RNA polymerase III transcription as a disease factor.

RNA polymerase (Pol) III is responsible for transcription of different noncoding genes in eukaryotic cells, whose RNA products have well-defined functions in translation and other biological processes for some, and functions that remain to be defined for others. For all of them, however, new functions are being described. For example, Pol III products have been reported to regulate certain proteins such as protein kinase R (PKR) by direct association, to constitute the source of very short RNAs with regulatory roles in gene expression, or to control microRNA levels by sequestration. Consistent with these many functions, deregulation of Pol III transcribed genes is associated with a large variety of human disorders. Here we review different human diseases that have been linked to defects in the Pol III transcription apparatus or to Pol III products imbalance and discuss the possible underlying mechanisms.

Structural Variants at the LMNB1 Locus: Deciphering Pathomechanisms in Autosomal Dominant Adult-Onset Demyelinating Leukodystrophy.

OBJECTIVE: We aimed to elucidate the pathogenic mechanisms underlying autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), and to understand the genotype/phenotype correlation of structural variants (SVs) in the LMNB1 locus. BACKGROUND: Since the discovery of 3D genome architectures and topologically associating domains (TADs), new pathomechanisms have been postulated for SVs, regardless of gene dosage changes. ADLD is a rare genetic disease associated with duplications (classical ADLD) or noncoding deletions (atypical ADLD) in the LMNB1 locus. METHODS: High-throughput chromosome conformation capture, RNA sequencing, histopathological analyses of postmortem brain tissues, and clinical and neuroradiological investigations were performed. RESULTS: We collected data from >20 families worldwide carrying SVs in the LMNB1 locus and reported strong clinical variability, even among patients carrying duplications of the entire LMNB1 gene, ranging from classical and atypical ADLD to asymptomatic carriers. We showed that patients with classic ADLD always carried intra-TAD duplications, resulting in a simple gene dose gain. Atypical ADLD was caused by LMNB1 forebrain-specific misexpression due to inter-TAD deletions or duplications. The inter-TAD duplication, which extends centromerically and crosses the 2 TAD boundaries, did not cause ADLD. Our results provide evidence that astrocytes are key players in ADLD pathology. INTERPRETATION: Our study sheds light on the 3D genome and TAD structural changes associated with SVs in the LMNB1 locus, and shows that a duplication encompassing LMNB1 is not sufficient per se to diagnose ADLD, thereby strongly affecting genetic counseling. Our study supports breaking TADs as an emerging pathogenic mechanism that should be considered when studying brain diseases. ANN NEUROL 2024;96:855-870.

Intragenic homozygous duplication in HEPACAM is associated with megalencephalic leukoencephalopathy with subcortical cysts type 2A.

Disease-causing variants in HEPACAM are associated with megalencephalic leukoencephalopathy with subcortical cysts 2A (MLC2A, MIM# 613,925, autosomal recessive), and megalencephalic leukoencephalopathy with subcortical cysts 2B, remitting, with or without impaired intellectual development (MLC2B, MIM# 613,926, autosomal dominant). These disorders are characterised by macrocephaly, seizures, motor delay, cognitive impairment, ataxia, and spasticity. Brain magnetic resonance imaging (MRI) in these individuals shows swollen cerebral hemispheric white matter and subcortical cysts, mainly in the frontal and temporal regions. To date, 45 individuals from 39 families are reported with biallelic and heterozygous variants in HEPACAM, causing MLC2A and MLC2B, respectively. A 9-year-old male presented with developmental delay, gait abnormalities, seizures, macrocephaly, dysarthria, spasticity, and hyperreflexia. MRI revealed subcortical cysts with diffuse cerebral white matter involvement. Whole-exome sequencing (WES) in the proband did not reveal any clinically relevant single nucleotide variants. However, copy number variation analysis from the WES data of the proband revealed a copy number of 4 for exons 3 and 4 of HEPACAM. Validation and segregation were done by quantitative PCR which confirmed the homozygous duplication of these exons in the proband and carrier status in both parents. To the best of our knowledge, this is the first report of an intragenic duplication in HEPACAM causing MLC2A.

De novo variants in POLR3B cause ataxia, spasticity, and demyelinating neuropathy.

POLR3B encodes the second-largest catalytic subunit of RNA polymerase III, an enzyme involved in transcription. Bi-allelic pathogenic variants in POLR3B are a well-established cause of hypomyelinating leukodystrophy. We describe six unrelated individuals with de novo missense variants in POLR3B and a clinical presentation substantially different from POLR3-related leukodystrophy. These individuals had afferent ataxia, spasticity, variable intellectual disability and epilepsy, and predominantly demyelinating sensory motor peripheral neuropathy. Protein modeling and proteomic analysis revealed a distinct mechanism of pathogenicity; the de novo POLR3B variants caused aberrant association of individual enzyme subunits rather than affecting overall enzyme assembly or stability. We expand the spectrum of disorders associated with pathogenic variants in POLR3B to include a de novo heterozygous POLR3B-related disorder.

A novel heterozygous TMEM63A variant in a familial case with early onset nystagmus, severe hypomyelination, and a favorable adult prognosis.

Heterozygous transmembrane protein 63A (TMEM63A) variants cause transient infantile hypomyelinating leukodystrophy-19, which features remarkable natural resolution of clinical and imaging findings during childhood. Previous reports have mainly described de novo variants lacking detailed familial cases. Herein, we describe the clinical course of familial cases with a TMEM63A variant. A 5-month-old girl presented with nystagmus, global hypotonia, and difficulty swallowing since birth. Brain magnetic resonance imaging at 1.5 and 5 months revealed diffuse hypomyelination. Her mother, maternal aunt, and grandfather had nystagmus and motor developmental delays in infancy, which resolved spontaneously during childhood. Compared with these cases, the proband's motor developmental delay was profound, and she was the only one with feeding difficulties, necessitating nasogastric tube feeding. Genetic testing revealed a heterozygous TMEM63A variant (NM_014698.3:c.1658G>A, p.(Gly553Asp)) in the proband and her family. This is the first three-generation familial report of a TMEM63A variant that provides insight into its history and heterogeneity.

A novel missense variant in HIKESHI: Clinical phenotype, in vitro functional testing, and potential for gene therapy.

A 7-month-old boy presented to our clinic with developmental delay, Magnetic Resonance Imaging (MRI) features of delayed myelination and diffusion restriction, and a homozygous variant of uncertain significance (c.4T>G, p.Phe2Val) in HIKESHI, a gene associated with autosomal-recessive hypomyelinating leukodystrophy 13. We hypothesized that the variant is disease-causing and aimed to rescue the cellular phenotype with vector-mediated gene replacement. HIKESHI mediates heat-induced nuclear accumulation of heat-shock proteins, including HSP70, to protect cells from stress. We generated skin fibroblasts from the proband and proband's mother (heterozygous) to compare protein expression and subcellular localization of HSP70 under heat stress conditions, and the effect of vector-mediated overexpression of HIKESHI in the proband's cells under the same heat stress conditions. Western blot analysis revealed absent HIKESHI protein from proband fibroblasts, contrasted with ample expression in parental cells. Under heat stress conditions, while the mother's cells displayed appropriate nuclear localization of HSP70, the proband's cells displayed impaired nuclear translocalization. When patient fibroblasts were provided exogenous HIKESHI, the transfected proband's cells showed restored heat-induced nuclear translocalization of HSP70 under conditions of heat stress. These functional data establish that the patient's variant is a pathogenic loss-of-function mutation, thus confirming a diagnosis of hypomyelinating leukodystrophy 13 and that vector-mediated gene replacement may be an effective treatment approach for patients with this disorder.

Exploring metabolic alterations in PYCR2 deficiency: Unveiling pathways and clinical presentations of hypomyelinating leukodystrophy 10.

Proline-5-carboxylate reductase 2, encoded by PYCR2 gene, is an enzyme that catalyzes the last step of proline synthesis from pyrroline-5-carboxylate synthetase to proline. PYCR2 gene defect causes hypomyelinating leukodystrophy 10. Up until now, to our knowledge around 38 patients with PYCR2 defect have been reported. Herein, we describe clinical, neuroradiological, biochemical findings, and metabolomic profiling of three new genetically related cases of PYCR2 defects from a large family. Cerebrospinal fluid (CSF) amino acid levels were measured and untargeted metabolomic profiling of plasma and CSF were conducted and evaluated together with the clinical findings in the patients. While plasma and CSF proline levels were found to be totally normal, untargeted metabolomic profiling revealed mild increases of glutamate, alpha-ketoglutarate, and l-glutamate semialdehyde and marked increases of inosine and xanthine. Our findings and all the previous reports suggest that proline auxotrophy is not the central disease mechanism. Untargeted metabolomics point to mild changes in proline pathway and also in purine/pyrimidine pathway.

Hypomyelination, hypodontia and craniofacial abnormalities in a Polr3b mouse model of leukodystrophy.

RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit. Here, we determined the impact of POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic Pdgfrα-Cre/ERT; R26-Stopfl-EYFP; Polr3bfl mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed. Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis. In summary, we characterized the Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions.

Aquaporin-4 and GPRC5B: old and new players in controlling brain oedema.

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema.

Transcriptional and metabolic effects of aspartate-glutamate carrier isoform 1 (AGC1) downregulation in mouse oligodendrocyte precursor cells (OPCs).

Aspartate-glutamate carrier isoform 1 (AGC1) is a carrier responsible for the export of mitochondrial aspartate in exchange for cytosolic glutamate and is part of the malate-aspartate shuttle, essential for the balance of reducing equivalents in the cells. In the brain, mutations in SLC25A12 gene, encoding for AGC1, cause an ultra-rare genetic disease, reported as a neurodevelopmental encephalopathy, whose symptoms include global hypomyelination, arrested psychomotor development, hypotonia and seizures. Among the biological components most affected by AGC1 deficiency are oligodendrocytes, glial cells responsible for myelination processes, and their precursors [oligodendrocyte progenitor cells (OPCs)]. The AGC1 silencing in an in vitro model of OPCs was documented to cause defects of proliferation and differentiation, mediated by alterations of histone acetylation/deacetylation. Disrupting AGC1 activity could possibly reduce the availability of acetyl groups, leading to perturbation of many biological pathways, such as histone modifications and fatty acids formation for myelin production. Here, we explore the transcriptome of mouse OPCs partially silenced for AGC1, reporting results of canonical analyses (differential expression) and pathway enrichment analyses, which highlight a disruption in fatty acids synthesis from both a regulatory and enzymatic stand. We further investigate the cellular effects of AGC1 deficiency through the identification of most affected transcriptional networks and altered alternative splicing. Transcriptional data were integrated with differential metabolite abundance analysis, showing downregulation of several amino acids, including glutamine and aspartate. Taken together, our results provide a molecular foundation for the effects of AGC1 deficiency in OPCs, highlighting the molecular mechanisms affected and providing a list of actionable targets to mitigate the effects of this pathology.

PPAR-γ activation enhances myelination and neurological recovery in premature rabbits with intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) results in periventricular inflammation, hypomyelination of the white matter, and hydrocephalus in premature infants. No effective therapy exists to prevent these disorders. Peroxisome proliferator activated receptor-γ (PPAR-γ) agonists reduce inflammation, alleviate free radical generation, and enhance microglial phagocytosis, promoting clearance of debris and red blood cells. We hypothesized that activation of PPAR-γ would enhance myelination, reduce hydrocephalus, and promote neurological recovery in newborns with IVH. These hypotheses were tested in a preterm rabbit model of IVH; autopsy brain samples from premature infants with and without IVH were analyzed. We found that IVH augmented PPAR-γ expression in microglia of both preterm human infants and rabbit kits. The treatment with PPAR-γ agonist or PPAR-γ overexpression by adenovirus delivery further elevated PPAR-γ levels in microglia, reduced proinflammatory cytokines, increased microglial phagocytosis, and improved oligodendrocyte progenitor cell (OPC) maturation in kits with IVH. Transcriptomic analyses of OPCs identified previously unrecognized PPAR-γ-induced genes for purinergic signaling, cyclic adenosine monophosphate generation, and antioxidant production, which would reprogram these progenitors toward promoting myelination. RNA-sequencing analyses of microglia revealed PPAR-γ-triggered down-regulation of several proinflammatory genes and transcripts having roles in Parkinson's disease and amyotrophic lateral sclerosis, contributing to neurological recovery in kits with IVH. Accordingly, PPAR-γ activation enhanced myelination and neurological function in kits with IVH. This also enhanced microglial phagocytosis of red blood cells but did not reduce hydrocephalus. Treatment with PPAR-γ agonist might enhance myelination and neurological recovery in premature infants with IVH.

A De Novo Splicing Mutation of STXBP1 in Epileptic Encephalopathy Associated with Hypomyelinating Leukodystrophy.

Deleterious variations in STXBP1 are responsible for early infantile epileptic encephalopathy type 4 (EIEE4, OMIM # 612164) because of its dysfunction in the central nervous system. The clinical spectrum of the neurodevelopmental delays associated with STXBP1 aberrations is collectively defined as STXBP1 encephalopathy (STXBP1-E), the conspicuous features of which are highlighted by early-onset epileptic seizures without structural brain anomalies. A girl was first diagnosed with unexplained disorders of movement and cognition, which later developed into STXBP1-E with unexpected leukoaraiosis and late onset of seizures. Genetic screening and molecular tests alongside neurological examinations were employed to investigate the genetic etiology and establish the diagnosis. A heterozygous mutation of c.37+2dupT at the STXBP1 splice site was identified as the pathogenic cause in the affected girl. The de novo mutation (DNM) did not result in any truncated proteins but immediately triggered mRNA degradation by nonsense-mediated mRNA decay (NMD), which led to the haploinsufficiency of STXBP1. The patient showed atypical phenotypes characterized by hypomyelinating leukodystrophy, and late onset of epileptic seizures, which had never previously been delineated in STXBP1-E. These findings strongly indicated that the haploinsufficiency of STXBP1 could also exhibit divergent clinical phenotypes because of the genetic heterogeneity in the subset of encephalopathies.

Mlc1-Expressing Perivascular Astrocytes Promote Blood-Brain Barrier Integrity.

In the mammalian brain, perivascular astrocytes (PAs) closely juxtapose blood vessels and are postulated to have important roles in the control of vascular physiology, including regulation of the blood-brain barrier (BBB). Deciphering specific functions for PAs in BBB biology, however, has been limited by the ability to distinguish these cells from other astrocyte populations. In order to characterize selective roles for PAs in vivo, a new mouse model has been generated in which the endogenous megalencephalic leukoencephalopathy with subcortical cysts 1 (Mlc1) gene drives expression of Cre fused to a mutated estrogen ligand-binding domain (Mlc1-T2A-CreERT2). This knock-in mouse model, which we term MLCT, allows for selective identification and tracking of PAs in the postnatal brain. We also demonstrate that MLCT-mediated ablation of PAs causes severe defects in BBB integrity, resulting in premature death. PA loss results in aberrant localization of Claudin 5 and -VE-Cadherin in endothelial cell junctions as well as robust microgliosis. Collectively, these data reveal essential functions for Mlc1-expressing PAs in regulating endothelial barrier integrity in mice and indicate that primary defects in astrocytes that cause BBB breakdown may contribute to human neurologic disorders.SIGNIFICANCE STATEMENT Interlaced among the billions of neurons and glia in the mammalian brain is an elaborate network of blood vessels. Signals from the brain parenchyma control the unique permeability properties of cerebral blood vessels known as the blood-brain barrier (BBB). However, we understand very little about the relative contributions of different neural cell types in the regulation of BBB functions. Here, we show that a specific subpopulation of astrocyte is essential for control of BBB integrity, with ablation of these cells leading to defects in endothelial cell junctions, BBB breakdown, and resulting neurologic deficits.

A novel role for MLC1 in regulating astrocyte-synapse interactions.

Loss of function of the astrocyte membrane protein MLC1 is the primary genetic cause of the rare white matter disease Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC), which is characterized by disrupted brain ion and water homeostasis. MLC1 is prominently present around fluid barriers in the brain, such as in astrocyte endfeet contacting blood vessels and in processes contacting the meninges. Whether the protein plays a role in other astrocyte domains is unknown. Here, we show that MLC1 is present in distal astrocyte processes, also known as perisynaptic astrocyte processes (PAPs) or astrocyte leaflets, which closely interact with excitatory synapses in the CA1 region of the hippocampus. We find that the PAP tip extending toward excitatory synapses is shortened in Mlc1-null mice. This affects glutamatergic synaptic transmission, resulting in a reduced rate of spontaneous release events and slower glutamate re-uptake under challenging conditions. Moreover, while PAPs in wildtype mice retract from the synapse upon fear conditioning, we reveal that this structural plasticity is disturbed in Mlc1-null mice, where PAPs are already shorter. Finally, Mlc1-null mice show reduced contextual fear memory. In conclusion, our study uncovers an unexpected role for the astrocyte protein MLC1 in regulating the structure of PAPs. Loss of MLC1 alters excitatory synaptic transmission, prevents normal PAP remodeling induced by fear conditioning and disrupts contextual fear memory expression. Thus, MLC1 is a new player in the regulation of astrocyte-synapse interactions.

Identification of the GlialCAM interactome: the G protein-coupled receptors GPRC5B and GPR37L1 modulate megalencephalic leukoencephalopathy proteins.

Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a type of vacuolating leukodystrophy, which is mainly caused by mutations in MLC1 or GLIALCAM. The two MLC-causing genes encode for membrane proteins of yet unknown function that have been linked to the regulation of different chloride channels such as the ClC-2 and VRAC. To gain insight into the role of MLC proteins, we have determined the brain GlialCAM interacting proteome. The proteome includes different transporters and ion channels known to be involved in the regulation of brain homeostasis, proteins related to adhesion or signaling as several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptor GPR37L1. Focusing on these two GPCRs, we could validate that they interact directly with MLC proteins. The inactivation of Gpr37l1 in mice upregulated MLC proteins without altering their localization. Conversely, a reduction of GPRC5B levels in primary astrocytes downregulated MLC proteins, leading to an impaired activation of ClC-2 and VRAC. The interaction between the GPCRs and MLC1 was dynamically regulated upon changes in the osmolarity or potassium concentration. We propose that GlialCAM and MLC1 associate with different integral membrane proteins modulating their functions and acting as a recruitment site for various signaling components as the GPCRs identified here. We hypothesized that the GlialCAM/MLC1 complex is working as an adhesion molecule coupled to a tetraspanin-like molecule performing regulatory effects through direct binding or influencing signal transduction events.

De novo EIF2AK1 and EIF2AK2 Variants Are Associated with Developmental Delay, Leukoencephalopathy, and Neurologic Decompensation.

EIF2AK1 and EIF2AK2 encode members of the eukaryotic translation initiation factor 2 alpha kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants in EIF2AK1 and EIF2AK2 have not been reported. Here, we describe the identification of nine unrelated individuals with heterozygous de novo missense variants in EIF2AK1 (1/9) or EIF2AK2 (8/9). Features seen in these nine individuals include white matter alterations (9/9), developmental delay (9/9), impaired language (9/9), cognitive impairment (8/9), ataxia (6/9), dysarthria in probands with verbal ability (6/9), hypotonia (7/9), hypertonia (6/9), and involuntary movements (3/9). Individuals with EIF2AK2 variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and proband-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate eukaryotic translation initiation factor 2 subunit 1 (EIF2S1, also known as EIF2α), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter (CACH/VWM), a leukodystrophy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate that EIF2AK2 missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.

Identification of POLR3B biallelic mutations-associated hypomyelinating leukodystrophy-8 in two siblings.

POLR3B gene encodes the 2nd largest catalytic subunit and affects the function of RNA polymerase III enzymes in transcription. Bi-allelic variants in POLR3B pathogenically cause hypomyelinating leukodystrophy-8 (HLD8). Herein, we recruited a family with two patients, who presented clinically with cerebellar atrophy, intellectual disability, hypogonadotropic hypogonadism, and visual problems. We identified the two affected siblings carrying the compound heterozygous variations (c.165_167del; c.1615G>T) in POLR3B by trio-whole-exome sequencing (trio-WES). The qPCR and western blot showed that both transcriptional and translational levels of the mutation (c.165_167del, p.I55_K56delinsM) were sharply attenuated. Following that, a thorough functional examination of a zebrafish line disrupted for human POLR3B validated the pathogenic effects of the two mutations. Our research broadens the spectrum of HLD8-related pathogenic POLR3B mutations and provides new molecular and animal evidence.

A novel variant of the POLR3A gene in a Chinese patient with POLR3-related leukodystrophy.

BACKGROUND: POLR3-related leukodystrophy is a group of rare neurodegenerative disorders characterized by degeneration of the white matter with different combinations of major clinical features. CASE: An 18-year-old lady was admitted for no menstruation since childhood. She gradually developed slight symptoms, such as choking after drinking water and unsteady walking in the last 2 years. Furthermore, her test scores and response capability were far lower than that of her peers. Physical examination revealed her to be of a slightly short stature, with stiff expressions and bilateral breast enlargement. She revealed clumsy movements when examined for ataxia, with an SARA score of 9. FINDINGS: The laboratory data revealed a decreased level of estradiol, FSH, and LH, with a MoCA score of 7. Conventional karyotype analysis revealed a 46 XX 9qh + karyotype. Ultrasound indicated primordial uterus (19 × 11 × 10 mm). Brain MRI showed bilateral cerebral hemisphere myelin dysplasia, brain atrophy, thin corpus callosum, and small pituitary gland with uneven reinforcement and enlarged ventricles. Exome sequencing exhibited two missense mutations in the POLR3A gene (c.3013C > T and c.1757C > T), which were inherited from her mother and father, respectively. CONCLUSION: Collectively, we identified novel compound heterozygous mutations of the POLR3A gene that caused POLR3A-related hypomyelinating leukodystrophy with hypogonadism in the patient combined with the clinical presentation, MRI brain pattern, and medical exome sequencing. TEACHING POINTS: The complexity of clinical phenotypes and heterogeneity of genotypes raise new challenges in genetic diagnoses. This study will further aid our understanding of POLR3A-related leukodystrophy and promote further analysis of phenotype-genotype correlations of related diseases.

yippee like 3 (ypel3) is a novel gene required for myelinating and perineurial glia development.

Hypomyelination, a neurological condition characterized by decreased production of myelin sheets by glial cells, often has no known etiology. Elucidating the genetic causes of hypomyelination provides a better understanding of myelination, as well as means to diagnose, council, and treat patients. Here, we present evidence that YIPPEE LIKE 3 (YPEL3), a gene whose developmental role was previously unknown, is required for central and peripheral glial cell development. We identified a child with a constellation of clinical features including cerebral hypomyelination, abnormal peripheral nerve conduction, hypotonia, areflexia, and hypertrophic peripheral nerves. Exome and genome sequencing revealed a de novo mutation that creates a frameshift in the open reading frame of YPEL3, leading to an early stop codon. We used zebrafish as a model system to validate that YPEL3 mutations are causative of neuropathy. We found that ypel3 is expressed in the zebrafish central and peripheral nervous system. Using CRISPR/Cas9 technology, we created zebrafish mutants carrying a genomic lesion similar to that of the patient. Our analysis revealed that Ypel3 is required for development of oligodendrocyte precursor cells, timely exit of the perineurial glial precursors from the central nervous system (CNS), formation of the perineurium, and Schwann cell maturation. Consistent with these observations, zebrafish ypel3 mutants have metabolomic signatures characteristic of oligodendrocyte and Schwann cell differentiation defects, show decreased levels of Myelin basic protein in the central and peripheral nervous system, and develop defasciculated peripheral nerves. Locomotion defects were observed in adult zebrafish ypel3 mutants. These studies demonstrate that Ypel3 is a novel gene required for perineurial cell development and glial myelination.

Structural basis for the dominant or recessive character of GLIALCAM mutations found in leukodystrophies.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a type of leukodystrophy characterized by white matter edema, and it is caused mainly by recessive mutations in MLC1 and GLIALCAM genes. These variants are called MLC1 and MLC2A with both types of patients sharing the same clinical phenotype. In addition, dominant mutations in GLIALCAM have also been identified in a subtype of MLC patients with a remitting phenotype. This variant has been named MLC2B. GLIALCAM encodes for an adhesion protein containing two immunoglobulin (Ig) domains and it is needed for MLC1 targeting to astrocyte-astrocyte junctions. Most mutations identified in GLIALCAM abolish GlialCAM targeting to junctions. However, it is unclear why some mutations behave as recessive or dominant. Here, we used a combination of biochemistry methods with a new developed anti-GlialCAM nanobody, double-mutants and cysteine cross-links experiments, together with computer docking, to create a structural model of GlialCAM homo-interactions. Using this model, we suggest that dominant mutations affect different GlialCAM-GlialCAM interacting surfaces in the first Ig domain, which can occur between GlialCAM molecules present in the same cell (cis) or present in neighbouring cells (trans). Our results provide a framework that can be used to understand the molecular basis of pathogenesis of all identified GLIALCAM mutations.