndrome, present in >75% of affected individuals. Gain of function at the channel level in developing neurons is likely an important mechanism of disease pathogenesis. ANN NEUROL 2020;88:348-362.

PIGT-CDG, a disorder of the glycosylphosphatidylinositol anchor: description of 13 novel patients and expansion of the clinical characteristics.

PURPOSE: To provide a detailed electroclinical description and expand the phenotype of PIGT-CDG, to perform genotype-phenotype correlation, and to investigate the onset and severity of the epilepsy associated with the different genetic subtypes of this rare disorder. Furthermore, to use computer-assisted facial gestalt analysis in PIGT-CDG and to the compare findings with other glycosylphosphatidylinositol (GPI) anchor deficiencies. METHODS: We evaluated 13 children from eight unrelated families with homozygous or compound heterozygous pathogenic variants in PIGT. RESULTS: All patients had hypotonia, severe developmental delay, and epilepsy. Epilepsy onset ranged from first day of life to two years of age. Severity of the seizure disorder varied from treatable seizures to severe neonatal onset epileptic encephalopathies. The facial gestalt of patients resembled that of previously published PIGT patients as they were closest to the center of the PIGT cluster in the clinical face phenotype space and were distinguishable from other gene-specific phenotypes. CONCLUSION: We expand our knowledge of PIGT. Our cases reaffirm that the use of genetic testing is essential for diagnosis in this group of disorders. Finally, we show that computer-assisted facial gestalt analysis accurately assigned PIGT cases to the multiple congenital anomalies-hypotonia-seizures syndrome phenotypic series advocating the additional use of next-generation phenotyping technology.

Characterization of glycosylphosphatidylinositol biosynthesis defects by clinical features, flow cytometry, and automated image analysis.

BACKGROUND: Glycosylphosphatidylinositol biosynthesis defects (GPIBDs) cause a group of phenotypically overlapping recessive syndromes with intellectual disability, for which pathogenic mutations have been described in 16 genes of the corresponding molecular pathway. An elevated serum activity of alkaline phosphatase (AP), a GPI-linked enzyme, has been used to assign GPIBDs to the phenotypic series of hyperphosphatasia with mental retardation syndrome (HPMRS) and to distinguish them from another subset of GPIBDs, termed multiple congenital anomalies hypotonia seizures syndrome (MCAHS). However, the increasing number of individuals with a GPIBD shows that hyperphosphatasia is a variable feature that is not ideal for a clinical classification. METHODS: We studied the discriminatory power of multiple GPI-linked substrates that were assessed by flow cytometry in blood cells and fibroblasts of 39 and 14 individuals with a GPIBD, respectively. On the phenotypic level, we evaluated the frequency of occurrence of clinical symptoms and analyzed the performance of computer-assisted image analysis of the facial gestalt in 91 individuals. RESULTS: We found that certain malformations such as Morbus Hirschsprung and diaphragmatic defects are more likely to be associated with particular gene defects (PIGV, PGAP3, PIGN). However, especially at the severe end of the clinical spectrum of HPMRS, there is a high phenotypic overlap with MCAHS. Elevation of AP has also been documented in some of the individuals with MCAHS, namely those with PIGA mutations. Although the impairment of GPI-linked substrates is supposed to play the key role in the pathophysiology of GPIBDs, we could not observe gene-specific profiles for flow cytometric markers or a correlation between their cell surface levels and the severity of the phenotype. In contrast, it was facial recognition software that achieved the highest accuracy in predicting the disease-causing gene in a GPIBD. CONCLUSIONS: Due to the overlapping clinical spectrum of both HPMRS and MCAHS in the majority of affected individuals, the elevation of AP and the reduced surface levels of GPI-linked markers in both groups, a common classification as GPIBDs is recommended. The effectiveness of computer-assisted gestalt analysis for the correct gene inference in a GPIBD and probably beyond is remarkable and illustrates how the information contained in human faces is pivotal in the delineation of genetic entities.

Rare GABRA3 variants are associated with epileptic seizures, encephalopathy and dysmorphic features.

Genetic epilepsies are caused by mutations in a range of different genes, many of them encoding ion channels, receptors or transporters. While the number of detected variants and genes increased dramatically in the recent years, pleiotropic effects have also been recognized, revealing that clinical syndromes with various degrees of severity arise from a single gene, a single mutation, or from different mutations showing similar functional defects. Accordingly, several genes coding for GABAA receptor subunits have been linked to a spectrum of benign to severe epileptic disorders and it was shown that a loss of function presents the major correlated pathomechanism. Here, we identified six variants in GABRA3 encoding the α3-subunit of the GABAA receptor. This gene is located on chromosome Xq28 and has not been previously associated with human disease. Five missense variants and one microduplication were detected in four families and two sporadic cases presenting with a range of epileptic seizure types, a varying degree of intellectual disability and developmental delay, sometimes with dysmorphic features or nystagmus. The variants co-segregated mostly but not completely with the phenotype in the families, indicating in some cases incomplete penetrance, involvement of other genes, or presence of phenocopies. Overall, males were more severely affected and there were three asymptomatic female mutation carriers compared to only one male without a clinical phenotype. X-chromosome inactivation studies could not explain the phenotypic variability in females. Three detected missense variants are localized in the extracellular GABA-binding NH2-terminus, one in the M2-M3 linker and one in the M4 transmembrane segment of the α3-subunit. Functional studies in Xenopus laevis oocytes revealed a variable but significant reduction of GABA-evoked anion currents for all mutants compared to wild-type receptors. The degree of current reduction correlated partially with the phenotype. The microduplication disrupted GABRA3 expression in fibroblasts of the affected patient. In summary, our results reveal that rare loss-of-function variants in GABRA3 increase the risk for a varying combination of epilepsy, intellectual disability/developmental delay and dysmorphic features, presenting in some pedigrees with an X-linked inheritance pattern.

Novel Mutations in the IRF6 Gene on the Background of Known Polymorphisms in Polish Patients With Orofacial Clefting.

OBJECTIVE: To examine the role of the IRF6 mutations in Polish families with Van der Woude syndrome and popliteal pterygium syndrome and to determine the effect of IRF6 single nucleotide polymorphisms (rs7552506, rs2013162, and rs2235375) on cleft lip and/or palate susceptibility. DESIGN: IRF6 mutation screening was performed by direct sequencing of all coding exons of the gene and their flanking intronic regions. Cosegregation analysis was performed to establish the relation of single nucleotide polymorphisms and cleft lip and/or palate phenotypes. PATIENTS: We screened the IRF6 gene in eight families with clinical recognition of Van der Woude syndrome and popliteal pterygium syndrome. RESULTS: In five families we identified pathogenic mutations, all affecting the DNA-binding or the protein-binding domain of IRF6. Two of the mutations were novel-a missense mutation Arg31Thr and a small deletion Trp40Glyfs*23. In most cases we found also a haplotype of three single nucleotide polymorphisms-rs7552506, rs2013162, and rs2235375. The association of the single nucleotide polymorphisms and cleft lip and/or palate susceptibility has been previously published. The variants did not cosegregate with phenotype in examined families nor did they cosegregate with pathogenic mutations. The single nucleotide polymorphisms were deemed not causative, due to their presence in unaffected family members. CONCLUSIONS: Two novel mutations (Arg31Thr and Trp40Glyfs*23) in the IRF6 gene were identified to be causative for Van der Woude and popliteal pterygium syndromes. In the present study no association between the single nucleotide polymorphisms rs7552506, rs2013162, and rs2235375 and the cleft lip and/or palate phenotype was found. The hypothesis, whether the haplotype of the three single nucleotide polymorphisms was correlated with IRF6 expression level, demands further investigation.

Variants in CUL4B are associated with cerebral malformations.

Variants in cullin 4B (CUL4B) are a known cause of syndromic X-linked intellectual disability. Here, we describe an additional 25 patients from 11 families with variants in CUL4B. We identified nine different novel variants in these families and confirmed the pathogenicity of all nontruncating variants. Neuroimaging data, available for 15 patients, showed the presence of cerebral malformations in ten patients. The cerebral anomalies comprised malformations of cortical development (MCD), ventriculomegaly, and diminished white matter volume. The phenotypic heterogeneity of the cerebral malformations might result from the involvement of CUL-4B in various cellular pathways essential for normal brain development. Accordingly, we show that CUL-4B interacts with WDR62, a protein in which variants were previously identified in patients with microcephaly and a wide range of MCD. This interaction might contribute to the development of cerebral malformations in patients with variants in CUL4B.