A novel de novo truncating variant in a Hungarian patient with CTNNB1 neurodevelopmental disorder.

PURPOSE: We aimed to elucidate the underlying disease in a Hungarian family, with only one affected family member, a 16-year-old male Hungarian patient, who developed global developmental delay, cognitive impairment, behavioral problems, short stature, intermittent headaches, recurrent dizziness, strabismus, hypermetropia, complex movement disorder and partial pituitary dysfunction. After years of detailed clinical investigations and careful pediatric care, the exact diagnosis of the patient and the cause of the disease was still unknown. METHODS: We aimed to perform whole exome sequencing (WES) in order to investigate whether the affected patient is suffering from a rare monogenic disease. RESULTS: Using WES, we identified a novel, de novo frameshift variant (c.1902dupG, p.Ala636SerfsTer12) of the catenin beta-1 (CTNNB1) gene. Assessment of the novel CTNNB1 variant suggested that it is a likely pathogenic one and raised the diagnosis of CTNNB1 neurodevelopmental disorder (OMIM 615,075). CONCLUSIONS: Our manuscript may contribute to the better understanding of the genetic background of the recently discovered CTNNB1 neurodevelopmental disorder and raise awareness among clinicians and geneticists. The affected Hungarian family demonstrates that based on the results of the clinical workup is difficult to establish the diagnosis and high-throughput genetic screening may help to solve these complex cases.

[Novel heterozygous STUB1 gene mutation causes SCA48 in a Hungarian patient].

Autosomal dominant cerebellar ataxias (ADCA), also known as spinocerebellar ataxias (SCA) are a group of progressive neurodegenerative diseases with remarkable clinical and genetic heterogeneity. In the last ten years 20 genes were identified in the background of SCAs. One of these genes was STUB1 (STIP1 homology and U-box containing protein 1) (chromosome 16p13, NM_005861.4) encoding a multifunctional E3 ubiquitine ligase (CHIP)1. In 2013, STUB1 was identified as a causative gene of autosomal recessive spinocerebellar ataxia 16 (SCAR16), but in 2018 Genis et al. published that heterozygous mutations of this gene can cause the autosomal dominantly inherited SCA48 as well1,2. 28 French, twelve Italian, three Belgian, two North-American, one Spanish, one Turkish, one Dutch, one German and one British SCA48 families have been reported so far2-9. Based on these publications, SCA48 is a late-onset, progressive disorder characterized by cerebellar dysfunction, cognitive impairment, psychiatric features, dysphagia, hyperreflexia, urinary tract symptoms and movement disorders including Parkinsonism, chorea, dystonia and rarely tremor. The brain MRI in all SCA48 patients demonstrated vermian and hemispheric cerebellar atrophy which was more pronounced in the posterior areas (lobules VI and VII) of the cerebellum in most of the cases2-9. Besides this, T2- weighted imaging (T2WI) hyperintensity of dentate nuclei (DN) was reported in some Italian patients10. Moreover, the most recent publication described alterations on DAT-scan imaging in some French families9. Neurophysiological examinations did not find any central or peripheral nervous system abnormalities2,3,5. Neuropathologic findings revealed definite cerebellar atrophy and cortical shrinkage with variable severity6,7. The histopathological assessment denoted Purkinje cell loss, p62-positive neuronal intranuclear inclusions in some cases and tau pathology in one patient6-7. In this paper we describe the clinical and genetic characterization of the first Hungarian SCA48 case with a novel heterozygous STUB1 gene missense mutation.

A Novel Homozygous Frameshift WDR81 Mutation associated with Microlissencephaly, Corpus Callosum Agenesis, and Pontocerebellar Hypoplasia.

Microlissencephaly is a brain malformation characterized by microcephaly and extremely simplified gyral pattern. It may be associated with corpus callosum agenesis and pontocerebellar hypoplasia. In this case report, we described two siblings, a boy and a girl, with this complex brain malformation and lack of any development. In the girl, exome sequencing of a gene set representing 4,813 genes revealed a homozygous AG deletion in exon 7 of the WDR81 gene, leading to a frameshift (c.4668_4669delAG, p.Gly1557AspfsTer16). The parents were heterozygous for this mutation. The boy died without proper genetic testing. Our findings expand the phenotypic and genotypic spectrum of WDR81 gene mutations.

Tremor as an early sign of hereditary spastic paraplegia due to mutations in ALDH18A1.

BACKGROUND: The ALDH18A1 gene is located at 10q24.1 and encodes delta-1-pyrroline-5-carboxylate synthetase (P5CS), a mitochondrial bifunctional enzyme that catalyzes the first two steps in de novo biosynthesis of proline, ornithine, citrulline, and arginine. ALDH18A1-related disorders have been classified into four groups, such as autosomal dominant and recessive hereditary spastic paraplegia (SPG9A and SPG9B, respectively), as well as autosomal dominant and recessive cutis laxa (ADCL3 and ARCL3A, respectively). Neurodegeneration is a characteristic feature of all groups. CASE REPORT: Here, we report a girl with compound heterozygous disease-causing variants (c.-28-2A>G and c.383G>A, p.Arg128His) in the ALDH18A1 gene, revealed by whole exome sequencing. The c.-28-2A>G variant in intron 1, inherited from the mother, is a novel mutation, while the c.383G>A variant in exon 4, inherited from the father, has already been reported. The patient presented with vigorous infantile tremor preceding progressive spastic paraplegia. Dysmorphic features included elongated face, deep-set ears, upturned nose, long philtrum and pointed chin. Intrauterine and postnatal growth retardation, microcephaly, global developmental delay and profound intellectual disability were also noticed. Blood fasting ammonia level, plasma proline, ornithine and arginine levels were normal, while citrulline level was slightly decreased. Brain MRI revealed moderate hypoplasia of the corpus callosum and reduction of white matter volume. CONCLUSIONS: The patient represents SPG9B, a rare form of autosomal recessive hereditary spastic paraplegias. The early onset tremor, preceding lower limb spasticity appears to be a unique early manifestation of neurodegeneration in this case.

Co-occurrence of mutations in FOXP1 and PTCH1 in a girl with extreme megalencephaly, callosal dysgenesis and profound intellectual disability.

Heterozygous disruptions in FOXP1 are responsible for developmental delay, intellectual disability and speech deficit. Heterozygous germline PTCH1 disease-causing variants cause Gorlin syndrome. We describe a girl with extreme megalencephaly, developmental delay and severe intellectual disability. Dysmorphic features included prominent forehead, frontal hair upsweep, flat, wide nasal bridge, low-set, abnormally modelled ears and post-axial cutaneous appendages on the hands. Brain MRI showed partial agenesis of the corpus callosum and widely separated leaves of the septum pellucidum. Exome sequencing of a gene set representing a total of 4813 genes with known relationships to human diseases revealed an already known heterozygous de novo nonsense disease-causing variant in FOXP1 (c.1573C>T, p.Arg525Ter) and a heterozygous novel de novo frameshift nonsense variant in PTCH1 (c.2834delGinsAGATGTTGTGGACCC, p.Arg945GlnfsTer22). The composite phenotype of the patient seems to be the result of two monogenic diseases, although more severe than described in conditions due to disease-causing variants in either gene.

Recessive mutations in POLR1C cause a leukodystrophy by impairing biogenesis of RNA polymerase III.

A small proportion of 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) or RNA polymerase III (POLR3)-related leukodystrophy cases are negative for mutations in the previously identified causative genes POLR3A and POLR3B. Here we report eight of these cases carrying recessive mutations in POLR1C, a gene encoding a shared POLR1 and POLR3 subunit, also mutated in some Treacher Collins syndrome (TCS) cases. Using shotgun proteomics and ChIP sequencing, we demonstrate that leukodystrophy-causative mutations, but not TCS mutations, in POLR1C impair assembly and nuclear import of POLR3, but not POLR1, leading to decreased binding to POLR3 target genes. This study is the first to show that distinct mutations in a gene coding for a shared subunit of two RNA polymerases lead to selective modification of the enzymes' availability leading to two different clinical conditions and to shed some light on the pathophysiological mechanism of one of the most common hypomyelinating leukodystrophies, POLR3-related leukodystrophy.

Spectrum of neurodevelopmental disabilities: a cohort study in hungary.

The spectrum of neurodevelopmental disabilities was studied in a cohort of patients in Hungary. A search for etiologies and assessment of the degree of intellectual disability were carried out. The study included 241 (131 boys) patients. Disability occurred without any prenatal, perinatal, and/or neonatal adverse events in 167 patients. They were classified into the following subgroups: genetic syndromes with recognized etiology, global developmental delay/intellectual disability in association with dysmorphic features but unknown etiology, global developmental delay/intellectual disability without dysmorphic features and recognized etiology, brain malformations, inborn errors of metabolism, leukoencephalopathies, epileptic syndromes, developmental language impairment, and neuromuscular disorders. Adverse events occurred in 74 children classified into subgroups such as cerebral palsy after delivery preterm or at term, and disabilities without cerebral palsy. The etiology was identified in 66.4%, and genetic diagnosis was found in 19.5%. Classification of neurodevelopmental disorders contribute to etiological diagnosis, proper rehabilitation, and genetic counseling.

Mutations in B9D1 and MKS1 cause mild Joubert syndrome: expanding the genetic overlap with the lethal ciliopathy Meckel syndrome.

Joubert syndrome is a clinically and genetically heterogeneous ciliopathy characterized by a typical cerebellar and brainstem malformation (the "molar tooth sign"), and variable multiorgan involvement. To date, 24 genes have been found mutated in Joubert syndrome, of which 13 also cause Meckel syndrome, a lethal ciliopathy with kidney, liver and skeletal involvement. Here we describe four patients with mild Joubert phenotypes who carry pathogenic mutations in either MKS1 or B9D1, two genes previously implicated only in Meckel syndrome.

Hydrogen is neuroprotective and preserves cerebrovascular reactivity in asphyxiated newborn pigs.

Hydrogen (H2) has been reported to neutralize toxic reactive oxygen species. Oxidative stress is an important mechanism of neuronal damage after perinatal asphyxia. We examined whether 2.1% H2-supplemented room air (H2-RA) ventilation would preserve cerebrovascular reactivity (CR) and brain morphology after asphyxia/reventilation (A/R) in newborn pigs. Anesthetized, ventilated piglets were assigned to one of the following groups: A/R with RA or H2-RA ventilation (A/R-RA and A/R-H2-RA; n = 8 and 7, respectively) and respective time control groups (n = 9 and 7). Asphyxia was induced by suspending ventilation for 10 min, followed by reventilation with the respective gases for 4 h. After euthanasia, the brains were processed for neuropathological examination. Pial arteriolar diameter changes to graded hypercapnia (5-10% CO2 inhalation), and NMDA (10(-4) M) were determined using the closed cranial window/intravital microscopy before and 1 h after asphyxia. Neuropathology revealed that H2-RA ventilation significantly reduced neuronal injury induced by A/R in virtually all examined brain regions including the cerebral cortex, the hippocampus, basal ganglia, cerebellum, and the brainstem. Furthermore, H2-RA ventilation significantly increased CR to hypercapnia after A/R (% vasodilation was 23 ± 4% versus 41 ± 9%, p < 0.05). H2-RA ventilation did not affect reactive oxygen species-dependent CR to NMDA. In summary, H2-RA could be a promising approach to reduce the neurologic deficits after perinatal asphyxia.

Epileptic seizures increase circulating endothelial cells in peripheral blood as early indicators of cerebral vascular damage.

Circulating endothelial cells (CECs) are nonhematopoetic mononuclear cells in peripheral blood that are dislodged from injured vessels during cardiovascular disease, systemic vascular disease, and inflammation. Their occurrence during cerebrovascular insults has not been previously described. Epileptic seizures cause the long-term loss of cerebrovascular endothelial dilator function. We hypothesized that seizures cause endothelial sloughing from cerebral vessels and the appearance of brain-derived CECs (BCECs), possible early indicators of cerebral vascular damage. Epileptic seizures were induced by bicuculline in newborn pigs; venous blood was then sampled during a 4-h period. CECs were identified in the fraction of peripheral blood mononuclear cells by the expression of endothelial antigens (CD146, CD31, and endothelial nitric oxide synthase) and by Ulex europeaus lectin binding. In control animals, few CECs were detected. Seizures caused a time-dependent increase in CECs 2-4 h after seizure onset. Seizure-induced CECs coexpress glucose transporter-1, a blood-brain barrier-specific glucose transporter, indicating that these cells originate in the brain vasculature and are thus BCECs. Seizure-induced BCECs cultured in EC media exhibited low proliferative potential and abnormal cell contacts. BCEC appearance during seizures was blocked by a CO-releasing molecule (CORM-A1) or cobalt protoporphyrin (heme oxygenase-1 inducer), which prevented apoptosis in cerebral arterioles and the loss of cerebral vascular endothelial function during the late postictal period. These findings suggest that seizure-induced BCECs are injured ECs dislodged from cerebral microvessels during seizures. The correlation between the appearance of BCECs in peripheral blood, apoptosis in cerebral vessels, and the loss of postictal cerebral vascular function suggests that BCECs are early indicators of late cerebral vascular damage.

Secretory phospholipase A2 inhibitor PX-18 preserves microvascular reactivity after cerebral ischemia in piglets.

Cerebral ischemia/reperfusion (I/R) results in cellular energy failure and dysfunction of the neurovascular unit that contribute to subsequent neuronal cell death in the neonate. PX-18 is a putative neuroprotective inhibitor of secretory phospholipase A(2) (sPLA(2)) but its in vivo testing has been limited by its poor solubility. Our purpose was to assess whether PX-18 preserved neuronal-vascular reactivity to I/R-sensitive endothelium-dependent (hypercapnia, bradykinin) and/or neuron-dependent (N-methyl-D-aspartate; NMDA) stimuli. To make the drug available for in vivo studies, PX-18 was formulated as a 3% nanosuspension applying high pressure homogenization. Newborn piglets (1-day old, n=40) were anesthetized and ventilated, and cerebrovascular reactivity to the above stimuli was determined by measuring changes in pial arteriolar diameters using the closed cranial window/intravital videomicroscopy technique. Intravenous infusion of PX-18 nanosuspension (6 mg/kg, 20 min) did not affect baseline arteriolar diameters, or hypercapnia-, bradykinin-, or NMDA-induced pial arteriolar vasodilation under normoxic conditions. Global cerebral ischemia (10 min) followed by 1 h of reperfusion significantly attenuated hypercapnia-, bradykinin-, and NMDA-induced vasodilation in untreated or vehicle-treated controls. However, PX-18 resulted in nearly full preservation of cerebrovascular reactivity to all these stimuli. In conclusion, inhibition of sPLA(2) by PX-18 improves neurovascular function both at the neuronal and the microvascular level following I/R. This effect of PX-18 likely contributes to its neuroprotective effect.

PACAP and VIP differentially preserve neurovascular reactivity after global cerebral ischemia in newborn pigs.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are neuroprotective in numerous models. Impairment of cerebrovascular reactivity (CR) contributes to ischemia/reperfusion (I/R)-induced neuronal damage. We tested whether PACAP and/or VIP preserve CR to I/R-sensitive dilator responses dependent on endothelial and/or neuronal function. Accordingly, changes in pial arteriolar diameters in response to hypercapnia (5-10% CO(2) ventilation) or topical N-methyl-d-aspartate (NMDA, 10(-4) M) were determined before and after I/R via intravital microscopy in anesthetized/ventilated piglets. Local pretreatment with non-vasoactive doses of PACAP (10(-8) M) and VIP (10(-9) M) prevented the attenuation of postischemic CR to hypercapnia; to 10% CO(2), the CR values were 27+/-8% vs 92+/-5% vs 88+/-13% (vehicle vs PACAP38 vs VIP, CR expressed as a percentage of the response before I/R, mean+/-SEM, n=8-8, p<0.05). PACAP, but not VIP, preserved CR to NMDA after I/R, with CR values of 31+/-10% vs 87+/-8% vs 35+/-12% (vehicle vs PACAP38 vs VIP, n=6-6). Unlike PACAP, VIP-induced vasodilation has not yet been investigated in the piglet. We tested whether VIP-induced arteriolar dilation was sensitive to inhibitors of cyclooxygenase (COX)-1 (SC-560, 1 mg/kg), COX-2 (NS-398, 1 mg/kg), indomethacin (5 mg/kg), and nitric oxide synthase (L-NAME, 15 mg/kg). VIP (10(-8)-10(-7)-10(-6) M, n=8) induced reproducible, dose-dependent vasodilation of 16+/-3%, 33+/-6%, and 70+/-8%. The response was unaffected by all drugs, except that the vasodilation to 10(-8) M VIP was abolished by SC-560 and indomethacin. In conclusion, PACAP and VIP differentially preserve postischemic CR; independent of their vasodilatory effect.

Seizure-induced alterations in cerebrovascular function in the neonate.

Epileptiform seizures are most common during the neonatal period, affecting at least 0.3% of term neonates and more than 10% of preterm neonates. The adverse impact of neonatal seizures on the long-term neurological outcome has been well documented, but their cerebrovascular consequences are rarely emphasized. The cerebral blood flow is controlled by the interaction of the vascular and parenchymal cells forming the neurovascular unit via multiple mediator systems that have unique features in the newborn. Seizures drastically affect the neurovascular unit, resulting in (1) dramatic increases in brain metabolism and cerebral blood flow during the ictal period, (2) disruption of the blood-brain barrier, (3) an acute loss of cerebral pressure autoregulation, and (4) a delayed impairment of cerebrovascular reactivity to various stimuli. Furthermore, seizures frequently accompany and potentially aggravate a pre-existing cerebrovascular insult. This review summarizes the current knowledge on how seizures affecting various cells in the neurovascular unit result in the observed alterations in cerebrovascular function in the neonate.

Acetazolamide induces indomethacin and ischaemia-sensitive pial arteriolar vasodilation in the piglet.

AIM: Acetazolamide (AZD) produces cerebral vasodilation. The underlying mechanism is unclear, but it is assumed to be largely due to CO2 retention and acidosis. We tested if cerebrovascular effects of AZD were similar to hypercapnia in the newborn pig. METHODS: We used the closed cranial window/intravital microscopy technique to determine pial arteriolar diameters simultaneously with laser-Doppler flowmetry (LDF) to monitor cortical blood perfusion. Anaesthetized (Na-thiopenthal +alpha-chloralose), ventilated, 1-day-old instrumented piglets (n=38) were divided into five experimental groups: time control (n=11), indomethacin, ibuprofen, Nomega-nitro-L-arginine methyl ester (L-NAME) treatments (1, 30, 15 mg/kg, i.v., n=6, 6, 4, respectively), and global ischaemia/reperfusion (I/R, 10 min induced by elevated intracranial pressure, n=11). Responses to 5-10% inhaled CO2 were recorded before and after the treatments, and then in a similar manner to AZD (10-20 mg/kg, i.v.). RESULTS: Hypercapnia and AZD produced pial arteriolar vasodilation and increases in cortical perfusion. Consistent with previous data, hypercapnia-induced changes were abolished by indomethacin, unaltered by ibuprofen and L-NAME and were significantly attenuated after I/R. AZD-induced vasodilation was also sensitive to indomethacin and I/R and was unaltered by ibuprofen or L-NAME. CONCLUSION: The mechanism of AZD-induced vasodilation appears to be similar/identical to hypercapnia, and pial arteriolar diameter changes reflect changes in cortical perfusion.

Cerebroprotective effects of the CO-releasing molecule CORM-A1 against seizure-induced neonatal vascular injury.

Endogenous CO, a product of heme oxygenase activity, has vasodilator and cytoprotective effects in the cerebral circulation of newborn pigs. CO-releasing molecule (CORM)-A1 (sodium boranocarbonate) is a novel, water-soluble, CO-releasing compound. We addressed the hypotheses that CORM-A1 1) can deliver CO to the brain and exert effects of CO on the cerebral microvasculature and 2) is cerebroprotective. Acute and delayed effects of topically and systemically administered CORM-A1 on cerebrovascular and systemic circulatory parameters were determined in anesthetized newborn pigs with implanted closed cranial windows. Topical application of CORM-A1 (10(-7)-10(-5) M) to the brain produced concentration-dependent CO release and pial arteriolar dilation. Systemically administered CORM-A1 (2 mg/kg ip or iv) caused pial arteriolar dilation and increased cortical cerebrospinal fluid CO concentration. Systemic CORM-A1 did not have acute or delayed effects on blood pressure, heart rate, or blood gases. Potential cerebroprotective vascular effects of CORM-A1 (2 mg/kg ip, 30 min before seizures) were tested 2 days after bicuculline-induced epileptic seizures (late postictal period). In control piglets, seizures reduced postictal cerebrovascular responsiveness to selective physiologically relevant vasodilators (bradykinin, hemin, and isoproterenol) indicative of cerebrovascular injury. In contrast, in CORM-A1-pretreated animals, no loss of postictal cerebrovascular reactivity was observed. We conclude that systemically administered CORM-A1 delivers CO to the brain, elicits the vasodilator and cytoprotective effects of CO in the cerebral circulation, and protects the neonatal brain from cerebrovascular injury caused by epileptic seizures.

Reventilation with room air or 100% oxygen after asphyxia differentially affects cerebral neuropathology in newborn pigs.

AIM: To test if reventilation with room air (RA) or 100% oxygen (O2) after asphyxia would differentially affect neuronal damage in different brain areas of newborn pigs. METHODS: Anaesthetized piglets were subjected to 10 min asphyxia (n=27) or served as time controls (n=7). Reventilation started with either RA or O2 for 1 h, and was continued with RA for an additional 1-3 h. Cortical or cerebellar blood flow was assessed with laser-Doppler flowmetry (LDF). Haematoxylin/eosin-stained sections from six brain regions were prepared for blinded neuropathological examination and scoring. RESULTS: Asphyxia resulted in significant neuronal damage compared to time controls in all areas examined except the pons. O2 ventilation elicited greater neuronal lesions in the hippocampus and the cerebellum but smaller damage in the basal ganglia compared to RA. The assessed physiological parameters including the LDF signals were similar in both ventilation groups, except for PaO2 in the first hour of reventilation (RA 75+/-5 mmHg, O2 348+/-57 mmHg; p<0.05). Interestingly, however, reactive hyperaemia was much higher in the O2-sensitive cerebellum as compared with the cortex (1101+/-227 vs 571+/-73; p<0.05, area under the curve). CONCLUSION: O2 toxicity after asphyxia was demonstrated in the piglet hippocampus and cerebellum but not in the cerebral cortex or basal ganglia. The observed regional differences may be associated with local haemodynamic factors.