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.

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

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

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.