SMA Patients Research Articles

N.P.2 03 Correlation between SMN2 copy number variations in expression of SMN2 mRNA and clinical outcome in SMA patients treated with phenylbutyrate and valproic acid

In humans, the survival motor neuron gene (SMN) is duplicated with telomeric copy (SMN1) and centromeric copy (SMN2). The differences between these genes result in alterations in their RNA expression patterns. SMN2 may occur in more than two copies per diploid genome. Studies of correlation between disease severity and the number of SMN2 copies have shown an inverse relationship. It was found, that some drugs can modify the pattern of SMN2 splicing in SMA patients towards increase in full-length SMN2 mRNA and amount of SMN protein.

Therapeutics Development for Spinal Muscular Atrophy

Spinal muscular atrophy is an autosomal recessive motor neuron disease that is the leading inherited cause of infant and early childhood mortality. Spinal muscular atrophy is caused by mutation of the telomeric copy of the survival motor neuron gene (SMN1), but all patients retain a centromeric copy of the gene, SMN2. SMN2 produces reduced amounts of full-length SMN mRNA, and spinal muscular atrophy likely results from insufficient levels of SMN protein in motor neurons. The SMN protein plays a well-established role in assembly of the spliceosome and may also mediate mRNA trafficking in the axon and nerve terminus of neurons. In patients, spinal muscular atrophy disease severity correlates inversely with increased SMN2 gene copy number and, in transgenic mice lacking endogenous SMN, increasing SMN2 gene copy number from two to eight prevents the SMA disease phenotype. These observations suggest that increasing SMN expression levels may be beneficial to SMA patients. Currently pursued therapeutic strategies for SMA include induction of SMN2 gene expression, modulation of splicing of SMN2-derived transcripts, stabilization of SMN protein, neuroprotection of SMN deficit neurons, and SMN1 gene replacement. Early clinical trials of candidate therapeutics are now ongoing in SMA patients. Clinical trials in this disease present a unique set of challenges, including the development of meaningful outcome measures and disease biomarkers.

415. Stimulating Full-Length SMN2 Expression by Delivering Bi-Functional RNAs Via a Viral Vector

Spinal Muscular Atrophy is an autosomal recessive neuromuscular disorder that is the leading genetic cause of infant mortality. SMA is caused by the homozygous absence of Survival Motor Neuron-1 (SMN1). In humans, a nearly identical copy gene is present called SMN2. SMN2 is retained in all SMA patients and encodes an identical protein compared to SMN1, however, SMN1 and SMN2 differ by a silent C to T transition at the 5' end of exon 7. This single nucleotide difference results in the production of an alternatively spliced isoform called SMN-delta7 that encodes an unstable and non-functional protein. Even though the majority of SMN2-derived transcripts lack exon 7, the presence of SMN2 represents a unique therapeutic target because SMN2 has the capacity to encode a fully functional protein. Here we describe an in vivo delivery system for short bi-functional RNAs that modulate SMN2 splicing. Bi-functional RNAs derive their name due to the presence of two functional domains: an RNA sequence that is complimentary to a specific cellular RNA (e.g. SMN exon 7); and an untethered RNA segment that serves as a sequence-specific binding platform for cellular splicing factors, such as SR proteins. Plasmid-based and recombinant adeno-associated virus vectors were developed that expressed bi-functional RNAs that stimulated SMN2 exon 7 inclusion and full-length SMN protein in patient fibroblasts. These experiments provide a mechanism to modulate splicing from a variety of genetic contexts and directly demonstrate a novel therapeutic approach for SMA.

Genotype-Phenotype correlation of SMN locus genes in spinal muscular atrophy patients from India

Spinal muscular atrophy has been classified into four groups based on the age of onset and clinical severity of the disease. Homozygous deletion in SMN1 gene causes the disease but the clinical severity may be modified by copy number of homologous gene SMN2 as well as the extent of deletion at SMN locus. In the view of scarcity of genotype and phenotype correlation data from India, this study has been undertaken to determine that correlation in SMA patients by using the SMN and NAIP genes and two polymorphic markers C212 and C272 located in this region. Two to four alleles of the markers C212 and C272 were observed in normal individuals. However, majority of Type I patients showed only one allele from both markers whereas in Type II and III patients, 2-3 alleles were observed. The SMN2 copy number in our type III patients showed that patients carry 3-5 copies of SMN2 gene. Our results suggest that extent of deletions encompassing H4F5, SMN1, NAIP and copy number of SMN2 gene can modify the SMA phenotype, thus accounting for the different clinical subtypes of the disease.

Study of Survival of Motor Neuron (SMN) and Neuronal Apoptosis Inhibitory Protein (NAIP) gene deletions in SMA patients

In view of the paucity of deletion studies of survival of motor neuron (SMN) and neuronal apoptosis inhibitor protein (NAIP) genes in Indian SMA patients, this study has been undertaken to determine the status of SMN1, SMN2 and NAIP gene deletions in Indian SMA patients. Clinically and neurophysiologically diagnosed SMA patients were included in the study. A gene deletion study was carried out in 45 proximal SMA patients and 50 controls of the same ethnic group. Both SMN1 and NAIP genes showed homozygous absence in 76% and 31% respectively in proximal SMA patients. It is proposed that the lower deletion frequency of SMN1 gene in Indian patients may be due to mutations present in other genes or population variation, which need further study.

Indoprofen Upregulates the Survival Motor Neuron Protein through a Cyclooxygenase-Independent Mechanism

Most patients with the pediatric neurodegenerative disease spinal muscular atrophy have a homozygous deletion of the survival motor neuron 1 (SMN1) gene, but retain one or more copies of the closely related SMN2 gene. The SMN2 gene encodes the same protein (SMN) but produces it at a low efficiency compared with the SMN1 gene. We performed a high-throughput screen of approximately 47,000 compounds to identify those that increase production of an SMN2-luciferase reporter protein, but not an SMN1-luciferase reporter protein. Indoprofen, a nonsteroidal anti-inflammatory drug (NSAID) and cyclooxygenase (COX) inhibitor, selectively increased SMN2-luciferase reporter protein and endogenous SMN protein and caused a 5-fold increase in the number of nuclear gems in fibroblasts from SMA patients. No other NSAIDs or COX inhibitors tested exhibited this activity.

Detection of novel mutations in the SMN Tudor domain in type I SMA patients.

The authors present a complete SMN gene analysis in four type I unrelated spinal muscular atrophy patients who retained one copy of the SMN1 gene. Two intragenic point mutations were identified in exon 3 (I116F, Q136E), affecting a very conserved region with the Tudor domain of SMN1. The remaining two patients showed no alterations in the SMN1 coding sequences although a transcription defect was detected in one of them, corroborating the existence of non-functional SMN1 genes.

Surgical treatment of scoliosis in spinal muscular atrophy

To describe the early and late postoperative data from SMA patients with surgical procedure. Clinical data and radiographic imaging from 14 SMA patients with surgical treatment of scoliosis were reviewed, and all were reassessed clinically with new spinal radiographs and a questionnaire. The mean follow-up were 22 months. The mean preoperative Cobb angle was 78.4 . All patients presented pelvic obliquity (mean 25.5 ) and 11 had cifosis. The mean age at time of surgery was 12 years and 3 months. All patients were treated with posterior spinal fusion with Luque-Galveston instrumentation in 12 and Cotrel-Dubousset instrumentation in 2. The average curve correction at the immediate postoperative was 64.3% for scoliosis and 36.4% for cifosis, with the pelvic obliquity correction of the 70.9%. The complications were liquoric fistula and infection earlier in one case, and lately wire looseness of T1 in 2 patients. It was detected mean lost of the correction at the final assessment of the 0.26 of the scoliosis and the 1.28 of the pelvic obliquity. Relatives and the patients related good improvement regarding to esthetic aspects, posture balance, body care, as well as respiratory problems. The spinal fusion for scoliosis in SMA patients has a satisfactory impact for esthetic, quality of life and respiratory function, with minimal lost of corrected deformities and few complications.

Spinal muscular atrophy type 1: management and outcomes.

Our objectives were to describe survival, hospitalization, speech, and outcomes related to respirator needs for spinal muscular atrophy type 1 (SMA1) patients, using noninvasive or tracheostomy ventilation. From 65 SMA patients referred to our clinic since 1996, we chose 56 SMA1 patients who developed respiratory failure before age 2 years. Patients either had tracheostomy tubes (group A), or used noninvasive ventilation and assisted coughing; a previously reported extubation protocol (group B) was used as needed. Sixteen patients underwent tracheostomy at 10.8 +/- 5.0 months of age, 33 were in group B, and 7 others died without life-support interventions. Compared to group B, group A patients had fewer hospitalizations until age 3 years, but more after age 5, and 15 of 16 lost all spontaneous breathing tolerance posttracheostomy and could not speak. One group A patient died at 16 months of age, and the others were 73.8 +/- 57 months of age (the oldest was 19 years old). Two group B patients died at 6 and 13 months, respectively, whereas the other 31 were 41.8 +/- 26.0 months (and up to 8.3 years) old. Three of 31 in group B required high-span positive inspiratory pressure plus positive end-expiratory pressure (PIP + PEEP) continuously with minimal tolerance for breathing on their own, and 4 could not communicate verbally.In conclusion, SMA type 1 children can survive beyond 2 years of age when offered tracheostomy or noninvasive respiratory support. The latter is associated with fewer hospitalizations after age 5 years, freedom from daytime ventilator use, and the ability to speak.

SMA carrier testing--validation of hemizygous SMN exon 7 deletion test for the identification of proximal spinal muscular atrophy carriers and patients with a single allele deletion.

To facilitate the detection of carriers of a hemizygous survival motor neuron (SMN) exon 7 deletion we have modified the quantitative SMN exon 7 assay described by McAndrew et al (1997). The major changes include quantitative analysis of the amount of SMN exon 7-specific fluorescently-labelled PCR product on an automated sequencer, and the monitoring of the completeness of a DraI digestion necessary to distinguish the PCR products of exons 7 of SMN and its copy gene. In our method the amount of SMN exon 7 PCR product is compared with the amount of a co-amplified PCR product of the retinoblastoma (RB1) exon containing a DraI restriction site. By co-amplification using the same primers of plasmids included in the reaction as internal standards containing SMN exon 7 with a 36-nucleotide deletion and RB1 exon 13 with a 19-nucleotide deletion, respectively, the relative amplification efficacy can be monitored. The assay has been validated in 63 ascertained carriers and 28 ascertained non-carriers. The sensitivity of the test is approximately 97%, the specificity approaches 100%. In four out of six SMA patients without a homozygous deletion we detected a hemizygous deletion. The implications of the use of this assay for carrier testing and for confirmation of the clinical diagnosis of SMA are discussed.

Detection of a de novo mutation in a family with SMA Type I: The importance of dosage testing

Type I spinal muscular atrophy is a devastating neurodegenerative disease with onset by 6 months of age and death by the age of two years, usually due to respiratory infection. Homozygous deletions of the SMNtel, but not the SMNcen, gene have been identified in 95% of SMA patients. These genes map to chromosomes 5p13 and are within a large inverted repeat which includes the NAIP gene, the telomeric copy of which is also deleted in a large proportion of SMA type I patients.We have investigated a family, in which there had been an affected child who was homozygously deleted for SMNtel and NAIP exon 5, to determine the disease status of a fetus. Analysis of cultured amniocytes indicated that the fetus is not homozygously deleted for SMNtel or NAIP. Several markers in the SMN/NAIP region were used to rule out maternal contamination of the amniocytes. During this process, it was noted that the fetus and the affected child had similar haplotypes but the paternal chromosome in the affected child carried a recombination. This recombination is associated with a deletion of several alleles of two multiallelic markers, AG1 and CATT. Dosage analysis revealed that the mother is an SMA earner with only one copy of SMNtel. In contrast, the father has 2 copies of SMNtel, suggesting that he is not a carrier of an SMNtel deletion. Rather, the paternal allele in the affected child probably resulted from a recombination event during which there was simultaneous deletion of the SMNtel gene along with some AG1 and CATT alleles. These data significantly lower the SMA recurrence risk for this family. This case highlights the necessity of using dosage information when assessing recurrence risk in SMA families.

Kinematic analysis of patients with spinal muscular atrophy during spontaneous breathing and mechanical ventilation.

Patients with infantile spinal muscular atrophy develop pectus excavatum along with a severe restrictive syndrome as a result of failure to expand the upper chest wall and lungs because of intercostal muscle weakness. By using an automatic motion analyzer to provide three-dimensional computer analyses of images sampled at 25 Hz, 9 spinal muscular atrophy Type II patients and 13 controls underwent kinematic analysis of thoracoabdominal movements partitioned into the upper thorax, lower thorax, and abdominal volume compartments. The analyses were performed during spontaneous breathing for the controls and during spontaneous breathing and while using mechanically assisted ventilation for the patients. Vital capacity, maximum inspiratory pressures, and nocturnal oxyhemoglobin saturation and transcutaneous carbon dioxide tensions were also measured for the patients. The kinematic data demonstrated a paradoxical ventilatory pattern for the spontaneously breathing SMA patients with the following inspiratory volume changes: upper thorax, -6.4+/-9.6%; lower thorax, 7.3+/-15.8%; abdominal, 99.1+/-21.3%. During mechanical ventilation, the compartmental volume changes were as follows: upper thorax, 13.5+/-6%; lower thorax, 13.7+/-7.9%; abdominal, 72.7+/-9.3%. This kinematic pattern is comparable with that seen in spontaneously breathing normal subjects. We conclude that mechanical ventilation can normalize kinematic volume changes during alveolar ventilation and that this might help deter loss of thoracic compliance caused by the chronic hypoventilation of the upper thoracic compartments. Kinematic analysis may be helpful for choosing the ventilation parameters to optimize therapeutic benefits.

A possible role of NAIP gene deletions in sex-related spinal muscular atrophy phenotype variation.

Childhood SMAs are common neuromuscular disorders, due to the occurrence of large genomic deletions encompassing the SMN gene and often extending to involve the NAIP gene. Although NAIP deletions are more frequently observed in patients affected by the acute form of the disease, it is not possible to establish an unambiguous correlation between deletion size and clinical severity. We have investigated the effects of gender on the association between NAIP gene deletion and disease severity. NAIP deletions were screened in 197 Italian SMA patients lacking SMN; the results obtained were correlated with disease severity in male and female samples separately. No significant relationship between deletion size and clinical phenotype was observed among male subjects, whereas in females the absence of NAIP was strongly associated with a severe phenotype (p <0.0001). These results provide a possible molecular explanation for the sex-dependent phenotype variation observed in SMA patients.

A multicopy transcription-repair gene, BTF2p44, maps to the SMA region and demonstrates SMA associated deletions.

The childhood-onset spinal muscular atrophies are a clinically heterogeneous group of autosomal recessive disorders characterized by selective degeneration of the anterior horn cells with subsequent weakness and atrophy of limb muscles. The disease locus has been mapped to a region of chromosome 5q13 characterized by genetic instability and DNA duplication. Among the duplicated genes in this region, SMNT (telomeric copy; survival motor neuron) is thought to be the major disease determining gene since it is missing in the majority of SMA patients and since small, intragenic mutations in the gene have been associated with the disorder. Approximately half of the severely affected SMA I patients are also missing both homologues of a neighboring gene, the neuronal apoptosis inhibitory protein (NAIP). These data indicate that loss of NAIP may affect disease severity and further, that the molecular events underlying the childhood-onset SMAs are complex, possibly involving multiple genes. We report a third multicopy gene in the SMA region, encoding the p44 subunit of basal transcription factor II (BTF2p44). One copy of this transcription-repair gene is deleted in at least 15% of all SMA cases.

Evidence for compound heterozygosity causing mild and severe forms of autosomal recessive spinal muscular atrophy.

Spinal muscular atrophy is an autosomal recessive disease of motor neurone degeneration which shows a variable phenotype. Two candidate genes show deletions in affected subjects but with no distinction between...

Unusual molecular findings in autosomal recessive spinal muscular atrophy.

All three types of autosomal recessive spinal muscular atrophy map to chromosome 5q11.2-q13.3 and are associated with deletions or mutations of the SMN (survival motor neurone) gene. The availability of a test to distinguish between the SMN gene and its nearly identical centromeric copy cBCD541 allows molecular diagnosis. We have analysed patients from 24 Belgian and 34 Turkish families for the presence or absence of a deletion in the SMN gene. A homozygous deletion in the SMN gene was seen in 90% of unrelated SMA patients. A non-radioactive SSCP assay allows for a semiquantitative analysis of the copy number of the centromeric and SMN genes. Hence, direct carrier detection has become feasible under certain conditions. We observed a phenotypically normal male, father of an SMA type I patient, presenting with only a single copy of the SMN gene and lacking both copies of the cBCD541 gene. This illustrates that a reduction of the total number of SMN and cBCD541 genes to a single SMN copy is compatible with normal life. In another SMA type I family, there is evidence for a de novo deletion of the centromeric gene in a normal sib. This observation illustrates the susceptibility of the SMA locus to de novo deletions and rearrangements.

Electrocardiographic findings in children with spinal muscular atrophy.

Most of the patients in this study with spinal muscular atrophy were found to have tremors of the isoelectric line in the electrocardiogram (ECG) tracings. There were a total of 47 cases of SMA (mean age 40.8 months). All three types of SMA had a similar incidence (about 80%) of tremors in the tracings (p = 0.885). In 7 cases the ECG tremors had an intermittent pattern. ECG tremors were commonly found in the majority of SMA patients and this finding, though non-specific, may suggest a possible SMA diagnosis.

Possible strategies for treatment of SMA patients: A neurobiologist's view

This paper discusses possible strategies that might prevent or alleviate muscle weakness of SMA patients and hence improve their condition. The strategies discussed are as follows. (1) Prevention of motoneurone death. To achieve this two main approaches have been applied. Firstly, trophic factors have been used to prevent motoneurone death after nerve injury and clinically in diseases such as motoneurone disease. The results of these attempts will be described. Secondly, the possibility that injured motoneurones die as a result of the excitotoxic effects of the excitatory transmitter glutamate will be explored. Evidence will be presented which indicates that blocking glutamate receptors can rescue injured motoneurones from death. (2) Replacement of lost motoneurones by embryonic grafts. Motoneurones from grafts of embryonic spinal cord have been shown to survive in the adult spinal cord and are able to reinnervate skeletal muscles. The potential and practical problems of this approach will be discussed. (3) Expansion or motor unit territory of surviving motoneurones. Such an expansion of the territory occupied by individual motor units can be achieved by encouraging sprouting and ensuring that the newly formed connections between the motoneurone and muscle fibres are maintained, so that individual motor units are capable of developing more force. Strategies to achieve such an expansion of motor unit territory will be described. Finally, combinations of some of these approaches are considered.

Survival Motor-Neuron Gene Transcript Analysis in Muscles from Spinal Muscular-Atrophy Patients

We have identified and characterized four different mRNA isoforms of the survival motor neuron (SMN) gene from skeletal muscle of 9 SMA patients and 15 unaffected controls. These isoforms appear to be generated by combinatorial splicing of both exons 5 and 7 of the SMN telomeric and centromeric gene copies. The full-size and the truncated SMN-1b isoforms of the telomeric SMN copy are significantly reduced in muscle of SMA patients, irrespective of the disease types. Our results suggest that multiple RNA splicing is operative in the two SMN-related genes and that SMN-related polypeptides may be active in the muscle.

Deletions in the survival motor neuron gene on 5q13 in autosomal recessive spinal muscular atrophy.

Autosomal recessive spinal muscular atrophy is a motor neuron disease which affects about 1 in 10,000 births. Recent evidence shows that the candidate region contains multiple copies of genes and pseudogenes and is characterised by genome instability. We have analysed the frequency of deletions in a recently characterised candidate survival motor neuron (SMN) gene. Our data confirm previous analyses and show that this gene is disrupted by deletion in SMA patients. The same deletion frequency is observed in the milder variants of the disease as in patients with the severe form. In addition, we observed one case of a new mutation in a family previously thought not to be segregating for a chromosome 5 linked form of SMA. This assay is a very good diagnostic for SMA although no direct correlation between phenotype and genotype is apparent and carrier status cannot be determined. The implications for the identification of the gene or genes causing the disease are discussed.