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SummaryThe controlled release of promoter-proximal paused RNA polymerase II (Pol II) into productive elongation is a major step in gene regulation. However, functional analysis of Pol II pausing is difficult because factors that regulate pause release are almost all essential. In this study, we identified heterozygous loss-of-function mutations inSUPT5H, which encodes SPT5, in individuals with β-thalassemia unlinked toHBBmutations. During erythropoiesis in healthy human cells, cell cycle genes were highly paused at the transition from progenitors to precursors. When the pathogenic mutations were recapitulated bySUPT5Hediting, Pol II pause release was globally disrupted, and the transition from progenitors to precursors was delayed, marked by a transient lag in erythroid-specific gene expression and cell cycle kinetics. Despite this delay, cells terminally differentiate, and cell cycle phase distributions normalize. Therefore, hindering pause release perturbs proliferation and differentiation dynamics at a key transition during erythropoiesis, revealing a role for Pol II pausing in the temporal coordination between the cell cycle and differentiation.

Mitochondria play critical roles in cellular metabolism, primarily by serving as the site of assembly and function of the oxidative phosphorylation (OXPHOS) machinery. The OXPHOS proteins are encoded by mitochondrial DNA (mtDNA) and nuclear DNA, which reside and are regulated within separate compartments. To unravel how the two gene expression systems collaborate to produce the OXPHOS complexes, the regulatory principles controlling the production of mtDNA-encoded proteins need to be elucidated. In this study, we performed a quantitative analysis of the mitochondrial messenger RNA (mt-mRNA) life cycle to determine which steps of gene expression experience strong regulatory control. Our analysis revealed that the high accumulation of mt-mRNA despite their rapid turnover was made possible by a 700-fold higher transcriptional output than nuclear-encoded OXPHOS genes. In addition, we observed that mt-mRNA processing and its association with the mitochondrial ribosome occur rapidly and that these processes are linked mechanistically. Based on these data, we developed a model of mtDNA expression that is predictive across human cell lines, revealing that differences in turnover and translation efficiency are the major contributors to mitochondrial-encoded protein synthesis. Applying this framework to a disease model of Leigh syndrome, French-Canadian type, we found that the disease-associated nuclear-encoded gene,LRPPRC, acts predominantly by stabilizing mt-mRNA. Our findings provide a comprehensive view of the intricate regulatory mechanisms governing mtDNA-encoded protein synthesis, highlighting the importance of quantitatively analyzing the mitochondrial RNA life cycle in order to decode the regulatory principles of mtDNA expression.

Mitochondrial oxidative phosphorylation (OXPHOS) complexes are assembled from proteins encoded by both nuclear and mitochondrial DNA. These dual-origin enzymes pose a complex gene regulatory challenge for cells, in which gene expression must be coordinated across organelles using distinct pools of ribosomes. How cells produce and maintain the accurate subunit stoichiometries for these OXPHOS complexes remains largely unknown. To identify genes involved in dual-origin protein complex synthesis, we performed FACS-based genome-wide screens analyzing mutant cells with unbalanced levels of mitochondrial- and nuclear-encoded subunits of cytochromecoxidase (Complex IV). We identified novel genes involved in OXPHOS biogenesis, including two uncharacterized genes:PREPLandNME6. We found that PREPL specifically regulates Complex IV biogenesis by interacting with mitochondrial protein synthesis machinery, while NME6, an uncharacterized nucleoside diphosphate kinase (NDPK), controls OXPHOS complex biogenesis through multiple mechanisms reliant on its NDPK domain. First, NME6 maintains local mitochondrial pyrimidine triphosphate levels essential for mitochondrial RNA abundance. Second, through stabilizing interactions with RCC1L, NME6 modulates the activity of mitoribosome regulatory complexes, leading to disruptions in mitoribosome assembly and mitochondrial RNA pseudouridylation. Taken together, we propose that NME6 acts as a link between compartmentalized mitochondrial metabolites and mitochondrial gene expression. Finally, we present these screens as a resource, providing a catalog of genes involved in mitonuclear gene regulation and OXPHOS biogenesis.

The late-onset cerebellar ataxias (LOCAs) have largely resisted molecular diagnosis.We sequenced the genomes of six persons with autosomal dominant LOCA who were members of three French Canadian families and identified a candidate pathogenic repeat expansion. We then tested for association between the repeat expansion and disease in two independent case-control series - one French Canadian (66 patients and 209 controls) and the other German (228 patients and 199 controls). We also genotyped the repeat in 20 Australian and 31 Indian index patients. We assayed gene and protein expression in two postmortem cerebellum specimens and two induced pluripotent stem-cell (iPSC)-derived motor-neuron cell lines.In the six French Canadian patients, we identified a GAA repeat expansion deep in the first intron ofA dominantly inherited deep intronic GAA repeat expansion in

Dissecting the myriad regulatory mechanisms controlling eukaryotic transcripts from production to degradation requires quantitative measurements of mRNA flow across the cell. We developed subcellular TimeLapse-seq to measure the rates at which RNAs are released from chromatin, exported from the nucleus, loaded onto polysomes, and degraded within the nucleus and cytoplasm. These rates varied substantially, yet transcripts from genes with related functions or targeted by the same transcription factors and RNA binding proteins flowed across subcellular compartments with similar kinetics. Verifying these associations uncovered roles for DDX3X and PABPC4 in nuclear export. For hundreds of genes, most transcripts were degraded within the nucleus, while the remaining molecules were exported and persisted with stable lifespans. Transcripts residing on chromatin for longer had extended poly(A) tails, whereas the reverse was observed for cytoplasmic mRNAs. Finally, a machine learning model identified additional molecular features that underlie the diverse life cycles of mammalian mRNAs.

Combinatorially, intron excision within a given nascent transcript could proceed down any of thousands of paths, each of which would expose different dynamic landscapes of cis-elements and contribute to alternative splicing. In this study, we found that post-transcriptional multi-intron splicing order in human cells is largely predetermined, with most genes spliced in one or a few predominant orders. Strikingly, these orders were conserved across cell types and stages of motor neuron differentiation. Introns flanking alternatively spliced exons were frequently excised last, after their neighboring introns. Perturbations to the spliceosomal U2 snRNA altered the preferred splicing order of many genes, and these alterations were associated with the retention of other introns in the same transcript. In one gene, early removal of specific introns was sufficient to induce delayed excision of three proximal introns, and this delay was caused by two distinct cis-regulatory mechanisms. Together, our results demonstrate that multi-intron splicing order in human cells is predetermined, is influenced by a component of the spliceosome, and ensures splicing fidelity across long pre-mRNAs.

During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3’-end cleavage, and polyadenylation. Here, we describe nanopore analysis of CO-transcriptional Processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing (NET-seq), which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3’ ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read–based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 days.

The late-onset cerebellar ataxias (LOCAs) have largely resisted molecular diagnosis.We sequenced the genomes of six persons with autosomal dominant LOCA who were members of three French Canadian families and identified a candidate pathogenic repeat expansion. We then tested for association between the repeat expansion and disease in two independent case-control series - one French Canadian (66 patients and 209 controls) and the other German (228 patients and 199 controls). We also genotyped the repeat in 20 Australian and 31 Indian index patients. We assayed gene and protein expression in two postmortem cerebellum specimens and two induced pluripotent stem-cell (iPSC)-derived motor-neuron cell lines.In the six French Canadian patients, we identified a GAA repeat expansion deep in the first intron of FGF14, which encodes fibroblast growth factor 14. Cosegregation of the repeat expansion with disease in the families supported a pathogenic threshold of at least 250 GAA repeats ([GAA]≥250). There was significant association between FGF14 (GAA)≥250 expansions and LOCA in the French Canadian series (odds ratio, 105.60; 95% confidence interval [CI], 31.09 to 334.20; P

Objective. The aim of the paper is to propose an all-in-one method based on magnetic resonance-supersonic shear wave imaging (MR-SSI) and proton resonance frequency shift (PRFS) to monitor high intensity focused ultrasound (HIFU) thermal ablations. Approach. Mechanical properties have been shown to be related to tissue damage induced by thermal ablations. Monitoring elasticity in addition to temperature changes may help in ensuring the efficacy and the accuracy of HIFU therapies. For this purpose, an MR-SSI method has been developed where the ultrasonic transducer is used for both mechanical wave generation and thermal ablation. Transient quasi-planar shear waves are generated using the acoustic radiation force, and their propagation is monitored in motion-sensitized phase MR images. Using a single-shot gradient-echo echo-planar-imaging sequence, MR images can be acquired at a sufficiently high temporal resolution to provide an update of PRFS thermometry and MR-SSI elastography maps in real time. Main results. The proposed method was first validated on a calibrated elasticity phantom, in which both the possibility to detect inclusions with different stiffness and repeatability were demonstrated. The standard deviation between the 8 performed measurements was 2% on the background of the phantom and 11%, at most, on the inclusions. A second experiment consisted in performing a HIFU heating in a gelatin phantom. The temperature increase was estimated to be 9 °C and the shear modulus was found to decrease from 2.9 to 1.8 kPa, reflecting the gel softening around the HIFU focus, whereas it remained steady in non-heated areas. Significance. The proposed MR-SSI technique allows monitoring HIFU ablations using thermometry and elastography simultaneously, without the need for an additional external mechanical exciter such as those used in MR elastography.

Recessive mutations in the ubiquitously expressed POLR3A and POLR3B genes are the most common cause of POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), a rare childhood-onset disorder characterized by deficient cerebral myelin formation and cerebellar atrophy. POLR3A and POLR3B encode the two catalytic subunits of RNA Polymerase III (Pol III), which synthesizes numerous small non-coding RNAs. We recently reported that mice homozygous for the Polr3a mutation c.2015G > A (p.Gly672Glu) have no neurological abnormalities and thus do not recapitulate the human POLR3-HLD phenotype. To determine if other POLR3-HLD mutations can cause a leukodystrophy phenotype in mouse, we characterized mice carrying the Polr3b mutation c.308G > A (p.Arg103His). Surprisingly, homozygosity for this mutation was embryonically lethal with only wild-type and heterozygous animals detected at embryonic day 9.5. Using proteomics in a human cell line, we found that the POLR3B R103H mutation severely impairs assembly of the Pol III complex. We next generated Polr3aG672E/G672E/Polr3b+/R103Hdouble mutant mice but observed that this additional mutation was insufficient to elicit a neurological or transcriptional phenotype. Taken together with our previous study on Polr3a G672E mice, our results indicate that missense mutations in Polr3a and Polr3b can variably impair mouse development and Pol III function. Developing a proper model of POLR3-HLD is crucial to gain insights into the pathophysiological mechanisms involved in this devastating neurodegenerative disease. Electronic supplementary material The online version of this article (10.1186/s13041-019-0479-7) contains supplementary material, which is available to authorized users.

RNA polymerase III (Pol III) is an essential enzyme responsible for the synthesis of several small noncoding RNAs, a number of which are involved in mRNA translation. Recessive mutations in POLR3A, encoding the largest subunit of Pol III, cause POLR3-related hypomyelinating leukodystrophy (POLR3–HLD), characterized by deficient central nervous system myelination. Identification of the downstream effectors of pathogenic POLR3A mutations has so far been elusive. Here, we used CRISPR-Cas9 to introduce the POLR3A mutation c.2554A→G (p.M852V) into human cell lines and assessed its impact on Pol III biogenesis, nuclear import, DNA occupancy, transcription, and protein levels. Transcriptomic profiling uncovered a subset of transcripts vulnerable to Pol III hypofunction, including a global reduction in tRNA levels. The brain cytoplasmic BC200 RNA (BCYRN1), involved in translation regulation, was consistently affected in all our cellular models, including patient-derived fibroblasts. Genomic BC200 deletion in an oligodendroglial cell line led to major transcriptomic and proteomic changes, having a larger impact than those of POLR3A mutations. Upon differentiation, mRNA levels of the MBP gene, encoding myelin basic protein, were significantly decreased in POLR3A-mutant cells. Our findings provide the first evidence for impaired Pol III transcription in cellular models of POLR3–HLD and identify several candidate effectors, including BC200 RNA, having a potential role in oligodendrocyte biology and involvement in the disease.

Background Cerebellar atrophy is a nonspecific imaging finding observed in a number of neurological disorders. Genetic ataxias associated with cerebellar atrophy are a heterogeneous group of conditions, rendering the approach to diagnosis challenging. Objectives To define the spectrum of genetic ataxias associated with cerebellar atrophy in a Canadian cohort and the diagnostic yield of exome sequencing for this group of conditions. Methods A total of 92 participants from 66 families with cerebellar atrophy were recruited for this multicenter prospective cohort study. Exome sequencing was performed for all participants between 2011 and 2017 as part of 1 of 2 national research programs, Finding of Rare Genetic Disease Genes or Enhanced Care for Rare Genetic Diseases in Canada. Results A genetic diagnosis was established in 53% of families (35/66). Pathogenic variants were found in 21 known genes, providing a diagnosis for 31/35 families (89%), and in 4 novel genes, accounting for 4/35 families (11%). Of the families, 31/66 (47%) remained without a genetic diagnosis. The most common diagnoses were channelopathies, which were established in 9/35 families (26%). Additional clinical findings provided useful clues to specific diagnoses. Conclusions We report on the high frequency of channelopathies as a cause of genetic ataxias associated with cerebellar atrophy and the utility of exome sequencing for this group of conditions.

Pseudouridylation is a common post‐transcriptional modification in RNA, but its functional consequences at the cellular level remain largely unknown. Using a proximity‐biotinylation assay, we identified a protein module in mitochondrial RNA granules, platforms for post‐transcriptional RNA modification and ribosome assembly, containing several proteins of unknown function including three uncharacterized pseudouridine synthases, TRUB2, RPUSD3, and RPUSD4. TRUB2 and RPUSD4 were previously identified as core essential genes in CRISPR/Cas9 screens. Depletion of the individual enzymes produced specific mitochondrial protein synthesis and oxidative phosphorylation assembly defects without affecting mitochondrial mRNA levels. Investigation of the molecular targets in mitochondrial RNA by pseudouridine‐Seq showed that RPUSD4 plays a role in the pseudouridylation of a single residue in the 16S rRNA, a modification that is essential for its stability and assembly into the mitochondrial ribosome, while TRUB2/RPUSD3 were similarly involved in pseudouridylating specific residues in mitochondrial mRNAs. These results establish essential roles for epitranscriptomic modification of mitochondrial RNA in mitochondrial protein synthesis, oxidative phosphorylation, and cell survival.

Understanding how splicing events are coordinated across numerous introns in metazoan RNA transcripts requires quantitative analyses of transient RNA processing events in living cells. We developed nanopore analysis of co-transcriptional processing (nano-COP), in which nascent RNAs are directly sequenced through nanopores, exposing the dynamics and patterns of RNA splicing without biases introduced by amplification. Long nano-COP reads reveal that, in human and Drosophila cells, splicing occurs after RNA polymerase II transcribes several kilobases of pre-mRNA, suggesting that metazoan splicing transpires distally from the transcription machinery. Inhibition of the branch-site recognition complex SF3B rapidly diminished global co-transcriptional splicing. We found that splicing order does not strictly follow the order of transcription and is associated with cis-acting elements, alternative splicing, and RNA-binding factors. Further, neighboring introns in human cells tend to be spliced concurrently, implying that splicing of these introns occurs cooperatively. Thus, nano-COP unveils the organizational complexity of RNA processing.

Human genes have numerous exons that are differentially spliced within pre-mRNA. Understanding how multiple splicing events are coordinated across nascent transcripts requires quantitative analyses of transient RNA processing events in living cells. We developed nanopore analysis of CO-transcriptional Processing (nano-COP), in which nascent RNAs are directly sequenced through nanopores, exposing the dynamics and patterns of RNA splicing without biases introduced by amplification. nano-COP showed that in both human andDrosophilacells, co-transcriptional splicing occurs after RNA polymerase II transcribes several kilobases of pre-mRNA, suggesting that metazoan splicing transpires distally from the transcription machinery. Inhibition of the branch-site recognition complex SF3B globally abolished co-transcriptional splicing in both species. Our findings revealed that splicing order does not strictly follow the order of transcription and is influenced by cis-regulatory elements. In human cells, introns with delayed splicing frequently neighbor alternative exons and are associated with RNA-binding factors. Moreover, neighboring introns in human cells tend to be spliced concurrently, implying that splicing occurs cooperatively. Thus, nano-COP unveils the organizational complexity of metazoan RNA processing.

Hereditary cerebellar ataxias and hereditary spastic paraplegias are clinically and genetically heterogeneous and often overlapping neurological disorders. Mutations in SPG7 cause the autosomal recessive spastic paraplegia type 7 (SPG7), but recent studies indicate that they are also one of the most common causes of recessive cerebellar ataxia. In Quebec, a significant number of patients affected with cerebellar ataxia and spasticity remain without a molecular diagnosis. We performed whole-exome sequencing in three French Canadian (FC) patients affected with spastic ataxia and uncovered compound heterozygous variants in SPG7 in all three. Sanger sequencing of SPG7 exons and exon/intron boundaries was used to screen additional patients. In total, we identified recessive variants in SPG7 in 22 FC patients belonging to 12 families (38.7% of the families screened), including two novel variants. The p.(Ala510Val) variant was the most common in our cohort. Cerebellar features, including ataxia, were more pronounced than spasticity in this cohort. These results strongly suggest that variants affecting the function of SPG7 are the fourth most common form of recessive ataxia in FC patients. Thus, we propose that SPG7 mutations explain a significant proportion of FC spastic ataxia cases and that this gene should be considered in unresolved patients.

SummaryPlatelet derived growth factor receptor α (PDGFRα) signaling is required for proliferation, commitment and maintenance of oligodendrocyte (OL) precursor cells (OPCs). PDGFRα signaling promotes OPC homeostasis and its attenuation signals OPC differentiation and maturation triggering the onset of myelination of the central nervous system (CNS). The initial steps of how PDGFRα signaling is attenuated are still poorly understood. Herein we show that decreased Protein Arginine MethylTransferase5 (PRMT5) expression, as occurs during OPC differentiation, is involved in the down-regulation of PDGFRα by modulating its cell surface bioavailability leading to its degradation in a Cbldependent manner. Mechanistically, loss of arginine methylation at R554 of the PDGFRα intracellular domain reveals a masked Cbl binding site at Y555. Physiologically, depletion of PRMT5 in OPCs results in severe CNS myelination defects. We propose that decreased PRMT5 activity initiates PDGFRα degradation to promote OL differentiation. More broadly, inhibition of PRMT5 may be used therapeutically to manipulate PDGFRα bioavailability.

Mandatory predonation reading materials inform donors about risk factors for transmissible disease, possible complications of donation and changes to the donation process. We aimed to assess the attention to predonation reading materials and factors which may affect attention.A national survey in 2008 of 18,108 blood donors asked about self-assessed attention to reading the materials. In face-to-face interviews, 441 donors completed additional questions about reading the materials and a literacy test. Qualitative interviews of 27 donors assessed their approach to reading.In the national survey, most of the first-time donors said they read all or most of the materials (90.9% first-time vs. 57.6% repeat donors, P0.001) and 66% vs. 23.1% reported reading them carefully (P0.001). In face-to-face interviews comparing those who read materials carefully, skimmed or did not read, most knew that donors are informed of positive transmissible disease test results (97.1%, 95.5, 98.0 P0.05), but fewer recalled seeing the definition of sex (77.2%, 56.9, 24.2 P0.001). Literacy was poor (30.5% frustration level, 60.3% instructional, 9.2% independent) but similar when those who read materials carefully, skimmed or did not read were compared (P0.05). Qualitative interviews showed that donors are reluctant to read any more than necessary and decide based on perceived importance or relevance.Attention to predonation reading materials tends to be better among first-time donors. The effectiveness is limited by low motivation to read, especially for repeat donors, as well as poor literacy.

Episodic ataxias (EAs) are a heterogeneous group of neurological disorders characterized by recurrent attacks of ataxia. Mutations in KCNA1 and CACNA1A account for the majority of EA cases worldwide. We recruited a two-generation family affected with EA of unknown subtype and performed whole-exome sequencing on two affected members. This revealed a novel heterozygous mutation c.211_212insA (p.I71NfsX27) leading to a premature stop codon in FGF14. Mutations in FGF14 are known to cause spinocerebellar ataxia type 27 (SCA27). Sanger sequencing confirmed segregation within the family. Our findings expand the phenotypic spectrum of SCA27 by underlining the possible episodic nature of this ataxia.

The qkI gene encodes a family of RNA binding proteins alternatively spliced at its 3′ end, giving rise to three major spliced isoforms: QKI-5, QKI-6 and QKI-7. Their expression is tightly regulated during brain development with nuclear QKI-5 being the most abundant during embryogenesis followed by QKI-6 and QKI-7 that peak during myelination. Previously, we generated a mouse conditional qkI allele where exon 2 is excised using Olig2-Cre resulting in QKI-deficient oligodendrocytes (OLs). These mice have dysmyelination and die at the third post-natal week. Herein, we performed a transcriptomic analysis of P14 mouse brains of QKI-proficient (QKI FL/FL;- ) and QKI-deficient (QKI FL/FL;Olig2-Cre ) OLs. QKI deficiency results in major global changes of gene expression and RNA processing with >1,800 differentially expressed genes with the top categories being axon ensheathment and myelination. Specific downregulated genes included major myelin proteins, suggesting that the QKI proteins are key regulators of RNA metabolism in OLs. We also identify 810 alternatively spliced genes including known QKI targets, MBP and Nfasc. Interestingly, we observe in QKI FL/FL;Olig2-Cre a switch in exon 2-deficient qkI mRNAs favoring the expression of the qkI-5 rather than the qkI-6 and qkI-7. These findings define QKI as regulators of alternative splicing in OLs including self-splicing.

Congenital myopathies define a heterogeneous group of neuromuscular diseases with neonatal or childhood hypotonia and muscle weakness. The genetic cause is still unknown in many patients, precluding genetic counselling and better understanding of the physiopathology. To identify novel genetic causes of congenital myopathies, exome sequencing was performed in three consanguineous families. We identified two homozygous frameshift mutations and a homozygous nonsense mutation in the mitogen-activated protein triple kinase ZAK. In total, six affected patients carry these mutations. Reverse transcription polymerase chain reaction and transcriptome analyses suggested nonsense mRNA decay as a main impact of mutations. The patients demonstrated a generalized slowly progressive muscle weakness accompanied by decreased vital capacities. A combination of proximal contractures with distal joint hyperlaxity is a distinct feature in one family. The low endurance and compound muscle action potential amplitude were strongly ameliorated on treatment with anticholinesterase inhibitor in another patient. Common histopathological features encompassed fibre size variation, predominance of type 1 fibre and centralized nuclei. A peculiar subsarcolemmal accumulation of mitochondria pointing towards the centre of the fibre was a novel histological hallmark in one family. These findings will improve the molecular diagnosis of congenital myopathies and implicate the mitogen-activated protein kinase (MAPK) signalling as a novel pathway altered in these rare myopathies.

Recessive mutations in the ubiquitously expressed POLR3A gene cause one of the most frequent forms of childhood-onset hypomyelinating leukodystrophy (HLD): POLR3-HLD. POLR3A encodes the largest subunit of RNA Polymerase III (Pol III), which is responsible for the transcription of transfer RNAs (tRNAs) and a large array of other small non-coding RNAs. In order to study the central nervous system pathophysiology of the disease, we introduced the French Canadian founder Polr3a mutation c.2015G > A (p.G672E) in mice, generating homozygous knock-in (KI/KI) as well as compound heterozygous mice for one Polr3a KI and one null allele (KI/KO). Both KI/KI and KI/KO mice are viable and are able to reproduce. To establish if they manifest a motor phenotype, WT, KI/KI and KI/KO mice were submitted to a battery of behavioral tests over one year. The KI/KI and KI/KO mice have overall normal balance, muscle strength and general locomotion. Cerebral and cerebellar Luxol Fast Blue staining and measurement of levels of myelin proteins showed no significant differences between the three groups, suggesting that myelination is not overtly impaired in Polr3a KI/KI and KI/KO mice. Finally, expression levels of several Pol III transcripts in the brain showed no statistically significant differences. We conclude that the first transgenic mice with a leukodystrophy-causing Polr3a mutation do not recapitulate the childhood-onset HLD observed in the majority of human patients with POLR3A mutations, and provide essential information to guide selection of Polr3a mutations for developing future mouse models of the disease. Electronic supplementary material The online version of this article (doi:10.1186/s13041-017-0294-y) contains supplementary material, which is available to authorized users.

Mutations in POLR3A encoding the largest subunit of RNA polymerase III (Pol III) were found to be responsible for the majority of cases presenting with three clinically overlapping hypomyelinating leukodystrophy phenotypes. We uncovered in three cases without POLR3A mutation recessive mutations in POLR3B, which codes for the second largest subunit of Pol III. Mutations in genes coding for Pol III subunits are a major cause of childhood-onset hypomyelinating leukodystrophies with prominent cerebellar dysfunction, oligodontia, and hypogonadotropic hypogonadism.

The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information. CLaP uses laser illumination to crosslink biotin onto the plasma membrane, coupled with streptavidin conjugates to label individual cells for genomic, cell-tracking, flow cytometry or ultra-microscopy applications. We show that the incorporated mark is stable, non-toxic, retained for several days, and transferred by cell division but not to adjacent cells in culture. To demonstrate the potential of CLaP for genomic applications, we combine CLaP with microfluidics-based single-cell capture followed by transcriptome-wide next-generation sequencing. Finally, we show that CLaP can also be exploited for inducing transient cell adhesion to substrates for microengineering cultures with spatially patterned cell types., Cell labelling in a non-invasive and genetic engineering-free manner is crucial to cell biology applications. Here the authors develop cell labelling via photobleaching (CLaP), that uses laser illumination to label individual cells for genomics, cell-tracking, flow cytometry or ultra-microscopy.

Mutations in GALC cause Krabbe disease. This autosomal recessive leukodystrophy generally presents in early infancy as a severe disorder, but sometimes manifests as a milder adult-onset disease with spastic paraplegia as the main symptom. We recruited a family with five affected individuals presenting with adult-onset predominant cerebellar ataxia with mild spasticity. Whole exome sequencing (WES) revealed one novel and one previously reported compound heterozygous variants in GALC. Magnetic resonance imaging (MRI) confirmed the presence of typical Krabbe features. Our findings expand the phenotypic spectrum of adult-onset Krabbe disease and demonstrate the usefulness of combining WES and pattern-specific MRI for the diagnosis of neurodegenerative diseases.

A small proportion of 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) or RNA polymerase III (POLR3)-related leukodystrophy cases are negative for mutations in the previously identified causative genes POLR3A and POLR3B. Here we report eight of these cases carrying recessive mutations in POLR1C, a gene encoding a shared POLR1 and POLR3 subunit, also mutated in some Treacher Collins syndrome (TCS) cases. Using shotgun proteomics and ChIP sequencing, we demonstrate that leukodystrophy-causative mutations, but not TCS mutations, in POLR1C impair assembly and nuclear import of POLR3, but not POLR1, leading to decreased binding to POLR3 target genes. This study is the first to show that distinct mutations in a gene coding for a shared subunit of two RNA polymerases lead to selective modification of the enzymes' availability leading to two different clinical conditions and to shed some light on the pathophysiological mechanism of one of the most common hypomyelinating leukodystrophies, POLR3-related leukodystrophy., Some RNA polymerase (POLR) 3-related leukodystrophy cases do not have the causal mutations in POLR3A and POLR3B. Here, by exome sequencing, the authors identify recessive mutations in POLR1C, a gene encoding a shared POLR1 and POLR3 subunit, impairing assembly and nuclear import of POLR3, but not POLR1.

Travel to malaria risk areas such as Mexico is a common source of donor deferral in Canada. On February 21st, 2011 the deferrable regions in Mexico were revised to permit donation if donors travelled to the state of Quintana Roo, Mexico, a popular ocean-side resort area.Canadian travel data and malaria deferral rates since 2007 were plotted to examine trends. Deferral records in one centre were accessed from January to April, 2011 to tabulate travel destinations of deferred donors immediately before and after the change.Travel to Mexico and the Caribbean accounts for 63% of general population travel, and travel to Mexico has been increasing (P0.05). Deferral for short-term malaria risk travel has a strong seasonal trend with peaks in the winter and troughs in the summer. Approximately 36,000 fewer donations were lost following the change, a reduction of 37% from the previous year. Deferrals in one centre increased for Caribbean/Central America after the change (P0.05) consistent with the seasonal trend, but decreased for Mexico (P0.05).Deferrals for malaria risk travel are substantial. Careful revision and refinement of risk areas of travel can significantly reduce the burden of deferral.

Objective To report a novel clinical and genetic presentation of a patient with 4H syndrome, which is a recently described leukodystrophy syndrome characterized by ataxia, hypomyelination, hypodontia, and hypogonadotropic hypogonadism. Design Case report. Setting University teaching hospital. Patient A 20-year-old male patient with 4H syndrome. Results The patient was found to have delayed tooth eruption and a late-onset growth hormone deficiency without overt growth failure. He was a compound heterozygote for the novel missense mutations R1005H and A1331T of POLR3A, which codes for the largest subunit of RNA polymerase III. Conclusion This is the first report of this type of leukodystrophy from southeastern Europe, which suggests that POLR3A mutations should be suspected in patients with hypomyelination and various central nervous system–based endocrine abnormalities.

Sir, Recently, Jobling et al. (2015) reported the identification of mutations in PMPCA in 17 patients from four families affected with autosomal recessive non-progressive cerebellar ataxia. A homozygous missense mutation in PMPCA (c.1129G>A, p.Ala377Thr) was uncovered in three families of Christian Lebanese Maronite origin, while compound heterozygous mutations (c.287C>T, p.Ser96Leu; c.1543G>A, p.Gly515Arg) were identified in a French patient (Jobling et al. , 2015). PMPCA encodes the alpha subunit of the mitochondrial processing peptidase (MPP), which cleaves the targeting peptide of nuclear-encoded mitochondrial precursor proteins upon their import into mitochondria (Teixeira and Glaser, 2013). Jobling et al. (2015) showed compelling evidence for the causality of PMPCA variants. First, they demonstrated co-segregation of the variants with the disease among the four families, including a large consanguineous family of 32 individuals. Moreover, they observed decreased levels of MPPα in lymphoblasts from two affected members compared to heterozygote carriers and healthy controls. Interestingly, they also showed impaired processing of frataxin (FXN), a mitochondrial protein that is a known substrate of MPP (Cavadini et al. , 2000; Schmucker et al. , 2008). The paper by Jobling et al. (2015) is the first to associate defects in PMPCA with a human disease, which suggests a new mechanism for the pathogenesis of non-progressive cerebellar ataxias. We wish to complement this study with our own identification of two brothers affected by a juvenile-onset recessive cerebellar ataxia caused by a homozygous mutation in PMPCA . These two patients were born of French Canadian parents who are distantly related (Fig. 1A). Considerable clinical variability was present between the two affected cases. Patient II.2 started developing impaired gait, dysarthria, dysmetria and mild distal atrophy during adolescence. He was diagnosed with a slowly progressive spinocerebellar ataxia. He did not have intellectual deficiency but had learning difficulties in school. Patient II.3 …

Objective: Search for mutations in the second largest subunit of RNA polymerase III (Pol III), POLR3B , in case negative for mutation in POLR3A . Background Leukodystrophies are a heterogeneous group of neurodegenerative disorders characterized by abnormal central nervous system white matter. It is estimated that at least 30% to 40% of patients with leukodystrophies remain without a precise diagnosis, despite extensive investigations. We recently demonstrated that mutations in POLR3A, encoding the largest subunit of RNA polymerase III (Pol III), were responsible for the majority of cases presenting with three clinically overlapping hypomyelinating leukodystrophy phenotypes. Design/Methods: We sequenced all exons, exon-intron boundaries and 39 and 59 UTR of POLR3B (NM_018082, hg19) on available genomic DNA from four 4H cases not found previously to carry POLR3A mutations. Results: We uncovered in four cases without POLR3A mutation, recessive mutations in POLR3B which codes for the second largest subunit of Pol III. All four cases were found to be compound heterozygote for mutations in POLR3B , with one missense mutation being common to all individuals. Conclusions: Mutations in genes coding for Pol III subunits are a major cause of childhood-onset hypomyelinating leukodystrophies typically characterized by prominent cerebellar dysfunction, oligodontia and hypogonadotropic hypogonadism. Supported by: Dr G Bernard received fellowship scholarships from the RMGA (Reseau de Medecine Genetique Appliquee) and FRSQ (Fonds de Recherche en Sante du Quebec). M. Tetreault received the Frederick Banting and Charles Best Doctoral scholarship from the CIHR (Canadian Institute of Health Research). This work was supported by Fondation sur les Leucodystrophies and the European Leukodystrophy Association (ELA). Disclosure: Dr. Tetreault has nothing to disclose. Dr. Choquet has nothing to disclose. Dr. Orcesi has nothing to disclose. Dr. Tonduti has nothing to disclose. Dr. Ballotin has nothing to disclose. Dr. Teichmann has nothing to disclose. Dr. Fribourg has nothing to disclose. Dr. Schiffmann has received personal compensation for activities with Amicus Therapeutics and Shire Human Genetic Therapies. Dr. Schiffmann has received research support from Shire Human Genetic Therapies, Amicus Therapeutics, and Genzyme Corporation. Dr. Brais has nothing to disclose. Dr. Vanderver has nothing to disclose. Dr. Bernard has received personal compensation for activities with Actelion Pharmaceuticals Canada Inc, Santhera, Venture in Research as a participant on advisory boards and research teams.