A sensitive assay for measuring SMN mRNA levels in peripheral blood and in muscle samples of patients affected with spinal muscular atrophy

Vézain, Myriam; Saugier-Veber, Pascale; Melki, Judith; Toutain, Annick; Bieth, Éric; Husson, Marie; Pédespan, Jean-Michel; Viollet, Louis; Pénisson-Besnier, I.; Fehrenbach, Séverine; Bou, Jacqueline; Frébourg, Thierry; Tosi, Mario

doi:10.1038/sj.ejhg.5201885

Cited by 33 publications

(47 citation statements)

References 34 publications

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“…We compared the SMN2-fl transcript levels and SMN2 gene number and did not find a significant correlation (Figure 3). Similar results were also found in the study by Simard et al, 23 whereas Sumner et al 22 and Vezain et al 24 found that SMN2 transcript levels are related to the number of SMN2 genes. The discrepancy among different studies can be at least partially accounted for by the use of relative quantification and of different endogenous internal standards that vary widely among different individuals.…”

Section: Discussionsupporting

confidence: 86%

“…However, possible variations of SMN transcripts/protein levels as evaluated in leukocytes may not reflect the real effect of pharmacological treatment in target tissues, such as spinal cord and, eventually, skeletal muscle. So far, some assays have been developed and validated for SMN2 transcript 14,[22][23][24] or protein [25][26] quantification. However, to date it has not been shown whether SMN full-length (SMN-fl) transcript or protein levels in leukocytes differ significantly among controls, carriers, and patients.…”

Section: Introductionmentioning

confidence: 99%

“…14,22 The reported SMN mRNA assays are mainly based on relative semiquantitative PCR in which transcript levels are determined by normalizing with respect to housekeeping gene transcript levels, used as endogenous controls. [22][23][24] However, it has been shown that the expression levels of these genes vary widely in the general population and/or can be putatively affected by pharmacological treatments or metabolic status, thus reducing the sensitivity of the earlier published assays. 14 Brichta et al 14 have developed a real-time PCR assay based on the measurement of SMN levels relative to the amount of RNA.…”

Section: Introductionmentioning

confidence: 99%

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SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

Tiziano¹,

Pinto²,

Fiori³

et al. 2009

Eur J Hum Genet

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Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous mutations of the SMN1 gene. Three forms of SMA are recognized (type I-III) on the basis of clinical severity. All patients have at least one or more (usually 2-4) copies of a highly homologous gene (SMN2), which produces insufficient levels of functional SMN protein, because of alternative splicing of exon 7. Recently, evidence has been provided that SMN2 expression can be enhanced by pharmacological treatment. However, no reliable biomarkers are available to test the molecular efficacy of the treatments. At present, the only potential biomarker is the dosage of SMN products in peripheral blood. However, the demonstration that SMN full-length (SMN-fl) transcript levels are reduced in leukocytes of patients compared with controls remains elusive (except for type I). We have developed a novel assay based on absolute real-time PCR, which allows the quantification of SMN1-fl/SMN2-fl transcripts. For the first time, we have shown that SMN-fl levels are reduced in leukocytes of type II-III patients compared with controls. We also found that transcript levels are related to clinical severity as in type III patients SMN2-fl levels are significantly higher compared with type II and directly correlated with functional ability in type II patients and with age of onset in type III patients. Moreover, in haploidentical siblings with discordant phenotype, the less severely affected individuals showed significantly higher transcript levels. Our study shows that SMN2-fl dosage in leukocytes can be considered a reliable biomarker and can provide the rationale for SMN dosage in clinical trials.

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Section: Discussionsupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

Tiziano¹,

Pinto²,

Fiori³

et al. 2009

Eur J Hum Genet

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“…Gene expression profiling data were validated on some targeted genes by quantitative multiplex RT-PCR (Quantitative Multiplex polymerase chain reaction of Fluorescent Fragments). 30 A high correlation between jFCj was obtained between multiplexed PCR and hybridization arrays (online supplementary Figure 1 . Identification parameters were: a cut-off identity score set at 25, tryptic peptides, one missed cleavage allowed, fragment mass tolerance set at 10 ppm, peptide tolerance set at 0.5 Da and took into account cysteine carbamidomethylation as fixed modification and methionine oxidation as variable modifications.…”

Section: Transcriptome Analysesmentioning

confidence: 94%

Major remodeling of brain microvessels during neonatal period in the mouse: A proteomic and transcriptomic study

Porte

Hardouin

Derambure

et al. 2016

J Cereb Blood Flow Metab

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Preterm infants born before 29 gestation weeks incur major risk of subependymal/intracerebral/intraventricular hemorrhage. In mice, neonate brain endothelial cells are more prone than adult cells to secrete proteases under glutamate challenge, and invalidation of the Serpine 1 gene is accompanied by high brain hemorrhage risk up to five days after birth. We hypothesized that the structural and functional states of microvessels might account for age-dependent vulnerability in mice up to five days after birth and might represent a pertinent paradigm to approach the hemorrhage risk window observed in extreme preterms. Mass spectrometry proteome analyses of forebrain microvessels at days 5, 10 and in adult mice revealed 899 proteins and 36 enriched pathways. Microarray transcriptomic study identified 5873 genes undergoing at least two-fold change between ages and 93 enriched pathways. Both approaches pointed towards extracellular matrix, cell adhesion and junction pathways, indicating delayed microvascular strengthening after P5. Furthermore, glutamate receptors, proteases and their inhibitors exhibited convergent evolutions towards excitatory aminoacid sensitivity and low proteolytic control likely accounting for vascular vulnerability in P5 mice. Thus, age vascular specificities must be considered in future therapeutic interventions in preterms. Data are available on ProteomeXchange (identifier PXD001718) and NCBI Gene-Expression-Omnibus repository (identification GSE67870).

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“…The different technical approaches utilized are analyzed in detail throughout this section. [121][122][123][124][125][126][127] A greater number of studies belong to the second category, as the majority of therapeutic approaches have aimed to increase SMN protein levels by increasing the level of SMN transcripts. An exception to this transcript ''rule'' is the modification of SMN protein stability, 117 where a change in SMN transcript levels is not expected.…”

Section: Smn Transcript Assaysmentioning

confidence: 99%

Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy

Cherry

Kobayashi

Lynes

et al. 2014

ASSAY and Drug Development Technologies

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A sensitive assay for measuring SMN mRNA levels in peripheral blood and in muscle samples of patients affected with spinal muscular atrophy

Cited by 33 publications

References 34 publications

SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

Major remodeling of brain microvessels during neonatal period in the mouse: A proteomic and transcriptomic study

Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy

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