Clin Genet 2016: 90: 127–133 © 2015 John Wiley & Sons A/S. Printed in Singapore. All rights reserved Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12712 Original Article Mutation analysis of genes within the dynactin complex in a cohort of hereditary peripheral neuropathies a a Tey S., Ahmad-Annuar A., Drew A.P., Shahrizaila N., Nicholson G.A., S. Tey , A. Ahmad-Annuar , Kennerson M.L. Mutation analysis of genes within the dynactin complex in A.P. Drewb, N. Shahrizailac, , a cohort of hereditary peripheral neuropathies. G.A. Nicholsonb d and Clin Genet 2016: 90: 127–133. © John Wiley & Sons A/S. Published by M.L. Kennersonb,d John Wiley & Sons Ltd, 2015 aDepartment of Biomedical Science, The cytoplasmic dynein–dynactin genes are attractive candidates for Faculty of Medicine, University of Malaya, b neurodegenerative disorders given their functional role in retrograde Kuala Lumpur, Malaysia, Northcott transport along neurons. The cytoplasmic dynein heavy chain (DYNC1H1) Neuroscience Laboratory, ANZAC Research Institute, and Sydney Medical gene has been implicated in various neurodegenerative disorders, and School, University of Sydney, Sydney, dynactin 1 (DCTN1) genes have been implicated in a wide spectrum of Australia, cDepartment of Medicine, disorders including motor neuron disease, Parkinson’s disease, spinobulbar Faculty of Medicine, University of Malaya, muscular atrophy and hereditary spastic paraplegia. However, the Kuala Lumpur, Malaysia, and dMolecular involvement of other dynactin genes with inherited peripheral neuropathies Medicine Laboratory, Concord Hospital, (IPN) namely, hereditary sensory neuropathy, hereditary motor neuropathy Sydney, Australia and Charcot–Marie–Tooth disease is under reported. We screened eight genes; DCTN1-6 and ACTR1A and ACTR1B in 136 IPN patients using Key words: Charcot–Marie–Tooth – whole-exome sequencing and high-resolution melt (HRM) analysis. Eight dynactin – hereditary motor non-synonymous variants (including one novel variant) and three neuropathies – hereditary sensory synonymous variants were identified. Four variants have been reported neuropathies – peripheral neuropathies previously in other studies, however segregation analysis within family members excluded them from causing IPN in these families. No variants of Corresponding author: Azlina disease significance were identified in this study suggesting the dynactin Ahmad-Annuar, Department of Biomedical Science, Faculty of genes are unlikely to be a common cause of IPNs. However, with the ease of Medicine, University of Malaya, 50603 querying gene variants from exome data, these genes remain worthwhile Kuala Lumpur, Malaysia. candidates to assess unsolved IPN families for variants that may affect the Tel.: +60 37 967 4948; function of the proteins. fax: +60 37 967 6600; e-mail: [email protected] Conflictofinterest Received 2 September 2015, revised The authors declare that they have no conflicts of interest. and accepted for publication 7 December 2015 Inherited peripheral neuropathies (IPN) are a large group complex in patients with a range of neurodegenerative of disorders that can manifest in motor and/or sensory disorders. Mutations in cytoplasmic dynein heavy chain, phenotypes and are increasingly recognized for their DYNC1H1 have been reported in several studies result- phenotypic and genetic heterogeneity. Over 80 genes ing in an axonal form of Charcot–Marie–Tooth (CMT) have been implicated in these disorders and despite the disease, intellectual disability and malformations of cor- wide diversity of function the phenotypes that result tical development (2–4). In addition, mouse mutants of from these mutated genes commonly share a progressive Dync1h1 show neurodegenerative phenotypes in keep- degeneration of the peripheral nerves (1). ing with a motor and sensory phenotype (5–7). Apart Next generation sequencing has expedited the from the heavy chain, within the cytoplasmic dynein identification of causal variants in genes within the complex there are other subunits including the dynein microtubule-associated dynein–dynactin motor protein intermediate chains 1 and 2 (DYNC1I1 and DYNC1I2), 127 Tey et al. light chains (DYNLL1–L2, DYNLRB1, DYNLT1 and Table 1. IPN families screened in this study. The subtype of IPN DYNLT3) and light intermediate chains (DYNC1LI1 and and the methods of candidate gene screening are shown. A total DYNC1LI2). Less is known about the involvement of of the 136 families were screened in which 76 were diagnosed these genes in neurodegenerative phenotypes in humans, with CMT, 29 with HSN and 31 with HMN although studies in mice have shown that a point muta- Number of patients screened tion in Dyncili1 causes anxiety-like behaviour (8). In addition, mutations in the Drosophila dynein light Classes of IPNs WES HRM intermediate chain genes give rise to defects in dendritic CMT (n = 76) morphogenesis through the Rab5 pathway and also in the CMT1 4 0 establishment of axonal and dendritic polarity (9, 10). CMT2 43 0 Recently we reported that genes within the cytoplasmic CMT4 0 1 dynein gene family (DYNC1I1, DYNC1I2, DYNLL1–L2, CMT with 21 DYNLRB1, DYNLT1 and DYNLT3, DYNC1LI1 and pyramidal signs DYNC1LI2) were not associated with IPNs (11). In this CMTX 25 0 study, we have investigated the role of genes within HSN (n = 29) the dynactin complex. Dynactin is a multi-subunit HSN1 1 27 protein complex that is required for a wide range of HSN2 0 1 cellular activities including mitosis and retrograde Other (n = 31) axonal transport. The dynactin complex comprises of HMN 14 0 subunits encoded by eight genes (DCTN1-6, ACTR1A HMNP 15 0 and ACTR1B). Structurally, the dynactin complex is HMNX 2 0 divided into two parts: the projecting side arm consists Total 106 30 of the p150Glued subunit (DCTN1), p22/24 (DCTN3) Gene screening All known IPN PMP22, MPZ, genes and all MFN2, GJB1 and p50/dynamitin (DCTN2); the Arp1 rod consists of a the Arp1 filaments (ACTR1A/1B) and heterotetrameric dynactin genes and all dynactin complex composed of p62 (DCTN4), p25 (DCTN5), p27 genes (except DCTN1) (DCTN6) and Arp11 (12, 13). The Drosophila mutants for the homolog of DCTN1, CMT, Charcot–Marie–Tooth disease; HSN, hereditary sensory p150Glued and mouse models with point mutations in neuropathy; HMN, hereditary motor neuropathy; HMNP, HMN Dctn1 have impaired cargo trafficking, whereas mice with pyramidal signs. overexpressing Dctn1 exhibit motor neuron degeneration aThe known IPN genes are listed (Table S2) and were excluded (14–16). In humans, mutations in DCTN1 are associated as a pathogenic cause in the patients screened by WES. with a form of human spinal muscular atrophy (SMA) and Perry Syndrome, frontotemporal dementia, amy- on the clinical presentation of the patients and family his- otrophic lateral sclerosis (ALS) and Parkinson disease tory. The index patients were further sub-classified into: (17–24). In addition, BICD2 which is an interacting part- demyelinating CMT (CMT1); axonal CMT (CMT2); ner of DCTN2 was recently reported to cause congenital autosomal recessive CMT (CMT4); CMT with pyra- SMA and hereditary spastic paraplegia (25, 26). midal signs; query CMTX and query HMNX as there Although DCTN1 has been implicated in various neu- was no male to male inheritance in these families and rological disorders, the other genes within the complex the females had a less severe phenotype; HMN with have not been studied in detail for a pathogenic role pyramidal signs; adult onset HSN (HSN1); and juvenile in neurodegenerative disease. We screened the coding onset HSN (HSN2) (Table 1). The 106 patients under- exons of the eight genes within the dynactin complex going WES had been screened for the common IPN using a combination of high-resolution melt (HRM) genes (PMP22, MPZ, MFN2 and GJB1) prior to this analysis and whole-exome sequencing (WES). Eleven study and no mutations were identified. Subsequent anal- variants including one novel variant were identified in ysis of the WES data from the 106 patients excluded six of the genes. However, the non-synonymous variants mutations in all the known IPN genes (listed in Table identified did not segregate with the disease phenotype S1, Supporting information). Samples not sent for WES and are probably have no functional effect on the disease. (28 index samples from HSN families, 1 index CMT4 sample and 1 index sample with CMT with pyramidal Materials and methods signs) were excluded for mutations in the common IPN genes prior to this study. In addition, the 29 HSN fam- Patient selection ilies had been excluded for SPTLC1 and SPTLC2 prior Index patients from 136 IPN families were recruited to this study. through the Neurogenetics Clinic Concord Hospital, Sydney. The cohort included 76 CMT patients, 29 Ethics hereditary sensory neuropathy (HSN) patients and 31 hereditary motor neuropathy (HMN) patients. The index Patient ascertainment and collection of blood samples patients were from families with two or more affected for DNA analysis were performed with informed consent individuals. The classification of the disease was based according to protocols approved by the Sydney Local 128 Dynactin genes and peripheral neuropathies Table 2. Dynactin subunits screened in this study Name Symbol Chromosome position Gene ID Number of exons Dynactin 1 (p150) DCTN1 2p13 NM_004082 32 Dynactin 2 (p50/dynamitin) DCTN2 12q13.3 NM_006400 14 Dynactin 3 (p22/p24) DCTN3 9p13 NM_024348 6 Dynactin 4 (p62) DCTN4 5q31-32 NM_001135643.1 14 Dynactin 5 (p25) DCTN5 16p12.2 NM_032486