Received: 5 April 2021 | Accepted: 17 June 2021 DOI: 10.1111/ene.15001 ORIGINAL ARTICLE Charcot–­Marie–­Tooth disease due to MORC2 mutations in Spain Rafael Sivera1 | Vincenzo Lupo2 | Marina Frasquet3,4,5 | Herminia Argente-­Escrig3,4,5 | Jorge Alonso-­Pérez6,7 | Jordi Díaz-­Manera5,6,7,8 | Luis Querol5,6,7 | María del Mar García-­Romero9,10 | Samuel Ignacio Pascual9,10 | Tania García-­Sobrino11 | Carmen Paradas12,13 | Juan Francisco Vázquez-­Costa3,4,5,15 | Nuria Muelas3,4,5 | Elvira Millet14 | Juan Jesús Vílchez3,4,5,15 | Carmen Espinós2 | Teresa Sevilla3,4,5,15 1 Department of Neurology, Hospital Francesc de Borja, Gandía, Spain 2 Unit of Rare Neurodegenerative Diseases Felipe, Centro de Investigación Príncipe, Valencia, Spain 3 Neuromuscular Diseases Unit, Department of Neurology, Hospital Universitari i Politècnic La Fe, Valencia, Spain 4 Neuromuscular and Ataxias Research Group, Instituto de Investigación Sanitaria La Fe, Valencia, Spain 5 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, Spain 6 Neuromuscular Diseases Unit, Department of Neurology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain 7 Universitat Autonoma de Barcelona, Barcelona, Spain 8 John Walton Muscular Dystrophy Research Center, Newcastle University Translational and Clinical Research Institute, Newcastle Upon Tyne, UK 9 Neuropaediatrics Department, Hospital Universitario La Paz, Madrid, Spain 10 Department of Pediatrics, Universidad Autónoma de Madrid, Madrid, Spain 11 Department of Neurology, Complexo Hospitalario Universitario de Santiago de Compostela, Santiago de Compostela, Spain 12 Department of Neurology, Hospital Universitario Virgen del Rocío, Sevilla, Spain 13 Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain 14 Neuromuscular Diseases Unit, Department of Clinical Neurophysiology, Hospital Universitari i Politècnic La Fe, Valencia, Spain 15 Department of Medicine, Universitat de València, Valencia, Spain Correspondence Abstract Teresa Sevilla, Hospital Universitari i Politècnic La Fe, Valencia, Centro de Background and purpose: MORC2 mutations have been described as a rare cause of ax- Investigación Biomédica en Red de onal Charcot–­Marie–­Tooth disease (CMT2Z). The aim of this work was to determine the Enfermedades Raras (CIBERER), Valencia, Universitat de Valencia, Avinguda de frequency and distribution of these mutations throughout Spain, to provide a compre- Fernando Abril Martorell, 106, 46026 hensive phenotypical description and, if possible, to establish a genotype–­phenotype València, Valencia. Email: m.teresa.sevilla@uv.es correlation. Methods: Retrospectively, data on patients diagnosed with CMT2Z in Spain were col- Funding information This collaborative joint project is awarded lected and clinical, electrophysiological and muscle imaging information were analysed. by IRDiRC and funded by the Instituto Results: Fifteen patients with CMT2Z were identified throughout Spain, seven of them de Salud Carlos III (ISCIII)—­Subdirección General de Evaluación y Fomento de belonging to a single kindred, whilst the rest were sporadic. The most common mutation la Investigación within the framework was p.R252W, and four new mutations were identified. Eleven patients were catego- of the National R+D+I Plan co-­funded with ERDF funds (Grants PI16/00403, rized as having a scapuloperoneal phenotype, with asymmetric muscle weakness, early PI16/00316, PI19/01178) and Río Hortega proximal upper limb involvement and frequent spontaneous muscular activity with distal contract to MF (Grant CM20/00152) This is an open access article under the terms of the Creat​ive Commo​ns Attri​bution-NonCo​mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2021 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology Eur J Neurol. 2021;28:3001–3011.  wileyonlinelibrary.com/journal/ene | 3001 | 14681331, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.15001 by Readcube (Labtiva Inc.), Wiley Online Library on [26/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3002 SIVERA et al. and Juan Rodés contract to JFVC (Grant sensory impairment and pes cavus, whilst two presented with a more classic length de- JR19/00030) and by the Generalitat Valenciana (Grant PROMETEO/2018/135 pendent sensory motor phenotype. This distinction was corroborated by the distribution and PROMETEO/2019/075). Part of the of muscle fatty infiltration in muscle imaging. Two other patients were classified as having equipment employed in this work has been funded by Generalitat Valenciana and a neurodevelopmental phenotype consisting in congenital or early onset, delay in motor co-­financed with ERDF funds (OP ERDF milestones, and global developmental delay in one of them. Nerve conduction studies of Comunitat Valenciana 2014-­2020). TS is part of the Inherited Neuropathy revealed an unequivocally axonal neuropathy with frequent spontaneous activity, and Consortium (INC), which is within the serum creatine kinase levels were increased in 50% of the patients. NCATS Rare Diseases Clinical Research Network (RDCRN) and is supported also Conclusions: MORC2 mutations are a rare cause of CMT in Spain, but in-­depth phenotyp- by the Muscular Dystrophy Association ing reveals a recognizable phenotypic spectrum that will be clinically relevant for future (MDA) and the Charcot–­Marie–­Tooth American Association (CMTA). MF, JD-­M , identification of this disease. LQ, CP, JFVC, NM, EM, JJV and TS are members of the European Reference KEYWORDS Network for Rare Neuromuscular Charcot–­Marie–­Tooth disease, CMT2Z, MORC2, Spain Diseases (ERN EURO-­NMD). I NTRO D U C TI O N learning difficulties, seizures or hearing defects [2]. Since then, sev- eral case reports and short clinical series have expanded and clarified Mutations in the gene microrchidia family CW-­type zinc finger 2 the phenotypic spectrum to include cerebellar atrophy, respiratory (MORC2) were first identified as a rare cause of axonal Charcot–­ insufficiency, dysphonia, microcephaly and retinal abnormalities [3–­ Marie–­Tooth disease (CMT2Z) in three Spanish kindred [1]. Affected 11]. Recently, MORC2 mutations have been associated with growth individuals in one family presented with hypotonia at birth, devel- retardation, short stature and cranial dysmorphism in many cases opmental delay, important limb weakness, scoliosis and respiratory without prominent neuromuscular involvement [12]. To date, 18 caus- insufficiency, whilst the rest presented with an axonal, motor and sen- ative amino acid changes in MORC2 have been reported, and there is sory asymmetric neuropathy with early proximal involvement. Almost clinical information of 89 patients, as detailed in Figure 1 and Table S1. simultaneously, the finding was replicated in four Australian families in The MORC2 protein is a member of the MORC protein family which the phenotype also included pyramidal signs and occasionally and has a GHKL-­t ype ATPase module composed of a GHKL-­t ype MORC2 Phenotype Reference Mutation p.T24I Neurodevelopmental 12 p.E27K Neurodevelopmental 12 p.S87L Neurodevelopmental 1 p.A88V Neurodevelopmental 12 p.R132C Neurodevelopmental 12 p.R132L Scapuloperoneal 4 p.A152P SM polyneuropathy This work p.E236G Scapuloperoneal 2 p.R252W Scapuloperoneal 1 p.R266S Neurodevelopmental 12 p.R319H Adult onset SMA 11 p.R319C SM polyneuropathy This work p.S388R Neurodevelopmental 12 p.Y394C Neurodevelopmental, 10 scapuloperoneal p.Q400R Scapuloperoneal 5 p.A406V Scapuloperoneal This work p.C407Y SM polyneuropathy 10 F I G U R E 1 MORC2 protein structure p.V413F Neurodevelopmental 12 and corresponding MORC2 mutations and reported phenotype. CC, coiled-­ p.T424R Neurodevelopmental 6 coil insertion; CW, zinc finger domain; p.A431V Scapuloperoneal 10 CD, coil domain; SMA, spinal muscular p.D466N Scapuloperoneal 7 atrophy [Colour figure can be viewed at p.D466G Scapuloperoneal This work wileyonlinelibrary.com] | 14681331, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.15001 by Readcube (Labtiva Inc.), Wiley Online Library on [26/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License CHARCOT–­MARIE–­TOOTH DISEASE DUE TO MORC2 MUTATIONS IN SPAIN 3003 ATPase domain (residues 1–­265), a transducer S5-­like domain (res- with whole exome sequencing as previously reported [1]. Two ad- idues 366–­494) and a coiled-­coil insertion CC1 (residues 282–­361), ditional families were then diagnosed by Sanger sequencing [1]. A a CW-­t ype finger domain, and three predicted coiled-­coil domains search for mutations in MORC2 using an in-­house gene panel that [13,14]. Causative mutations have been detected in all the domains employs a hybridization-­based targeted sequencing with Agilent that compose the ATPase module. The most frequent location is the probes (Agilent technologies) to capture the coding exons and GHL-­ATPase domain, where the c.754C>T (p.R252W) change can be flanking intronic sequences of 121 inherited peripheral neuropa- found, which represents 41% of the cases published. The pathway thy genes (Table S2) and sequencing on an Illumina MiSeq was by which these changes alter the function of MORC2 and can cause performed in the rest of the cases. Variant analysis from next peripheral neuropathy has been subject to much debate and many generation sequencing data was carried out using standard proce- investigations and includes transcriptional dysregulation, HUSH-­ dures. For a comprehensive impact analysis of the novel variants, mediated repression, destabilization of the ATPase-­CW module, VarSome (https://varso​me.com/) which evaluates them according altered expression of homeobox genes etc. [15,16]. There does not to the American College of Medical Genetics guidelines was used seem to be a clear correlation between the affected domain and the [17]. Genotype–­phenotype correlation and final classification of phenotype, although most of the variants have been detected in very the variants were discussed in a multidisciplinary setting including few subjects (Figure 1, Table S1). CMT clinicians and a geneticist specialized in inherited peripheral The aim of the study was to determine the distribution and neuropathies. All the variants reported in this work were confirmed frequency of causative MORC2 mutations in Spain, to provide a by Sanger sequencing and further investigated by segregation anal- comprehensive phenotypical description and to try to establish a ysis if possible. genotype–­phenotype correlation. Clinical assessments PATI E NT S A N D M E TH O D S Clinical assessments were collected using a standardized history Patients and symptom questionnaire. Examinations included strength using the standard Medical Research Council (MRC) scale, pinprick, vibra- This cross-­sectional retrospective observational study includes all tory and positional sensory modalities, reflexes, as well as a general CMT patients with causative MORC2 mutations evaluated at five and neurological examination. The severity of the neuropathy was centres throughout Spain. Patients were identified in a nationwide evaluated with the CMT neuropathy score version 1 (CMTNSv1) as register of hereditary neuropathies developed in 2012 as part of a significant proportion of these patients had not undergone the the Spanish Registry of Neuromuscular Diseases project which was neurophysiological radial nerve testing which is mandatory for the designed in accordance with current Spanish law on biomedical re- second version [18,19]. The CMT examination score was used for search and data protection. The Registry is reviewed annually to patients without nerve conduction studies. safeguard the quality of the information and data included in the Registry have been used in previous published studies. Also, a call for collaboration was issued in the Neuromuscular Work Group of Nerve conduction studies the Spanish Neurology Society and the genetic database of Centro de Investigación Principe Felipe (CIPF) in Valencia was reviewed. Motor and sensory nerve conduction velocities (CVs) were per- formed using standard techniques. Temperature was controlled dur- ing the procedure and kept at more than 32℃. Compound muscle Standard protocol approvals, registrations and action potential amplitudes (CMAPs), sensory nerve action potential patient consents amplitudes (SNAPs), conduction velocities and distal latencies were recorded. Needle electromyography was performed in proximal and All patients and relatives included in the present study signed in- distal muscles of the upper and lower limbs. Spontaneous muscular formed consent, and the research protocols were approved by the activity was graded from 0 to ++++; motor unit potential amplitude, respective institutional board of the Ethics Committees of the cor- duration and phases were analysed. responding hospitals. Photographs and videos of the patients were taken and reproduced only after specific informed consent. Muscle magnetic resonance imaging Genetic studies Muscle magnetic resonance imaging (MRI) was performed in the upper and lower limbs if possible. The protocol employed was the In all cases the molecular studies were performed in the CIPF in same as described previously, and muscle fatty substitution was Valencia. The MORC2 mutations of the first kindred were studied graded from 0 to 4 according to the modified Mercuri score as | 14681331, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.15001 by Readcube (Labtiva Inc.), Wiley Online Library on [26/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3004 SIVERA et al. follows: 0, no fat signal in muscle; 1, traces of increased signal inten- Clinical picture sity on the T1-­weighted MRI sequences; 2, increased T1-­weighted signal intensity with beginning confluence in less than 50% of the Fifteen CMT2Z patients were included, and the clinical characteris- muscle; 3, increased T1-­weighted signal intensity with beginning tics of 14 are detailed in Table 1. Part of the clinical information of confluence in more than 50% of the muscle; 4, entire muscle re- families fCMT-­197, fCMT-­237 and fCMT-­438 have been previously placed by increased density of connective tissue and fat [20,21]. reported but are detailed and updated in this work [1]. Probands Heat maps of muscle fatty infiltration were developed. were detected throughout Spain, without a clear geographical cluster. To categorize the clinical characteristics into phenotypic groups, Laboratory analysis whether the predominant impairment was neurodevelopmental or neuromuscular was taken into account, and in the latter case Blood tests were performed after overnight fasting and at least 5 whether muscle weakness was length dependent or asymmetric days without any physical exertion. They included basic biochemical with early shoulder girdle involvement. analysis and creatine kinase (CK) activity in the serum. The upper The 11 patients with the p.R252W, p.A406V and p.D466G limit CK levels were defined by each laboratory and hyperCKemia changes were classified as having a scapuloperoneal phenotype, was considered when the value was >1.5 times the upper limit of reminiscent of the description published by Davidenkow in 1927 normal according to the European Federation of Neurological [23]. Symptom onset appeared in the first two decades except for Societies guidelines [22]. Median CK values were determined if more one asymptomatic 17-­year-­old carrier. The most frequent initial than one was available. symptoms were walking difficulties, tripping and cramps, the for- mer present in 78%. Weakness always began distally in the lower limbs but spread to intrinsic hand muscles (usually between 20 and R E S U LT S 30 years of age) and subsequently to proximal muscles of the upper more than the lower limbs. Proximal muscle involvement was het- Genetic analysis erogeneous, weakness being more prominent in pelvic and espe- cially in shoulder girdle muscles, whilst knee and elbow extensors A total of seven MORC2 missense mutations were identified in were relatively preserved (mean MRC scores of shoulder abduction nine families with CMT (Table 1). All the genetic studies were vs. elbow flexo-­extension were 3.2 vs. 4.1), as seen in Figure 2. Neck performed in the CIPF in Valencia. 389 CMT2 probands had to extension was weak in 6/10 patients. Spontaneous muscular activity be screened to detect these subjects; in most of them causa- was frequently observed, mostly in shoulder girdle muscles (Videos tive GDAP1, GJB1 and MFN changes had already been ruled out. S1–­S3) but also in other areas like the thigh muscles or even the The most common change was the NM_001303257:c.754C>T tongue of patient III:3 of fCMT-­397 (Video S4). Distal limb sensory (p.R252W), which was detected in a seven-­member kindred and impairment was observed early in the course of the disease, as were in two sporadic patients. Two other previously reported mutations pes cavus and reduced tendon reflexes. Two patients reported hear- were observed, the NM_001303257:c.260C>T (p.S87L) and the ing loss, and the two most severely affected also reported urinary NM_001303257:c.1181A>G (p.Y394C) [1,10,12]. incontinence. No central nervous system features were observed, The other four variants were detected for the first time like pyramidal signs, learning difficulties or seizures. in this work: the NM_001303257:c.955C>T (p.R319C) and The cases harbouring the p.R319C and p.A152P changes were NM_001303257:c.1397A>G (p.D466G) changes were predicted as classified as having a sensory-­motor length dependent polyneurop- deleterious by eight different pathogenic predictions in the VarSome athy phenotype. Patient S1247, harbouring the p.R319C change, re- platform and, moreover, they are located in the same codon where ported an onset at 30 years with stumbling and distal lower limb previous pathogenic mutations have been reported (p.R319H and weakness. Mild proximal weakness in the lower limbs appeared p.D466N, respectively) [7,11]; the NM_001303257:c.1217C>T during the course of the disease, as well as in the intrinsic hand (p.A406V) mutation was predicted as deleterious by five different com- muscles but not in the shoulder girdle. Sensory impairment was putational predictions and it segregates with the disease—­the affected prominent, especially in the lower limbs needing a walker for sta- father harboured the variant in heterozygous state whilst two healthy ble ambulation, and tendon reflexes were asymmetrically reduced. siblings were negative for this change. Therefore, the novel missense Finally, patient S1238 (p.A152P—­VUS) had a disease onset in the changes p.R319C, p.D466G and p.A406V were considered as new first decade with stumbling, foot deformities and distal lower limb MORC2 pathogenic mutations. Finally, the NM_001303257:c.455C>G weakness. Disease progression was relatively slow until the fifth or (p.A152P) variant was predicted as benign by 11 in silico programs in sixth decade, with clear worsening of the distal weakness and devel- VarSome and it appears in 1/250364 alleles in the gnomAD database opment of intrinsic hand muscles, and proximal lower limb weakness (https://gnomad.broad​insti​tute.org/); no other family members were but no proximal upper limb weakness. All sensory modalities were available for segregation analysis. Thus it was classified as a variant of impaired throughout the disease. Pes cavus, scoliosis and upper limb uncertain significance (VUS). tremor were observed. TA B L E 1 Clinical characteristics. Motor Sensory Reflexes MORC2 CMTNS Functional Patient mutation Phenotype Onset (years) Cramps Age UL LL UL LL UL LL CMTES situation (#) CK Other fCMT-­438 p.S87L ND Hypotonia at birth No 6 3/3 4/2 –­ –­ 0 0 –­/–­ Wheelchair –­ Microcephaly, scoliosis fCMT-­237 p.R252W SP Stumbling, LL weakness –­ 41 2/2 3/2 T, P, V T, P, V 0 0 30/22 Wheelchair –­ II:1 (5–­10) (58) fCMT-­237 p.R252W SP Difficulty walking, cramps (5) Yes 75 2/2 2/2 T, P, V All 0 0 31/28 Wheelchair 219 Incontinence, II:3 (60) hypoacusis fCMT-­237 p.R252W SP Difficulty walking, cramps (3) Yes 47 4/3 4/2 T, P, V T, P, V 0 0 24/20 Walking stick 381 Incontinence, III:1 (31) tremor fCMT-­237 p.R252W SP LL weakness (16) Yes 51 4/2 4/2 T, P, V T, P, V 0 0 27/22 Walking stick 118 III:4 (35) fCMT-­237 p.R252W SP Cramps (5) Yes 20 5/4 5/4 V P, V + 0 11/9 Running –­ IV:3 difficulty CHARCOT–­MARIE–­TOOTH DISEASE DUE TO MORC2 MUTATIONS IN SPAIN fCMT-­237 p.R252W SP Asymptomatic, mild cramps Yes 17 5/5 5/4 Normal V ++ Achilles 0 –­/2 Difficulty heel –­ IV:4 Patellar + walking fCMT-­197 p.R252W SP Delay motor milestones (2) Yes 45 2/2 2/2 T, P, V T, P, V 0 0 32/29 Wheelchair 210 Incontinence (39) fCMT-­4 0 p.R252W SP Stumbling (5) Yes 26 5/4 5/2 N T 0 0 9/7 No aids 804 S1247 p.R319C SMP Stumbling, LL weakness (30) Yes 65 5/4-­ 4/2 T, P, V All Left 0 0 23/21 Walker (65) 350 right + S1325 p.Y394C ND Delay motor and mental No 46 4/3 2/2 Normal P, V 0 0 25/18 Wheelchair –­ Intellectual development (36) disability fCMT-­397 p.A406V SP LL weakness, cramps (13) Yes 45 2/3 3/4 Normal T, P, V 0 0 18/15 Wheelchair 555 (45) S1150 p.D466G SP Proximal LL weakness (25) No 68 2/3 2/0 T, P, V T, P, V 0 0 22/18 Walker (58) –­ Hyperhidrosis, tremor S1238 p.A152Pa SMP Stumbling, foot deformities Yes 75 5/4 4/4 –­ All 0 0 15/13 Crutches (60) 246 Tremor, (5–­10) scoliosis Abbreviations: #, age at which the aid use started; CK, maximum creatine kinase in serum expressed in U/l; CMT, Charcot–­Marie–­Tooth; CMTNS, CMT neuropathy score version 1; CMTES: CMT examination score; LL, lower limbs; ND, neurodevelopmental phenotype; SMP, sensory motor length dependent polyneuropathy; SP, scapuloperoneal phenotype; UL, upper limbs. Sensory modalities: P, pain; Po, positional; T, touch; V, vibration. a This change is classified as a variant of uncertain significance at the moment. | 3005 14681331, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.15001 by Readcube (Labtiva Inc.), Wiley Online Library on [26/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 14681331, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.15001 by Readcube (Labtiva Inc.), Wiley Online Library on [26/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3006 SIVERA et al. (a) (c) (d) (b) F I G U R E 2 Characteristic ‘scapuloperoneal’ phenotype in patient fCMT-­397 at age 44. (a) Asymmetric scapular winging and atrophy of trapezius and deltoideus. (b) Patient is incapable of raising his arms over his shoulders because of weakness of the scapular girdle muscles. (c) Hand atrophy, especially in thenar muscles. (d) Bilateral atrophy of the lower legs [Colour figure can be viewed at wileyonlinelibrary.com] The patients harbouring the p.S87L and p.Y394C changes and occasional respiratory support. Microcephaly was detected were classified as having a neurodevelopmental disorder pheno- at birth, but subsequent cognitive and language development has type. The patient with the p.Y394C change (S1325) had an onset been practically normal. in early childhood consisting in developmental motor and cogni- tive delay. He managed to walk at a late age, but proximal weak- ness appeared in his 30s and soon after he became wheelchair Electrophysiological studies bound. At examination there was important lower limb weakness (2/5 proximally and distally) and upper limb involvement (4/5 Nerve conduction studies and needle electromyography were per- proximally and 2–­3/5 distally) with mild asymmetry. Intellectual formed in 13 patients and the findings are presented in Table 2. disability was also prominent, but no seizures, microcephaly or fa- Changes were relatively uniform in all phenotypic groups. Motor cial dysmorphism were observed. Patient III:3 of fCMT-­4 38, har- and sensory CVs were within normal range in practically all the bouring the p.S87L mutation, presented with hypotonia at birth nerves that were explored. CMAPs were reduced globally, but and was able to stand up at 24 months as was reported previously especially in the lower limb nerves. SNAPs were also decreased [1]. At 6 years of age, she is wheelchair dependent but able to throughout the whole series, even when studied early in the course take a few lateral steps, and on examination there is prominent of the disease. In most patients, the decrease in CMAPs was not neck and limb girdle weakness (3/5 global MRC score) as well as homogeneous amongst the different nerves of the upper and lower of the distal muscles of the limbs, with relative sparing of knee limbs. The median nerve appeared to be affected earlier and to a and elbow flexion-­e xtension (4/5). She also suffers from kypho- greater degree than the ulnar nerve: mean median CMAP was 4 mV scoliosis treated with a corset, needs nutritional supplementation (0.1–­8 mV) and mean ulnar CMAP was 7.9 mV (0.5–­15.5 mV). In the TA B L E 2 Nerve conduction studies Needle electromyography Ulnar Median Posterior MORC2 CV/ CV/ Peroneal tibial CV/ Ulnar CV/ Median Sural CV/ Peroneal Spontaneous Patient mutation Age CMAP CMAP CV/CMAP CMAP SNAP CV/SNAP SNAP CV/SNAP activity Distribution MUP morphology fCMT-­4 38 p.S87L 2 –­ –­ 31.7/0.7 –­ –­ –­ 56/3.3 –­ –­ –­ Denervation fCMT-­237 II:1 p.R252W 41 –­ NR NR –­ –­ NR NR –­ Fa Biceps, deltoid Increased A, P fCMT-­237 II:3 p.R252W 75 58/4.5 –­ NR –­ 55/1.2 –­ NR –­ Fi, PSW Triceps Increased A, P, D fCMT-­237 p.R252W 47 61.7/10 55/7.8 43.3/0.7 47.6/13.4 NR NR NR NR Fa +, M +++ AT, ADM Increased A, P, D III:1 fCMT-­237 p.R252W 51 58/4.3 NR/2.2 37/2.3 42.5/5.2 –­ NR NR –­ No –­ Increased A, P III:4 fCMT-­237 p.R252W 20 54.7/8.2 55/6.6 46.8/3.5 50/10.3 53.6/3.1 NR 52/1.2 NR Fa ++, M ++ Deltoid, ADM, Increased A, P, D CHARCOT–­MARIE–­TOOTH DISEASE DUE TO MORC2 MUTATIONS IN SPAIN IV:3 AT fCMT-­197 p.R252W 22 62/15.5 56/8 57/0.7 42/8.4 46/0.7 NR 39/0.8 NR M +, Fi ++, Deltoid, biceps, Increased A, P, D PSW ++ ADM, AT fCMT-­4 40 p.R252W 25 –­ 57.8/12 NR –­ 52/2.3 52/4.6 NR –­ –­ –­ –­ S1247 p.R319C 64 50/7.1 NR/0.4 NR/0.1 NR 45.8/3.8 35.6/0.8 42/1 38.3/0.9 Fi +, PSW + AT Increased A, P, D S1325 p.Y394C 45 –­/0.5 NR –­ –­ NR –­ –­ –­ –­ –­ –­ fCMT-­397 p.A406V 38 65/11 66.7/0.1 28.6/0.4 48.2/4.2 57/0.3 51.6/1 NR NR Fa +, M +, Deltoid, ADM, Increased A, P, D Fi + Ga, gluteus S1150 p.D466G 47 56/4.6 49/1.3 –­ –­ NR NR –­ –­ PSW + Biceps, AT Increased A, P, D a S1238 p.A152P 75 37/10 42/5.6 36/0.6 34/0.9 36/9 37/13 31/4 –­ Fi ++, PSW AT, Ga Increased A, P, D ++ Abbreviations: –­, not performed; A, amplitude; ADM, abductor digiti minimi; AT, anterior tibialis; CMAP, compound muscle action potential, expressed in mV; CV, conduction velocity, expressed in m/s; D, duration; Fa, fasciculation; Fi, fibrillation; Ga, gastrocnemius; M, myokymia; MUP, motor unit potential; NR, not recordable; P, polyphasia; PSW, positive sharp wave; SNAP, sensory nerve action potential, expressed in mV. a This change is classified as a variant of uncertain significance at the moment. | 3007 14681331, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.15001 by Readcube (Labtiva Inc.), Wiley Online Library on [26/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 14681331, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.15001 by Readcube (Labtiva Inc.), Wiley Online Library on [26/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3008 SIVERA et al. lower limbs, there was also asymmetry between the CMAPs of the and of fCMT-­237 III:1 and fCMT-­397 in Figure 3 [1]. All stud- more affected peroneal nerve and the tibial nerve. Needle electro- ies were performed in patients with a scapuloperoneal clinical myography was performed in 11 patients and spontaneous muscu- presentation except S1247 and in all the studied subjects there lar activity was detected in nine, consisting mostly of fibrillation and was detectable muscle fatty infiltration and atrophy. Short T1 positive sharp waves, but also fasciculations and myokymia. They inversion recovery hyperintensities were only observed in the were prominent in the upper limb muscles (deltoideus, biceps, ab- gastrocnemius of one patient. Heat maps of fatty substitution ductor digiti minimi) but also present in lower limb muscles of 4/9 are detailed in Figure 3b. In patients with the scapuloperoneal patients. Motor unit potentials were unequivocally classified as neu- phenotype the pattern of muscle fatty infiltration was not length rogenic, with increase in amplitude, duration and number of phases. dependent as the intrinsic muscles of the feet were less infil- trated than the muscles of the calf in 4/5 cases (Figure 3a). In the latter, there was less fatty infiltration in the deep posterior Muscle magnetic resonance compartment of the calf compared to the rest of the compart- ments (graded 2.4 vs. 3.4 with the modified Mercuri scale). In Muscle MRI was performed in seven patients; the images of the thigh, early and frequent fatty substitution in semimembra- fCMT-­237 III:4 and fCMT-­197 can be consulted in previous work, nosus and semitendinosus muscles could be detected, and in the (a) 1 3 5 2 4 6 (b) - 0 - 1 2 - - - - - - - - - - - - - 3 - - - - - - - - - - - - - 4 Gastrocnemius medialis Gastrocnemius lateralis Mulfudus iliocostalis Intrinsic foot muscles Rhomboideus major Vastus intermedius Semimembranosus Extensor digitorum Gluteus mínimum Rectus abdominis Gluteus maximus Serratus anterior Tibialis posterior Flexor digitorum Biceps femoralis Semitendinosus Lassimus dorsi Gluteus medius Supraespinosus Tibialis anterior Vastus medialis Vastus lateralis Rectus femoris Subescapularis Infraespinosus Triceps brachii Biceps brachii Deltoideus Peronealis Pectoralis Trapecius Adductor Sartorius Gracilis Soleus Psoas F I G U R E 3 (a) (1), (2) Scapular girdle and upper limb muscle MRI of patient III:3 fCMT-­397 at age 38. Note the atrophy and fatty infiltration in the serratus anterior, triceps and biceps brachii [1] as well as in the deltoideus and subscapularis [2]. (a) (3), (4), (5) Lower limb muscle MRI of patient fCMT-­237 III:1 at age 48. In the calf there is relative preservation of the deep posterior muscular compartment of the calf and the intrinsic foot muscles compared to the superficial posterior and antero-­lateral compartments. In the thigh, note the initial fatty infiltration in the semimembranosus and semitendinosus muscles. (6) Thigh muscle MRI of patient S1150, age 69, with prominent fatty infiltration of all muscles except the adductor longus. (b) Heat map showing muscle imaging involvement by muscle MRI: (1) fCMT-­237 III:1, age 48, p.R252W; (2) fCMT-­237 III:4, age 51, p.R252W; (3) fCMT-­237 IV:3, age 20, p.R252W; (4) fCMT-­197, age 45, p.R252W; (5) S1247, age 64, p.R319C; (6) fCMT-­397 III:3, age 38, p.A406V; (7) S1150, age 69, p.D466G. In each patient fatty substitution is graded 0–­4 on the right and left side. –­, not performed | 14681331, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.15001 by Readcube (Labtiva Inc.), Wiley Online Library on [26/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License CHARCOT–­MARIE–­TOOTH DISEASE DUE TO MORC2 MUTATIONS IN SPAIN 3009 latter stages of the disease there was relative preservation of the phenotype which in our series corresponds to the patients with the adductor muscles, especially the adductor longus. In the pelvic p.R252W, p.A406V and p.D466G changes. girdle muscles, the gluteus medius and minimus were frequently This group had a symptom onset in the first two decades with infiltrated, as were the deltoideus, trapecius and subescapularis distal lower limb weakness and frequent cramps. Weakness spread in the shoulder girdle, but the forearm and hand muscles were early onto the distal upper limbs and subsequently to pelvic and not studied. Left–­r ight asymmetry, although mild, was present in shoulder girdle muscles, and neck extensors, whilst knee and elbow all the studies performed. These findings correlated with abnor- extensors were relatively preserved (Figure 2). Visible spontaneous mal muscular strength on clinical examination in all cases. The muscular activity in proximal upper limb muscles was quite frequent muscle MRI performed in subject S1247 confirmed the clinical (Videos), as were foot deformities (pes cavus and Achilles tendon length dependent weakness with an almost exclusive fatty infil- shortening) and sensory impairment from an early age. CNS signs tration of distal muscles without proximal involvement except for like hyperreflexia, learning difficulties or seizures which had previ- the semimembranosus. ously been reported [2] were not detected. There are other CMT subtypes with asymmetric muscle weak- ness, like the newly described repeat expansion in VWA1, but the HyperCkemia combination of early proximal upper limb involvement and frequent spontaneous muscular activity with clear sensory impairment make Creatine kinase serum levels were determined in eight patients and this phenotypic subtype quite characteristic [24]. It is hypothesized values are shown in Table 1. The mean values for blood CK were that it may fit the description published by Davidenkow in 1927 and increased (360.4 mg/dl, range 118–­8 04 mg/dl). CK levels did not named scapuloperoneal amyotrophy [23]. He described sporadic and correlate with clinical phenotype, severity or disease progression; dominant families with ‘a peculiar form of Charcot–­Marie disease with in fact the highest levels were detected in a young patient (26 years) typical distribution of atrophy in the lower limbs… on the other hand with a mild-­to-­moderate phenotype (CMTNS 15). the atrophy of the upper extremities was localized proximally and in- volved the muscles of the shoulder girdle and partially those of the hu- merus, whilst hypesthesia showed a characteristic distal distribution’. DISCUSSION The muscle MRI in this clinical group revealed a recognizable pat- tern of fatty substitution and atrophy concordant with the clinical Here a compilation of the CMT2Z patients detected throughout examination. There was preference of fatty infiltration in the calf mus- Spain is presented, accounting for one of the largest clinical series to cles over the intrinsic foot muscles and asymmetry between muscle date. This comprehensive phenotypic description allows the clinical groups, being more prominent in the anterolateral and superficial pos- and genetic spectrum of this disease to be delineated. The descrip- terior compartment in the calf, in certain muscles of the thigh (semi- tion of disease-­causing MORC2 variants is relatively recent and cur- membranosus and semitendinosus) and shoulder girdle (deltoideus, rent reported patients are detailed in Table S1. trapezius and subscapularis), whilst mostly sparing the anterolateral The most frequent underlying mutation is the p.R252W change, compartment of the thigh and the adductor muscles. In any case these which was detected in one-­third of the index cases in our series and findings need to be replicated and extended to include patients who 41% of those published, and affects the GHL-­ATPase domain, where represent the whole phenotypic spectrum of MORC2 mutations. 9/18 of the causative MORC2 mutations have been described. Three In the patients harbouring two other missense mutations pathogenic novel mutations (p.R319C, p.A406V and p.D466G) and (p.A152P and p.R319C) the predominant feature was also neuro- one VUS (p.A152P) were detected in our series; the latter also affects muscular, but weakness appeared with a more typical length depen- the ATPase domain. The p.R319C affects the coil–­coil domain whilst dent distribution, more concordant with a classic CMT2 phenotype. the other two (p.A406V and p.D466G) are located in the transducer Proximal weakness was symmetric and appeared only in the lower S5-­like domain between the coil–­coil and the zinc finger domain. This limbs and late in the course of the disease. Both had important domain seems to be a hotspot for mutations as eight other changes sensory impairment, one of them with disabling walking instability in this location had already been reported to cause disease (Figure 1). and the other with upper limb tremor. The muscle MRI performed In any case, there does not seem to be a clear correlation between in one of these patients confirmed the length dependent distal the affected domain and the phenotypic spectrum, as changes in the predominance. same domain, or even in the same position as p.R132C and p.R132L, On the other hand, two patients developed a severe form of the may cause quite different clinical presentations. disease with prominent neurodevelopmental and neuromuscular The phenotypic spectrum of these mutations includes both neu- impairment. The p.S87L change has been described in four other romuscular and central nervous system (CNS) impairment (Figure 1, patients who also presented with hypotonia and muscle weakness Table S1). An attempt was made to delineate phenotypic categories at birth, developing generalized weakness and sensory impairment according to the predominant feature (CNS neurodevelopmental [4,12]. Intellectual disability and facial dysmorphism were not de- vs. neuromuscular) and the muscle weakness distribution. The most tected in our patient but have been previously associated with distinctive clinical presentation was perhaps the scapuloperoneal this mutation [12]. The p.Y394C change is a clear example of the | 14681331, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ene.15001 by Readcube (Labtiva Inc.), Wiley Online Library on [26/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3010 SIVERA et al. phenotypic variability and overlap between the different phenotypic and editing (equal). Marina Frasquet: Data curation (equal); for- groups. Our patient presented with developmental motor delay and mal analysis (equal); writing—­review and editing (equal). Herminia intellectual disability associated with prominent progressive weak- Argente-­E scrig: Data curation (equal); formal analysis (equal); ness, being wheelchair bound since his 30s, but this same change has writing—­review and editing (equal). Jorge Alonso-­Pérez: Data cu- been previously described as a VUS in a sporadic Japanese patient ration (equal); formal analysis (equal); writing—­review and editing with mild neuropathic features, and subsequently in two sporadic (equal). Jordi Diaz-­Manera: Data curation (equal); formal analysis patients, one with prominent progressive, asymmetric limb weak- (equal); writing—­review and editing (equal). Luis Querol: Data cu- ness and elevated CK levels, and the other with a mild intellectual ration (equal); formal analysis (equal); writing—­review and editing disability, hypoacusia and spasticity [10,12]. (equal). Maria del Mar García-­Romero: Data curation (equal); for- Nerve conduction studies confirmed an unequivocally axonal mal analysis (equal); writing—­review and editing (equal). Samuel sensory and motor neuropathy irrespective of clinical categories or Ignacio Pascual-­Pascual: Data curation (equal); formal analysis genotypes. Sensory lower limb nerves were severely affected even in (equal); writing—­review and editing (equal). Tania García-­S obrino: patients with mild motor conduction abnormalities; in fact the lower Data curation (equal); formal analysis (equal); writing—­review limb SNAPs were not recordable or were severely reduced through- and editing (equal). Carmen Paradas: Data curation (equal); for- out the series. CMAP reduction was heterogeneous, being more pro- mal analysis (equal); writing—­review and editing (equal). Juan nounced in the peroneal nerve compared to the tibial nerve, which is Francisco Vázquez Costa: Data curation (equal); formal analysis concordant with the pattern of fatty infiltration detected in the calf (equal); writing—­review and editing (equal). Nuria Muelas Gómez: muscle MRI of most scapuloperoneal patients. Needle electromy- Data curation (equal); formal analysis (equal); writing—­review and ography revealed frequent spontaneous muscular activity predomi- editing (equal). Elvira Millet: Data curation (equal); formal analysis nantly in upper limb muscles, and this may be related to the frequently (equal); writing—­review and editing (equal). Juan J Vílchez: Data increased serum CK levels. Although the early proximal involvement curation (equal); formal analysis (equal); writing—­review and edit- and CK elevation may raise the issue of concomitant muscular dam- ing (equal). Carmen Espinós: Data curation (equal); formal analy- age, an in-­depth review of the electrophysiological and pathological sis (equal); writing—­review and editing (equal). Teresa Sevilla: features of muscle and nerve biopsies in our patients and those re- Conceptualization (lead); funding acquisition (equal); project ad- ported so far reveal an unequivocal neuropathic injury [1,2,6,10]. ministration (equal); supervision (lead); validation (equal); writing—­ MORC2 is a ubiquitous protein that has been shown to be highly original draft (equal); writing—­review and editing (lead). expressed in both embryonic and adult human neural tissues, as well as in glia [15,16]. Loss of function murine models of Morc2 are lethal, with DATA AVA I L A B I L I T Y S TAT E M E N T clear abnormalities in neural system development (https://dmdd.org. The data that support the findings of this study are available from uk/mutan​ts/Morc2a), and Morc2 expression has been seen to peak at the corresponding author upon reasonable request. early stages of development which is concordant with the neurodevel- opmental features described in some patients [16]. Although there have ORCID been advances in understanding the way in which MORC2 dysfunction Rafael Sivera https://orcid.org/0000-0003-2810-1180 can alter neural development or produce neuron damage, the complete Vincenzo Lupo https://orcid.org/0000-0002-3774-9854 physiopathological mechanisms by which some MORC2 changes alter Marina Frasquet https://orcid.org/0000-0001-7206-5362 the protein function in the early stages of neural development whilst Herminia Argente-­Escrig https://orcid.org/0000-0001-8537-1318 others occur in a more mature nervous system are still to be clarified, Jorge Alonso-­Pérez https://orcid.org/0000-0001-8866-5186 and this may be one of the keys to better understand the phenotypic Tania García-­Sobrino https://orcid.org/0000-0001-8067-431X spectrum in this disease. Juan Francisco Vázquez-­Costa https://orcid. org/0000-0002-3043-7938 AC K N OW L E D G E M E N T S Nuria Muelas https://orcid.org/0000-0002-2349-7481 The commitment of the families studied is deeply appreciated. Carmen Espinós https://orcid.org/0000-0003-4435-1809 Teresa Sevilla https://orcid.org/0000-0003-4716-2667 D I S C LO S U R E S None. REFERENCES 1. Sevilla T, Lupo V, Martinez-­Rubio D, et al. Mutations in the AU T H O R C O N T R I B U T I O N S MORC2 gene cause axonal Charcot–­Marie–­Tooth disease. Brain. 2016;139:62-­72. Rafael Sivera: Conceptualization (equal); data curation (lead); for- 2. Albulym OM, Kennerson ML, Harms MB, et al. MORC2 mutations mal analysis (equal); funding acquisition (supporting); investiga- cause axonal Charcot–­Marie–­Tooth disease with pyramidal signs. tion (equal); methodology (equal); validation (equal); visualization Ann Neurol. 2016;79:419-­427. (equal); writing—­original draft (lead); writing—­review and edit- 3. 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