Biopolym. Cell. 2010; 26(1):51-55.
Genomics, Transcriptomics and Proteomics
Implementation of the quantitative Real-Time PCR for the molecular-genetic diagnostics of spinal muscular atrophy
1Soloviov O. O., 1Livshits G. B., 1Podlesnaya S. S., 1Livshits L. A.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680


Aim. To develop an easy and reliable assay for quantitative analysis of the SMN1 gene exon 7 copy number with Real-Time PCR and a SYBR Green dye which can be used as a test-system for spinal muscular atrophy (SMA) diagnostics. Methods. For the quantification the SMN1 gene exon 7 copies we have used the approach, which is based on the comparison of ratio between PCR amplification of the genomic DNA sample and that of an internal standard (Albumin gene) for each subject tested. For the development and validation of the assay we tested the DNA samples from ten patients with SMA (homozygous deletion of the exon 7 in the SMN1 gene) which were previously analyzed using standard PCR-RFLP method and 42 control DNA samples from: 29 heterozygous carriers of the deletion of the exon 7 in the SMN1 gene, 13 individuals without SMN1 deletion, which were previously analyzed using linkage analysis of 2AE9.1 (D5S557) and LAS96 (D5S681) polymorphic microsatellite loci, and 10 samples from individuals of the general population. The results were calculated using standard Livak method (2–ΔΔCt method). Results. The mean ± SD of the 2–ΔΔCt ratios for the carriers of the heterozygous deletion of the exon 7 in the SMN1 gene is 0.475 ± 0.091; and for the controls – 0.909 ± 0.068. The results obtained don’t show overlapping between 2–Ct ratios at the carriers of the SMN1 heterozygous deletion and individuals without it (t =3.84, p > 0.05). Conclusions. This method can be used as a basis for creating the test-system for SMA DNA diagnostics, especially for the carrier screening.
Keywords: spinal muscular atrophy, SMN1 gene, deletion, Real-Time PCR


[1] Ekshiian A. Iu., Livshits L. A., Bychkova A. M., Afanas'eva N. A., Bariliak I. R. A molecular genetic analysis of spinal muscular atrophy (SMA) in families at high risk from different regions of Ukraine. Tsitol. Genet. 1997 31, N 6 P. 75–81.
[2] Munsat T. L., Davies K. E. International SMA consortium meeting Neuromuscul. Disord 1992 2, N 5–6 P. 423–428.
[3] Lefebvre S., Burglen L., Reboullet S., Clermont O., Burlet P., Viollet L., Benichou B., Cruaud C., Millasseau P., Zeviani M., Le Paslier D., Frezal J., Cohen D., Weissenbach J., Munnich A., Melki J. Identification and characterization of a spinal muscular atrophy-determining gene. Cell 1995; 80, N 1 P. 155–165.
[4] Melki J., Lefebvre S., Burglen L., Burlet P., Clermont O., Millasseau P., Reboullet S., Benichou B., Zeviani M., Le Paslier D., Cohen D., Weissenbach J., Munnich A. De novo and inherited deletions of the 5q13 region in spinal muscular atrophies. Science 1994 264, N 5164 P. 1474–1477.
[5] Lorson C. L., Hahnen E., Androphy E. J., Wirth B. A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy Proc. Nat. Acad. Sci. USA 1999 96, N 11 P. 6307–6311.
[6] Monani U. R., Lorson C. L., Parsons D. W., Prior T. W., Androphy E. J., Burghes A. H., McPherson J. D. A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2 Hum. Mol. Genet 1999 8, N 7 P. 1177–1183.
[7] Livshits G. B., Hryshchenko N. V., Podlesna S. S., Ekshiian A. Iu. Molecular-genetic analysis of spinal muscular atrophy in Ukrainian patients. Int. Neurol. J. 2009; 26, N 4 P. 20–22.
[8] Ogino S., Leonard D., Rennert H., Wilson R. B. Spinal muscular atrophy genetic testing experience at an academic medical center. J. Mol. Diagn 2002 4, N 1 P. 53–58.
[9] McAndrew P. E., Parsons D. W., Simard L. R., Rochette C., Ray P. N., Mendell J. R., Prior T. W., Burghes A. H. Identification of proximal spinal muscular atrophy carriers and patients by analysis of SMNT and SMNC gene copy number Am. J. Hum. Genet 1997 60, N 6 P. 1411–1422.
[10] Ogino S., Leonard D. G. B., Rennert H., Gao S., Wilson R. B. Heteroduplex formation in SMN gene dosage analysis J. Mol. Diagn 2001 3, N 4 P. 150–157.
[11] Maniatis T., Fritsch E. F., Sambrook J. Molecular cloning: a laboratory manual New York: Cold Spring Harbor Lab. publ., 1982 545 p.
[12] Kim S. W., Lee K. S., Jin H. S., Lee T. M., Koo S. K., Lee Y. J., Jung S. C. Rapid detection of duplication/deletion of the PMP22 gene in patients with Charcot-Marie-Tooth disease type 1A and hereditary neuropathy with liability to pressure palsy by real-time quantitative PCR using SYBR Green I dye. J. Korean. Med. Sci 2003; 18, N 5 P. 727–732.
[13] Livak K. J., Schmittgen T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–CT method Methods 2001 25 P. 402–408.
[14] Chen K. L., Wang Y. L., Rennert H., Joshi I., Mills J. K., Leonard D. G., Wilson R. B. Duplications and de novo deletions of the SMNt gene demonstrated by fluorescence-based carrier testing for spinal muscular atrophy. Am. J. Med. Genet 1999 85, N 5 P. 463–469.