A De Novo Mutation in the β-Tubulin Gene TUBB4A Results in the Leukoencephalopathy Hypomyelination with Atrophy of the Basal Ganglia and Cerebellum
2013; Elsevier BV; Volume: 92; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2013.03.018
ISSN1537-6605
AutoresCas Simons, Nicole I. Wolf, Nathan McNeil, Ljubica Caldovic, Joseph M. Devaney, Asako Takanohashi, Joanna Crawford, Kelin Ru, Sean M. Grimmond, David S. Miller, Davide Tonduti, Johanna Schmidt, Robert S. Chudnow, Rudy Van Coster, Lieven Lagae, Jill Kisler, Jürgen Sperner, Marjo S. van der Knaap, Raphael Schiffmann, Ryan J. Taft, Adeline Vanderver,
Tópico(s)RNA and protein synthesis mechanisms
ResumoHypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a rare hereditary leukoencephalopathy that was originally identified by MRI pattern analysis, and it has thus far defied all attempts at identifying the causal mutation. Only 22 cases are published in the literature to date. We performed exome sequencing on five family trios, two family quartets, and three single probands, which revealed that all eleven H-ABC-diagnosed individuals carry the same de novo single-nucleotide TUBB4A mutation resulting in nonsynonymous change p.Asp249Asn. Detailed investigation of one of the family quartets with the singular finding of an H-ABC-affected sibling pair revealed maternal mosaicism for the mutation, suggesting that rare de novo mutations that are initially phenotypically neutral in a mosaic individual can be disease causing in the subsequent generation. Modeling of TUBB4A shows that the mutation creates a nonsynonymous change at a highly conserved asparagine that sits at the intradimer interface of α-tubulin and β-tubulin, and this change might affect tubulin dimerization, microtubule polymerization, or microtubule stability. Consistent with H-ABC’s clinical presentation, TUBB4A is highly expressed in neurons, and a recent report has shown that an N-terminal alteration is associated with a heritable dystonia. Together, these data demonstrate that a single de novo mutation in TUBB4A results in H-ABC. Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a rare hereditary leukoencephalopathy that was originally identified by MRI pattern analysis, and it has thus far defied all attempts at identifying the causal mutation. Only 22 cases are published in the literature to date. We performed exome sequencing on five family trios, two family quartets, and three single probands, which revealed that all eleven H-ABC-diagnosed individuals carry the same de novo single-nucleotide TUBB4A mutation resulting in nonsynonymous change p.Asp249Asn. Detailed investigation of one of the family quartets with the singular finding of an H-ABC-affected sibling pair revealed maternal mosaicism for the mutation, suggesting that rare de novo mutations that are initially phenotypically neutral in a mosaic individual can be disease causing in the subsequent generation. Modeling of TUBB4A shows that the mutation creates a nonsynonymous change at a highly conserved asparagine that sits at the intradimer interface of α-tubulin and β-tubulin, and this change might affect tubulin dimerization, microtubule polymerization, or microtubule stability. Consistent with H-ABC’s clinical presentation, TUBB4A is highly expressed in neurons, and a recent report has shown that an N-terminal alteration is associated with a heritable dystonia. Together, these data demonstrate that a single de novo mutation in TUBB4A results in H-ABC. Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC [MIM 612438]) is a rare leukodystrophy described by Van der Knaap et al. in 2002.1van 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 To date, only 22 individual cases have been reported. It is characterized by variable onset (from infancy to childhood), developmental delay, extrapyramidal movement disorders (dystonia, choreoathetosis, rigidity, opisthotonus, and oculogyric crises), progressive spastic tetraplegia, ataxia, and, more rarely, seizures.1van 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 MRI plays a fundamental role in the diagnostic work-up because neuroradiological findings are pathognomonic; diagnostic criteria include the combination of hypomyelination, cerebellar atrophy, and absence or disappearance of the putamen,1van 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 all features that have been comprehensively confirmed in a recent autopsy case.2van der Knaap M.S. Linnankivi T. Paetau A. Feigenbaum A. Wakusawa K. Haginoya K. Köhler W. Henneke M. Dinopoulos A. Grattan-Smith P. et al.Hypomyelination with atrophy of the basal ganglia and cerebellum: follow-up and pathology.Neurology. 2007; 69: 166-171Crossref PubMed Scopus (48) Google Scholar The disease appears to be sporadic in nature given that there are no previously published sibling groups. The lack of familial groups has thus far foiled attempts at identifying a causative mutation or rare variant. It has also been somewhat uncertain whether H-ABC represents a single disorder or the common neuroradiologic manifestations of a heterogeneous group of disorders. The reported finding of low levels of 5-methyltetrahydrofolate in the cerebrospinal fluid (CSF) of one affected individual resulted in a therapeutic trial with folinic acid, which led to an improvement.3Mercimek-Mahmutoglu S. Stockler-Ipsiroglu S. Cerebral folate deficiency and folinic acid treatment in hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) syndrome.Tohoku J. Exp. Med. 2007; 211 (author reply 97): 95-96Crossref PubMed Scopus (17) Google Scholar, 4Mercimek-Mahmutoglu S. van der Knaap M.S. Baric I. Prayer D. Stoeckler-Ipsiroglu S. Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). Report of a new case.Neuropediatrics. 2005; 36: 223-226Crossref PubMed Scopus (22) Google Scholar It was then suggested that H-ABC could be related to cerebral folate deficiency3Mercimek-Mahmutoglu S. Stockler-Ipsiroglu S. Cerebral folate deficiency and folinic acid treatment in hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) syndrome.Tohoku J. Exp. Med. 2007; 211 (author reply 97): 95-96Crossref PubMed Scopus (17) Google Scholar, 4Mercimek-Mahmutoglu S. van der Knaap M.S. Baric I. Prayer D. Stoeckler-Ipsiroglu S. Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). Report of a new case.Neuropediatrics. 2005; 36: 223-226Crossref PubMed Scopus (22) Google Scholar and, conversely, that because CSF analysis was normal in some affected individuals, H-ABC could be considered a symptomatic representation of heterogeneous disorders.5Wakusawa K. Uematsu M. Tsuchiya S. Haginoya K. Blau N. The cerebrospinal fluid level of 5-methylterahydrofolate in a Japanese boy with hypomyelination with atrophy of the basal ganglia and cerebellum.Tohoku J. Exp. Med. 2007; 213: 373Crossref PubMed Scopus (3) Google Scholar Additionally, an H-ABC-affected individual with a partial response to L-dopa has been reported,6Wakusawa K. Haginoya K. Kitamura T. Togashi N. Ishitobi M. Yokoyama H. Higano S. Onuma A. Nara T. Iinuma K. Effective treatment with levodopa and carbidopa for hypomyelination with atrophy of the basal ganglia and cerebellum.Tohoku J. Exp. Med. 2006; 209: 163-167Crossref PubMed Scopus (15) Google Scholar and a 21-month-old boy with Down syndrome and a neuroradiologic presentation consistent with H-ABC has been described.7Narumi Y. Shiihara T. Yoshihashi H. Sakazume S. van der Knaap M.S. Nishimura-Tadaki A. Matsumoto N. Fukushima Y. Hypomyelination with atrophy of the basal ganglia and cerebellum in an infant with Down syndrome.Clin. Dysmorphol. 2011; 20: 166-167Crossref PubMed Scopus (3) Google Scholar The advent of exome-sequencing technology provides the opportunity to study individual H-ABC cases and to establish whether this disorder is indeed a single monogenic entity or a clustering of heterogeneous leukoencephalopathies. To understand the genetic underpinning of H-ABC, we recruited affected individuals and their family members via the Myelin Disorders Bioregistry Project or the Amsterdam Database of Unclassified Leukoencephalopathies with approval from the institutional review board at Children’s National Medical Center, the Baylor Neurogenetic Institute, VU University Medical Center, or the University of Queensland. Written informed consent was obtained for each study participant. Genomic DNA samples were collected from blood samples provided to the biorepositories, and in the case of family LD_0638, additional genomic DNA was isolated from buccal and saliva samples. A total of 11 individuals from ten unrelated families fulfilling the MRI criteria for H-ABC diagnosis were included in this study (Figure 1 and Table 1). All affected individuals presented in infancy or early childhood with predominant motor dysfunction, which often manifested as delayed acquisition of milestones or unsteady walking. Most of these individuals had deterioration of motor skills, often marked initially by hemidystonia. Gait progressively deteriorated in all cases, and independent and even supported ambulation was lost over time. Language and cognitive development appeared relatively preserved, although over time, dysarthria increasingly impaired communication. Receptive language was often normal: many children functioned at age-appropriate levels.Table 1Clinical Manifestation of Individuals with H-ABCIndividualLD_0313.0LD_0345.0LD_0440.0LD_0605.0LD_0638.0ALD_0638.0BHA04HA07HA23HA27HA107GendermalefemalemalefemalemalemalefemalemalemalemalefemaleEthnicitywhiteArabwhiteHispanicAsianAsianwhitewhitewhitewhitewhiteAge at presentation1.5 years1 years3 years9 months2 years4.5 years1 years20 months6 months2.5 years2 yearsCurrent age11 years7 years39 years30 years5 years8 years23 years21 years19 years29 years6 yearsPresenting signmild gait instability; unclear speechgait instability; delayed speechmotormotormild gait instability; unclear speechdystonia and dysarthriadelayed motor development; hypotoniadelayed motor development; spasticitydelayed motor development; hypotoniaprogressive motor problemsdelayed speech development; lack of motor developmental progressInitial motor developmentdelayed; best skill was unsupported walking at 17 months; normal fine motor skillsdelayed; best skill was the ability to take a few unsupported steps at 1 yeardelayeddelayeddelayed; best skill was unsupported walking at 2 yearsnormaldelayed; able to stand at 12 months; walked with support at 24 months; walked a few steps without support at 33 monthsnormal; sat at 11 months; pulled to stand at 12 months; walked with support 18 monthsdelayed; sat at 18 months; walked with support at 3.5 yearsnormal; walked without support at 16 months; walked on toesnormal; walked without support at 13 months; wide-based gaitOnset of motor deterioration4.5 years; hemidystonia2 years3 years7 years4.5 years; hemidystonia4.5 years; hemidystonia7 years2 years; deterioration of hand function6 years2.5 years4 yearsLoss of supported walking ability5 years2 years10 years10 yearssupported walking at last examination at 5.5 yearssupported walking at last examination at 7.5 years8 years9 years?14 years4 yearsSpasticity+++++++++++Ataxia+++−−−+++++Tremor−−++−++++++Choreoathetosis+−+−−−+++−−Dystonia+++−+++++++Rigidity−++−+++++++Dysarthria+++++++++++Oculomotor abnormalitiesnystagmus; oculomotor apraxia; hypometric saccadesnonenonenonehypometric saccadesnoneoculomotor apraxianormalnormalrotatory and pendular nystagmusnoneVisionnormalnormalnormalnormalnormalnormaldecreasednormalnormalnormalnormalHearingnormalnormalnormalnormalnormalnormalnormalnormalnormalsensorineural deafness (homozygous otoa deletion); cochlear implantnormalCognitive decline++++−++mild intellectual disabilitymild intellectual disability−−Language developmentinitially normal; loss of most spontaneous speech over time; normal receptive skillsdelayed; best skill was five to ten intelligible words; normal receptive language skillsdelayed; single words until age 6 years; better receptive skillsdelayed; single words until age 7 years; better receptive skillsnormalinitially normal, although great loss of intelligibility over timeinitially normal; 2 word sentences at 2 years; increasing dysarthriadelayed; increasing dysarthriaspoke syllables; no active speech; uses computer-assisted communicationalmost normal at 6 years; increasing dysarthria; loss of active speech at 12 yearsno active speech; uses signs and computer-assisted communicationEpilepsy−−−−−−−−−−+Individual LD_0440.0 was described by van der Knaap et al. in 2002 (individual 7),1van 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 and individual LD_0605.0 was described by van der Knaap et al. in 2007 (individual 10).2van der Knaap M.S. Linnankivi T. Paetau A. Feigenbaum A. Wakusawa K. Haginoya K. Köhler W. Henneke M. Dinopoulos A. Grattan-Smith P. et al.Hypomyelination with atrophy of the basal ganglia and cerebellum: follow-up and pathology.Neurology. 2007; 69: 166-171Crossref PubMed Scopus (48) Google Scholar Open table in a new tab Individual LD_0440.0 was described by van der Knaap et al. in 2002 (individual 7),1van 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 and individual LD_0605.0 was described by van der Knaap et al. in 2007 (individual 10).2van der Knaap M.S. Linnankivi T. Paetau A. Feigenbaum A. Wakusawa K. Haginoya K. Köhler W. Henneke M. Dinopoulos A. Grattan-Smith P. et al.Hypomyelination with atrophy of the basal ganglia and cerebellum: follow-up and pathology.Neurology. 2007; 69: 166-171Crossref PubMed Scopus (48) Google Scholar To identify the disease-causing mutation or variant, we performed exome sequencing on each of the 11 affected individuals, the unaffected parents in seven of the families, and one unaffected sibling. In brief, exomes were captured with the SeqCap EZ Human Exome Library v.3.0 and sequenced on an Illumina HiSeq 2000 with the 100 bp paired-end read-sequencing protocol at the Queensland Centre for Medical Genomics or the VU University Medical Center sequencing center. Reads were aligned to the reference human genome (UCSC Genome Browser hg19) with the Burrows-Wheeler Aligner (BWA),8Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26648) Google Scholar and downstream processing of sequence data was done with Picard v.1.8, SAMtools v.0.1.18,9Cerritelli S.M. Crouch R.J. Ribonuclease H: the enzymes in eukaryotes.FEBS J. 2009; 276: 1494-1505Crossref PubMed Scopus (495) Google Scholar and the Genome Analysis Toolkit (GATK) v.2.2.8.10McKenna A. Hanna M. Banks E. Sivachenko A. Cibulskis K. Kernytsky A. Garimella K. Altshuler D. Gabriel S. Daly M. DePristo M.A. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14776) Google Scholar Variants (SNPs and indels) were identified with GATK according to version four of the GATK Best Practice Variant Detection guide11DePristo M.A. Banks E. Poplin R. Garimella K.V. Maguire J.R. Hartl C. Philippakis A.A. del Angel G. Rivas M.A. Hanna M. et al.A framework for variation discovery and genotyping using next-generation DNA sequencing data.Nat. Genet. 2011; 43: 491-498Crossref PubMed Scopus (7101) Google Scholar or Varscan v.2.2.5.12Koboldt D.C. Chen K. Wylie T. Larson D.E. McLellan M.D. Mardis E.R. Weinstock G.M. Wilson R.K. Ding L. VarScan: variant detection in massively parallel sequencing of individual and pooled samples.Bioinformatics. 2009; 25: 2283-2285Crossref PubMed Scopus (870) Google Scholar Variants were annotated with the use of Annovar13Wang K. Li M. Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (7872) Google Scholar with UCSC Known Genes models, and known polymorphisms were identified with dbSNP135, 1000 Genomes (April 30, 2012, release), and the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP) Exome Variant Server (ESP6500 release), and minor allele frequencies were recorded from each data set. Subsequent analysis and identification of candidate variants was performed with an in-house workflow incorporating the annotated variant data and pedigree information. We produced 8–30 Gb of sequence for each individual, yielding a mean depth of 65-fold coverage and an average of 95% of target bases sequenced at least 18 times (Table S1, available online). A single heterozygous de novo mutation in TUBB4A (MIM 602662; RefSeq accession number NM_006087) was identified in all affected individuals (Table 2; Sanger sequencing validation is shown in Figure S1) but was absent from all unaffected parents and siblings with the exception of the mother in family LD_0638 (more below). This mutation—c.745G>A (RefSeq NM_006087.2) (g.6495765C>T) (RefSeq NC_000019.9)—is not present in dbSNP135, the 1000 Genomes Project database, or the NHLBI Exome Variant Server, consistent with its presumed pathogenicity and the relative population incidence of H-ABC.Table 2H-ABC-Associated TUBB4A GenotypesFamilyIndividualAffectedGenotypeWild-Type Reads (G)Alternative Reads (A)LD_0313LD_0313.0AyesG/A132118LD_0313.1noG/G1389LD_0313.2noG/G766LD_0345LD_0345.0AyesG/A5955LD_0345.1noG/G555LD_0345.2noG/G1052LD_0440LD_0440.0AyesG/A11481LD_0440.01noG/G1322LD_0440.1noG/G664LD_0440.2noG/G2462LD_0605LD_0605.0AyesG/A127121LD_0605.1noG/G1737LD_0605.2noG/G1444LD_0638LD_0638.0AyesG/A124124LD_0638.0ByesG/A140110LD_0638.1noG/A19354LD_0638.2noG/G2412HA04HA04yesG/A4340HA07HA07yesG/A4140HA23HA23yesG/A4635HA27HA27yesG/A4242HA28noG/G501HA29noG/G500HA107HA107yesG/A5165HA108noG/G1270HA109noG/G1180 Open table in a new tab TUBB4A (also known as TUBB4 and TUBB5) is a member of the highly conserved β-tubulin protein family that forms heterodimers with α-tubulins and then in turn forms copolymers that assemble into microtubules, an essential component of the cytoskeleton. TUBB4A is primarily expressed in the nervous system,14Lee M.G. Loomis C. Cowan N.J. Sequence of an expressed human beta-tubulin gene containing ten Alu family members.Nucleic Acids Res. 1984; 12: 5823-5836Crossref PubMed Scopus (78) Google Scholar, 15Su A.I. Wiltshire T. Batalov S. Lapp H. Ching K.A. Block D. Zhang J. Soden R. Hayakawa M. Kreiman G. et al.A gene atlas of the mouse and human protein-encoding transcriptomes.Proc. Natl. Acad. Sci. USA. 2004; 101: 6062-6067Crossref PubMed Scopus (2848) Google Scholar, 16Hersheson J. Mencacci N.E. Davis M. Macdonald N. Trabzuni D. Ryten M. Pittman A. Paudel R. Kara E. Fawcett K. et al.Mutations in the autoregulatory domain of β-tubulin 4a cause hereditary dystonia.Ann. Neurol. 2012; (Published online December 13, 2012)https://doi.org/10.1002/ana.23832Crossref PubMed Scopus (127) Google Scholar and its role in H-ABC is supported by several studies showing that neurological disorders characterized by abnormal neuronal migration, differentiation, and axon guidance and maintenance have been attributed to mutations in the α-tubulin- and β-tubulin-encoding genes TUBA1A (MIM 602529), TUBA8 (MIM 605742), TUBB2B (MIM 612850), and TUBB3 (MIM 602661).17Tischfield M.A. Cederquist G.Y. Gupta Jr., M.L. Engle E.C. Phenotypic spectrum of the tubulin-related disorders and functional implications of disease-causing mutations.Curr. Opin. Genet. Dev. 2011; 21: 286-294Crossref PubMed Scopus (153) Google Scholar, 18Cushion T.D. Dobyns W.B. Mullins J.G. Stoodley N. Chung S.K. Fry A.E. Hehr U. Gunny R. Aylsworth A.S. Prabhakar P. et al.Overlapping cortical malformations and mutations in TUBB2B and TUBA1A.Brain. 2013; 136: 536-548Crossref PubMed Scopus (110) Google Scholar Indeed, an autosomal-dominant mutation (c.4C>G [p.Arg2Gly]) in TUBB4A was recently identified in an extended family affected by dystonia type 4 (DYT4 [MIM 128101]; Figure 2A).16Hersheson J. Mencacci N.E. Davis M. Macdonald N. Trabzuni D. Ryten M. Pittman A. Paudel R. Kara E. Fawcett K. et al.Mutations in the autoregulatory domain of β-tubulin 4a cause hereditary dystonia.Ann. Neurol. 2012; (Published online December 13, 2012)https://doi.org/10.1002/ana.23832Crossref PubMed Scopus (127) Google Scholar This disorder is characterized by a “whispering” dysphonia, generalized dystonia, and gait ataxia with onset in the second to fourth decade. In contrast to individuals with H-ABC, however, individuals with DYT4 are reported to have a normal MRI.20Wilcox R.A. Winkler S. Lohmann K. Klein C. Whispering dysphonia in an Australian family (DYT4): a clinical and genetic reappraisal.Mov. Disord. 2011; 26: 2404-2408Crossref PubMed Scopus (50) Google Scholar Although H-ABC is a hypomyelinating leukoencephalopathy, it is distinguished from other disorders in this class by the presence of abnormalities of the deep gray nuclei, suggesting neuronal involvement. Further pathologic studies will be necessary to establish whether the H-ABC-related TUBB4A mutation results in cytoskeletal abnormalities in neurons and glia. The TUBB4A c.745G>A mutation (RefSeq NM_006087.2) identified in this study causes a nonsynonymous change to an aspartic acid (p.Asp249Asn) (Figure 2A) that is highly conserved in all β-tubulins spanning from yeast to primates (Figure 2B),19Löwe J. Li H. Downing K.H. Nogales E. Refined structure of α β-tubulin at 3.5 A resolution.J. Mol. Biol. 2001; 313: 1045-1057Crossref PubMed Scopus (991) Google Scholar and it is predicted to be “probably damaging” (score 1.0) by PolyPhen-2 and “damaging” (score 0.001) by SIFT.21Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. Kondrashov A.S. Sunyaev S.R. A method and server for predicting damaging missense mutations.Nat. Methods. 2010; 7: 248-249Crossref PubMed Scopus (9290) Google Scholar, 22Kumar P. Henikoff S. Ng P.C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.Nat. Protoc. 2009; 4: 1073-1081Crossref PubMed Scopus (5006) Google Scholar Asp249 is located within the TUBB4A T7 loop, which interacts with the guanosine triphosphate (GTP) nucleotide bound to the N-site of the α-tubulin and is important for the longitudinal interaction between tubulins (Figure 2C).19Löwe J. Li H. Downing K.H. Nogales E. Refined structure of α β-tubulin at 3.5 A resolution.J. Mol. Biol. 2001; 313: 1045-1057Crossref PubMed Scopus (991) Google Scholar Additionally, Asp249 forms a salt bridge with the β-tubulin N-terminal residue Arg2 (altered in DYT4), an interaction that is likely to be important for positioning of T7-loop residues that interact with the α-tubulin-bound GTP (Figure 2D). We predict that disruption of the interaction between Asp249 and Arg2 might lead to inefficient dimerization, reduced microtubule polymerization, or reduced microtubule stability. Intriguingly, this alteration has been identified in other β-tubulin isotypes or subfamily members in other animal species. For example, a dominant p.Asp249Asn substitution has been described in a hematopoietic-specific β-tubulin isotype in Cavalier King Charles spaniels, where it is responsible for the inherited disease macrothrombocytopenia.23Davis B. Toivio-Kinnucan M. Schuller S. Boudreaux M.K. Mutation in beta1-tubulin correlates with macrothrombocytopenia in Cavalier King Charles Spaniels.J. Vet. Intern. Med. 2008; 22: 540-545Crossref PubMed Scopus (64) Google Scholar In Caenorhabditis elegans, a c.745G>A (p.Asp249Asn) mutation in the β-tubulin-encoding gene mec-7 leads to a dominant loss-of-touch-sensitivity phenotype caused by disrupted migration of the specialized touch receptor neuron cells.24Savage C. Xue Y. Mitani S. Hall D. Zakhary R. Chalfie M. Mutations in the Caenorhabditis elegans beta-tubulin gene mec-7: effects on microtubule assembly and stability and on tubulin autoregulation.J. Cell Sci. 1994; 107: 2165-2175PubMed Google Scholar It is likely that the H-ABC-associated TUBB4A mutation might similarly disrupt neuronal growth or axonal function. Given the severity of H-ABC and its seemingly sporadic presentation, it had been previously proposed that this disease was likely to be caused by a de novo mutation.2van der Knaap M.S. Linnankivi T. Paetau A. Feigenbaum A. Wakusawa K. Haginoya K. Köhler W. Henneke M. Dinopoulos A. Grattan-Smith P. et al.Hypomyelination with atrophy of the basal ganglia and cerebellum: follow-up and pathology.Neurology. 2007; 69: 166-171Crossref PubMed Scopus (48) Google Scholar In our family cohort study, however, one family (LD_0638) includes siblings diagnosed with H-ABC. To resolve this apparent incongruity, we performed a detailed investigation of the exome-sequencing data from this family quartet. It revealed that, as expected, the father (LD_0638.2) is homozygous for the reference base and that both affected children (LD_0638.0A and LD_0638.0B) are heterozygous for the TUBB4A mutation. The initial SNP call for the mother (LD_0638.1), however, was annotated as “heterozygous,” despite the fact that she is asymptomatic, suggesting either that we were incorrect about the pathogenicity of the TUBB4A mutation or that some other subtle genetic process was confounding our results. Detailed inspection of the sequence data derived from the mother revealed 193 reads that supported the wild-type allele, whereas only 54 reads supported the mutant allele. This ratio of 3.6:1 is substantially higher than the ratios observed in any of the 11 heterozygous H-ABC-affected individuals in this study (1.0:1–1.4:1; Table 2). Given the high read depth for all individuals at this locus (minimum of 50-fold coverage), these results suggest that individual LD_0638.1 could be mosaic for the c.745G>A variant. To confirm mosaicism, we collected additional DNA samples from saliva and buccal cells of individuals LD_0638.1 and LD_0638.2. A 216 nt genomic fragment centered on the c.745G>A mutation was amplified with primers 5′-CAACGAGGCACTCTACGACA-3′ and 5′-CTGGTCAGGGGTGCGAAG-3′, and 1 ng of each PCR product was prepared for sequencing with the Nextera XT Library Preparation Kit. Sequencing of the pooled libraries was completed according to the manufacturer’s recommendations with the MiSEQ v.2 instrument and the MiSeq Reagent Kit, which generated paired 150 bp reads. Reads were aligned to the reference human genome (UCSC Genome Browser hg19) with the BWA tool with default parameters.8Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26648) Google Scholar We obtained more than 1,000,000-fold coverage over the TUBB4A de novo mutation site in each sample tested (Table 3). The results showed that in the asymptomatic mother (LD_0638.1), the c.745G>A allele was present in 25% of reads from blood, 29% of reads from buccal cell DNA, and 26% of reads from salivary DNA, suggesting a level of mosaicism between 50% and 58% (Table 3). These results suggest that rare de novo mutations that are initially phenotypically neutral in a mosaic individual can be disease causing in the subsequent generation if they are inherited.Table 3Amplicon Sequencing of Family LD_0638 Shows that LD_0638.1 Is Mosaic for the TUBB4A c.745G>A VariantIndividualAffectedDNA SourceWild-Type Reads (G)Alternative Reads (A)Percentage of Alternative ReadsLD_0638.1noblood1,339,079446,65225%buccal1,023,413426,82129%saliva1,003,089354,85926%LD_0638.2noblood1,386,04815800%buccal1,071,5897010%saliva991,8801,0110%LD_0638.0Ayesblood838,773785,68448%LD_0638.0Byesblood680,222637,58548% Open table in a new tab Individuals with H-ABC have cerebellar atrophy, basal ganglia degeneration with a predilection for the putamen, and a striking lack of cerebral myelin development (hypomyelination). Intriguingly, expression data of TUBB4A in normal human brain samples suggest that it has its highest expression in the cerebellum, putamen, and supratentorial white matter.16Hersheson J. Mencacci N.E. Davis M. Macdonald N. Trabzuni D. Ryten M. Pittman A. Paudel R. Kara E. Fawcett K. et al.Mutations in the autoregulatory domain of β-tubulin 4a cause hereditary dystonia.Ann. Neurol. 2012; (Published online December 13, 2012)https://doi.org/10.1002/ana.23832Crossref PubMed Scopus (127) Google Scholar Although individuals with DYT4 share many phenotypic characteristics (including dysphonia, dystonia, and ataxia) with those with H-ABC, MRI features such as hypomyelination and disappearance of the putamen over time are not reported. The p.Arg2Gly alteration causing DYT4 is within the MREI (Met-Arg-Glu-Ile) autoregulatory motif of β-tubulin proteins; this motif is responsible for regulating the abundance of β-tubulins and their encoding mRNA in the cell, which might be a partial explanation for the different presentations of H-ABC and DYT4. All individuals who have the H-ABC phenotype and who have been tested thus far demonstrate a single mutation, but the finding of TUBB4A mutations in individuals with DYT4 suggests that other mutations in this gene could result in a phenotype of primary dystonia with or without involvement of the cerebral white matter. We hypothesize that the single de novo TUBB4A mutation identified in individuals with H-ABC affects gene function in a dominant-negative fashion and leads to the loss of, or inefficient, dimerization of microtubules. This prediction, however, requires further validation in models of disease and in human material. Given TUBB4A expression in neuronal cells and previous pathologic descriptions,2van der Knaap M.S. Linnankivi T. Paetau A. Feigenbaum A. Wakusawa K. Haginoya K. Köhler W. Henneke M. Dinopoulos A. Grattan-Smith P. et al.Hypomyelination with atrophy of the basal ganglia and cerebellum: follow-up and pathology.Neurology. 2007; 69: 166-171Crossref PubMed Scopus (48) Google Scholar we also hypothesize that the H-ABC-related TUBB4A mutation results in a primary disturbance of neurons and the secondary involvement of glial cells. The finding of TUBB4A mutations provides further insight into the complex interplay among cellular cytoskeleton, function, and glial-neuronal interactions. This study was supported by the National Institutes of Health Intramural Program, the Myelin Disorders Bioregistry Project, ZonMw TOP grant 91211005, the Institute for Molecular Bioscience Core Sequencing facility, the NeCTAR Genomics Virtual Lab (G.V.L.), and a University of Queensland Foundation Research Excellence Award. A.V. is supported by K08NS060695. R.J.T. is supported by an Australian Research Council Discovery Early Career Research Award and is a consultant to Isis Pharmaceuticals. Download .pdf (.5 MB) Help with pdf files Document S1. Figure S1 and Table S1 The URLs for data presented herein are as follows:NHLBI Exome Sequencing Project (ESP) Exome Variant Server, http://evs.gs.washington.edu/EVS/Online Mendelian Inheritance in Man (OMIM), http://www.omim.orgRefSeq, http://www.ncbi.nlm.nih.gov/RefSeqUCSC Genome Browser, http://genome.ucsc.eduVarScan, http://varscan.sourceforge.net
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