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Novel mutations in the PRX and the MTMR2 genes are responsible for unusual Charcot-Marie-Tooth disease phenotypes
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Neuromuscular Disorders 21 (2011) 543–550 www.elsevier.com/locate/nmd
Novel mutations in the PRX and the MTMR2 genes are responsible for unusual Charcot-Marie-Tooth disease phenotypes Sonia Nouioua a,b, Tarik Hamadouche b,c, Benoit Funalot d, Rafae¨lle Bernard e, Nora Bellatache a, Radia Bouderba a,b, Djamel Grid f, Salima Assami a,b, Traki Benhassine b,g, Nicolas Levy e, Jean-Michel Vallat d, Meriem Tazir a,b,⇑ a
Service de Neurologie, CHU Mustapha Bacha, Algiers, Algeria b Laboratoire de Neurosciences, Universite´ d’Alger, Algeria c Laboratoire de Biologie Mole´culaire, Universite´ M’hamed Bougara, Boumerdes, Algeria d Centre de re´fe´rence «neuropathies pe´riphe´riques rares», Service et Laboratoire de Neurologie, CHU de Limoges, Limoges, France e Inserm UMR_S 910, Ge´ne´tique Me´dicale et Ge´nomique Fonctionnelle, Faculte´ de Me´decine de Marseille, Universite´ de la Me´diterrane´e, 13005 Marseille, France f Ge´ne´thon, 91000 Evry, France g Laboratoire de ge´ne´tique, FSB, Universite´ Bab Ezzouar, Algiers, Algeria Received 16 March 2011; received in revised form 19 April 2011; accepted 27 April 2011
Abstract Autosomal recessive Charcot-Marie-Tooth diseases, relatively common in Algeria due to high prevalence of consanguineous marriages, are clinically and genetically heterogeneous. We report on two consanguineous families with demyelinating autosomal recessive Charcot-Marie-Tooth disease (CMT4) associated with novel homozygous mutations in the MTMR2 gene, c.331dupA (p.Arg111LysfsX24) and PRX gene, c.1090C>T (p.Arg364X) respectively, and peculiar clinical phenotypes. The three patients with MTMR2 mutations (CMT4B1 family) had a typical phenotype of severe early onset motor and sensory neuropathy with typical focally folded myelin on nerve biopsy. Associated clinical features included vocal cord paresis, prominent chest deformities and claw hands. Contrasting with the classical presentation of CMT4F (early-onset Dejerine–Sottas phenotype), the four patients with PRX mutations (CMT4F family) had essentially a late age of onset and a protracted and relatively benign evolution, although they presented marked spine deformities. These observations broaden the spectrum of clinical phenotypes associated with these two CMT4 forms. Ó 2011 Elsevier B.V. All rights reserved. Keywords: ARCMT; CMT4B1; CMT4F; MTMR2 and PRX genes; Vocal cord palsy; Skeletal deformities
1. Introduction Families with recessively inherited forms of CharcotMarie-Tooth disease (CMT) are relatively common in Algeria, as in other countries around the Mediterranean basin, due to high prevalence of consanguineous marriages. ⇑ Corresponding author at: Service de Neurologie, CHU Mustapha Bacha, Place du 1er Mai, Algiers 16000, Algeria. Tel./fax: +213 21 23 56 40. E-mail address: [email protected] (M. Tazir).
0960-8966/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2011.04.013
As with the dominant forms, it is usual to distinguish the demyelinating forms of autosomal recessive CharcotMarie-Tooth disease (ARCMT), designated CMT4 or ARCMT1, from the axonal forms (ARCMT2). ARCMT forms generally have an earlier onset and a more severe disease course [1] although they are clinically and genetically heterogeneous. Indeed, demyelinating ARCMT (CMT4) are due to mutations of several genes such as GDAP1 (CMT4A), MTMR2 (CMT4B1), MTMR13 (CMT4B2), SH3TC2 (CMT4C), NDRG1 (CMT4D), EGR2 (CMT4E), PRX (CMT4F), FGD4
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(CMT4H) and FIG4 (CMT4J). The clinical and/or the histopathological phenotypes often suggest that a specific gene may be mutated, which helps in some cases to orientate molecular testing [2]. CMT4B1, first reported in a large Italian family with 10 affected members, is characterized clinically by a severe early onset motor and sensory neuropathy, and pathologically by the presence of focally folded myelin [3,4]. The mechanisms of these histological lesions are so far unknown. Using a positional-cloning strategy in unrelated CMT4B1 patients, mutations occurring in the gene MTMR2, encoding myotubularin-related protein-2, a dual specificity phosphatase, were identified [5]. The CMT4F locus was mapped to 19q13.1-13.3 in a large consanguineous Lebanese family with a severe Dejerine–Sottas-like phenotype [6]. Mutations in the periaxin gene (PRX) were identified in this family [7] and in three other unrelated patients with severe CMT4 [8]. To date, 16 nonsense or frameshift PRX mutations have been reported in CMT4F patients [7–14]. The PRX gene code for L-periaxin and S-periaxin. Both periaxins are thought to be essential for the formation and maintenance of peripheral nerve myelin [7]. We report on 2 families with rare forms of demyelinating ARCMT, harbouring novel mutations in the myotubularin-related protein (MTMR2) and the periaxin (PRX) gene respectively, both families presenting peculiar clinical phenotypes. 2. Patients and methods Between 1998 and 2007, we explored overall 50 Algerian families (102 patients) with phenotypes suggestive of CMT. There were two autosomal dominant CMT families (five cases), 01 CMTX family (two cases), 6 spinal CMT (13 cases). It is noteworthy that 28 ARCMT families (37 patients) were not linked to known CMT loci. Axonal forms including ARCMT2 due to the c.892C>T mutation in the LMNA gene with a founder effect [15,16] were predominant (8 ARCMT2A families, 24 cases), whereas CMT4 (ARCMT1) forms were rare and included three families with CMT4C [17], one family with CMT4B1 and one family with CMT4F. The present report is devoted to these two last families. All assessed subjects gave informed consent to take part in this study which was approved by the Ministry of Health, Health Ethic Committee, Algeria. Seven patients of these two families, their parents and all the healthy siblings were assessed and examined for the presence of motor and sensory deficits, areflexia, cerebellar ataxia, nystagmus, dysarthria, tremor, foot deformities, scoliosis, pyramidal signs, and cranial nerve involvement. The Mini Mental State Examination (MMSE) was used to assess cognitive functions in adults. Children were assessed using the Brunet–Lezine scale of infant development. The propositi and most affected patients had also undergone serum albumin level testing, lipid electrophoresis, creatine kinase, hexosaminidase, and
aryl-sulfatase blood testing and CSF immunoelectrophoresis analysis. Electrophysiological analysis was performed in all patients. Nerve conduction studies were performed with surface stimulation and recording electrodes in patients, their parents and some unaffected siblings. Motor nerve conduction velocities (MNCVs) in the median, ulnar and the peroneal nerves were recorded. Antidromic sensory compound action potential was recorded from the median and sural nerves. Electromyography of the tibialis anterior and the first dorsalis interosseous muscle was carried out with a concentric needle electrode in patients. For analysis of the nerve biopsies, fascicles of the superficial peroneal nerves were divided into several pieces. One of them was fixed in formaldehyde (10%) embedded in paraffin and sections were stained using conventional methods. The other fascicles were fixed in buffered glutaraldehyde, processed and embedded in epon. Transverse semi-thin sections were stained with toluidine blue and ultrathin sections were stained with uranyl acetate and lead citrate and analysed in a Philips CM10 electron microscope. MTMR2 and PRX coding regions and exon/intron boundaries were amplified using previously described primers and amplification conditions [5,6]. Samples were purified using the QIAquick PCR purification kit (Qiagen) and then sequenced on both strands by use of the BigDyeÒ Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) and separated on an ABI PRISM DNA Sequencer 3100 (Applied Biosystems). Sequences were compared to the reference sequences (GenBank accession number NM_181882 for PRX and NM_016156 for MTMR2). 3. Results Clinical, electrophysiological and molecular data are summarised in Table 1. The parents and the healthy siblings in both families were unaffected. In family 1 (F1), the three affected sibs were born from consanguineous parents (Fig. 1) and had an early age of onset (1–2 years). All had a predominantly motor neuropathy with a steppage gait and distal limb weakness and wasting (Table 1). Moreover, they all had difficulties to use properly their deformed claw hands (Fig. 1). This distal motor and sensory neuropathy was associated to breathing difficulties, chronic stridor but a normal voice, facial paresis and convex and concave chest deformities (Fig 1). F1P2 and F1P3 patients presented a marked stridor during inspiration leading to dyspnea and abdominal respiration. The intensity of the stridor decreased in the older patient (F1P1) after the age of 5 years but he had a marked impairment of respiratory function (vital capacity: 40%) at the age of 14. Endoscopic examination showed severe adduction vocal cord paralysis in the three sibs. Cognitive functions were normal in all of them. Cerebrospinal fluid protein assessed in the propositus was markedly elevated to 110 mg/dl. All other biological tests were normal.
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Table 1 Clinical and molecular data.
Age (year)/gender Age of onset (year) Onset symptoms Muscle wasting UL Muscle wasting LL
F1P1
F1P2
F1P3
F2P1
F2P2
F2P3
F2P4
16/M 2 Stridor Falls Distal ++
12/M 2 Stridor
9/M 1 Stridor and chest deformity Proximal ++ Distal ++ Proximal ++ Distal +++ Proximal 4 Distal : 2–4 Proximal: 4 Distal: 2–3 Chest+++
22/M 12 Unstable gait and scoliosis Distal ++
20/M 10 Unstable gait
17/M 10 Unstable gait
15/F 7 IUnstable gait
Distal ++
Distal ++
Distal ++
Distal ++
Distal ++
Distal ++
Distal ++
Distal: 3–4
Distal: 3–4
Distal: 3–4
Distal: 4
Distal: 3–4
Distal: 2–4
Distal: 3–4
Distal:3–4
Kyphoscoliosis Kyphoscoliosis Kyphoscoliosis +++ ++ +++ Pes equinovarus+ Pes cavus+ Pes cavus+ Pes cavus+ Claw hands+++
Kyphoscoliosis ++ Pes cavus+
Absent Steppage
Absent Steppage
Absent Ataxic
Proximal+ Distal +++ Distal: 3–4
Weakness UL (MRC) Weakness LL (MRC) Skeletal deformities
Chest++
DTRs Gait
Pes equinovarus+++ Claw hands++ Absent Steppage
Sensory ataxia Touch and Pain sensation Vibration sense
Position sense Vocal cord paresis Median MNCV (m/s) Median Ampl (mV) CMTNS Gene Mutations F1: MTMR2 F2: PRX
Distal: 1–3
Proximal+ Distal ++ Proximal ++ Distal +++ Proximal 4 Distal: 2–4 Proximal: 4 Distal: 2–4 Chest+++ Claw hands+
No Reduced UL + LL Moderately reduced distally in UL + LL Normal Yes 13
No Normal
No Normal
Moderately reduced distally in UL + LL Normal Yes 15
Moderately reduced distally in UL + LL Normal Yes 19,6
Yes Reduced UL + LL Severely reduced UL + LL Akinesthesia No 9
1.8 24 c.331dupA
1.3 23 c.331dupA
1 25 c.331dupA
0.7 19 c.1090C>T
Absent Ataxic Mild steppage Yes Reduced UL + LL Severely reduced UL + LL Akinesthesia No 8
Absent Ataxic
Absent Ataxic
Yes Reduced UL + LL Moderately reduced distally in LL Akinesthesia No 5
Yes Reduced UL + LL Moderately reduced distally in LL Akinesthesia No 6
0.5 20 c.1090C>T
0.3 19 c.1090C>T
0.4 19 c.1090C>T
p.Arg111LysfsX24 p.Arg111LysfsX24 p.Arg111LysfsX24 p.Arg364STOP p.Arg364STOP p.Arg364STOP
p.Arg364STOP
MRC assessed muscles: UL proximal muscles: Supraspinatus, deltoide, biceps brachii, triceps brachii. UL distal muscles: Opponens pollicis, Extensor pollicis brevis, flexor pollicis brevis, palmar and dorsal interossei, palmaris longus and brevis, extensor digitorum. LL proximal muscles: Psoas, quadriceps femoris, biceps femoris, gluteus maximus. LL distal muscles: Tibialis anterieur, fibularis longus, gastrocnemius, extensor digitorum brevis, flexor hallucis longus, extensor digitorum longus, flexor digitorum longus. Abbreviations: + = Mild; ++ = moderate, +++ = severe; CMTNS = Charcot-Marie-Tooth Neuropathy Score; DTR = deep tendon reflex; UL = upper limbs, LL = lower limbs; MNCV = motor nerve conduction velocity; MRC = Medical Research Council scale; F1: family1; F2: family2.
In family 2 (F2), the four affected sibs were born from consanguineous parents (Fig. 2). Mean age at onset was 10 ± 2 years. All had normal cognitive functions. Initial symptoms were spine deformities and gait instability in all patients. Indeed, instability and disequilibrium on walking were described as the first symptoms by family members. Examination showed that sensory ataxia was the main sign including a Romberg sign, hypopallesthesia, akinesthesia and instability on walking. Steppage gait was not present, even many years after the onset, in almost all the CMT4F patients, although they could not walk on their heels. Kyposcoliosis was also present in all the patients and motor disability was mild in all of them. Indeed, patient 1 (propositus, Fig. 2) had difficulties to run and
to achieve sport at school. At last exam, he was 22 year old and attended university. Patient 2 had the same symptoms and received surgical treatment for kyphoscoliosis. Patient 3 had the same clinical picture with more severe spine and thoracic deformities. Patient 4 a 15 year-old girl, had moderate spine and feet deformities but also presented pyramidal signs and bilateral hypoacousia. Cerebral MRI showed bilateral acoustic neurofibroma and multiple cerebral meningiomas in patient 4 and was normal in the 3 others. In both families, nerve conduction velocities study indicated a demyelinating polyneuropathy with ulnar and median MNCV below 20 m/s, absence of motor peroneal nerve potentials and absence of sensory action potentials in the median and sural nerves. In family 2, median and
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Fig. 1. MTMR2 mutation segregating in family 1 pedigree and patients’ photography.
ulnar MNCV were dramatically decreased (below 10 m/s) and potential amplitudes were below 1 mV. Median, ulnar and peroneal MNCV were in the normal range in parents and assessed unaffected siblings of both families. Electromyography recording, performed only in patients, suggested a chronic neurogenic pattern in the explored muscles. Mean Charcot-Marie-Tooth neuropathy score (CMTNS) was 24 ± 1 in MTMR2 family whereas in PRX family it was 19 ± 0.5. F1P2 and F2P4 had a nerve biopsy before molecular study. In F1P2 (CMT4B1family), electron microscopy study of superficial peroneal nerve biopsy showed a significant loss of myelinated fibers and important myelin inand outfoldings in the majority of large diameter fibers (Fig. 3). There were also numerous demyelination-remyelination lesions with thin myelin sheaths and frequent onion bulb formations. This aspect thus, led us to suspect CMT4B1 and search for MTMR2 gene mutation. In F2P4 (CMT4F family), nerve biopsy study showed severe loss of myelinated and unmyelinated fibers and rare myelin outfoldings. The remaining fibers were surrounded by onion-bulb Schwann cell proliferations (Figs. 4 and 5).
For family 2 (PRX mutated), all loci responsible for CMT were first excluded by linkage analysis, whereas for family 1 (MTMR2 mutated), only CMT2B1 (LMNA gene) was initially excluded, before performing nerve biopsy.In family 1, a homozygous insertion of adenine was detected in MTMR2 exon 4: c.331dupA (p.Arg111LysfsX24) in the three patients. The parents and the paternal grand-mother were heterozygous carriers of this mutation ( Fig. 1). In family 2, The 4 patients harbored a novel homozygous c.1090C>T (p.Arg364X) mutation in PRX exon 7. The father and one healthy brother were heterozygous carriers of the same mutation (Fig. 2). The mother was not available for DNA testing. So, in both families, the segregation of the mutated alleles was consistent with the autosomal recessive inheritance of the disorder. One hundred control chromosomes from 50 unrelated individuals from the Algerian population have also been tested without the identification of any of these two mutations. 4. Discussion We reported on two families with autosomal recessive demyelinating forms of Charcot-Marie-Tooth disease
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Fig. 2. MTMR2 mutation segregating in family 1 pedigree and patients’ photography.
Fig. 3. F1P2. Electron micrograph. Transverse section shows severe axonal loss and onion-bulb Schwann cell proliferations; the myelin sheaths present aberrant myelin in and out-foldings.(Bar = 2 lm).
Fig. 4. F2P4. Electron micrograph. Transverse section shows severe rarefaction of myelinated fibers. One remaining fiber is surrounded by onion-bulb Schwann cell proliferations.
(CMT4B1 and CMT4F) harbouring novel mutations in the MTMR2 and PRX genes, respectively. As we know, consanguinity increases the risk of autosomal recessive
disorders (such as CMT4B1 and CMT4F). In both families, patients are homozygous carriers of a mutation probably inherited from a common ancestor. Affected
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Fig. 5. F2P4. Electron micrograph. Transverse section. This axon (A) is completely demyelinated and surrounded by onion-bulb proliferations of Schwann cells.
family members in both kindreds demonstrated unusual clinical features contrasting with the signs and symptoms observed in previously reported patients. A limited number of clinical reports on patients with CMT4B1, which is characterized by the presence of a great number of myelin outfoldings on nerve biopsy, have been published since the identification of the MTMR2 mutations in 2 Italian and 2 Saudi Arabian inbred families [5]. Thus, only 3 publications reported on 5 CMT4B1 cases with novel mutations [18–20]. All the last reported cases (Table 2) had an early age of onset, as was the case in our patients, and a severe distal motor and sensory neuropathy with proximal lower limbs weakness. Nevertheless, stridor and vocal cord paresis have been observed in only one reported case [18]. These distressing signs have also been reported in some CMT2C and CMT4A patients [21–23]. Moreover, early chest deformities, not mentioned elsewhere, were prominent in our patients. They were
probably the result of breathing difficulties due to severe vocal cord palsy for which laser treatment was considered and scheduled. Although skeletal deformities, including claw hands and chest deformities which add more severity to the clinical picture, were not taken into account in the CMTNS [24], it was indeed severe in the MTMR2 family [23–25]. In contrast to the normal CSF protein found in the only one assessed MTMR2 patient [20], it was markedly elevated in our MTMR2 propositus. The focal myelin outfoldings is not a specific morphologic change, as apart from CMT4B1 and CMT4B2 it has been observed in some other neuropathies, but it could be considered as a hallmark of CMT4B1.This aspect was missing in only one previously reported case, for which only onion bulbs and focal myelin thickening were described [20]. In our patient, this typical aspect of the nerve biopsy helped to orientate molecular study directly towards MTMR2 screening. In contrast to the majority of the previously reported CMT4F patients, which had an early onset of the disease and a Dejerine–Sottas phenotype, our patients with PRX mutations had a relatively late age of onset and a protracted evolution. As we know, age of onset is not easy to determine in chronic neurodegenerative disorders and especially CMT diseases. This has been underlined in a previous CMT study [15]. In this PRX family, age of onset was variable and was noticed earlier in the youngest sibs but still much later than usually mentioned in reported CMT4F cases. These last already described cases are characterized by very early onset, often with delayed motor milestones, but subsequent slow clinical progression [9–14]. The four patients were displaying a moderate sensory ataxia, a mild distal motor disability but prominent spine deformities. Scoliosis or kyphoscoliosis which could appear in CMT patients after one or two decade of evolution is probably caused by a
Table 2 CMT4B1 reported cases. CMT 4 B1 patients
Houlden et al.
Verny et al.
Parman et al.
This study
N° of patients Age at onset (months) Age (years) AR demyelinating sensorymotor PNP Vocal cord palsy/stridor Other cranial nerve palsy Respiratory difficulties
2 13 Case1: 16; case2: 5 Yes
1 Birth 18 Yes
2 Delayed motor milestones Case1: 21; case2: 24 Yes
3 24 Case 1: 15; case2: 11; case 3: 8 Yes
No Dysphagia Yes
No Hypophonia No
Yes Facial paresis Yes
Skeletal deformities
Yes (case 1) Dysarthria (case2) Yes (case 1) No (case 2) No
Pes equinovarus
Nerve biopsy
Myelin outfoldings
Chest deformities, pes equinovarus, claw hands Onion bulbs, myelin outfoldings
MTMR2 mutations
Case 1:G308A (Gly103 glu) Case2: delA324(frameshift)
Onion bulbs, myelin outfoldings G1749A (W583X)
Case1: pescavus, claw hands Case2: pescavus Case1: onion bulbs, focal myelin thickening Case 1: 446 bp insert (insert 47AA) Case 2: 841_844delATCA(frameshift X290)
c.331dupA (p.Arg111LysfsX24)
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motor deficit in the paravertebral muscles. Prominent kyphoscoliosis which used to be considered as a clinical hallmark of CMT4C [17], was the most important disability in this CMT4F family. It was one of the earlier signs and among first motives for seeking medical care in this family. One patient had beneficial spine surgical treatment; the three others were scheduled for medical rehabilitation and /or spine surgical orthopedics. According to the validated CMTNS scores given in Shy and al. study [24], family 2 had a moderate mean CMTNS (19) whereas a severe mean CMTNS (22) was observed in a recently reported PRX family [14], although disease duration was much longer in the later. One patient (F2P4) had bilateral acoustic neurofibroma and multiple cerebral meningiomas. The association of CMT1A and neurofibromatosis type 1 (NF1) has been reported [25] and it was concluded that 2 common autosomal dominant conditions (CMT1 and NF1) can occur in the same individual, not because of an underlying single molecular defect, but rather, secondary to a chance phenomenon. In our case, it could be also a fortuitous association because none of the other sibs had similar abnormalities. These CMT4F patients had nearly a normal life but they required constant care mainly for the spine and feet deformities. Although CMT4F disease is characterized by morphologic changes encompassing loss of axons, focally folded myelin (which are much less numerous than in CMT4B) and formation of typical basal lamina onion bulbs, in our CMT4F patient there were mainly a dramatic loss of myelinated and unmyelinated fibers and some onion bulb Schwann cell proliferation. Consistent with previous data, the new PRX mutation described here is a nonsense mutation, located in exon 7. The majority of previously reported mutations are also localized in exon 7 and lead to a truncated L-periaxin [7–14]. Although unusual phenotype and clinical heterogeneity are typical for ARCMT, these two families had further atypical clinical signs not previously reported, mainly a marked stridor leading to impairment of respiratory function and various chest deformities in CMT4B1 patients, and a late age of onset together with prominent spine deformities in CMT4F patients. Moreover these two families harbored two novel mutations in MTMR2 and PRX genes respectively. In summary, the 2 reported CMT4 families with novel mutations in the MTMR2 and the PRX genes had peculiar clinical phenotypes. The CMT4B1 patients had vocal cord paresis and prominent chest deformities and the CMT4F patients had essentially a late age of onset, a protracted and relatively benign evolution but major kyphoscoliosis. These observations broaden the spectrum of clinical phenotypes associated with these two CMT4 syndromes. Furthermore, the clinical heterogeneity observed in CMT4B1 and CMT4F patients reported so far has been also described in other CMT4 families, especially CMT4H [26], demonstrating a variable functional impairment in CMT4 patients, which may be correlated to mutations of variable severities in a given gene. However, clinical
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heterogeneity was also observed in patients affected by axonal forms of ARCMT due to the same mutation, as was the case in ARCMT2A where all patients harbored the homozygous c.892C>T mutation in the LMNA gene [15]. It is clear that for both axonal and demyelinating forms of ARCMT, the observation of particular combinations of clinical and histological features is often helpful to effectively orientate molecular tests. Acknowledgments We thank very much Dorra Hmida and Michel Koenig for their contribution.This work received support from the Ministe`re de la Sante´, de la Population et de la Re´forme Hospitalie`re (Algeria), the Ministe`re de l’Enseignement Supe´rieur et de la Recherche Scientifique (Algeria) and the Association Francßaise contre les Myopathies (AFM). References [1] Harding AE, Thomas PK. Autosomal recessive forms of hereditary motor and sensory neuropathy. J Neurol Neurosurg Psychiatry 1980;43:669–78. [2] Vallat JM, Tazir M, Magdelaine C, Sturtz F, Grid D. Autosomalrecessive Charcot-Marie-Tooth diseases. J Neuropathol Exp Neurol 2005;64:363–70. [3] Quattrone A, Gambardella A, Bono F, et al. Autosomal recessive hereditary motor and sensory neuropathy with focally folded myelin sheaths: clinical, electrophysiologic, and genetic aspects of a large family. Neurology 1996;46:1318–24. [4] Bolino A, Brancolini V, Bono F, et al. Localization of a gene responsible for autosomal recessive demyelinating neuropathy with focally folded myelin sheaths to chromosome 11q23 by homozygosity mapping and haplotype sharing. Hum Mol Genet 1996;5:1051–4. [5] Bolino A, Muglia M, Conforti FL, et al. Charcot-Marie-Tooth type 4B is caused by mutations in the gene encoding myotubularin-related protein-2. Nat Genet 2000;25:17–9. [6] Delague V, Bareil C, Tuffery S, et al. Mapping of a new locus for autosomal recessive demyelinating Charcot-Marie-Tooth disease to 19q13.1-13.3 in a large consanguineous Lebanese family: exclusion of MAG as a candidate gene. Am J Hum Genet 2000;67:236–43. [7] Guilbot A, Williams A, Ravise N, et al. A mutation in periaxin is responsible for CMT4F an autosomal recessive form of CharcotMarie-Tooth disease. Hum Mol Genet 2001;10:415–21. [8] Boerkoel CF, Takashima H, Stankiewicz P, et al. Periaxin mutations cause recessive Dejerine-Sottas neuropathy. Am J Hum Genet 2001;68:325–33, Note: Erratum: Am. J Hum Genet 2001; 68: 557. [9] Takashima H, Boerkoel CF, De Jonghe P, et al. Periaxin mutations cause a broad spectrum of demyelinating neuropathies. Ann Neurol 2002;51:709–15. [10] Kijima K, Numakura C, Shirahata E, et al. Periaxin mutation causes early-onset but slow progressive Charcot-Marie-Tooth disease. J Hum Genet 2004;49:376–9. [11] Kabzin´ska D, Drac H, Sherman DL, et al. Charcot-Marie-Tooth type 4F disease caused by S399fsX410 mutation in the PRX gene. Neurology 2006;66:745–7. [12] Bara´nkova´ L, Siskova´ D, Hu¨hne K, et al. A 71-nucleotide deletion in the periaxin gene in a Romani patient with early-onset slowly progressive demyelinating CMT. Eur J Neurol 2008;15:548–51. [13] Auer-Grumbach M, Fischer C, Papic´ L, et al. Two novel mutations in the GDAP1 and PRX genes in early onset Charcot-Marie-Tooth syndrome. Neuropediatrics 2008;39:33–8.
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[20] Parman Y, Battaloglu E, Baris I, et al. Clinicopathological and genetic study of early-onset demyelinating neuropathy. Brain 2004;127:2540–50. [21] Donaghy M, Kennett R. Varying occurrence of vocal cord paralysis in a family with autosomal dominant hereditary motor and sensory neuropathy. J Neurology 1999;246:552–5. [22] Mc Entagart ME, Reid SL, Irrthum A. Confirmation of a hereditary motor and sensory neuropathy II C locus at chromosome 12q23-q24. Ann Neurol 2005;57:293–7. [23] Cuesta A, Pedrola L, Sevilla T. The gene encoding gangliosideinduced differentiation-associated protein 1 is mutated in axonal Charcot-Marie-Tooth type 4 A disease. Nat Genet 2002;30:22–5. [24] Shy ME, Blake J, Krajewski K, et al. Reliability and validity of the CMT neuropathy score as a measure of disability. Neurology 2005;64:1209–14. [25] Lupski JR, Pentao L, Williams LL, Patel PI. Stable inheritance of the CMT1A DNA duplication in two patients with CMT1 and NF1. Am J Med Genet 1993;45:92–6. [26] Fabrizi GM, Taioli F, Cavallaro T, et al. Further evidence that mutations in FGD4/frabin cause Charcot-Marie-Tooth disease type 4H. Neurology 2009;72:1160–4.
Neuromuscular Disorders 21 (2011) 543–550 www.elsevier.com/locate/nmd
Novel mutations in the PRX and the MTMR2 genes are responsible for unusual Charcot-Marie-Tooth disease phenotypes Sonia Nouioua a,b, Tarik Hamadouche b,c, Benoit Funalot d, Rafae¨lle Bernard e, Nora Bellatache a, Radia Bouderba a,b, Djamel Grid f, Salima Assami a,b, Traki Benhassine b,g, Nicolas Levy e, Jean-Michel Vallat d, Meriem Tazir a,b,⇑ a
Service de Neurologie, CHU Mustapha Bacha, Algiers, Algeria b Laboratoire de Neurosciences, Universite´ d’Alger, Algeria c Laboratoire de Biologie Mole´culaire, Universite´ M’hamed Bougara, Boumerdes, Algeria d Centre de re´fe´rence «neuropathies pe´riphe´riques rares», Service et Laboratoire de Neurologie, CHU de Limoges, Limoges, France e Inserm UMR_S 910, Ge´ne´tique Me´dicale et Ge´nomique Fonctionnelle, Faculte´ de Me´decine de Marseille, Universite´ de la Me´diterrane´e, 13005 Marseille, France f Ge´ne´thon, 91000 Evry, France g Laboratoire de ge´ne´tique, FSB, Universite´ Bab Ezzouar, Algiers, Algeria Received 16 March 2011; received in revised form 19 April 2011; accepted 27 April 2011
Abstract Autosomal recessive Charcot-Marie-Tooth diseases, relatively common in Algeria due to high prevalence of consanguineous marriages, are clinically and genetically heterogeneous. We report on two consanguineous families with demyelinating autosomal recessive Charcot-Marie-Tooth disease (CMT4) associated with novel homozygous mutations in the MTMR2 gene, c.331dupA (p.Arg111LysfsX24) and PRX gene, c.1090C>T (p.Arg364X) respectively, and peculiar clinical phenotypes. The three patients with MTMR2 mutations (CMT4B1 family) had a typical phenotype of severe early onset motor and sensory neuropathy with typical focally folded myelin on nerve biopsy. Associated clinical features included vocal cord paresis, prominent chest deformities and claw hands. Contrasting with the classical presentation of CMT4F (early-onset Dejerine–Sottas phenotype), the four patients with PRX mutations (CMT4F family) had essentially a late age of onset and a protracted and relatively benign evolution, although they presented marked spine deformities. These observations broaden the spectrum of clinical phenotypes associated with these two CMT4 forms. Ó 2011 Elsevier B.V. All rights reserved. Keywords: ARCMT; CMT4B1; CMT4F; MTMR2 and PRX genes; Vocal cord palsy; Skeletal deformities
1. Introduction Families with recessively inherited forms of CharcotMarie-Tooth disease (CMT) are relatively common in Algeria, as in other countries around the Mediterranean basin, due to high prevalence of consanguineous marriages. ⇑ Corresponding author at: Service de Neurologie, CHU Mustapha Bacha, Place du 1er Mai, Algiers 16000, Algeria. Tel./fax: +213 21 23 56 40. E-mail address: [email protected] (M. Tazir).
0960-8966/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2011.04.013
As with the dominant forms, it is usual to distinguish the demyelinating forms of autosomal recessive CharcotMarie-Tooth disease (ARCMT), designated CMT4 or ARCMT1, from the axonal forms (ARCMT2). ARCMT forms generally have an earlier onset and a more severe disease course [1] although they are clinically and genetically heterogeneous. Indeed, demyelinating ARCMT (CMT4) are due to mutations of several genes such as GDAP1 (CMT4A), MTMR2 (CMT4B1), MTMR13 (CMT4B2), SH3TC2 (CMT4C), NDRG1 (CMT4D), EGR2 (CMT4E), PRX (CMT4F), FGD4
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(CMT4H) and FIG4 (CMT4J). The clinical and/or the histopathological phenotypes often suggest that a specific gene may be mutated, which helps in some cases to orientate molecular testing [2]. CMT4B1, first reported in a large Italian family with 10 affected members, is characterized clinically by a severe early onset motor and sensory neuropathy, and pathologically by the presence of focally folded myelin [3,4]. The mechanisms of these histological lesions are so far unknown. Using a positional-cloning strategy in unrelated CMT4B1 patients, mutations occurring in the gene MTMR2, encoding myotubularin-related protein-2, a dual specificity phosphatase, were identified [5]. The CMT4F locus was mapped to 19q13.1-13.3 in a large consanguineous Lebanese family with a severe Dejerine–Sottas-like phenotype [6]. Mutations in the periaxin gene (PRX) were identified in this family [7] and in three other unrelated patients with severe CMT4 [8]. To date, 16 nonsense or frameshift PRX mutations have been reported in CMT4F patients [7–14]. The PRX gene code for L-periaxin and S-periaxin. Both periaxins are thought to be essential for the formation and maintenance of peripheral nerve myelin [7]. We report on 2 families with rare forms of demyelinating ARCMT, harbouring novel mutations in the myotubularin-related protein (MTMR2) and the periaxin (PRX) gene respectively, both families presenting peculiar clinical phenotypes. 2. Patients and methods Between 1998 and 2007, we explored overall 50 Algerian families (102 patients) with phenotypes suggestive of CMT. There were two autosomal dominant CMT families (five cases), 01 CMTX family (two cases), 6 spinal CMT (13 cases). It is noteworthy that 28 ARCMT families (37 patients) were not linked to known CMT loci. Axonal forms including ARCMT2 due to the c.892C>T mutation in the LMNA gene with a founder effect [15,16] were predominant (8 ARCMT2A families, 24 cases), whereas CMT4 (ARCMT1) forms were rare and included three families with CMT4C [17], one family with CMT4B1 and one family with CMT4F. The present report is devoted to these two last families. All assessed subjects gave informed consent to take part in this study which was approved by the Ministry of Health, Health Ethic Committee, Algeria. Seven patients of these two families, their parents and all the healthy siblings were assessed and examined for the presence of motor and sensory deficits, areflexia, cerebellar ataxia, nystagmus, dysarthria, tremor, foot deformities, scoliosis, pyramidal signs, and cranial nerve involvement. The Mini Mental State Examination (MMSE) was used to assess cognitive functions in adults. Children were assessed using the Brunet–Lezine scale of infant development. The propositi and most affected patients had also undergone serum albumin level testing, lipid electrophoresis, creatine kinase, hexosaminidase, and
aryl-sulfatase blood testing and CSF immunoelectrophoresis analysis. Electrophysiological analysis was performed in all patients. Nerve conduction studies were performed with surface stimulation and recording electrodes in patients, their parents and some unaffected siblings. Motor nerve conduction velocities (MNCVs) in the median, ulnar and the peroneal nerves were recorded. Antidromic sensory compound action potential was recorded from the median and sural nerves. Electromyography of the tibialis anterior and the first dorsalis interosseous muscle was carried out with a concentric needle electrode in patients. For analysis of the nerve biopsies, fascicles of the superficial peroneal nerves were divided into several pieces. One of them was fixed in formaldehyde (10%) embedded in paraffin and sections were stained using conventional methods. The other fascicles were fixed in buffered glutaraldehyde, processed and embedded in epon. Transverse semi-thin sections were stained with toluidine blue and ultrathin sections were stained with uranyl acetate and lead citrate and analysed in a Philips CM10 electron microscope. MTMR2 and PRX coding regions and exon/intron boundaries were amplified using previously described primers and amplification conditions [5,6]. Samples were purified using the QIAquick PCR purification kit (Qiagen) and then sequenced on both strands by use of the BigDyeÒ Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) and separated on an ABI PRISM DNA Sequencer 3100 (Applied Biosystems). Sequences were compared to the reference sequences (GenBank accession number NM_181882 for PRX and NM_016156 for MTMR2). 3. Results Clinical, electrophysiological and molecular data are summarised in Table 1. The parents and the healthy siblings in both families were unaffected. In family 1 (F1), the three affected sibs were born from consanguineous parents (Fig. 1) and had an early age of onset (1–2 years). All had a predominantly motor neuropathy with a steppage gait and distal limb weakness and wasting (Table 1). Moreover, they all had difficulties to use properly their deformed claw hands (Fig. 1). This distal motor and sensory neuropathy was associated to breathing difficulties, chronic stridor but a normal voice, facial paresis and convex and concave chest deformities (Fig 1). F1P2 and F1P3 patients presented a marked stridor during inspiration leading to dyspnea and abdominal respiration. The intensity of the stridor decreased in the older patient (F1P1) after the age of 5 years but he had a marked impairment of respiratory function (vital capacity: 40%) at the age of 14. Endoscopic examination showed severe adduction vocal cord paralysis in the three sibs. Cognitive functions were normal in all of them. Cerebrospinal fluid protein assessed in the propositus was markedly elevated to 110 mg/dl. All other biological tests were normal.
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Table 1 Clinical and molecular data.
Age (year)/gender Age of onset (year) Onset symptoms Muscle wasting UL Muscle wasting LL
F1P1
F1P2
F1P3
F2P1
F2P2
F2P3
F2P4
16/M 2 Stridor Falls Distal ++
12/M 2 Stridor
9/M 1 Stridor and chest deformity Proximal ++ Distal ++ Proximal ++ Distal +++ Proximal 4 Distal : 2–4 Proximal: 4 Distal: 2–3 Chest+++
22/M 12 Unstable gait and scoliosis Distal ++
20/M 10 Unstable gait
17/M 10 Unstable gait
15/F 7 IUnstable gait
Distal ++
Distal ++
Distal ++
Distal ++
Distal ++
Distal ++
Distal ++
Distal: 3–4
Distal: 3–4
Distal: 3–4
Distal: 4
Distal: 3–4
Distal: 2–4
Distal: 3–4
Distal:3–4
Kyphoscoliosis Kyphoscoliosis Kyphoscoliosis +++ ++ +++ Pes equinovarus+ Pes cavus+ Pes cavus+ Pes cavus+ Claw hands+++
Kyphoscoliosis ++ Pes cavus+
Absent Steppage
Absent Steppage
Absent Ataxic
Proximal+ Distal +++ Distal: 3–4
Weakness UL (MRC) Weakness LL (MRC) Skeletal deformities
Chest++
DTRs Gait
Pes equinovarus+++ Claw hands++ Absent Steppage
Sensory ataxia Touch and Pain sensation Vibration sense
Position sense Vocal cord paresis Median MNCV (m/s) Median Ampl (mV) CMTNS Gene Mutations F1: MTMR2 F2: PRX
Distal: 1–3
Proximal+ Distal ++ Proximal ++ Distal +++ Proximal 4 Distal: 2–4 Proximal: 4 Distal: 2–4 Chest+++ Claw hands+
No Reduced UL + LL Moderately reduced distally in UL + LL Normal Yes 13
No Normal
No Normal
Moderately reduced distally in UL + LL Normal Yes 15
Moderately reduced distally in UL + LL Normal Yes 19,6
Yes Reduced UL + LL Severely reduced UL + LL Akinesthesia No 9
1.8 24 c.331dupA
1.3 23 c.331dupA
1 25 c.331dupA
0.7 19 c.1090C>T
Absent Ataxic Mild steppage Yes Reduced UL + LL Severely reduced UL + LL Akinesthesia No 8
Absent Ataxic
Absent Ataxic
Yes Reduced UL + LL Moderately reduced distally in LL Akinesthesia No 5
Yes Reduced UL + LL Moderately reduced distally in LL Akinesthesia No 6
0.5 20 c.1090C>T
0.3 19 c.1090C>T
0.4 19 c.1090C>T
p.Arg111LysfsX24 p.Arg111LysfsX24 p.Arg111LysfsX24 p.Arg364STOP p.Arg364STOP p.Arg364STOP
p.Arg364STOP
MRC assessed muscles: UL proximal muscles: Supraspinatus, deltoide, biceps brachii, triceps brachii. UL distal muscles: Opponens pollicis, Extensor pollicis brevis, flexor pollicis brevis, palmar and dorsal interossei, palmaris longus and brevis, extensor digitorum. LL proximal muscles: Psoas, quadriceps femoris, biceps femoris, gluteus maximus. LL distal muscles: Tibialis anterieur, fibularis longus, gastrocnemius, extensor digitorum brevis, flexor hallucis longus, extensor digitorum longus, flexor digitorum longus. Abbreviations: + = Mild; ++ = moderate, +++ = severe; CMTNS = Charcot-Marie-Tooth Neuropathy Score; DTR = deep tendon reflex; UL = upper limbs, LL = lower limbs; MNCV = motor nerve conduction velocity; MRC = Medical Research Council scale; F1: family1; F2: family2.
In family 2 (F2), the four affected sibs were born from consanguineous parents (Fig. 2). Mean age at onset was 10 ± 2 years. All had normal cognitive functions. Initial symptoms were spine deformities and gait instability in all patients. Indeed, instability and disequilibrium on walking were described as the first symptoms by family members. Examination showed that sensory ataxia was the main sign including a Romberg sign, hypopallesthesia, akinesthesia and instability on walking. Steppage gait was not present, even many years after the onset, in almost all the CMT4F patients, although they could not walk on their heels. Kyposcoliosis was also present in all the patients and motor disability was mild in all of them. Indeed, patient 1 (propositus, Fig. 2) had difficulties to run and
to achieve sport at school. At last exam, he was 22 year old and attended university. Patient 2 had the same symptoms and received surgical treatment for kyphoscoliosis. Patient 3 had the same clinical picture with more severe spine and thoracic deformities. Patient 4 a 15 year-old girl, had moderate spine and feet deformities but also presented pyramidal signs and bilateral hypoacousia. Cerebral MRI showed bilateral acoustic neurofibroma and multiple cerebral meningiomas in patient 4 and was normal in the 3 others. In both families, nerve conduction velocities study indicated a demyelinating polyneuropathy with ulnar and median MNCV below 20 m/s, absence of motor peroneal nerve potentials and absence of sensory action potentials in the median and sural nerves. In family 2, median and
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Fig. 1. MTMR2 mutation segregating in family 1 pedigree and patients’ photography.
ulnar MNCV were dramatically decreased (below 10 m/s) and potential amplitudes were below 1 mV. Median, ulnar and peroneal MNCV were in the normal range in parents and assessed unaffected siblings of both families. Electromyography recording, performed only in patients, suggested a chronic neurogenic pattern in the explored muscles. Mean Charcot-Marie-Tooth neuropathy score (CMTNS) was 24 ± 1 in MTMR2 family whereas in PRX family it was 19 ± 0.5. F1P2 and F2P4 had a nerve biopsy before molecular study. In F1P2 (CMT4B1family), electron microscopy study of superficial peroneal nerve biopsy showed a significant loss of myelinated fibers and important myelin inand outfoldings in the majority of large diameter fibers (Fig. 3). There were also numerous demyelination-remyelination lesions with thin myelin sheaths and frequent onion bulb formations. This aspect thus, led us to suspect CMT4B1 and search for MTMR2 gene mutation. In F2P4 (CMT4F family), nerve biopsy study showed severe loss of myelinated and unmyelinated fibers and rare myelin outfoldings. The remaining fibers were surrounded by onion-bulb Schwann cell proliferations (Figs. 4 and 5).
For family 2 (PRX mutated), all loci responsible for CMT were first excluded by linkage analysis, whereas for family 1 (MTMR2 mutated), only CMT2B1 (LMNA gene) was initially excluded, before performing nerve biopsy.In family 1, a homozygous insertion of adenine was detected in MTMR2 exon 4: c.331dupA (p.Arg111LysfsX24) in the three patients. The parents and the paternal grand-mother were heterozygous carriers of this mutation ( Fig. 1). In family 2, The 4 patients harbored a novel homozygous c.1090C>T (p.Arg364X) mutation in PRX exon 7. The father and one healthy brother were heterozygous carriers of the same mutation (Fig. 2). The mother was not available for DNA testing. So, in both families, the segregation of the mutated alleles was consistent with the autosomal recessive inheritance of the disorder. One hundred control chromosomes from 50 unrelated individuals from the Algerian population have also been tested without the identification of any of these two mutations. 4. Discussion We reported on two families with autosomal recessive demyelinating forms of Charcot-Marie-Tooth disease
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Fig. 2. MTMR2 mutation segregating in family 1 pedigree and patients’ photography.
Fig. 3. F1P2. Electron micrograph. Transverse section shows severe axonal loss and onion-bulb Schwann cell proliferations; the myelin sheaths present aberrant myelin in and out-foldings.(Bar = 2 lm).
Fig. 4. F2P4. Electron micrograph. Transverse section shows severe rarefaction of myelinated fibers. One remaining fiber is surrounded by onion-bulb Schwann cell proliferations.
(CMT4B1 and CMT4F) harbouring novel mutations in the MTMR2 and PRX genes, respectively. As we know, consanguinity increases the risk of autosomal recessive
disorders (such as CMT4B1 and CMT4F). In both families, patients are homozygous carriers of a mutation probably inherited from a common ancestor. Affected
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Fig. 5. F2P4. Electron micrograph. Transverse section. This axon (A) is completely demyelinated and surrounded by onion-bulb proliferations of Schwann cells.
family members in both kindreds demonstrated unusual clinical features contrasting with the signs and symptoms observed in previously reported patients. A limited number of clinical reports on patients with CMT4B1, which is characterized by the presence of a great number of myelin outfoldings on nerve biopsy, have been published since the identification of the MTMR2 mutations in 2 Italian and 2 Saudi Arabian inbred families [5]. Thus, only 3 publications reported on 5 CMT4B1 cases with novel mutations [18–20]. All the last reported cases (Table 2) had an early age of onset, as was the case in our patients, and a severe distal motor and sensory neuropathy with proximal lower limbs weakness. Nevertheless, stridor and vocal cord paresis have been observed in only one reported case [18]. These distressing signs have also been reported in some CMT2C and CMT4A patients [21–23]. Moreover, early chest deformities, not mentioned elsewhere, were prominent in our patients. They were
probably the result of breathing difficulties due to severe vocal cord palsy for which laser treatment was considered and scheduled. Although skeletal deformities, including claw hands and chest deformities which add more severity to the clinical picture, were not taken into account in the CMTNS [24], it was indeed severe in the MTMR2 family [23–25]. In contrast to the normal CSF protein found in the only one assessed MTMR2 patient [20], it was markedly elevated in our MTMR2 propositus. The focal myelin outfoldings is not a specific morphologic change, as apart from CMT4B1 and CMT4B2 it has been observed in some other neuropathies, but it could be considered as a hallmark of CMT4B1.This aspect was missing in only one previously reported case, for which only onion bulbs and focal myelin thickening were described [20]. In our patient, this typical aspect of the nerve biopsy helped to orientate molecular study directly towards MTMR2 screening. In contrast to the majority of the previously reported CMT4F patients, which had an early onset of the disease and a Dejerine–Sottas phenotype, our patients with PRX mutations had a relatively late age of onset and a protracted evolution. As we know, age of onset is not easy to determine in chronic neurodegenerative disorders and especially CMT diseases. This has been underlined in a previous CMT study [15]. In this PRX family, age of onset was variable and was noticed earlier in the youngest sibs but still much later than usually mentioned in reported CMT4F cases. These last already described cases are characterized by very early onset, often with delayed motor milestones, but subsequent slow clinical progression [9–14]. The four patients were displaying a moderate sensory ataxia, a mild distal motor disability but prominent spine deformities. Scoliosis or kyphoscoliosis which could appear in CMT patients after one or two decade of evolution is probably caused by a
Table 2 CMT4B1 reported cases. CMT 4 B1 patients
Houlden et al.
Verny et al.
Parman et al.
This study
N° of patients Age at onset (months) Age (years) AR demyelinating sensorymotor PNP Vocal cord palsy/stridor Other cranial nerve palsy Respiratory difficulties
2 13 Case1: 16; case2: 5 Yes
1 Birth 18 Yes
2 Delayed motor milestones Case1: 21; case2: 24 Yes
3 24 Case 1: 15; case2: 11; case 3: 8 Yes
No Dysphagia Yes
No Hypophonia No
Yes Facial paresis Yes
Skeletal deformities
Yes (case 1) Dysarthria (case2) Yes (case 1) No (case 2) No
Pes equinovarus
Nerve biopsy
Myelin outfoldings
Chest deformities, pes equinovarus, claw hands Onion bulbs, myelin outfoldings
MTMR2 mutations
Case 1:G308A (Gly103 glu) Case2: delA324(frameshift)
Onion bulbs, myelin outfoldings G1749A (W583X)
Case1: pescavus, claw hands Case2: pescavus Case1: onion bulbs, focal myelin thickening Case 1: 446 bp insert (insert 47AA) Case 2: 841_844delATCA(frameshift X290)
c.331dupA (p.Arg111LysfsX24)
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motor deficit in the paravertebral muscles. Prominent kyphoscoliosis which used to be considered as a clinical hallmark of CMT4C [17], was the most important disability in this CMT4F family. It was one of the earlier signs and among first motives for seeking medical care in this family. One patient had beneficial spine surgical treatment; the three others were scheduled for medical rehabilitation and /or spine surgical orthopedics. According to the validated CMTNS scores given in Shy and al. study [24], family 2 had a moderate mean CMTNS (19) whereas a severe mean CMTNS (22) was observed in a recently reported PRX family [14], although disease duration was much longer in the later. One patient (F2P4) had bilateral acoustic neurofibroma and multiple cerebral meningiomas. The association of CMT1A and neurofibromatosis type 1 (NF1) has been reported [25] and it was concluded that 2 common autosomal dominant conditions (CMT1 and NF1) can occur in the same individual, not because of an underlying single molecular defect, but rather, secondary to a chance phenomenon. In our case, it could be also a fortuitous association because none of the other sibs had similar abnormalities. These CMT4F patients had nearly a normal life but they required constant care mainly for the spine and feet deformities. Although CMT4F disease is characterized by morphologic changes encompassing loss of axons, focally folded myelin (which are much less numerous than in CMT4B) and formation of typical basal lamina onion bulbs, in our CMT4F patient there were mainly a dramatic loss of myelinated and unmyelinated fibers and some onion bulb Schwann cell proliferation. Consistent with previous data, the new PRX mutation described here is a nonsense mutation, located in exon 7. The majority of previously reported mutations are also localized in exon 7 and lead to a truncated L-periaxin [7–14]. Although unusual phenotype and clinical heterogeneity are typical for ARCMT, these two families had further atypical clinical signs not previously reported, mainly a marked stridor leading to impairment of respiratory function and various chest deformities in CMT4B1 patients, and a late age of onset together with prominent spine deformities in CMT4F patients. Moreover these two families harbored two novel mutations in MTMR2 and PRX genes respectively. In summary, the 2 reported CMT4 families with novel mutations in the MTMR2 and the PRX genes had peculiar clinical phenotypes. The CMT4B1 patients had vocal cord paresis and prominent chest deformities and the CMT4F patients had essentially a late age of onset, a protracted and relatively benign evolution but major kyphoscoliosis. These observations broaden the spectrum of clinical phenotypes associated with these two CMT4 syndromes. Furthermore, the clinical heterogeneity observed in CMT4B1 and CMT4F patients reported so far has been also described in other CMT4 families, especially CMT4H [26], demonstrating a variable functional impairment in CMT4 patients, which may be correlated to mutations of variable severities in a given gene. However, clinical
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