PATIENTS AND METHODS

Patients

The pedigree is depicted in fig 1. Nine affected males were examined. Diagnosis of DSMA was based on neurological examination and supported by electrophysiological and histopathological studies, according to the aforementioned guidelines.⁸ All studies were performed following informed consent.

Eletrophysiological studies

In all affected patients, sensory nerve conduction studies were performed in the median, ulnar, superficial peroneal, and sural nerves. Motor nerve conduction studies were performed in the median, ulnar, and peroneal nerves. Electromyography was performed in the deltoid, vastus lateralis, and anterior tibial muscles.

Muscle and nerve biopsy

A muscle biopsy from the right gastrocnemius was obtained from the proband, IV-36. The material was snap frozen in liquid nitrogen and processed for routine histology and histochemistry studies. A sural nerve biopsy was performed in patient IV-45 for light microscopy and transmission electron microscopy studies.

Genotyping and linkage analysis

DNA was isolated from peripheral blood of informative family members using the “salting out” precipitation method.¹⁶ Before linkage analysis, we excluded spinal bulbar muscular atrophy and X linked Charcot-Marie-Tooth as candidate genes.

Linkage analysis with 48 microsatellite markers from the human chromosome, spaced at approximately 5 cM, were selected from the ABI Prism Linkage Mapping sets (Applied Biosystems, Foster City, CA, USA). Analysis was performed for 30 genotyped family members: 10 affected male patients, 6 normal men, 11 obligate carriers, and 3 normal women (fig 2). These microsatellite markers were amplified by PCR using the protocol recommended by the mapping set protocols. The amplified DNA fragments were separated by electrophoresis in a capillary system containing polymer 4 (POP04) in an ABI310 automated DNA sequencer. The analysis was performed using the software programs GeneScan and Genotyper (Applied Biosystems).

For the fine mapping of the region, based on two informative recombinant events (individuals IV-9 and IV-48, figs 1 and 2), nine microsatellite markers between DXS1216 and DXS1196 were selected.¹⁷ These were amplified by radioative PCR using α³² dCTP in a 10 μl reaction volume. The PCR products were separated by size on a 6.5% polyacrylamide, 7.5 mol/l urea denaturing gel, dried in the system Gel Dryer (Model 583 (Biorad) and exposed to x ray films (Kodak XOmat).

Two-point lod score linkage analysis between a putative disease locus and each marker were calculated, under the assumption of X linked recessive inheritance, with a gene frequency of 0.00001 and equal male and female recombination values. Linkage analysis was performed using the MLINK, ILINK and LINKMAP programs of the LINKAGE package (version 5.1) as implemented in the program FASTLINK.^18,19 The order and the distance between markers on the X chromosome was estimated according to the Marshfield map (http://research.marshfieldclinic.org/genetics/).

RESULTS

Clinical findings and diagnostic tests

The proband, currently aged 26 years, (fig 3) was 2 years old when first ascertained. Clinical history was similar in affected male relatives (table 2). In six of nine patients who were personally examined, age of onset varied from 1 to 10 years and the first detected symptom was foot deformity (pes cavus or varus); gait instability was reported in two other individuals. Subsequently, distal lower limb weakness and atrophy were observed, and finally the hands were affected. Despite the large clinical variability, the disease progression was very slow in all individuals and independent gait was maintained even late in life. No cognitive, pyramidal, or sensitivity impairment was detected and cranial nerves were unaffected. All obligate carriers were clinically normal.

Nerve conduction studies and electromyography were perfomed in all patients. Compound muscle action potential (CMAP) amplitude was reduced in seven patients and motor conduction velocity was slightly reduced in five. Sensory nerve conduction was always normal. Electromyography disclosed chronic denervation in seven patients. In two young individuals (IV-11 and IV-52), neurophysiological evaluation was normal. Serum creatine kinase, tested in two patients (IV-36 and IV-48), was in the normal range. Nerve biopsy, performed in patient IV-45, was normal, while muscle histopathology for the proband showed a neurogenic pattern.

Linkage analysis

Haplotype analysis revealed a conserved disease associated haplotype between markers DXS991 and DXS1106 (fig 2). Two point lod score linkage analysis revealed a maximum lod score of 5.74 at θ = 0.00 with the marker DXS986 (table 3).

Two informative recombination events were identified: one in an unaffected member (IV-9) between markers DXS991 and DXS990, and another in an affected patient (IV-48) between markers DXS991 and DXS986. Further analysis of these two recombinants was performed with 14 microsatellite markers: DXS1216, DXS8111, DXS1275, DXS8031, DXS8107, DXS8052, DXS8046, DXS8060, DXS8092, DXS986, DXS1002, DXS1196, DXS1217, and DXS990. It confirmed the presence of the disease gene in a 4.3 cM region between markers DXS8046 and DXS990 that corresponds to a physical distance of 21.4 Mb on the Marshfield map.

DISCUSSION

Here we describe a new form of X linked distal spinal muscular atrophy in a white Brazilian family. In all affected patients who were examined, the phenotype was similar to CMT, with distal weakness and atrophy in lower limbs, particularly in the tibioperoneal compartment, and presence of pes cavus. At onset, the most constant change was foot deformity, and diagnosis at this stage could be very difficult. Muscle atrophy and weakness in upper limbs, mainly in the hands, occurred later. Disease progression was very slow and variable.

DSMA presenting with the phenotype of peroneal muscular atrophy should be distinguished from the demyelinating and axonal types of CMT disease (CMT1 and CMT2, respectively). Although in DSMA there is no involvement of the sensory fibres, clinical differential diagnosis between CMT1 and CMT2 may be impossible, as about 60% of CMT patients (particularly CMT2) have no clinical sensory loss.² DSMA may be distinguished from CMT1 and CMT2 by the presence of normal sensory conduction velocities and action potentials, and normal findings on sural nerve biopsy.^3,21

In the present family, spinal bulbar muscular atrophy and the X linked form of CMT were ruled out at an early phase of study. Electrophysiological studies revealed a distal neurogenic EMG pattern in seven of nine affected individuals. Two clinically affected individuals who were younger than 10 years old had normal EMG and nerve conduction results. Sensory conduction velocities and potential amplitudes were normal in all patients. Muscle biopsy demonstrated a mixed neurogenic and myogenic pattern while the nerve biopsy of the sural nerve showed no abnormality.

Linkage analysis with microsatellite markers along the X chromosome allowed the mapping of this X linked DSMA * (DSMAX) gene to Xq13.1–q21. A maximum lod score of 5.74 at θ = 0.00 with the marker DXS986 strongly supports linkage of the disease gene to the above region. In addition, recombination haplotype analysis in one normal (IV-9) and one affected (IV-48) individual revealed that the gene is located in a 4.3 cM genetic interval between the markers DXS8046 and DXS990. A systematic search of public available databases (http://research.marshfieldclinic.org/genetics/http://ncbi.nlm.nih.gov/mapview/; and http://www.ensembl.org/) has identified 120 known and 162 predicted genes in this region, several of them expressed in the nervous system. It is of interest that an X linked form of spastic paraplegia has been mapped to the same region.²³ As there are many examples of mutations in a gene resulting in different phenotypes (for example, the caveolin, dysferlin, and lamin A–C genes), the hypothesis that this form of spastic paraplegia and X linked recessive distal spinal muscular atrophy may be allelic cannot be ruled out.

In conclusion, we have mapped a new locus for DSMA at Xq13.1–Xq21. Clinical manifestations start in the first decade of life and progression is slow, with affected individuals remaining ambulatory even when elderly.