Demyelinating Charcot–Marie–Tooth Neuropathy Associated with FBLN5 Mutations

ORIGINAL ARTICLE

Demyelinating Charcot–Marie–Tooth neuropathy associated with FBLN5 mutations

D. Safka Brozkovaa , T. Stojkovicb, J. Haberlovaa, R. Mazanecc, R. Windhagerd, P. Fernandes Roseneggerd, S. Hackerd,S.Zuchner€ e, A. Kochanski f, S. Leonard-Louisg, B. Francouh, P. Latouri, J. Senderekj, P. Seemana and M. Auer-Grumbachd

aDNA Laboratory, Department of Paediatric Neurology, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, Czech Republic; bCentre de Ref erence des Maladies Neuromusculaires Nord/Est/Ile de France, Institut de Myologie, APHP, G-H Pitie-Salp etri^ ere, Paris, France; cDepartment of Neurology, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, Czech Republic; dDepartment of Orthopaedics and Trauma Surgery, Medical University of Vienna, Vienna, Austria; eDr John T. Macdonald Foundation Department of Human Genetics, John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL, USA; fNeuromuscular Unit, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland; gUnite de Pathologie Neuromusculaire, Centre de Ref erence des Maladies Neuromusculaires Nord/Est/Ile de France, APHP, G-H Pitie-Salp etri^ ere, Paris; hService de Gen etique Moleculaire, Pharmacogen etique et Hormonologie, APHP, Hopital^ Kremlin-Bicetre,^ Paris; iService de Biochimie et Biologie Moleculaire Grand Est, CHU de Lyon, GH Est, Bron, France; and jDepartment of Neurology, Friedrich-Baur-Institute, LMU Munich, Munich, Germany

Keywords: Background and purpose: Charcot–Marie–Tooth disease type 1 (CMT1) is a Charcot–Marie–Tooth group of autosomal dominantly inherited demyelinating sensorimotor neu- neuropathy, inherited ropathies. Symptoms usually start in the first to second decade and include peripheral neuropathy, distal muscle weakness and wasting, sensory disturbances and foot deformities. demyelinating The most frequent cause is a duplication of PMP22 whilst point mutations in neuropathy, autosomal PMP22 and other genes are rare causes. Recently, FBLN5 mutations have dominant, FBLN5 been reported in CMT1 families. Methods: Individuals with FBLN5-associated CMT1 were compiled from Received 12 December 2019 clinical and research genetic testing laboratories. Clinical data were extracted Accepted 29 July 2020 from medical records or obtained during patients’ visits at our centres or primary care sites. European Journal of Results: Nineteen CMT1 families containing 38 carriers of three different Neurology 2020, 0: 1–7 FBLN5 missense variants were identified and a mutational hotspot at > doi:10.1111/ene.14463 c.1117C T (p.Arg373Cys) was confirmed. Compared to patients with the common PMP22 duplication, individuals with FBLN5 variants had a later age of diagnosis (third to fifth decade) and less severely reduced motor median nerve conduction velocities (around 31 m/s). The most frequent clinical presentations were prominent sensory disturbances and painful sensations, often as initial symptom and pronounced in the upper limbs, contrasting with rather mild to

moderate motor deficits. EUROPEAN JOURNAL OF NEUROLOGY Conclusions: Our study confirms the relevance of FBLN5 mutations in CMT1. It is proposed to include FBLN5 in the genetic work-up of individuals suspected with CMT1, particularly when diagnosis is established beyond the first and second decade and comparably moderate motor deficits contrast with early and marked sensory involvement. FBLN5-associated CMT1 has a recognizable clinical phenotype and should be referred to as CMT1H according to the current classification scheme.

Correspondence: M. Auer-Grumbach, Department of Orthopedics and Trauma Surgery, Medical University of Vienna, Vienna, 1090, Austria (tel.: +4314040040820; fax: +43 (0)1 40400-40910; e-mail: [email protected]).

CZ9, including one previously reported family [8]), Introduction seven from Austria (AT1–AT7, including two previ- Charcot–Marie–Tooth type 1 (CMT1), also known as ously reported families [6]), two from France (FR1, hereditary motor and sensory neuropathy type I FR2) and one from Germany (GE1). All participants (HMSN I), is an autosomal dominantly inherited con- were examined by experienced neurologists and neuro- dition, characterized by slowly progressive distal mus- physiologists at their primary care centres or our aca- cle weakness and wasting, mild to moderate sensory demic institutions. disturbances and pes cavus foot deformity. Disease Pathogenic variants in other CMT1 genes (PMP22 onset is usually during school age or adolescence or duplication; point mutations in PMP22, MPZ, less commonly later [1]. Histopathological and electro- LITAF) and in the gene for X-linked dominant CMT physiological studies in CMT1 reveal peripheral ner- (GJB1) had been excluded in most index patients prior vous system (PNS) demyelination and decreased to this study. Mutations in FBLN5 (NM_006329.3) motor nerve conduction velocities (NCVs) (<38 m/s were detected by Sanger sequencing, multi-gene panel for upper limb motor nerves [2]). By far the most fre- sequencing or whole-exome sequencing as reported quent genetic subtype is CMT1A, caused by a 1.4 Mb previously [10,11]. To compare haplotypes between duplication encompassing the PMP22 gene [3,4]). families carrying the same FBLN5 variants, dinu- Point mutations in PMP22 and several other genes cleotide short tandem repeat (STR) markers flanking (MPZ, LITAF, EGR2, NEFL and PMP2) are much the FBLN5 gene were analysed. Sequences were poly- rarer causes of CMT1 and have been classified as merase chain reaction (PCR) amplified using fluores- CMT1B–CMT1G [5]. In 2011, mutations in FBLN5, cently labelled primers and lengths of PCR products encoding a member of the fibulin family of extracellu- were determined by capillary electrophoresis and fluo- lar matrix proteins, were reported in Austrian CMT1 rescence detection on an ABI 3130 Genetic Analyzer families [6] and causality of FBLN5 mutations was (Applied Biosystems, Foster City, CA, USA). subsequently confirmed in single Czech, Chinese and The study was approved by the ethics committee of Japanese families [7–9]. Patients usually presented the University Hospital Motol in Prague and the with late-onset demyelinating sensorimotor neuropa- Medical University of Vienna. All procedures were thy with mild to moderate motor deficits but rather conducted in accordance with the Declaration of Hel- prominent sensory disturbances and neuropathic pain sinki. All participants gave written informed consent [6–9]. for involvement in the study. In this study, clinical and electrophysiological data are reported of 38 individuals from 19 families from Results Austria, the Czech Republic, France and Germany, carrying three different FBLN5 variants. A review of records from our clinical and research genetic testing laboratories yielded information on 19 families afflicted with CMT1, who carried three differ- Patients and methods ent FBLN5 missense variants (Figs 1a and S1). All Thirty-eight individuals with FBLN5 variants from 19 variants were private or ultra-rare in large public families afflicted with CMT1 were identified. Nine databases which are commonly used to infer popula- families originated from the Czech Republic (CZ1– tion-level frequencies of potentially disease-causing

Figure 1 FBLN5 variants identified in this study. (a) Simplified pedigrees of CMT1 families with FBLN5 variants: white symbols, unaffected; black symbols, affected; grey symbols, subjectively healthy although pes cavus, diminished or absent deep tendon reflexes or NCV studies indicate peripheral neuropathy. FBLN5 genotypes are given below the pedigree symbols (+, mutation; À, wild type). No pedigree information was available for French family FR1 with reportedly autosomal dominant disease. (b) Schematic drawing of FBLN5 highlighting functional domains [green, signal peptide; light blue, modified calcium-binding epidermal growth factor (cbEGF)- like domain; blue, cbEGF-like domain; red, fibulin-type-C-terminal region; orange, RGD motif]. Positions of missense variants causing age-related macular degeneration, cutis laxa and CMT1 are shown. Blue stars indicate the position of nonsense variants p.Arg284* and p.Glu391* and a dotted horizontal line represents an in-frame duplication which causes cutis laxa. (c) Conservation of amino acid residues mutated in FBLN5-related CMT. Multiple sequence alignment of partial FBLN5 protein sequences from different species and surrounding residues mutated in FBLN5-related CMT. Residues on a black background are strictly conserved. Residues on a grey background represent amino acids of similar physicochemical properties. (d) Comparison of disease-associated haplotypes in families with the c.1117C>T (p.Arg373Cys) FBLN5 variant. The position (GRCh37/hg19) of STR markers on chromosome 14q32 used in this study is given in centromeric (cen) to q terminal (qter) orientation. The localization of the FBLN5 mutation is also shown. Allele sizes of STR markers are given in base pairs.

(a) AT1 p.Arg373Cys AT2 p.Arg373Cys AT3 p.Arg373Cys AT4 p.Arg373Cys

+/– +/– +/– +/–

+/– +/– +/– +/– +/– +/–

AT5 p.Arg373Cys AT6 p.Arg373Cys AT7 p.Arg373Cys +/– +/–

+/– +/– FR2 p.Asp329Val CZ1 p.Arg373Cys

+/– +/– +/– +/–

CZ2 p.Arg373Cys +/– +/– +/–

–/– +/– +/– +/– CZ3 p.Arg373Cys +/– +/– CZ4 p.Arg373Cys CZ5 p.Arg373Cys +/– +/–

+/– +/– CZ9 p.Arg373Cys

+/– +/– +/– GE1 p.Arg331His

CZ6 p.Arg373Cys CZ7 p.Arg331His CZ8 p.Arg331His +/–

+/–

+/– +/– +/– +/–

(b) Macular degeneration (d) R71G A363T V60L P87S Q124P I169T G267S R351W G412E c.1117C>T D14S1015 D14S291 D14S995 * D14S1016 C144W G202R S227P * D329VR331H R373C C217R cen qter Cutis laxa CMT1 Duplication of 161 aa (cbEGF 2–5) 91.3 91.6 92.4 92.7 92.9 Mb FBLN5

T 5 6 (c) 1 5 > 1 1 9 9 C 0 0 D329V R331H R373C 2 9 7 1 1 S S 1 S S 4 4 1 4 4 1 1 1 1 1 . D D c D D Families AT1-6 212 266 T 262 222 Families CZ1-3 214 270 T 264 218 Family CZ4 212 270 T 264 224 Family CZ5 214 268 T 264 222

© 2020 European Academy of Neurology 4 D. SAFKA BROZKOVA ET AL. variants (Table S1). Variants affected conserved patients had been referred to neurological examination amino acid residues in the sixth calcium-binding epi- for suspected carpal tunnel syndrome (CTS). In many dermal growth factor (cbEGF)-like domain and the younger patients, motor involvement was absent, partic- adjacent fibulin-type-C-terminal region [12,13] ularly in the upper extremities, but prevalence increased (Fig. 1b and c). At least 8/10 in silico prediction pro- with age and all or almost all patients aged 51 years or grams and ≥2/3 conservation scores classified all three older had motor deficits in lower (11/11) and upper (9/ variants as pathogenic (Table S1). Pedigree informa- 11) extremities (Fig. 2d, Table S2). All patients tion indicated familial aggregation of peripheral neu- remained ambulant and maintained purposeful hand ropathy in 17 families whilst two index patients use (Fig. 2f, Table S2). Seven probands aged ≤30 years, presented clinically as isolated cases. Segregation of who had been recruited for segregation studies or in FBLN5 variants with the CMT1 phenotype was con- whom demyelinating neuropathy was an incidental find- firmed (variant observed in three or more affected ing triggering genetic testing for a potential hereditary individuals) in three families and was suggestive (vari- cause, were subjectively healthy and had a normal neu- ant observed in two affected individuals) in nine fami- rological examination except for diminished or missing lies (Fig. 1a). In the remaining families, no genetic deep tendon reflexes in 6/7 and foot deformities in 5/7 testing results of relatives were available. individuals. None of the 38 individuals reported in this The c.1117C>T (p.Arg373Cys) FBLN5 mutation study had hyperelastic skin. One patient aged 81 years was observed in a total of 15 families from Austria, had age-related macular degeneration. the Czech Republic and France (Fig. 1a). As previ- Sensory and motor NCVs in upper and lower limbs ously shown [8], the disease-associated haplotype were uniformly slow. Median motor NCVs were in observed in Austrian families with the FBLN5 the demyelinating range (below the cut-off value of c.1117C>T variant was different from the haplotype in 38 m/s [2]) in 27/31 individuals carrying FBLN5 vari- Czech FBLN5 c.1117C>T pedigrees. Analysis of STR ants and were slightly higher in the remaining four markers flanking FBLN5 confirmed that two further subjects (median 31 m/s, range 22.0–42.6 m/s) pedigrees of Czech origin shared a disease-associated (Fig. 2g, Table S2). Overall, motor NCV slowing was FBLN5 haplotype with the previously reported Czech considerably less prominent than in 23 randomly family. Reconstruction of disease-associated haplo- selected CMT1A cases. types in another two Czech families was consistent with additional independent mutational events Discussion (Figs 1d and S2). Two Czech families carrying the c.992G>A (p.Arg331His) variant (Fig. 1a) were found Nineteen families afflicted with CMT1 are reported to share the same disease-associated haplotype (data and FBLN5 missense variants are detected as the most not shown). likely cause in each of them. According to the current Most patients with FBLN5-related CMT1 (22/26) classification scheme of CMT neuropathies, it is pro- were diagnosed in the third to fifth decade (median posed to categorize FBLN5-related CMT1 as 39 years, range 16–56 years), which was considerably CMT1H. There are several lines of evidence that the later than the age of diagnosis in a series of 24 randomly three FBLN5 variants p.Asp329Val, p.Arg331His and selected individuals with CMT1 due to the common p.Arg373Cys reported here are pathogenic: first, they PMP22 duplication (CMT1A) (Fig. 2a, Table S2). are absent or ultra-rare in public databases; second, Most frequently observed presenting symptoms were they affect highly conserved residues, are predicted to foot deformities (18/25), upper limb sensory distur- be deleterious by several algorithms and are located in bances which often affected the hands (19/30), lower the same region of the protein; third, variants segre- limb sensory disturbances (11/30), lower limb motor def- gated with HMSN I in families whenever tested; icits (11/30) and painful sensations in upper limbs (10/ fourth, the observation that FBLN5 variants occurred 30) (Fig. 2b, Table S2). In retrospect, many patients in several unrelated families and always caused the had foot deformities (Fig. 2c, Table S2) since childhood same particular CMT1 phenotype further supports without physical limitations so that no further diagnos- their pathogenic relevance. Finally, the p.Arg373Cys tic work-up for a peripheral neuropathy had been inves- variant has been replicated in CMT1 patients from tigated until other symptoms appeared. Amongst all China and Japan with unlikely relationship to our patients sensory disturbances were early and common cases of European ancestry [7,9]. Although the vari- complaints, often pronounced in the upper extremities ants p.Asp329Val and p.Arg331His have not been (Fig. 2d, Table S2) and frequently described as unpleas- replicated in independent cohorts and are assessed as ant and painful sensations (Fig. 2e, Table S2). Because ‘variants of uncertain significance’ according to the of marked sensory problems in the hands, several American College of Medical Genetics and Genomics

(b) Foot deformity (18/25) Sensory UL (19/30) 60 (a) Sensory LL (11/30) 50 Motor LL (11/30) (10/30) 40 Pain UL Pain LL (5/30) 30 Motor UL (2/30) 20 0 25 50 75 100 Prevalence of symptoms Age at diagnosis (years) Age at diagnosis 10 at age of diagnosis (%) 0 FBLN5 CMT1A (e) Painful sensaons No pain Only UL (c) Only LL Both UL and LL (n = 25)

(f) Severity of motor deﬁcits UL Mild weakness Moderate weakness Severe weakness (d) (0/12) Complete paralysis Motor UL (4/12) (9/11) (n = 13)

(1/12) Severity of motor deﬁcits LL Motor LL (7/12) Abnormal gait, no support (11/11) Walks with unilateral support (4/12) Walks with bilateral support Sensory UL (9/12) Loss of ambulaon (10/11) (n = 19) (3/12) Sensory LL (7/12) (7/11) (g) 50 (7/9) Asymptomac Aﬀected Foot deformity (9/10) (10/10)

(m/s) 40

0 25 50 75 100 NCV Prevalence of symptoms 30 in diﬀerent age groups (%) motor

15-30 years 31-50 years >50 years 20 Median 10 FBLN5 CMT1A

Figure 2 Clinical and electrophysiological findings in FBLN5-related CMT1 (CMT1H). (a) Distribution of age at diagnosis in 26 patients with FBLN5 variants compared to age at diagnosis in 24 randomly selected CMT1A patients (PMP22 duplication). Median, quartiles and whiskers corresponding to 1.5 times interquartile range are shown. (b) Prevalence of symptoms at age of diagnosis. Sev- eral patients presented with more than one symptom and thus the summed-up numbers can exceed the total count of cases. LL, lower limbs; UL, upper limbs. (c) Variable severity of foot deformities. Clinical photographs of patients carrying the p.Arg373Cys FBLN5 variant. (d) Prevalence of symptoms in different age groups. Both affected and asymptomatic individuals were considered if relevant clinical information was available. Several patients presented with more than one symptom and thus the summed-up numbers exceed the total counts of cases in the respective groups. (e) Prevalence of painful sensations in upper and lower extremities. All patients with clinically manifest neuropathy for whom sufficient clinical information was available were included. (f) Degree of severity of upper and lower limb motor involvement. All patients with upper or lower limb motor manifestation and sufficient clinical information were considered. (g) Motor NCV of the nervus medianus of 31 individuals with FBLN5 variants compared to values observed in 23 randomly selected CMT1A patients (PMP22 duplication). The dotted horizontal line represents the threshold of 38 m/s commonly used to dis- criminate demyelinating and axonal CMT. Median, quartiles and whiskers corresponding to 1.5 times interquartile range are shown.

criteria [14], taken overall the above evidence seems were commonly diagnosed based on complaints mani- strong enough to classify them as disease-related. festing in the third to ﬁfth decade. Although motor Charcot–Marie–Tooth is typically diagnosed during involvement was generally mild to moderate, upper school age or adolescence, motor symptoms are usu- limbs were almost as frequently involved as lower ally more prominent in lower than upper extremities limbs (82% vs. 100% in older patients). Moreover, and sensory disturbances are comparably mild in most marked sensory disturbances and painful sensations aﬀected individuals [1]. In contrast, CMT1H patients were early and common complaints, often pronounced

© 2020 European Academy of Neurology 6 D. SAFKA BROZKOVA ET AL. in the upper extremities, leading to a tentative diagno- independently in several families, which always caused sis of CTS in several patients. It is proposed that the the same consistent, recognizable CMT1 phenotype. At combination of prominent sensory disturbances, com- the moment, conclusions on the proportion of FBLN5- parably moderate motor deficits and slow motor related CMT1 amongst CMT1 cases cannot be drawn NCVs should prompt genetic testing of FBLN5.In as cases with unexplained suspected CMT1 have not our series, values for median nerve motor NCVs were yet been systematically screened for FBLN5 variants in above the threshold of 38 m/s for CMT1 (but still our laboratories. Generally, it is not possible to extrap- below the normal range) in a small proportion of indi- olate our results to populations of other ancestries as viduals (4/31). This suggests that FBLN5 should also variant frequencies may vary in different populations. be considered in the group of patients with ‘intermedi- However, based on the observation of FBLN5 variants ate’ CMT, which is defined by median nerve motor in Chinese and Japanese CMT1 families and recogni- NCVs in the range 25–45 m/s [5]. tion of a possible mutation hotspot on c.1117, FBLN5 Several genes involved in the pathogenesis of variants are expected to be associated with CMT1 in demyelinating CMT encode proteins that either func- other populations as well. Systematic genetic testing, tion as structural myelin proteins or regulate molecu- e.g. by including FBLN5 in diagnostic multi-gene pan- lar and cellular events believed to control myelin els, will allow appropriate recognition of the contribu- build-up and maintenance [15]. Conversely, FBLN5 is tion of CMT1H to the entirety of CMT1 cases. a secreted extracellular matrix protein [12,13] and its role in the PNS is currently not well understood. Amongst other functions, extracellular matrix cues Acknowledgements control Rho GTPase signalling pathways to provide The participating families and the referring physicians localized, context-dependent responses to the cellular are thanked for their cooperation. MAG received microenvironment [16,17]. Notably, Rho GTPase sig- funds from the FWF (P27634FW) and the OeNB nalling has been shown to be essential for PNS myeli- (16880), DSB and PS from the MZCR grant no. 16- nation [18,19] and deficiency of a Rho GTPase 31921A and DRO 00064203, AK from the National regulator results in demyelinating CMT [20–22]. Science Centre Poland grant No.UMO-2016/23/B/ FBLN5 mutations have been previously shown to NZ3/02035 and JS from the Fritz-Thyssen-Stiftung cause autosomal dominant age-related macular degen- (Az.10.15.1.021MN) and the BMBF (01GM1511B). eration (AMD) [23] and autosomal dominant and TS is a member of the ERN neuromuscular team. recessive cutis laxa [24,25]. In our study, none of the probands who were younger than 60 years com- plained about visual problems and only one out of Disclosure of conflict of interest five individuals aged 60 years or older had AMD, The authors declare no financial or other conflicts of consistent with either a merely accidental finding and interest. non-association of CMT1H variants with AMD or incomplete penetrance even at an advanced age. There was also no consistent association between CMT1- Data availability statement related FBLN5 variants and connective tissue Data supporting the findings of this study are avail- disorders although one patient in our series died of a able from the corresponding author upon reasonable ruptured aortic aneurysm and a clinically affected cou- request. sin of another patient had suspected subarachnoid haemorrhage at age 30. These observations might well be incidental and there is currently insufficient level of Supporting Information evidence to recommend baseline screening for vascular Additional Supporting Information may be found in abnormalities in CMT1H. Different clinical outcomes the online version of this article: of FBLN5 variants are supposed to be related to the mode of inheritance, type of mutation or affected pro- Appendix S1 tein domains. Although it is noted that the three CMT1-related missense variants cluster in one portion References of the protein, further reports of patients and variants are needed to establish a link between clinical out- 1. Harding AE, Thomas PK. The clinical features of hereditary motor and sensory neuropathy types I and II. come and a specific set of variants. Brain 1980; 103: 259–280. In essence, three convincing FBLN5 mutations were 2. Dyck PJ, Lambert EH. Lower motor and primary sen- found, including one variant (p.Arg373Cys) arising sory neuron diseases with peroneal muscular atrophy. I.

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