Autosomal recessive axonal Charcot^Marie ^Tooth disease (ARCMT2): phenotype ^ genotype correlations in 13 Moroccan families

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1 doi: /brain/awm014 Brain (2007), 130,1062^1075 Autosomal recessive axonal Charcot^Marie ^Tooth disease (ARCMT2): phenotype ^ genotype correlations in 13 Moroccan families Ahmed Bouhouche, 1 Nazha Birouk, 2 Hamid Azzedine, 4 Ali Benomar, 1 Garry Durosier, 4 Dorothe e Ente, 4 Marie-Paule Muriel, 4 Merle Ruberg, 4 Ilham Slassi, 3 Mohamed Yahyaoui, 1 Odile Dubourg, 4,5 Reda Ouazzani 2 and Eric LeGuern 4,6 1 Laboratoire de Neuroge ne tique, Service de Neurologie B, Ho pital des Spe cialite s, 2 Service de Neurophysiologie clinique, Ho pital des Spe cialite s, Rabat, 3 Service de Neurologie, Ho pital Ibn Rochd, Casablanca, Morocco, 4 UMR679 INSERM - Universite Paris VI (Pierre et Marie Curie), Ho pital de la Pitie -Salpe trie're, 5 Laboratoire de Neuropathologie Raymond Escourolle, Ho pital de la Pitie -Salpe trie're, AP-HP and 6 Laboratoire de neuroge ne tique mole culaire et cellulaire, De partement de ge ne tique, cytoge ne tique et embryologie, Ho pital de la Pitie -Salpe trie're, Paris, France The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors. Correspondence to: Ahmed Bouhouche, Laboratoire de Neuroge ne tique, Service de Neurologie B, Ho pital des Spe cialite s, BP 6402, Rabat Morocco and Hamid Azzedine, INSERM U679 Ba t. Pharmacie, Ho pital de la Salpe trie're, 47 Bd de l Ho pital 75651, Paris, France azzedine.hamid@yahoo.fr Charcot^Marie^Tooth disease is a genetically heterogeneous group of hereditary motor and sensory neuropathies. Three loci for the axonal autosomal recessive subgroup (ARCMT2) have been reported in 1q21 (CMT2B1, LMNA), 8q21 (CMT4A and CMT2K,GDAP1) and19q13(cmt2b2).we report here a clinical, electrophysiological, pathological and genetic study in 13 Moroccan families with ARCMT2 phenotypes. Clinical and electrophysiological examinations were performed in all index cases and 64 at-risk relatives. Thirty-one patients were clinically affected. A peroneal nerve biopsy was obtained from three patients. Four families were linked to the 1q21 locus, all had the LMNA R298C mutation. Six families were linked to the 8q21 locus, all had the GDAP1 S194X mutation. Founder effects for both mutations were suggested by the analysis of microsatellite markers close to the genes. The three remaining families were excluded from the three known loci. The electrophysiological findings were consistent with an axonal neuropathy. The clinical data show that in CMT2B1 the disease began most often in the second decade and progressed gradually from distal to proximal muscles. Three of our patients with the longest disease durations (424 years) had also severe impairment in the scapular muscles. Reported here for the first time, this might be a hallmark of CMT2B1. Patients with CMT4A/2K had onset most often before the age of 2 years. Most had severe clubfoot from the beginning, one of the hallmarks of CMT4A/2K. None of our patients with CMT4A/2K had vocal cord paralysis. The clinical phenotype of the three families that are not linked to the three known loci presented some particularities that were not seen in those with known genetic defects. One family was characterized by late onset of the disease (420 years) or a mild neuropathy that was diagnosed only when the family was examined. In a second family, dorsal scoliosis was the most prominent symptom. In the third family, symptoms began in the second decade with a moderate neuropathy associated with a pronounced scoliosis. These families illustrate the extent of clinical and genetic heterogeneity in ARCMT2. Keywords: Charcot^Marie^Tooth disease;lmna gene; GDAP1 gene; peripheral neuropathy; founder effect Abbreviations: ARCMT2 ¼ autosomal recessive axonal Charcot^Marie ^Tooth disease; CMAP ¼ compound muscle action potential; DML ¼ distal motor latency; MNCV ¼ motor nerve conduction velocity; SNAP ¼ sensory nerve action potential Received November 28, Revised December 24, Accepted January 15, Advance Access publication March 8, 2007 ß The Author (2007). Published by Oxford University Press on behalf ofthe Guarantors of Brain. Allrights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 ARCMT2 phenotype ^ genotype correlations Brain (2007), 130, 1062^ Introduction The hereditary motor and sensory neuropathies, also known as Charcot Marie Tooth disease [(CMT (MIM )], are a heterogeneous group of disorders affecting the peripheral nervous system. Two major types have been distinguished on the basis of electrophysiological criteria, a demyelinating form (CMT1) and an axonal form (CMT2), in which median motor nerve conduction velocities (MNCV) are 535 and 440 m/s, respectively (Bouche et al., 1983). Three loci for the autosomal recessive axonal form (ARCMT2) have been reported, and two of the diseasecausing genes have been identified. The first locus [CMT2B1 (MIM )] was mapped on chromosome 1q21.2 in a large inbred Moroccan family by Bouhouche et al. (1999), and a R298C mutation in the LMNA [MIM ] gene was found to be responsible for the disease in Algerian families with linkage to the same locus (De Sandre-Giovannoli et al., 2002). Mutations in the LMNA gene were also reported to be responsible for dilated cardiomyopathy (Fatkin et al. 1999), autosomal dominant (Bonne et al., 1999) and autosomal recessive Emery Dreifuss muscular dystrophy (Raffaele di Barletta et al., 2000), limb-girdle muscular dystrophy (Muchir et al., 2000), familial partial lipodystrophy (Shackleton et al., 2000), mandibuloacral dysplasia (Shen et al., 2003), Hutchinson Gilford progeria syndrome (Eriksson et al., 2003; De Sandro-Giovannoli et al., 2003a), Werner s syndrome (Chen et al., 2003) and recently for restrictive dermopathy (Navarro et al., 2004), depending on the functional domain of the LMNA protein affected. A second locus [CMT2B2 (MIM )] was assigned to chromosome 19q13.3 in a Costa Rican family with a moderate phenotype (Leal et al., 2001), but the responsible gene has not yet been identified. The third locus was originally mapped to chromosome 8q21.3 in a Tunisian family with a severe AR demyelinating neuropathy, designated CMT4A [MIM ] (Ben Othman et al., 1993). Mutations in ganglioside-induced differentiation-associated protein-1 gene [GDAP1 (MIM )] were shown to be responsible for AR demyelinating (CMT4A) (Baxter et al., 2002), AR axonal (CMT2K [MIM ]) (Cuesta et al., 2002; Azzedine et al., 2003b; Birouk et al., 2003; Stojkovic et al., 2004; Claramunt et al., 2005; Kabzinska et al., 2005), as well as intermediate ARCMT [MIM ] (Nelis et al., 2002; Senderek et al., 2003). In the present study we performed a clinical, electrophysiological, pathological and genetic study in 13 Moroccan families with ARCMT2 phenotypes, in order to find phenotype genotype correlations that could aid diagnostic strategies and prognosis. Material and methods Families Thirteen families from Morocco were diagnosed in the Service de Neurophysiologie clinique, Hôpital des Spécialités, Rabat as having ARCMT2. The pedigrees of these families are shown in Fig. 1. In 12 families, the parents were unaffected suggesting that the clinical phenotype segregated with an autosomal recessive (AR) mode of inheritance. However, in Family MAD-008 (Fig. 1A), many loops of consanguinity led us to consider this mode of inheritance despite transmission of the disease from an affected father (individual 32) to his sons. Eleven of the families were consanguineous, and the parents in the other two families originated from the same tribe. Families MAD-008 and AIT-011 have been already described in previous studies by Bouhouche et al. (1999) and Birouk et al. (2003), respectively. Peripheral venous blood samples were obtained from patients and relatives after informed consent was given according to the Helsinki Convention. The genomic DNA was extracted by standard procedures. Clinical study All index cases and 64 at-risk relatives from the 13 consanguineous Moroccan families were examined for the presence of motor and sensory loss, areflexia, foot deformities, scoliosis and other associated signs such as nerve hypertrophy, tremor, ataxia, pyramidal signs, cranial nerve involvement and dementia. Disease severity was evaluated in terms of ability to walk and run and to use the hands in daily tasks. Thirty-one patients were clinically affected. All other family members were found to be normal on clinical and electrophysiological examination. Electrophysiological study Electrophysiological examinations were performed in all patients and at-risk relatives. Nerve conduction velocities were recorded with surface stimulating and recording electrodes. Median and ulnar compound muscle action potentials (CMAPs) were recorded from the abductor pollicis brevis and the abductor digiti quinti, respectively, with stimulation at the wrist and elbow. The peroneal CMAP was recorded from the extensor digitorum brevis with stimulation at the ankle and knee. Distal motor latency (DML), MNCV and distal CMAP amplitude were measured. Median and ulnar sensory nerve action potentials (SNAPs) were obtained orthodromically at the wrist, following stimulation of the third and the fifth digits, respectively. The sural SNAP was recorded antidromically at the ankle following stimulation of the calf. SNAP amplitudes and sensory nerve conduction velocity were measured. Electromyography was performed with a concentric needle electrode in the tibialis anterior (TA), the vastus lateralis (VL), the first dorsalis interosseus (FDI), the extensor carpi radialis (ECR) and the deltoid muscles. Pathological study A superficial peroneal nerve biopsy was obtained with informed consent from patients AIT , MAD and OUI (Fig. 1). The specimen was fixed in buffered glutaraldehyde, post-fixed in osmium tetroxide and embedded in epoxy resin. Semi-thin sections of 0.5 mm were stained with toluidine blue for light microscope examination. Ultra-thin sections were contrasted with lead citrate and uranyl acetate for electron microscopy.

3 1064 Brain (2007), 130,1062^1075 A. Bouhouche et al. Fig. 1 Pedigrees of families with ARCMT2. Black and white symbols correspond to affected and unaffected members, respectively. Members who were included in the study are marked with a star. (A) families with the S194X mutation in the GDAP gene; (B) families with the R298C mutation in the LMNA gene; (C) families without any linkage to the three known loci.

4 ARCMT2 phenotype ^ genotype correlations Brain (2007), 130,1062^ Genotyping, homozygosity mapping and linkage analysis Genotyping was performed with microsatellite markers D1S2715, D1S2777, D1S2624 and D1S506 for the 1q21.2 locus, with D8S530, D8S286, D8S1705 and D8S1757 markers for the 8q21.2 locus and with D19S879, D19S604, D19S867 and D19S907 markers for the 19q13.3 locus. Genotyping was performed with the additional markers D1S2721 and D8S551 to study the founder effect of the S194X and R298C mutations, respectively. Genomic DNA was amplified using fluorescent-labelled primers in a 15 ml reaction volume containing 15 pmol of each primer, 3 mm of each dntp, 1.5 ml 10 PCR buffer and 0.6 U AmpliTaq Gold DNA polymerase (ABI). Samples were incubated in an ABI 2400 thermocycler for 30 s at 94 C, 30 s at C depending on the primer pair and 30 s at 72 C for 35 cycles. PCR products were pooled and loaded with GeneScan 400HD size standard onto a 4% acrylamide gel in an ABI 3100 automated DNA sequencer. The data was collected and analysed using the ABI Genescan (version 3.1) and Genotyper (version 2.0) software of Applied Biosystems. Haplotypes were constructed manually and homozygosity regions were looked for. Pairwise and multipoint LOD scores were calculated using the MLINK and LINKMAP programs of the FASTLINK package (Lathrop et al., 1985; Shaffer et al., 1994) and Allegro software (Gudbjartsson et al., 2000). A fully penetrant autosomal recessive trait with a disease allele frequency of and equal recombination rates in males and females was assumed for all three loci. Allele frequencies for microsatellite markers were considered to be equal. Mutation analysis In families with linkage to the CMT2K/4A (8q21.1) locus, the exons of the GADP1 gene were amplified by PCR and both strands were sequenced with the Bigdye TM Terminator Reaction Kit (Applied Biosystems) according to the manufacturer s instructions using an automated ABI 3100 DNA sequencer. Data were collected and analysed with SeqScape software (Applied Biosystems). For families with linkage to the CMT2B1 locus, we systematically searched for the 892C! T mutation in the LMNA gene using the restriction digest assay described by De Sandre- Giovannoli et al. (2002). Exon 5 of LMNA gene was amplified by PCR with the LMNA5-R and LMNA5-F primers. The PCR products were then digested by the restriction endonuclease AciI, run on a 2% agarose gel, stained with ethidium bromide and visualized under UV fluorescence. Segregation in the families was verified by the same technique, and DNAs from probands in each family were then sequenced and analysed to confirm the mutation. Results Genetic data Four families, LAT-014, MAD-008, OMA-025 and ZOU-046, had significant cumulative LOD scores at CMT2B1 locus (Table 1A). The affected individuals of each family shared a common homozygosity region around the LMNA gene (Fig. 2). The same 892C! T mutation in exon 5 of the LMNA gene was found to segregate in all four families causing an Arg! Cys substitution at the highly conserved amino acid 298. The presence of the mutation was also confirmed by sequence analysis. This mutation was already reported in Algerian families (De Sandro- Giovannoli et al., 2002; Tazir et al., 2004). Six families (AIT-011, CHA-028, RMI-039, ELM-058, KAD-063 and ARA-068) had significant cumulative LOD scores for all the microsatellite markers at the 8q21.2 locus (Table 1B). The patients from each family shared a common homozygosity region around the GDAP1 gene (Fig. 2). Homozygous 581C! G mutations were found in all 11 patients from the 6 families. This mutation converts a codon specifying serine to a nonsense codon (S194X) leading to an absent or a truncated protein. This mutation has been already reported in a Spanish (Cuesta et al., 2002), a Tunisian (Baxter et al., 2002) and two Moroccan families (Azzedine et al., 2003b; Birouk et al., 2003). Penetrance of the two mutations S194X and R298C was complete, since all the individuals carrying the mutation at the homozygous state were affected. All the relatives carrying mutation (heterozyotes) of all families were normal at clinical and electrophysiological examination confirming that the inherited trait is recessive in our series. To evaluate the frequency of each mutation in the Moroccan population, DNA samples from at least 157 Moroccan controls were sequenced for exon 5 of LMNA and exon 5 of GDAP1 genes. The R194X mutation (GDAP1) was found in 2/157 controls (2/314 Table 1A Two -point LOD score values (at ¼ 0) of the four CMT2B1 families Microsatellite markers LMNA Families D1S2715 D1S2777 D1S2624 D1S506 LAT MAD OMA ZOU P of LOD scores Table 1B Two -point LOD score values (at ¼ 0) of the six CMT4A/2K families. Families Microsatellite markers GDAP1 D8S530 D8S286 D8S1705 D8S1757 AIT ARA 68 NI NI NI NI CHA ELM KAD RMI P of LOD scores NI ¼ none informative.

5 1066 Brain (2007),130,1062^1075 A.Bouhoucheet al. chromosomes) giving a mutation frequency of 0.6% and a 1.2% frequency of heterozygous individuals in the Moroccan population. In contrast, no R298C mutations (LMNA) were found in 165 controls (330 chromosomes). The three remaining families, AMI-027, MOU-023 and OUI-030, had pairwise LOD scores at ¼ 0 (Table 1C) less than the threshold of 2 for all markers used, excluding linkage to chromosomes 1q21.2, 8q21.1 and 19q13. These results were confirmed by haplotype reconstruction (data not shown) and multipoint LOD scores calculations (Fig. 3). Fig. 2 Segregating haplotypes encompassing the LMNA (Top)and the GDAP1 (Bottom) genes in the four and six families, respectively. The homozygous haplotype shared by the affected individuals in each group is highlighted. Ordered markers are indicated from the centromere (left) to the telomere(right). For each microsatellite, alleles are indicated by numbers. T and G correspond to the mutated nucleotide in the LMNA and GDPA1 genes, respectively. Clinical and electrophysiological data Patients with the homozygous S194X mutations in the GDAP1 gene The clinical data concerning patients with homozygous S194X mutation in the GDAP1 gene are shown in Table 2. Onset was early in childhood in all patients (range 1 6 years; mean: years). One patient (AIT ) had hypotonia at birth and delayed development of early motor milestones. All the others walked at the normal age, but eight had difficulty walking from the beginning, in particular because of serious foot deformities. Symptoms consisted of distal weakness and wasting of legs, predominantly in peroneal muscles, with severe foot deformities of the pes equinovarus type in nine cases. Most of the patients had total areflexia and loss of proprioception in Table 1C Two-point LOD scores for the three known loci of ARCMT2 1q21.2, 8q21.1 and 19q13.3 in MOU- 023, AMI-027 and OUI-030 families Markers Family LOD scores at recombination fraction () D1S2715 MOU AMI OUI D1S2777 MOU AMI OUI D1S2624 MOU AMI OUI D1S506 MOU AMI OUI D8S530 MOU AMI OUI D8S286 MOU AMI OUI D8S1705 MOU AMI OUI D8S1757 MOU AMI OUI D19S879 MOU AMI OUI D19S867 MOU AMI OUI

6 ARCMT2 phenotype ^ genotype correlations Brain (2007), 130, 1062^ Fig. 3 Multipoint linkage analysis graphs for the three known loci in three families. Linkage was excluded from the CMT2B1, CMT2K/4A and CMT2B2 loci. lower limbs. Weakness in the upper limbs, particularly in the hand muscles, developed later in the first decade, claw-like were observed in six patients. Nine patients developed severe weakness of the proximal muscles with loss of autonomy; four became wheelchair-bound at ages 4, 11, 15 and 16 years, respectively, and the remaining five needed crutches to walk. Six patients had mild scoliosis. There was no cranial nerve involvement, cerebellar or pyramidal signs. Clinically enlarged nerves were not obvious in any of the patients. Intellectual function was normal in all cases. The results of the electrophysiological examination are given in Table 3. No motor or sensory response could be elicited in five patients. Motor action potentials were not obtained in the lower limbs in any of the patients because of severe muscle atrophy. CMAP amplitudes in the upper limbs were greatly reduced, DML were delayed in three patients and MNCV of median and ulnar nerves were slightly reduced (means: and m/s, respectively). The lowest MNCV of the median nerve (35 m/s) was found in two patients who also had very reduced CMAP amplitudes. SNAPs were obtained in five patients, but the amplitude was much reduced in all five. Needle EMG examinations showed a reduced recruitment pattern in the proximal muscles in two patients. Voluntary activity was absent in distal muscles. These observations were consistent with an axonal form of CMT disease. Patients with homozygous R289C mutations in the LMNA gene The clinical findings concerning 14 patients from 4 families with a homozygous R289C mutation in the LMNA gene are shown in Table 4. The mean age at onset was years (range 8 20 years). Except for twin brothers who had their first symptoms at age 8 years, all patients had onset during the second decade. None had hypotonia at birth, and all walked at the normal age. Distal muscle weakness and atrophy affected both upper and lower limbs in nine patients and was limited to the lower limbs in five. Four patients from the same family (MAD-008) had important proximal muscle atrophy of the pelvic and scapular girdle. A few patients had sensory impairment of either pain and touch or proprioception, predominantly in the lower limbs. Most of patients had total areflexia, but two patients from the same family (OMA-025) had only ankle jerk areflexia. Foot deformities were moderate or absent; only five patients had moderate scoliosis. The severity was variable even in the same family. Four patients from family MAD-008 were wheelchair-dependent and had claw-like, two patients needed help walking but had moderate impairment of the hands, eight patients walked abnormally but autonomously and had no functional disability of the hands and one patient walked normally and had normal hands. There was no cranial nerve involvement or cerebellar or pyramidal signs. Intellectual function was normal in all cases. The electrophysiological findings, shown in Table 5, are consistent with an axonal form of CMT. The mean MNCV was m/s (range 36 66) for the median nerve, m/s (range 32 64) for the ulnar nerve and

7 1068 Brain (2007), 130,1062^1075 A.Bouhoucheet al. Table 2 Clinical findings in patients with ARCMT neuropathy due to homozygous S194X mutations in the GDAP1 gene Case No Age at onset (years) Hypotonia atbirth Yes No No No No No No No No No No Age of walking 4 years 12 months 518 months 518 months 12 months 12 months 12 months 518 months 518 months 12 months 12 months Age at examination (years) Disease duration (years) Distal motor deficit UL þ LL ULþ LL ULþ LL UL þ LL UL þll UL þll ULþ LL ULþ LL UL þll LL LL Proximal motor deficit UL þ LL LL No No LL No No ULþ LL UL þll No No Distal muscle atrophy UL þ LL ULþ LL ULþ LL UL þ LL UL þll UL þll ULþ LL ULþ LL UL þll LL LL Proximal muscle atrophy UL þ LL No No No No No No ULþ LL UL þll No No Sensory loss Pain and touch LL No No No No No ^ No LL No No Proprioception LL LL LL LL No No ^ UL þ LL LL No No Areflexia UL þ LL ULþ LL ULþ LL UL þ LL LL UL þll LL ULþ LL UL þll LL LL Foot deformities Moderate Severe Severe Severe Moderate No Severe Severe Severe Severe No Scoliosis Yes Yes Yes Yes No No No No No No No Functional disability UL Claw-like Moderate Moderate Claw-like No Moderate Claw-like Claw-like Claw-like Claw-like Claw-like Wheelchair bound Wheelchair bound with aid with aid with a cane with a cane with a cane LL Wheelchair bound UL ¼ upper limbs; LL ¼ lower limbs m/s (range ) for the peroneal nerve. Only one patient (MAD ) had values lower than 40 m/s. The CMAP amplitudes in the upper limbs were either reduced (in five patients) or normal (in nine patients). A motor response from the peroneal nerve could not be elicited in five patients. The peroneal nerve MNCV and CMAP amplitudes were normal in three patients. Sensory responses were abolished in most patients in both the upper and lower limbs. Needle electromyography showed a neurogenic pattern in both distal and proximal muscles, predominantly in the lower limbs. Families without linkage to known loci Three families with six affected individuals were not associated to 1q21.2, 8q21.1 or 19q13.3. As shown in Table 6, ages at onset were in the second decade. Two individuals had no functional disability, and were diagnosed when the family was examined. They had, at least, ankle jerk areflexia. The four remaining patients had distal muscle weakness and atrophy predominantly in the lower limbs, without impairment of proximal muscles. Sensory loss was found in two patients only and foot deformities were absent or moderate. The hallmark in two families (MOU-023 and OUI-030) was a pronounced scoliosis, which was the first symptom in patient MOU Functional disability was absent in the hands in all patients and was moderate in the lower limbs in most cases. The most affected patients, with the longest disease durations (20 years), needed only one cane for walking. There was no sign of central nervous system involvement in any patient. The electrophysiological data are given in Table 7. MNCV and CMAP amplitudes of the median and ulnar nerves were normal in all patients. DMLs were slightly prolonged in the upper limbs in four cases. Peroneal nerve motor responses were abolished in two patients, normal in one case and showed slowed conduction in three cases. SNAPs were abolished in all four limbs (two cases), reduced in all four limbs (two patients) or reduced in the lower limbs only (two patients). The needle EMG examination showed a neurogenic pattern in the distal muscles in all cases. All these data are consistent with an axonal neuropathy. Pathological data One patient from each of the following families had a nerve biopsy: AIT-011 with a mutation in the GDAP1 gene, MAD-008 with a mutation in the LMNA gene and OUI-030 with no linkage to any known loci. The results are shown in Fig. 4. Individual AIT underwent a nerve biopsy at age 15 years, after a disease duration of 14 years. His sensory nerve was severely affected. There was a complete loss of large myelinated fibres. The remaining fibres all had small diameters (57 mm) and there were few onion bulb formations. The myelin sheaths were normal with no

8 ARCMT2 phenotype ^ genotype correlations Brain (2007), 130,1062^ Table 3 Motor and sensory nerve conduction and needle electromyography findings in patients with ARCMT neuropathy due to homozygous S194X mutation in the GDAP1 gene Case No Motor nerve conduction Median nerve DML (ms) MNCV (m/s) CMAP amplitude (mv) Cubital DML (ms) ^ 2.8 MNCV (m/s) ^ 3.2 CMAP amplitude (mv) ^ 53 SPE DML (ms) MNCV (m/s) CMAP amplitude (mv) Sensory nerve action potential amplitude (mv) Median nerve Ulnar nerve ^ ^ 0 0 ^ 0 Suralnerve Needle EMG TA ^ ^ ^ NVA ^ ^ ^ NVA ^ ^ ^ First DIO ^ ^ ^ RRP þþ ^ ^ ^ ^ ^ ^ ^ ECR ^ ^ ^ RRP þþþ ^ ^ ^ ^ ^ ^ ^ Deltoid ^ ^ ^ RRP þ ^ ^ ^ RRPþþ ^ ^ ^ DML ¼ distal motor latency; MNCV ¼motor nerve conduction velocity; CMAP ¼ compound muscle action potential; TA ¼ tibialis anterior; DIO ¼ dorsal interosseus; ECR ¼ extensor carpi radialis. The 0 value was attributed to abolished motor or sensory potentials; NVA ¼ no voluntary activity; RRP ¼ reduced recruitment pattern; þ, þþ and þþþ for slight, moderate and important RRP, respectively. evidence of decompaction or redundant loops. Rare clusters of regeneration were observed, which were either typical or of the pseudo-onion bulb type. These were either clusters surrounded by concentrically arranged Schwann cell processes or a single fibre surrounded by Schwann cell processes enclosing unmyelinated axons. The nerve biopsy in individual MAD was obtained at age 18 years, after a disease duration of 9 years. The sensory nerve was less affected than in individual AIT The loss of myelinated fibres was less severe, but large diameter fibres (48 mm) were most affected. Myelin thickness was normal, with no onion bulb formations, nor abnormal myelin sheaths. There were many clusters of regeneration consisting of groups of several small fibres with thin myelin sheaths. Unmyelinated fibres were not obviously abnormal. Individual OUI was biopsied at age 21 years, after 5 years of disease evolution. His sensory nerve was the most severely affected of the three patients. There were almost no myelinated fibres left and the few remaining fibres were very small in diameter (1 2 mm). These myelin sheaths appeared normal, and their thickness was appropriate to the diameter of the axons. There were no onion bulb formations. Some fibres were actively degenerating. There were no clusters of myelinated fibres. Unmyelinated fibres appeared in small clusters surrounded by Schwann cell cytoplasm. Discussion Since 1996, 86 families with hereditary motor and sensory neuropathy (HMSN) types I and II were examined in the Neurophysiology Department in Rabat (Morocco). Forty had a pattern of inheritance compatible with autosomal recessive transmission. ARCMT therefore seems to be more frequent (46%) than autosomal dominant CMT in Morocco. Thirteen of the 40 ARCMT families (32%) had an axonal form of the disease, whereas the 27 remaining families (68%) had a demyelinating neuropathy. These data show that demyelinating forms are twice as frequent as axonal types in autosomal recessive CMT in Morocco. We thus had the rare opportunity of analysing, in the same department, a set of 13 large consanguineous families with axonal ARCMT caused by mutations in different genes. This allowed us to look for phenotype genotype correlations, potentially useful for guiding the molecular diagnosis and evaluating the prognosis. The vast majority (10/13) of our families with axonal ARCMT have mutations in the known GDAP1 and LMNA genes. There are only three (23%) families in our series for which the causative gene has not yet been localized and identified. Allelic homogeneity seems to be complete in Morocco, since all four CMT2B1 families (31%) have the same R289C mutation in LMNA, and six CMT2K/4A families (46%) have the same S194X mutation in GDAP1.

9 1070 Brain (2007), 130,1062^1075 A.Bouhoucheet al. Table 4 Clinical findings in patients with ARCMT neuropathy due to R289C mutations in the LMNA gene Case No Age at onset (years) Hypotonia at birth No No No No No No No No No No No No No No Age of walking Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Age at examination (years) Disease duration (years) Distal motor deficit UL þ LL UL þ LL UL þ LL LL UL þ LL LL UL þ LL UL þ LL UL þ LL LL LL LL UL þ LL UL þ LL Proximal motor deficit ULþ LL ULþ LL ULþ LL LL LL LL UL þ LL LL LL No No No LL LL Distal muscle atrophy ULþ LL ULþ LL ULþ LL LL ULþ LL UL þ LL LL LL UL þ LL LL LL LL UL þ LL UL þ LL Proximal muscle atrophy ULþ LL ULþ LL ULþ LL No LL LL LL No No No No No No LL Sensory loss Pain and touch UL þ LL UL þ LL No No No No No No No No LL LL No LL Proprioception No UL þ LL UL þ LL No LL LL No No No No No LL No No Areflexia UL þ LL UL þ LL UL þ LL UL þ LL UL þ LL UL þ LL UL þ LL LL UL þ LL LL AJ AJ UL þ LL LL Foot deformities Moderate Moderate Moderate No No No No Moderate Moderate Moderate No Moderate Moderate Severe Scoliosis No Yes Yes Yes Yes No No Yes No No No No No No Functional disability No No No Moderate Moderate No Moderate No Moderate No Claw-like Claw-like Claw-like UL Claw-like with a cane Normal Wheelchair bound with crutches Wheelchair bound Wheelchair bound LL Wheelchair bound UL ¼ upper limbs; LL ¼ lower limbs; AJ ¼ ankle jerk. Homozygosity or compound heterozygosity for the S194X mutation in the GDAP1 gene has already been associated with the axonal form of the disease (CMT2K) (Cuesta et al., 2002; Nelis et al., 2002; Azzeddine et al., 2003b; Birouk et al., 2003). However, Baxter et al. (2002) reported one family with this mutation, in which the neuropathy was considered to be of the demyelinating type. No S194X mutation was found in the 27 Moroccan families with an AR demyelinating neuropathy. Since mutations in GDAP1 have been reported to cause axonal or demyelinating neuropathy, all GDAP1 exons should be screened in these families. All the six families with the S194X mutation shared a common haplotype around the GDAP1 gene suggesting a founder effect for this mutation in Morocco (Fig. 2). Genotyping of additional markers is needed to verify this hypothesis and to date the mutation. From a diagnostic point of view, since the frequency of the heterozygous carriers of the S194X mutation is 1.2% and the rate of consanguinity is 29% in Morocco (Bendriss et al., 2006), the proportion of affected individuals (homozygous for this mutation) is likely to be high in CMT2K/4A in this country. This mutation will now be given priority in molecular diagnoses of CMT2K/4A in Moroccan families. The R289C mutation is the only LMNA gene mutation described in CMT2B1 and has already been reported in many families from Algeria (Chaouch et al., 2003; Tazir et al., 2004). This is the first report of mutations in families originating outside this country. A common haplotype around the LMNA gene in the four families suggests a founder effect (Fig. 2). The absence of the R298C mutation in a Moroccan control population could be due to a private mutation in Morocco that has not had time to diffuse into the general population. As all the other known CMT2B1 families (7 from Tazir et al., 2004, 4 from Chaouch et al., 2003 and 13 from Azzedine et al., 2002, 2003a) originate from Algeria, the other most likely explanation is that this mutation appeared first in Algeria before arriving in Morocco. In order to verify this hypothesis, further studies including the Algerian families with the LMNA mutation are necessary to date the mutation and help to analyse the migration of populations in North West Africa. From a physiopathological point of view, the absence of genetic heterogeneity makes it possible to study the intraand inter-familial variability of the phenotype in this population without taking into account the effects of different mutations on the protein level. The electrophysiological findings for all patients from the 13 families studied were consistent with an axonal neuropathy, since the median nerve MNCV, when recordable, was above 40 m/s with reduced CMAP and SNAP amplitudes except for patients MAD and CHA who had MNCVs of 36 and 35 m/s with reduced CMAP amplitudes of 3.5 and 0.4 mv, respectively. The clinical data show that patients with a mutation in the LMNA gene have variable ages at onset (from 8 to

10 Table 5 Motor and sensory nerve conduction and needle electromyography findings in patients with ARCMT neuropathy due to R289C mutation in the LMNA gene Case No Motor nerve conduction Median nerve DML (ms) ^ MNCV (m/s) CMAPamplitude (mv) Cubital DML (ms) ^ MNCV (m/s) CMAPamplitude (mv ) SPE DML (ms) MNCV (m/s) CMAP amplitude (mv) Sensory nerve action potentials amplitude (mv) Median nerve Ulnar nerve ^ 0 ^ Suralnerve Needle EMG TA ^ ^ ^ RRPþþþ RRPþþþ ^ RRPþþþ RRPþþþ RRPþþþ RRPþþþ ^ RRPþþ RRPþþþ RRPþþ Vastus lateralis ^ ^ ^ RRPþþ RRPþþ ^ RRPþþþ RRPþþþ RRPþþþ ^ ^ RRPþþ RRPþþ ^ First DIO ^ ^ ^ Normal RRPþ ^ RRPþþþ RRPþþ RRPþþ Normal ^ RRPþ RRPþþ RRPþþ ECR ^ ^ ^ ^ ^ ^ 6 Normal Normal Normal ^ ^ ^ ^ Deltoid ^ ^ ^ ^ RRPþ ^ Normal Normal Normal ^ ^ ^ Normal ^ DML ¼ distal motor latency; MNCV ¼motor nerve conduction velocity; CMAP ¼ compound muscle action potential; TA ¼ tibialis anterior; DIO ¼ dorsal interosseus; ECR ¼ extensor carpi radialis. The 0 value was attributed to abolished motor or sensory potentials; NVA ¼ no voluntary activity; RRP ¼ reduced recruitment pattern; þ, þþ and þþþ for slight, moderate and important RRP, respectively. ARCMT2 phenotype ^ genotype correlations Brain (2007), 130,1062^

11 1072 Brain (2007), 130,1062^1075 A.Bouhoucheet al. Table 6 Clinical findings in patients with ARCMT neuropathy due to an unknown genetic defect Case No Age at onset (years) 14 NFD NFD Hypotonia at birth No No No No No No Age of walking Normal Normal Normal Normal Normal Normal Age at examination (years) Disease duration (years) 2 ^ ^ Distal motor deficit UL þ LL No UL þ LL UL þ LL LL No Proximal motor deficit LL No No No No No Distal muscle atrophy LL No UL þ LL UL þ LL LL No Proximal muscle atrophy No No No No No No Sensory loss Pain and touch No No LL No No No Proprioception No No UL þ LL LL No No Areflexia UL þ LL LL UL þ LL UL þ LL AJ AJ Foot deformities No No Moderate Moderate No Moderate Scoliosis Severe X-ray diagnosed Severe Severe No No Functional disability UL No No No No No No LL Normal walk No with a cane No NFD ¼ no functional disability; UL ¼ upper limbs; LL ¼ lower limbs; AJ ¼ ankle jerk. Table 7 Motor and sensory nerve conduction and needle electromyography findings in patients with ARCMT neuropathy due to an unknown genetic defect Case No Motor nerve conduction Median nerve DML (ms) MNCV (m/s) CMAP amplitude (mv) Cubital DML (ms) MNCV (m/s) CMAP amplitude (mv) SPE DML (ms) MNCV (m/s) CMAP amplitude (mv) Sensory nerve action potentials amplitude (mv) Median nerve Ulnar nerve ^ Suralnerve Needle EMG TA RRPþ Normal RRPþþþ RRPþþþ NVA RRPþþ Vastus lateralis Normal ^ ^ ^ ^ ^ First DIO Normal Normal RRPþ RRP þþ Normal Normal ECR ^ ^ RRPþ RRP þ Normal ^ Deltoid Normal ^ ^ ^ ^ ^ DML ¼ distal motor latency; MNCV ¼motor nerve conduction velocity; CMAP ¼ compound muscle action potential; TA ¼ tibialis anterior; DIO ¼ dorsal interosseus; ECR ¼ extensor carpi radialis. The 0 value was attributed to abolished motor or sensory potentials; NVA ¼ no voluntary activity; RRP ¼ reduced recruitment pattern; þ, þþ and þþþ for slight, moderate and important RRP, respectively. 20 years), but that the disease began most often in the second decade and progressed gradually from distal to proximal muscles. The evolution was similar to that reported in Algerian families (Chaouch et al., 2003; Tazir et al., 2004). However, we noted that three of our patients with the longest disease durations (more than 24 years) had severe impairment, not only in the pelvic but also in the scapular muscles, which has not yet been reported and might be a hallmark of CMT2B1. Patients had minor or no foot deformities. The disease severity was variable in the families with LMNA mutations as described by Tazir et al. (2004). In our largest family with nine affected members (Family MAD-008), five of whom had disease durations of more than 20 years, one patient (MAD ) still walked

12 ARCMT2 phenotype ^ genotype correlations Brain (2007), 130, 1062^ Fig. 4 Nerve biopsies. Electron micrograph of transverse sections of the superficial peroneal nerve. A, B, C: biopsy from patient AIT showing a small myelinated fibre with a normal myelin sheath (A), an onion bulb formation with concentric cytoplasmic Schwann cell processes around a myelinated fibre (B) and a cluster of regeneration formed by a group of three myelinated fibres (C); D, E, F: biopsy from patient MAD showing at low magnification the loss of myelinated fibres and the absence of onion bulb formations (D), the normal appearance of myelin sheaths (E) and one cluster of regeneration, which were numerous throughout the nerve biopsy (F); G, H: biopsy from patient OUI showing a very small diameter fibre with a normal myelin sheath (G) and clusters of unmyelinated fibres (H). and had a moderate phenotype, whereas the others were either wheelchair-bound or needed help walking. Patients with a mutation in the GDAP1 gene had an earlier onset, most often before the age of 2 years, as in the previously reported families with GDAP1 gene mutations (Baxter et al., 2002; Nelis et al., 2002; Sevilla et al., 2003; Azzedine et al., 2003b; Boerkoel et al., 2003; De Sandre Giovannoli et al., 2003b; Sendereck et al., 2003; Claramunt et al., 2005). Most of our patients with GDAP1 S194X mutation had severe clubfoot from the beginning, as in the patients reported by Sendereck et al. (2003). This seems to be one of the hallmarks of ARCMT with GDAP1 mutations. Disease progression was severe in all patients, with involvement of proximal muscles of the lower limbs and hands toward the end of the first decade. Most patients could no longer walk without help in the second decade. The clinical phenotype of ARCMT with GDAP1 mutations seems to be homogeneous since a similar disease course was described in all reported cases (Benothman et al., 1993; Baxter et al., 2002; Cuesta et al., 2002; Nelis et al., 2002; Boerkoel et al., 2003; De Sandre Giovannoli et al., 2003b). However, we did not notice vocal cord paralysis in any of our patients, as observed in a few families of Spanish or Moroccan ancestry (Cuesta et al., 2002; Sevilla et al., 2003; Azzedine et al., 2003b). The clinical phenotype of the three families that are not linked to the three known loci presented some particularities that were not seen in those with known genetic defects. Family AMI-027 was characterized by late onset of the disease (420 years) or a mild neuropathy that was diagnosed only when the family was examined. In family MOU-023, dorsal scoliosis was the most

13 1074 Brain (2007), 130,1062^1075 A.Bouhoucheet al. prominent symptom. In family OUI-030, symptoms began in the second decade with a moderate neuropathy associated with a pronounced scoliosis. These families illustrate the extent of clinical and genetic heterogeneity in ARCMT2. Histopathological examination of a distal sensory nerve in one patient from each group showed an axonal process, characterized by loss of myelinated fibres, especially those of large diameter, associated with different degrees of axonal regeneration. However, there were some differences between the three biopsies. Although fibre loss was severe in all cases, it was less pronounced in patient MAD with the R289C mutation in the LMNA gene, and was associated with numerous clusters of regenerating axons. This contrasts with the finding of Chaouch et al. (2003), who noted the absence of regenerative clusters in the nerve of a patient with this mutation. The presence of axonal sprouting in our patient may explain why sensory impairment was not observed clinically, although sensory potentials were found to be abolished on electrophysiological examination, due to the loss of large diameter fibres. The loss of myelinated fibres was more severe in patient AIT with the S194X mutation in the GDAP1 gene. Axonal regeneration in this nerve was unusual because of the presence of pseudo-onion bulb formations instead of classical clusters. Some true onion bulb formations, indicative of demyelination remyelination, were also observed. These pathological features are very similar to those described by Sevilla et al. (2003). In individual OUI , belonging to a family without linkage to the three known loci, there were almost no myelinated fibres left; only fibres with very small diameters (1 2 mm) and clusters of unmyelinated fibres were observed. These lesions represent a late stage of axonal degeneration and may explain why the patient had more clinical sensory impairment than the other biopsied patients, including loss of pain and touch sensation in the lower limbs and decreased proprioception in both upper and lower limbs. In conclusion, these results suggest that Moroccan ARCMT2 patients with late involvement of proximal muscles and intra-familial variability of disease severity should first be tested for the R298C mutation in the LMNA gene, whereas patients with early or congenital onset, severe disability and pronounced foot deformities should be tested for the S194X mutation in exon 5 of GDAP1 gene, even if the neuropathy is not associated with vocal cord paralysis. However, these mutations do not account for all ARCMT2 patients. Other genes/loci remain to be discovered. Acknowledgements We would like to convey our gratitude to the family members for their participation in this study. 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