Genetic heterogeneity of motor neuropathies · neuropathies in a large cohort of patients from the North of England. Methods: Detailed neurologic and electrophysiologic assessments

Boglarka Bansagi MDHelen Griffin PhDRoger G Whittaker MD

PhDThalia Antoniadi PhDTeresinha Evangelista

MDJames Miller MD PhDMark Greenslade PhDNatalie Forester PhDJennifer Duff PhDAnna BradshawStephanie Kleinle PhDVeronika Boczonadi PhDHannah Steele MDVenkateswaran Ramesh

MDEdit Franko MD PhDAngela Pyle PhDHanns Lochmuumlller MD

PhDPatrick F Chinnery MD

FRCP FMedSciRita Horvath MD PhD

Correspondence toDr HorvathRitaHorvathnclacuk

Supplemental dataat Neurologyorg

Genetic heterogeneity of motorneuropathies

ABSTRACT

Objective To study the prevalence molecular cause and clinical presentation of hereditary motorneuropathies in a large cohort of patients from the North of England

Methods Detailed neurologic and electrophysiologic assessments and next-generation paneltesting or whole exome sequencing were performed in 105 patients with clinical symptoms ofdistal hereditary motor neuropathy (dHMN 64 patients) axonal motor neuropathy (motorCharcot-Marie-Tooth disease [CMT2] 16 patients) or complex neurologic disease predominantlyaffecting the motor nerves (hereditary motor neuropathy plus 25 patients)

Results The prevalence of dHMN is 214 affected individuals per 100000 inhabitants (95confidence interval 162ndash266) in the North of England Causative mutations were identified in26 out of 73 index patients (356) The diagnostic rate in the dHMN subgroup was 325which is higher than previously reported (20) We detected a significant defect of neuromus-cular transmission in 7 cases and identified potentially causative mutations in 4 patients withmultifocal demyelinating motor neuropathy

Conclusions Many of the genes were shared between dHMN and motor CMT2 indicating identicaldisease mechanisms therefore we suggest changing the classification and including dHMN also asa subcategory of Charcot-Marie-Tooth disease Abnormal neuromuscular transmission in somegenetic forms provides a treatable target to develop therapies Neurologyreg 2017881226ndash1234

GLOSSARYCI 5 confidence interval CMT 5 Charcot-Marie-Tooth disease dHMN 5 distal hereditary motor neuropathy HMN 5 hered-itary motor neuropathy LED 5 lower extremity dominance MAF 5 minor allele frequency NCS 5 nerve conduction studiesNGS 5 next-generation sequencing NMJ 5 neuromuscular junction SMA 5 spinal muscular atrophy SMARD1 5 spinalmuscular atrophy with respiratory distress WES 5 whole exome sequencing

Distal hereditary motor neuropathies (dHMN) are clinically and genetically heterogeneous dis-orders caused by lower motor neuron dysfunction1 The classic phenotype of dHMN is a length-dependent motor weakness and atrophy initially affecting the intrinsic foot muscles and theperoneal compartment of the leg frequently leading to foot deformities such as pes cavus pesplanus and clawing of the toes2 The majority of the cases show a slowly progressive diseasecourse gradually involving proximal leg muscles or affecting intrinsic hand muscles Variable ageat onset diverse clinical course and associated neurologic features complicate the pheno-type and aid the clinical subclassification Congenital or infantile-onset forms of weaknessand atrophy of the feet and legs with no or very slow disease progression were grouped asspinal muscular atrophy with lower extremity dominance (SMA-LED) Other clinicalsubgroups of dHMN were classified on the basis of additional features such as diaphrag-matic paralysis (dHMN-IV) upper limb predominance (dHMN-V) SMA with respiratory

From the MRC Centre for Neuromuscular Diseases and John Walton Muscular Dystrophy Research Centre Institute of Genetic Medicine (BBHG TE JD AB VB HS EF AP HL PFC RH) and Institute of Neuroscience (RGW JM) Newcastle University Newcastleupon Tyne Bristol Genetics Laboratory (TA MG NF) Pathology Sciences North Bristol NHS Trust Southmead Hospital Medical GeneticCenter (SK) Munich Germany Department of Paediatric Neurology (VR) Royal Victoria Infirmary Newcastle upon Tyne FoundationHospitals NHS Trust Nuffield Department of Clinical Neurosciences (EF) University of Oxford and Department of Clinical Neurosciences(PFC) Cambridge Biomedical Campus University of Cambridge UK

Go to Neurologyorg for full disclosures Funding information and disclosures deemed relevant by the authors if any are provided at the end of the articleThe Article Processing Charge was paid by Medical Research CouncilWellcome Trust

This is an open access article distributed under the terms of the Creative Commons Attribution License 40 (CC BY) which permits unrestricteduse distribution and reproduction in any medium provided the original work is properly cited

1226 Copyright copy 2017 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology

ordf 2017 American Academy of Neurology Unauthorized reproduction of this article is prohibited

distress (SMARD1 or dHMN-VI) adult-onsetwith vocal cord palsy (dHMN-VII) and pyra-midal features or X-linked inheritance3

Gene identification in dHMN was previ-ously based on linkage studies in rare extendedfamilies However recent development ofnext-generation technology enabled studyingisolated patients or small families and a largenumber of new disease genes and novel pheno-types have been discovered4 Compared to the7 genes and 13 chromosomal loci 10 yearsago56 to date we acknowledge around 30genes responsible for autosomal dominantrecessive and X-linked forms of dHMN Toidentify the molecular cause of motor neurop-athies we performed detailed clinical investi-gations and state of the art genetic studies ina large population-based cohort from theNorth of England

METHODS Patients Between 2010 and 2015 we selected

105 patients out of 461 diagnosed with inherited neuropathies

in our genetic neuropathy clinic at Newcastle Hospitals NHS

Foundation which is the catchment area of a total population

of 299 million Inclusion of patients was based on the presence

of a motor neuropathyneuronopathy with no or only subclinical

sensory changes on electrophysiology Acquired causes were

excluded by detailed laboratory analysisno response on immuno-

suppressive therapy Some affected family members of patients

with a confirmed genetic diagnosis were included in this cohort

even if no electrophysiologic data were available

Standard protocol approvals registrations and patientconsents Participants provided written informed consent

approved by local research ethics committees for all experiments

using human participants and for photographs that may be

published

Electrophysiology We performed electrophysiologic examina-

tion in 90 patients according to standard techniques and inter-

preted by the same expert neurophysiologists Eight family

members did not undergo electrophysiologic studies 7 patients

were tested at different hospitals Motor and sensory nerve con-

duction studies (NCS) were performed and qualitative and quan-

titative analysis of motor unit potentials and spontaneous activity

were assessed on EMG Patient classification into study groups

was based on these analyses Additional assessment of repetitive

nerve stimulation and single-fiber EMG were carried out in 27

patients and the percentage increment or decrement was

calculated EMG was performed using Natus Neurology

(Pleasanton CA) disposable 30-G concentric needles with

a bandpass range of 10ndash10000 Hz For single-fiber EMG

studies the low pass filter was increased to 2000 Hz and the

percentage of fiber pairs showing increased jitter or blocking was

calculated

Molecular genetic analysis DNA was obtained from periph-

eral blood Initially PMP22 deletionduplication MFN2 andGJB1 were excluded in all patients The multigene panel assay

utilizing next-generation sequencing (NGS) was carried out in 46

index patients in the Bristol Genetics Laboratory following the

UK Genetic Testing Network approved criteria Variant

classification was based on Association for Clinical Genetic

Science Practice Guidelines (2013) We performed whole exome

sequencing (WES) in 40 patients DNA was fragmented exome-

enriched and sequenced (Illumina [San Diego CA] TruSeq 62 Mb

and HiSeq 2000 100 bp paired-end reads) Bioinformatic analysis

included duplicate sequence read removal with FastUniq7

alignment to UCSC hg19 with BWA8 variant detection with

FreeBayes and variant annotation with ANNOVAR Variants

were annotated as exonicsplicing excluding synonymous variants

with a rare minor allele frequency (MAF 001) in the context of

genotype (heterozygous MAF 0001 homozygous MAF 001)

in several databases downloaded via ANNOVAR (ExAC

NHLBI_ESP6500 cg69) and also in 281 in-house exomes

Protein prediction and evolutionary sequence conservation

algorithms downloaded via ANNOVAR were used to analyze the

in silico effects following the guidelines of the American College of

Medical Genetics and Genomics9 Variants were defined as

confirmed pathogenic if they were previously shown to be

pathogenic or if they were novel variants in a known neuropathy-

associated gene predicted to be deleterious (affect protein structure

or function highly conserved) and segregated with the disease in at

least 1 additional affected family member10 Highly conserved in

silico deleterious rare sequence variants of known or novel genes

were determined as possible pathogenic if segregation studies

involving at least one additional affected family member could not

be carried out Putative pathogenic variants were confirmed by

Sanger sequencing and were tested for segregation

RESULTS Clinical presentation We identified 105patients from 73 families with inherited motor neurop-athies Based on neurologic and electrophysiologicfindings all patients were classified into 1 of 3 sub-groups dHMN motor predominant Charcot-Marie-Tooth disease (CMT2) (motor CMT2) and motorneuropathy with additional neurologic symptoms(hereditary motor neuropathy [HMN] plus)Diagnosis of dHMN was based on preserved sensorynerve studies with normal or reduced compoundmotor unit action potentials or neurogenic changeson EMG examination2 Patients with dHMN butwith decreased sensory nerve action potentialsindicative of an accompanying sensory axonopathywere grouped as motor CMT2 We considered HMNplus when motor neuropathy was the leading featurebut was accompanied by other neurologic symptomsNCS data of all patients are available on request

All patients seen in our center were from ourcatchment area (299 million people in 2011 UK cen-sus) leading to a minimum prevalence of dHMN as214 affected individuals per 100000 inhabitants(95 confidence interval [CI] 162ndash266)

Sixty-four out of 105 patients (609) had dHMNSixteen patients had motor CMT2 (152) and 25HMN plus (238) (figures 1ndash3) There was male pre-dominance in all phenotypic groups Symptoms startedat a younger age in dHMN (mean 16 years) and HMNplus (mean 176 years) compared to motor CMT2(mean 238 years) The main inheritance pattern wasautosomal dominant in CMT2 (710 families [70])

Neurology 88 March 28 2017 1227


while isolated cases were more frequent in HMN plusThe dHMN group consisted of an almost equal num-ber of dominant families (n5 16) and isolated patientswith negative or unavailable family history (n 5 20)

We identified causative mutations in 26 out of 73families providing a 356 mutation detection rate(figure 2) Candidate gene sequencing based on clin-ical presentation led to the molecular diagnosis in

Figure 1 Clinical heterogeneity of different forms of hereditary motor neuropathy (HMN)

Distal hereditary motor neuropathy (dHMN) (top 2 rows) motor Charcot-Marie-Tooth disease 2 (CMT2) (third row) and HMN plus (bottom row) (A) patients 2and 3 GARS (B) patient 19 IGHMBP2 (C) patient 23 TRPV4 (D) patient 32 IGHMBP2 (E) not yet diagnosed dHMN (F) patient 18 DYNC1H1 (G) patient10 SYT2 (H) patient 16 BICD2 (I J) not yet diagnosed spinal muscular atrophy lower extremity dominance (K) patient 42 AARS (L) patient 39 AARS (M)family 22 DNM2 (N) patient 51 FUS (O) patient 53 ATP7A (P) patient 57 TBX5 (Q) patient 58 STAT5B (R) not yet diagnosed HMN plus

1228 Neurology 88 March 28 2017


5 out of 105 patients only (47) while 12 indexpatients were diagnosed by NGS panel from 46 per-formed tests (26) and a further 18 index patientsby WES (total 40 analysis) (45) There were nonovel shared genesmutations in the exome negativecasesMutation spectrum in dHMN We achieved the possi-ble genetic diagnosis in 1740 families which re-sulted in 425 detection rate (figure 2) Variantswere considered confirmed pathogenic in 13 indexcases (325) 7 of them carried known pathogenicdHMN mutations while in 6 families novel delete-rious variants were detected in known genes andsegregated with the phenotype (table e-1A atNeurologyorg)

A previously reported heterozygous c421AGp(Lys141Glu) mutation in HSPB8 presented witha progressive lower limb predominant phenotype(family 1) We found 2 novel GARS mutations in 2independent families (families 2ndash3) The heterozy-gous c647AG p(His216Arg) was detected ina mother and her son with predominant upperextremity involvement (dHMN-V) (figure 1A) Thec1528AC p(Lys510Gln) mutation cosegregatedin 3 generations with dominant distal motor neurop-athy affecting both upper and lower extremities Elec-trophysiologic examination revealed mildly increasedjitter in one patient consistent with a motor neurop-athy and significantly increased jitter with blocking inanother patient One patient with dHMN involving

Figure 2 Identified genes in our hereditary motor neuropathy (HMN) patient cohort

(A) Spectrum and distribution of mutated genes detected in our cohort within the 3 phenotypic groups (B) Overlapping clinical phenotypes related to theidentified genes and key additional clinical features associated Upper motor neuron involvement ALS 5 amyotrophic lateral sclerosis



both upper and lower limbs carried the previouslyreported c1834GA p(Val612Met) mutation inDYNC1H1 (family 7) (figure 1F) A rapidly progres-sive dHMN in a 15-year-old boy was due to the denovo previously reported c1126AG p(Met376V-al) mutation in MFN2 (family 12)11 The heterozy-gous c2119CT p(Arg707Trp) MFN2 mutationwas found in a 70- year-old man with late-onsetdHMN (family 13)12

The homozygous frameshift c292_303delin-sATGCT p(Gly98fs) mutation in IGHMBP2 led

to SMARD1 in 2 siblings of consanguineous Pakis-tani origin (family 8) (figure 1B) A brother andsister from another family with childhood-onset dis-tal SMA and lack of respiratory involvement carrieda heterozygous c1813CT p(Arg605) nonsenseIGHMBP2 mutation which was hemizygous in thecDNA (family 9)13

We have previously reported 6 patients with non-progressive neuropathy and fatigable weakness carryingc923CT p(Pro308Leu) in SYT2 (family 4) (figure1G)14 5 patients with distal congenital nonprogessive

Figure 3 Diagnostic flow chart and mutation detection rates in our hereditary motor neuropathy (HMN) cohort

CMT 5 Charcot-Marie-Tooth disease dHMN 5 distal hereditary motor neuropathy WES 5 whole exome sequencing



SMA carrying c320CT p(Ser107Leu) in BICD2(families 5ndash6) (figure 1H)15 and 2 patients withTRPV4 mutations associated with childhood-onsetscapuloperoneal SMA and metatropic dysplasia (family10) (figure 1C) or adult-onset dHMN (family 11)16

Possibly causative mutations were detected in 4families (table e-1B) The heterozygous novelc2752C T p(Arg918Cys) IGHMBP2 variantwas detected in 3 siblings with motor neuropathywithout respiratory dysfunction (family 14) All pre-diction tools predicted this change pathogenic and itwas absent in the ExAC database although we couldnot identify a second mutation in this family Thesingle heterozygous c767CG p(Ala256Gly)IGHMBP2 variant was detected in a 35-year-oldboy with compatible phenotype (family 15) (figure1D) In 3 members of a dominant dHMN pedigree(family 16) we found a novel heterozygous DHTKD1variant c628GT p(Ala210Ser) In a young femalepatient (family 17) we identified a heterozygous novelc1949GA p(Tyr650Cys) sequence change inARHGEF10

Mutation spectrum in motor CMT2Motor CMT2 wasconsidered in 16 patients from 10 families Weachieved 70 mutation detection rate in this groupby concluding molecular diagnosis in 7 CMT2 fam-ilies (figure 2) (table e-2) We identified the recurrentc986GA p(Arg329His) AARS mutation in 6 pa-tients from 4 families (families 18ndash21) (figure 1 Kand L)17 In 3 members of a dominant family withearly-onset intermediate motor neuropathy splithand deformity and hearing loss we detected thepreviously described c1739TC p(Met580Thr)DNM2 mutation (family 22) (figure 1M)18 Afemale patient with upper limb weakness carried thepathogenic c1403GA p(Arg468His) MFN2mutation (family 23)12 Exome sequencing of malemonozygotic twins with early-onset severe motorpredominant neuropathy revealed the recentlyreported heterozygous c754CT p(Arg252Trp)mutation in the MORC2 gene (family 24)19

Mutation spectrum in HMN plus families Twenty-fivepatients from 23 families with predominant motor neu-ropathy had additional neurologic features Confirmedpathogenic mutations were detected in 6 patients(26) and a probable disease cause was identified in a fur-ther 5 index patients (217) (figure 2 table e-3A) Theheterozygous c1529AG p(Lys510Arg) FUSmutationwas detected in a 52-year-oldmanwith asymmetric lowerlimb motor neuropathy The progression was rapid withevolving motor neuron disease frontal dementia andloss of ambulation within half a year (family 27) (figure1N) WES identified a hemizygous c2279AGp(Tyr760Cys) novel ATP7A variant with upperand lower motor neuron symptoms (family 29)

(figure 1O)20 We detected compound heterozygousc916GA p(Gly306Arg) and c1016TC p(Leu339Pro) mutations in SLC52A2 (family 25)21 anda homozygous c96_99dupATCC p(Pro34Ilefs25)mutation in the c12orf65 gene (family 26)22 Distalmotor neuropathy with ataxia and dystonia wasassociated with c3823CT p(Arg1275Cys) inDCTN1 (family 28)23 Compound heterozygousc1580CG p(Ser527) and c6781CAp(Leu2261Ile) mutations in SACS caused juvenile-onset motor neuropathy ataxia and spasticity in a 71-year-old man (family 30)24 Possibly causative mutationswere detected in 5 families (table e-3B) A 17-year-oldman with distal motor neuropathy and optic atrophy(CMT type 6) carried a single previously reportedheterozygous c2386CT p(Gln796) mutation inFIG425 Heterozygous FIG4 mutations have beenassociated with adult-onset amyotrophic lateral sclerosisbut without optic atrophy26 We found the heterozygousc1371CG p(Phe457Leu) mutation in SLC52A3 ina 19-year-old man with Brown-Vialetto-Van-Laeresyndrome who responded well to riboflavin therapyHowever we could not identify a second mutationeven by analyzing WES for copy number variationswithin this gene (family 32) A 19-year-old man withshoulder girdle weakness commonly seen in Holt-Oram syndrome carried the previously reportedc331GT p(Asp111Tyr) TBX5 mutation (family33 figure 1P)27 We identified the novel homozygousc944TG p(Glu315Ala) mutation in thetranscriptional factor STAT5B in a sibling pair froma consanguineous family with growth retardationdysmorphic face and motor neuropathy (family 34)(figure 1Q) Asymmetric motor neuron weaknesscranial nerve involvement pyramidal signs andmultifocal motor neuropathy with conduction blockswere observed in a man who carried a novel de novoc269TC p(Phe90Ser) mutation in PTEN (family35)

DISCUSSION Although HMNs are suggested to berare we identified 105 patients in the North of Eng-land with a population of 299 million We previ-ously determined that the minimum prevalence ofCMT in the North of England is 118 per 100000individuals2829 Here we report that the minimumprevalence of dHMN in the same population is214 affected individuals per 100000 inhabitants(95 CI 162ndash266) which is higher than suggestedearlier2930

Previous studies indicated that the molecular causecan be identified in the vast majority of patients withdemyelinating CMT however the detection rate hasbeen much lower in dHMN and motor CMT2(20)3 We identified potentially pathogenic muta-tions in 479 (3573 families) which consisted of



confirmed mutations in 356 and possibly causativevariants in an additional 123 Previously indHMN the detection rate was 15ndash2035631

We confirmed genetic diagnosis in 325 (1340families) of dHMN and a likely causative mutationwas identified in an additional 10 (figure 2) whichis higher than in previous studies (tables e-4ndashe-6)

dHMN is strictly considered as the pure motorend of CMT many patients show minor sensoryabnormalities16 and mutations in the same genecan cause both dHMN and CMT2231 HMN maybe complicated by SMA spastic paraplegia or otherneurologic abnormalities1632 Affected pathwayslinked to dHMN include DNARNA metabolismprotein translation and synthesis stress response apo-ptosis axonal guidance intracellular traffickingand synaptic activity (figure 4) Mitochondrial abnor-malities are common in our cohort in all forms ofmotor neuropathies and contribute to the symp-toms by altering mitochondrial fusionfission(MFN2 DNM2 SLC25A46) axonal transport(HSPB1 HSPB8) protein synthesis (C12orf65SACS) or cofactors (SLC52A23)

SMA-LED has been introduced to characterize thecongenital or early childhood-onset nonprogressiveatrophy and weakness of the legs affecting both distaland proximal muscles caused by altered motor neurondevelopment or function3 The clinical presentation

may overlap between spinal and distal motor neurondysfunction Correction of deformities is importantsince these patients do not deteriorate in the later dis-ease course We identified 17 patients with clinicalsymptoms resembling SMA-LED carrying mutationsin SYT2 BICD2 and DYNC1H1 and no causativemutation was identified in 4 patients (figure 1 IndashJ)No major common pathway can be highlighted by thepathomechanism of these genes while different muta-tions in the same gene (such as DYNC1H1) may alsocause different clinical presentations

We reported that mutations in synaptic proteinscan cause nonprogressive motor neuropathy and fati-gable weakness with presynaptic neuromuscular junc-tion (NMJ) defect (SYT2)15 which can benefit from34 diaminopyridine treatment33 Abnormal neuro-muscular transmission has been shown in Drosophilaand mouse models of GARS mutations3435 Further-more a genetic defect of the presynaptic cholinetransport leads to HMN due to SLC5A7mutations36

Based on these findings we studied neuromusculartransmission in 27 patients and substantial abnormal-ities (over and above that expected in the context ofimmature NMJs formed during denervation and re-innervation) were detected in 7 cases harboring dif-ferent gene mutations Further detailed studies of theNMJ are warranted including genetically character-ized patients Neuromuscular transmission can be

Figure 4 Pathomechanisms of genes reported in hereditary motor neuropathy (HMN)



modified by several already available compoundswhich have been broadly used in congenital myas-thenic syndromes but not in hereditary neuropathiesWe suggest performing repetitive stimulation and sin-gle fiber EMG as part of routine electrophysiologicstudies in patients with HMN in order to uncoverpotentially treatable transmission defects

We identified a subgroup of patients with inheritedmotor neuropathy resembling multifocal demyelinat-ing motor neuropathy or nodo-paranodopathy Talipesand pure motor nerve involvement were caused bya novel mutation in ARHGEF10 Slow nerve conduc-tion velocities have been implicated in ARHGEF10mutations and a 10-bp deletion in Leonberger dogsled to pelvic muscle weakness stepping gait and recur-rent laryngeal nerve palsy37

A previously reported pathogenic mutation inTBX5 was detected in a patient with demyelinatingmultifocal motor neuropathy TBX5 participates intranscriptional regulatory cascades and its pathogenicmutations located within the DNA binding T-boxdomain manifest in a rare condition called Holt-Oram syndrome with deformity of upper limbs carpaltunnel syndrome and cardiac anomalies27 We suggestthatTBX5may cause demyelinating neuropathy affect-ing predominantly the motor nerves No cardiac symp-toms were detected in our patient We detected a novelpossibly causative STAT5B mutation in a sibling pairwith motor neuropathy ptosis short stature and dys-morphic facial features Homozygous mutations inSTAT5B a signal transducer and activator of transcrip-tion have been reported in only a handful of patientsworldwide all manifesting with postnatal growth retar-dation and occasional immunologic alterations38

Impaired STAT5B signaling may lead to aberrantperipheral myelination through cyclin D1 overexpres-sion and may have an effect on neuronal growth anddifferentiation39 Finally we detected a possibly causa-tive de novo PTEN mutation in a patient with patchymotor neuropathy focal demyelination hamartoma-like skin lesions and autism spectrum disorder PTENa lipid phosphatase inhibits phosphoinositide 3-kinase which is also impaired in several forms ofCMT (FIG4 FGD4 MTMR2 MTMR13SBF2) Ithas been implicated in peripheral neural plasticity axo-nal outgrowth and hypermyelination ExperimentalPTEN suppression in mice resulted in progressiveperipheral neuropathy with tomacula formation andmyelin outfoldings40

The identification of pathogenic mutations in thesame genes in dHMN and motor CMT indicate thatdHMN should not be classified as a different diseasegroup The detection of mutations in rare diseasegenes not implicated in neuropathy to date suggestthat the neuropathy can be part of a more complexgenetic disease and key clinical signs are important

to recognize these diseases These overlapping pheno-types highlight basic biological pathways in peripheralnerves that may be targeted to develop therapies forpatients with inherited motor neuropathies

AUTHOR CONTRIBUTIONSBoglarka Bansagi drafting the manuscript study concept and design

acquisition analysis and interpretation of data Helen Griffin analysis

and interpretation of data Roger Whittaker analysis and interpretation

of data Thalia Antoniadi analysis and interpretation of data Teresinha

Evangelista analysis and interpretation of data James Miller analysis

and interpretation of data Mark Greenslade analysis and interpretation

of data Natalie Forester analysis and interpretation of data Jennifer

Duff analysis and interpretation of data Anna Bradshaw analysis and

interpretation of data Stephanie Kleinle analysis and interpretation of

data Veronika Boczonadi analysis and interpretation of data Hannah

Steele analysis and interpretation of data Venkatateswaran Ramesh

analysis and interpretation of data Edit Franko analysis and interpreta-

tion of data Angela Pyle analysis and interpretation of data Hanns

Lochmuumlller analysis and interpretation of data critical correction of

the manuscript Patrick F Chinnery analysis and interpretation of data

critical correction of the manuscript Rita Horvath study funding

draftingrevision of the manuscript study concept and design acquisi-

tion analysis and interpretation of data

ACKNOWLEDGMENTThe authors thank the members of the clinical team at the John Walton

Muscular Dystrophy Research Centre (Newcastle) who may have seen some

of the patients included in this cohort the Northern Genetic Service and the

UKGTN for providing diagnostic testing for some patients in this collective

and the Medical Research Council (MRC) Centre for Neuromuscular Dis-

eases Biobank Newcastle and EuroBioBank for supporting this project

STUDY FUNDINGRH is a Wellcome Trust Investigator (109915Z15Z) who receives

support from the Medical Research Council (UK) (MRN0254311)

the European Research Council (309548) the Wellcome Trust Path-

finder Scheme (201064Z16Z) and the Newton Fund (UKTurkey

MRN0273021) BB is a Medical Research Council (MRC)ndashfunded

Clinical Research Fellow and receives support from the MRC Centre for

Neuromuscular Diseases HL receives funding from the European

Union Seventh Framework Programme (FP72007ndash2013) under grant

agreement no 305444 (RD-Connect) and 305121 (Neuromics) PFC

is a Wellcome Trust Senior Fellow in Clinical Science (101876Z13Z)

and a UK NIHR Senior Investigator who receives support from the

MRCMitochondrial Biology Unit (MC_UP_15012) the Wellcome Trust

Centre for Mitochondrial Research (096919Z11Z) the MRC (UK)

Centre for Translational Muscle Disease (G0601943) EU FP7 TIRCON

and the NIH Research (NIHR) Biomedical Research Centre based at

Cambridge University Hospitals NHS Foundation Trust and the Univer-

sity of Cambridge The views expressed are those of the authors and not

necessarily those of the NHS the NIHR or the Department of Health

DISCLOSUREThe authors report no disclosures relevant to the manuscript Go to

Neurologyorg for full disclosures

Received September 5 2016 Accepted in final form January 6 2017

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HMSN II) and distal hereditary motor neuropathy (distal



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8 Li H Durbin R Fast and accurate short read alignment

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ACMG clinical laboratory standards for next-generation

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in undiagnosed inherited and sporadic ataxias Brain 2015

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and missense mutations in IGHMBP2 cause Charcot-

Marie Tooth disease type 2 Am J Hum Genet 201495

590ndash601

14 Herrmann DN Horvath R Sowden JE et al Synaptotag-

min 2 mutations cause an autosomal-dominant form of

Lambert-Eaton myasthenic syndrome and nonprogressive

motor neuropathy Am J Hum Genet 201495332ndash339

15 Bansagi B Griffin H Ramesh V et al The pSer107Leu

in BICD2 is a mutation ldquohot spotrdquo causing distal spinal

muscular atrophy Brain 2015138e391

16 Evangelista T Bansagi B Pyle A et al Phenotypic vari-

ability of TRPV4-related neuropathies Neuromuscul Dis-

ord 201525516ndash521

17 Bansagi B Antoniadi T Burton-Jones S et al Genotype-

phenotype correlations in AARS-related neuropathy in

a cohort of patients from the United Kingdom and

Ireland J Neurol 20152621899ndash1908

18 Haberlova J Mazanec R Ridzon P et al Phenotypic

variability in a large Czech family with a dynamin 2-asso-

ciated Charcot-Marie-Tooth neuropathy J Neurogenet

201125182ndash188

19 Albulym OM Kennerson ML Harms MB et al MORC2mutations cause axonal Charcot-Marie-Tooth disease with

pyramidal signs Ann Neurol 201679419ndash427

20 Bansagi B Lewis-Smith DJ Pal E et al Phenotypic con-

vergence of Menkes and Wilsonrsquos disease Neurol Genet

20162e119

21 Foley AR Menezes MP Pandraud A et al Treatable

childhood neuronopathy caused by mutations in riboflavin

transporter RFVT2 Brain 201413744ndash56

22 Pyle A Ramesh V Bartsakoulia M et al Behrrsquos syndrome is

typically associated with disturbed mitochondrial translation

and mutations in the C12orf65 gene J Neuromuscul Dis

2014155ndash63

23 Daud D Griffin H Douroudis K et al Whole exome

sequencing and the clinician we need clinical skills and

functional validation in variant filtering J Neurol 2015

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24 Yu-Wai-Man P Pyle A Griffin H et al Abnormal retinal

thickening is a common feature among patients with

ARSACS-related phenotypes Br J Ophthalmol 201498

711ndash713

25 DiVincenzo C Elzinga CD Medeiros AC et al The

allelic spectrum of Charcot-Marie-Tooth disease in over

17000 individuals with neuropathy Mol Genet Genomic

Med 20142522ndash529

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28 Foley C Schofield I Eglon G et al Charcot-Marie-Tooth

disease in Northern England J Neurol Neurosurg Psychi-

atry 201283572ndash573

29 Norwood FL Harling C Chinnery PF et al Prevalence of

genetic muscle disease in Northern England in-depth analysis

of a muscle clinic population Brain 20091323175ndash3186

30 Pearn J Incidence prevalence and gene frequency studies

of chronic childhood spinal muscular atrophy J Med

Genet 197815409ndash413

31 Dierick I Baets J Irobi J et al Relative contribution of

mutations in genes for autosomal dominant distal heredi-

tary motor neuropathies a genotype-phenotype correla-

tion study Brain 20081311217ndash1227

32 Van Den Bosch L Timmerman V Genetics of motor neu-

ron disease Curr Neurol Neurosci Rep 20066423ndash431

33 Whittaker RG Herrmann DN Bansagi B et al Electro-

physiologic features of SYT2 mutations causing a treatable

neuromuscular syndrome Neurology 2015851964ndash1971

34 Ermanoska B Motley WW Leitatildeo-Gonccedilalves R et al CMT-

associated mutations in glycyl- and tyrosyl-tRNA synthetases

exhibit similar pattern of toxicity and share common genetic

modifiers in Drosophila Neurobiol Dis 201468180ndash189

35 Sleigh JN Grice SJ Burgess RW et al Neuromuscular

junction maturation defects precede impaired lower motor

neuron connectivity in Charcot-Marie-Tooth type 2D

mice Hum Mol Genet 2014232639ndash2650

36 Barwick KE Wright J Al-Turki S et al Defective

presynaptic choline transport underlies hereditary motor

neuropathy Am J Hum Genet 2012911103ndash1107

37 Ekenstedt KJ Becker D Minor KM et al An ARHGEF10

deletion is highly associated with a juvenile-onset inherited

polyneuropathy in Leonberger and Saint Bernard dogs

PLoS Genet 201410e1004635

38 Pugliese-Pires PN Tonelli CA Dora JM et al A novel

STAT5B mutation causing GH insensitivity syndrome asso-

ciated with hyperprolactinemia and immune dysfunction in

two male siblings Eur J Endocrinol 2010163349ndash355

39 Kalita A Gupta S Singh P et al IGF-1 stimulated upre-

gulation of cyclin D1 is mediated via STAT5 signaling

pathway in neuronal cells IUBMB Life 201365462ndash471

40 Goebbels S Oltrogge JH Wolfer S et al Genetic disrup-

tion of Pten in a novel mouse model of tomaculous neu-

ropathy EMBO Mol Med 20124486ndash499



DOI 101212WNL00000000000037722017881226-1234 Published Online before print March 1 2017Neurology

Boglarka Bansagi Helen Griffin Roger G Whittaker et al Genetic heterogeneity of motor neuropathies

This information is current as of March 1 2017

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ISSN 0028-3878 Online ISSN 1526-632XWolters Kluwer Health Inc on behalf of the American Academy of Neurology All rights reserved Print1951 it is now a weekly with 48 issues per year Copyright Copyright copy 2017 The Author(s) Published by

reg is the official journal of the American Academy of Neurology Published continuously sinceNeurology

distress (SMARD1 or dHMN-VI) adult-onsetwith vocal cord palsy (dHMN-VII) and pyra-midal features or X-linked inheritance3

Gene identification in dHMN was previ-ously based on linkage studies in rare extendedfamilies However recent development ofnext-generation technology enabled studyingisolated patients or small families and a largenumber of new disease genes and novel pheno-types have been discovered4 Compared to the7 genes and 13 chromosomal loci 10 yearsago56 to date we acknowledge around 30genes responsible for autosomal dominantrecessive and X-linked forms of dHMN Toidentify the molecular cause of motor neurop-athies we performed detailed clinical investi-gations and state of the art genetic studies ina large population-based cohort from theNorth of England

METHODS Patients Between 2010 and 2015 we selected

105 patients out of 461 diagnosed with inherited neuropathies

in our genetic neuropathy clinic at Newcastle Hospitals NHS

Foundation which is the catchment area of a total population

of 299 million Inclusion of patients was based on the presence

of a motor neuropathyneuronopathy with no or only subclinical

sensory changes on electrophysiology Acquired causes were

excluded by detailed laboratory analysisno response on immuno-

suppressive therapy Some affected family members of patients

with a confirmed genetic diagnosis were included in this cohort

even if no electrophysiologic data were available

Standard protocol approvals registrations and patientconsents Participants provided written informed consent

approved by local research ethics committees for all experiments

using human participants and for photographs that may be

published

Electrophysiology We performed electrophysiologic examina-

tion in 90 patients according to standard techniques and inter-

preted by the same expert neurophysiologists Eight family

members did not undergo electrophysiologic studies 7 patients

were tested at different hospitals Motor and sensory nerve con-

duction studies (NCS) were performed and qualitative and quan-

titative analysis of motor unit potentials and spontaneous activity

were assessed on EMG Patient classification into study groups

was based on these analyses Additional assessment of repetitive

nerve stimulation and single-fiber EMG were carried out in 27

patients and the percentage increment or decrement was

calculated EMG was performed using Natus Neurology

(Pleasanton CA) disposable 30-G concentric needles with

a bandpass range of 10ndash10000 Hz For single-fiber EMG

studies the low pass filter was increased to 2000 Hz and the

percentage of fiber pairs showing increased jitter or blocking was

calculated

Molecular genetic analysis DNA was obtained from periph-

eral blood Initially PMP22 deletionduplication MFN2 andGJB1 were excluded in all patients The multigene panel assay

utilizing next-generation sequencing (NGS) was carried out in 46

index patients in the Bristol Genetics Laboratory following the

UK Genetic Testing Network approved criteria Variant

classification was based on Association for Clinical Genetic

Science Practice Guidelines (2013) We performed whole exome

sequencing (WES) in 40 patients DNA was fragmented exome-

enriched and sequenced (Illumina [San Diego CA] TruSeq 62 Mb

and HiSeq 2000 100 bp paired-end reads) Bioinformatic analysis

included duplicate sequence read removal with FastUniq7

alignment to UCSC hg19 with BWA8 variant detection with

FreeBayes and variant annotation with ANNOVAR Variants

were annotated as exonicsplicing excluding synonymous variants

with a rare minor allele frequency (MAF 001) in the context of

genotype (heterozygous MAF 0001 homozygous MAF 001)

in several databases downloaded via ANNOVAR (ExAC

NHLBI_ESP6500 cg69) and also in 281 in-house exomes

Protein prediction and evolutionary sequence conservation

algorithms downloaded via ANNOVAR were used to analyze the

in silico effects following the guidelines of the American College of

Medical Genetics and Genomics9 Variants were defined as

confirmed pathogenic if they were previously shown to be

pathogenic or if they were novel variants in a known neuropathy-

associated gene predicted to be deleterious (affect protein structure

or function highly conserved) and segregated with the disease in at

least 1 additional affected family member10 Highly conserved in

silico deleterious rare sequence variants of known or novel genes

were determined as possible pathogenic if segregation studies

involving at least one additional affected family member could not

be carried out Putative pathogenic variants were confirmed by

Sanger sequencing and were tested for segregation

RESULTS Clinical presentation We identified 105patients from 73 families with inherited motor neurop-athies Based on neurologic and electrophysiologicfindings all patients were classified into 1 of 3 sub-groups dHMN motor predominant Charcot-Marie-Tooth disease (CMT2) (motor CMT2) and motorneuropathy with additional neurologic symptoms(hereditary motor neuropathy [HMN] plus)Diagnosis of dHMN was based on preserved sensorynerve studies with normal or reduced compoundmotor unit action potentials or neurogenic changeson EMG examination2 Patients with dHMN butwith decreased sensory nerve action potentialsindicative of an accompanying sensory axonopathywere grouped as motor CMT2 We considered HMNplus when motor neuropathy was the leading featurebut was accompanied by other neurologic symptomsNCS data of all patients are available on request

All patients seen in our center were from ourcatchment area (299 million people in 2011 UK cen-sus) leading to a minimum prevalence of dHMN as214 affected individuals per 100000 inhabitants(95 confidence interval [CI] 162ndash266)

Sixty-four out of 105 patients (609) had dHMNSixteen patients had motor CMT2 (152) and 25HMN plus (238) (figures 1ndash3) There was male pre-dominance in all phenotypic groups Symptoms startedat a younger age in dHMN (mean 16 years) and HMNplus (mean 176 years) compared to motor CMT2(mean 238 years) The main inheritance pattern wasautosomal dominant in CMT2 (710 families [70])
















































































































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200413195ndash202






20127e52249



251754ndash1760








138276ndash283






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590ndash601










ord 201525516ndash521








201125182ndash188





20162e119







2014155ndash63




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Reprints











































































































200413195ndash202






20127e52249



251754ndash1760








138276ndash283






348g459




590ndash601










ord 201525516ndash521








201125182ndash188





20162e119







2014155ndash63




2621673ndash1677




711ndash713









































































Reprints





































































































200413195ndash202






20127e52249



251754ndash1760








138276ndash283






348g459




590ndash601










ord 201525516ndash521








201125182ndash188





20162e119







2014155ndash63




2621673ndash1677




711ndash713









































































Reprints





























































































200413195ndash202






20127e52249



251754ndash1760








138276ndash283






348g459




590ndash601










ord 201525516ndash521








201125182ndash188





20162e119







2014155ndash63




2621673ndash1677




711ndash713









































































Reprints




















































































200413195ndash202






20127e52249



251754ndash1760








138276ndash283






348g459




590ndash601










ord 201525516ndash521








201125182ndash188





20162e119







2014155ndash63




2621673ndash1677




711ndash713









































































Reprints










































































200413195ndash202






20127e52249



251754ndash1760








138276ndash283






348g459




590ndash601










ord 201525516ndash521








201125182ndash188





20162e119







2014155ndash63




2621673ndash1677




711ndash713









































































Reprints













200413195ndash202






20127e52249



251754ndash1760








138276ndash283






348g459




590ndash601










ord 201525516ndash521








201125182ndash188





20162e119







2014155ndash63




2621673ndash1677




711ndash713









































































Reprints






















Reprints




Documents

Genetic heterogeneity of motor neuropathies · neuropathies in a large cohort of patients from the North of England. Methods: Detailed neurologic and electrophysiologic assessments