Quantification of SMN1 and SMN2 genes by capillary electrophoresis for diagnosis of spinal muscular atrophy

Abstract

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.

Selective Neuromuscular Denervation in Taiwanese Severe SMA Mouse Can Be Reversed by Morpholino Antisense Oligonucleotides

“…To generate severe SMA mice, the Smn -/- ; SMN2 tg/tg mice were crossbred to heterozygous Smn knockout mice ( Smn +/- ; SMN 2 0/0 ) to generate 50% severe SMA mice ( Smn -/- ; SMN 2 tg/0 ) and 50% control littermates (control) ( Smn +/- ; SMN 2 tg/0 ). We confirmed two SMN2 copies in severe SMA mice ( Smn -/- ; SMN 2 tg/0 ) and four SMN2 copies in mild mice ( Smn -/- ; SMN 2 tg/tg ) by capillary electrophoresis as described previously [ 32 ]. The SMN2 and Smn knockout alleles were genotyped by PCR analyses of tail DNA [ 31 ].…”

Identification of bidirectional gene conversion between SMN1 and SMN2 by simultaneous analysis of SMN dosage and hybrid genes in a Chinese population

“…Amplification of the KRIT1 and CYBB genes was used as endogenous controls for comparison. Quantification of the copy number of SMN1 and SMN2 genes was accomplished by CE (Beckman P/ACE MDQ system; Fullerton, CA, USA), and the quantitative accuracy of this method was also validated by another method, multiplex ligation-dependent probe amplification (MLPA), as previously described [13,14]. All test samples were analyzed in triplicate.…”

“…Recently, we have developed a capillary electrophoresis (CE) assay to analyze SMN gene dosages [13,14]. In this study, we quantified SMN1 and SMN2 gene dosage in three Chinese population groups to investigate the role of SMN gene conversion in the equilibrium of these two highly homologous genes.…”

Large-Scale Population Carrier Screening for Spinal Muscular Atrophy in Israel—Effect of Ethnicity on the False-Negative Rate

“…These methods enable an efficient detection of a homozygous absence of SMN1 in the majority of patients, but a clear distinction between carriers and noncarriers cannot always be achieved. For the determination of the exact SMN gene copy number, several quantitative real-time polymerase chain reaction approaches using LightCycler or TaqMan technology as well as denaturing high-performance liquid chromatography and capillary electrophoresis have been developed (Feldkotter et al, 2002;Anhuf et al, 2003;Su et al, 2005;Wang et al, 2008).…”