Quantitative Analyses of SMN1 and SMN2 Based on Real-Time LightCycler PCR: Fast and Highly Reliable Carrier Testing and Prediction of Severity of Spinal Muscular Atrophy

Abstract

Spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans, caused by homozygous absence of the survival motor neuron gene 1 (SMN1). SMN2, a copy gene, influences the severity of SMA and may be used in somatic gene therapy of patients with SMA in the future. We present a new, fast, and highly reliable quantitative test, based on real-time LightCycler PCR that amplifies either SMN1 or SMN2. The SMN1 copies were determined and validated in 329 carriers and controls. The specificity of the test is 100%, whereas the sensitivity is 96.2%. The quantitative analysis of SMN2 copies in 375 patients with type I, type II, or type III SMA showed a significant correlation between SMN2 copy number and type of SMA as well as duration of survival. Thus, 80% of patients with type I SMA carry one or two SMN2 copies, and 82% of patients with type II SMA carry three SMN2 copies, whereas 96% of patients with type III SMA carry three or four SMN2 copies. Among 113 patients with type I SMA, 9 with one SMN2 copy lived <11 mo, 88/94 with two SMN2 copies lived <21 mo, and 8/10 with three SMN2 copies lived 33-66 mo. On the basis of SMN2 copy number, we calculated the posterior probability that a child with homozygous absence of SMN1 will develop type I, type II, or type III SMA.

Baseline results of the NeuroNEXT spinal muscular atrophy infant biomarker study

“…Disease severity spans a wide range of phenotypes divided into five categories based upon maximal motor function: type 0, (neonates who present with severe hypotonia often with history of decreased fetal movements), type 1 (never sit independently), type 2 (sit but never stand independently), type 3 (ambulatory children), and type 4 (ambulatory adults) 9, 1011, 12, 13, 14. Thus, SMN2 copy number is a prognostic biomarker that predicts future clinical outcome.…”

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

“…[7][8][9] SMN2 copy number can also vary, and patients with high copy number often have a milder phenotype. [10][11][12] At present, there is no effective treatment for SMA. Some therapeutic approaches aim to increase the amount of SMN protein produced by SMN2 through promoter activation, reduction of exon 7 alternative splicing, or both.…”

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

“…However, the correlation is not absolute and patients with three SMN2 copies may suffer type I, II or III SMA. 11,12 As the differences between SMN1 and SMN2 are related to the complete transcript and the amount of protein, drugs capable of increasing FL-SMN expression and SMN protein may have therapeutic effects for SMA patients. 13 Histone deacetylases inhibitors (HDACi), for example, increase acetylation of histones and other proteins 14 and this hyperacetylation relaxes the tertiary structure of chromatin, facilitating access of the transcriptional machinery to target genes.…”