Recessive mutations in POLR1C cause a leukodystrophy by impairing biogenesis of RNA polymerase III

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

Biallelic mutations in valyl-tRNA synthetase gene VARS are associated with a progressive neurodevelopmental epileptic encephalopathy.

Aminoacyl-tRNA synthetases (ARSs) function to transfer amino acids to cognate tRNA molecules, which are required for protein translation. To date, biallelic mutations in 31 ARS genes are known to cause recessive, early-onset severe multi-organ diseases. VARS encodes the only known valine cytoplasmic-localized aminoacyl-tRNA synthetase. Here, we report seven patients from five unrelated families with five different biallelic missense variants in VARS. Subjects present with a range of global developmental delay, epileptic encephalopathy and primary or progressive microcephaly. Longitudinal assessment demonstrates progressive cortical atrophy and white matter volume loss. Variants map to the VARS tRNA binding domain and adjacent to the anticodon domain, and disrupt highly conserved residues. Patient primary cells show intact VARS protein but reduced enzymatic activity, suggesting partial loss of function. The implication of VARS in pediatric neurodegeneration broadens the spectrum of human diseases due to mutations in tRNA synthetase genes

Solving inherited white matter disorder etiologies in the neurology clinic: Challenges and lessons learned using next-generation sequencing

IntroductionRare neurodevelopmental disorders, including inherited white matter disorders or leukodystrophies, often present a diagnostic challenge on a genetic level given the large number of causal genes associated with a range of disease subtypes. This study aims to demonstrate the challenges and lessons learned in the genetic investigations of leukodystrophies through presentation of a series of cases solved using exome or genome sequencing.MethodsEach of the six patients had a leukodystrophy associated with hypomyelination or delayed myelination on MRI, and inconclusive clinical diagnostic genetic testing results. We performed next generation sequencing (case-based exome or genome sequencing) to further investigate the genetic cause of disease.ResultsFollowing different lines of investigation, molecular diagnoses were obtained for each case, with patients harboring pathogenic variants in a range of genes including TMEM106B, GJA1, AGA, POLR3A, and TUBB4A. We describe the lessons learned in reaching the genetic diagnosis, including the importance of (a) utilizing proper multi-gene panels in clinical testing, (b) assessing the reliability of biochemical assays in supporting diagnoses, and (c) understanding the limitations of exome sequencing methods in regard to CNV detection and region coverage in GC-rich areas.DiscussionThis study illustrates the importance of applying a collaborative diagnostic approach by combining detailed phenotyping data and metabolic results from the clinical environment with advanced next generation sequencing analysis techniques from the research environment to increase the diagnostic yield in patients with genetically unresolved leukodystrophies

L'hypométhylation de la région promotrice du gène Transmembrane Protease Serine 3 D variant (TMPRSS3-D) est la cause potentielle de sa surexpression dans le cancer ovarien épithélial

La base moléculaire de la progression du cancer ovarien epithelial (COE) est encore peu comprise. Récemment, l'importance de la perturbation épigénétique de la régulation de gènes par l'hypométhylation globale du génome a commencé à être plus appréciée. L'hypométhylation est très peu étudiée dans le COE bien que l'on ait déjà montré qu'il existe une relation entre hypométhylation de l'ADN et la progression, l'invasion tumorale ainsi que la formation de métastases. Cette étude vise à identifier des gènes qui pourraient être activés par l'hypométhylation de leur région promotrice, ce qui contribuerait à la progression tumorale. Afin d'identifier de nouveaux gènes hypométhylés, quatre lignées cellulaires ovariennes (Skov3, Tovll2, Tov21 et Ovcar3) ont été traitées avec un donneur de groupement méthyl, du S-Adenosylmethionine (AdoMet), afin d'inhiber la déméthylation de l'ADN. Plusieurs gènes sous-exprimés après traitement ont été identifiés par l'analyse de l'expression génique globale en utilisant la technique de micropuces à ADN. Cette analyse a permis d'identifier le gène TMPRSS3 comme une cible potentielle d'hypométhylation dans le COE. Le gène TMPRSS3-D est connu pour son implication dans la migration et invasion dans le COE. Le statut de méthylation de la région promotrice du gène TMPRSS3 a été vérifié par analyse Methylation-Spécific PCR (MSP) et par Bisulfite Specific PCR (BSP). L'évaluation de son rôle potentiel dans l'étiologie du COE a été faite par la technique d'interférence à l'ARN. L'analyse d'expression génique globale en utilisant le clone où l'expression de TMPRSS3-D a été supprimée et son contrôle correspondant a permis de mieux comprendre l'effet moléculaire de TMPRSS3-D. Pris ensemble, les résultats présentés dans ce mémoire confirment le rôle potentiel du gène TMPRSS3-D dans l'invasion et métastase du COE et plus important, nos données indiquent que la surexpression de TMPRSS3-D dans le COE est due aux mécanismes épigénétiques reliés à l'hypométhylation de sa région promotrice

Dystonia in RNA Polymerase III-Related Leukodystrophy

Objectives: To identify the prevalence of dystonia in a RNA Polymerase III (POLR3)-related leukodystrophy patient cohort and to further characterize their dystonic features. Background: POLR3-related leukodystrophy is a hypomyelinating leukodystrophy characterized by neurological and non-neurological features. Dystonia remains a challenging and under-recognized feature. Methods: A retrospective chart review was performed in a cohort of 20 patients for whom videos of a standardized neurological examination were available. Patients were recruited at the Montreal Children's Hospital of the McGill University Health Center and the Myelin Disorders Bioregistry Project. Families were consented at the initial assessment and the following data was recorded: age and symptoms at clinical presentation, investigations, causal gene and mutation(s), type and severity of dystonia, and treatment response when needed. Standardized examination videos were reviewed by three independent reviewers and scored using the Global Dystonia Scale. Results: 10 males and 10 females were included in this study; 12/20 had POLR3A mutations, while 8/20 had POLR3B mutations; 19/20 patients had documented dystonia, with 3/19 requiring therapy. There was a good response in two patients to a single agent, and a poor response in one patient to three agents; the majority had mild-to-moderate multifocal dystonia without a functional impact. Conclusions: Dystonia is a common, yet underdiagnosed, slowly progressive manifestation of POLR3-related leukodystrophy, and in most cases has limited-to-no functional impact. When treatment is needed, good response to typically used medication may occur. Further studies are needed to assess evolution of dystonia over time, patients’ functional outcome, and response to therapy (when needed)

Large exonic deletions in POLR3B gene cause POLR3-related leukodystrophy

POLR3-related (or 4H) leukodystrophy is an autosomal recessive disorder caused by mutations in POLR3A or POLR3B and is characterized by neurological and non-neurological features. In a small proportion of patients, no mutation in either gene or only one mutation is found. Analysis of the POLR3B cDNA revealed a large deletion of exons 21–22 in one case and of exons 26–27 in another case. These are the first reports of long deletions causing POLR3-related leukodystrophy, suggesting that deletions and duplications in POLR3A or POLR3B should be investigated in patients with a compatible phenotype, especially if one pathogenic variant has been identified

Table_1_Biallelic pathogenic variants in POLR3D alter tRNA transcription and cause a hypomyelinating leukodystrophy: A case report.xlsx

RNA polymerase III-related leukodystrophy (POLR3-related leukodystrophy) is a rare, genetically determined hypomyelinating disease arising from biallelic pathogenic variants in genes encoding subunits of RNA polymerase III (Pol III). Here, we describe the first reported case of POLR3-related leukodystrophy caused by biallelic pathogenic variants in POLR3D, encoding the RPC4 subunit of Pol III. The individual, a female, demonstrated delays in walking and expressive and receptive language as a child and later cognitively plateaued. Additional neurological features included cerebellar signs (e.g., dysarthria, ataxia, and intention tremor) and dysphagia, while non-neurological features included hypodontia, hypogonadotropic hypogonadism, and dysmorphic facial features. Her MRI was notable for diffuse hypomyelination with myelin preservation of early myelinating structures, characteristic of POLR3-related leukodystrophy. Exome sequencing revealed the biallelic variants in POLR3D, a missense variant (c.541C > T, p.P181S) and an intronic splice site variant (c.656-6G > A, p.?). Functional studies of the patient’s fibroblasts demonstrated significantly decreased RNA-level expression of POLR3D, along with reduced expression of other Pol III subunit genes. Notably, Pol III transcription was also shown to be aberrant, with a significant decrease in 7SK RNA and several distinct tRNA genes analyzed. Affinity purification coupled to mass spectrometry of the POLR3D p.P181S variant showed normal assembly of Pol III subunits yet altered interaction of Pol III with the PAQosome chaperone complex, indicating the missense variant is likely to alter complex maturation. This work identifies biallelic pathogenic variants in POLR3D as a novel genetic cause of POLR3-related leukodystrophy, expanding the molecular spectrum associated with this disease, and proposes altered tRNA homeostasis as a factor in the underlying biology of this hypomyelinating disorder.</p