MSL2 variants lead to a neurodevelopmental syndrome with lack of coordination, epilepsy, specific dysmorphisms, and a distinct episignature.

Epigenetic dysregulation has emerged as an important etiological mechanism of neurodevelopmental disorders (NDDs). Pathogenic variation in epigenetic regulators can impair deposition of histone post-translational modifications leading to aberrant spatiotemporal gene expression during neurodevelopment. The male-specific lethal (MSL) complex is a prominent multi-subunit epigenetic regulator of gene expression and is responsible for histone 4 lysine 16 acetylation (H4K16ac). Using exome sequencing, here we identify a cohort of 25 individuals with heterozygous de novo variants in MSL complex member MSL2. MSL2 variants were associated with NDD phenotypes including global developmental delay, intellectual disability, hypotonia, and motor issues such as coordination problems, feeding difficulties, and gait disturbance. Dysmorphisms and behavioral and/or psychiatric conditions, including autism spectrum disorder, and to a lesser extent, seizures, connective tissue disease signs, sleep disturbance, vision problems, and other organ anomalies, were observed in affected individuals. As a molecular biomarker, a sensitive and specific DNA methylation episignature has been established. Induced pluripotent stem cells (iPSCs) derived from three members of our cohort exhibited reduced MSL2 levels. Remarkably, while NDD-associated variants in two other members of the MSL complex (MOF and MSL3) result in reduced H4K16ac, global H4K16ac levels are unchanged in iPSCs with MSL2 variants. Regardless, MSL2 variants altered the expression of MSL2 targets in iPSCs and upon their differentiation to early germ layers. Our study defines an MSL2-related disorder as an NDD with distinguishable clinical features, a specific blood DNA episignature, and a distinct, MSL2-specific molecular etiology compared to other MSL complex-related disorders.

Hypomyelination with Atrophy of Basal Ganglia and Cerebellum (HABC) Due to UFM1 Mutation in Roma Patients - Severe Early Encephalopathy with Stridor and Severe Hearing and Visual Impairment. A Single Center Experience.

BACKGROUND: Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a neurodegenerative disease with neurodevelopmental delay, motor, and speech regression, pronounced extrapyramidal syndrome, and sensory deficits due to TUBB4A mutation. In 2017, a severe variant was described in 16 Roma infants due to mutation in UFM1. OBJECTIVE: The objective of this study is to expand the clinical manifestations of H-ABC due to UFM1 mutation and suggest clues for clinical diagnosis. METHODOLOGY: Retrospective analysis of all 9 cases with H-ABC due to c.-273_-271delTCA mutation in UFM1 treated during 2013-2020 in a Neuropediatric Ward in Plovdiv, Bulgaria. RESULTS: Presentation is no later than 2 months with inspiratory stridor, impaired sucking, swallowing, vision and hearing, and reduced active movements. By the age of 10 months, a monomorphic disease was observed: microcephaly (6/9), malnutrition (5/9), muscle hypertonia (9/9) and axial hypotonia (4/9), progressing to opisthotonus (6/9), dystonic posturing (5/9), nystagmoid ocular movements (6/9), epileptic seizures (4/9), non-epileptic spells (3/9). Dysphagia (7/9), inspiratory stridor (9/9), dyspnea (5/9), bradypnea (5/9), apnea (2/9) were major signs. Vision and hearing were never achieved or lost by 4-8 mo. Neurodevelopment was absent or minimal with subsequent regression after 2-5 mo. Brain imaging revealed cortical atrophy (7/9), atrophic ventricular dilatation (4/9), macrocisterna magna (5/9), reduced myelination (6/6), corpus callosum atrophy (3/6) and abnormal putamen and caput nuclei caudati. The age at death was between 8 and 18 mo. CONCLUSION: Roma patients with severe encephalopathy in early infancy with stridor, opisthotonus, bradypnea, severe hearing and visual impairment should be tested for the Roma founder mutation of H-ABC in UFM1.

Association of NGF and Mitochondrial Respiration with Autism Spectrum Disorder.

BACKGROUND: NGF is a molecule with a pleiotropic role, affecting neuro-immune functions, energy homeostasis, and synaptic plasticity. The mechanisms of NGF-induced neuronal differentiation are well established, but its effect on mitochondria in autism spectrum disorder (ASD) is still unclear. We hypothesize that NGF-induced neuronal development requires large amounts of energy, and mitochondria in ASD are overloaded to meet the new functional requirements. METHOD: The study includes primary diagnosed ASD children. Peripheral blood mononuclear cells (PBMCs) and plasma were obtained from both patients and typically developing children (TDC). PBMCs were analyzed with Seahorse XFp, and plasma NGF protein levels were measured. RESULTS: We detected nearly 50% higher NGF levels and approximately 40% elevation in spare respiratory capacity in ASD compared to TDC. These findings are consistent with the observed difference in maximal respiration, which was also significantly higher in the patient group. Both mitochondrial respiration and NGF plasma levels exhibit a strong potential to discriminate children with ASD from TDC. CONCLUSIONS: This study is the first to link elevated NGF with mitochondrial respiration and altered energy homeostasis in ASD. High NGF correlates with basic bioenergetic signatures that may be used as a screening tool to improve early diagnosis and clinical follow-up in ASD.

Clinical significance and role of coinfections with respiratory pathogens among individuals with confirmed severe acute respiratory syndrome coronavirus-2 infection.

Introduction: This study aimed to determine the prevalence, viral profile, and clinical features of coinfections with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and other respiratory viruses. Methods: Nasopharyngeal samples and clinical data of 221 hospitalized patients and 21 outpatients were collected and analyzed. Real-time reverse transcription-polymerase chain reaction was used to detect SARS-CoV-2, influenza virus, respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza virus (PIV) 1,2,3, rhinovirus (RV), adenovirus (AdV), bocaviruses (BoV), and seasonal coronaviruses (OC43, 229E, NL63, and HKU1). Viral load was determined by capillary electrophoresis. Results: From November 2020 to mid-March 2022, 242 SARS-CoV-2 positive patients were tested for seasonal respiratory viruses, and 24 (9.9%) cases of coinfections were detected. The distribution of viruses involved in cases of coinfections were as follows: HMPV (n = 6; 25%), RSV (n = 4;16.7%), AdV (n = 4; 16.7%), BoV (n = 4; 16.7%), PIV3 (n = 2; 8.3%), influenza A (H3N2; n = 2; 8.3%), RV (n = 1; 4.62%), and RV+BoV (n = 1; 4.62%). The proportion of detected coinfections with SARS-CoV-2 was highest in children aged 0-5 years (59%), followed by those >65 years (33%). In specimens with detected coinfection, the viral load of influenza was higher than that of SARS-CoV-2, and the mean viral load of SARS-CoV-2 was higher than that of the other respiratory viruses. C-reactive protein (CRP) and lymphocytes count in co-infected patients >65 years of age were on average higher than in children <16 years of age (mean CRP of 161.8 ± 133.1 mg/L; 19.7 ± 3.09% vs. mean 6.9 ± 8.9 mg/L, 0.9 ± 3.1%; p < 0.01). Patients >65 years of age co-infected with SARS-CoV-2 and other respiratory viruses had longer hospital stays than those <16 years of age (mean 9 ± 3.96 days vs. 5.44 ± 1.89 days; p = 0.025). The combination of AdV and SARS-CoV-2 is fatal for patients aged >65 years. Conclusion: In patients aged >65 years, coinfection with SARS CoV-2 and other respiratory viruses, together with concomitant diseases, causes worsening of the clinical picture and complications, and can be fatal. Screening of patients with SARS CoV-2 for other respiratory viruses is needed to select appropriate treatments and prevent a fatal outcome of the disease.

Cellular Bioenergetic and Metabolic Changes in Patients with Autism Spectrum Disorder.

BACKGROUND: Although Autism Spectrum Disorder (ASD) is considered a heterogeneous neurological disease in childhood, a growing body of evidence associates it with mitochondrial dysfunction explaining the observed comorbidities. INTRODUCTION: The aim of this study is to identify variations in cellular bioenergetics and metabolism dependent on mitochondrial function in ASD patients and healthy controls using Peripheral Blood Mononuclear Cells (PBMCs). We hypothesized that PBMCs may reveal the cellular pathology and provide evidence of bioenergetic and metabolic changes accompanying the disease. METHODS: PBMC from children with ASD and a control group of the same age and gender were isolated. All patients underwent an in-depth clinical evaluation. A well-characterized cohort of Bulgarian children is selected. Bioenergetic and metabolic studies of isolated PBMCs are performed with a Seahorse XFp analyzer. RESULTS: Our data show that PBMCs from patients with ASD have increased respiratory reserve capacity (by 27.5%), increased maximal respiration (by 67%) and altered adaptive response to oxidative stress induced by DMNQ. In addition, we demonstrate а strong dependence on fatty acids and impaired ability to reprogram cell metabolism. The listed characteristics are not observed in the control group. These results can contribute to a better understanding of the underlying causes of ASD, which is crucial for selecting a successful treatment. CONCLUSION: The current study, for the first time, provides a functional analysis of cell bioenergetics and metabolic changes in a group of Bulgarian patients with ASD. It reveals physiological abnormalities that do not allow mitochondria to adapt and meet the increased energetic requirements of the cell. The link between mitochondria and ASD is not yet fully understood, but this may lead to the discovery of new approaches for nutrition and therapy.

First case of Roma ethnic origin with Andermann syndrome: A novel frameshift mutation in exon 20 of SLC12A6 gene.

Andermann syndrome (AS) is caused by mutation of SLC12A6 gene. It comprises severe progressive sensory and motor neuropathy with early onset, varying degree of agenesis of corpus callosum (ACC) and mental retardation. AS occurs occasionally among population outside the northeastern Quebec-Saguenay-Lac- St-Jean and Charlevoix regions, inhabited by French Canadians. None of the described patients were of Roma ethnic origin. We present an 8-month-old infant of Roma ethnic origin with AS, caused by a novel frame shift mutation c.2604delT,p.(Asp868GlufsTer11) in exon 20 of SLC12A6 gene. Our case presented with several atypical findings: clinical presentation resembling "spinal muscular atrophy plus" syndrome; tongue fasciculations, which are not reported in the literature; early contractures of the wrists; normal motor action potentials and preserved sensory action potentials. Our patient is the first of Roma origin from nonconsanguineous parents, which suggests that this mutation might be widespread in the Roma population, although screening for this mutation in 140 alleles from Roma individuals originating from the same geographic region did not reveal further carriers, implying the mutation is rare. We recommend that Roma patients presenting with the clinical phenotype of AS should be tested for this mutation primarily.

Epilepsy in Children with Autistic Spectrum Disorder.

The comorbidity of autistic spectrum disorder (ASD) and epilepsy has been widely discussed but many questions still remain unanswered. The aim of this study was to establish the occurrence of epilepsy among children with ASD to define the type of epileptic seizures and syndromes, the age of onset of epilepsy, EEG abnormalities, the used antiepileptic drugs and the therapeutic responses for seizures and autistic behavior, as well as to find some correlations between epilepsy and gender, etiology and intellectual disability (ID). A retrospective study of medical files of 59 patients (aged 1⁻18 years) with ASD during a 5-year period was performed. ASD diagnosis was based on the DSM-5 diagnostic criteria. The patients were examined with a detailed medical history, physical and neurological examination, as well as some additional functional, imaging, laboratory and genetic investigations ASD etiology was syndromic in 9, probable syndromic in 9, and idiopathic in 41 children. ID was established in 90% of ASD children, and epilepsy in 44.4%. The onset of epilepsy prevailed before 7 years of age. The most common seizure types were focal with or without secondary generalization (53.4%). Focal epileptiform EEG abnormalities prevailed. Therapeutic response to seizures was good: 58% were seizure-free, while 27% had >50% seizure reduction but no improvement in autistic behavior. There was no correlation between epilepsy and either occurrence or degree of ID. There was a correlation between the frequency of epileptic seizures and the degree of ID. There was no significant difference among epilepsy rates in different etiologic, gender, and ID groups, probably because of the high percentage of ID and because this was a hospital-based study. Our study showed a significant percentage of epilepsy in ASD population and more than 1/4 were of symptomatic etiology. Those could be managed with specific treatments based on the pathophysiology of the gene defect.

Developmental performance of 5-year-old Bulgarian children-An example of translational neuroscience in practice.

RATIONALE, AIMS, AND OBJECTIVES: Developmental testing is essential for early recognition of the various developmental impairments. The tools used should be composed of items that are age specific, adapted, and standardized for the population they are applied to. The achievements of neurosciences, medicine, psychology, pedagogy, etc. are applied in the elaboration of a comprehensive examination tool that should screen all major areas of development. The key age of 5 years permits identification of almost all major developmental disabilities leaving time for therapeutic intervention before school entrance. The aim of the research is to evaluate the developmental performance of 5-year-old Bulgarian children using the approach of translation neuroscience. METHOD: A comprehensive test program was developed composed of 89 items grouped in the following domains: fine and gross motor development, coordination and balance, central motor neuron disturbances, language development and articulation, perception, attention and behavior, visual acuity, and strabismus. The overall sample comprises 434 children of mean age 63.5 months (SD-3.7). Male to female ratio is 1:1.02. From this group, 390 children are between 60 and 71 months of age. The children are examined in 51 kindergartens in 21 villages and 18 cities randomly chosen in southern Bulgaria. Eight children were excluded from the final analysis because they fulfilled less than 50% of the test items (7 children did not cooperate and 1 child was with autistic spectrum disorder). RESULTS: The items with abnormal response in less than 5% of the children are 43. The items with abnormal response in 6% to 35% of the children are 37. The items with high abnormal response (more than 35%) rate are only 9. CONCLUSIONS: The test is an example of a translational approach in neuroscience. On one hand, it is based on the results of several sciences studying growth and development from different perspective. On the other hand, the results from the present research may be implemented in other fields of child development-education, psychology, speech and language therapy, and intervention programs.

Founder p.Arg 446* mutation in the PDHX gene explains over half of cases with congenital lactic acidosis in Roma children.

Investigation of 31 of Roma patients with congenital lactic acidosis (CLA) from Bulgaria identified homozygosity for the R446* mutation in the PDHX gene as the most common cause of the disorder in this ethnic group. It accounted for around 60% of patients in the study and over 25% of all CLA cases referred to the National Genetic Laboratory in Bulgaria. The detection of a homozygous patient from Hungary and carriers among population controls from Romania and Slovakia suggests a wide spread of the mutation in the European Roma population. The clinical phenotype of the twenty R446* homozygotes was relatively homogeneous, with lactic acidosis crisis in the first days or months of life as the most common initial presentation (15/20 patients) and delayed psychomotor development and/or seizures in infancy as the leading manifestations in a smaller group (5/20 patients). The subsequent clinical picture was dominated by impaired physical growth and a very consistent pattern of static cerebral palsy-like encephalopathy with spasticity and severe to profound mental retardation seen in over 80% of cases. Most patients had a positive family history. We propose testing for the R446* mutation in PDHX as a rapid first screening in Roma infants with metabolic acidosis. It will facilitate and accelerate diagnosis in a large proportion of cases, allow early rehabilitation to alleviate the chronic clinical course, and prevent further affected births in high-risk families.