De novo ARF3 variants cause neurodevelopmental disorder with brain abnormality.

An optimal Golgi transport system is important for mammalian cells. The adenosine diphosphate (ADP) ribosylation factors (ARF) are key proteins for regulating cargo sorting at the Golgi network. In this family, ARF3 mainly works at the trans-Golgi network (TGN), and no ARF3-related phenotypes have yet been described in humans. We here report the clinical and genetic evaluations of two unrelated children with de novo pathogenic variants in the ARF3 gene: c.200A > T (p.Asp67Val) and c.296G > T (p.Arg99Leu). Although the affected individuals presented commonly with developmental delay, epilepsy and brain abnormalities, there were differences in severity, clinical course and brain lesions. In vitro subcellular localization assays revealed that the p.Arg99Leu mutant localized to Golgi apparatus, similar to the wild-type, whereas the p.Asp67Val mutant tended to show a disperse cytosolic pattern together with abnormally dispersed Golgi localization, similar to that observed in a known dominant negative variant (p.Thr31Asn). Pull-down assays revealed that the p.Asp67Val had a loss-of-function effect and the p.Arg99Leu variant had increased binding of the adaptor protein, Golgi-localized, γ-adaptin ear-containing, ARF-binding protein 1 (GGA1), supporting the gain of function. Furthermore, in vivo studies revealed that p.Asp67Val transfection led to lethality in flies. In contrast, flies expressing p.Arg99Leu had abnormal rough eye, as observed in the gain-of-function variant p.Gln71Leu. These data indicate that two ARF3 variants, the possibly loss-of-function p.Asp67Val and the gain-of-function p.Arg99Leu, both impair the Golgi transport system. Therefore, it may not be unreasonable that they showed different clinical features like diffuse brain atrophy (p.Asp67Val) and cerebellar hypoplasia (p.Arg99Leu).

Genetic and clinical landscape of childhood cerebellar hypoplasia and atrophy.

PURPOSE: Cerebellar hypoplasia and atrophy (CBHA) in children is an extremely heterogeneous group of disorders, but few comprehensive genetic studies have been reported. Comprehensive genetic analysis of CBHA patients may help differentiating atrophy and hypoplasia and potentially improve their prognostic aspects. METHODS: Patients with CBHA in 176 families were genetically examined using exome sequencing. Patients with disease-causing variants were clinically evaluated. RESULTS: Disease-causing variants were identified in 96 of the 176 families (54.5%). After excluding 6 families, 48 patients from 42 families were categorized as having syndromic associations with CBHA, whereas the remaining 51 patients from 48 families had isolated CBHA. In 51 patients, 26 aberrant genes were identified, of which, 20 (76.9%) caused disease in 1 family each. The most prevalent genes were CACNA1A, ITPR1, and KIF1A. Of the 26 aberrant genes, 21 and 1 were functionally annotated to atrophy and hypoplasia, respectively. CBHA+S was more clinically severe than CBHA-S. Notably, ARG1 and FOLR1 variants were identified in 2 families, leading to medical treatments. CONCLUSION: A wide genetic and clinical diversity of CBHA was revealed through exome sequencing in this cohort, which highlights the importance of comprehensive genetic analyses. Furthermore, molecular-based treatment was available for 2 families.

Autopsy Report of a Woman with Infantile Alexander Disease Who Survived 39 Years.

Patients with infantile Alexander disease (AxD) usually do not survive beyond their early teens without life support care because of progressive central hypoventilation. We present the autopsy report of a woman with infantile AxD carrying an R239C mutation in the glial fibrillary acidic protein gene, who survived 39 years. She presented with psychomotor retardation in infancy and regressed after age 5. Brain computed tomography scans showed bilateral low frontal white matter density. She became quadriplegic with bulbar palsy and was intellectually handicapped after a measles infection at age 7. Tube feeding was introduced because of dysphagia at age 15. Noninvasive positive pressure ventilation was required due to central hypoventilation in her early thirties. She died of neurogenic respiratory failure at 39 years. Autopsy findings revealed a markedly atrophic brain (709 g, -6.0 standard deviation), especially in the frontal lobe, cerebellum, and brainstem portions. We found demyelination, gliosis, and cystic lesions throughout the brain, and we saw Rosenthal fibers accumulating in the perivascular spaces. We also identified a variety of abnormalities in other organs such as pancreatic necrosis, completely desquamated epithelium in the lower esophagus and stomach, foreign-body giant cells in the colon submucosa, glomerular sclerosis, and multiple bladder stones. This is the first autopsied case report of a patient with infantile AxD with long survival, who showed not only central nervous system characteristic findings, but also unexpected pathological changes in other organs.

Mutations in the glutaminyl-tRNA synthetase gene cause early-onset epileptic encephalopathy.

Aminoacylation is the process of attaching amino acids to their cognate tRNA, and thus is essential for the translation of mRNA into protein. This direct interaction of tRNA with amino acids is catalyzed by aminoacyl-tRNA synthetases. Using whole-exome sequencing, we identified compound heterozygous mutations [c.169T>C (p.Tyr57His) and c.1485dup (p.Lys496*)] in QARS, which encodes glutaminyl-tRNA synthetase, in two siblings with early-onset epileptic encephalopathy (EOEE). Recessive mutations in QARS, including the loss-of-function missense mutation p.Tyr57His, have been reported to cause intractable seizures with progressive microcephaly. The p.Lys496* mutation is novel and causes truncation of the QARS protein, leading to a deletion of part of the catalytic domain and the entire anticodon-binding domain. Transient expression of the p.Lys496* mutant in neuroblastoma 2A cells revealed diminished and aberrantly aggregated expression, indicating the loss-of-function nature of this mutant. Together with the previous report, our data suggest that abnormal aminoacylation is one of the underlying pathologies of EOEE.

Refinement of the deletion in 8q22.2-q22.3: the minimum deletion size at 8q22.3 related to intellectual disability and epilepsy.

Kuechler et al. [2011] reported five patients with interstitial deletions in 8q22.2-q22.3 who had intellectual disability, epilepsy, and dysmorphic features. We report on a new patient with the smallest overlapping de novo deletion in 8q22.3 and refined the phenotype. The proposita was an 8-year-old girl, who developed seizures at 10 months, and her epileptic seizure became severe and difficult to control with antiepileptic drugs. She also exhibited developmental delay and walked alone at 24 months. She was referred to us for evaluation for developmental delay and epilepsy at the age of 8 years. She had intellectual disability (IQ 37 at 7 years) and autistic behavior, and spoke two word sentences at 8 years. She had mild dysmorphic features, including telecanthus and thick vermilion of the lips. Array comparative genomic hybridization detected a 1.36 Mb deletion in 8q22.3 that encompassed RRM2B and NCALD, which encode the small subunit of p53-inducible ribonucleotide reductase and neurocalcin delta in the neuronal calcium sensor family of calcium-binding proteins, respectively. The minimum overlapping region between the present and previously reported patients is considered to be a critical region for the phenotype of the deletion in 8q22.3. We suggest that the deletion in 8q22.3 may represent a clinically recognizable condition, which is characterized by intellectual disability and epilepsy.

[Clinical profile of persistent generalized muscle contraction following the insult of developing brain].

OBJECTIVE: Persons with severe motor and intellectual disabilities (SMID) caused by injury to the developing brain sometimes present generalized hypertonia in a specific position with extreme muscle overactivity persisting for most of the time during wakefulness. This "persistent generalized muscle contraction" is often associated with bad humor, sleep disturbance, hyperhidrosis, wasting, elevation of serum creatine kinase levels, regular daytime use of hypnotic or sedative medication, and the necessity to maintain the neck or hip in a flexed position manually. The aim of this study is to elucidate the clinical profile of this condition. METHODS: We retrospectively examined the medical records and brain imaging data of 66 SMID patients in the state of persistent generalized muscle contraction. RESULTS: Most patients could be classified into 2 major categories on the basis of clinical presentation and brain imaging: (A) those with premature birth and bilateral lesion of globus pallidus interna (kernicterus) (n = 16), and (B) those with various widespread bilateral basal ganglia/thalamic and/or cerebral lesions such as hypoxia-ischemia, acute encephalopathy, malformation, etc (n = 50). Group A assumed an asymmetrical tonic-neck-reflex-like position, torsion of the trunk, fluctuation of hypertonia, and better mental development. Three of them exhibited extreme hypertonia resembling status dystonicus. Group B often exhibited persistent and fixed retroflexion of the neck and trunk or opisthotonus. Drugs such as oral muscular relaxants were ineffective in both groups. Injection of botulinum toxin into the cervical and paravertebral muscles partially alleviated symptoms. CONCLUSIONS: Persistent generalized muscle contraction in SMID has at least two different types. Group A has characteristics of severe dystonic hypertonia that could lead to status dystonicus. Group B might have peculiar characteristics of muscle overactivity triggered by wakefulness or discomfort, which probably results from inability to achieve spontaneous muscle relaxation.

De novo mutations in SLC35A2 encoding a UDP-galactose transporter cause early-onset epileptic encephalopathy.

Early-onset epileptic encephalopathies (EOEE) are severe neurological disorders characterized by frequent seizures accompanied by developmental regression or retardation. Whole-exome sequencing of 12 patients together with five pairs of parents and subsequent Sanger sequencing in additional 328 EOEE patients identified two de novo frameshift and one missense mutations in SLC35A2 at Xp11.23, respectively. The three patients are all females. X-inactivation analysis of blood leukocyte DNA and mRNA analysis using lymphoblastoid cells derived from two patients with a frameshift mutation indicated that only the wild-type SLC35A2 allele was expressed in these cell types, at least in part likely as a consequence of skewed X-inactivation. SLC35A2 encodes a UDP-galactose transporter (UGT), which selectively supplies UDP-galactose from the cytosol to the Golgi lumen. Transient expression experiments revealed that the missense mutant protein was correctly localized in the Golgi apparatus. In contrast, the two frameshift mutant proteins were not properly expressed, suggesting that their function is severely impaired. Defects in the UGT can cause congenital disorders of glycosylation. Of note, no abnormalities of glycosylation were observed in three serum glycoproteins, which is consistent with favorably skewed X-inactivation. We hypothesize that a substantial number of neurons might express the mutant SLC35A2 allele and suffer from defective galactosylation, resulting in EOEE.

Diagnostic utility of whole exome sequencing in patients showing cerebellar and/or vermis atrophy in childhood.

Cerebellar and/or vermis atrophy is recognized in various types of childhood disorders with clinical and genetic heterogeneity. Although careful evaluation of clinical features and neuroimaging can lead to correct diagnosis of disorders, their diagnosis is sometimes difficult because clinical features can overlap with each other. In this study, we performed family-based whole exome sequencing of 23 families including 25 patients with cerebellar and/or vermis atrophy in childhood, who were unable to be diagnosed solely by clinical examination. Pathological mutations of seven genes were found in ten patients from nine families (9/23, 39.1 %): compound heterozygous mutations in FOLR1, C5orf42, POLG, TPP1, PEX16, and de novo mutations in CACNA1A, and ITPR1. Patient 1A with FOLR1 mutations showed extremely low concentration of 5-methyltetrahydrofolate in the cerebrospinal fluid and serum, and Patient 6 with TPP1 mutations demonstrated markedly lowered tripeptidyl peptidase 1 activity in leukocytes. Furthermore, Patient 8 with PEX16 mutations presented a mild increase of very long chain fatty acids in the serum as supportive data for genetic diagnosis. The main clinical features of these ten patients were nonspecific and mixed, and included developmental delay, intellectual disability, ataxia, hypotonia, and epilepsy. Brain MRI revealed both cerebellar and vermis atrophy in eight patients (8/10, 80 %), vermis atrophy/hypoplasia in two patients (2/10, 20 %), and brainstem atrophy in one patient (1/10, 10 %). Our data clearly demonstrate the utility of whole exome sequencing for genetic diagnosis of childhood cerebellar and/or vermis atrophy.

Molecular analysis of the genes causing recessive demyelinating Charcot-Marie-Tooth disease in Japan.

Charcot-Marie-Tooth disease (CMT), the most common hereditary neuropathy, has been classified into two types, demyelinating and axonal types. We previously analyzed the genes causing dominant demyelinating CMT in 227 Japanese patients to identify the genetic background, but could not find any mutations in 110 patients. To investigate the frequency of patients with autosomal recessive demyelinating CMT (CMT4) mutations, we analyzed the coding sequence of known causative genes of CMT4 in 103 demyelinating CMT patients, excluding seven patients owing to lack of specimens. We found one patient with a GDAP1 mutation, one patient with an MTMR2 mutation, two patients with SH3TC2/KIAA1985 mutations and three patients with FGD4 mutations. Twelve patients, including five previously detected patients with PRX mutations, were diagnosed as CMT4, accounting for 5.5% of demyelinating CMT. In the patient with GDAP1 mutation, only one mutation inherited from his mother was detected by genomic sequencing. Analysis by reverse transcription polymerase chain reaction using messenger RNA (mRNA) from the patient's leukocytes revealed the absence of transcription from the allele inherited from his father, suggesting the existence of one more mutation leading to a lack or destabilization of mRNA. Most patients carrying CMT4 gene mutations present with early-onset and slowly progressive symptoms, which may be associated with the function of mutants. We could not identify the disease-causing gene in 96 patients (about 45%). Further studies including studies with next-generation sequencers will be required to identify the causative gene in Japanese CMT.

A child with three episodes of reversible splenial lesion.

In this study, we report the case of an 8-year-old girl who had three episodes of reversible splenial lesion of the corpus callosum (SCC) in 2 years. Vomiting, hypoglycemia, and fever were followed by altered consciousness and diminished muscle tone. In each episode, the clinical manifestations and abnormalities detected during magnetic resonance imaging resolved in 2 weeks. Transient alteration of vision and spike discharges revealed by interictal electroencephalogram implied the SCC lesions were related to epileptic activities. At follow-up, the patient had not presented with SCC lesions or altered consciousness for more than 4 years after undergoing carbamazepine treatment. Our case is the first report of a patient who presented with three episodes of reversible splenial lesion.

Atypical clinical course in two patients with GNB1 variants who developed acute encephalopathy.

INTRODUCTION: Variants in the GNB1 gene, which encodes the ß1 subunit of a trimeric G protein, can cause moderate to severe psychomotor retardation. Acute encephalopathies have also been observed in patients with central nervous system abnormalities; however, severe neurological sequelae have not previously been reported. CASE PRESENTATIONS: Patient 1 was a Japanese female with a de novo GNB1 variant (c.284 T > C). At 8 months old she contracted influenza A and developed generalized convulsions. In the acute phase, brain magnetic resonance imaging (MRI) findings indicated acute encephalopathy; diffuse cerebral atrophy was present 1 month later. Although multidisciplinary treatment was administered, she had severe neurological sequelae including spastic tetraplegia, severe intellectual disabilities, and refractory epilepsy. Patient 2 was a Japanese male with a de novo GNB1 variant (c.239 T > C). He experienced an unexplained respiratory arrest aged 17 years; refractory convulsions developed. Brain MRI at 1 month showed bilateral basal ganglia high intensities; at 3 months, diffuse cerebral cortex and white matter atrophy was observed. Despite multidisciplinary treatment, he developed severe spastic tetraplegia and mental regression. DISCUSSION: We report two patients with GNB1 variants who had acute lesions on brain MRI and unexpected disease courses. In such patients with acute neurological deterioration, multidisciplinary treatment is required; patients should also be carefully observed for progression to acute encephalopathy.

Contiguous deletion of SLC6A8 and BAP31 in a patient with severe dystonia and sensorineural deafness.

We report here a 6-year-old boy exhibiting severe dystonia, profound intellectual and developmental disability with liver disease, and sensorineural deafness. A deficient creatine peak in brain (1)H-MR spectroscopy and high ratio of creatine/creatinine concentration in his urine lead us to suspect a creatine transporter (solute carrier family 6, member 8; SLC6A8) deficiency, which was confirmed by the inability to take up creatine into fibroblasts. We found a large ~19 kb deletion encompassing exons 5-13 of SLC6A8 and exons 5-8 of the B-cell receptor-associated protein (BAP31) gene. This case is the first report in which the SLC6A8 and BAP31 genes are both deleted. The phenotype of BAP31 mutations has been reported only as a part of Xq28 deletion syndrome or contiguous ATP-binding cassette, sub-family D, member 1 (ABCD1)/DXS1375E (BAP31) deletion syndrome [MIM ID #300475], where liver dysfunction and sensorineural deafness have been suggested to be attributed to the loss of function of BAP31. Our case supports the idea that the loss of BAP31 is related to liver dysfunction and hearing loss.

A case of Sjögren-Larsson syndrome with minimal MR imaging findings facilitated by proton spectroscopy.

We present a 5-year-old girl who was ultimately diagnosed with Sjögren-Larsson syndrome (SLS). Although her MRI findings were minimal compared to previously published cases, prominent and characteristic abnormal lipid peaks on single-voxel proton MR spectroscopy ((1)H-MRS) facilitated the diagnosis. This case emphasizes the importance and usefulness of (1)H-MRS in diagnosing SLS.

[Clinical characteristics of acute encephalopathies associated with influenza H1N1-2009 in children].

We report 12 cases of acute encephalopathy associated with influenza H1N1-2009 treated according to Japanese guideline (2009). In all 12 cases, electroencephalogram presented diffuse or localized high-amplitude slow waves. Brain CT and MRI showed abnormalities in 4 and 6 cases, respectively. We used hypothermia therapy for 5 patients. One patient showed impairment in short term memory, while the rest of the patients showed no sequelae. These 12 cases presented here suggest the early recognition and therapy according to the newly proposed guideline may reduce severe sequelae and mortality by acute encephalopathy associated with influenza H1N1-2009.

Elevation of brain gamma-aminobutyric acid levels is associated with vigabatrin-associated brain abnormalities on magnetic resonance imaging.

OBJECTIVE: Vigabatrin (VGB) is an effective antiseizure medication for West syndrome. It works by irreversibly inhibiting gamma-aminobutyric acid (GABA) transaminase and increasing central GABA levels. Vigabatrin-associated brain abnormalities on magnetic resonance imaging (VABAM) are an adverse effect of VGB that has only been reported in children, but the pathophysiology of this effect is unknown. In this study, we evaluated the relationship of VGB and brain GABA levels as well as the association between VABAM and GABA. METHODS: For the 15 consecutive pediatric patients treated with VGB, free GABA, glutamine, and glutamate in cerebrospinal fluid (CSF) and plasma, GABA to creatine and phosphocreatine (Cr) peak ratio (GABA/Cr), glutamine (Gln)/Cr, and glutamate (Glu)/Cr as quantified by proton MR spectroscopy (1H-MRS) were retrospectively examined. GABA/Cr was compared with in-house normal pediatric controls. Differences in the levels of each metabolite in VABAM and non-VABAM cases were also examined. RESULTS: Thirteen of the included subjects underwent magnetic resonance imaging (MRI) and 1H-MRS, and two of them presented VABAM; both cases were very-low-birth-weight preterm infants with post-hemorrhagic hydrocephalus. CSF levels of free GABA were significantly higher in VABAM (5.26 ± 1.95 µmol/L) than in non-VABAM (0.59 ± 0.63 µmol/L) cases (P = 0.03). The GABA/Cr was significantly higher in patients with VGB (0.34 ± 0.16) than in pediatric controls (0.20 ± 0.05) (P = 0.02). The GABA/Cr tended to be higher in VABAM (0.48 ± 0.10) than in non-VABAM cases (0.31 ± 0.15) (P = 0.31). SIGNIFICANCE: Elevated brain GABA levels were observed in VABAM patients, suggesting its involvement in the pathogenesis of this condition. In particular, a marked increase in CSF-free GABA was characteristic. Although the elevation of the GABA/Cr is mild, it may be useful for early identification of patients with risk of VABAM.

Infection-associated decrease of serum creatine kinase levels in Fukuyama congenital muscular dystrophy.

BACKGROUND: Marked decreases in serum creatine kinase levels have been noted in Duchenne and Becker muscular dystrophies as rare complications of autoimmune or autoinflammatory diseases. SUBJECTS AND METHODS: The influence of systemic inflammation on serum creatine kinase levels was reviewed from the charts of three subjects with Fukuyama congenital muscular dystrophy. RESULTS: A total of 30 infectious events were identified. Elevated serum C-reactive protein levels coincided with decreased creatine kinase levels on 19 occasions. In one subject, administration of 2 mg/kg/d prednisolone for bronchial asthma resulted in a decrease in creatine kinase level on six other occasions. CONCLUSION: Apart from an increase in endogenous cortisol secretion, certain inflammation-related molecules could play a role in mitigating muscle cell damage in Fukuyama congenital muscular dystrophy during febrile infectious episodes. Corticosteroids may be a promising agent for the treatment of muscular symptoms in this disorder.

Epilepsy in Christianson syndrome: Two cases of Lennox-Gastaut syndrome and a review of literature.

Christianson syndrome (CS) is an X-linked intellectual disorder caused by mutations in the SLC9A6 gene. Clinical features of CS include an inability to speak, truncal ataxia, postnatal microcephaly, hyperkinesis, and epilepsy. Almost all patients with CS develop drug-resistant epilepsy-its most serious complication. We report two cases of CS with drug-resistant epilpesy associated with the Lennox-Gastaut syndrome (LGS). One patient experienced generalized tonic seizures since 9 months of age with cognitive regression, which evolved to include atonic seizures at the age of 7 years. Electroencephalography (EEG) showed generalized slow spike-wave complexes and generalized paroxysmal fast activity. Seizures remained drug-resistant despite multiple anti-seizure drugs. The second patient experienced generalized tonic seizures since the age of 17 months and arrested development. EEG showed generalized slow spike-wave complexes, with frequent atonic seizures since the age of 6 years. Electrical status epilepticus during slow-wave sleep (ESES) developed at the age of 7 years. Our cases illustrate that CS may cause LGS in addition to other developmental and epileptic encephalopathies of the neonatal and infantile period. We suggest that generalized tonic or tonic-clonic seizures and generalized slow spike-wave complexes in interictal EEG be included as potential electroclinical features of epilepsy in CS.

De novo CACNA1G variants in developmental delay and early-onset epileptic encephalopathies.

INTRODUCTION: Variants of CACNA1G, which encodes CaV3.1, have been reported to be associated with various neurological disorders. METHODS: Whole-exome sequencing of genomic DNA from 348 Japanese patients with neurodevelopmental disorders and their parents was conducted, and de novo variants of CACNA1G were extracted. The electrophysiological properties of each mutant channel were investigated by voltage-clamp and current-clamp analyses of HEK293T cells overexpressing these channels. RESULTS: Two patients diagnosed with Rett syndrome and West syndrome were found to have known pathological CACNA1G mutations reported in cerebellar ataxia cohorts: c.2881G > A, p.Ala961Thr and c.4591A > G, p.Met1531Val, respectively. One patient with Lennox-Gastaut syndrome was revealed to harbor a previously unreported heterozygous variant: c.3817A > T, p.Ile1273Phe. Clinical symptoms of the two patients with known mutations included severe developmental delay without acquisition of the ability to walk independently. The patient with a potentially novel mutation showed developmental delay, intractable seizures, and mild cerebral atrophy on MRI, but the severity of symptoms was milder than in the former two cases. Electrophysiological study using HEK293T cells demonstrated significant changes of T-type Ca2+ currents by p.Ala961Thr and p.Met1531Val SNVs, which were likely to enhance oscillation of membrane potential at low frequencies. In contrast, p.Ile1273Phe showed no significant effects in our electrophysiological evaluations, with its pathogenesis remaining undetermined. CONCLUSION: De novo variants of CACNA1G explain some neurodevelopmental disorders. Our study further provides information to understand the genotype-phenotype correlations of patients with CACNA1G mutations.