Rapid diagnosis of spinal muscular atrophy using denaturing high-performance liquid chromatography.
OBJECTIVE
To introduce the application of denaturing high-performance liquid chromatography (DHPLC) in the diagnosis of childhood type spinal muscular atrophy (SMA).


METHODS
Exon 7 and flanking area of survival motor neuron (SMN) gene were amplified by PCR in 1 standard sample, 25 normal individuals and 25 patients with SMA. The PCR products were then directly loaded onto the DHPLC system after denaturing and annealing. Different DNA segments were separated by changing the concentration of buffer A relative to that of buffer B.


RESULTS
Different DNA segments were separable on the DHPLC chromatogram. Three peaks including SMN1/SMN2 heteroduplex peak, SMN2 homoduplex peak and SMN1 homoduplex peak were detected in 23 out of 25 normal individuals. Only SMN1 homoduplex peak was detected in 2 normal individuals and the standard sample, indicating the deletion of SMN2 On the contrary, only the SMN2 homoduplex peak was detected in 22 out of 25 patients with SMA, indicating deletion of SMN1. The three peaks as those of normal individuals were detected in the other 3 patients, indicating no SMN1 or SMN2 deletion.


CONCLUSIONS
As a new technology for diagnosing SMA, DHPLC is sensitive, accurate, rapid and convenient.
Chen
Wan-jin
WJ
Department of Neurology, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, 350005, PR China.
Wu
Zhi-ying
ZY
Wang
Ning
N
Lin
Min-ting
MT
Mu-Rong
Shen-xing
SX
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005091:Exons / Q000235:genetics; D006801:Humans; D009134:Muscular Atrophy, Spinal / Q000175:diagnosis* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D055532:SMN Complex Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics; D055540:Survival of Motor Neuron 2 Protein
D055532:SMN Complex Proteins; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein