Clinical manifestation of CDKL5 deficiency disorder and identified mutations in a cohort of Slovak patients.

CDKL5 deficiency disorder (CDD) is an independent clinical entity associated with early-onset encephalopathy, which is often considered the type of epileptic encephalopathy with CDKL5 mutation also found in children diagnosed with early-onset seizure (Hanefeld) type of Rett syndrome, epileptic spasms, West syndrome, Lennox-Gastaut syndrome, or autism. Since early seizure onset is a prominent feature, in this study, a cohort of 54 unrelated patients consisting of 26 males and 28 females was selected for CDKL5 screening, with seizures presented before 12 months of age being the only clinical criterion. Five patients were found to have pathogenic or likely pathogenic variants in CDKL5 while 1 was found to have a variant of uncertain significance (p.L522V). Although CDKL5 variants are more frequently identified in female patients, we identified three male and three female patients (11.1 %, 6/54) in this study. Missense variant with unknown inheritance (p.L522V), de novo missense variant (p.E60 K), two de novo splicing (IVS15 + 1G > A, IVS16 + 2 T > A), and one de novo nonsense variant p.W125* were identified using Sanger sequencing. Whole exome analysis approach revealed de novo frameshift variant c.1247_1248delAG in a mosaic form in one of the males. Patient clinical features are reviewed and compared to those previously described in related literature. Variable clinical features were presented in CDKL5 positive patients characterised in this study. In addition to more common features, such as early epileptic seizures, severe intellectual disability, and gross motor impairment, inappropriate laughing/screaming spells and hypotonia appeared at the age of 1 year in all patients, regardless of the type of CDKL5 mutation or sex. All three CDKL5 positive males from our cohort were initially diagnosed with West syndrome, which suggests that the CDKL5 gene mutations are a significant cause of West syndrome phenotype, and also indicate the overlapping characteristics of these two clinical entities.

A Study among the Genotype, Functional Alternations, and Phenotype of 9 SCN1A Mutations in Epilepsy Patients.

Mutations in the voltage-gated sodium channel Nav1.1 (SCN1A) are linked to various epileptic phenotypes with different severities, however, the consequences of newly identified SCN1A variants on patient phenotype is uncertain so far. The functional impact of nine SCN1A variants, including five novel variants identified in this study, was studied using whole-cell patch-clamp recordings measurement of mutant Nav1.1 channels expressed in HEK293T mammalian cells. E78X, W384X, E1587K, and R1596C channels failed to produce measurable sodium currents, indicating complete loss of channel function. E788K and M909K variants resulted in partial loss of function by exhibiting reduced current density, depolarizing shifts of the activation and hyperpolarizing shifts of the inactivation curves, and slower recovery from inactivation. Hyperpolarizing shifts of the activation and inactivation curves were observed in D249E channels along with slower recovery from inactivation. Slower recovery from inactivation was observed in E78D and T1934I with reduced current density in T1934I channels. Various functional effects were observed with the lack of sodium current being mainly associated with severe phenotypes and milder symptoms with less damaging channel alteration. In vitro functional analysis is thus fundamental for elucidation of the molecular mechanisms of epilepsy, to guide patients' treatment, and finally indicate misdiagnosis of SCN1A related epilepsies.

Structural and Functional Impact of Seven Missense Variants of Phenylalanine Hydroxylase.

The molecular genetics of well-characterized inherited diseases, such as phenylketonuria (PKU) and hyperphenylalaninemia (HPA) predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene, is often complicated by the identification of many novel variants, often with no obvious impact on the associated disorder. To date, more than 1100 PAH variants have been identified of which a substantial portion have unknown clinical significance. In this work, we study the functionality of seven yet uncharacterized PAH missense variants p.Asn167Tyr, p.Thr200Asn, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, p.Ala342Pro, and p.Ile406Met first identified in the Czech PKU/HPA patients. From all tested variants, three of them, namely p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met, exerted residual enzymatic activity in vitro similar to wild type (WT) PAH, however, when expressed in HepG2 cells, their protein level reached a maximum of 72.1% ± 4.9%, 11.2% ± 4.2%, and 36.6% ± 7.3% compared to WT PAH, respectively. Remaining variants were null with no enzyme activity and decreased protein levels in HepG2 cells. The chaperone-like effect of applied BH4 precursor increased protein level significantly for p.Asn167Tyr, p.Asp229Gly, p.Ala342Pro, and p.Ile406Met. Taken together, our results of functional characterization in combination with in silico prediction suggest that while p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met PAH variants have a mild impact on the protein, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, and p.Ala342Pro severely affect protein structure and function.