New mutations in KCNT2 gene causing early infantile epileptic encephalopathy type 57: Case study and literature review.

PURPOSE: Early infantile epileptic encephalopathy (EIEE) 57 belongs to a group of encephalopathies with early-onset and characterised by severe electroencephalogram abnormalities, seizures, developmental delay and intellectual disability. METHOD: We carried out Whole Exome analysis using Next Generation Sequencing (NGS) and bioinformatic analysis performed to find mutation associated with the patient phenotypes. The effect of the mutation on protein structure analysed by PolyPhen2 and Swissmodel ExPASy. RESULTS: In this study, we evaluated two unrelated Turkish males diagnosed with EIEE type 57 to investigate the genetic cause of this disease. Whole exome sequencing revealed mutations in KCN2 gene, which is a member of Potassium channels (KCN) gene family associated with epileptic encephalopathies. Two mutations, c.545A>T (p.Asn182Ile and c.2638C>A (p.Leu880Met) were reported here as a novel mutation. CONCLUSIONS: Our findings implicate the genotype-phenotype correlation of these mutations. Furthermore, the computational analysis showed their effect on protein binding site and function suggesting their role in the development of early infantile epileptic encephalopathy 57.

The New CIC Mutation Associates with Mental Retardation and Severity of Seizure in Turkish Child with a Rare Class I Glucose-6-Phosphate Dehydrogenase Deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive disease that causes acute or chronic hemolytic anemia and potentially leads to severe jaundice in response to oxidative agents. Capicua transcriptional repressor (CIC) is an important gene associated with mental retardation, autosomal dominant 45. Affiliated tissues including skin, brain, bone, and related phenotypes are intellectual disability and seizures. Clinical, biochemical, and whole exome analysis are carried out in a Turkish family. Mutation analysis of G6PD and CIC genes by Sanger sequencing in the whole family was carried out to reveal the effect of these mutations on the patient's clinical outcome. Here, we present the case of epilepsy in an 8-year-old child with a hemizygous variation in G6PD gene and heterozygous mutation in CIC gene, resulting in focal epileptiform activity and hypsarrhythmia in electroencephalography (EEG), seizures, psychomotor retardation, speech impairment, intellectual disability, developmental regression, and learning difficulties. Whole exome sequencing confirmed the diagnosis of X-linked increased susceptibility for hemolytic anemia due to G6PD deficiency and mental retardation type 45 due to CIC variant, which explained the development of epileptic seizures. Considering CIC variant and relevant relation with the severity and course of the disease, G6PD mutations sustained through the family are defined as hereditary. Our findings could represent the importance of variants found in G6PD as well as CIC genes linked to the severity of epilepsy, which was presumed based on the significant changes in protein configuration.

A novel mutation in the SERAC1 gene correlates with the severe manifestation of the MEGDEL phenotype, as revealed by whole-exome sequencing.

The condition 3-methylglutaconic aciduria (3-MGA) with deafness, encephalopathy and Leigh-like (MEGDEL) syndrome, also known as 3-MGA IV, is one of a group of five rare metabolic disorders characterized by mitochondrial dysfunction, resulting in a series of phenotypic abnormalities. It is a rare, recessive inherited disorder with a limited number of cases reported worldwide; hence, it is important to study each case to understand its genetic complexity. An impaired activity of serine active site-containing protein 1 (SERAC1), caused by mutations, leads to defects in phosphatidylglycerol remodelling, which is important for mitochondrial function and intracellular cholesterol trafficking. In the present study, the patients (two male siblings of consanguineous Turkish parents) were analysed, whose multisystem dysfunctions, including an elevated 3-MGA concentration in early age, hearing loss and Leigh-like syndrome as determined by MRI, were consistent with MEGDEL syndrome. A novel mutation in the SERAC1 gene, in the upstream lipase domain, c.1015G>C (p.Gly339Arg) mutation located on exon 10 of the SERAC1, was identified and predicted to cause protein dysfunction. Furthermore, the results pointed towards a possible association between this mutation and the severity of MEGDEL syndrome.

ASC-1 Is a Cell Cycle Regulator Associated with Severe and Mild Forms of Myopathy.

OBJECTIVE: Recently, the ASC-1 complex has been identified as a mechanistic link between amyotrophic lateral sclerosis and spinal muscular atrophy (SMA), and 3 mutations of the ASC-1 gene TRIP4 have been associated with SMA or congenital myopathy. Our goal was to define ASC-1 neuromuscular function and the phenotypical spectrum associated with TRIP4 mutations. METHODS: Clinical, molecular, histological, and magnetic resonance imaging studies were made in 5 families with 7 novel TRIP4 mutations. Fluorescence activated cell sorting and Western blot were performed in patient-derived fibroblasts and muscles and in Trip4 knocked-down C2C12 cells. RESULTS: All mutations caused ASC-1 protein depletion. The clinical phenotype was purely myopathic, ranging from lethal neonatal to mild ambulatory adult patients. It included early onset axial and proximal weakness, scoliosis, rigid spine, dysmorphic facies, cutaneous involvement, respiratory failure, and in the older cases, dilated cardiomyopathy. Muscle biopsies showed multiminicores, nemaline rods, cytoplasmic bodies, caps, central nuclei, rimmed fibers, and/or mild endomysial fibrosis. ASC-1 depletion in C2C12 and in patient-derived fibroblasts and muscles caused accelerated proliferation, altered expression of cell cycle proteins, and/or shortening of the G0/G1 cell cycle phase leading to cell size reduction. INTERPRETATION: Our results expand the phenotypical and molecular spectrum of TRIP4-associated disease to include mild adult forms with or without cardiomyopathy, associate ASC-1 depletion with isolated primary muscle involvement, and establish TRIP4 as a causative gene for several congenital muscle diseases, including nemaline, core, centronuclear, and cytoplasmic-body myopathies. They also identify ASC-1 as a novel cell cycle regulator with a key role in cell proliferation, and underline transcriptional coregulation defects as a novel pathophysiological mechanism. ANN NEUROL 2020;87:217-232.

Revealing the function of a novel splice-site mutation of CHD7 in CHARGE syndrome.

Most cases of CHARGE syndrome are sporadic and autosomal dominant. CHD7 is a major causative gene of CHARGE syndrome. In this study, we screened CHD7 in two Turkish patients demonstrating symptoms of CHARGE syndrome such as coloboma, heart defect, choanal atresia, retarded growth, genital abnomalities and ear anomalies. Two mutations of CHD7 were identified including a novel splice-site mutation (c.2443-2A>G) and a previously known frameshift mutation (c.2504_2508delATCTT). We performed exon trapping analysis to determine the effect of the c.2443-2A>G mutation at the transcriptional level, and found that it caused a complete skip of exon 7 and splicing at a cryptic splice acceptor site. Our current study is the second study demonstrating an exon 7 deficit in CHD7. Results of previous studies suggest that the c.2443-2A>G mutation affects the formation of nasal tissues and the neural retina during early development, resulting in choanal atresia and coloboma, respectively. The findings of the present study will improve our understanding of the genetic causes of CHARGE syndrome.

Novel POC1A mutation in primordial dwarfism reveals new insights for centriole biogenesis.

POC1A encodes a WD repeat protein localizing to centrioles and spindle poles and is associated with short stature, onychodysplasia, facial dysmorphism and hypotrichosis (SOFT) syndrome. These main features are related to the defect in cell proliferation of chondrocytes in growth plate. In the current study, we aimed at identifying the molecular basis of two patients with primordial dwarfism (PD) in a single family through utilization of whole-exome sequencing. A novel homozygous p.T120A missense mutation was detected in POC1A in both patients, a known causative gene of SOFT syndrome, and confirmed using Sanger sequencing. To test the pathogenicity of the detected mutation, primary fibroblast cultures obtained from the patients and a control individual were used. For evaluating the global gene expression profile of cells carrying p.T120A mutation in POC1A, we performed the gene expression array and compared their expression profiles to those of control fibroblast cells. The gene expression array analysis showed that 4800 transcript probes were significantly deregulated in cells with p.T120A mutation in comparison to the control. GO term association results showed that deregulated genes are mostly involved in the extracellular matrix and cytoskeleton. Furthermore, the p.T120A missense mutation in POC1A caused the formation of abnormal mitotic spindle structure, including supernumerary centrosomes, and changes in POC1A were accompanied by alterations in another centrosome-associated WD repeat protein p80-katanin. As a result, we identified a novel mutation in POC1A of patients with PD and showed that this mutation causes the formation of multiple numbers of centrioles and multipolar spindles with abnormal chromosome arrangement.

Whole-exome sequencing revealed two novel mutations in Usher syndrome.

Usher syndrome is a clinically and genetically heterogeneous autosomal recessive inherited disorder accompanied by hearing loss and retinitis pigmentosa (RP). Since the associated genes are various and quite large, we utilized whole-exome sequencing (WES) as a diagnostic tool to identify the molecular basis of Usher syndrome. DNA from a 12-year-old male diagnosed with Usher syndrome was analyzed by WES. Mutations detected were confirmed by Sanger sequencing. The pathogenicity of these mutations was determined by in silico analysis. A maternally inherited deleterious frameshift mutation, c.14439_14454del in exon 66 and a paternally inherited non-sense c.10830G>A stop-gain SNV in exon 55 of USH2A were found as two novel compound heterozygous mutations. Both of these mutations disrupt the C terminal of USH2A protein. As a result, WES revealed two novel compound heterozygous mutations in a Turkish USH2A patient. This approach gave us an opportunity to have an appropriate diagnosis and provide genetic counseling to the family within a reasonable time.

Mutations in the voltage-gated potassium channel gene KCNH1 cause Temple-Baraitser syndrome and epilepsy.

Temple-Baraitser syndrome (TBS) is a multisystem developmental disorder characterized by intellectual disability, epilepsy, and hypoplasia or aplasia of the nails of the thumb and great toe. Here we report damaging de novo mutations in KCNH1 (encoding a protein called ether à go-go, EAG1 or KV10.1), a voltage-gated potassium channel that is predominantly expressed in the central nervous system (CNS), in six individuals with TBS. Characterization of the mutant channels in both Xenopus laevis oocytes and human HEK293T cells showed a decreased threshold of activation and delayed deactivation, demonstrating that TBS-associated KCNH1 mutations lead to deleterious gain of function. Consistent with this result, we find that two mothers of children with TBS, who have epilepsy but are otherwise healthy, are low-level (10% and 27%) mosaic carriers of pathogenic KCNH1 mutations. Consistent with recent reports, this finding demonstrates that the etiology of many unresolved CNS disorders, including epilepsies, might be explained by pathogenic mosaic mutations.

Poikiloderma with neutropenia: genotype-ethnic origin correlation, expanding phenotype and literature review.

Poikiloderma with neutropenia (PN), is a rare genodermatosis associated with patognomic features of poikiloderma and permanent neutropenia. Three common recurrent mutations of related gene, USB1, were considered to be associated with three different ethnic origins. The most common recurrent mutation, c.531delA, has been detected in seven Caucasian patients in the literature. In this paper, we present review of all patients from the literature and report two additional patients of Turkish ancestry with the diagnosis of PN. The diagnosis of these two PN patients were made clinically and confirmed by molecular analysis which detected the most common recurrent mutation, c.531delA. Genotype-ethnic origin correlation hypothesis, therefore, has been strengthened with this result. Short stature in PN, is a common finding, which until now has never been treated with growth hormone (GH). One of our patients is the first patient with attempted treatment of short stature via GH administration. Finally, both of our patients had high-pitched voice and vocal cord nodules which might be considered as additional clinical findings not associated with PN before.

Human CLP1 mutations alter tRNA biogenesis, affecting both peripheral and central nervous system function.

CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.R140H) in five unrelated families, leading to a loss of CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex, largely reduced pre-tRNA cleavage activity, and accumulation of linear tRNA introns. The affected individuals develop severe motor-sensory defects, cortical dysgenesis, and microcephaly. Mice carrying kinase-dead CLP1 also displayed microcephaly and reduced cortical brain volume due to the enhanced cell death of neuronal progenitors that is associated with reduced numbers of cortical neurons. Our data elucidate a neurological syndrome defined by CLP1 mutations that impair tRNA splicing. Reduction of a founder mutation to homozygosity illustrates the importance of rare variations in disease and supports the clan genomics hypothesis.

Therapeutic plasma exchange for malignant refractory status epilepticus: a case report.

BACKGROUND: Refractory status epilepticus is a prolongation of status epilepticus despite anticonvulsant therapy with two or three medications in proper doses; it is defined as malignant status epilepticus if it takes weeks or months. Intravenous immunoglobulin, high-dose steroids, magnesium infusion, pyridoxine, hypothermia, ketogenic diet, electroconvulsive therapy, and surgical therapy are the other treatment options for status epilepticus. PATIENT: Our 5-year-old male patient was hospitalized at our pediatric intensive care unit because of status epilepticus secondary to meningoencephalitis. No response could be obtained with many medical and nonmedical therapies in our patient, who developed malignant status epilepticus with unknown etiology. Therapeutic plasma exchange was applied as convulsions continued. RESULT: Ours is the first child for whom therapeutic plasma exchange was successfully applied because of malignant refractory status epilepticus secondary to meningoencephalitis. CONCLUSION: Therapeutic plasma exchange may be a treatment option for children with refractory status epilepticus following presumed meningoencephalitis.

Keutel syndrome: report of two novel MGP mutations and discussion of clinical overlap with arylsulfatase E deficiency and relapsing polychondritis.

Keutel syndrome is a rare, autosomal recessive disorder characterized by diffuse cartilage calcification, peripheral pulmonary artery stenosis, midface retrusion, and short distal phalanges. To date, 28 patients from 18 families have been reported, and five mutations in the matrix Gla protein gene (MGP) have been identified. The matrix Gla protein (MGP) is a vitamin K-dependent extracellular protein that functions as a calcification inhibitor through incompletely understood mechanisms. We present the clinical manifestations of three affected siblings from a consanguineous Turkish family, in whom we detected the sixth MGP mutation (c.79G>T, which predicts p.E27X) and a fourth unrelated patient in whom we detected the seventh MGP mutation, a partial deletion of exon 4. Both mutations predict complete loss of MGP function. One of the patients presented initially with a working diagnosis of relapsing polychondritis. Clinical features suggestive of Keutel syndrome were also observed in one additional unrelated patient who was later found to have a deletion of arylsulfatase E, consistent with a diagnosis of X-linked recessive chondrodysplasia punctata. Through a discussion of these cases, we highlight the clinical overlap of Keutel syndrome, X-linked chondrodysplasia punctata, and the inflammatory disease relapsing polychondritis.

The effect of genetic polymorphisms of cytochrome P450 CYP2C9, CYP2C19, and CYP2D6 on drug-resistant epilepsy in Turkish children.

BACKGROUND AND OBJECTIVE: Despite the availability of several antiepileptic drugs, drug resistance remains one of the major challenges in epilepsy therapy. Genetic factors are known to play a significant role in the prognosis and treatment of epilepsy. The aim of this study was to determine the frequencies of alleles for CYP2C9, CYP2C19, and CYP2D6 genes in Turkish children with epilepsy, and to investigate the relationship between the genetic polymorphism of these genes with multiple drug resistance in epilepsy patients. METHODS: We genotyped 132 epileptic patients (60 drug resistant and 72 drug responsive) and 55 healthy controls for six single nucleotide polymorphisms (SNPs) in CYP2C9, CYP2C19, and CYP2D6. Genotype, allele, and haplotype frequencies were compared between groups. RESULTS: The frequencies of CYP2C9*3/*3 genotype and CYP2C9*3 allele, and the haplotype CCGG (CYP2C9*2 C>T, CYP2C9*3 A>C, and CYP2C19*2 G>A, CYP2C19* G>A) were significantly higher in drug-resistant versus -responsive patients. CONCLUSION: Our results demonstrated the important role of the CYP2C9*3 allelic variant in preventing epilepsy patients from developing drug resistance. These data suggest that CYP2C9, CYP2C19, and CYP2D6 SNPs and haplotypes may affect the response to antiepileptic drugs.

The drug-transporter gene MDR1 C3435T and G2677T/A polymorphisms and the risk of multidrug-resistant epilepsy in Turkish children.

One-third of all individuals with epilepsy are resistant to antiepileptic drug (AED) treatment. Antiepileptic treatment response has been suggested to be modulated by genetic polymorphisms of drug efflux transporters. Several polymorphic variants within the multidrug resistance 1 (MDR1) gene, which encodes the major transmembrane efflux transporter P-glycoprotein, have been proposed to be associated with AED resistance in epilepsy patients. The aim of this study was to evaluate the effect of C3435T and G2677T/A polymorphisms of MDR1 on AED resistance in Turkish children with epilepsy. MDR1 C3435T and G2677T/A were genotyped in 152 patients with epilepsy, classified as drug-resistant in 69 and drug-responsive in 83. Genotypes of the C3435T and G2677T/A polymorphisms were determined by polymerase chain reaction followed by restriction fragment length polymorphism. Genotype and allele frequencies of C3435T and G2677T/A polymorphisms of the MDR1 gene did not differ between drug-resistant and drug-responsive epilepsy patients. Our results suggest that MDR1 C3435T and G2677T/A polymorphisms are not associated with AED resistance in Turkish epileptic patients. To clarify the exact clinical implication of the MDR1 polymorphisms on the multidrug resistance in epilepsy, further investigations in various ethnic populations would be necessary.

Melatonin attenuates phenytoin sodium-induced DNA damage.

Phenytoin sodium (PHT Na(+)) is a potent antiepileptic drug against epileptic seizures and is used as a prophylactic treatment in traumatic brain injury. PHT Na(+) leads to the formation of reactive oxygen species (ROS), and DNA is a crucial molecular target of ROS-initiated toxicity. Melatonin and its metabolites possess free-radical-scavenging activity. We therefore designed this study to investigate the potential protective effect of melatonin against PHT Na(+)-induced DNA damage by using the comet assay in a rat model in vivo. Thirty-three 3-month-old male Wistar rats were divided into five groups of control treated with isotonic sodium chloride (a single injection of isotonic sodium chloride and 100 µL in drinking water for 10 days), ethanol treated (in drinking water for 10 days containing 100 µL of ethanol in each 300-mL drinking bottle), melatonin treated (4 mg/kg body weight [b.w.] intraperitoneally [i.p.] at the start, in drinking water for 10 days), PHT Na(+) treated (a single i.p. injection of 50 mg/kg) and PHT Na(+) (50 mg/kg b.w., single i.p.) and melatonin (4 mg/kg b.w. i.p. at the start and 4 mg/kg in drinking water for 10 days) cotreated. To determine the protective effects of melatonin, the comet assay was performed using lymphocytes isolated in different time intervals (0, 15, 30, 45 and 60 minutes) from each group of animals. On days 1, 3, 7 and 10, blood samples were taken and the comet assay technique was performed. Our present data suggest that melatonin reversed PHT Na(+)-induced DNA damage.

A novel EFNB1 mutation in a patient with craniofrontonasal syndrome and right hallux duplication.

Craniofrontonasal syndrome (CFNS, MIM #304110) is a rare X-linked dominant developmental disorder that shows paradoxically greater severity in affected females than in affected males. Our female patient with frontonasal dysplasia, craniosynostosis and additional malformations was consistent with CFNS. EFNB1, which encodes a member of the ephrin family of transmembrane ligands for Eph receptor tyrosine kinases, is the only gene in which mutation is known to cause CFNS. Here, we describe 402T>C, a novel de novo mutation on EFNB1. This mutation results in substitution of highly conserved isoleucine at 134th residue to threonine.

MicroRNA profiling in lymphocytes and serum of tyrosinemia type-I patients.

Tyrosinemia type-I results from lack of fumarylacetoacetate hydrolase (FAH), which is a liver enzyme and also shown to be present in lymphocytes, fibroblasts, and cultured amniotic fluid cells. In young infants, symptoms of untreated Tyrosinemia type-I are restricted to severe liver involvement. Later in the first year; however, it is known to be present with liver and renal tubular dysfunction associated with growth failure and rickets. MicroRNAs are small regulatory RNAs that function post-transcriptionally. They target commonly 3'-UTR of the mRNAs and inhibit protein expression by either blocking the synthesis or causing degradation of the mRNAs. MiRNA deregulation was observed in a variety of pathologic conditions but their roles in metabolic diseases were remained unsolved. We studied 6 patients with classical phenotypes of Tyrosinemia type-I. To identify possible miRNAs targeting FAH transcripts, microarray profiling of 961 miRNAs for lymphocytes and serum is performed. Computational algorithms are used for prediction of putative mRNA-miRNA interactions. A number of deregulated miRNAs, targeting the non-conserved sites on FAH transcripts were found. Besides, there are some miRNAs that are similarly altered both in lymphocytes and serum, possibly contributing to the disease phenotype. Since miRNAs may have an active role in the enzymatic pathway of tyrosine catabolism, characterizing miRNA profile in fibroblasts of tyrosinemia patients is also important because miRNAs would have distinctive role in disease pathogenesis and they are promising for future therapeutic studies.