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.

Congenital coenzyme Q5-linked pathology: causal genetic association, core phenotype, and molecular mechanism.

Coenzyme Q5 (COQ5), a C-methyltransferase, modifies coenzyme Q10 (COQ10) during biosynthesis and interacts with polyA-tail regulating zinc-finger protein ZC3H14 in neural development. Here, we present a fifth patient (a third family) worldwide with neurodevelopmental and physiological symptoms including COQ10 deficiency. Our patient harbors one novel c.681+1G>A and one recurrent p.Gly118Ser variant within COQ5. The patient's mRNA profile reveals multiple COQ5 splice-variants. Subsequently, we comprehensively described patient's clinical features as compared to phenotype and symptoms of other known congenital coenzyme Q5-linked cases. A core spectrum of COQ5-associated symptoms includes reduced COQ10 levels, intellectual disability, encephalopathy, cerebellar ataxia, cerebellar atrophy speech regression/dysarthria, short stature, and developmental delays. Our patient additionally displays dysmorphia, microcephaly, and regressive social faculties. These results formally establish causal association of biallelic COQ5 mutation with pathology, outline a core COQ5-linked phenotype, and identify mRNA mis-splicing as the molecular mechanism underlying all COQ5 variant-linked pathology to date.

A Zebrafish/Drosophila Dual System Model for Investigating Human Microcephaly.

Microcephaly presents in neurodevelopmental disorders with multiple aetiologies, including bi-allelic mutation in TUBGCP2, a component of the biologically fundamental and conserved microtubule-nucleation complex, γ-TuRC. Elucidating underlying principles driving microcephaly requires clear phenotype recapitulation and assay reproducibility, areas where go-to experimental models fall short. We present an alternative simple vertebrate/invertebrate dual system to investigate fundamental TUBGCP2-related processes driving human microcephaly and associated developmental traits. We show that antisense morpholino knockdown (KD) of the Danio rerio homolog, tubgcp2, recapitulates human TUBGCP2-associated microcephaly. Co-injection of wild type mRNA pre-empts microcephaly in 55% of KD zebrafish larvae, confirming causality. Body shortening observed in morphants is also rescued. Mitotic marker (pH3) staining further reveals aberrantly accumulated dividing brain cells in microcephalic tubgcp2 KD morphants, indicating that tubgcp2 depletion disrupts normal mitosis and/or proliferation in zebrafish neural progenitor brain cells. Drosophila melanogaster double knockouts (KO) for TUBGCP2 homologs Grip84/cg7716 also develop microcephalic brains with general microsomia. Exacerbated Grip84/cg7716-linked developmental aberration versus single mutations strongly suggests interactive or coinciding gene functions. We infer that tubgcp2 and Grip84/cg7716 affect brain size similarly to TUBGCP2 and recapitulate both microcephaly and microcephaly-associated developmental impact, validating the zebrafish/fly research model for human microcephaly. Given the conserved cross-phyla homolog function, the data also strongly support mitotic and/or proliferative disruption linked to aberrant microtubule nucleation in progenitor brain cells as key mechanistic defects for human microcephaly.

De Novo ACTG1 Variant Expands the Phenotype and Genotype of Partial Deafness and Baraitser-Winter Syndrome.

Actin molecules are fundamental for embryonic structural and functional differentiation; γ-actin is specifically required for the maintenance and function of cytoskeletal structures in the ear, resulting in hearing. Baraitser-Winter Syndrome (B-WS, OMIM #243310, #614583) is a rare, multiple-anomaly genetic disorder caused by mutations in either cytoplasmically expressed actin gene, ACTB (ß-actin) or ACTG1 (γ-actin). The resulting actinopathies cause characteristic cerebrofrontofacial and developmental traits, including progressive sensorineural deafness. Both ACTG1-related non-syndromic A20/A26 deafness and B-WS diagnoses are characterized by hypervariable penetrance in phenotype. Here, we identify a 28th patient worldwide carrying a mutated γ-actin ACTG1 allele, with mildly manifested cerebrofrontofacial B-WS traits, hypervariable penetrance of developmental traits and sensorineural hearing loss. This patient also displays brachycephaly and a complete absence of speech faculty, previously unreported for ACTG1-related B-WS or DFNA20/26 deafness, representing phenotypic expansion. The patient's exome sequence analyses (ES) confirms a de novo ACTG1 variant previously unlinked to the pathology. Additional microarray analysis uncover no further mutational basis for dual molecular diagnosis in our patient. We conclude that γ-actin c.542C > T, p.Ala181Val is a dominant pathogenic variant, associated with mildly manifested facial and cerebral traits typical of B-WS, hypervariable penetrance of developmental traits and sensorineural deafness. We further posit and present argument and evidence suggesting ACTG1-related non-syndromic DFNA20/A26 deafness is a manifestation of undiagnosed ACTG1-related B-WS.

Bilateral Pallidal Stimulation in a Family With Myoclonus Dystonia Syndrome Due to a Mutation in the Sarcoglycan Gene.

OBJECTIVES: The study aimed to present a family with myoclonus dystonia (M-D) syndrome due to a mutation in the epsilon sarcoglycan gene (SGCE). Three members of the family suffered from treatment-refractory severe myoclonic jerks of the neck, trunk, and upper extremities. The mild dystonic symptoms recognized as cervical dystonia or truncal dystonia affected all individuals. The efficacy of pharmacotherapy, including anticholinergic, dopaminergic, and serotoninergic drugs, has failed. One individual developed an alcohol dependency and suffered from alcoholic epilepsy. MATERIALS AND METHODS: The patients were referred for stereotactic surgery. All individuals underwent bilateral implantation of deep brain stimulation (DBS) leads into the posteroventrolateral segment of the globus pallidus internus (GPi). Surgeries were uneventful. The formal preoperative objective assessment included the Unified Myoclonus Rating Scale (UMRS) and the Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS). The postoperative UMRS and BFMDRS assessments were done only under continuous stimulation at 3, 6, and 12 months after the surgery and at the last available follow-up ranging from 6 to 15 months (mean, 10 months follow-up). RESULTS: At the last follow-up visit, the rest and action parts of UMRS were improved by 93.3% and 88.2%, respectively, when compared to the baseline scores. The motor and disability scales of BFMDRS were improved by 77% and 43% at the last follow-up visit compared to the baseline BFMDRS scores. There were no hardware or stimulation-induced complications over the follow-up period. Positive social adjustment allowed two patients to regain jobs and one patient continued his education and hobbies. CONCLUSION: Our experience gathered in three individuals in the family with a mutation in SGCE indicates that bilateral GPi DBS can be an effective and safe treatment for disabling pharmacological resistant, intractable M-D syndrome.

Exome Sequencing Reveals Novel Variants and Expands the Genetic Landscape for Congenital Microcephaly.

Congenital microcephaly causes smaller than average head circumference relative to age, sex and ethnicity and is most usually associated with a variety of neurodevelopmental disorders. The underlying etiology is highly heterogeneous and can be either environmental or genetic. Disruption of any one of multiple biological processes, such as those underlying neurogenesis, cell cycle and division, DNA repair or transcription regulation, can result in microcephaly. This etiological heterogeneity manifests in a clinical variability and presents a major diagnostic and therapeutic challenge, leaving an unacceptably large proportion of over half of microcephaly patients without molecular diagnosis. To elucidate the clinical and genetic landscapes of congenital microcephaly, we sequenced the exomes of 191 clinically diagnosed patients with microcephaly as one of the features. We established a molecular basis for microcephaly in 71 patients (37%), and detected novel variants in five high confidence candidate genes previously unassociated with this condition. We report a large number of patients with mutations in tubulin-related genes in our cohort as well as higher incidence of pathogenic mutations in MCPH genes. Our study expands the phenotypic and genetic landscape of microcephaly, facilitating differential clinical diagnoses for disorders associated with most commonly disrupted genes in our cohort.

Genetics of Parkinson's disease in the Polish population.

INTRODUCTION: Genetic forms of Parkinson's disease (PD) often cluster in different ethnic groups and may present with recognisable unique clinical manifestations. Our aim was to summarise the current state of knowledge regarding the genetic causes of PD and describe the first Polish patient with SNCA duplication. METHODOLOGY: We searched the electronic database, PubMed, for studies between January 1995 and June 2020 that evaluated genetics in Polish patients with PD, using the search terms 'Parkinson's disease, 'Polish', 'genetics', 'mutations', and 'variants'. RESULTS: In total, 73 publications were included in the review; 11 genes responsible for monogenic forms and 19 risk factor genes have been analysed in the Polish population. Pathogenic variants were reported in four monogenic genes (LRRK2, PRKN, PINK1, and SNCA). Eight genes were associated with PD risk in the Polish population (GBA, TFAM, NFE2L2, MMP12, HLA-DRA, COMT, MAOB, and DBH). Multiplex ligation-dependent probe amplification and Sanger sequencing in PRKN, PINK1, DJ1, LRRK2, and SNCA revealed SNCA duplication in a 43-year-old Polish patient with PD examined by movement disorder specialists. CONCLUSION: Only a limited number of positive results have been reported in genes previously associated with PD in the Polish population. In the era of personalised medicine, it is important to report on genetic findings in specific populations.

Comprehensive genomic analysis of patients with disorders of cerebral cortical development.

Malformations of cortical development (MCDs) manifest with structural brain anomalies that lead to neurologic sequelae, including epilepsy, cerebral palsy, developmental delay, and intellectual disability. To investigate the underlying genetic architecture of patients with disorders of cerebral cortical development, a cohort of 54 patients demonstrating neuroradiologic signs of MCDs was investigated. Individual genomes were interrogated for single-nucleotide variants (SNV) and copy number variants (CNV) with whole-exome sequencing and chromosomal microarray studies. Variation affecting known MCDs-associated genes was found in 16/54 cases, including 11 patients with SNV, 2 patients with CNV, and 3 patients with both CNV and SNV, at distinct loci. Diagnostic pathogenic SNV and potentially damaging variants of unknown significance (VUS) were identified in two groups of seven individuals each. We demonstrated that de novo variants are important among patients with MCDs as they were identified in 10/16 individuals with a molecular diagnosis. Three patients showed changes in known MCDs genes  and a clinical phenotype beyond the usual characteristics observed, i.e., phenotypic expansion, for a particular known disease gene clinical entity. We also discovered 2 likely candidate genes, CDH4, and ASTN1, with human and animal studies supporting their roles in brain development, and 5 potential candidate genes. Our findings emphasize genetic heterogeneity of MCDs disorders and postulate potential novel candidate genes involved in cerebral cortical development.

The mutation responsible for torsion dystonia type 1 shows the ability to stimulate intracellular aggregation of mutant huntingtin.

OBJECTIVE: Introduction: Torsion dystonia type 1 is the most common form of early-onset primary dystonia. Previous reports have suggested that torsin 1A, a protein mutated in this disease, might function as a chaperone that prevents the toxic aggregation of misfolded polypeptides. The aim of the study: The aim of this study was to verify the chaperone function of torsin 1A by investigating its ability to prevent the aggregation of huntingtin model peptides. PATIENTS AND METHODS: Materials and methods: N-terminal mutant huntingtin fragments of different length were co-expressed in neuronal HT-22 and non-neuronal HeLa cells with either the wild-type or mutant (ΔE302/303) torsin 1A protein. The transfected cells were immunostained and analyzed for the presence of huntingtin aggregates using fluorescence microscopy. RESULTS: Results: The immunofluorescence analysis of huntingtin subcellular distribution within the transfected cells showed no significant difference between the huntingtin aggregation levels in cells co-expressing the wild-type torsin 1A and in control cells co-transfected with an empty vector. Instead, it was the increased level of huntingtin aggregation in the presence of the torsion dystonia-causing ΔE302/303 mutant that reached statistical significance in both neuronal and non-neuronal cells. CONCLUSION: Conclusions: Either torsin 1A does not function as a chaperone protein or huntingtin is not an efficient substrate for such a hypothetical chaperone activity. However, the ability of mutant torsin 1A to stimulate the accumulation of aggregation-prone polypeptides might constitute an important source of ΔE302/303 pathogenicity and thus a potential target for future therapy.

[Torsin 1A and the pathomechanism of torsion dystonia type 1]. / Funkcja torsyny 1A w patomechanizmie dystonii torsyjnej typu 1.

Torsin 1A is a protein mutated in torsion dystonia type 1, a hereditary neurological disorder of early onset and variable clinical picture. The basic cellular function of torsin 1A, a polypeptide localized predominantly in the endoplasmic reticulum and nuclear envelope, remains unknown, although the protein is suspected of being involved in many different cellular processes, including regulating a proper structure and function of nuclear envelope, contributing to the synaptic vesicular trafficking, or assisting in proper folding of misfolded proteins. This review summarizes the current state of knowledge regarding the potential functions of torsin 1A in the context of hypothetical pathomechanisms responsible for torsion dystonia type 1.

Gene conversion between cationic trypsinogen (PRSS1) and the pseudogene trypsinogen 6 (PRSS3P2) in patients with chronic pancreatitis.

Mutations of the human cationic trypsinogen gene (PRSS1) are frequently found in association with hereditary pancreatitis. The most frequent variants p.N29I and p.R122H are recognized as disease-causing mutations. Three pseudogene paralogs in the human trypsinogen family, including trypsinogen 6 (PRSS3P2), carry sequence variations in exon 3 that mimic the p.R122H mutation. In routine genetic testing of patients with chronic pancreatitis, we identified in two unrelated individuals similar gene conversion events of 24-71 nucleotides length between exon 3 of the PRSS1 (acceptor) and PRSS3P2 (donor) genes. The converted allele resulted in three nonsynonymous alterations c.343T>A (p.S115T), c.347G>C (p.R116P), and c.365_366delinsAT (p.R122H). Functional analysis of the conversion triple mutant revealed markedly increased autoactivation resulting in high and sustained trypsin activity in the presence of chymotrypsin C. This activation phenotype was identical to that of the p.R122H mutant. In addition, cellular secretion of the triple mutant from transfected HEK 293T cells was increased about twofold and this effect was attributable to mutation p.R116P. Our observations confirm and extend the notion that recombination events between members of the trypsinogen family can generate high-risk PRSS1 alleles. The pathogenic phenotype of the novel conversion is explained by a unique combination of increased trypsinogen activation and secretion.

Glucose transporter type 1 deficiency due to SLC2A1 gene mutations--a rare but treatable cause of metabolic epilepsy and extrapyramidal movement disorder; own experience and literature review.

THE AIM: To present the molecular and clinical characteristics of three children with glucose deficiency syndrome, an inborn rare metabolic disease, caused by mutations in the SLC2A1 gene. MATERIAL AND METHODS: The investigation was carried out in three children: two girls and one boy showing symptoms of GLUT1 deficiency syndrome (GLUT1-DS). They were referred for SLC2A1 gene analysis. RESULTS: The presence of mutations in all of them was confirmed. Only point mutations were identified, two missenses p.Gly132Ser, p.Arg212Cys and amino acid insertion p.Ser_Val227insValProPro. In two cases the mutations arose de novo, one was heritable of paternal origin. CONCLUSIONS: GLUT1-DS shows high clinical variability. It should be suspected in children of any age presenting with single features or a combination of any form of intractable epilepsy with seizures of various types, movement disorders and paroxysmal events, especially triggered by exercise, exertion, or fasting, and any unexplainable neurological deterioration. The basic diagnostic hallmarks of GLUT1-DS are CSF hypoglycorrhachia and lowered CSF/Blood serum glucose ratio. This is why lumbar punction should be considered more frequently than it is in practice being performed nowadays. Antiepileptic drug treatment may be ineffective or even potentially detrimental. Early identification and molecular confirmation of GLUT1-DS is important, because this is a metabolic disorder and patients should as soon as possible primarily be treated with a ketogenic diet.

Intrafamilial variability of the primary dystonia DYT6 phenotype caused by p.Cys5Trp mutation in THAP1 gene.

Mutations localized in THAP1 gene, locus 18p11.21 have been reported as causative of primary dystonia type 6 (DYT6). Disease which is characterized mainly by focal dystonia, frequently involving the craniocervical region, however associated also with early-onset generalized dystonia and spasmodic dysphonia. Here we report a novel mutation in the THAP1 gene identified in a Polish family with DYT6 phenotype - the c.15C>G substitution in exon 1 introducing the missense mutation p.Cys5Trp within the N-terminal THAP domain. The mutation was described in two generations, in patients showing a broad spectrum of focal and generalized dystonia symptoms of variable onset. Our results indicate that certain mutations in the THAP1 gene may lead to primary dystonia with remarkable intrafamilial clinical variability.

Novel A18T and pA29S substitutions in α-synuclein may be associated with sporadic Parkinson's disease.

OBJECTIVE: Mutations in the α-synuclein-encoding gene SNCA are considered as a rare cause of Parkinson's disease (PD). Our objective was to examine the frequency of the SNCA point mutations among PD patients of Polish origin. METHODS: Detection of the known SNCA point mutations A30P (c.88G>C), E46K (c.136G>A) and A53T (c.157A>T) was performed either using the Sequenom MassArray iPLEX platform or by direct sequencing of the SNCA exons 2 and 3. As the two novel substitutions A18T (c.52G>A) and A29S (c.85G>T) were identified, their frequency in a control population of Polish origin was assessed and in silico analysis performed to investigate the potential impact on protein structure and function. RESULTS: We did not observe the previously reported point mutations in the SNCA gene in our 629 PD patients; however, two novel potentially pathogenic substitutions A18T and A29S were identified. Each variant was observed in a single patient presenting with a typical late-onset sporadic PD phenotype. Although neither variant was observed in control subjects and in silico protein analysis predicts a damaging effect for A18T and pA29S substitutions, the lack of family history brings into question the true pathogenicity of these rare variants. CONCLUSIONS: Larger population based studies are needed to determine the pathogenicity of the A18T and A29S substitutions. Our findings highlight the possible role of rare variants contributing to disease risk and may support further screening of the SNCA gene in sporadic PD patients from different populations.

Mutations in the PLP1 gene residue p. Gly198 as the molecular basis of Pelizeaus-Merzbacher phenotype.

Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia type 2 (SPG2) are rare X-linked allelic disorders caused by mutations in the PLP1 gene, encoding the main component of myelin, proteolipid protein 1 (PLP1). Various types of mutations, acting through different molecular mechanism, cause the diseases. Duplications of variable size at Xq22.2, containing the entire PLP1, are responsible for more than 50% of PMD cases. Other causes of PMD include point mutations, gene deletions and triplications. There is a spectrum of PLP1-related disorders with some correlation between the type of mutation and phenotype. Generally the missense mutations cause the more severe forms of the disease, the most common PLP1 duplications, result in the classical PMD whereas deletions and null mutations in mild form of PMD and SPG2. We present a patient with c.593G>A substitution in the exon 4 of the PLP1 gene causing a novel missense mutation p.Gly198Asp, finally diagnosed as PMD but showing an atypical MRI picture.

Nuclear import and export signals of human cohesins SA1/STAG1 and SA2/STAG2 expressed in Saccharomyces cerevisiae.

BACKGROUND: Human SA/STAG proteins, homologues of the yeast Irr1/Scc3 cohesin, are the least studied constituents of the sister chromatid cohesion complex crucial for proper chromosome segregation. The two SA paralogues, SA1 and SA2, show some specificity towards the chromosome region they stabilize, and SA2, but not SA1, has been shown to participate in transcriptional regulation as well. The molecular basis of this functional divergence is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In silico analysis indicates numerous putative nuclear localization (NLS) and export (NES) signals in the SA proteins, suggesting the possibility of their nucleocytoplasmic shuttling. We studied the functionality of those putative signals by expressing fluorescently tagged SA1 and SA2 in the yeast Saccharomyces cerevisiae. Only the N-terminal NLS turned out to be functional in SA1. In contrast, the SA2 protein has at least two functional NLS and also two functional NES. Depending on the balance between these opposing signals, SA2 resides in the nucleus or is distributed throughout the cell. Validation of the above conclusions in HeLa cells confirmed that the same N-terminal NLS of SA1 is functional in those cells. In contrast, in SA2 the principal NLS functioning in HeLa cells is different from that identified in yeast and is localized to the C-terminus. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration of the possibility of non-nuclear localization of an SA protein. The reported difference in the organization between the two SA homologues may also be relevant to their partially divergent functions. The mechanisms determining subcellular localization of cohesins are only partially conserved between yeast and human cells.

Novel de novo large deletion in cystic fibrosis transmembrane conductance regulator gene results in a severe cystic fibrosis phenotype.

We identified c.1521_1523delCTT and c.1679+94_2619+986del8118 in trans in a 6-year-old boy with a severe cystic fibrosis phenotype. The first deletion was inherited maternally, but the latter had arisen de novo.

Incidence of spinal muscular atrophy in Poland--more frequent than predicted?

BACKGROUND: The application of molecular methods has enhanced and enlarged the diagnostics of spinal muscular atrophy (SMA) and its carriership. It allows for reliable epidemiological studies which are of importance to demography and genetic counseling. METHODS: This study sought to evaluate the incidence of SMA in Poland, on the basis of the prevalence of the SMN1 gene deletion carrier state in the general population, as well as an analysis of all cases of SMA diagnosed in the years 1998-2005. RESULTS: The prevalence of the SMN1 gene deletion carrier state was estimated at 1 per 35 persons (17/600), yielding an incidence of SMA equal to 1 per 4,900. By contrast, the incidence of SMA based on the results of the meta-analysis was an estimated 1 per 7,127 in Warsaw and 1 per 9,320 persons across Poland, suggesting that some cases of SMA remain undiagnosed. SMA1 predominated among the diagnoses, accounting for 69% of all cases. CONCLUSION: A high prevalence of the SMN1 deletion carrier state in the general population indicates that SMA could be a more frequent disease than is predicted by the epidemiological data regarding diagnosed cases.

[Gene mapping in 14 families with X-linked nonspecific mental retardation]. / Mapowanie genów w 14 rodzinach z niespecyficzna niepelnosprawoscia intelektualna sprzezona z chromosomem X.

UNLABELLED: Mental retardation affects 2-3% of the population. The identification of nonspecific X-linked mental retardation genes represents a challenge of considerable medical and scientific importance. AIM: An attempt to identify new genes and mutations in known genes in 14 families with nonspecific X-linked mental retardation. MATERIAL AND METHODS: Linkage analysis with microsatellite markers was performed in 14 families with mental retardation segregating as an X-linked feature. Significant lod score (> 2) was obtained only for 2 families, due to insufficient number of analyzed families' members. Known MRX genes located in the linkage intervals were analysed. RESULTS: Analysis of selected known MRX genes enabled identification of pathogenic mutations in 3 out of 14 families. Sequencing of further candidate genes is in progress. In all families the critical region and the number of genes to analyze was significantly narrowed. CONCLUSIONS: Linkage analysis in families with mental retardation segregating as an X-linked feature is still a considerable approach leading to identification of new genes, and mutations in known genes. It is a first step of identification of disease background, even in small families with lod score < 2.