Quantification of SMN1 and SMN2 genes by capillary electrophoresis for diagnosis of spinal muscular atrophy.
We present the first CE method for the separation and quantification of SMN1 and SMN2 genes. Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder deleted or mutated in SMN1 gene and retained at least one copy of SMN2 gene. However, these two genes are highly homologous, differentiation and quantification of SMN1 and SMN2 are therefore required in diagnosis to identify SMA patients and carriers. We developed a fluorescence-labeled conformation-sensitive CE method to quantitatively analyze PCR products covering the variable position in the SMN1/SMN2 genes using a copolymer solution composed of hydroxyethylcellulose and hydroxypropylcellulose. The DNA samples included 24 SMA patients, 52 parents of SMA patients (obligatory carriers), and 255 controls. Those 331 samples were blind analyzed to evaluate the method, and the results compared with those obtained using denaturing HPLC (DHPLC). Validation of accuracy was performed by comparing the results with those of DHPLC. Nine of total samples showed different results. Diagnosis of one fetus DNA among them was related to abortion or not, which was further confirmed by gel electrophoresis and DNA sequencing. Our method showed good coincidence with them, and proved the misdiagnosis of DHPLC. This simple and reliable CE method is a powerful tool for clinical genotyping of large populations to detect carriers and SMA patients.
Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University, and Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
Chun-Chi
CC
Wang
Jan-Gowth
JG
Chang
Jerome
J
Ferrance
Hsin-Yi
HY
Chen
Chung-Yee
CY
You
Yung-Fu
YF
Chang
Yuh-Jyh
YJ
Jong
Shou-Mei
SM
Wu
Chao-Hung
CH
Yeh
D017362:Cyclic AMP Response Element-Binding Protein / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D009134:Muscular Atrophy, Spinal / Q000175:diagnosis* / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000032:analysis*; D016133:Polymerase Chain Reaction; D016601:RNA-Binding Proteins / Q000032:analysis*; D055532:SMN Complex Proteins; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein
D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; 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