Artigo Acesso aberto Revisado por pares

Mutations in POLR3A and POLR3B Encoding RNA Polymerase III Subunits Cause an Autosomal-Recessive Hypomyelinating Leukoencephalopathy

2011; Elsevier BV; Volume: 89; Issue: 5 Linguagem: Inglês

10.1016/j.ajhg.2011.10.003

ISSN

1537-6605

Autores

Hirotomo Saitsu, Hitoshi Osaka, Masayuki Sasaki, Jun‐ichi Takanashi, K. Hamada, Akio Yamashita, Hidehiro Shibayama, Masaaki Shiina, Yukiko Kondo, Kiyomi Nishiyama, Yoshinori Tsurusaki, Noriko Miyake, Hiroshi Doi, Kazuhiro Ogata, Ken Inoue, Naomichi Matsumoto,

Tópico(s)

RNA and protein synthesis mechanisms

Resumo

Congenital hypomyelinating disorders are a heterogeneous group of inherited leukoencephalopathies characterized by abnormal myelin formation. We have recently reported a hypomyelinating syndrome characterized by diffuse cerebral hypomyelination with cerebellar atrophy and hypoplasia of the corpus callosum (HCAHC). We performed whole-exome sequencing of three unrelated individuals with HCAHC and identified compound heterozygous mutations in POLR3B in two individuals. The mutations include a nonsense mutation, a splice-site mutation, and two missense mutations at evolutionally conserved amino acids. Using reverse transcription-PCR and sequencing, we demonstrated that the splice-site mutation caused deletion of exon 18 from POLR3B mRNA and that the transcript harboring the nonsense mutation underwent nonsense-mediated mRNA decay. We also identified compound heterozygous missense mutations in POLR3A in the remaining individual. POLR3A and POLR3B encode the largest and second largest subunits of RNA Polymerase III (Pol III), RPC1 and RPC2, respectively. RPC1 and RPC2 together form the active center of the polymerase and contribute to the catalytic activity of the polymerase. Pol III is involved in the transcription of small noncoding RNAs, such as 5S ribosomal RNA and all transfer RNAs (tRNA). We hypothesize that perturbation of Pol III target transcription, especially of tRNAs, could be a common pathological mechanism underlying POLR3A and POLR3B mutations. Congenital hypomyelinating disorders are a heterogeneous group of inherited leukoencephalopathies characterized by abnormal myelin formation. We have recently reported a hypomyelinating syndrome characterized by diffuse cerebral hypomyelination with cerebellar atrophy and hypoplasia of the corpus callosum (HCAHC). We performed whole-exome sequencing of three unrelated individuals with HCAHC and identified compound heterozygous mutations in POLR3B in two individuals. The mutations include a nonsense mutation, a splice-site mutation, and two missense mutations at evolutionally conserved amino acids. Using reverse transcription-PCR and sequencing, we demonstrated that the splice-site mutation caused deletion of exon 18 from POLR3B mRNA and that the transcript harboring the nonsense mutation underwent nonsense-mediated mRNA decay. We also identified compound heterozygous missense mutations in POLR3A in the remaining individual. POLR3A and POLR3B encode the largest and second largest subunits of RNA Polymerase III (Pol III), RPC1 and RPC2, respectively. RPC1 and RPC2 together form the active center of the polymerase and contribute to the catalytic activity of the polymerase. Pol III is involved in the transcription of small noncoding RNAs, such as 5S ribosomal RNA and all transfer RNAs (tRNA). We hypothesize that perturbation of Pol III target transcription, especially of tRNAs, could be a common pathological mechanism underlying POLR3A and POLR3B mutations. Congenital hypomyelinating disorders form a heterogeneous group of central nervous system leukoencephalopathies that is characterized by abnormal myelin formation. Although these conditions are readily recognized by brain magnetic resonance imaging (MRI), many cases are not diagnosed correctly.1Schiffmann R. van der Knaap M.S. Invited article: an MRI-based approach to the diagnosis of white matter disorders.Neurology. 2009; 72: 750-759Crossref PubMed Scopus (379) Google Scholar Several syndromes affecting myelination, such as hypomyelination with hypodontia and hypogonadotropic hypogonadism (4H) syndrome (MIM 612440) and hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) (MIM 612438), have been described.2Timmons M. Tsokos M. Asab M.A. Seminara S.B. Zirzow G.C. Kaneski C.R. Heiss J.D. van der Knaap M.S. Vanier M.T. Schiffmann R. Wong K. Peripheral and central hypomyelination with hypogonadotropic hypogonadism and hypodontia.Neurology. 2006; 67: 2066-2069Crossref PubMed Scopus (76) Google Scholar, 3Wolf N.I. Harting I. Boltshauser E. Wiegand G. Koch M.J. Schmitt-Mechelke T. Martin E. Zschocke J. Uhlenberg B. Hoffmann G.F. et al.Leukoencephalopathy with ataxia, hypodontia, and hypomyelination.Neurology. 2005; 64: 1461-1464Crossref PubMed Scopus (70) Google Scholar, 4Wolf N.I. Harting I. Innes A.M. Patzer S. Zeitler P. Schneider A. Wolff A. Baier K. Zschocke J. Ebinger F. et al.Ataxia, delayed dentition and hypomyelination: a novel leukoencephalopathy.Neuropediatrics. 2007; 38: 64-70Crossref PubMed Scopus (49) Google Scholar, 5van der Knaap M.S. Naidu S. Pouwels P.J. Bonavita S. van Coster R. Lagae L. Sperner J. Surtees R. Schiffmann R. Valk J. New syndrome characterized by hypomyelination with atrophy of the basal ganglia and cerebellum.AJNR Am. J. Neuroradiol. 2002; 23: 1466-1474PubMed Google Scholar We have recently reported a hypomyelinating syndrome characterized by diffuse cerebral hypomyelination with cerebellar atrophy and hypoplasia of the corpus callosum (HCAHC).6Sasaki M. Takanashi J. Tada H. Sakuma H. Furushima W. Sato N. Diffuse cerebral hypomyelination with cerebellar atrophy and hypoplasia of the corpus callosum.Brain Dev. 2009; 31: 582-587Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Individuals with HCAHC do not show hypodontia or atrophy of the basal ganglia, which are observed in 4H syndrome and H-ABC; however, diffuse hypomyelination, atrophy, or hypoplasia of the cerebellum and corpus callosum are overlapping features of these three syndromes, suggesting that there might be a common underlying pathological mechanism. Here, we report on four individuals with HCAHC from three unrelated families (Figure 1A ; Table 1). Clinical information and peripheral blood or saliva samples were obtained from the family members after obtaining written informed consent. Experimental protocols were approved by the Institutional Review Board of Yokohama City University. To identify pathogenic mutations, we performed whole-exome sequencing of three probands from three unrelated families (individuals 1, 3, and 4). DNAs were captured with the SureSelect Human All Exon 50Mb Kit (Agilent Technologies, Santa Clara, CA) and sequenced with one lane per sample on an Illumina GAIIx (Illumina, San Diego, CA) with 108 bp paired-end reads. Image analysis and base calling were performed by sequence control software real-time analysis and CASAVA software v1.7 (Illumina). A total of 90,014,368 (individual 1), 86,942,264 (individual 3), and 92,168,758 (individual 4) paired-end reads were obtained and aligned to the human reference genome sequence (GRCh37/hg19) with MAQ7Li H. Ruan J. Durbin R. Mapping short DNA sequencing reads and calling variants using mapping quality scores.Genome Res. 2008; 18: 1851-1858Crossref PubMed Scopus (2015) Google Scholar and NextGENe software v2.00 with sequence condensation by consolidation (SoftGenetics, State College, PA). This approach resulted in more than 88% of target exomes being covered by ten reads or more (see Table S1, available online). Single nucleotide variants (SNVs) were called with MAQ and NextGENe. Small insertions and deletions were detected with NextGENe. Called SNVs were annotated with SeattleSeq Annotation.Table 1Clinical Features of the IndividualsClinical FeaturesIndividual 1Individual 2Individual 3Individual 4GenesPOLR3BPOLR3BPOLR3BPOLR3AMutations, DNAc.1857-2A>C, c.2303G>Ac.1857-2A>C, c.2303G>Ac.1648C>T, c.2778C>Gc.2690T>A, c.3013C>TMutations, proteinp.Asn620_Lys652del, p.Arg768Hisp.Asn620_Lys652del, p.Arg768Hisp.Arg550X, p.Asp926Glup.Ile897Asn, p.Arg1005CysGenderMFFMCurrent age (years)27301617Intellectual disabilitymildmildmoderatemildCognitive regression––––Seizures––––Initial motor developmentnormalnormalnormalnormalAge of onset (years)3324Motor deterioration–––+Wheelchair use–––+Optic atrophy––––Myopia++–+Nystagmus++––Abnormal pursuit+++–Vertical gaze limitation+++–Dysphagia––+–Hypersalivation––––Cerebellar signs++++Tremor–+++Babinski refex––––Spasticity––mild–Peripheral nerve involvement––––Nerve biopsyNANANANAHypodontia––––Hypogonadism++––NA is an abbreviation for not available. Open table in a new tab NA is an abbreviation for not available. We adopted a prioritization scheme to identify the pathogenic mutation in each individual, similar to the approach taken by recent studies (Table S2).8Doi H. Yoshida K. Yasuda T. Fukuda M. Fukuda Y. Morita H. Ikeda S. Kato R. Tsurusaki Y. Miyake N. et al.Exome sequencing reveals a homozygous SYT14 mutation in adult-onset, autosomal-recessive spinocerebellar ataxia with psychomotor retardation.Am. J. Hum. Genet. 2011; 89: 320-327Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 9Pierce S.B. Walsh T. Chisholm K.M. Lee M.K. Thornton A.M. Fiumara A. Opitz J.M. Levy-Lahad E. Klevit R.E. King M.C. Mutations in the DBP-deficiency protein HSD17B4 cause ovarian dysgenesis, hearing loss, and ataxia of Perrault Syndrome.Am. J. Hum. Genet. 2010; 87: 282-288Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 10Gilissen C. Arts H.H. Hoischen A. Spruijt L. Mans D.A. Arts P. van Lier B. Steehouwer M. van Reeuwijk J. Kant S.G. et al.Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome.Am. J. Hum. Genet. 2010; 87: 418-423Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar First, we excluded the variants registered in the dbSNP131 or 1000 Genome Project from all the detected variants. Then, SNVs commonly detected by MAQ and NextGENe analyses were selected as highly confident variants; 364 to 374 SNVs of nonsynonymous (NS) or canonical splice-site (SP) changes, along with 113 to 124 small insertions or deletions (indels), were identified per individual. We also excluded variants found in our 55 in-house exomes, which are derived from 12 healthy individuals and 43 individuals with unrelated diseases, reducing the number of candidate variants to ∼250 per individual. Assuming that HCAHC is an autosomal-recessive disorder based on two affected individuals in one pedigree (individuals 1 and 2), we focused on rare heterozygous variants that are not registered in the dbSNP or in our in-house 55 exomes. We surveyed all genes in each individual for two or more NS, SP, or indel variants. We found three to eight candidate genes per individual (Table S2). Among them, only POLR3B encoding RPC2, the second largest subunit of RNA Polymerase III (Pol III), was common in two individuals (individuals 1 and 3). The inheritance of the variants in POLR3B (transcript variant 1, NM_018082.5) was examined by Sanger sequencing. In individual 1, we confirmed that a canonical splice-site mutation (c.1857-2A>C [p.Asn620_Lys652del]), 2 bp upstream of exon 18, was inherited from his father, and that a missense mutation (c.2303G>A [p.Arg768His]) in exon 21 were inherited from his mother (Figure 1A). The two mutations were also present in an affected elder sister (individual 2) but not present in a healthy elder brother. In individual 3, we confirmed that a nonsense mutation (c.1648C>T [p.Arg550X]) in exon 16 was inherited from her father and that a missense mutation (c.2778C>G [p.Asp926Glu]) in exon 24 was inherited from her mother (Figure 1A). The two mutations were not present in a healthy younger brother. To examine the mutational effects of c.1857-2A>C and c.1648C>T, reverse transcription PCR and sequencing with total RNA extracted from lymphoblastoid cells derived from the individuals was performed as previously described.11Saitsu H. Kato M. Okada I. Orii K.E. Higuchi T. Hoshino H. Kubota M. Arai H. Tagawa T. Kimura S. et al.STXBP1 mutations in early infantile epileptic encephalopathy with suppression-burst pattern.Epilepsia. 2010; 51: 2397-2405Crossref PubMed Scopus (117) Google Scholar We demonstrated that the c.1857-2A>C mutation caused deletion of exon 18 from the POLR3B mRNA (Figures 2A–2C ), resulting in an in-frame 33 amino acid deletion (p.Asn620_Lys652del) from RPC2 (Figure 1B). In addition, the mutated transcript harboring the nonsense mutation (c.1648C>T) was found to be expressed at a much lower level compared with the wild-type transcript (Figure 2D). The expression level of the mutated transcript was increased after treatment with 30 μM cycloheximide (CHX),11Saitsu H. Kato M. Okada I. Orii K.E. Higuchi T. Hoshino H. Kubota M. Arai H. Tagawa T. Kimura S. et al.STXBP1 mutations in early infantile epileptic encephalopathy with suppression-burst pattern.Epilepsia. 2010; 51: 2397-2405Crossref PubMed Scopus (117) Google Scholar which inhibits nonsense-mediated mRNA decay (NMD), indicating that the mutant transcript underwent NMD (Figure 2D). The two missense mutations (p.Arg768His and p.Asp926Glu) found in the three individuals occurred at evolutionary conserved amino acids (Figure 1B). Among the other candidate genes in individuals 1 and 3, MSLN (MIM 601051), encoding mesothelin isoform 1 preproprotein that is cleaved into megakaryocyte potentiating factor and mesothelin, is a potential candidate in the family of individual 1 as its homozygous variant segregated with the phenotype; however, it is expressed in epithelial mesotheliomas, and the mutation affects less conserved amino acid (Table S3). The other candidate genes' variants did not cosegregate with the phenotype. Thus, mutations in POLR3B are most likely to cause HCAHC in two families. In individual 4, in whom no POLR3B mutations were found, there were six candidate genes for an autosomal-recessive model. Among them, POLR3A (MIM 614258, GenBank accession number NM_007055.3), harboring two missense mutations, appeared to be a primary candidate because it encodes the largest subunit of Pol III (RPC1) (Figure 1A and Table S2). By Sanger sequencing, we confirmed that a missense mutation (c.2690T>A [p.Ile897Asn]) in exon 20 was inherited from his father and that another missense mutation (c.3013C>T [p.Arg1005Cys]) in exon 23 was inherited from his mother (Figure 1A). The two mutations were not present in a healthy younger sister. The two missense mutations (p.Ile897Asn and p.Arg1005Cys) occurred at relatively conserved amino acids (Figure 1B). In total, we found four mutations in POLR3B and two mutations in POLR3A. Evaluation of the missense mutations by PolyPhen-2 program showed that three mutations (p.Arg768His, p.Asp926Glu, and p.Ile897Asn) were probably damaging and that p.Arg1005Cys is tolerable. The c.2303G>A mutation (POLR3B) was found in one allele out of 540 Japanese control chromosomes. The remaining five mutations were not detected in 540 Japanese control chromosomes, indicating that the mutations are very rare in the Japanese population. Among the other candidate genes in individuals 4, IGSF10, a member of immunoglobulin superfamily, is a potential candidate because its variants segregated with the phenotype (Table S3); however, considering a close relationship between POLR3A and POLR3B, and the fact that POLR3A mutations have been recently reported in hypomyelinating leukodystrophy (see below),12Bernard G. Chouery E. Putorti M.L. Tetreault M. Takanohashi A. Carosso G. Clement I. Boespflug-Tanguy O. Rodriguez D. Delague V. et al.Mutations of POLR3A Encoding a Catalytic Subunit of RNA Polymerase Pol III Cause a Recessive Hypomyelinating Leukodystrophy.Am. J. Hum. Genet. 2011; 89: 415-423Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar POLR3A abnormality is the most plausible culprit for HCAHC in individual 4. The structure of Pol III13Jasiak A.J. Armache K.J. Martens B. Jansen R.P. Cramer P. Structural biology of RNA polymerase III: subcomplex C17/25 X-ray structure and 11 subunit enzyme model.Mol. Cell. 2006; 23: 71-81Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 14Fernández-Tornero C. Böttcher B. Riva M. Carles C. Steuerwald U. Ruigrok R.W. Sentenac A. Müller C.W. Schoehn G. Insights into transcription initiation and termination from the electron microscopy structure of yeast RNA polymerase III.Mol. Cell. 2007; 25: 813-823Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar and Pol II15Cramer P. Bushnell D.A. Kornberg R.D. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.Science. 2001; 292: 1863-1876Crossref PubMed Scopus (966) Google Scholar, 16Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution.Science. 2001; 292: 1876-1882Crossref PubMed Scopus (744) Google Scholar is highly homologous, especially in the largest subunits. Thus, we extrapolated the mutations of RPC1 or RPC2 onto the structure of yeast Pol II (Protein Data Bank [PDB] accession number 3GTP)17Wang D. Bushnell D.A. Huang X. Westover K.D. Levitt M. Kornberg R.D. Structural basis of transcription: backtracked RNA polymerase II at 3.4 angstrom resolution.Science. 2009; 324: 1203-1206Crossref PubMed Scopus (185) Google Scholar (Figure 1C). RPB1 and RPB2 subunits of yeast Pol II are homologous to RPC1 and RPC2 of Pol III, respectively. Asn620_Lys652 in RPC2 corresponds to Tyr679_Lys712 in RPB2. The deletion of Asn620_Lys652 (Tyr679_Lys712) would destroy a structural core of RPB2, leading to loss of RPB2 function. In addition, Arg768 (Arg852 in RPB2) interacts with the main-chain carbonyl group of Arg70 of the RPB12 subunit, and Asp926 (Asp1009 in RPB2) interacts with the side chain of Arg48 of the RPB10 subunit of Pol II (Figure 1D). Arg768His (Arg852His) and Asp926Glu (Asp1009Glu) substitutions are considered to disturb these subunit interactions, leading to dysfunction of the polymerase. Therefore, structural prediction suggests that the mutations in POLR3B (RPC2) could affect Pol III function. On the other hand, Ile897 and Arg1005 in RPC1 correspond to Val863 and Arg1036 in RPB1, respectively. Ile897 (Val863) has hydrophobic interactions with Leu170 and Pro176 of the RPB5 subunit and with Phe900 (Phe866) of the RPB1 subunit of Pol II (Figure 1E). Ile897Asn (Val863Asn) substitution is likely to disturb this interaction. Arg1005 (Arg1036) stabilizes interaction between RPB1 and RPB8 subunits (Figure 1F). The Arg1005Cys (Arg1036Cys) substitution appears to make this interaction unstable. Thus mutations in POLR3A are also predicted to affect Pol III function. Clinical features of individuals with POLR3A or POLR3B mutations are presented in Table 1. MRI revealed high-intensity areas in the white matter in T2-weighted images, cerebellar atrophy, and a hypoplastic corpus callosum in all four individuals (Figure 3). Individuals 1 and 2 showed an extremely similar clinical course. They developed normally during their early infancy, i.e., walking unaided at 15 and 14 months, and uttering a few words at 12 and 13 months, respectively. After the age of 3, individual 1 presented with unstable walking and frequent stumbling and falling down, and individual 2 became poor at exercise. They both had severe myopia (corrected visual acuity of 0.7 and 0.5 at most, respectively). They graduated from elementary, junior high, and high schools with poor records, and the intelligence quotient (IQ) of individual 2 was 52 (WAIS-III). In individual 1, unstable walking was prominent at around 18 years, and he could not ride a bicycle because of ataxia; however, he could drive an automobile. Amenorrhea was noted in individual 2, and was successfully treated by hormone therapy. Individual 1 showed several signs of hypogonadism, including absence of underarm and mustache hair, thin pubic hair (Tanner II), and serum levels of testosterone, follicle stimulating hormone, and luteinizing hormone that were below normal for age 27. Neurological examination of both individuals revealed mild horizontal nystagmus, slowing of smooth-pursuit eye movement, and gaze limitation, especially in vertical gazing, hypotonia, mildly exaggerated deep-tendon reflex (patellar and Achilles tendon reflex) with negative Babinski reflex, and cerebellar signs and symptoms, including ataxic speech, wide-based ataxic gait, dysdiadochokinesis, and dysmetria. Clinical information for individual 3 has been reported previously.6Sasaki M. Takanashi J. Tada H. Sakuma H. Furushima W. Sato N. Diffuse cerebral hypomyelination with cerebellar atrophy and hypoplasia of the corpus callosum.Brain Dev. 2009; 31: 582-587Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Additional findings are as follows: slowing of smooth-pursuit eye movement, gaze limitation in vertical gazing, normal auditory brain responses (ABR), cerebral symptoms with mild spasticity, and intellectual disability (an IQ of 43 according to the WISC-III test), and no myopia but hypermetropic astigmatism. She showed no deterioration besides a mild dysphagia and walks herself to a school for the disabled. Individual 4 developed normally during his early infancy, had normal head control at 3 months, was speaking a few words at 12 months, and was walking unaided at 14 months. His parents noted mild tremors around 4 years. He had normal stature, weight, and head circumference. Although he had severe myopia, his eye movement was smooth with no limitation or nystagmus. He had sensory neuronal deafness on the left side. He showed normal muscle tone and had no spasticity or rigidity. His tendon reflexes were slightly elevated with a negative Babinski reflex. Cerebellar signs were noted; expressive ataxic explosive speech, intension tremor, poor finger to nose test, dysdiadochokinesis, dysmetria, and wide-based ataxic gait. His intelligence quotient was 57 (according to the WISC-III test). His peripheral nerve conduction velocity was within the normal range and his ABR showed normal responses on the right side. He suffered motor deterioration around age 14 and became wheelchair bound. In this study, we successfully identified compound heterozygous mutations in POLR3A and POLR3B in individuals with HCAHC. Very recently, Bernard et al.12Bernard G. Chouery E. Putorti M.L. Tetreault M. Takanohashi A. Carosso G. Clement I. Boespflug-Tanguy O. Rodriguez D. Delague V. et al.Mutations of POLR3A Encoding a Catalytic Subunit of RNA Polymerase Pol III Cause a Recessive Hypomyelinating Leukodystrophy.Am. J. Hum. Genet. 2011; 89: 415-423Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar reported that POLR3A mutations cause three overlapping leukodystrophies, including 4H syndrome, suggesting that HCAHC is, at least in part, within a wide clinical spectrum caused by POLR3A mutations. The p.Arg1005Cys mutation was shared between individual 9 in their report and our individual 4. All 19 individuals with POLR3A mutations showed progressive upper motor neuron dysfunction and cognitive regression. In addition, individual 9 showed abnormal eye movement, hypodontia, and hypogonadism. None of these features were recognized in our individual 4; these differences further support phenotypic variability of POLR3A mutations.12Bernard G. Chouery E. Putorti M.L. Tetreault M. Takanohashi A. Carosso G. Clement I. Boespflug-Tanguy O. Rodriguez D. Delague V. et al.Mutations of POLR3A Encoding a Catalytic Subunit of RNA Polymerase Pol III Cause a Recessive Hypomyelinating Leukodystrophy.Am. J. Hum. Genet. 2011; 89: 415-423Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar Given the phenotypic similarities among 4H syndrome, HCAHC, and H-ABC, there is a possibility that H-ABC is also allelic and caused by recessive mutations in either POLR3A or POLR3B. Pol III consists of 17 subunits and is involved in the transcription of small noncoding RNAs, such as 5S ribosomal RNA (rRNA), U6 small nuclear RNA (snRNA), 7SL RNA, RNase P, RNase MRP, short interspersed nuclear elements (SINEs), and all transfer RNAs (tRNAs). Pol III-transcribed genes are classified into three types based on promoter elements and transcription factors. 5S rRNA is a solo type I gene. Type II genes include tRNA, 7SL RNA, and SINEs. Type III genes include U6 snRNA, RNase P, and RNase MRP.18Oler A.J. Alla R.K. Roberts D.N. Wong A. 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Cell growth- and differentiation-dependent regulation of RNA polymerase III transcription.Cell Cycle. 2010; 9: 3687-3699Crossref PubMed Scopus (51) Google Scholar In zebrafish, polr3b mutant larvae that have a deletion of 41 conserved amino acids (Δ239-279) from the Rpc2 protein showed a proliferation deficit in multiple tissues, including intestine, endocrine pancreas, liver, retina and terminal branchial arches.21Yee N.S. Gong W. Huang Y. Lorent K. Dolan A.C. Maraia R.J. Pack M. Mutation of RNA Pol III subunit rpc2/polr3b Leads to Deficiency of Subunit Rpc11 and disrupts zebrafish digestive development.PLoS Biol. 2007; 5: e312Crossref PubMed Scopus (54) Google Scholar In the mutants, the expression levels of tRNA were significantly reduced, whereas the level of 5S rRNA expression was not changed, suggesting that this polr3b mutation can differentially affect Pol III target promoters.21Yee N.S. Gong W. Huang Y. Lorent K. Dolan A.C. Maraia R.J. Pack M. Mutation of RNA Pol III subunit rpc2/polr3b Leads to Deficiency of Subunit Rpc11 and disrupts zebrafish digestive development.PLoS Biol. 2007; 5: e312Crossref PubMed Scopus (54) Google Scholar RPC2 contributes to the catalytic activity of the polymerase and forms the active center of the polymerase together with the largest subunit, RPC1.22Werner M. Thuriaux P. Soutourina J. Structure-function analysis of RNA polymerases I and III.Curr. Opin. Struct. Biol. 2009; 19: 740-745Crossref PubMed Scopus (40) Google Scholar Thus, it is reasonable to consider that mutations in POLR3A and POLR3B cause overlapping phenotypes. Indeed, three individuals with POLR3B mutations showed diffuse cerebral hypomyelination, atrophy of the cerebellum and corpus callosum, and abnormal eye movements that overlap with POLR3A abnormalities.12Bernard G. Chouery E. Putorti M.L. Tetreault M. Takanohashi A. Carosso G. Clement I. Boespflug-Tanguy O. Rodriguez D. Delague V. et al.Mutations of POLR3A Encoding a Catalytic Subunit of RNA Polymerase Pol III Cause a Recessive Hypomyelinating Leukodystrophy.Am. J. Hum. Genet. 2011; 89: 415-423Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar Furthermore, two out of three individuals showed hypogonadism, suggesting a common pathological mechanism between POLR3A and POLR3B mutations. In the zebrafish polr3b mutants there were no defects of the central nervous system other than a reduced size of the retina, probably reflecting species differences; however, the reduced level of tRNA in the polr3b mutants raises the possibility that defects of tRNA transcription by Pol III could be a common pathological mechanism underlying POLR3A and POLR3B mutations. 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Genet. 2006; 38: 197-202Crossref PubMed Scopus (295) Google Scholar Thus, it is very likely that regulation of tRNA expression is essential for development and maintenance of myelination in both central and peripheral nervous systems. An interesting clinical feature of POLR3B mutations is the absence of motor deterioration. All three individuals with POLR3B mutations could walk without support at ages 16, 27, and 30, whereas individual 3 with POLR3A mutations had motor deterioration around age 14. Bernard et al.12Bernard G. Chouery E. Putorti M.L. Tetreault M. Takanohashi A. Carosso G. Clement I. Boespflug-Tanguy O. Rodriguez D. Delague V. et al.Mutations of POLR3A Encoding a Catalytic Subunit of RNA Polymerase Pol III Cause a Recessive Hypomyelinating Leukodystrophy.Am. J. Hum. Genet. 2011; 89: 415-423Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar also reported progressive upper motor neuron dysfunction and cognitive regression in individuals with POLR3A mutations. Thus, there is a possibility that phenotypes caused by POLR3A mutations could be more severe and progressive than POLR3B mutant phenotypes. Identification of a greater number of cases with POLR3B mutations is required to confirm this hypothesis. In conclusion, our data, together with that of a previous report,12Bernard G. Chouery E. Putorti M.L. Tetreault M. Takanohashi A. Carosso G. Clement I. Boespflug-Tanguy O. Rodriguez D. Delague V. et al.Mutations of POLR3A Encoding a Catalytic Subunit of RNA Polymerase Pol III Cause a Recessive Hypomyelinating Leukodystrophy.Am. J. Hum. Genet. 2011; 89: 415-423Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar demonstrate that mutations in Pol III subunits cause overlapping autosomal-recessive hypomyelinating disorders. Establishment of an animal model will facilitate our understanding of the pathophysiology of the multiple defects caused by Pol III mutations. We would like to thank all the individuals and their families for their participation in this study. This work was supported by research grants from the Ministry of Health, Labour, and Welfare (H.S., H.O., M.S., J.T., N. Miyake, K.I. and N. Matsumoto), the Japan Science and Technology Agency (N. Matsumoto), a Grant-in-Aid for Scientific Research on Innovative Areas (Foundation of Synapse and Neurocircuit Pathology) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (N. Matsumoto), a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (H.O., N. Matsumoto), a Grant-in-Aid for Young Scientist from Japan Society for the Promotion of Science (H.S.). This work has been done at Advanced Medical Research Center, Yokohama City University. Download .pdf (.1 MB) Help with pdf files Document S1. Three Tables The URLs for data presented herein are as follows:ClustalW, http://www.genome.jp/tools/clustalw/dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/Ensembl, http://uswest.ensembl.org/index.htmlGenBank, http://www.ncbi.nlm.nih.gov/Genbank/Online Mendelian Inheritance in Man, http://www.omim.orgPolyPhen-2, http://genetics.bwh.harvard.edu/pph2/Protein Data Bank, http://www.pdb.org/pdb/home/home.doPyMOL, http://www.pymol.org/SeattleSeq Annotation, http://gvs.gs.washington.edu/SeattleSeqAnnotation/

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