Exosomes populate the cerebrospinal fluid of preterm infants with post-haemorrhagic hydrocephalus

Background Preterm infants are at risk of germinal matrix haemorrhage-intraventricular haemorrhage (GMH-IVH) which leads to post-haemorrhagic hydrocephalus (PHH) in 30% of infants; this is associated with moderate-severe neurodevelopmental impairment and confers significant risk of cerebral palsy. There are however no predictive indicators of the severity or long-term outcome after GMH-IVH. In recent years, endosome-derived extracellular vesicles (EVs) or exosomes have been isolated from biofluids and shown to mediate intercellular communication via selective enrichment in proteins and micro-RNAs. Methods This study aimed to isolate and characterise EVs from the cerebrospinal fluid (CSF) of 3 preterm infants with PHH using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) with immunogold protein labelling, and micro-RNA analysis. Results NTA of unaltered CSF revealed a heterogeneous and dynamic population of EVs. Exosomal-sized EVs were isolated by differential ultracentrifugation and TEM confirmed the presence of CD63+ and CD81+ exosomes. The micro-RNAs miR-9, miR-17, miR-26a, miR-124 and miR-1911 were detected within the exosome-enriched fraction and profiled over time. Conclusion This is the first reported characterisation of exosomes from the CSF of preterm infants with post-haemorrhagic hydrocephalus

The miRNA transcriptome of cerebrospinal fluid in preterm infants reveals the signaling pathways that promote reactive gliosis following cerebral hemorrhage

IntroductionGerminal Matrix-Intraventricular Haemorrhage (GM-IVH) is one of the most common neurological complications in preterm infants, which can lead to accumulation of cerebrospinal fluid (CSF) and is a major cause of severe neurodevelopmental impairment in preterm infants. However, the pathophysiological mechanisms triggered by GM-IVH are poorly understood. Analyzing the CSF that accumulates following IVH may allow the molecular signaling and intracellular communication that contributes to pathogenesis to be elucidated. Growing evidence suggests that miRs, due to their key role in gene expression, have a significant utility as new therapeutics and biomarkers.MethodsThe levels of 2,083 microRNAs (miRs) in 15 CSF samples from 10 infants with IVH were measured using miRNA whole transcriptome sequencing. Gene ontology (GO) and miR family analysis were used to uncover dysregulated signalling which were then validated in vitro in human foetal neural progenitor cells treated with IVH-CSF.ResultsFive hundred eighty-seven miRs were differentially expressed in the CSF extracted at least 2 months after injury, compared to CSF extracted within the first month of injury. GO uncovered key pathways targeted by differentially expressed miRs including the MAPK cascade and the JAK/STAT pathway. Astrogliosis is known to occur in preterm infants, and we hypothesized that this could be due to abnormal CSF-miR signaling resulting in dysregulation of the JAK/STAT pathway – a key controller of astrocyte differentiation. We then confirmed that treatment with IVH-CSF promotes astrocyte differentiation from human fetal NPCs and that this effect could be prevented by JAK/STAT inhibition. Taken together, our results provide novel insights into the CSF/NPCs crosstalk following perinatal brain injury and reveal novel targets to improve neurodevelopmental outcomes in preterm infants

Prevalence of DDC genotypes in patients with aromatic L-amino acid decarboxylase (AADC) deficiency and in silico prediction of structural protein changes

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive genetic disorder affecting the biosynthesis of dopamine, a precursor of both norepinephrine and epinephrine, and serotonin. Diagnosis is based on the analysis of CSF or plasma metabolites, AADC activity in plasma and genetic testing for variants in the DDC gene. The exact prevalence of AADC deficiency, the number of patients, and the variant and genotype prevalence are not known. Here, we present the DDC variant (n = 143) and genotype (n = 151) prevalence of 348 patients with AADC deficiency, 121 of whom were previously not reported. In addition, we report 26 new DDC variants, classify them according to the ACMG/AMP/ACGS recommendations for pathogenicity and score them based on the predicted structural effect. The splice variant c.714+4A>T, with a founder effect in Taiwan and China, was the most common variant (allele frequency = 32.4%), and c.[714+4A>T];[714+4A>T] was the most common genotype (genotype frequency = 21.3%). Approximately 90% of genotypes had variants classified as pathogenic or likely pathogenic, while 7% had one VUS allele and 3% had two VUS alleles. Only one benign variant was reported. Homozygous and compound heterozygous genotypes were interpreted in terms of AADC protein and categorized as: i) devoid of full-length AADC, ii) bearing one type of AADC homodimeric variant or iii) producing an AADC protein population composed of two homodimeric and one heterodimeric variant. Based on structural features, a score was attributed for all homodimers, and a tentative prediction was advanced for the heterodimer. Almost all AADC protein variants were pathogenic or likely pathogenic

Prevalence of DDC genotypes in patients with aromatic L-amino acid decarboxylase (AADC) deficiency and in silico prediction of structural protein changes

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive genetic disorder affecting the biosynthesis of dopamine, a precursor of both norepinephrine and epinephrine, and serotonin. Diagnosis is based on the analysis of CSF or plasma metabolites, AADC activity in plasma and genetic testing for variants in the DDC gene. The exact prevalence of AADC deficiency, the number of patients, and the variant and genotype prevalence are not known. Here, we present the DDC variant (n = 143) and genotype (n = 151) prevalence of 348 patients with AADC deficiency, 121 of whom were previously not reported. In addition, we report 26 new DDC variants, classify them according to the ACMG/AMP/ACGS recommendations for pathogenicity and score them based on the predicted structural effect. The splice variant c.714+4A>T, with a founder effect in Taiwan and China, was the most common variant (allele frequency = 32.4%), and c.[714+4A>T];[714+4A>T] was the most common genotype (genotype frequency = 21.3%). Approximately 90% of genotypes had variants classified as pathogenic or likely pathogenic, while 7% had one VUS allele and 3% had two VUS alleles. Only one benign variant was reported. Homozygous and compound heterozygous genotypes were interpreted in terms of AADC protein and categorized as: i) devoid of full-length AADC, ii) bearing one type of AADC homodimeric variant or iii) producing an AADC protein population composed of two homodimeric and one heterodimeric variant. Based on structural features, a score was attributed for all homodimers, and a tentative prediction was advanced for the heterodimer. Almost all AADC protein variants were pathogenic or likely pathogenic

SLC6A3-Related Dopamine Transporter Deficiency Syndrome

Clinical characteristicsSLC6A3-related dopamine transporter deficiency syndrome (DTDS) is a complex movement disorder with a continuum that ranges from classic early-onset DTDS (in the first 6 months) to atypical later-onset DTDS (in childhood, adolescence, or adulthood). Classic DTDS. Infants typically manifest nonspecific findings (irritability, feeding difficulties, axial hypotonia, and/or delayed motor development) followed by a hyperkinetic movement disorder (with features of chorea, dystonia, ballismus, orolingual dyskinesia). Over time, affected individuals develop parkinsonism-dystonia characterized by bradykinesia (progressing to akinesia), dystonic posturing, distal tremor, rigidity, and reduced facial expression. Limitation of voluntary movements leads to severe motor delay. Episodic status dystonicus, exacerbations of dystonia, and secondary orthopedic, gastrointestinal, and respiratory complications are common. Many affected individuals appear to show relative preservation of intellect with good cognitive development. Atypical DTDS. Normal psychomotor development in infancy and early childhood is followed by later-onset manifestations of parkinsonism-dystonia with tremor, progressive bradykinesia, variable tone, and dystonic posturing. The long-term outcome of this form is currently unknown.Diagnosis/testingThe diagnosis of SLC6A3-related DTDS is established in a proband with characteristic clinical, laboratory, and imaging findings and biallelic pathogenic variants in SLC6A3 identified by molecular genetic testing.ManagementTreatment of manifestations: Treatment to control chorea and dyskinesia in early stages of the disease includes tetrabenazine and benzodiazepines. Dystonia is more difficult to control and treatment often includes pramipexole and ropinirole as first-line agents; adjuncts such as trihexyphenidyl, baclofen, gabapentin, and clonidine for severe dystonia; and chloral hydrate and benzodiazepines for exacerbations of dystonia or status dystonicus. Prevention of secondary complications: Regular physiotherapy to reduce the risk of contractures; early referral for management of feeding difficulties; use of influenza vaccine, prophylactic antibiotics, and chest physiotherapy for patients prone to chest infections, especially in the winter months. Surveillance: Evaluation every six to 12 months for early evidence of hip dislocation and/or spinal deformity; regular assessment of swallowing to evaluate risk for aspiration; regular nutrition assessment to ensure adequate caloric intake. Agents/circumstances to avoid: Although the dopamine agonists bromocriptine and pergolide could be considered, the associated increased risk of pulmonary, retroperitoneal, and pericardial fibrosis makes them less desirable than the newer dopamine agonists. Drugs with anti-dopaminergic side effects (e.g., some antihistamines, sedatives, and dimenhydrinate) may exacerbate the movement disorder. For the treatment of vomiting, anti-emetics such as the anti-serotoninergic agents (e.g., ondansetron) potentially have fewer side effects than other agents.Genetic counselingSLC6A3-related DTDS is inherited in an autosomal recessive manner. At conception, each sib of an affected individual has a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Once the SLC6A3 pathogenic variants have been identified in an affected family member, carrier testing for at-risk relatives and prenatal and preimplantation genetic testing are possible

Development of a UK phase-2 clinical trial of 4'-phosphopantetheine for pantothenate kinase associated neurodegeneration

Background: Pantothenate kinase-associated neurodegeneration (PKAN), a Neurodegeneration with Brain Iron Accumulation (NBIA) disorder, is an inborn error of vitamin B5-coenzyme A (CoA) metabolism caused by biallelic mutations in the PANK2 gene. Children with classic PKAN present at a mean age of 3.4 years, often with gait difficulties and clumsiness; the atypical form presents later in adolescence or early adult life and progresses more slowly. PKAN is characterized by a severe movement disorder, cognitive and neuropsychiatric involvement, and pathological iron accumulation in the basal ganglia; these cause profound disability and risk of premature mortality. There are no proven disease-modifying treatments for PKAN. / Proposed therapy: PKAN is caused by functional loss of the mitochondrially-located PANK2 enzyme which phosphorylates vitamin B5 (pantothenate) in the first step of CoA metabolism. CoA is essential for the tri-carboxylic acid cycle, fatty acid oxidation and synthesis, amino acid metabolism and neurotransmitter synthesis, serving more than 9% of mammalian biochemical reactions. 4’-phosphopantetheine (4’-PPT) is the endogenous precursor to CoA and is found in all cells and many foods. In cultured human cells and the Pank2-knock out mouse model, administration of 4’-PPT corrects disease-specific phenotypes and does not cause toxicity. / Trial design: We will undertake a phase-2 clinical trial of enteral 4’-PPT administered once per day to 24 participants aged 1–25 years. A 6-month placebo-controlled, double-blinded, dose-ranging phase will be followed by an 18-month open-label, single-dose phase. Primary outcome measures are of safety and tolerability with regular blood test and side effect monitoring. The secondary outcome is response to treatment of a blood biomarker measuring the expression of CoASY mRNA, the last enzyme in the CoA synthesis pathway. Exploratory tertiary outcomes include questionnaire and examination-based disease rating scales, activity of daily living scales, measures of dystonia and quality of life, and functional ophthalmological assessments