POLR3B Gene Research Articles

POLR3B de novo variants are a rare cause of infantile myoclonic epilepsy

PurposeTo report on a new phenotype in a patient carrying a novel, undescribed de novo variant in POLR3B, affected by generalized myoclonic epilepsy and neurodevelopmental disorder, without neuropathy. It is known that biallelic pathogenic variants in POLR3B cause hypomyelinating leukodystrophy-8, and heterozygous de novo variants are described in association to a phenotype characterized by predominantly demyelinating sensory-motor peripheral neuropathy, ataxia, spasticity, intellectual disability and epilepsy, in which the peripheral neuropathy is often the main clinical presentation. MethodsWe collected clinical, electrophysiological and neuroimaging data from the affected subject and performed a Trio-Clinical Exome Sequencing. ResultsWe detected a de novo novel heterozygous missense variant c.1132A>G in POLR3B (NM_018082.6) that was considered as likely pathogenic following ACMG criteria. We also consulted our custom genomic database of a total of 1485 patients that were genetically analysed from 2018 for epilepsy, and found no other de novo variants in the POLR3B gene. ConclusionWe hypothesize a possible genotype-phenotype correlation, particularly regarding epilepsy. We also provide a review of the literature about the previously described POLR3B heterozygous patients, with particular attention to the epileptic phenotype, underlining the association between POLR3B and early onset myoclonic epilepsy, which can represent the main manifestation of the disease at its onset.

Uncertain significance mutation in the POLR3B gene in a Syrian boy with leukodystrophy: a case report.

A 4-year-old boy presented in the neurologic clinic with delayed psychomotor development and progressive neurologic symptoms that started from the age of 20 months. Physical examination revealed ataxic features and a global development delay. The MRI was significant for hypomyelination. The most common causes of leukodystrophy were rolled out. He was referred to an inherited metabolic disease specialist under suspect of inborn metabolic errors because of laboratory analysis, which showed elevated levels of lactic acid, pyruvate, 4-Hydroxy-Phenylactic acid, 3-Hydroxy propionic acid, and decreased levels of PCO2, HCO3, total CO2, 25-Hydroxyvitamin D. These results were unspecific and mitochondrial disease was highly suspected. However, the genetic study was requested to get a defined diagnosis and treatment; the whole exon sequencing result showed a homozygous variant of uncertain significance mutation; related to an amino acid change from Ile to Thr at position 1002 in the POLR3B gene, which helped us to reveal the final diagnosis, and the genetic counseling were recommended for the next pregnancies. POLR3-related Leukodystrophy is a very rare disease. The early diagnosis should be raised depending on clinical history and MRI findings after other conditions were rolled out, and the confirmed diagnosis depends on the genetic study.

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

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

Intellectual disability associated with craniofacial dysmorphism due to POLR3B mutation and defect in spliceosomal machinery

BackgroundIntellectual disability (ID) is a clinically important disease and a most prevalent neurodevelopmental disorder. The etiology and pathogenesis of ID are poorly recognized. Exome sequencing revealed a homozygous missense mutation in the POLR3B gene in a consanguineous family with three Intellectual disability with craniofacial anomalies patients. POLR3B gene encoding the second largest subunit of RNA polymerase III.MethodsWe performed RNA sequencing on blood samples to obtain insights into the biological pathways influenced by POLR3B mutation. We applied the results of our RNA-Seq analysis to several gene ontology programs such as ToppGene, Enrichr, KEGG.ResultsA significant decrease in expression of several spliceosomal RNAs, ribosomal proteins, and transcription factors was detected in the affected, compared to unaffected, family members.ConclusionsWe hypothesize that POLR3B mutation dysregulates the expression of some important transcription factors, ribosomal and spliceosomal genes, and impairments in protein synthesis and splicing mediated in part by transcription factors such as FOXC2 and GATA1 contribute to impaired neuronal function and concurrence of intellectual disability and craniofacial anomalies in our patients. Our study highlights the emerging role of the spliceosome and ribosomal proteins in intellectual disability.

The First Korean Siblings With Adult-Onset 4H Leukodystrophy Related to Nonsynonymous POLR3B Mutations.

Objectives4H leukodystrophy is a rare autosomal recessive hypomyelinating disorder characterized by several combinations of motor dysfunction, abnormal dentition, and ophthalmic and endocrine abnormalities. To date, only a single Korean case report of pediatric leukodystrophy caused by the POLR1C sequence variation has been published, while there are no reports on the POLR3B, POLR3A, or POLR3K variants.MethodsGenetic tests of Korean sibling pairs with primary amenorrhea due to normosmic isolated hypogonadotropic hypogonadism and cognitive or behavioral symptoms were performed by whole-exome sequencing (WES). The WES results were validated by direct Sanger sequencing.ResultsWe identified biallelic variations in the POLR3B gene of p.Tyr685* and p.Tyr746Cys, which have not been associated with 4H leukodystrophy. Both sequence variants lie in the hybrid-binding domain of the protein RPC2. The protein structure analysis predicted that cysteine substitution of the phylogenetically conserved amino acid tyrosine can cause destabilization.DiscussionThe siblings reported are the first POLR3B-related hypomyelinating leukodystrophy cases in Korea. Our report expands the mutational spectrum of 4H leukodystrophy and suggests that it is mandatory to consider its diagnostic possibility in adult patients presenting with primary amenorrhea and mild cognitive or behavioral symptoms.

The leukodystrophy mutation Polr3b R103H causes homozygote mouse embryonic lethality and impairs RNA polymerase III biogenesis

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.

POLR3B-associated leukodystrophy: clinical, neuroimaging and molecular-genetic analyses in four patients: clinical heterogeneity and novel mutations in POLR3B gene.

White matter disorders represent a spectrum of neurological diseases frequently associated with an unfavourable prognosis and a delay in diagnostics. We report the broad phenotypic spectrum of a rare hypomyelinating leukodystrophy and three novel mutations. Further, we aim to explore the role of the combined clinical and neuroimaging diagnostic approach in the era of whole exome sequencing. We present a clinical, neuroimaging and molecular-genetic characterisation of four patients from three families suffering from a rare genetic leukoencephalopathy. Two severely affected siblings (P1, P2) manifested a profound developmental delay, cerebellar symptomatology, microcephaly, failure to thrive, short stature and delayed teeth eruption with oligodontia. The other two patients (P3, P4), on the contrary, suffer from substantially less serious impairment with mild to moderate developmental delay and cerebellar symptomatology, delayed teeth eruption, or well-manageable epilepsy. In all four patients, magnetic resonance revealed cerebellar atrophy and supratentorial hypomyelination with T2-weight hypointensities in the areas of the ventrolateral thalamic nuclei, corticospinal tract and the dentate nuclei. Using whole-exome sequencing in P1, P2 and P3, and targeted sequencing in P4, pathogenic variants were disclosed in POLR3B, a gene encoding one of 17 subunits of DNA-dependent RNA polymerase III - all patients were compound heterozygotes for point mutations. Three novel mutations c.727A>G (p.Met243Val) and c.2669G>A (p.Arg890His) (P1, P2), and c.1495G>A (p.Met499Val) (P3) were found. Magnetic resonance revealed the characteristic radiological pattern of POLR3-leukodystrophies in our patients. The diagnosis of POLR3-associated leukodystrophies can be significantly accelerated using the combined clinical and neuroradiological recognition pattern. Therefore, it is of crucial importance to raise the awareness of this rare disorder among clinicians. Molecular-genetic analyses are indispensable for a swift diagnosis confirmation in cases of clear clinical suspicion, and for diagnostic search in patients with less pronounced symptomatology. They represent an invaluable tool for unravelling the complex genetic background of heritable white matter disorders.

A novel homozygous mutation in POLR3A gene causing 4H syndrome: a case report

Background4H syndrome is a congenital hypomyelinating leukodystrophy characterized by hypodontia, hypomyelination and hypogonadotropic hypogonadism belonging to the Pol III-related leukodystrophies which arise due to mutations in the POLR3A or POLR3B gene. The clinical presentation is of neurodevelopmental delay or regression with ataxia, dystonia, nystagmus, delayed deciduous dentition and abnormal order of eruption of teeth. MRI brain shows a characteristic hypomyelination pattern. Several mutations have been described in the implicated genes but there are no reports on mutations seen in patients from India.Case presentationWe report a 1½ year old girl, only child of a non-consanguinous couple who presented with delayed developmental milestones and delayed dentition. On physical examination she had downward slanting palpebral fissures, low set ears, smooth philtrum, hypodontia, prominent body hair and clitoromegaly. There was prominent horizontal nystagmus, hypertonia of both upper and lower limbs, exaggerated deep tendon jerks and flexor planter response. She had not attained complete head control and required support to sit. She showed absent waves on brainstem evoked response audiometry and her fundus examination showed bilateral optic atrophy with prolongation of P100 latencies on visual evoked potentials. MRI Brain showed hyperintensity of entire white matter with involvement of the internal and external capsule, frontal deep white matter and corpus callosum. Her karyotype was 46 XX and her endocrinal profile was unremarkable. Clinical exome sequencing identified an unreported mutation in the POLR3A gene. The same mutation was identified by Sanger sequencing in heterozygous state in both parents. The child is being managed with physiotherapy and developmental therapy. She has been provided with hearing aids and started on speech therapy. Parents were provided anticipatory guidance and genetic counselling about autosomal recessive nature of inheritance, risk of recurrence and need for follow-up.Conclusion4H syndrome is a rare congenital hypomyelinating leukodystrophy inherited as an autosomal recessive disorder due to mutations in the POLR3A and POLR3B gene. Delay or regression of milestones, abnormalities in dentition and endocrinal perturbations are its hallmark. A novel mutation in the POLR3A gene resulting in amino acid substitution of arginine for glutamine at codon 808 (p.R808Q) was detected in exon 18 in our case.

The RNA polymerase III subunit Polr3b is required for the maintenance of small intestinal crypts in mice.

Background & AimsThe continuously self-renewing mammalian intestinal epithelium, with high cellular turnover, depends on adequate protein synthesis for its proliferative capacity. RNA polymerase III activity is related closely to cellular growth and proliferation. Here, we studied the role of Polr3b, a large RNA polymerase III subunit, in the mammalian intestinal epithelium.MethodsWe derived mice with an intestinal epithelium-specific hypomorphic mutation of the Polr3b gene, using VillinCre-mediated gene ablation. Phenotypic consequences of the Polr3b mutation on the intestinal epithelium in mice were assessed using histologic and molecular methodologies, including genetic lineage tracing.ResultsThe Polr3b mutation severely reduced survival and growth in mice during the first postnatal week, the period when the expansion of the intestinal epithelium, and thus the requirement for protein synthesis, are highest. The neonatal intestinal epithelium of Polr3bloxP/loxP;VillinCre mice was characterized by areas with reduced proliferation, abnormal epithelial architecture, loss of Wnt signaling, and a dramatic increase in apoptotic cells in crypts. Genetic lineage tracing using Polr3bLoxP/LoxP;Rosa26-lox-stop-lox-YFP;VillinCre mice showed that in surviving mutant mice, Polr3b-deficient dying crypts were replaced progressively by Cre-escaper cells that had retained wild-type Polr3b function. In addition, enteroids cultured from Polr3bloxP/loxP;VillinCre mice showed reduced proliferative activity and increased apoptosis.ConclusionsWe provide evidence for an essential role of the RNA polymerase III subunit Polr3b in orchestrating the maintenance of the intestinal crypt during early postnatal development in mice.

Brain Magnetic Resonance Imaging (MRI) Pattern Recognition in Pol III-Related Leukodystrophies

Pol III-related leukodystrophies are caused by mutations in POLR3A and POLR3B genes and all share peculiar imaging and clinical features. The objectives of this study are (1) to define the neuroradiologic pattern in a cohort of POLR3A and POLR3B subjects and (2) to compare the neuroradiologic pattern of Pol III-related leukodystrophies with other hypomyelinating disorders. The magnetic resonance imaging (MRI) examinations of 13 patients with POLR3A and POLR3B mutations and of 14 patients with other hypomyelinating disorders were analyzed. All the subjects with Pol III-related leukodystrophies presented hypomyelination associated with T2 hypointensity of the thalami and/or the pallida. Twelve subjects (92%) presented T2 hypointensity of the optic radiations. Cerebellar atrophy was observed in most patients (92%). The combination of the analyzed criteria identified patients with Pol III-related leukodystrophies with a sensitivity of 84.6% and a specificity of 92.9%.

Mutations in POLR3A and POLR3B are a major cause of hypomyelinating leukodystrophies with or without dental abnormalities and/or hypogonadotropic hypogonadism

BackgroundLeukodystrophies are a heterogeneous group of inherited neurodegenerative disorders characterised by abnormal central nervous system white matter. Mutations in POLR3A and POLR3B genes were recently reported to cause four clinically...

Retinoic Acid and Endothelin-3 Reciprocally Regulate Enteric Nervous System Precursor Survival, Differentiation, and Plexus Formation

Background and aims: RNA polymerase III transcribes small non-coding RNAs, indispensable for protein synthesis and cell growth control, especially in fast self-renewing tissues such as the intestinal epithelium. Recently, the importance of the RNA polymerase III complex in digestive development was demonstrated using zebrafish genetics. However, the role of RNA polymerase III in mammalian organisms has not been studied so far. We investigated the effects of disruption of the RNA polymerase III complex on the mouse intestinal epithelium, using tissue-specific gene mutation of the Polr3b gene, encoding the second largest subunit of the RNA polymerase III complex. Medhods: The conditional Polr3b mutant allele, with LoxP sites flanking exon 10 of the Polr3b gene, was derived in mouse embryonic stem cells via homologous recombination, allowing for ablation of exon 10 in Polr3bloxP/loxP upon Cre recombinase activation in Polr3bloxP/loxP; VillinCre mice. Results: Polr3bloxP/ loxP;Villin-Cre mice showed nearly 50% lower survival rate within the first postnatal week and significantly lower body weight. Surviving mutants developed severely disorganized intestinal epithelia, characterized by villus branching, and increased number and expansion of crypts. Furthermore, the intestinal epithelium of Polr3b mutant mice showed a remarkable increase in proliferative crypt regions and disorganized cellular migration, suggesting impaired cellular turnover in mice with disrupted RNA polymerase III complex. Polr3b mutant mice did not display major abnormalities in enterocyte or Goblet cell differentiation. However, Paneth cells in three-week old Polr3bLoxP/LoxP;VillinCre mice were not confined to the crypts, but expanded into the villus region. Surprisingly, mis-positioned Paneth cells resembled Goblet cell by morphology, and expressed both lysozyme and mucin 2, Paneth cell and Goblet cell markers, respectively. Gene profile analysis of newborn Polr3bloxP/ loxP;Villin-Cre mice demonstrated reduced expression of the Paneth cell genetic program. Overall, the strongest phenotype was apparent in three week-old Polr3b mutant mice. After the weaning period, the surviving of Polr3b mutant mice caught up in weight with their littermates; however, increased proliferative activity and disorganization of the intestinal epithelium persisted in adult Polr3bloxP/loxP;Villin-Cre mice. Conclusion: These data show that the RNA polymerase III complex regulates development of the intestinal epithelium and Paneth cell maturation. The RNA polymerase III complex plays an essential role in cell cycle dynamics of the intestinal epithelium inmice. Our future studies will focus on addressing the question whether targeting of RNA polymerase III is feasible for treatment of cancer.

RNA Polymerase III is Required for Intestinal Epithelial Development and Paneth Cell Maturation in Mice

Background and aims: RNA polymerase III transcribes small non-coding RNAs, indispensable for protein synthesis and cell growth control, especially in fast self-renewing tissues such as the intestinal epithelium. Recently, the importance of the RNA polymerase III complex in digestive development was demonstrated using zebrafish genetics. However, the role of RNA polymerase III in mammalian organisms has not been studied so far. We investigated the effects of disruption of the RNA polymerase III complex on the mouse intestinal epithelium, using tissue-specific gene mutation of the Polr3b gene, encoding the second largest subunit of the RNA polymerase III complex. Medhods: The conditional Polr3b mutant allele, with LoxP sites flanking exon 10 of the Polr3b gene, was derived in mouse embryonic stem cells via homologous recombination, allowing for ablation of exon 10 in Polr3bloxP/loxP upon Cre recombinase activation in Polr3bloxP/loxP; VillinCre mice. Results: Polr3bloxP/ loxP;Villin-Cre mice showed nearly 50% lower survival rate within the first postnatal week and significantly lower body weight. Surviving mutants developed severely disorganized intestinal epithelia, characterized by villus branching, and increased number and expansion of crypts. Furthermore, the intestinal epithelium of Polr3b mutant mice showed a remarkable increase in proliferative crypt regions and disorganized cellular migration, suggesting impaired cellular turnover in mice with disrupted RNA polymerase III complex. Polr3b mutant mice did not display major abnormalities in enterocyte or Goblet cell differentiation. However, Paneth cells in three-week old Polr3bLoxP/LoxP;VillinCre mice were not confined to the crypts, but expanded into the villus region. Surprisingly, mis-positioned Paneth cells resembled Goblet cell by morphology, and expressed both lysozyme and mucin 2, Paneth cell and Goblet cell markers, respectively. Gene profile analysis of newborn Polr3bloxP/ loxP;Villin-Cre mice demonstrated reduced expression of the Paneth cell genetic program. Overall, the strongest phenotype was apparent in three week-old Polr3b mutant mice. After the weaning period, the surviving of Polr3b mutant mice caught up in weight with their littermates; however, increased proliferative activity and disorganization of the intestinal epithelium persisted in adult Polr3bloxP/loxP;Villin-Cre mice. Conclusion: These data show that the RNA polymerase III complex regulates development of the intestinal epithelium and Paneth cell maturation. The RNA polymerase III complex plays an essential role in cell cycle dynamics of the intestinal epithelium inmice. Our future studies will focus on addressing the question whether targeting of RNA polymerase III is feasible for treatment of cancer.