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

Abstract

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.

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

“…We found similar results also in our previous study. 33 The main difference with the cohort included in the present study is related to the long duration of the disease of our patients and to the associated complications, which may impair the clinical evaluation. Moreover, to our knowledge, longitudinal data regarding SMN level variations with age are not available.…”

“…A multivariate model was used to take account of the influence of other covariates. Because of small sample size and non-normal distribution of SMN transcript levels, 33 the non-parametric Kruskal-Wallis 'ANOVA' by ranks (KW) and Mann-Whitney U-test (MW) …”

“…SMN2 full length (SMN2-fl), lacking exon 7 (SMN-del7) and total (SMN-fl plus SMN-del7, SMN-tot) transcript levels were assessed by absolute real-time PCR (Tiziano et al 33 and Angelozzi et al, in preparation). In patients with the G287R variant, full-length transcripts were determined by an alternative set of Taqman MGB probe and primers.…”

“…In patients with the G287R variant, full-length transcripts were determined by an alternative set of Taqman MGB probe and primers. 33 GAPDH transcript levels were determined as quality control for RT-PCR and real-time PCR.…”

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

“…The recent availability of an absolute quantification method for SMN transcripts may help to further categorize patients according to their responder status. 47 Moreover, in another report, induced pluripotent stem cells generated from skin fibroblasts from a child with SMA were differentiated into motor neurons. 48 This new model of motor neurons is a promising resource to study SMA disease mechanisms and to further predict the response to drugs that upregulate SMN2 expression.…”