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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., (© 2024 The Author(s). Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive rare disease condition in which audiological deficit is also observed. A 4-year-old male child with PMD underwent an audiological evaluation. The results suggested normal middle ear and outer hair cells functioning, with only peak I of the auditory brainstem response present until 30 dBnHL. Further, the cervical vestibular evoked myogenic potential showed delayed latencies with normal amplitudes. In this case report, we attempt to explain the audio-vestibular test results and correlate them with the pathophysiology. This is the first report on the cervical vestibular myogenic potentials in patients with PMD.

Claudin-25 (CLDN-25), also known as Claudin containing domain 1, is an uncharacterized claudin family member. It has less conserved amino acid sequences when compared to other claudins. It also has a very broad tissue expression profile and there is currently a lack of functional information from murine knockout models. Here, we report a de novo missense heterozygous variant in CLDN25 (c. 745G>C, p. A249P) found in a patient diagnosed with Pelizaeus-Merzbacher-like leukodystrophy and presenting with symptoms such as delayed motor development, several episodes of tonic absent seizures and generalized dystonia. The variant protein does not localize to the cell-cell borders where it would normally be expected to be expressed. Amino acid position 249 is located 4 amino acids from the C-terminal end of the protein where most claudin family members have a conserved binding motif for the key scaffolding protein ZO-1. However, CLDN-25 does not contain this motif. Here, we show that the C-terminal end of CLDN-25 is required for its junctional localization in a ZO-1 independent manner. The A249P mutant protein as well as a deletion mutant lacking its last 5 C-terminal amino acids also failed to localize to the cell-cell border in vitro. Intriguingly, cellular knockout of CLDN25, in vitro, appeared to increase the integrity of the tight junction between 2 contacting cells, while driving highly unusual increased movement of solutes between cells. We propose that the barrier function of CLDN-25 is akin to a decoy claudin, whereby decreasing its expression in "leaky" epithelial cells and endothelial cells will drive dynamic changes in the adhesion and interaction capacity of cell-cell contact points. While it remains unclear how this de novo CLDN-25 mutant induces leukodystrophy, our findings strongly suggest that this mutation induces haploinsufficiency of CLDN-25. Elucidating the function of this uncharacterized claudin protein will lead to a better understanding of the role of claudin proteins in health and disease., (© The Author(s) 2024. Published by Oxford University Press.)

Pelizaeus-Merzbacher disease (PMD) is caused by mutations in the proteolipid protein 1 (PLP1) gene encoding proteolipid protein (PLP). As a major component of myelin, mutated PLP causes progressive neurodegeneration and eventually death due to severe white matter deficits. Medical care has long been limited to symptomatic treatments, but first-in-class PMD therapies with novel mechanisms now stand poised to enter clinical trials. Here, we review PMD disease mechanisms and outline rationale for therapeutic interventions, including PLP1 suppression, cell transplantation, iron chelation, and intracellular stress modulation. We discuss available preclinical data and their implications on clinical development. With several novel treatments on the horizon, PMD is on the precipice of a new era in the diagnosis and treatment of patients suffering from this debilitating disease., Competing Interests: Declaration of interests P.J.T. and M.S.E. are inventors on issued and pending patent claims filed by Case Western Reserve University covering methods of PLP1 suppression. Case Western Reserve University has licensed intellectual property related to PLP1 suppression to Ionis Pharmaceuticals. P.J.T. is Chair of the Scientific Advisory Board (volunteer position) for the Pelizaeus–Merzbacher Disease Foundation., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Genetic alterations in the PLP1 gene, i.e. point mutations and duplications, are associated with demyelinating disease Pelizaeus-Merzbacher. Here, we describe the generation of a human iPSC line harboring a PLP1 variant in codon 33 which leads to an amino acid change from cysteine to tyrosine. The established PLP1 C33Y iPSC line enables the study of PMD pathophysiology by investigating various cell types and -characteristics in our developed protocol for bioengineered neuronal organoids (BENOs) 1 ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Two preclinical studies using mouse models of Pelizeaus-Merzbacher disease (PMD) have revealed the potential therapeutic effects of curcumin. In this study, we examined the effects of curcumin in patients with PMD., Methods: We conducted a study administering an open-label oral bioavailable form of curcumin in nine patients genetically confirmed to have PMD (five to 20 years; mean 11 years) for 12 months (low doses for two months followed by high doses for 10 months). We evaluated changes in clinical symptoms as the primary end point using two scales, Gross Motor Function Measure (GMFM) and the PMD Functional Disability Score (PMD-FDS). The level of myelination by brain magnetic resonance imaging (MRI) and the electrophysiological state by auditory brainstem response (ABR) were evaluated as secondary end points. The safety and tolerability of oral curcumin were also examined., Results: Increase in GMFM and PMD-FDS were noted in five and three patients, respectively, but overall, no statistically significant improvement was demonstrated. We found no clear improvement in their brain MRI or ABR. No adverse events associated with oral administration of curcumin were observed., Conclusions: Although we failed to demonstrate any significant therapeutic effects of curcumin after 12 months, its tolerability and safety were confirmed. This study does not exclude the possibility of therapeutic effects of curcumin, and a trial of longer duration should be considered to compare the natural history of the disease with the effects of curcumin., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ken Inoue reports that measurement of serum curcumin concentration was provided by Theravalues Corporation (Japan)., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Hypomyelinating leukodystrophies are a subset of genetic white matter diseases characterized by insufficient myelin deposition during development. MRI patterns are used to identify hypomyelinating disorders, and genetic testing is used to determine the causal genes implicated in individual disease forms. Clinical course can range from severe, with patients manifesting neurologic symptoms in infancy or early childhood, to mild, with onset in adolescence or adulthood. This chapter discusses the most common hypomyelinating leukodystrophies, including X-linked Pelizaeus-Merzbacher disease and other PLP1-related disorders, autosomal recessive Pelizaeus-Merzbacher-like disease, and POLR3-related leukodystrophy. PLP1-related disorders are caused by hemizygous pathogenic variants in the proteolipid protein 1 (PLP1) gene, and encompass classic Pelizaeus-Merzbacher disease, the severe connatal form, PLP1-null syndrome, spastic paraplegia type 2, and hypomyelination of early myelinating structures. Pelizaeus-Merzbacher-like disease presents a similar clinical picture to Pelizaeus-Merzbacher disease, however, it is caused by biallelic pathogenic variants in the GJC2 gene, which encodes for the gap junction protein Connexin-47. POLR3-related leukodystrophy, or 4H leukodystrophy (hypomyelination, hypodontia, and hypogonadotropic hypogonadism), is caused by biallelic pathogenic variants in genes encoding specific subunits of the transcription enzyme RNA polymerase III. In this chapter, the clinical features, disease pathophysiology and genetics, imaging patterns, as well as supportive and future therapies are discussed for each disorder., (Copyright © 2024 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Objective: Xq chromosome duplication with complex rearrangements is generally acknowledged to be associated with neurodevelopmental disorders, such as Pelizaeus-Merzbacher disease (PMD) and MECP2 duplication syndrome. For couples who required a PGT-M (pre-implantation genetic testing for monogenic disease) for these disorders, junction-specific PCR is useful to directly detect pathogenic variants. Therefore, pre-clinical workup for PGT-M requires the identification of the junction of duplicated segments in PMD and MECP2 duplication syndrome, which is generally difficult., Methods: In this report, we used nanopore long-read sequencing targeting the X chromosome using an adaptive sampling method to identify breakpoint junctions in disease-causing triplications., Results: By long-read sequencing, we successfully identified breakpoint junctions in one PMD case with PLP1 triplication and in another MECP2 triplication case in a single sequencing run. Surprisingly, the duplicated region involving MECP2 was inserted 45 Mb proximal to the original position. This inserted region was confirmed by FISH analysis. With the help of precise mapping of the pathogenic variant, we successfully re-established STR haplotyping for PGT-M and avoided any potential misinterpretation of the pathogenic allele due to recombination., Conclusion: Long-read sequencing with adaptive sampling in a PGT-M pre-clinical workup is a beneficial method for identifying junctions of chromosomal complex structural rearrangements., (© 2023 John Wiley & Sons Ltd.)

Background: Clinical and genetic features were analyzed in five pedigrees with Pelizaeus-Merzbacher-like disease (PMLD) to provide bases for genetic counseling and prenatal diagnosis., Conclusions: Six patients from five pedigrees were diagnosed with PMLD based on their clinical data. Six GJC2 novel mutations were found in this study, expanding the spectrum of GJC2 mutations. This is the second group of GJC2 mutations reported from six Chinese patients with PMLD., Methods: Clinical data including medical history, physical signs, and auxiliary examinations were collected from six patients and their family numbers in five pedigrees with PMLD. Polymerase chain reaction and sequence analysis were used to amplify GJC2 and PLP1 alterations, while multiplex ligation-dependent probe amplification (MLPA) was performed to detect PLP1 dosage changes. The gene mutations were diagnosed for further analysis of the genetic features., Results: A total of seven GJC2 mutations were identified in these patients, including two novel missense mutations (c.217C>T, p.Pro73Ser; c.1199C>A, p.Ala400Glu), one nonsense mutation (c.735C>A, p.Cys245X), three novel frameshift mutations (c.579delC, p.Gly193fsX17 and c.1296_1297insG, p.Gly433fsX59; c.689delG, p.Gly230AlafsX241), and one known missense mutation (c.814T>G, p.Tyr272Asp). Compound heterozygotes were found for P1-3, while homozygotes were found for P4-6 that were inherited from their parents with normal phenotypes except for P5 and P6, respectively. The c.814T>G (p.Tyr272Asp) mutation in P5 was de novo. A c.1199C>A (p.Ala400Glu) homozygous mutation in GJC2 was identified in P6. A heterozygous variation was found in his father and the wild type was seen in his mother.

Pelizaeus-Merzbacher disease is a rare X-linked leukodystrophy accompanied by central nervous system hypomyelination with a spectrum of clinical phenotypes. This is the first survey of caregivers of individuals with Pelizaeus-Merzbacher disease to investigate the presenting symptoms, path to diagnosis, identity and impact of most bothersome symptoms, and needs that future treatment should address. One hundred participants completed the survey. Results from this survey demonstrate that the majority of Pelizaeus-Merzbacher disease symptoms manifest before 2 years of age and commonly include deficits in gross and fine motor skills, speech, and communication. Caregivers rated difficulty crawling, standing, or walking as the most bothersome symptoms due to Pelizaeus-Merzbacher disease, with constipation and difficulty with sleep, manual dexterity, and speech and communication rated nearly as high. The most important treatment goals for caregivers were improved mobility and communication. The survey findings present a caregiver perspective of the impact of symptoms in Pelizaeus-Merzbacher disease and provide helpful guidance to affected families, physicians, and drug developers on the often-long path to diagnosis and the unmet medical needs of this patient population.

Hypomyelinating leukodystrophies comprise a subclass of genetic disorders with deficient myelination of the CNS white matter. Here we report four unrelated families with a hypomyelinating leukodystrophy phenotype harbouring variants in TMEM163 (NM_030923.5). The initial clinical presentation resembled Pelizaeus-Merzbacher disease with congenital nystagmus, hypotonia, delayed global development and neuroimaging findings suggestive of significant and diffuse hypomyelination. Genomic testing identified three distinct heterozygous missense variants in TMEM163 with two unrelated individuals sharing the same de novo variant. TMEM163 is highly expressed in the CNS particularly in newly myelinating oligodendrocytes and was recently revealed to function as a zinc efflux transporter. All the variants identified lie in highly conserved residues in the cytoplasmic domain of the protein, and functional in vitro analysis of the mutant protein demonstrated significant impairment in the ability to efflux zinc out of the cell. Expression of the mutant proteins in an oligodendroglial cell line resulted in substantially reduced mRNA expression of key myelin genes, reduced branching and increased cell death. Our findings indicate that variants in TMEM163 cause a hypomyelinating leukodystrophy and uncover a novel role for zinc homeostasis in oligodendrocyte development and myelin formation., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Oligodendrocytes, the myelinating cells of the central nervous system, possess great potential for disease modeling and cell transplantation-based therapies for leukodystrophies. However, caveats to oligodendrocyte differentiation protocols ( Ehrlich et al., 2017; Wang et al., 2013; Douvaras and Fossati, 2015) from human embryonic stem and induced pluripotent stem cells (iPSCs), which include slow and inefficient differentiation, and tumorigenic potential of contaminating undifferentiated pluripotent cells, are major bottlenecks towards their translational utility. Here, we report the rapid generation of human oligodendrocytes by direct lineage conversion of human dermal fibroblasts (HDFs). We show that the combination of the four transcription factors OLIG2, SOX10, ASCL1 and NKX2.2 is sufficient to convert HDFs to induced oligodendrocyte precursor cells (iOPCs). iOPCs resemble human primary and iPSC-derived OPCs based on morphology and transcriptomic analysis. Importantly, iOPCs can differentiate into mature myelinating oligodendrocytes in vitro and in vivo. Finally, iOPCs derived from patients with Pelizaeus Merzbacher disease, a hypomyelinating leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene, showed increased cell death compared with iOPCs from healthy donors. Thus, human iOPCs generated by direct lineage conversion represent an attractive new source for human cell-based disease models and potentially myelinating cell grafts., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Leukodystrophies are a heterogenous group of genetic disorders, characterised by abnormal development of cerebral white matter. Pelizaeus-Merzbacher disease is caused by mutations in PLP1, encoding major myelin-resident protein required for myelin sheath assembly. We report a missense variant p.(Ala109Asp) in MAL as causative for a rare, hypomyelinating leukodystrophy similar to Pelizaeus-Merzbacher disease. MAL encodes a membrane proteolipid that directly interacts with PLP1, ensuring correct distribution during myelin assembly. In contrast to wild-type MAL, mutant MAL was retained in the endoplasmic reticulum but was released following treatment with 4-phenylbutyrate. Proximity-dependent identification of wild-type MAL interactants implicated post-Golgi vesicle-mediated protein transport and protein localisation to membranes, whereas mutant MAL interactants suggested unfolded protein responses. Our results suggest that mislocalisation of MAL affects PLP1 distribution, consistent with known pathomechanisms for hypomyelinating leukodystrophies., (© 2022. The Author(s).)

Pelizaeus-Merzbacher-like disease type 1 (PMLD1) is a hypomyelinating disorder arising in patients with mutations in GJC2, encoding Connexin47 (Cx47). PMLD1 causes nystagmus, cerebellar ataxia, spasticity and changes in CNS white matter detected by MRI. At least one mutation (p.I33M) yields a much milder phenotype, spastic paraplegia type 44 (SPG44). Cx47 contributes to gap junction communication channels between oligodendrocytes (OLs), the myelinating cells in the central nervous system (CNS), and between OLs and astrocytes. Prior studies in cell lines have shown that PMLD1 mutants such as p.P87S display defective protein trafficking, intracellular retention in the ER and loss-of-function. Here we show that when expressed in primary OLs, three PMLD1 associated mutants (p.P87S, p.Y269D and p.M283T) show ER retention of Cx47 and evidence of activation of the cellular stress (unfolded protein response, UPR) and apoptotic pathways. On the other hand, the milder SPG44 associated mutation p.I33M shows a wild-type-like subcellular distribution and no activation of the UPR or apoptotic pathways. These studies provide new insight into a potential element of toxic gain of function underlying the mechanism of PMLD1 that should help guide future therapeutic approaches., (Copyright © 2022. Published by Elsevier Inc.)

Background: The natural history and genotype-phenotype correlation of Pelizaeus-Merzbacher disease (PMD) of Chinese patients has been rarely reported., Method: Patients who met the criteria for PMD were enrolled in our study. Genomic analysis was conducted by multiplex ligation probe amplification (MLPA) and Sanger or whole-exome sequencing (WES). Natural history differences and genotype-phenotype correlations were analyzed., Result: A total of 111 patients were enrolled in our follow-up study. The median follow-up interval was 53 m (1185). Among PMD patients, developmental delay was the most common sign, and nystagmus and hypotonia were the most common initial symptoms observed. A total of 78.4% of the patients were able to control their head, and 72.1% could speak words. However, few of the patients could stand (9.0%) or walk (4.5%) by themselves. Nystagmus improved in more than half of the patients, and hypotonia sometimes deteriorated to movement disorders. More PLP1 point mutations patients were categorized into severe group, while more patients with PLP1 duplications were categorized into mild group (p < 0.001). Compared to patients in mild groups, those in the severe group had earlier disease onset and had acquired fewer skills at a later age., Conclusion: PMD patients have early disease onset with nystagmus and hypotonia followed by decreased nystagmus and movement disorders, such as spasticit. Patients with PLP1 duplication were more likely to be categorized into the mild group, whereas patients with point mutations were more likely to be categorized into the severe group., (© 2022. The Author(s).)

Among the hypomyelinating leukodystrophies, Pelizaeus-Merzbacher disease (PMD) is a representative disorder. The disease is caused by different types of PLP1 mutations, among which PLP1 duplication accounts for ∼70% of the mutations. Previous studies have shown that PLP1 duplications lead to PLP1 retention in the endoplasmic reticulum (ER); in parallel, recent studies have demonstrated that PLP1 duplication can also lead to mitochondrial dysfunction. As such, the respective roles and interactions of the ER and mitochondria in the pathogenesis of PLP1 duplication are not clear. In both PLP1 patients' and healthy fibroblasts, we measured mitochondrial respiration with a Seahorse XF Extracellular Analyzer and examined the interactions between the ER and mitochondria with super-resolution microscopy (spinning-disc pinhole-based structured illumination microscopy, SD-SIM). For the first time, we demonstrated that PLP1 duplication mutants had closer ER-mitochondrion interfaces mediated through structural and morphological changes in both the ER and mitochondria-associated membranes (MAMs). These changes in both the ER and mitochondria then led to mitochondrial dysfunction, as reported previously. This work highlights the roles of MAMs in bridging PLP1 expression in the ER and pathogenic dysfunction in mitochondria, providing novel insight into the pathogenicity of mitochondrial dysfunction resulting from PLP1 duplication. These findings suggest that interactions between the ER and mitochondria may underlie pathogenic mechanisms of hypomyelinating leukodystrophies diseases at the organelle level., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pelizaeus-Merzbacher-Like Disease 1 is a genetic disorder affecting the central nervous system with an autosomal recessive inheritance pattern. It is a rare genetic disorder that affects the central nervous system. In this report, we demonstrated the clinical and paraclinical features of an Iranian consanguine pedigree with suspected hypomyelinating leukodystrophy, without any defined diagnosis. The proband, a 15-month-old girl, visited the Razi pathobiology and medical genetic laboratory of Karaj, where the study was conducted in 2020. Following whole-exome sequencing analysis of the proband and segregation analysis, a novel pathogenic mutation was discovered. GJC2 (NM&#95;020435.4):c.1096dupG was found to be homozygous in the proband and heterozygous in both parents. This mutation was in the coding region of the protein, which results in D366Gfs*126 (p.Asp366GlyfsTer126). The site of mutation was at the 3' region of the connexin superfamily domain. The frameshift results in a different peptide sequence of the C-terminal and extended protein. Our findings led to the diagnosis of the proband's disease as Pelizaeus-Merzbacher-Like Disease 1 and led to the end of the diagnostic odyssey. We provided effective genetic counseling through the identification of a novel pathogenic mutation in gap junction protein C2 in this family and suggested preimplantation genetic diagnosis for the next pregnancy. Furthermore, our findings confirmed the association of GJC2 mutations with PMLD1. This discovery added to the repertoire of genetic mutations of Pelizaeus-Merzbacher-Like Disease 1. This knowledge could be applied for expanded carrier screening of other families, especially for Iranian consanguine marriages., (Copyright: © Iranian Journal of Medical Sciences.)

Monoallelic mutations on TMEM63A have been recently reported as cause of a previously unrecognized disorder named "infantile-onset transient hypomyelination". Clinical and neuroradiological presentation is described as highly similar to Pelizaeus-Merzbacher Disease but evolution over time was surprisingly benign with a progressive spontaneous improving course. We report on a new TMEM63A-mutated girl. The clinical picture was similar to the one already described except for the presence of recurrent episodes of unilateral eyelid twitching, and for the evidence of spinal cord involvement on MRI. These are interesting findings helping in distinguishing this condition from classic PMD since early disease stages. However, additional observations are needed to confirm if these are common features of this condition., (© 2021. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

The blood-brain barrier is a dynamic endothelial cell barrier in the brain microvasculature that separates the blood from the brain parenchyma. Specialized brain endothelial cells, astrocytes, neurons, microglia and pericytes together compose the neurovascular unit and interact to maintain blood-brain barrier function. A disturbed brain barrier function is reported in most common neurological disorders and may play a role in disease pathogenesis. However, a comprehensive overview of how the neurovascular unit is affected in a wide range of rare disorders is lacking. Our aim was to provide further insights into the neuropathology of the neurovascular unit in leukodystrophies to unravel its potential pathogenic role in these diseases. Leukodystrophies are monogenic disorders of the white matter due to defects in any of its structural components. Single leukodystrophies are exceedingly rare, and availability of human tissue is unique. Expression of selective neurovascular unit markers such as claudin-5, zona occludens 1, laminin, PDGFRβ, aquaporin-4 and α-dystroglycan was investigated in eight different leukodystrophies using immunohistochemistry. We observed tight junction rearrangements, indicative of endothelial dysfunction, in five out of eight assessed leukodystrophies of different origin and an altered aquaporin-4 distribution in all. Aquaporin-4 redistribution indicates a general astrocytic dysfunction in leukodystrophies, even in those not directly related to astrocytic pathology or without prominent reactive astrogliosis. These findings provide further evidence for dysfunction in the orchestration of the neurovascular unit in leukodystrophies and contribute to a better understanding of the underlying disease mechanism.

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2 -deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90β (Hsp90β) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90β-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.

Limited access to human oligodendrocytes impairs better understanding of oligodendrocyte pathology in myelin diseases. Here, we describe a method to robustly convert human fibroblasts directly into oligodendrocyte-like cells (dc-hiOLs), which allows evaluation of remyelination-promoting compounds and disease modeling. Ectopic expression of SOX10, OLIG2, and NKX6.2 in human fibroblasts results in rapid generation of O4 + cells, which further differentiate into MBP + mature oligodendrocyte-like cells within 16 days. dc-hiOLs undergo chromatin remodeling to express oligodendrocyte markers, ensheath axons, and nanofibers in vitro, respond to promyelination compound treatment, and recapitulate in vitro oligodendroglial pathologies associated with Pelizaeus-Merzbacher leukodystrophy related to PLP1 mutations. Furthermore, DNA methylome analysis provides evidence that the CpG methylation pattern significantly differs between dc-hiOLs derived from fibroblasts of young and old donors, indicating the maintenance of the source cells' "age." In summary, dc-hiOLs represent a reproducible technology that could contribute to personalized medicine in the field of myelin diseases., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in RNA polymerase III (Pol III) cause hypomeylinating leukodystrophy (HLD) and neurodegeneration in humans. POLR3A and POLR3B, the two largest Pol III subunits, together form the catalytic center and carry the majority of disease alleles. Disease-causing mutations include invariant and highly conserved residues that are predicted to negatively affect Pol III activity and decrease transcriptional output. A subset of HLD missense mutations in POLR3A cluster in the pore region that provides nucleotide access to the Pol III active site. These mutations were engineered at the corresponding positions in the Saccharomyces cerevisiae homolog, Rpc160, to evaluate their functional deficits. None of the mutations caused a growth or transcription phenotype in yeast. Each mutation was combined with a frequently occurring pore mutation, POLR3A G672E, which was also wild-type for growth and transcription. The double mutants showed a spectrum of phenotypes from wild-type to lethal, with only the least fit combinations showing an effect on Pol III transcription. In one slow-growing temperature-sensitive mutant the steady-state level of tRNAs was unaffected, however global tRNA synthesis was compromised, as was the synthesis of RPR1 and SNR52 RNAs. Affinity-purified mutant Pol III was broadly defective in both factor-independent and factor-dependent transcription in vitro across genes that represent the yeast Pol III transcriptome. Thus, the robustness of yeast Rpc160 to single Pol III leukodystrophy mutations in the pore domain can be overcome by a second mutation in the domain., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A family with a history of Pelizaeus-Merzbacher disease (PMD) received prenatal diagnosis of PLP1 gene duplication in a fetus using a single nucleotide polymorphism (SNP) array. A 27-year-old pregnant woman was referred for genetic counseling due to her four-year-old son being diagnosed with a suspected classic type of PMD. Amniocentesis was performed at 18 and 3/7 weeks of gestation, and the SNP array was carried out on DNA from the mother, her affected son, and fetus, then further confirmed by multiplex ligation-dependent probe amplification (MLPA). Cytogenetic analysis of the fetus showed 46,XY. SNP array analysis revealed that the male fetus did not carry PLP1 gene duplication but the affected boy did, and the mother was a carrier for the duplication of the PLP1 gene. All SNP array results were further confirmed by MLPA. SNP array and MLPA analyses of peripheral blood verified the nonduplication of the PLP1 gene in the infant after birth. At present, the child (without PLP1 duplication) is developing normally. This study preliminarily suggests that SNP array is a sensitive and accurate technology for identifying PLP1 duplication and is feasible for reliable diagnosis, including for the prenatal diagnosis of PMD resulting from PLP1 duplication.

Objective: To explore the genotypic and phenotypic characteristics of a child with congenital Pelizaeus-Merzbacher disease., Methods: Clinical, imaging and genetic characteristics of the child were retrospectively analyzed., Results: The patient manifested significantly reduced muscle tension, apparent tremor of eyeballs, and retardation of motor development after birth. Cranial MRI at 6-month-old showed no myelinization of brain white matter, though no pathogenic variant was detected by clinical exome sequencing. A copy number variation was found at chrX: 102 192 246-103 045 526. The duplication has spanned approximately 853 kb and was recorded in the Decipher database to be associated with Pelizaeus-Merzbacher disease. Quantitative PCR confirmed that the duplication has derived from his mother., Conclusion: Reduced muscle tension, nystagmus, poor motor development, and absence of myelinization of white matter should alert the diagnosis of congenital type Pelizaeus-Merzbacher disease. Both sequence variant and copy number variation should be searched upon genetic testing. A clear diagnosis is required for genetic counseling.

Autosomal dominant leukodystrophy (ADLD) is a fatal, progressive adult-onset disease characterized by widespread central nervous system (CNS) demyelination and significant morbidity. The late age of onset together with the relatively slow disease progression provides a large therapeutic window for the disorder. However, no treatment exists for ADLD, representing an urgent and unmet clinical need. We have previously shown that ADLD is caused by duplications of the lamin B1 gene causing increased expression of the lamin B1 protein, a major constituent of the nuclear lamina, and demonstrated that transgenic mice with oligodendrocyte-specific overexpression of lamin B1 exhibit temporal and histopathological features reminiscent of the human disease. As increased levels of lamin B1 are the causative event triggering ADLD, approaches aimed at reducing lamin B1 levels and associated functional consequences represent a promising strategy for discovery of small-molecule ADLD therapeutics. To this end, we have created an inducible cell culture model of lamin B1 overexpression and developed high-content analysis in connection with multivariate analysis to define, analyze, and quantify lamin B1 expression and its associated abnormal nuclear phenotype in mouse embryonic fibroblasts (MEFs). The assay has been optimized to meet high-throughput screening (HTS) criteria in multiday variability studies. To control for batch-to-batch variation in the primary MEFs, we have implemented a screening strategy that employs sentinel cells to avoid costly losses during HTS. We posit the assay will identify bona fide suppressors of lamin B1 pathophysiology as candidates for development into potential therapies for ADLD.

Mutations in PLP1, the gene that encodes proteolipid protein (PLP), result in failure of myelination and neurological dysfunction in the X-chromosome-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD) 1,2 . Most PLP1 mutations, including point mutations and supernumerary copy variants, lead to severe and fatal disease. Patients who lack PLP1 expression, and Plp1-null mice, can display comparatively mild phenotypes, suggesting that PLP1 suppression might provide a general therapeutic strategy for PMD 1,3-5 . Here we show, using CRISPR-Cas9 to suppress Plp1 expression in the jimpy (Plp1 jp ) point-mutation mouse model of severe PMD, increased myelination and restored nerve conduction velocity, motor function and lifespan of the mice to wild-type levels. To evaluate the translational potential of this strategy, we identified antisense oligonucleotides that stably decrease the levels of Plp1 mRNA and PLP protein throughout the neuraxis in vivo. Administration of a single dose of Plp1-targeting antisense oligonucleotides in postnatal jimpy mice fully restored oligodendrocyte numbers, increased myelination, improved motor performance, normalized respiratory function and extended lifespan up to an eight-month end point. These results suggest that PLP1 suppression could be developed as a treatment for PMD in humans. More broadly, we demonstrate that oligonucleotide-based therapeutic agents can be delivered to oligodendrocytes in vivo to modulate neurological function and lifespan, establishing a new pharmaceutical modality for myelin disorders.

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A-related leukodystrophy, Peroxisomal biogenesis disorders, POLR3-related Leukodystrophy, Vanishing White Matter, and Pelizaeus-Merzbacher Disease. Despite the relative frequency of these conditions, carrier-screening laboratories regularly test only 20 of the 55 leukodystrophy-related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening., (© 2020 Wiley Periodicals, Inc.)

We established a human induced pluripotent stem cells (hiPSC) line (MPIi006-A) from fibroblasts of a 20-year-old male Pelizaeus-Merzbacher disease (PMD) patient with a hemizygous 643C>T mutation in proteolipid protein 1 (PLP1) gene using a retroviral delivery of OCT4, SOX2, KLF4 and c-MYC. The MPIi006-A iPSC line carried the mutation, displayed typical iPSC morphology, expressed pluripotent stem cell makers, exhibited normal karyotype and were capable of differentiating into cells representative of three germ layers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Pelizaeus-Merzbacher disease is a fatal X-linked leukodystrophy caused by mutations in the PLP1 gene, which is expressed in the CNS by oligodendrocytes. Disease onset, symptoms and mortality span a broad spectrum depending on the nature of the mutation and thus the degree of CNS hypomyelination. In the absence of an effective treatment, direct cell transplantation into the CNS to restore myelin has been tested in animal models of severe forms of the disease with failure of developmental myelination, and more recently, in severely affected patients with early disease onset due to point mutations in the PLP1 gene, and absence of myelin by MRI. In patients with a PLP1 duplication mutation, the most common cause of Pelizaeus-Merzbacher disease, the pathology is poorly defined because of a paucity of autopsy material. To address this, we examined two elderly patients with duplication of PLP1 in whom the overall syndrome, including end-stage pathology, indicated a complex disease involving dysmyelination, demyelination and axonal degeneration. Using the corresponding Plp1 transgenic mouse model, we then tested the capacity of transplanted neural stem cells to restore myelin in the context of PLP overexpression. Although developmental myelination and axonal coverage by endogenous oligodendrocytes was extensive, as assessed using electron microscopy (n = 3 at each of four end points) and immunostaining (n = 3 at each of four end points), wild-type neural precursors, transplanted into the brains of the newborn mutants, were able to effectively compete and replace the defective myelin (n = 2 at each of four end points). These data demonstrate the potential of neural stem cell therapies to restore normal myelination and protect axons in patients with PLP1 gene duplication mutation and further, provide proof of principle for the benefits of stem cell transplantation for other fatal leukodystrophies with 'normal' developmental myelination., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Pelizaeus-Merzbacher disease (PMD) is a rare X-linked leukodystrophy caused by mutations in the proteolipid protein 1 gene (PLP1) which is specifically expressed on the myelin sheath of oligodendrocytes. We established an induced pluripotent stem cell (iPSC) line (ZJUi005-A) from peripheral blood mononuclear cells of an 18-year-old male PMD patient with a novel hemizygous c.437T>C mutation in PLP1 gene using episomal reprogramming plasmids. The ZJUi005-A iPSC line carried the PLP1 mutation, expressed pluripotency markers, exhibited normal karyotype and showed differentiation potential in vitro., Competing Interests: Declaration of Competing Interest All the authors declared there is no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Mechanically activated (MA) ion channels convert physical forces into electrical signals. Despite the importance of this function, the involvement of mechanosensitive ion channels in human disease is poorly understood. Here we report heterozygous missense mutations in the gene encoding the MA ion channel TMEM63A that result in an infantile disorder resembling a hypomyelinating leukodystrophy. Four unrelated individuals presented with congenital nystagmus, motor delay, and deficient myelination on serial scans in infancy, prompting the diagnosis of Pelizaeus-Merzbacher (like) disease. Genomic sequencing revealed that all four individuals carry heterozygous missense variants in the pore-forming domain of TMEM63A. These variants were confirmed to have arisen de novo in three of the four individuals. While the physiological role of TMEM63A is incompletely understood, it is highly expressed in oligodendrocytes and it has recently been shown to be a MA ion channel. Using patch clamp electrophysiology, we demonstrated that each of the modeled variants result in strongly attenuated stretch-activated currents when expressed in naive cells. Unexpectedly, the clinical evolution of all four individuals has been surprisingly favorable, with substantial improvements in neurological signs and developmental progression. In the three individuals with follow-up scans after 4 years of age, the myelin deficit had almost completely resolved. Our results suggest a previously unappreciated role for mechanosensitive ion channels in myelin development., (Copyright © 2019 American Society of Human Genetics. All rights reserved.)

Pelizaeus-Merzbacher disease (PMD) is an X-linked leukodystrophy caused by mutations in Proteolipid Protein 1 (PLP1), encoding a major myelin protein, resulting in profound developmental delay and early lethality. Previous work showed involvement of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways, but poor PLP1 genotype-phenotype associations suggest additional pathogenetic mechanisms. Using induced pluripotent stem cell (iPSC) and gene-correction, we show that patient-derived oligodendrocytes can develop to the pre-myelinating stage, but subsequently undergo cell death. Mutant oligodendrocytes demonstrated key hallmarks of ferroptosis including lipid peroxidation, abnormal iron metabolism, and hypersensitivity to free iron. Iron chelation rescued mutant oligodendrocyte apoptosis, survival, and differentiationin vitro, and post-transplantation in vivo. Finally, systemic treatment of Plp1 mutant Jimpy mice with deferiprone, a small molecule iron chelator, reduced oligodendrocyte apoptosis and enabled myelin formation. Thus, oligodendrocyte iron-induced cell death and myelination is rescued by iron chelation in PMD pre-clinical models., (Copyright © 2019. Published by Elsevier Inc.)

Inherited white matter disorders of the central nervous system frequently are degenerative and progressive clinical entities. They are classified into myelin disorders, including hypomyelination, dysmyelination, demyelination, and myelin vacuolization, but also astrocytopathies, leuko-axonopathies, microgliopathies, and leuko-vasculopathies. Hypomyelinating leukodystrophy is the main feature of Pelizaeus-Merzbacher disease (PMD) and Pelizaeus-Merzbacher-like disease (PMLD1). PMD- and PMLD1-affected patients display comparable neurological symptoms, including psychomotor developmental delay, spasticity, nystagmus, impairment of cognitive skills, sensorineural hearing loss, and different ophthalmological disabilities. While clinical features overlap, PMD and PMLD1 can be distinguished on the molecular genetic level. PMD is caused by mutations in the gene encoding for the proteolipid protein 1 (PLP1), whereas PMLD1 is associated with mutations in the gene encoding for the gap junction protein gamma 2 (GJC2). Here we present novel compound-heterozygous mutations in the GJC2 gene identified in two, unrelated infantile patients affected with PMLD1. The heterozygous frameshift mutations c.392dupC, p.H132Afs*6 and c.989delC, p.P330Rfs*141 were found in the first patient. The heterozygous nonsense variant c.291C>G, p.Y97*, as well as the heterozygous missense variant c.716T>C, p.V239A were detected in the second patient. All four variants were predicted to be damaging for structure and/or function of the GJC2 protein. Combinations of these genetic variants likely are pathogenic and resulted in the PMLD1-phenotype in the investigated children. In conclusion, our clinical and molecular findings confirmed the genotype-phenotype relationship between mutations in the GJC2 and PMLD1. The novel mutations of GJC2 described herein will help to further understand the pathogenic mechanism underlying PMLD1.

Competing Interests: The authors declare no conflicts of interest.

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive disorder caused by abnormalities in the gene PLP1. Most females harboring heterozygous PLP1 abnormalities are basically asymptomatic. However, as a result of abnormal patterns of X-chromosome inactivation, it is possible for some female carriers to be symptomatic. Whole-exome sequencing of a female patient with unknown spastic paraplegia was performed to obtain a molecular diagnosis. As a result, a de novo heterozygous single-nucleotide deletion in PLP1 [NM&#95;000533.5(PLP1&#95;v001):c.783del; p.Thr262Leufs*20] was identified. RNA sequencing was performed in a patient-derived lymphoblastoid cell line, confirming mono-allelic expression of the mutated allele and abnormal inactivation of the wild-type allele. The patient-derived lymphoblastoid cell line was then treated with VX680 or 5azadC, which resulted in restored expression of the wild-type allele. These two agents thus have the potential to reverse inappropriately-skewed inactivation of the X-chromosome.

Copy number increase or decrease of certain dosage-sensitive genes may cause genetic diseases with distinct phenotypes, conceptually termed genomic disorders. The most common cause of Pelizaeus-Merzbacher disease (PMD), an X-linked hypomyelinating leukodystrophy, is genomic duplication encompassing the entire proteolipid protein 1 (PLP1) gene. Although the exact molecular and cellular mechanisms underlying PLP1 duplication, which causes severe hypomyelination in the central nervous system, remain largely elusive, PLP1 overexpression is likely the fundamental cause of this devastating disease. Here, we investigated if adeno-associated virus-mediated (AAV-mediated) gene-specific suppression may serve as a potential cure for PMD by correcting quantitative aberrations in gene products. We developed an oligodendrocyte-specific Plp1 gene suppression therapy using artificial microRNA under the control of human CNP promoter in a self-complementary AAV (scAAV) platform. A single direct brain injection achieved widespread oligodendrocyte-specific Plp1 suppression in the white matter of WT mice. AAV treatment in Plp1-transgenic mice, a PLP1 duplication model, ameliorated cytoplasmic accumulation of Plp1, preserved mature oligodendrocytes from degradation, restored myelin structure and gene expression, and improved survival and neurological phenotypes. Together, our results provide evidence that AAV-mediated gene suppression therapy can serve as a potential cure for PMD resulting from PLP1 duplication and possibly for other genomic disorders.

Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) is a non-catalytic component of the multi-tRNA synthetase complex which catalyzes the ligation of amino acids to the correct tRNAs. Pathogenic variants in several aminoacyl-tRNA synthetases genes have been linked to various neurological disorders, including leukodystrophies and pontocerebellar hypoplasias (PCH). To date, loss-of-function variants in AIMP1 have been associated with hypomyelinating leukodystrophy-3 (MIM 260600). Here, we report a novel frameshift AIMP1 homozygous variant (c.160delA,p.Lys54Asnfs) in a child with pontocerebellar hypoplasia and simplified gyral pattern, a phenotype not been previously described with AIMP1 variants, thus expanding the phenotypic spectrum. AIMP1 should be included in diagnostic PCH gene panels.

The term "cerebral palsy mimic" is used to describe a number of neurogenetic disorders that may present with motor symptoms in early childhood, resulting in a misdiagnosis of cerebral palsy. Cerebral palsy describes a heterogeneous group of neurodevelopmental disorders characterized by onset in infancy or early childhood of motor symptoms (including hypotonia, spasticity, dystonia, and chorea), often accompanied by developmental delay. The primary etiology of a cerebral palsy syndrome should always be identified if possible. This is particularly important in the case of genetic or metabolic disorders that have specific disease-modifying treatment. In this article, we discuss clinical features that should alert the clinician to the possibility of a cerebral palsy mimic, provide a practical framework for selecting and interpreting neuroimaging, biochemical, and genetic investigations, and highlight selected conditions that may present with predominant spasticity, dystonia/chorea, and ataxia. Making a precise diagnosis of a genetic disorder has important implications for treatment, and for advising the family regarding prognosis and genetic counseling. © 2019 International Parkinson and Movement Disorder Society., (© 2019 International Parkinson and Movement Disorder Society.)

Pelizaeus-Merzbacher disease (PMD) represents a group of disorders known as hypomyelinating leukodystrophies, which are characterized by abnormal development and maintenance of myelin in the central nervous system. PMD is caused by different types of mutations in the proteolipid protein 1 (PLP1) gene, which encodes a major myelin membrane lipoprotein. These mutations in the PLP1 gene result in distinct cellular and molecular pathologies and a spectrum of clinical phenotypes. In this chapter, I discuss the historical aspects and current understanding of the mechanisms underlying how different PLP1 mutations disrupt the normal process of myelination and result in PMD and other disorders.

Pelizaeus Merzbacher Disease is a rare X-linked central nervous system disease involving the proteolipid protein 1 gene. Patients exhibit signs for instance nystagmus, hypotonia, ataxia. We report a three-year-old female patient with chief compliant of developmental delay. On physical examination, patient was alert but had poor eye contact while sitting in a stroller. Since no chromosomal evaluation was performed, a chromosomal microarray testing was performed. Review of geneticist report indicated that patient carries a deletion of at least 2.26 Mb within cytogenetic band Xq22.1 to Xq22.2 which is known to contain 39 genes. Out of the 39 genes, proteolipid protein 1 is associated with known clinical disorder; Pelizaeus Merzbacher Disease. Our case highlights the second only known female with Pelizaeus Merzbacher Disease due to deletions of proteolipid protein 1 gene. For a patient with developmental delay, the importance of performing genetic testing and/or radiological imaging early on is strongly recommended. Keywords: deletion; female; Genetic testing; Pelizaeus Merzbacher Disease; Proteolipid Protein 1.

Pelizaeus-Merzbacher disease (PMD) is a fatal X-linked disorder caused by loss of myelinating oligodendrocytes and consequent hypomyelination. The underlying cellular and molecular dysfunctions are not fully defined, but therapeutic enhancement of oligodendrocyte survival could restore functional myelination in patients. Here we generated pure, scalable quantities of induced pluripotent stem cell-derived oligodendrocyte progenitor cells (OPCs) from a severe mouse model of PMD, Plp1 jimpy . Temporal phenotypic and transcriptomic studies defined an early pathological window characterized by endoplasmic reticulum (ER) stress and cell death as OPCs exit their progenitor state. High-throughput phenotypic screening identified a compound, Ro 25-6981, which modulates the ER stress response and rescues mutant oligodendrocyte survival in jimpy, in vitro and in vivo, and in human PMD oligocortical spheroids. Surprisingly, increasing oligodendrocyte survival did not restore subsequent myelination, revealing a second pathological phase. Collectively, our work shows that PMD oligodendrocyte loss can be rescued pharmacologically and defines a need for multifactorial intervention to restore myelination., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Case Presentation: Pelizaeus Merzbacher Disease (PMD) is an X-linked developmental defect of myelination that causes childhood chronic spastic encephalopathy. Its genetic etiology can be either a duplication (or other gene dosage alterations) or a punctual mutation at the PLP1 locus. Clinically, it presents with developmental delay, nystagmus and, spasticity, supported by neuroimaging in which the defect of myelination is evident. We present a series of seven Colombian patients diagnosed with this leucodystrophy, describing their genotypic and phenotypic characteristics and heterogeneity., Clinical Findings: All patients included were male, 6 months to 16 years of age. Mean age at onset of symptoms was 8 months. Mean age at diagnosis was 5 years 5 months, being classic PMD most frequently diagnosed, as compared to the connatal phenotype. All cases had a primary diagnosis of developmental delay on 100%, and in 28.7% of cases, early onset nystagmus was described. 85.7% of patients had spasticity, 71.4% cerebellar signs, 57.0% hypotonia, and 28.5% had an abnormal movement disorder. Only three patients were able to achieve gait, though altered. In the two patients who had a diagnosis of connatal PMD maturational ages in danger zones according to the WHO Abbreviated Scale of Psychosocial Development were documented. All cases had abnormalities in neuroimages., Molecular Analysis and Results: Molecular studies were used in the majority of the cases to confirm the diagnosis (85.7 %). For two cases molecular confirmation was not considered necessary given their affected male brothers had already been tested. PLP1 gene dosage alterations (duplications) were found in 28.5 % of the patients (two siblings), whereas three different single nucleotide variants were detected., Clinical Relevance: According to these findings, as authors we propose the diagnostic algorithm in Colombian population to begin on a high clinical suspicion, followed by paraclinical extension, moving on to the molecular confirmation by using approaches to simultaneously sequence the PLP1 gene in order to detect point mutations and in/dels and performing a copy number variation analysis for the detection of gene dosage alterations., Competing Interests: Conflict of interests The authors declare that they have no competing interests

Background: Epilepsy regroups a common and diverse set of chronic neurological disorders that are characterized by spontaneous, unprovoked, and recurrent epileptic seizures. Epilepsies have a highly heterogeneous background with a strong genetic contribution and various mode of inheritance. X-linked epilepsy usually manifests as part of a syndrome or epileptic encephalopathy. The variability of clinical manifestations of X-linked epilepsy may be attributed to several factors including the causal genetic mutation, making diagnosis, genetic counseling and treatment decisions difficult. We report the description of a Moroccan family referred to our genetic department with X-linked epileptic seizures as the only initial diagnosis., Case Presentation: Knowing the new contribution of Next-Generation Sequencing (NGS) for clinical investigation, and given the heterogeneity of this group of disorders we performed a Whole-Exome Sequencing (WES) analysis and co-segregation study in several members of this large family. We detected a novel pathogenic PLP1 missense mutation c.251C > A (p.Ala84Asp) allowing us to make a diagnosis of Pelizaeus-Merzbacher Disease for this family., Conclusion: This report extends the spectrum of PLP1 mutations and highlights the diagnostic utility of NGS to investigate this group of heterogeneous disorders.

Pelizaeus-Merzbacher disease (PMD) is an X-linked disorder caused by mutations in the PLP1 gene, which encodes the proteolipid protein of myelinating oligodendroglia. PMD exhibits phenotypic variability that reflects its considerable genotypic heterogeneity, but all forms of the disease result in central hypomyelination associated with early neurologic dysfunction, progressive deterioration, and ultimately death. PMD has been classified into three major subtypes, according to the age of presentation: connatal PMD, classic PMD, and transitional PMD, combining features of both connatal and classic forms. Two other less severe phenotypes were subsequently described, including the spastic paraplegia syndrome and PLP1-null disease. These disorders may be associated with duplications, as well as with point, missense, and null mutations within the PLP1 gene. A number of clinically similar Pelizaeus-Merzbacher-like disorders (PMLD) are considered in the differential diagnosis of PMD, the most prominent of which is PMLD-1, caused by misexpression of the GJC2 gene encoding connexin-47. No effective therapy for PMD exists. Yet, as a relatively pure central nervous system hypomyelinating disorder, with limited involvement of the peripheral nervous system and little attendant neuronal pathology, PMD is an attractive therapeutic target for neural stem cell and glial progenitor cell transplantation, efforts at which are now underway in a number of centers internationally., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Pelizaeus-Merzbacher disease (PMD) is a rare Mendelian disorder characterised by central nervous system hypomyelination. PMD typically manifests in infancy or early childhood and is caused by mutations in proteolipid protein-1 (PLP1). However, variants in several other genes including gap junction protein gamma 2 (GJC2) can also cause a similar phenotype and are referred to PMD-like disease (PMLD). Whole-exome sequencing in two siblings presenting with clinical symptoms of PMD revealed a homozygous variant in the arginyl-tRNA synthetase (RARS) gene: NM&#95;002887.3: c.[5A>G] p.(Asp2Gly). Subsequent screening of a PMD cohort without a genetic diagnosis identified an unrelated individual with novel compound heterozygous variants including a missense variant c.[1367C>T] p.(Ser456Leu) and a de novo deletion c.[1846&#95;1847delTA] p.(Tyr616Leufs*6). Protein levels of RARS and the multi-tRNA synthetase complex into which it assembles were found to be significantly reduced by 80 and 90% by western blotting and Blue native-PAGE respectively using patient fibroblast extracts. As RARS is involved in protein synthesis whereby it attaches arginine to its cognate tRNA, patient cells were studied to determine their ability to proliferate with limiting amounts of this essential amino acid. Patient fibroblasts cultured in medium with limited arginine at 30 °C and 40 °C, showed a significant decrease in fibroblast proliferation (P<0.001) compared to control cells, suggestive of inefficiency of protein synthesis in the patient cells. Our functional studies provide further evidence that RARS is a PMD-causing gene.

Hypomyelinating leukodystrophies are a group of neurodevelopmental disorders that affect proper formation of the myelin sheath in the central nervous system. They are characterized by developmental delay, hypotonia, spasticity, and variable intellectual disability. We used whole exome analysis to study the molecular basis of hypomyelinating leukodystrophy in two sibs from a consanguineous family. A homozygous mutation, c.3068+5G>A, was identified in the ATRN gene, with the consequent insertion of an intronic sequence into the patients' cDNA and a predicted premature termination of the ATRN polypeptide. ATRN encodes Attractin, which was previously shown to play a critical role in central myelination. Several spontaneous ATRN rodent mutants exhibited impaired myelination which was attributed to oxidative stress and accelerated apoptosis. ATRN can now be added to the growing list of genes associated with hypomyelinating leukodystrophy. The disease seems to be confined to the CNS; however, given the young age of our patients, longer follow-up may be required.

Genomic disorders are the clinical conditions manifested by submicroscopic genomic rearrangements including copy number variants (CNVs). The CNVs can be identified by array-based comparative genomic hybridization (aCGH), the most commonly used technology for molecular diagnostics of genomic disorders. However, clinical aCGH only informs CNVs in the probe-interrogated regions. Neither orientational information nor the resulting genomic rearrangement structure is provided, which is a key to uncovering mutational and pathogenic mechanisms underlying genomic disorders. Long-range polymerase chain reaction (PCR) is a traditional approach to obtain CNV breakpoint junction, but this method is inefficient when challenged by structural complexity such as often found at the PLP1 locus in association with Pelizaeus-Merzbacher disease (PMD). Here we introduced 'capture and single-molecule real-time sequencing' (cap-SMRT-seq) and newly developed 'asymmetry linker-mediated nested PCR walking' (ALN-walking) for CNV breakpoint sequencing in 49 subjects with PMD-associated CNVs. Remarkably, 29 (94%) of the 31 CNV breakpoint junctions unobtainable by conventional long-range PCR were resolved by cap-SMRT-seq and ALN-walking. Notably, unexpected CNV complexities, including inter-chromosomal rearrangements that cannot be resolved by aCGH, were revealed by efficient breakpoint sequencing. These sequence-based structures of PMD-associated CNVs further support the role of DNA replicative mechanisms in CNV mutagenesis, and facilitate genotype-phenotype correlation studies. Intriguingly, the lengths of gained segments by CNVs are strongly correlated with clinical severity in PMD, potentially reflecting the functional contribution of other dosage-sensitive genes besides PLP1. Our study provides new efficient experimental approaches (especially ALN-walking) for CNV breakpoint sequencing and highlights their importance in uncovering CNV mutagenesis and pathogenesis in genomic disorders., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)