Spinal muscular atrophy

Background & objectives: Distal upper limb spinal muscular atrophy (SMA) is an uncommon segmental variant of SMA. The condition is usually sporadic, affects males more often than females, and manifests late in the second decade of life, remaining confined to the upper limbs. We examined four patients with this form of SMA in order to determine if they carried homozygous deletion mutations in the survival motor neuron (SMN) or neuronal apoptosis inhibitory protein (NAIP) genes that underlie proximal SMA.

Methods: The four patients with distal upper limb SMA were analysed clinically, electrophysiologically and biochemically. Genomic DNA from each of the patients was analysed by restriction enzyme digestion of polymerase chain reaction (PCR) amplification products, as well single stranded conformation polymorphism (SSCP), to detect deletion events of selected exons of the SMN and NAIP genes.

Results: The clinical phenotype of the four patients, together with the biochemical and electrophysiological studies, confirmed a diagnosis of distal upper limb SMA. The molecular studies excluded homozygous deletion mutations in these patients as causative of their phenotype.

Interpretation & conclusion: The genetic component underlying distal upper limb SMA appears not to involve mutations that are common in proximal SMA patients. It is possible that genes other than SMN and NAIP may be involved, while somatic mosaicism of SMN gene mutations could be implicated in the segmental nature of distal upper limb SMA.

Key words Distal upper limb SMA - NAIP gene - SMN gene - spinal monomelic amyotrophy

Distal upper limb SMA is a disorder of the anterior horn cells which affects mainly the distal portions of one or both upper limbs-4. This disorder is sporadic and affects adolescents and young adults. The majority of patients are between the ages of 18 and 22 yr and there is a very strong unexplained male preponderance 3.4 Early reports of the condition came from India and Japan, but it has been described in young people from many other parts of the world as well1-11 the largest series comprises 71 patients 4. Familial forms are uncommon and anecdotal family reports are documented in the literature 12,13.

Clinically, the patients begin with wasting and weakness of one hand and forearm, which then affects the other side, usually asymmetrically. Rarely, the more proximal muscles are affected later in the course of the disease with progression usually ceasing within a year or two and then remaining static. Nerve conduction studies are usually normal, while motor unit potentials are large and polyphasic, suggesting denervation and reinnervation3,4. As the process is distal, muscle biopsies are rarely obtained. Computed tomography (CT) and magnetic resonance imaging (MRI) scans of the cervical spinal cord often show atrophy of the cord with flattening of the anterior areas leading to the classification by some authors of monomelic amyotrophy14-16. However, others have shown forward displacement of the cervical dural sac and compressive flattening of the lower cervical cord during neck flexion17,18. These observations have been taken to support the hypothesis that the disorder is a type of cervical myelopathy. However, the mechanism underlying the pathophysiology remains unclear.

In terms of the molecular studies that have been performed on SMA patients, most work has concerned proximal SMA. Three forms of this disorder (SMA types 1, 11 and 111) map to chromosome 5q 11.2-13.3. Two candidate genes have been reported in this region. The first, designated SMN, exists as two nearly identical copies: a telomeric (SMNtel) and a centromeric (SMNcen) copy that differ by only two base pairs, one in exon 7 and the other in exon 819,20. The two genes can be discriminated by a simple PCR assay involving SSCP analysis or enzymatic digestion of the amplification products19.21. The lack of amplification of exon 7, or exons 7 and 8, of the SMN gene (SMNtel) has been found in > 90 per cent of all proximal SMA patients19. In the case of the NAIP gene, partial or complete homozygous deletion of exons 5 and 6 has been found in 45 per cent of SMA type I patients, and 18 per cent of SMA type II/III patients22. A variable number of partially deleted forms of the NAIP gene also map to Sq 13.1 and it has been suggested that the deletion of the NAIP gene plays a role in the severity of symptoms in SMA patients, though this association is not clear22. Gene conversion events have been found in SMA types II and III which lead to fewer SMNtel copies compared with the more severe type I patients thus supporting a genotype/phenotype correlation23. These events, as well as point mutations that have been found in the SMNtel gene in a few SMA patients24, underscores the important role that the SMNtel gene plays in the disease. Given the lack of molecular genetic studies of distal SMA patients, we report here the clinical and molecular analysis of four patients with this rarer form of SMA in order to exclude deletion mutations in the SMN and NAIP genes.

Material & Methods

Clinical evaluation: We saw 23 patients with apparent SMA in the period January 1987 to January 1997 at the neuromuscular clinic of the Muscular Dystrophy Society of Bombay, Mumbai, India. The patients underwent detailed history and clinical examination, routine haemogram, serum creatine kinase (CK) estimation, electrophysiological studies, magnetic resonance imaging when indicated, and genetic studies. Electrophysiology was performed using the Medlec system and standard cutaneous electrode placements. The patients were divided into four sub-categories of SMA according to their phenotype. Eleven were considered to be autosomal recessive SMA types 1/11/II/III; six had distal upper limb SMA, four had adult onset proximal SMA; and two patients had adult onset distal SMA. We excluded two of the six distal upper limb SMA patients as the duration of their illness was a few months only and one could not be sure about the spread of the weakness. All the four patients included in the study had the disease for more than five years and the disease had stopped progressing well before the study was conducted.

DNA based testing.for SMA: DNA was extracted from whole blood using the Puregene DNA extraction kit (Gentra Systems Inc.). All the DNA samples were adjusted to a concentration of 0.5mg/ ml. Exon 7 of the SMN gene was amplified with the intron 6 primer RI 11 (5' AGA CTA TCA ACT TAA TTT CTG ATC A 3') and a mismatch primer X7-Dra (5' CCT TCC TTC TTT TTG ATT TTG TTT 3)21 . Amplification involved 35 cycles of one minute at 94 deg C, one minute at 55 deg C, and one minute at 72 deg C using Taq DNA polymerase (Gibco BRL) in a reaction buffer recommended by the manufacturer. PCR products were subsequently digested to completeness with Dral. Exon 8 of the SMN gene was amplified with primers 541C 960 (5' GTA ATA ACC AAA TGC AAT GTG AA3') and 54 1C 1120 (5' CTA CAA CAC CCT TCT CAC AG3')21. The same PCR conditions were used as those for exon 7, except for an annealing temperature of 59 deg C instead of 55 deg C. PCR products were subsequently digested with Ddel. Electrophoresis was carried out in 2.5 per cent agarose gels that were subsequently stained with ethidium bromide and fragments visualised under UV light. Exon 5 deletions in the NAIP gene, and SSCP analysis of exons 7 and 8 of the SMN gene, were undertaken using previously published protocols 19,25.

Results

Of the six patients with distal upper limb SMA, we selected four patients for this study (summarised in the Table). The other two patients were not included as the duration of the disease process was only a few months and hence it was difficult to be certain about the segmental non progressive nature of the condition. The age at onset was between 16 and 18 yr and the duration of symptoms was between 5-7.3 yr. The clinical presentation of these patients involved weakness and wasting of the upper limbs only, with the distal muscles being weaker. The weakness involved the intrinsic muscles of the hands and the forearms, which tended to be asymmetrical, the proximal shoulder and arm muscles were not affected. Serum creatine kinase levels were normal or mildly elevated (112-263 IU) and were lower with longer duration of symptoms. Electrophysiological examination showed evidence of chronic partial denervation and reinnervation only in the upper limb segments. Cervical spine magnetic resonance images (MRIs) showed flattening of the anterior area of the spinal cord in keeping with the anterior horn cell loss. These studies confirmed the diagnosis of distal upper limb SMA.

DNA samples from these four patients were analysed to determine if they had homozygous deletions in exons 7 and 8 of the SMN gene. Restriction enzyme digestion and SSCP analysis both showed that all four distal SMA patients carried no homozygous deletion of these exons (Figs I and 2). The analysis of exon 5 of the NAIP gene also showed no homozygous deletions in the four patients (data not presented). These data exclude the most common mutations found in proximal SMA patients as causative of the pathology found in the four patients with distal upper limb SMA.

Discussion

The DNA based analysis of these patients involved the screening for deletions in two genes, SMNteI and NAIP, which have been found to be responsible for proximal SMA. No homozygous deletions of exons 7 and 8 of the SMNtel gene were found in the four patients, while no deletions in exon 5 of the NAIP gene were detected. This latter observation is not surprising given that deletions in the NAIP gene appear to correlate with the most severe form of SMA, namely type I, with disease onset in utero or within the first few months of life22.

DiDonato et al26 have reported that 15 per cent of SMA types II/III patients had lost SMNtel exon 7, but not exon 8. Further analysis of these patients showed this observation to be a consequence of a gene conversion event in which a portion of the SMNtel gene was replaced by a portion of the SMNcen gene.These workers suggested that gene conversion is a common event in proximal SMA and is associated with a milder form of the disease. In our study gene dosage was not studied due to the difficulty in quantitating the number of exon copies of SMNcen and SMNtel, however, all the distal upper limb SMA patients carried both exons 7 and 8 of the SMNtel and SMNcen genes.

The exclusion of the most common mutation events in the known candidate genes for SMA in the four patients we have studied leaves unanswered the question as to the genetic component that may underlie this disorder. The clinical presentation of these patients has features that can be found in segmental neurofibromatosis (NF)27 and arrested SMA29. In terms of the former, distal upper limb SMA may reflect somatic mutation in the SMNtel gene, but only in the cervical spinal cord, hence giving rise to a segmental manifestation. The disorder may also.be related to that of arrested SMA, where SMA disease progression ceases. The apparent sporadic nature of distal upper limb SMA, and a lack of family history precludes simple linkage studies to locate the disease-causing gene. Further studies of affected individuals would therefore benefit more from a candidate gene approach, while the possible role of modifier genes, other than the SMNcen gene, could be explored.

Acknowledgment

The authors acknowledge the support of the Muscular Dystrophy Society of Bombay, and the financial support of LabServices of Auckland Hospital. NGL was supported by the Australian National Health and Medical Research Council.

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Madhuri R. Hegde*+$, Belinda Chong*, Catherine Stevenson**, Nigel G. Laing**, Satish Khadilkar++ & Donald R. Love *+

*Molecular Genetics Laboratory, LabPlus, Auckland Hospital, Auckland, New Zealand, +Molecular Genetics & Development Group, School of Biological Sciences, University of Auckland, Auckland, , New Zealand, **Department of Neuropathology, Royal Perth Hospital, Perth, Western Australia, Australia & ++MRC Wing, Bombay Hospital, Mumbai, India

Received July 27, 2001

$ Present address : Diagnostic Sequencing Laboratory, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA

Reprint requests: Dr Donald R. Lowe, School of Biological Sciences, University of Auckland, Private Bag 92019 Aukland, New Zealand

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