Preclinical validation of a multiplex real-time assay to quantify SMN mRNA in patients with SMA

Abstract

Objective: To determine whether survival motor neuron (SMN) expression was stable over time.

Methods: We developed a multiplex real-time reverse transcriptase (RT)-PCR assay to quantify SMN transcripts in preclinical blood samples from 42 patients with spinal muscular atrophy (SMA) drawn for three time points per patient; most blood samples were shipped to a centralized laboratory.

Results: We obtained a sufficient amount (9.7 +/- 5.6 microg) of good-quality total RNA, and RNAs were stable for up to a 3-year interval. This allowed RNA samples collected during a 9- to 12-month period to be analyzed in a single run, thus minimizing interexperimental variability. SMN expression was stable over time; intersample variability for baseline measures, collected during a 17-month interval, was less than 15% for 38 of 42 SMA patients analyzed. This variability was well below the 1.95-fold increase in full-length SMN (flSMN) transcripts detected in SMA fibroblasts treated with 10 mM valproic acid.

Conclusion: Real-time quantification of SMN messenger RNA expression may be a biomarker that is amenable to multicenter SMA clinical trials.

Figure 1

Relationship between flSMN transcript quantity and SMN2 copy number. Quantification of flSMN was carried out by multiplex RT-PCR using PGK1 as an endogenous control. SMN2 copy number was evaluated as previously described.

Figure 2

Effect of VPA treatment of type I SMA fibroblast cells. SMA type I fibroblast cell line 3831 was treated with 0, 1, or 10 mM VPA (medium, light, and dark gray cylinders, respectively), and the amount of flSMN (A) or Δ7SMN (B) transcripts was determined using either PGK1, GUSB, or PPIA as endogenous controls. Data are reported as means ± SEM of two experiments.