Dataset Information

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

ABSTRACT:

Objectives

4H 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.

Methods

Genetic 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.

Results

We 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.

Discussion

The 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.

SUBMITTER: Yang HJ

PROVIDER: S-EPMC9007423 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Publications

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

Yang Hui-Jun HJ Park Gyeongmin G Nam-Goong Il Seong IS Ahn Jun-Woo JW Weon Young Cheol YC

Neurology. Genetics 20220413 3

<h4>Objectives</h4>4H 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 <i>POLR1C</i> sequence variation has been published, while there are no reports on the <i>POLR3B</i>, <i>POLR3A</i>, or <i>POLR3K</i> variants.<h4>Methods</h4>Genetic tests of Korean sibling pairs wit ...[more]

PMID: 35434302

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

Project description:4H leukodystrophy is a rare genetic disorder classically characterized by hypomyelination, hypodontia and hypogonadotropic hypogonadism. With the discovery that 4H is caused by mutations that affect RNA polymerase III, mainly involved in the transcription of small non-coding RNAs, patients with atypical presentations with mainly a neuronal phenotype were also identified. Pathomechanisms of 4H brain abnormalities are still unknown and research is hampered by a lack of preclinical models. We aimed to identify cells and pathways that are affected by 4H mutations using induced pluripotent stem cell models. RNA sequencing analysis on induced pluripotent stem cell-derived cerebellar cells revealed several differentially expressed genes between 4H patients and control samples, including reduced ARX expression. As ARX is involved in early brain and interneuron development, we studied and confirmed interneuron changes in primary tissue of 4H patients. Subsequently, we studied interneuron changes in more depth and analysed induced pluripotent stem cell-derived cortical neuron cultures for changes in neuronal morphology, synaptic balance, network activity and myelination. We showed a decreased percentage of GABAergic synapses in 4H, which correlated to increased neuronal network activity. Treatment of cultures with GABA antagonists led to a significant increase in neuronal network activity in control cells but not in 4H cells, also pointing to lack of inhibitory activity in 4H. Myelination and oligodendrocyte maturation in cultures with 4H neurons was normal, and treatment with sonic hedgehog agonist SAG did not improve 4H related neuronal phenotypes. Quantitative PCR analysis revealed increased expression of parvalbumin interneuron marker ERBB4, suggesting that the development rather than generation of interneurons may be affected in 4H. Together, these results indicate that interneurons are involved, possibly parvalbumin interneurons, in disease mechanisms of 4H leukodystrophy.