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.

Figure 1

Fluorescence versus cycle number, melting peaks, and sequence of new point mutation in SMN2. a,SYBR Green I fluorescence plot versus cycle number resulting from amplification of genomic DNA (external standard; st 0.5, st 1, st 2, st 3) of an unaffected individual carrying two SMN1 copies and homozygous absence of SMN2. In addition, four negative controls (three patients with SMA with homozygous absence of SMN1 and one water control) were tested. Slope = −4.112, error = 0.0279, r=-1.00. b, SYBR Green I fluorescence plot versus cycle number resulting from amplification of genomic DNA (external standard; st 1, st 2, st 4, st 8) of a patient with SMA with four SMN2 copies and homozygous absence of SMN1. In addition, four negative controls (three normal individuals with homozygous absence of SMN2 and one water control) were analyzed. Slope = −4.057, error = 0.0469, r=-1.00. c, Normalized SYBR Green I fluorescence plot of the external standard (st) for SMN1 and samples of carriers (C) and controls (ctr) with one, two, or three SMN1 copies. Each copy number was detected in three different DNA samples. Slope = −3.860, error = 0.0213, r=-1.00. d, Normalized SYBR Green I fluorescence plot of the external standard (st) for SMN2 and of patients with type I, type II, and type III SMA with one, two, three, or four SMN2 copies. Slope = − 3,819, error = 0.0181, r=-1.00. e, Melting-curve analysis of SMN2, with hybridization probe indicating the mutation 892G→C. The DNA of the patient with SMA with the homozygous 892G→C mutation in SMN2 shows a lower melting temperature (m/m, T_m=58.0°C) than an SMA patient with the wild-type SMN2 sequence (w/w, T_m=64.5°C). Patients with SMA with heterozygous genotype (w/m) display two peaks. f, New intragenic SMN2 mutation 892G→C in patients with SMA, found by sequencing the DNA samples with melting peaks at 58.0°C. Reverse nucleotide sequences obtained from direct sequencing of genomic PCR products from patients 4437 (heterozygous) and 4929 (homozygous ) for the mutation 892G→C; arrows indicate the positions of the mutation.

Figure 2

Plot of the average SMN1 values for carriers (N=124), noncarriers (unaffected sibs of patients with SMA) (N=65), and controls (N=140).

Figure 3

Diagram of the frequency of patients with type I, type II, and type III SMA versus SMN2 copy number.

Figure 4

a, Age of survival of children with type I SMA, correlated with the average SMN2 value. Circles correspond to one SMN2 copy, squares to two SMN2 copies, and triangles to three SMN2 copies. The line represents a trendline for the age of survival versus the SMN2 copy number. b, Kaplan-Meier curves show the probabilities of survival for patients with type I SMA who carry one, two, or three SMN2 copies.