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.

Figures

Figure 1

(a) Day-to-day variations of SMN-fl, SMN1-fl, and SMN2-fl transcript levels in seven controls (ctrl 1–7) and of SMN2-fl 2 type III patients (pt 1–6). SMN2-fl level fluctuations were similar to that expected for experimental variability, whereas in controls SMN1-fl levels and, consequently, SMN-fl levels varied up to threefold. (b) GAPDH transcript level fluctuations were in the range of experimental variability.

Figure 2

SMN2-fl levels in patients vs SMN-fl in controls. SMN2-fl transcript levels in patients are significantly reduced compared with SMN-fl levels in controls. Also median SMN2-fl levels in patients are significantly lower compared with controls when considering type II and III patients separately. Type II patients showed significantly lower SMN2-fl transcript levels compared with type III subjects.

Figure 3

SMN2-fl transcript levels are not related to SMN2 gene copy number. Although patients with four SMN2 copies showed higher median SMN2-fl levels compared with individuals with three SMN2, this difference was not statistically significant.

Figure 4

Correlation between SMN2-fl levels and clinical severity (a) Transcript levels are related to the age of onset of type III patients: patients with SMN2-fl levels <58 mol/ng of total RNA showed a threefold higher risk of disease onset below the age of 3 years (type IIIa). (b) In type II patients (ranging: 2.5–12 years), a linear correlation was found between SMN2-fl levels and Hammersmith functional scale scores. Dots indicate SMN2-fl levels and the corresponding functional score for individual patients; the black line is the graphic representation of the equation describing the linear regression model; the dark gray lines indicate 95% confidence interval and the outer light gray lines are 95% prediction limits for new observations. (c) Patients with a functional score ≤20 showed significantly lower SMN2-fl levels. (d) In haploidentical sib pairs with discordant SMA phenotype, the less severely affected sib (white columns) showed significantly higher transcript levels compared with the more severely affected one (black columns), whereas phenotypically concordant sibs (gray columns) had similar SMN2-fl levels. However, SMN2nn-fl transcript quantification is not predictive of phenotypic severity in individual cases, as less severely affected patients may have higher transcript levels. Error bars indicate SD of repeated experiments