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

Institute of Medical Genetics, Catholic University, Largo Francesco Vito, 1, 00168 Rome, Italy.
European journal of human genetics: EJHG (Impact Factor: 4.35). 08/2009; 18(1):52-8. DOI: 10.1038/ejhg.2009.116
Source: PubMed


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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    • "The quantification of the Chit1 and AMCase mRNA levels and the comparison of those levels with the levels of well-known reference genes are important steps in gaining insight into the in vivo regulation of mammalian chitinases. Recently, real-time RT-PCR has been used to quantify mRNA levels in many gene expression studies [17]–[20] because this method is sufficiently sensitive to detect mRNA from even a single cell. Real-time PCR commonly involves the normalization of the expression levels of the gene of interest with those of the housekeeping genes that are thought to be consistently expressed in all of the samples. "
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    ABSTRACT: Chitinases hydrolyze the β-1-4 glycosidic bonds of chitin, a major structural component of fungi, crustaceans and insects. Although mammals do not produce chitin or its synthase, they express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). These mammalian chitinases have attracted considerable attention due to their increased expression in individuals with a number of pathological conditions, including Gaucher disease, Alzheimer's disease and asthma. However, the contribution of these enzymes to the pathophysiology of these diseases remains to be determined. The quantification of the Chit1 and AMCase mRNA levels and the comparison of those levels with the levels of well-known reference genes can generate useful and biomedically relevant information. In the beginning, we established a quantitative real-time PCR system that uses standard DNA produced by ligating the cDNA fragments of the target genes. This system enabled us to quantify and compare the expression levels of the chitinases and the reference genes on the same scale. We found that AMCase mRNA is synthesized at extraordinarily high levels in the mouse stomach. The level of this mRNA in the mouse stomach was 7- to 10-fold higher than the levels of the housekeeping genes and was comparable to that the level of the mRNA for pepsinogen C (progastricsin), a major component of the gastric mucosa. Thus, AMCase mRNA is a major transcript in mouse stomach, suggesting that AMCase functions as a digestive enzyme that breaks down polymeric chitin and as part of the host defense against chitin-containing pathogens in the gastric contents. Our methodology is applicable to the quantification of mRNAs for multiple genes across multiple specimens using the same scale.
    PLoS ONE 11/2012; 7(11):e50381. DOI:10.1371/journal.pone.0050381 · 3.23 Impact Factor
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    • "Amplified SMN1 and SMN2 were obtained using normal control cDNA and primers SMN-F (5′-GCT GAT GCT TTG GGA AGT ATG TTA-3′) and SMN-R (5′-TCA ACT GCC TCA CCA CCG TGC TGG-3′), specific for exons 6 and 8, respectively. The primer pair for amplification of GAPDH was GAPDH_exst-F and GAPDH_exst-R, as previously described [26]. Amplicons for SMN1/SMN2 (395 bp) and GAPDH (133 bp) were cloned into the pGEM-T Easy cloning vector (Promega, USA). "
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    • "Besides fold changes, gene expressions can also be evaluated for a known quantity of cDNA [11] or RNA [12,13] as previously tried. In comparison to RNA, cDNAs are more stable during dilution procedure as observed in environmental samples [14]. "
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    Plant Methods 03/2012; 8(1):9. DOI:10.1186/1746-4811-8-9 · 3.10 Impact Factor
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