SMN protein analysis in fibroblast, amniocyte and CVS cultures from spinal muscular atrophy patients and its relevance for diagnosis

Patrizi, Anna Letizia; Tiziano, Francesco Danilo; Zappata, Stefania; Donati, Maria Alice; Neri, Giovanni; Brahe, Christina

doi:10.1038/sj.ejhg.5200286

Cited by 47 publications

(42 citation statements)

References 26 publications

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“…Levels of FL-SMN in the type I sample were lower than levels from the type II and III samples, in agreement with previous studies. [29][30][31][32] Control samples (with two SMN1 and two SMN2 copies) showed an FL/D7 ratio of around 6-9, whereas SMA samples showed values ranging from 1.13 to 2.6. Interestingly, in the SMA patients, we found that lymphoblast ratios were slightly higher than those for fibroblasts.…”

Section: Discussionmentioning

confidence: 99%

Treatment of spinal muscular atrophy cells with drugs that upregulate SMN expression reveals inter- and intra-patient variability

et al. 2011

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Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by mutations in the SMN1 gene. The homologous copy (SMN2) is always present in SMA patients. SMN1 gene transcripts are usually full-length (FL), but exon 7 is spliced out in a high proportion of SMN2 transcripts (delta7) (D7). Advances in drug therapy for SMA have shown that an increase in SMN mRNA and protein levels can be achieved in vitro. We performed a systematic analysis of SMN expression in primary fibroblasts and EBV-transformed lymphoblasts from seven SMA patients with varying clinical severity and different SMN1 genotypes to determine expression differences in two accessible tissues (skin and blood). The basal expression of SMN mRNA FL and D7 in fibroblasts and lymphoblasts was analyzed by quantitative real-time PCR. The FL-SMN and FL/D7 SMN ratios were higher in control cells than in patients. Furthermore, we investigated the response of these cell lines to hydroxyurea, valproate and phenylbutyrate, drugs previously reported to upregulate SMN2. The response to treatments with these compounds was heterogeneous. We found both intra-patient and inter-patient variability even within haploidentical siblings, suggesting that tissue and individual factors may affect the response to these compounds. To optimize the stratification of patients in clinical trials, in vitro studies should be performed before enrolment so as to define each patient as a responder or non-responder to the compound under investigation.

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Section: Discussionmentioning

confidence: 99%

Treatment of spinal muscular atrophy cells with drugs that upregulate SMN expression reveals inter- and intra-patient variability

et al. 2011

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“…In fact, SMN protein levels are reduced in spinal cord and cell cultures from SMA patients, and correlate inversely with phenotypic severity. [7][8][9] SMN2 copy number can also vary, and patients with high copy number often have a milder phenotype. [10][11][12] At present, there is no effective treatment for SMA.…”

Section: Introductionmentioning

confidence: 99%

Clinical and molecular cross-sectional study of a cohort of adult type III spinal muscular atrophy patients: clues from a biomarker study

Tiziano¹,

Lomastro²,

Pietro³

et al. 2012

Eur J Hum Genet

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Proximal spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations of the SMN1 gene. Based on severity, three forms of SMA are recognized (types I-III). All patients usually have 2-4 copies of a highly homologous gene (SMN2), which produces insufficient levels of functional survival motor neuron (SMN) protein due to the alternative splicing of exon 7. The availability of potential candidates to the treatment of SMA has raised a number of issues, including the availability of biomarkers. This study was aimed at evaluating whether the quantification of SMN2 products in peripheral blood is a suitable biomarker for SMA. Forty-five adult type III patients were evaluated by Manual Muscle Testing, North Star Ambulatory Assessment scale, 6-min walk test, myometry, forced vital capacity, and dual X-ray absorptiometry. Molecular assessments included SMN2 copy number, levels of full-length SMN2 (SMN2-fl) transcripts and those lacking exon 7 and SMN protein. Clinical outcome measures strongly correlated to each other. Lean body mass correlated inversely with years from diagnosis and with several aspects of motor performance. SMN2 copy number and SMN protein levels were not associated with motor performance or transcript levels. SMN2-fl levels correlated with motor performance in ambulant patients. Our results indicate that SMN2-fl levels correlate with motor performance only in patients preserving higher levels of motor function, whereas motor performance was strongly influenced by disease duration and lean body mass. If not taken into account, the confounding effect of disease duration may impair the identification of potential SMA biomarkers.

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“…RNA concentrations were determined using the GeneQuant II spectrophotometer (Pharmacia) and 2 mg RNA were used for first-strand cDNA synthesis, as described elsewhere. 11 Real time RT-PCR was performed using the ABI PRISM 7700 Sequence Detector System (Applied Biosystems, Foster City, CA, USA). SMN fl mRNA was amplified using a forward primer in SMN exon 6 (5 0 -GCT GAT GCT TTG GGA AGT ATG TTA-3 0 ) and a reverse primer in SMN exon 7 (5 0 -CAC CTT CCT TCT TTT TGA TTT TGT C-3 0 ); the sequence of the Taqman probe, localized in exon 6, was 5 0 FAM-TTT CAT GGT ACA TGA GTG GCT ATC ATA CTG GCT ATT AT-TAMRA 3 0 .…”

Section: Methodsmentioning

confidence: 99%

“…11 The antibodies were anti-SMN (dilution 1/5000, Transduction Laboratories), anti-b-tubulin (dilution 1/12 000) and anti-actin polyclonal antibody (dilution 1/2000, SigmaAldrich), anti-mouse horseradish peroxidase-conjugated (dilution 1/2000) and anti-rabbit horseradish peroxidaseconjugated (dilution 1/2000, Amersham Pharmacia Biosciences). Western blot quantification was performed as described; 11 SMN -actin -tubulin ratios were determined and normalized to untreated samples.…”

Section: Western Blot Analysismentioning

confidence: 99%

Phenylbutyrate increases SMN expression in vitro: relevance for treatment of spinal muscular atrophy

et al. 2003

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Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease, characterized by degeneration of the anterior horn cells of the spinal cord. SMA presents with a highly variable phenotype ranging from very severe to mild (type I-III). No cure for SMA is available at present. All forms of SMA are caused by homozygous loss of the functional survival motor neuron (SMN1) gene. However, all patients have one or more copies of the SMN2 gene, nearly identical to SMN1. Both genes encode the SMN protein but the level produced by SMN2 is insufficient to protect from disease. Increasing SMN2 gene expression could be of considerable therapeutic importance. The aim of this study was to assess whether SMN2 gene expression can be increased by 4-phenylbutyrate (PBA). Fibroblast cell cultures from 16 SMA patients affected by different clinical severities were treated with PBA, and full-length SMN2 transcripts were measured by real-time PCR. In all cell cultures, except one, PBA treatment caused an increase in full-length SMN2 transcripts, ranging from 50 to 160% in type I and from 80 to 400% in type II and III cultures. PBA was found also effective in enhancing SMN protein levels and the number of SMN-containing nuclear structures (gems). These data show that SMN expression is considerably increased by PBA, and suggest that the compound, owing also to its favorable pharmacological properties, could be a good candidate for the treatment of SMA.

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SMN protein analysis in fibroblast, amniocyte and CVS cultures from spinal muscular atrophy patients and its relevance for diagnosis

Cited by 47 publications

References 26 publications

Treatment of spinal muscular atrophy cells with drugs that upregulate SMN expression reveals inter- and intra-patient variability

Treatment of spinal muscular atrophy cells with drugs that upregulate SMN expression reveals inter- and intra-patient variability

Clinical and molecular cross-sectional study of a cohort of adult type III spinal muscular atrophy patients: clues from a biomarker study

Phenylbutyrate increases SMN expression in vitro: relevance for treatment of spinal muscular atrophy

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