Genetic heterogeneity in giant axonal neuropathy: an Algerian family not linked to chromosome 16q24.1

Neuromuscular Disorders 12 (2002) 849–852 www.elsevier.com/locate/nmd

Genetic heterogeneity in giant axonal neuropathy: an Algerian family not linked to chromosome 16q24.1 M. Tazir a,*, J.M. Vallat b, P. Bomont c, R. Zemmouri a, P. Sindou b, S. Assami a, S. Nouioua a, T. Hammadouche d, D. Grid e, M. Koenig c a

Service de Neurologie, C.H.U Mustapha Bacha, Place du 1 er Mai, 16000 Alger, Algeria b Service de Neuropathologie, CHR Limoges, France c Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire 67404 Illkirch cedex, C.U de Strasbourg, France d Laboratoire de Biologie Mole´culaire, Institut Pasteur d’Algerie, Alger, Algeria e Ge´ne´thon III, Evry, France Received 29 May 2001; received in revised form 10 October 2001; accepted 22 January 2002

Abstract Giant axonal neuropathy is a rare severe autosomal recessive childhood disorder affecting both the peripheral nerves and the central nervous system. Peripheral nerves characteristically show giant axonal swellings filled with neurofilaments. The giant axonal neuropathy gene was localised by homozygosity mapping to chromosome 16q24.1 and identified as encoding a novel, ubiquitously expressed cytoskeletal protein named gigaxonin. We describe a consanguineous Algerian family with three affected sibs aged 16, 14 and 12 years who present a mild demyelinating sensory motor neuropathy, hypoacousia and kyphoscoliosis which was moderate in the two elder patients, severe in the third one, with no sign of central nervous system involvement and normal cerebral magnetic resonance imaging. This clinical picture is different from the classical severe form, with kinky hairs and early onset of central nervous system involvement and from the less severe form, with protracted course and late involvement of central nervous system. Nerve biopsy showed a moderate loss of myelinated fibers and several giant axons with thin or absent myelin, filled with neurofilaments. This neuropathological aspect is similar to the previously described families linked to the gigaxonin gene. Genetic study in this family showed absence of linkage to chromosome 16q24.1, indicating for the first time, a genetic heterogeneity in giant axonal neuropathy. We propose to call this form of giant axonal neuropathy giant axonal neuropathy 2, and to use the name of giant axonal neuropathy 1 for the form linked to 16q24.1. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Giant axonal neuropathy; Gigaxonin; Giant axonal neuropathy 1; Giant axonal neuropathy 2

1. Introduction Giant axonal neuropathy (GAN) is a severe autosomal recessive childhood disorder affecting both the peripheral nerves and the central nervous system (CNS), first described in 1972 [1,2]. It is characterised by a progressive motor and sensory neuropathy associated with cerebellar and pyramidal signs, mental retardation and electroencephalographic and magnetic resonance imaging (MRI) abnormalities [3– 6]. Most reported cases presented kinky hairs. Peripheral nerve biopsies show the hallmark of the disease: giant axonal swellings filled with neurofilaments associated with moderate axonal loss. The GAN gene was localised by homozygosity mapping to chromosome 16q24.1 [7–9] * Corresponding author. Tel./fax: 1213-21-235-640. E-mail addresses: [email protected] (M. Tazir), [email protected] (M. Tazir).

and identified as encoding a novel, ubiquitously expressed cytoskeletal protein named gigaxonin [10]. Here, we describe a consanguineous Algerian family with three affected sibs that is not linked to chromosome 16q24.1.

2. Patients and methods 2.1. Clinical evaluation All the members of this family were examined by two of the authors (MT.RZ) The three patients underwent ophthalmological, cardiological, neuropsychological and imaging investigations.

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2.2. Electrophysiological studies

Table 2 Electrophysiological data a

The electrophysiological recordings of the three patients included electroencephalogram, electromyogram (EMG), nerve conduction velocities (NCV) and evoked potentials. Median (elbow-wrist), ulnar (elbow-wrist), peroneal (head of fibula-ankle) MNCV, distal latencies and muscle action potential amplitude were registered with surface electrodes. Sensory action potentials were recorded antidromically from the median nerve and the sural nerve. Needle EMG examination was performed on tibial and first interosseous muscles. Evoked potentials exploration included visual, brainstem auditory evoked response (BAER) and somatosensory evoked potentials (SEP). 2.3. Pathology RH, the oldest patient underwent peroneal superficial nerve biopsy. For analysis of nerve biopsy, fascicles of the superficial peroneal nerve were fixed in formaldehyde (10%) and embedded in paraffin. Serial sections were stained using conventional methods. Other fascicles were fixed in buffered glutaraldehyde, processed and embedded in epon. Semi-thin sections were stained with phenylenediamine and ultrathin sections were stained with uranyl acetate and lead citrate and viewed in a Philips CM10 electron microscope. 2.4. Linkage analysis Blood samples of the patients, parents and one healthy sib (#7) were obtained for diagnosis by linkage analysis. DNA was extracted according to standard procedures. Five polymorphic markers, located within 1 cm from the GAN locus [7,9], were analysed. Marker D16S3098 is located 50 kb away from the GAN gene [10]. Polymerase chain reaction (PCR) reactions were performed using one of the primers labelled with 32P. Annealing temperature for PCR was 28C below the lowest Tm of the two primers [9]. The PCR products were separated on 6% denaturing polyacrylamide gels and visualised by autoradiography. 3. Results 3.1. Clinical and electrophysiological findings The family originated from Algiers suburbs. The healthy

Motor nerves Median

Ulnar

Patients

HR

NR

OR

HR

NR

OR

Amplitude (mV) Distal (ms) Conduction Velocity (m/s)

1.6 12 17.5

1 11 16

1.9 7.5 13.5

2.7 7.8 30

1.5 9.6 17.8

0.7 6 17

a

Abbreviations: mV: millivolts; ms: milliseconds; and m/s: metres per second.

parents were first cousins and had six children. Among them HR, NR and OR were first seen at 4, 10 and 7 years of age. The propositus and oldest patient (HR) had difficulty in walking and running. When first seen in our department at 14, she could not run steadily but could walk alone with a mild steppage gait. Examination showed hypoacousia and signs of distal motor and sensory neuropathy with pes cavus and moderate kyphoscoliosis (Table 1). The second and the third patients (NR and OR, respectively) were asymptomatic until age 10 and 7 when a scoliosis was discovered on chest X-rays after an examination for bronchitis. At age of 12 and 10 years respectively, they presented a mild distal motor and sensory neuropathy, hypoacousia and moderate (NR) and severe (OR) scoliosis. (Table 1). Electrophysiological studies showed markedly decreased median and ulnar motor nerve conduction velocities, absent peroneal motor potential (Table 2), absent sensory nerves potentials (median and sural) and an EMG suggesting a neurogenic pattern. These data were compatible with motor and sensory demyelinating neuropathy, leading to a first diagnosis of hereditary motor and sensory neuropathy type I, (HMSN type I) modified after the nerve biopsy results. Visual evoked potentials were normal, whereas BAER showed an abnormal first wave, compatible with bilateral cochlear nerve lesion and SEP showed a normal central conduction. Electroencephalographic recording was normal in all patients. Cerebrospinal fluid (CSF) studies of the propositus showed moderately elevated proteins (160 mg/100 ml) with normal cell count. Ophthalmological, and cardiological examination were normal and none of the sibs had kinky hairs.

Table 1 Clinical data a Patient Sex

Age of onset (years)

Age at last exam (years)

Quadriareflexia

Distal muscular atrophy

Distal sensory impairment

Foot deformity

Scoliosis

Others signs

HR NR OR

4 10 7

16 14 12

1 1 1

LL LL –

LL LL –

PC PC PC

1 1 1

Hypoacousia Hypoacousia Hypoacousia

a

F F M

Abbreviations: LL: lower limbs; and PC: pes cavus.

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various configurations which push the endoplasmic reticulum and mitochondria toward the periphery near the axolemma. In some myelinated and unmyelinated fibers an increase in filament density may occur without swelling. Moreover, one could observe quite a few lesions of demyelination and remyelination associated with discrete onion bulb proliferation. 3.3. Linkage analysis

Fig. 1. EM microphotograph: at low magnification, ultrastrcutural examination confirms enlarged axon. Close to it onion bulb proliferation around a demyelinated axon is present. (Bar: 2 mm).

Haplotype analysis of the family with markers closely linked to the GAN gene allowed to exclude linkage to this region. Indeed, patient 6 did not share the same paternal haplotype with his two affected sisters, while the unaffected brother 7 shared both haplotypes with the two affected sisters (Fig. 3). In order to exclude sample mix up, blood of individuals 6 and 7 were resampled and tested with marker GAAT2C08, which was informative. The same results were obtained. Since markers LC8 and GAAT2C08 are respectively proximal and distal to the

Cerebral MRI was normal in all of them and they had normal mental status and frequented public school. 3.2. Histopathologic study There is only a slight reduction in the number of myelinated fibers. On semi-thin section several giant axons are seen in all the fascicles examined; the myelin sheaths around them are either absent or unusually thin. These axonal dilatations are segmental. On ultrastructural examination they are constituted of intense proliferation of neurofilaments (12.2 ^ 2.2 nm in diameter) (Figs. 1 and 2) in

Fig. 2. EM microphotograph: at higher magnification neurofilaments that randomly swirl and stream in various configurations through the axoplasm are clearly seen. (Bar: 1 mm).

Fig. 3. Pedigree of the Giant Axonal Neuropathy family and 16q24.1 genotyping results: microsatellite markers are indicated on the left and are shown from top to bottom in their pter-qter ordering. Allele numbering is as in Cavalier et al. [9] and Bomont et al. [10]. Allele 0 of GAAT 2C08 is a new allele, one repeat (GAAT) larger than allele 1. The GAN gene occupies the 110 kb interval between markers LC6 and D16S3098 (assembly of the human genome, August 2001 update, http://genome.ucsc.edu/). Both parents are informative for one marker on each side of the gene. The results exclude the possibility of a recombination between the informative markers and the gene. The haplotypes shared by the two affected girls 4 (HR) and 5 (NR) are boxed. Identical haplotype sharing with the healthy brother 7 (MR), the distinct paternal haplotype of the affected boy 6 (OR) and heterozygosity for three close markers (D16S3098, GAAT2C08 and D16S505) in the patients clearly exclude linkage to the GAN gene.

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GAN gene, this excludes a causative role of this gene in the neuropathy affecting the family. In addition, the patients are heterozygous for three of the five polymorphic markers tested, a result not compatible with linkage given the first degree consanguinity of the parents. 4. Discussion This consanguineous family with three affected siblings and healthy parents is the first GAN family reported not linked to GAN gene on chromosome 16q24. The clinical picture of these three patients is characterised by a mild motor and sensory neuropathy with pes cavus, moderate to severe scoliosis, and hypoacousia without any sign of CNS involvement. This family presented the mildest clinical picture of GAN described so far, with scoliosis as a prominent sign in two patients. Scoliosis is not unusually encountered in HMSN but rarely as presenting or prominent sign, except in demyelinating neuropathies due to mutation at the CMT4C locus [11–13]. Severely decreased nerve conduction velocities and EMG results indicated the presence of a demyelinating motor and sensory neuropathy, whereas the usual presentation found in previous GAN cases was axonal neuropathy. In the cases described by Ben Hamida et al. [14], decreased MNCV were found only in the oldest patients, suggesting secondary demyelination. This clinical and electrophysiological picture is different from the classical severe form with kinky hairs and early onset of CNS involvement [4–6] and from the less severe form with protracted course and late involvement of CNS [14–17]. Families with these two clinical forms of GAN had been linked to the gigaxonin gene [10]. The phenotypic variability can be attributed to different mutations with the two mildest cases being due to missense mutations (R15S and R138H) outside from the conserved BTB and Kelch domains. Nerve biopsy studies in our case showed a moderate loss of myelinated fibers and several giant axons with thin or absent myelin, and filled with neurofilaments. This neuropathological aspect is similar to the previously described families linked to gigaxonin gene. Besides GAN clinical heterogeneity [10] the absence of linkage to the gigaxonin gene of this GAN family clearly demonstrates genetic heterogeneity of this disease. We propose to call this form of giant axonal neuropathy, GAN2, and to use the name of GAN1 for the forms linked to 16q24.1, as suggested by Ben Hamida et al. [7]. The family reported in this study should be instrumental for the identification of the GAN2 locus.

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