Genome and RNA sequencing were essential to reveal cryptic intronic variants associated to defective ATP6AP1 mRNA processing.

The diagnosis of Mendelian disorders has notably advanced with integration of whole exome and genome sequencing (WES and WGS) in clinical practice. However, challenges in variant interpretation and uncovered variants by WES still leave a substantial percentage of patients undiagnosed. In this context, integrating RNA sequencing (RNA-seq) improves diagnostic workflows, particularly for WES inconclusive cases. Additionally, functional studies are often necessary to elucidate the impact of prioritized variants on gene expression and protein function. Our study focused on three unrelated male patients (P1-P3) with ATP6AP1-CDG (congenital disorder of glycosylation), presenting with intellectual disability and varying degrees of hepatopathy, glycosylation defects, and an initially inconclusive diagnosis through WES. Subsequent RNA-seq was pivotal in identifying the underlying genetic causes in P1 and P2, detecting ATP6AP1 underexpression and aberrant splicing. Molecular studies in fibroblasts confirmed these findings and identified the rare intronic variants c.289-233C > T and c.289-289G > A in P1 and P2, respectively. Trio-WGS also revealed the variant c.289-289G > A in P3, which was a de novo change in both patients. Functional assays expressing the mutant alleles in HAP1 cells demonstrated the pathogenic impact of these variants by reproducing the splicing alterations observed in patients. Our study underscores the role of RNA-seq and WGS in enhancing diagnostic rates for genetic diseases such as CDG, providing new insights into ATP6AP1-CDG molecular bases by identifying the first two deep intronic variants in this X-linked gene. Additionally, our study highlights the need to integrate RNA-seq and WGS, followed by functional validation, in routine diagnostics for a comprehensive evaluation of patients with an unidentified molecular etiology.

Management of seizures in patients with primary mitochondrial diseases: consensus statement from the InterERNs Mitochondrial Working Group.

BACKGROUND AND PURPOSE: Primary mitochondrial diseases (PMDs) are common inborn errors of energy metabolism, with an estimated prevalence of one in 4300. These disorders typically affect tissues with high energy requirements, including heart, muscle and brain. Epilepsy may be the presenting feature of PMD, can be difficult to treat and often represents a poor prognostic feature. The aim of this study was to develop guidelines and consensus recommendations on safe medication use and seizure management in mitochondrial epilepsy. METHODS: A panel of 24 experts in mitochondrial medicine, pharmacology and epilepsy management of adults and/or children and two patient representatives from seven countries was established. Experts were members of five different European Reference Networks, known as the Mito InterERN Working Group. A Delphi technique was used to allow the panellists to consider draft recommendations on safe medication use and seizure management in mitochondrial epilepsy, using two rounds with predetermined levels of agreement. RESULTS: A high level of consensus was reached regarding the safety of 14 out of all 25 drugs reviewed, resulting in endorsement of National Institute for Health and Care Excellence guidelines for seizure management, with some modifications. Exceptions including valproic acid in POLG disease, vigabatrin in patients with γ-aminobutyric acid transaminase deficiency and topiramate in patients at risk for renal tubular acidosis were highlighted. CONCLUSIONS: These consensus recommendations describe our intent to improve seizure control and reduce the risk of drug-related adverse events in individuals living with PMD-related epilepsy.

Developmental outcome of electroencephalographic findings in SYNGAP1 encephalopathy.

SYNGAP1 haploinsufficiency results in a developmental and epileptic encephalopathy (DEE) causing generalized epilepsies accompanied by a spectrum of neurodevelopmental symptoms. Concerning interictal epileptiform discharges (IEDs) in electroencephalograms (EEG), potential biomarkers have been postulated, including changes in background activity, fixation-off sensitivity (FOS) or eye closure sensitivity (ECS). In this study we clinically evaluate a new cohort of 36 SYNGAP1-DEE individuals. Standardized questionnaires were employed to collect clinical, electroencephalographic and genetic data. We investigated electroencephalographic findings, focusing on the cortical distribution of interictal abnormalities and their changes with age. Among the 36 SYNGAP1-DEE cases 18 presented variants in the SYNGAP1 gene that had never been previously reported. The mean age of diagnosis was 8 years and 8 months, ranging from 2 to 17 years, with 55.9% being male. All subjects had global neurodevelopmental/language delay and behavioral abnormalities; 83.3% had moderate to profound intellectual disability (ID), 91.7% displayed autistic traits, 73% experienced sleep disorders and 86.1% suffered from epileptic seizures, mainly eyelid myoclonia with absences (55.3%). A total of 63 VEEGs were revised, observing a worsening of certain EEG findings with increasing age. A disorganized background was observed in all age ranges, yet this was more common among older cases. The main IEDs were bilateral synchronous and asynchronous posterior discharges, accounting for ≥50% in all age ranges. Generalized alterations with maximum amplitude in the anterior region showed as the second most frequent IED (≥15% in all age ranges) and were also more common with increasing age. Finally, diffuse fast activity was much more prevalent in cases with 6 years or older. To the best of our knowledge, this is the first study to analyze EEG features across different age groups, revealing an increase in interictal abnormalities over infancy and adolescence. Our findings suggest that SYNGAP1 haploinsufficiency has complex effects in human brain development, some of which might unravel at different developmental stages. Furthermore, they highlight the potential of baseline EEG to identify candidate biomarkers and the importance of natural history studies to develop specialized therapies and clinical trials.

Metabolic characterization of neurogenetic disorders involving glutamatergic neurotransmission.

The study of inborn errors of neurotransmission has been mostly focused on monoamine disorders, GABAergic and glycinergic defects. The study of the glutamatergic synapse using the same approach than classic neurotransmitter disorders is challenging due to the lack of biomarkers in the CSF. A metabolomic approach can provide both insight into their molecular basis and outline novel therapeutic alternatives. We have performed a semi-targeted metabolomic analysis on CSF samples from 25 patients with neurogenetic disorders with an important expression in the glutamatergic synapse and 5 controls. Samples from patients diagnosed with MCP2, CDKL5-, GRINpathies and STXBP1-related encephalopathies were included. We have performed univariate (UVA) and multivariate statistical analysis (MVA), using Wilcoxon rank-sum test, principal component analysis (PCA), and OPLS-DA. By using the results of both analyses, we have identified the metabolites that were significantly altered and that were important in clustering the respective groups. On these, we performed pathway- and network-based analyses to define which metabolic pathways were possibly altered in each pathology. We have observed alterations in the tryptophan and branched-chain amino acid metabolism pathways, which interestingly converge on LAT1 transporter-dependency to cross the blood-brain barrier (BBB). Analysis of the expression of LAT1 transporter in brain samples from a mouse model of Rett syndrome (MECP2) revealed a decrease in the transporter expression, that was already noticeable at pre-symptomatic stages. The study of the glutamatergic synapse from this perspective advances the understanding of their pathophysiology, shining light on an understudied feature as is their metabolic signature.

Global Impairment of Immediate-Early Genes Expression in Rett Syndrome Models and Patients Linked to Myelination Defects.

Rett syndrome (RTT) is a severe neurodevelopmental disease caused almost exclusively by mutations to the MeCP2 gene. This disease may be regarded as a synaptopathy, with impairments affecting synaptic plasticity, inhibitory and excitatory transmission and network excitability. The complete understanding of the mechanisms behind how the transcription factor MeCP2 so profoundly affects the mammalian brain are yet to be determined. What is known, is that MeCP2 involvement in activity-dependent expression programs is a critical link between this protein and proper neuronal activity, which allows the correct maturation of connections in the brain. By using RNA-sequencing analysis, we found several immediate-early genes (IEGs, key mediators of activity-dependent responses) directly bound by MeCP2 at the chromatin level and upregulated in the hippocampus and prefrontal cortex of the Mecp2-KO mouse. Quantification of the IEGs response to stimulus both in vivo and in vitro detected an aberrant expression pattern in MeCP2-deficient neurons. Furthermore, altered IEGs levels were found in RTT patient's peripheral blood and brain regions of post-mortem samples, correlating with impaired expression of downstream myelination-related genes. Altogether, these data indicate that proper IEGs expression is crucial for correct synaptic development and that MeCP2 has a key role in the regulation of IEGs.

Leigh syndrome is the main clinical characteristic of PTCD3 deficiency.

Mitochondrial translation defects are a continuously growing group of disorders showing a large variety of clinical symptoms including a wide range of neurological abnormalities. To date, mutations in PTCD3, encoding a component of the mitochondrial ribosome, have only been reported in a single individual with clinical evidence of Leigh syndrome. Here, we describe three additional PTCD3 individuals from two unrelated families, broadening the genetic and phenotypic spectrum of this disorder, and provide definitive evidence that PTCD3 deficiency is associated with Leigh syndrome. The patients presented in the first months of life with psychomotor delay, respiratory insufficiency and feeding difficulties. The neurologic phenotype included dystonia, optic atrophy, nystagmus and tonic-clonic seizures. Brain MRI showed optic nerve atrophy and thalamic changes, consistent with Leigh syndrome. WES and RNA-seq identified compound heterozygous variants in PTCD3 in both families: c.[1453-1G>C];[1918C>G] and c.[710del];[902C>T]. The functional consequences of the identified variants were determined by a comprehensive characterization of the mitochondrial function. PTCD3 protein levels were significantly reduced in patient fibroblasts and, consistent with a mitochondrial translation defect, a severe reduction in the steady state levels of complexes I and IV subunits was detected. Accordingly, the activity of these complexes was also low, and high-resolution respirometry showed a significant decrease in the mitochondrial respiratory capacity. Functional complementation studies demonstrated the pathogenic effect of the identified variants since the expression of wild-type PTCD3 in immortalized fibroblasts restored the steady-state levels of complexes I and IV subunits as well as the mitochondrial respiratory capacity. Additionally, minigene assays demonstrated that three of the identified variants were pathogenic by altering PTCD3 mRNA processing. The fourth variant was a frameshift leading to a truncated protein. In summary, we provide evidence of PTCD3 involvement in human disease confirming that PTCD3 deficiency is definitively associated with Leigh syndrome.

Diagnosis of Genetic White Matter Disorders by Singleton Whole-Exome and Genome Sequencing Using Interactome-Driven Prioritization.

BACKGROUND AND OBJECTIVES: Genetic white matter disorders (GWMD) are of heterogeneous origin, with >100 causal genes identified to date. Classic targeted approaches achieve a molecular diagnosis in only half of all patients. We aimed to determine the clinical utility of singleton whole-exome sequencing and whole-genome sequencing (sWES-WGS) interpreted with a phenotype- and interactome-driven prioritization algorithm to diagnose GWMD while identifying novel phenotypes and candidate genes. METHODS: A case series of patients of all ages with undiagnosed GWMD despite extensive standard-of-care paraclinical studies were recruited between April 2017 and December 2019 in a collaborative study at the Bellvitge Biomedical Research Institute (IDIBELL) and neurology units of tertiary Spanish hospitals. We ran sWES and WGS and applied our interactome-prioritization algorithm based on the network expansion of a seed group of GWMD-related genes derived from the Human Phenotype Ontology terms of each patient. RESULTS: We evaluated 126 patients (101 children and 25 adults) with ages ranging from 1 month to 74 years. We obtained a first molecular diagnosis by singleton WES in 59% of cases, which increased to 68% after annual reanalysis, and reached 72% after WGS was performed in 16 of the remaining negative cases. We identified variants in 57 different genes among 91 diagnosed cases, with the most frequent being RNASEH2B, EIF2B5, POLR3A, and PLP1, and a dual diagnosis underlying complex phenotypes in 6 families, underscoring the importance of genomic analysis to solve these cases. We discovered 9 candidate genes causing novel diseases and propose additional putative novel candidate genes for yet-to-be discovered GWMD. DISCUSSION: Our strategy enables a high diagnostic yield and is a good alternative to trio WES/WGS for GWMD. It shortens the time to diagnosis compared to the classical targeted approach, thus optimizing appropriate management. Furthermore, the interactome-driven prioritization pipeline enables the discovery of novel disease-causing genes and phenotypes, and predicts novel putative candidate genes, shedding light on etiopathogenic mechanisms that are pivotal for myelin generation and maintenance.

Liver-Directed Adeno-Associated Virus-Mediated Gene Therapy for Mucopolysaccharidosis Type VI.

BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is an inherited multisystem lysosomal disorder due to arylsulfatase B (ARSB) deficiency that leads to widespread accumulation of glycosaminoglycans (GAG), which are excreted in increased amounts in urine. MPS VI is characterized by progressive dysostosis multiplex, connective tissue and cardiac involvement, and hepatosplenomegaly. Enzyme replacement therapy (ERT) is available but requires life-long and costly intravenous infusions; moreover, it has limited efficacy on diseased skeleton and cardiac valves, compromised pulmonary function, and corneal opacities. METHODS: We enrolled nine patients with MPS VI 4 years of age or older in a phase 1/2 open-label gene therapy study. After ERT was interrupted, patients each received a single intravenous infusion of an adeno-associated viral vector serotype 8 expressing ARSB. Participants were sequentially enrolled in one of three dose cohorts: low (three patients), intermediate (two patients), or high (four patients). The primary outcome was safety; biochemical and clinical end points were secondary outcomes. RESULTS: The infusions occurred without severe adverse events attributable to the vector, meeting the prespecified end point. Participants in the low and intermediate dose cohorts displayed stable serum ARSB of approximately 20% of the mean healthy value but returned to ERT by 14 months after gene therapy because of increased urinary GAG. Participants in the high-dose cohort had sustained serum ARSB of 30% to 100% of the mean healthy value and a modest urinary GAG increase that did not reach a concentration at which ERT reintroduction was needed. In the high-dose group, there was no clinical deterioration for up to 2 years after gene therapy. CONCLUSIONS: Liver-directed gene therapy for participants with MPS VI did not have a dose-limiting side-effect and adverse event profile; high-dose treatment resulted in ARSB expression over at least 24 months with preliminary evidence of disease stabilization. (Funded by the Telethon Foundation ETS, the European Commission Seventh Framework Programme, and the Isaac Foundation; ClinicalTrials.gov number, NCT03173521; EudraCT number, 2016-002328-10.)

Pediatric Gaucher disease with intermediate type 2-3 phenotype associated with parkinsonian features and levodopa responsiveness.

INTRODUCTION: Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by a deficiency of acid ß-glucosidase encoded by the GBA gene. In patients with GD, childhood onset parkinsonian features have been rarely described. METHODS: Twin siblings with GD are described, including clinical follow-up and treatment response. Bone marrow, enzyme activity studies and genotyping were performed. RESULTS: By age 9 months, symptoms at onset were thrombocytopenia and splenomegaly. By age 2, hypokinesia, bradykinesia and oculomotor apraxia were observed. By age 5 a complete rigid hypokinetic syndrome was stablished in both patients, including bradykinesia, tremor and rigidity. Treatment with imiglucerase, miglustat, ambroxol and levodopa were performed. Levodopa showed a good response with improvement in motor and non-motor skills. Foamy cells were found in the bone marrow study. Glucocerebrosidase activity was 28% and 26%. Sanger sequencing analysis identified a missense mutation and a complex allele (NP_000148: p.[(Asp448His)]; [(Leu422Profs*4)]) in compound heterozygosity in GBA gene. CONCLUSIONS: Two siblings with neuronopathic GD with an intermediate form between type 2 and 3, with a systemic and neurological phenotype are described. The complex neurological picture included a hypokinetic-rigid and tremor syndrome that improved with levodopa treatment. These conditions together have not been previously described in pediatric GD. We suggest that in children with parkinsonian features, lysosomal storage disorders must be considered, and a levodopa trial must be performed. Moreover, this report give support to the finding that GBA and parkinsonian features share biological pathways and highlight the importance of lysosomal mechanisms in parkinsonism pathogenesis, what might have therapeutic implications.

Unraveling Molecular Pathways Altered in MeCP2-Related Syndromes, in the Search for New Potential Avenues for Therapy.

Methyl-CpG-binding protein 2 (MeCP2) is an X-linked epigenetic modulator whose dosage is critical for neural development and function. Loss-of-function mutations in MECP2 cause Rett Syndrome (RTT, OMIM #312750) while duplications in the Xq28 locus containing MECP2 and Interleukin-1 receptor-associated kinase 1 (IRAK1) cause MECP2 duplication syndrome (MDS, OMIM #300260). Both are rare neurodevelopmental disorders that share clinical symptoms, including intellectual disability, loss of speech, hand stereotypies, vasomotor deficits and seizures. The main objective of this exploratory study is to identify novel signaling pathways and potential quantitative biomarkers that could aid early diagnosis and/or the monitoring of disease progression in clinical trials. We analyzed by RT-PCR gene expression in whole blood and microRNA (miRNA) expression in plasma, in a cohort of 20 females with Rett syndrome, 2 males with MECP2 duplication syndrome and 28 healthy controls, and correlated RNA expression with disease and clinical parameters. We have identified a set of potential biomarker panels for RTT diagnostic and disease stratification of patients with microcephaly and vasomotor deficits. Our study sets the basis for larger studies leading to the identification of specific miRNA signatures for early RTT detection, stratification, disease progression and segregation from other neurodevelopmental disorders. Nevertheless, these data will require verification and validation in further studies with larger sample size including a whole range of ages.

Five new cases of syndromic intellectual disability due to KAT6A mutations: widening the molecular and clinical spectrum.

BACKGROUND: Pathogenic variants of the lysine acetyltransferase 6A or KAT6A gene are associated with a newly identified neurodevelopmental disorder characterized mainly by intellectual disability of variable severity and speech delay, hypotonia, and heart and eye malformations. Although loss of function (LoF) mutations were initially reported as causing this disorder, missense mutations, to date always involving serine residues, have recently been associated with a form of the disorder without cardiac involvement. RESULTS: In this study we present five new patients, four with truncating mutations and one with a missense change and the only one not presenting with cardiac anomalies. The missense change [p.(Gly359Ser)], also predicted to affect splicing by in silico tools, was functionally tested in the patient's lymphocyte RNA revealing a splicing effect for this allele that would lead to a frameshift and premature truncation. CONCLUSIONS: An extensive revision of the clinical features of these five patients revealed high concordance with the 80 cases previously reported, including developmental delay with speech delay, feeding difficulties, hypotonia, a high bulbous nose, and recurrent infections. Other features present in some of these five patients, such as cryptorchidism in males, syndactyly, and trigonocephaly, expand the clinical spectrum of this syndrome.

Safety of drug use in patients with a primary mitochondrial disease: An international Delphi-based consensus.

Clinical guidance is often sought when prescribing drugs for patients with primary mitochondrial disease. Theoretical considerations concerning drug safety in patients with mitochondrial disease may lead to unnecessary withholding of a drug in a situation of clinical need. The aim of this study was to develop consensus on safe medication use in patients with a primary mitochondrial disease. A panel of 16 experts in mitochondrial medicine, pharmacology, and basic science from six different countries was established. A modified Delphi technique was used to allow the panellists to consider draft recommendations anonymously in two Delphi rounds with predetermined levels of agreement. This process was supported by a review of the available literature and a consensus conference that included the panellists and representatives of patient advocacy groups. A high level of consensus was reached regarding the safety of all 46 reviewed drugs, with the knowledge that the risk of adverse events is influenced both by individual patient risk factors and choice of drug or drug class. This paper details the consensus guidelines of an expert panel and provides an important update of previously established guidelines in safe medication use in patients with primary mitochondrial disease. Specific drugs, drug groups, and clinical or genetic conditions are described separately as they require special attention. It is important to emphasise that consensus-based information is useful to provide guidance, but that decisions related to drug prescribing should always be tailored to the specific needs and risks of each individual patient. We aim to present what is current knowledge and plan to update this regularly both to include new drugs and to review those currently included.

Molecular characterization of Spanish patients with MECP2 duplication syndrome.

MECP2 duplication syndrome (MDS) is an X-linked neurodevelopmental disorder characterized by a severe to profound intellectual disability, early onset hypotonia and diverse psycho-motor and behavioural features. To date, fewer than 200 cases have been published. We report the clinical and molecular characterization of a Spanish MDS cohort that included 19 boys and 2 girls. Clinical suspicions were confirmed by array comparative genomic hybridization and multiplex ligation-dependent probe amplification (MLPA). Using, a custom in-house MLPA assay, we performed a thorough study of the minimal duplicated region, from which we concluded a complete duplication of both MECP2 and IRAK1 was necessary for a correct MDS diagnosis, as patients with partial MECP2 duplications lacked some typical clinical traits present in other MDS patients. In addition, the duplication location may be related to phenotypic severity. This observation may provide a new approach for genotype-phenotype correlations, and thus more personalized genetic counselling.

Comprehensive Analysis of GABAA-A1R Developmental Alterations in Rett Syndrome: Setting the Focus for Therapeutic Targets in the Time Frame of the Disease.

Rett syndrome, a serious neurodevelopmental disorder, has been associated with an altered expression of different synaptic-related proteins and aberrant glutamatergic and γ-aminobutyric acid (GABA)ergic neurotransmission. Despite its severity, it lacks a therapeutic option. Through this work we aimed to define the relationship between MeCP2 and GABAA.-A1 receptor expression, emphasizing the time dependence of such relationship. For this, we analyzed the expression of the ionotropic receptor subunit in different MeCP2 gene-dosage and developmental conditions, in cells lines, and in primary cultured neurons, as well as in different developmental stages of a Rett mouse model. Further, RNAseq and systems biology analysis was performed from post-mortem brain biopsies of Rett patients. We observed that the modulation of the MeCP2 expression in cellular models (both Neuro2a (N2A) cells and primary neuronal cultures) revealed a MeCP2 positive effect on the GABAA.-A1 receptor subunit expression, which did not occur in other proteins such as KCC2 (Potassium-chloride channel, member 5). In the Mecp2+/- mouse brain, both the KCC2 and GABA subunits expression were developmentally regulated, with a decreased expression during the pre-symptomatic stage, while the expression was variable in the adult symptomatic mice. Finally, the expression of the gamma-aminobutyric acid (GABA) receptor-related synaptic proteins from the postmortem brain biopsies of two Rett patients was evaluated, specifically revealing the GABA A1R subunit overexpression. The identification of the molecular changes along with the Rett syndrome prodromic stages strongly endorses the importance of time frame when addressing this disease, supporting the need for a neurotransmission-targeted early therapeutic intervention.

New insights into genetic variant spectrum and genotype-phenotype correlations of Rubinstein-Taybi syndrome in 39 CREBBP-positive patients.

BACKGROUND: Rubinstein-Taybi syndrome (RSTS) is a rare congenital disorder characterized by broad thumbs and halluces, intellectual disability, distinctive facial features, and growth retardation. Clinical manifestations of RSTS are varied and overlap with other syndromes' phenotype, which makes clinical diagnosis challenging. CREBBP is the major causative gene (55%-60% of the cases), whereas pathogenic variants found in EP300 represent the molecular cause in 8% of RSTS patients. A wide range of CREBBP pathogenic variants have been reported so far, including point mutations (30%-50%) and large deletions (10%). METHODS: The aim of this study was to characterize the CREBBP genetic variant spectrum in 39 RSTS patients using Multiplex Ligation-dependent Probe Amplification and DNA sequencing techniques (Sanger and Trio-based whole-exome sequencing). RESULTS: We identified 15 intragenic deletions/duplications, ranging from one exon to the entire gene. As a whole, 25 de novo point variants were detected: 4 missense, 12 nonsense, 5 frameshift, and 4 splicing pathogenic variants. Three of them were classified as of uncertain significance and one of the patients carried two different variants. CONCLUSION: Seventeen of the 40 genetic variants detected were reported for the first time in this work contributing, thus, to expand the molecular knowledge of this complex disorder.

X chromosome inactivation does not necessarily determine the severity of the phenotype in Rett syndrome patients.

Rett syndrome (RTT) is a severe neurological disorder usually caused by mutations in the MECP2 gene. Since the MECP2 gene is located on the X chromosome, X chromosome inactivation (XCI) could play a role in the wide range of phenotypic variation of RTT patients; however, classical methylation-based protocols to evaluate XCI could not determine whether the preferentially inactivated X chromosome carried the mutant or the wild-type allele. Therefore, we developed an allele-specific methylation-based assay to evaluate methylation at the loci of several recurrent MECP2 mutations. We analyzed the XCI patterns in the blood of 174 RTT patients, but we did not find a clear correlation between XCI and the clinical presentation. We also compared XCI in blood and brain cortex samples of two patients and found differences between XCI patterns in these tissues. However, RTT mainly being a neurological disease complicates the establishment of a correlation between the XCI in blood and the clinical presentation of the patients. Furthermore, we analyzed MECP2 transcript levels and found differences from the expected levels according to XCI. Many factors other than XCI could affect the RTT phenotype, which in combination could influence the clinical presentation of RTT patients to a greater extent than slight variations in the XCI pattern.

Celia's encephalopathy and c.974dupG in BSCL2 gene: a hidden change in a known variant.

Celia's encephalopathy (progressive encephalopathy with/without lipodystrophy (PELD)) is a childhood neurodegenerative disorder with a fatal prognosis before the age of 10, due to the variant c.985C>T in the BSCL2 gene that causes a cryptic splicing site leading to skipping of exon 7. For years, different authors have reported cases of congenital generalized lipodystrophy due to the variant c.974dupG in BSCL2 associated with neurological manifestations of variable severity, although some of them clearly superimposable to PELD. To identify the molecular mechanisms responsible for these neurological alterations in two patients with c.974dupG. Clinical characterization, biochemistry, and neuroimaging studies of two girls carrying this variant. In silico analysis, PCR amplification, and BSCL2 cDNA sequencing. BSCL2-201 transcript expression, which lacks exon 7, by qPCR in fibroblasts from the index case, from a healthy child as a control and from two patients with PELD, and in leukocytes from the index case and her parents. One with a severe encephalopathy including a picture of intellectual deficiency, severe language impairment, myoclonic epilepsy, and lipodystrophy as described in PELD, dying at 9 years and 9 months of age. The other 2-year-old patient showed incipient signs of neurological involvement. In silico and cDNA sequencing studies showed that variant c.974dupG gives rise to skipping of exon 7. The expression of BSCL2-201 in fibroblasts was significantly higher in the index case than in the healthy child, although less than in the case with homozygous PELD due to c.985C>T variant. The expression of this transcript was approximately half in the healthy carrier parents of this patient. The c.974dupG variant leads to the skipping of exon 7 of the BSCL2 gene and is responsible for a variant of Celia's encephalopathy, with variable phenotypic expression.

PLA2G6-associated neurodegeneration: New insights into brain abnormalities and disease progression.

INTRODUCTION: PLA2G6-associated neurodegeneration (PLAN) comprises a continuum of three phenotypes with overlapping clinical and radiologic features. METHODS: Observational clinical study in a cohort of infantile and childhood onset PLAN patients and genetic analysis of the PLA2G6 gene. We analysed chronological evolution in terms of age at onset and disease course through a 66-item questionnaire. We performed qualitative and quantitative assessment of MRI abnormalities and searched for clinical and radiological phenotype and genotype correlations. RESULTS: Sixteen PLAN patients (mean age: 10.2 years, range 3-33) were evaluated, with a median onset (years) of signs/symptoms as follows: neurological regression (1.5), oculomotor abnormalities (1.5), hypotonia (1.8), gait loss (2.2), pyramidal signs (3.0), axonal neuropathy (3.0), dysphagia (4.0), optic atrophy (4.0), psychiatric symptoms (4.0), seizures (5.9), joint contractures (6.0), dystonia (8.0), bladder dysfunction (13.0) and parkinsonism (15.0). MRI assessment identified cerebellar atrophy (19/19), brain iron deposition (10/19), clava hypertrophy (8/19) and T2/FLAIR hyperintensity of the cerebellar cortex (6/19). The mid-sagittal vermis relative diameter (MVRD) correlated with age at onset of clinical variants, meaning that the earlier the onset, the more severe the cerebellar atrophy. All patients harboured missense, nonsense and frameshift mutations in PLA2G6, including four novel variants. CONCLUSIONS: Cerebellar atrophy was a universal radiological sign in infantile and childhood onset PLAN, and correlated with the severity of the phenotype. Iron accumulation within the globus pallidum and substantia nigra was also a common and strikingly uniform feature regardless of the phenotype.

Elosulfase alfa for mucopolysaccharidosis type IVA: Real-world experience in 7 patients from the Spanish Morquio-A early access program.

There is a growing interest in evaluating the effectiveness of enzyme replacement therapy (ERT) with elosulfase alfa in patients with mucopolysaccharidosis type IVA (MPS-IVA) under real-world conditions. We present the experience of seven pediatric MPS-IVA patients from the Spanish Morquio-A Early Access Program. Efficacy was evaluated based on the distance walked in the 6-min walking test (6-MWT) and the 3-min-stair-climb-test (3-MSCT) at baseline and after 8 months of ERT treatment. Additionally, urinary glycosaminoglycans were measured, and a molecular analysis of a GALNS mutation was performed. The health-related quality of life was evaluated using the EuroQoL (EQ)-5D-5 L. The distance walked according to the 6-MWT ranged from 0 to 325 m at baseline and increased to 12-300 m after 8 months with elosulfase alfa (the walked distance improved in all patients except one). An increase was observed for the two patients who had to use a wheelchair. Improvements were also observed for the 3-MSCT in four patients, whereas two patients showed no changes. Three patients showed an improvement in the EQ-VAS score, whereas the scores of three patients remained stable. Regarding urinary glycosaminoglycans measurements, an irregular response was observed. Our results showed overall improvement in endurance and functionality after 8 months of elosulfase alfa treatment in a heterogeneous subset of MPS IVA patients with severe clinical manifestations managed in a real-world setting.