Exome Sequencing Identifies a DYNC1H1 Mutation in a Large Pedigree with Dominant Axonal Charcot-Marie-Tooth Disease
2011; Elsevier BV; Volume: 89; Issue: 2 Linguagem: Inglês
10.1016/j.ajhg.2011.07.002
ISSN1537-6605
AutoresMichael N. Weedon, Robert Hastings, Richard Caswell, Weijia Xie, Konrad Paszkiewicz, Thalia Antoniadi, Maggie Williams, Cath King, Lynn Greenhalgh, Ruth Newbury‐Ecob, Sian Ellard,
Tópico(s)Cellular Mechanics and Interactions
ResumoCharcot-Marie-Tooth disease is characterized by length-dependent axonal degeneration with distal sensory loss and weakness, deep-tendon-reflex abnormalities, and skeletal deformities. It is caused by mutations in more than 40 genes. We investigated a four-generation family with 23 members affected by the axonal form (type 2), for which the common causes had been excluded by Sanger sequencing. Exome sequencing of three affected individuals separated by eight meioses identified a single shared novel heterozygous variant, c.917A>G, in DYNC1H1, which encodes the cytoplasmic dynein heavy chain 1 (here, novel refers to a variant that has not been seen in dbSNP131or the August 2010 release of the 1000 Genomes project). Testing of six additional affected family members showed cosegregation and a maximum LOD score of 3.6. The shared DYNC1H1 gene variant is a missense substitution, p.His306Arg, at a highly conserved residue within the homodimerization domain. Three mouse models with different mutations within this domain have previously been reported with age-related progressive loss of muscle bulk and locomotor ability. Cytoplasmic dynein is a large multisubunit motor protein complex and has a key role in retrograde axonal transport in neurons. Our results highlight the importance of dynein and retrograde axonal transport in neuronal function in humans. Charcot-Marie-Tooth disease is characterized by length-dependent axonal degeneration with distal sensory loss and weakness, deep-tendon-reflex abnormalities, and skeletal deformities. It is caused by mutations in more than 40 genes. We investigated a four-generation family with 23 members affected by the axonal form (type 2), for which the common causes had been excluded by Sanger sequencing. Exome sequencing of three affected individuals separated by eight meioses identified a single shared novel heterozygous variant, c.917A>G, in DYNC1H1, which encodes the cytoplasmic dynein heavy chain 1 (here, novel refers to a variant that has not been seen in dbSNP131or the August 2010 release of the 1000 Genomes project). Testing of six additional affected family members showed cosegregation and a maximum LOD score of 3.6. The shared DYNC1H1 gene variant is a missense substitution, p.His306Arg, at a highly conserved residue within the homodimerization domain. Three mouse models with different mutations within this domain have previously been reported with age-related progressive loss of muscle bulk and locomotor ability. Cytoplasmic dynein is a large multisubunit motor protein complex and has a key role in retrograde axonal transport in neurons. Our results highlight the importance of dynein and retrograde axonal transport in neuronal function in humans. Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder and has an estimated prevalence of 1 in 2500. It is a chronic motor and sensory polyneuropathy characterized by distal muscle weakness and atrophy that might be associated with sensory loss, depressed tendon reflexes, and pes cavus. There are two main forms: the demyelinating type 1 that affects myelin (CMT1) and the axonal type 2 affecting the nerve axon (CMT2). Autosomal-dominant inheritance is most common, but autosomal-recessive and X-linked forms are also seen. There is considerable genetic heterogeneity, and more than 40 genes or loci have been identified.1Patzkó A. Shy M.E. Update on Charcot-Marie-Tooth disease.Curr. Neurol. Neurosci. Rep. 2011; 11: 78-88Crossref PubMed Scopus (106) Google Scholar We studied a four-generation family with 23 members affected (Figure 1) with CMT2, characterized by delayed motor milestones and/or an abnormal gait. Sanger sequencing excluded the genes commonly associated with CMT2; MPZ (MIM 159440), NEFL (MIM 162280), MFN2 (MIM 608507), and LMNA (MIM 150330). Male to male transmission excluded GJB1 (MIM 304040), and after sequencing of PMP22 (MIM 601097) failed to identify a pathogenic mutation, we employed a whole-exome sequencing strategy. Exonic sequences from three affected individuals (IV-2, -7, and -14) separated by eight meioses were enriched from genomic DNA with Agilent's SureSelect whole-exome kit (version 1) as recommended by the manufacturers except that gDNA fragmentation was carried out with a Diagenode Bioruptor. After hybridization reactions, captured DNA was amplified with 12 cycles of PCR then sequenced on an Illumina GAII sequencer with 76 bp paired-end reads. We used Novoalign (Novocraft Technologies) to align sequence reads to the hg19 reference genome and removed any duplicate reads from subsequent analyses. Seventy-six percent of targeted Consensus Coding Sequence project (CCDS) exonic bases were covered with at least 20 reads with an average coverage of 70× (Table S1, available online). We used Samtools2Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (24672) Google Scholar to call SNPs and indels. Annovar3Wang K. Li M. Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (6208) Google Scholar was used for annotation of variants, and we filtered variants by using dbSNP131, 1000 Genomes (August 2010 release), and our in-house (exome data from 11 individuals of European descent) databases. Informed consent was obtained from all participants. We assumed a rare autosomal-dominant model of inheritance, and after filtering, we found the three individuals had 177, 192, and 199 novel heterozygous variants (here, novel refers to a variant that has not been seen in dbSNP131or the August 2010 release of the 1000 Genomes project). annotated as missense, nonsense, frameshift, or splice site (Table 1 and Table S2). Four to six variants were shared by each pair, but only one variant in DYNC1H1 (NM_001376.4 [MIM 600112]) was shared by all three (the number expected on the basis of the number of meioses separating these individuals). Testing of six additional affected family members by Sanger sequencing showed cosegregation with a maximum LOD score of 3.6 (see Figure 1). The variant was not present in 322 ethnically matched control chromosomes. The shared DYNC1H1 variant is a missense change, p.His306Arg (c.917A>G), at a highly conserved residue (Figure S1) within the homodimerization domain of cytoplasmic dynein heavy chain 1.Table 1Novel Heterozygous Missense, Nonsense, Frameshift, or Splice-Site VariantsIndividualsSharedIV-2IV-7IV-14IV-2 and IV-7IV-2 and IV-14IV-7 and IV-14All1771921996541Number of novel heterozygous variants in the exome of each sequenced individual and the number shared across the individuals. Here, “novel” refers to a variant that has not been seen in dbSNP131, the August 2010 release of the 1000 Genomes project, or 11 in-house control exomes processed with the same laboratory and analysis pipeline. Open table in a new tab Number of novel heterozygous variants in the exome of each sequenced individual and the number shared across the individuals. Here, “novel” refers to a variant that has not been seen in dbSNP131, the August 2010 release of the 1000 Genomes project, or 11 in-house control exomes processed with the same laboratory and analysis pipeline. Cytoplasmic dynein is a large multisubunit motor protein complex that has a range of cellular functions. In particular, it is the primary motor protein responsible for retrograde axonal transport in neurons—the movement of cargo such as organelles from the cell periphery to the cell body along cytoskeletal microtubules. A homodimer of the 532 kDa cytoplasmic dynein heavy chain 1 forms the core of dynein and is responsible for the protein complex binding to and moving along microtubules.4Banks G.T. Fisher E.M. Cytoplasmic dynein could be key to understanding neurodegeneration.Genome Biol. 2008; 9: 214Crossref PubMed Scopus (23) Google Scholar Other subunits of dynein include the intermediate, light-intermediate, and light chains, thought to be responsible for interacting with cargo and maintaining the stability of the complex.4Banks G.T. Fisher E.M. Cytoplasmic dynein could be key to understanding neurodegeneration.Genome Biol. 2008; 9: 214Crossref PubMed Scopus (23) Google Scholar Our results are corroborated by previous animal studies that have implicated disruption of Dync1h1 in neuropathic disease. Legs at odd angles (Loa)5Hafezparast M. Klocke R. Ruhrberg C. Marquardt A. Ahmad-Annuar A. Bowen S. Lalli G. Witherden A.S. Hummerich H. Nicholson S. et al.Mutations in dynein link motor neuron degeneration to defects in retrograde transport.Science. 2003; 300: 808-812Crossref PubMed Scopus (554) Google Scholar, Cramping 1 (Cra1)5Hafezparast M. Klocke R. Ruhrberg C. Marquardt A. Ahmad-Annuar A. Bowen S. Lalli G. Witherden A.S. Hummerich H. Nicholson S. et al.Mutations in dynein link motor neuron degeneration to defects in retrograde transport.Science. 2003; 300: 808-812Crossref PubMed Scopus (554) Google Scholar and Sprawling (Swl)6Chen X.J. Levedakou E.N. Millen K.J. Wollmann R.L. Soliven B. Popko B. Proprioceptive sensory neuropathy in mice with a mutation in the cytoplasmic Dynein heavy chain 1 gene.J. Neurosci. 2007; 27: 14515-14524Crossref PubMed Scopus (120) Google Scholar are mouse phenotypes that arose from N-ethyl-N-nitrosourea (ENU) or radiation-induced mutagenesis. Heterozygous mice have age-related progressive loss of muscle bulk and locomotor ability without a major reduction in life span.5Hafezparast M. Klocke R. Ruhrberg C. Marquardt A. Ahmad-Annuar A. Bowen S. Lalli G. Witherden A.S. Hummerich H. Nicholson S. et al.Mutations in dynein link motor neuron degeneration to defects in retrograde transport.Science. 2003; 300: 808-812Crossref PubMed Scopus (554) Google Scholar, 6Chen X.J. Levedakou E.N. Millen K.J. Wollmann R.L. Soliven B. Popko B. Proprioceptive sensory neuropathy in mice with a mutation in the cytoplasmic Dynein heavy chain 1 gene.J. Neurosci. 2007; 27: 14515-14524Crossref PubMed Scopus (120) Google Scholar Although there are differences between the mouse models, and indeed different pathogenesis suggested for the same mouse model by different researchers,7Courchesne S.L. Pazyra-Murphy M.F. Lee D.J. Segal R.A. Neuromuscular junction defects in mice with mutation of dynein heavy chain 1.PLoS ONE. 2011; 6: e16753Crossref PubMed Scopus (18) Google Scholar, 8Braunstein K.E. Eschbach J. Ròna-Vörös K. Soylu R. Mikrouli E. Larmet Y. René F. De Aguilar J.L. Loeffler J.P. Müller H.P. et al.A point mutation in the dynein heavy chain gene leads to striatal atrophy and compromises neurite outgrowth of striatal neurons.Hum. Mol. Genet. 2010; 19: 4385-4398Crossref PubMed Scopus (44) Google Scholar the mouse phenotype is broadly similar to that observed in our family. Affected patients typically have delayed motor milestones and/or an abnormal gait along with early-onset slowly progressive distal lower limb weakness and wasting with pes cavus deformity (see Figure S2). Clinical features of the best characterized family members are shown in Table 2. Upper limb involvement is less common, and ambulation is usually maintained through adulthood. Nerve conduction studies are within the normal range, sural nerve biopsies demonstrated minimal changes suggestive of increased axon degeneration, and muscle biopsies show findings consistent with secondary muscle involvement due to denervation (see Table 3). Transient paresthesia and neuropathic lower limb pains are also reported by several family members, typically those more severely affected. Although the core features consistent with CMT2 are seen across the family, there is significant variability in other findings: reflexes might be lost or preserved; fine touch, vibration sense, and proprioception are retained in many family members but lost in some; atypical features, such as predominant proximal muscle involvement, periscapular wasting and weakness, spinal and hip problems, and a broad-based waddling gait, are also found in a few.Table 2Clinical Features of Selected Family MembersPatientPresentationFeaturesII 10delayed motor milestones; recurrent surgery to feet and ankles in childhoodreduced power and wasting in distal upper and lower limbs; pes cavus; reduced proprioception, normal vibration sense; reflexes normalII 13pes cavus at birth; recurrent surgery to feet and ankles in childhooddistal lower limb weakness and wasting; pes cavus; reduced proprioception, pin-prick, fine touch and vibration sense; reflexes normal; significant neuropathic pain; depression and paraphrenia; extrapyramidal features probably due to antipsychoticsIII 4delayed motor milestones and recurrent fallsmild distal lower limb weakness and wasting; pes cavus; reduced fine touch, other sensory modalities normal; reflexes normalIII 7presented at age 11 with difficulties runningmild distal lower limb weakness and paresthesia; pes cavus; all sensory modalities reduced; reduced reflexesIII 9presented at age 14 with frequent fallsdistal upper and lower limb weakness and wasting (LL > UL); pes cavus; all sensory modalities reduced; reduced ankle reflexesIII 11talipes at birth; delayed motor milestonesdistal lower limb weakness and wasting; pes cavus; retained reflexes and sensation; retinoblastomaIII 14delayed motor milestonesbroad-based gait; distal lower limb weakness and wasting; reduced reflexes; sensory modalities retained; mild intention tremor; strabismusIII 17abnormal gait in early childhoodminimally reduced power and wasting in distal lower limbs; normal reflexes and sensation; no pes cavusIII 18delayed motor milestones; speech delayproximal and distal lower limbs weakness and wasting and scapula wasting; pes cavus and bilateral foot drop; reflexes reduced; paraesthesia but objectively normal sensation; significant neuropathic pain and back painIII 21delayed motor milestones and learning difficultiesproximal and distal lower limb weakness and wasting; reduced reflexes; normal sensory modality testing; lumbar lordosis and reduced hip movements; behavioral problems including school exclusionsIV 2abnormal gait and falls in early childhood; global developmental delaydistal lower limb weakness; no pes cavus; normal reflexes and sensationIV 7delayed motor milestones; speech delay and learning difficultiesbroad-based gait; distal lower limb weakness and wasting; no pes cavus; reduced reflexes; reduced proprioception, other sensory modalities normal; lumbar lordosis and reduced hip movements; Achilles-tendon-lengthening operationsIV 14delayed motor milestonesWaddling gait; proximal lower limb weakness more dominant than distal lower limb weakness; no pes cavus; reduced knee reflexes; sensory modalities normal; lumbar lordosis Open table in a new tab Table 3Clinical Investigation ResultsPatientAge at StudyMotorSensoryElectromyographySural Nerve BiopsyMuscle BiopsyRight PeronealRight TibialRight SuralRight Sup PeronealAmplitude (mV)Velocity (m/s)Amplitude (mV)Velocity (m/s)Pk-Pk (μV)Velocity (m/s)Pk-Pk (μV)Velocity (m/s)II 1029––3.85462039––chronic denervation––II 1331––4.3511241––central neuronal atrophy–severe connective tissue replacementII 1536normalnormalnormalnormalnormalnormalnormalnormal–indolent axon degenerative process with some evidence of fiber regeneration consistent with a diagnosis of HMSN type 2–III 4252.056.68.9–20.342.913.345.5normal––III 14285.444.27.139.68.637.77.840vastus medialis MUPs enlarged otherwise normal––III 1720normalnormalnormalnormalnormalnormalnormalnormal–––III 18222.94411.5–7281535mild degree of large fiber axonal sensory motor peripheral neuropathy––III 212normalnormalnormalnormalnormalnormalnormalnormalinterference pattern nonspecifically abnormal, occasional units up to 6 mV––IV 75not performednot performednot performednot performednot performednot performednot performednot performed––atrophic fibers consistent with chronic partial denervation Open table in a new tab The mouse mutations of Dync1h1 (NM_030238.2; c.1739T>A [p.Phe580Tyr] in Loa; c.3164A>G [p.Tyr1055Cys] in Cra; and a 9 bp deletion, c.3119_3127delGCATAGTGA [p.(Gly1040_Thr1043delinsAla)], in Swl) also affect residues within the homodimerization region of the dynein stem domain (Figure 2). The embryonic lethality of the homozygous Dync1h1 null mouse but normal phenotype of heterozygous null mice9Harada A. Takei Y. Kanai Y. Tanaka Y. Nonaka S. Hirokawa N. Golgi vesiculation and lysosome dispersion in cells lacking cytoplasmic dynein.J. Cell Biol. 1998; 141: 51-59Crossref PubMed Scopus (261) Google Scholar raises the possibility of a dominant-negative effect for the neuropathic mutations. Given the multiple functions of dynein, the exact mechanism by which these mutations cause disease is still unclear. One recent study of the Cra mouse suggested an impact on synapse structure at neuromuscular junctions,7Courchesne S.L. Pazyra-Murphy M.F. Lee D.J. Segal R.A. Neuromuscular junction defects in mice with mutation of dynein heavy chain 1.PLoS ONE. 2011; 6: e16753Crossref PubMed Scopus (18) Google Scholar whereas another proposed an effect on striatal neurons.8Braunstein K.E. Eschbach J. Ròna-Vörös K. Soylu R. Mikrouli E. Larmet Y. René F. De Aguilar J.L. Loeffler J.P. Müller H.P. et al.A point mutation in the dynein heavy chain gene leads to striatal atrophy and compromises neurite outgrowth of striatal neurons.Hum. Mol. Genet. 2010; 19: 4385-4398Crossref PubMed Scopus (44) Google Scholar However, a recent report with single-molecule and live-imaging techniques in the Loa mouse strongly suggested that defects in dynein motor processivity play a key role in disease causation.10Ori-McKenney K.M. Xu J. Gross S.P. Vallee R.B. A cytoplasmic dynein tail mutation impairs motor processivity.Nat. Cell Biol. 2010; 12: 1228-1234Crossref PubMed Scopus (126) Google Scholar Measurement of retrograde run-lengths (periods of uninterrupted motion) showed a marked reduction in the ability of mutant dynein to move along microtubules both in vitro and in vivo.10Ori-McKenney K.M. Xu J. Gross S.P. Vallee R.B. A cytoplasmic dynein tail mutation impairs motor processivity.Nat. Cell Biol. 2010; 12: 1228-1234Crossref PubMed Scopus (126) Google Scholar There was also evidence of altered interaction between the dynein motor and stem domains, and this interaction raises the possibility of motor domain miscoordination.10Ori-McKenney K.M. Xu J. Gross S.P. Vallee R.B. A cytoplasmic dynein tail mutation impairs motor processivity.Nat. Cell Biol. 2010; 12: 1228-1234Crossref PubMed Scopus (126) Google Scholar This reduction in processivity could explain the observed neuropathy phenotype because the neurons most affected will be those with long axons such as the motor and sensory neurons. Mutations in other axonal transport genes cause related neuronal diseases. Autosomal-dominant mutations in the p150 subunit of dynactin (also known as dynein activator) cause a form of motor neuron disease (MIM 601143)11Puls I. Jonnakuty C. LaMonte B.H. Holzbaur E.L. Tokito M. Mann E. Floeter M.K. Bidus K. Drayna D. Oh S.J. et al.Mutant dynactin in motor neuron disease.Nat. Genet. 2003; 33: 455-456Crossref PubMed Scopus (768) Google Scholar and a heterozygous KIF1B (MIM 605995) mutation results in CMT2A (MIM 118210).12Zhao C. Takita J. Tanaka Y. Setou M. Nakagawa T. Takeda S. Yang H.W. Terada S. Nakata T. Takei Y. et al.Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bbeta.Cell. 2001; 105: 587-597Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar KIF1B is a member of the kinesin superfamily of motor proteins that are involved in anterograde axonal transport (taking cargo from the cell body to the periphery of the neurons). Mutations in other kinesin genes have been shown to cause neuronal disease, for example KIF5A (MIM 602821) mutations cause hereditary spastic paraplegia (MIM 604187).13Reid E. Kloos M. Ashley-Koch A. Hughes L. Bevan S. Svenson I.K. Graham F.L. Gaskell P.C. Dearlove A. Pericak-Vance M.A. et al.A kinesin heavy chain (KIF5A) mutation in hereditary spastic paraplegia (SPG10).Am. J. Hum. Genet. 2002; 71: 1189-1194Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar Our findings provide further evidence of the importance of motor proteins and in particular retrograde axonal transport in the normal functioning of neurons. Our work has used exome sequencing in selected individuals from a large pedigree to identify a disease-causing mutation. Charcot-Marie-Tooth disease can be caused by mutations in over 40 genes, and screening with Sanger sequencing is costly and time consuming. DYNC1H1 (14q32.31) is not within a known region of linkage for CMT. An alternative approach,14Ostergaard P. Simpson M.A. Brice G. Mansour S. Connell F.C. Onoufriadis A. Child A.H. Hwang J. Kalidas K. Mortimer P.S. et al.Rapid identification of mutations in GJC2 in primary lymphoedema using whole exome sequencing combined with linkage analysis with delineation of the phenotype.J. Med. Genet. 2011; 48: 251-255Crossref PubMed Scopus (69) Google Scholar, 15Wang J.L. Yang X. Xia K. Hu Z.M. Weng L. Jin X. Jiang H. Zhang P. Shen L. Guo J.F. et al.TGM6 identified as a novel causative gene of spinocerebellar ataxias using exome sequencing.Brain. 2010; 133: 3510-3518Crossref PubMed Scopus (211) Google Scholar linkage analysis followed by candidate gene Sanger sequencing, utilized in other studies would still have required the sequencing of at least the 78 exons of DYNC1H1. In summary, we have identified a mutation in DYNC1H1 that causes Charcot-Marie-Tooth disease by using exome sequencing in a large dominant pedigree. The mouse models also harboring mutations in the homodimerization domain of cytoplasmic dynein heavy chain 1 support the pathogenicity of the human DYNC1H1 mutation and give valuable insights to pathophysiology. These results highlight the importance of dynein and retrograde axonal transport in neuronal function in humans. We would like to express our gratitude to the participating family and to Alex Moorhouse and Andy Brash for technical assistance. Sequence analysis of MFN2 was performed by the Institute of Neurology (London, UK). Download .pdf (.19 MB) Help with pdf files Document S1. Two Figures and Two Tables The URLs for data presented herein are as follows:1000 Genomes, http://www.1000genomes.org/Online Mendelian Inheritance in Man (OMIM), http://www.omim.org
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