Cross-sectional quantitative analysis of the natural history of TUBA1A and TUBB2B tubulinopathies.

PURPOSE: TUBA1A and TUBB2B tubulinopathies are rare neurodevelopmental disorders characterized by cortical and extracortical malformations and heterogenic phenotypes. There is a need for quantitative clinical endpoints that will be beneficial for future diagnostic and therapeutic trials. METHODS: Quantitative natural history modeling of individuals with TUBA1A and TUBB2B tubulinopathies from clinical reports and database entries of DECIPHER and ClinVar. Main outcome measures were age at disease onset, survival, and diagnostic delay. Phenotypical, neuroradiological, and histopathological features were descriptively illustrated. RESULTS: Mean age at disease onset was 4 (TUBA1A) and 6 months (TUBB2B), respectively. Mortality was equally estimated with 7% at 3.2 (TUBA1A) and 8.0 years (TUBB2B). Diagnostic delay was significantly higher in TUBB2B (12.3 years) compared with TUBA1A tubulinopathy (4.2 years). We delineated the isotype-dependent clinical, neuroradiological, and histopathological phenotype of affected individuals and present brain malformations associated with epilepsy and an unfavorable course of disease. CONCLUSION: The natural history of tubulinopathies is defined by the genotype and associated brain malformations. Defined data on estimated survival, diagnostic delay, and disease characteristics of TUBA1A and TUBB2B tubulinopathy will help to raise disease awareness and encourage future clinical trials to optimize genetic testing, family counseling, and supportive care.

Refining Genotypes and Phenotypes in KCNA2-Related Neurological Disorders.

Pathogenic variants in KCNA2, encoding for the voltage-gated potassium channel Kv1.2, have been identified as the cause for an evolving spectrum of neurological disorders. Affected individuals show early-onset developmental and epileptic encephalopathy, intellectual disability, and movement disorders resulting from cerebellar dysfunction. In addition, individuals with a milder course of epilepsy, complicated hereditary spastic paraplegia, and episodic ataxia have been reported. By analyzing phenotypic, functional, and genetic data from published reports and novel cases, we refine and further delineate phenotypic as well as functional subgroups of KCNA2-associated disorders. Carriers of variants, leading to complex and mixed channel dysfunction that are associated with a gain- and loss-of-potassium conductance, more often show early developmental abnormalities and an earlier onset of epilepsy compared to individuals with variants resulting in loss- or gain-of-function. We describe seven additional individuals harboring three known and the novel KCNA2 variants p.(Pro407Ala) and p.(Tyr417Cys). The location of variants reported here highlights the importance of the proline(405)-valine(406)-proline(407) (PVP) motif in transmembrane domain S6 as a mutational hotspot. A novel case of self-limited infantile seizures suggests a continuous clinical spectrum of KCNA2-related disorders. Our study provides further insights into the clinical spectrum, genotype-phenotype correlation, variability, and predicted functional impact of KCNA2 variants.

Prenatal phenotype of PNKP-related primary microcephaly associated with variants affecting both the FHA and phosphatase domain.

Biallelic PNKP variants cause heterogeneous disorders ranging from neurodevelopmental disorder with microcephaly/seizures to adult-onset Charcot-Marie-Tooth disease. To date, only postnatal descriptions exist. We present the first prenatal diagnosis of PNKP-related primary microcephaly. Pathological examination of a male fetus in the 18th gestational week revealed micrencephaly with extracerebral malformations and thus presumed syndromic microcephaly. A recessive disorder was suspected because of previous pregnancy termination for similar abnormalities. Prenatal trio-exome sequencing identified compound heterozygosity for the PNKP variants c.498G>A, p.[(=),0?] and c.302C>T, p.(Pro101Leu). Segregation confirmed both variants in the sister fetus. Through RNA analyses, we characterized exon 4 skipping affecting the PNKP forkhead-associated (FHA) and phosphatase domains (p.Leu67_Lys166del) as the predominant effect of the paternal c.498G>A variant. We retrospectively investigated two unrelated individuals diagnosed with biallelic PNKP-variants to compare prenatal/postnatal phenotypes. Both carry the splice donor variant c.1029+2T>C in trans with a variant in the FHA domain (c.311T>C, p.(Leu104Pro); c.151G>C, p.(Val51Leu)). RNA-seq showed complex splicing for c.1029+2T>C and c.151G>C. Structural modeling revealed significant clustering of missense variants in the FHA domain with variants generating structural damage. Our clinical description extends the PNKP-continuum to the prenatal stage. Investigating possible PNKP-variant effects using RNA and structural modeling, we highlight the mutational complexity and exemplify a PNKP-variant characterization framework.

Efficacy, Tolerability, and Retention of Antiseizure Medications in PRRT2-Associated Infantile Epilepsy.

Background and Objectives: Pathogenic variants in PRRT2, encoding for the proline-rich transmembrane protein 2, were identified as the main cause of self-limiting sporadic and familial infantile epilepsy. Reported data on treatment response to antiseizure medications (ASMs) in defined monogenic epilepsies are limited. The aim of this study was to evaluate the treatment response of ASMs in children with monogenic PRRT2-associated infantile epilepsy. Methods: A multicenter, retrospective, cross-sectional cohort study was conducted according to the Strengthening the Reporting of Observational Studies in Epidemiology criteria. Inclusion criteria were occurrence of infantile seizures and genetic diagnosis of likely pathogenic/pathogenic PRRT2 variants. Results: Treatment response data from 52 individuals with PRRT2-associated infantile epilepsy with a total of 79 treatments (defined as each use of an ASM in an individual) were analyzed. Ninety-six percent (50/52) of all individuals received ASMs. Levetiracetam (LEV), oxcarbazepine (OXC), valproate (VPA), and phenobarbital (PB) were most frequently administered. Sodium channel blockers were used in 22 individuals and resulted in seizure freedom in all but 1 child, who showed a reduction of more than 50% in seizure frequency. By contrast, treatment with LEV was associated with worsening of seizure activity in 2/25 (8%) treatments and no effect in 10/25 (40%) of treatments. LEV was rated significantly less effective also compared with VPA and PB. The retention rate for LEV was significantly lower compared with all aforementioned ASMs. No severe adverse events were reported, and no discontinuation of treatment was reported because of side effects. Discussion: In conclusion, a favorable effect of most ASMs, especially sodium channel blockers such as carbamezepine and OXC, was observed, whereas the efficacy and the retention rate of LEV was lower in PRRT2-associated childhood epilepsy. Tolerability in these young children was good for all ASMs reported in the cohort. Classification of Evidence: This study provides Class IV evidence that in individuals with PRRT2-associated infantile epilepsy, sodium channel blockers are associated with reduced seizure frequency but levetiracetam is not.

Complementing the phenotypical spectrum of TUBA1A tubulinopathy and its role in early-onset epilepsies.

TUBA1A tubulinopathy is a rare neurodevelopmental disorder associated with brain malformations as well as early-onset and intractable epilepsy. As pathomechanisms and genotype-phenotype correlations are not completely understood, we aimed to provide further insights into the phenotypic and genetic spectrum. We here present a multicenter case series of ten unrelated individuals from four European countries using systematic MRI re-evaluation, protein structure analysis, and prediction score modeling. In two cases, pregnancy was terminated due to brain malformations. Amongst the eight living individuals, the phenotypic range showed various severity. Global developmental delay and severe motor impairment with tetraparesis was present in 63% and 50% of the subjects, respectively. Epilepsy was observed in 75% of the cases, which showed infantile onset in 83% and a refractory course in 50%. One individual presented a novel TUBA1A-associated electroclinical phenotype with evolvement from early myoclonic encephalopathy to continuous spike-and-wave during sleep. Neuroradiological features comprised a heterogeneous spectrum of cortical and extracortical malformations including rare findings such as cobblestone lissencephaly and subcortical band heterotopia. Two individuals developed hydrocephalus with subsequent posterior infarction. We report four novel and five previously published TUBA1A missense variants whose resulting amino acid substitutions likely affect longitudinal, lateral, and motor protein interactions as well as GTP binding. Assessment of pathogenic and benign variant distributions in synopsis with prediction scores revealed sections of variant enrichment and intolerance to missense variation. We here extend the clinical, neuroradiological, and genetic spectrum of TUBA1A tubulinopathy and provide insights into residue-specific pathomechanisms and genotype-phenotype correlations.

Exploring two-photon optogenetics beyond 1100 nm for specific and effective all-optical physiology.

Two-photon (2-P) all-optical approaches combine in vivo 2-P calcium imaging and 2-P optogenetic modulations. Here, firstly, we combined in vivo juxtacellular recordings and GCaMP6f-based 2-P calcium imaging in mouse visual cortex to tune our detection algorithm towards a 100% specific identification of action potential-related calcium transients. Secondly, we minimized photostimulation artifacts by using extended-wavelength-spectrum laser sources for optogenetic stimulation. We achieved artifact-free all-optical experiments performing optogenetic stimulation from 1100 nm to 1300 nm. Thirdly, we determined the spectral range for maximizing efficacy until 1300 nm. The rate of evoked transients in GCaMP6f/C1V1-co-expressing cortical neurons peaked already at 1100 nm. By refining spike detection and defining 1100 nm as the optimal wavelength for artifact-free and effective GCaMP6f/C1V1-based all-optical physiology, we increased the translational value of these approaches, e.g., for the development of network-based therapies.

Deep-Phenotyping the Less Severe Spectrum of PIGT Deficiency and Linking the Gene to Myoclonic Atonic Seizures.

The two aims of this study were (i) to describe and expand the phenotypic spectrum of PIGT deficiency in affected individuals harboring the c.1582G>A; p.Val528Met or the c.1580A > G; p.Asn527Ser variant in either homozygous or compound heterozygous state, and (ii) to identify potential genotype-phenotype correlations and any differences in disease severity among individuals with and without the PIGT variants. The existing literature was searched to identify individuals with and without the two variants. A detailed phenotypic assessment was performed of 25 individuals (both novel and previously published) with the two PIGT variants. We compared severity of disease between individuals with and without these PIGT variants. Twenty-four individuals carried the PIGT variant Val528Met in either homozygous or compound heterozygous state, and one individual displayed the Asn527Ser variant in a compound heterozygous state. Disease severity in the individual with the Asn527Ser variant was compatible with that in the individuals harboring the Val528Met variant. While individuals without the Asn527Ser or Val528Met variant had focal epilepsy, profound developmental delay (DD), and risk of premature death, those with either of the two variants had moderate to severe DD and later onset of epilepsy with both focal and generalized seizures. Individuals homozygous for the Val528Met variant generally became seizure-free on monotherapy with antiepileptic drugs, compared to other PIGT individuals who were pharmaco-resistant. Two patients were diagnosed with myoclonic-atonic seizures, and a single patient was diagnosed with eyelid myoclonia. Our comprehensive analysis of this large cohort of previously published and novel individuals with PIGT variants broadens the phenotypical spectrum and shows that both Asn527Ser and Val528Met are associated with a milder phenotype and less severe outcome. Our data show that PIGT is a new candidate gene for myoclonic atonic epilepsy. Our genotype-phenotype correlation will be useful for future genetic counseling. Natural history studies of this mild spectrum of PIGT-related disorder may shed light on hitherto unknown aspects of this rare disorder.

Clinical spectrum and treatment outcome of 95 children with continuous spikes and waves during sleep (CSWS).

OBJECTIVE: Continuous spikes and waves during sleep (CSWS) is an epileptic encephalopathy characterized by generalised epileptiform activity and neurocognitive dysfunction. Causes and outcome are diverse and treatment is mainly empirical. METHODS: Retrospective descriptive analysis of clinical and EEG data of children with CSWS diagnosed between 1998 and 2018 at the University Hospital Heidelberg. RESULTS: Ninety-five children were included with a median age at diagnosis of 5.4 years. A structural/metabolic aetiology was found in 43.2%, genetic alterations in 17.9%, while it remained unknown in 38.9%. The proportion of patients with genetic aetiology increased from 10.3% (1998-2007) to 22.8% (2008-2018). On average, each patient received 5 different treatments. CSWS was refractory in >70% of cases, steroids and neurosurgery were most effective. No difference was observed between children with CSWS or Near-CSWS (Spike-Wave-Index 40-85%). CONCLUSIONS: Our cohort confirms CSWS as an age-dependent epileptic encephalopathy. Structural brain abnormalities were most frequent, but genetic causes are increasingly identified. More specific criteria for the diagnosis and treatment goals should be elaborated and implemented based on evidence. SIGNIFICANCE: This study is the largest monocentric observational study on treatment effects in children with CSWS, providing data for diagnostic and therapeutic decisions.

The Phenotypic Spectrum of PRRT2-Associated Paroxysmal Neurologic Disorders in Childhood.

Pathogenic variants in PRRT2, encoding the proline-rich transmembrane protein 2, have been associated with an evolving spectrum of paroxysmal neurologic disorders. Based on a cohort of children with PRRT2-related infantile epilepsy, this study aimed at delineating the broad clinical spectrum of PRRT2-associated phenotypes in these children and their relatives. Only a few recent larger cohort studies are on record and findings from single reports were not confirmed so far. We collected detailed genetic and phenotypic data of 40 previously unreported patients from 36 families. All patients had benign infantile epilepsy and harbored pathogenic variants in PRRT2 (core cohort). Clinical data of 62 family members were included, comprising a cohort of 102 individuals (extended cohort) with PRRT2-associated neurological disease. Additional phenotypes in the cohort of patients with benign sporadic and familial infantile epilepsy consist of movement disorders with paroxysmal kinesigenic dyskinesia in six patients, infantile-onset movement disorders in 2 of 40 individuals, and episodic ataxia after mild head trauma in one girl with bi-allelic variants in PRRT2. The same girl displayed a focal cortical dysplasia upon brain imaging. Familial hemiplegic migraine and migraine with aura were reported in nine families. A single individual developed epilepsy with continuous spikes and waves during sleep. In addition to known variants, we report the novel variant c.843G>T, p.(Trp281Cys) that co-segregated with benign infantile epilepsy and migraine in one family. Our study highlights the variability of clinical presentations of patients harboring pathogenic PRRT2 variants and expands the associated phenotypic spectrum.

Thirty Years of Orphan Drug Legislation and the Development of Drugs to Treat Rare Seizure Conditions: A Cross Sectional Analysis.

BACKGROUND: Epilepsy is a serious chronic health condition with a high morbidity impairing the life of patients and afflicted families. Many epileptic conditions, especially those affecting children, are rare disorders generating an urgent medical need for more efficacious therapy options. Therefore, we assessed the output of the US and European orphan drug legislations. METHODS: Quantitative analysis of the FDA and EMA databases for orphan drug designations according to STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) criteria. RESULTS: Within the US Orphan Drug Act 40 designations were granted delivering nine approvals, i.e. clobazam, diazepam viscous solution for rectal administration, felbamate, fosphenytoin, lamotrigine, repository corticotropin, rufinamide, topiramate, and vigabatrin. Since 2000 the EMA granted six orphan drug designations whereof two compounds were approved, i.e. rufinamide and stiripentol. In the US, two orphan drug designations were withdrawn. Orphan drugs were approved for conditions including Lennox-Gastaut syndrome, infantile spasms, Dravet syndrome, and status epilepticus. Comparing time to approval for rufinamide, which was approved in the US and the EU to treat rare seizure conditions, the process seems faster in the EU (2.2 years) than in the US (4.3 years). CONCLUSION: Orphan drug development in the US and in the EU delivered only few molecular entities to treat rare seizure disorders. The development programs focused on already approved antiepileptic drugs or alternative pharmaceutical formulations. Most orphan drugs approved in the US are not approved in the EU to treat rare seizures although some were introduced after 2000 when the EU adopted the Orphan Drug Regulation.