Skip to main content
SpringerLink
Log in

Robust quantification of the SMN gene copy number by real-time TaqMan PCR

  • Original Article
  • Published:
Neurogenetics Aims and scope Submit manuscript

An Erratum to this article was published on 24 February 2009

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by mutation or deletion of the survival motor neuron gene 1 (SMN1). The highly homologous gene, SMN2, is present in all patients, but it cannot compensate for loss of SMN1. SMN2 differs from SMN1 by a few nucleotide changes, but a C → T transition in exon 7 leads to exon skipping. As a result, most transcripts from the SMN2 gene lack exon 7. Although SMN1 is the disease-determining gene, the number of SMN2 copies appears to modulate SMA clinical phenotypes. Thus, determining the SMN copy number is important for clinical diagnosis and prognosis. We have developed a quantitative real-time TaqMan polymerase chain reaction assay for both the SMN1 and SMN2 genes, in which reliable copy number determination was possible on deoxyribonucleic acid samples obtained by two different isolation methods and from two different sources (human blood and skin fibroblasts). For SMN1, allele specificity was attained solely by addition of an allele-specific forward primer and, for SMN2, by addition of a specific forward primer and a nonextending oligonucleotide (SMN1 blocker) that reduced nonspecific amplification from SMN1 to a negligible level. We validated the reliability of this real-time polymerase chain reaction approach and found that the coefficient of variation for all the gene copy number measurements was below 10%. Quantitative analysis of the SMN copy number in SMA fibroblasts by this approach showed deletion of SMN1 and an inverse correlation between the SMN2 copy number and severity of the disease..

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Emery AE (1991) Population frequencies of inherited neuromuscular diseases—a world survey. Neuromuscul Disord 1:19–29

    Article  CAS  PubMed  Google Scholar 

  2. Pearn JH (1973) The gene frequency of acute Werdnig–Hoffmann disease (SMA type 1). A total population survey in North-East England. J Med Genet 10:260–265

    Article  CAS  PubMed  Google Scholar 

  3. Pearn J (1978) Incidence, prevalence, and gene frequency studies of chronic childhood spinal muscular atrophy. J Med Genet 15:409–413

    Article  CAS  PubMed  Google Scholar 

  4. Ogino S, Leonard DG, Rennert H, Ewens WJ, Wilson RB (2002) Genetic risk assessment in carrier testing for spinal muscular atrophy. Am J Med Genet 110:301–307

    Article  PubMed  Google Scholar 

  5. Munsat TL, Davies KE (1992) International SMA consortium meeting. Neuromuscul Disord 2:423–428

    Article  CAS  PubMed  Google Scholar 

  6. Lefebvre S, Burglen L, Reboullet S, Clermont O, Burlet P, Viollet L, Benichou B, Cruaud C, Millasseau P, Zeviani M et al (1995) Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80:155–165

    Article  CAS  PubMed  Google Scholar 

  7. Cartegni L, Krainer AR (2002) Related articles, disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat Genet 30:377–384

    Article  CAS  PubMed  Google Scholar 

  8. Lorson CL, Hahnen E, Androphy EJ, Wirth B (1999) Related articles, a single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc Natl Acad Sci USA 96:6307–6311

    Article  CAS  PubMed  Google Scholar 

  9. Wirth B, Herz M, Wetter A, Moskau S, Hahnen E, Rudnik-Schoneborn S, Wienker T, Zerres K (1999) Quantitative analysis of survival motor neuron copies: identification of subtle SMN1 mutations in patients with spinal muscular atrophy, genotype–phenotype correlation, and implications for genetic counseling. Am J Hum Genet 64:1340–1356

    Article  CAS  PubMed  Google Scholar 

  10. Burghes AH (1997) When is a deletion not a deletion? When it is converted. Am J Hum Genet 61:9–15

    Article  CAS  PubMed  Google Scholar 

  11. Lefebvre S, Burlet P, Liu Q, Bertrandy S, Clermont O, Munnich A, Dreyfuss G, Melki J (1997) Correlation between severity and SMN protein level in spinal muscular atrophy. Nat Genet 16:265–269

    Article  CAS  PubMed  Google Scholar 

  12. Feldkotter M, Schwarzer V, Wirth R, Wienker TF, Wirth B (2002) Quantitative analyses of SMN1 and SMN2 based on real-time lightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. Am J Hum Genet 70:358–368

    Article  CAS  PubMed  Google Scholar 

  13. Wirth B, Brichta L, Schrank B, Lochmuller H, Blick S, Baasner A, Heller R (2006) Mildly affected patients with spinal muscular atrophy are partially protected by an increased SMN2 copy number. Hum Genet 119:422–428

    Article  CAS  PubMed  Google Scholar 

  14. Monani UR, Sendtner M et al (2000) The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(−/−) mice and results in a mouse with spinal muscular atrophy. Hum Mol Genet 9:333–339

    Article  CAS  PubMed  Google Scholar 

  15. Chang JG, Hsieh-Li HM, Jong YJ, Wang NM, Tsai CH, Li H (2001) Treatment of spinal muscular atrophy by sodium butyrate. Proc Natl Acad Sci USA 98:9808–9813

    Article  CAS  PubMed  Google Scholar 

  16. Andreassi C, Jarecki J, Zhou J, Coovert DD, Monani UR, Chen X, Whitney M, Pollok B, Zhang M, Androphy E, Burghes AH (2001) Aclarubicin treatment restores SMN levels to cells derived from type I spinal muscular atrophy patients. Hum Mol Genet 10:2841–2849

    Article  CAS  PubMed  Google Scholar 

  17. Sumner CJ, Huynh TN, Markowitz JA, Perhac JS, Hill B, Coovert DD, Schussler K, Chen X, Jarecki J, Burghes AH, Taylor JP, Fischbeck KH (2003) Valproic acid increases SMN levels in spinal muscular atrophy patient cells. Ann Neurol 54:647–654

    Article  CAS  PubMed  Google Scholar 

  18. Brichta L, Hofmann Y, Hahnen E, Siebzehnrubl FA, Raschke H, Blumcke I, Eyupoglu IY, Wirth B (2003) Valproic acid increases the SMN2 protein level: a well-known drug as a potential therapy for spinal muscular atrophy. Hum Mol Genet 12:2481–2489

    Article  CAS  PubMed  Google Scholar 

  19. van der Steege G, Grootscholten PM, van der Vlies P, Draaijers TG, Osinga J, Cobben JM, Scheffer H, Buys CH (1995) PCR-based DNA test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy. Lancet 345:985–986

    Article  CAS  PubMed  Google Scholar 

  20. Chan V, Yip B, Yam I, Au P, Lin CK, Wong V, Chan TK (2004) Carrier incidence for spinal muscular atrophy in southern Chinese. J Neurol 251:1089–1093

    Article  CAS  PubMed  Google Scholar 

  21. Anhuf D, Eggermann T, Rudnik-Schoneborn S, Zerres K (2003) Determination of SMN1 and SMN2 copy number using TaqMan technology. Human Mutat 22:74–78

    Article  CAS  Google Scholar 

  22. Pyatt RE, Prior TW (2006) A feasibility study for the newborn screening of spinal muscular atrophy. Genet Med 8:428–437

    Article  PubMed  Google Scholar 

  23. Lee TM, Kim SW, Lee KS, Jin HS, Koo SK, Jo I, Kang S, Jung SC (2004) Quantitative analysis of SMN1 gene and estimation of SMN1 deletion carrier frequency in Korean population based on real-time PCR. J Korean Med Sci 19:870–873

    Article  CAS  PubMed  Google Scholar 

  24. Wang W, Dimatteo D et al (2005) Increased susceptibility of spinal muscular atrophy fibroblasts to camptothecin-induced cell death. Mol Genet Metab 85:38–45

    Article  CAS  PubMed  Google Scholar 

  25. Mimault C, Giraud G, Courtois V, Cailloux F, Boire JY, Dastugue B, Boespflug-Tanguy O (1999) Proteolipoprotein gene analysis in 82 patients with sporadic Pelizaeus–Merzbacher Disease: duplications, the major cause of the disease, originate more frequently in male germ cells, but point mutations do not. Am J Hum Genet 65:360–369

    Article  CAS  PubMed  Google Scholar 

  26. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  27. Coovert DD, Le TT et al (1997) The survival motor neuron protein in spinal muscular atrophy. Hum Mol Genet 6:1205–1214

    Article  CAS  PubMed  Google Scholar 

  28. Martin Y, Valero A, Castillo E, Pascual SI, Hernandez-Chico C (2002) Genetic study of SMA patients without homozygous SMN1 deletions: identification of compound heterozygotes and characterisation of novel intragenic SMN1 mutations. Hum Genet 110:257–263

    Article  CAS  PubMed  Google Scholar 

  29. Sun Y, Grimmler M, Schwarzer V, Schoenen F, Fischer U, Wirth B (2005) Molecular and functional analysis of intragenic SMN1 mutations in patients with spinal muscular atrophy. Human Mutat 25:64–71

    Article  CAS  Google Scholar 

  30. Wirth B (2000) An update of the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA). Human Mutat 15:228–237

    Article  CAS  Google Scholar 

  31. Celi FS, Cohen MM, Antonarakis SE, Wertheimer E, Roth J, Shuldiner AR (1994) Determination of gene dosage by a quantitative adaptation of the polymerase chain reaction (gd-PCR): rapid detection of deletions and duplications of gene sequences. Genomics 21:304–310

    Article  CAS  PubMed  Google Scholar 

  32. Ogino S, Leonard DG, Rennert H, Gao S, Wilson RB (2001) Heteroduplex formation in SMN gene dosage analysis. J Mol Diagnostics 3:150–157

    CAS  Google Scholar 

  33. McAndrew PE, Parsons DW, Simard LR, Rochette C, Ray PN, Mendell JR, Prior TW, Burghes AH (1997) Identification of proximal spinal muscular atrophy carriers and patients by analysis of SMNT and SMNC gene copy number. Am J Hum Genet 60:1411–1422

    Article  CAS  PubMed  Google Scholar 

  34. Gerard B, Ginet N, Matthijs G, Evrard P, Baumann C, Da Silva F, Gerard-Blanluet M, Mayer M, Grandchamp B, Elion J (2000) Genotype determination at the survival motor neuron locus in a normal population and SMA carriers using competitive PCR and primer extension. Human Mutat 16:253–263

    Article  CAS  Google Scholar 

  35. Ogino S, Wilson RB (2002) Quantification of PCR bias caused by a single nucleotide polymorphism in SMN gene dosage analysis. J Mol Diagnostics 4:185–190

    CAS  Google Scholar 

  36. Ogino S, Wilson RB (2002) Genetic testing and risk assessment for spinal muscular atrophy (SMA). Hum Genet 111:477–500

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Dr. Brunhilde Wirth for the externally validated DNA standards, Priscilla Moses for help with cell cultures, and Drs. Robert Mason, Andre Salama, and Jeffery Twiss for critical comments on this work. Some patient cell lines were provided by a MRDD research center grant, HD024061, at Johns Hopkins University. This work was supported by Nemours and a COBRE grant award from the NIH (1 P20 RR020173-01) to support the Center for Pediatric Research at the Alfred I. duPont Hospital for Children.

Author information

Authors and Affiliations

  1. Nemours Biomedical Research, Alfred I. duPont Hospital for Children, P.O. Box 269, Wilmington, DE, 19899, USA

    Ilsa Gómez-Curet, Karyn G. Robinson, Vicky L. Funanage, Mena Scavina & Wenlan Wang

  2. Department of Pediatrics, Thomas Jefferson University, Philadelphia, PA, USA

    Vicky L. Funanage & Wenlan Wang

  3. Departments of Neurology and Pediatrics, Johns Hopkins University, Baltimore, MD, USA

    Thomas O. Crawford

Authors
  1. Ilsa Gómez-Curet
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Karyn G. Robinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vicky L. Funanage
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas O. Crawford
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mena Scavina
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wenlan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wenlan Wang.

Additional information

An erratum to this article can be found at http://dx.doi.org/10.1007/s10048-009-0181-5

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gómez-Curet, I., Robinson, K.G., Funanage, V.L. et al. Robust quantification of the SMN gene copy number by real-time TaqMan PCR. Neurogenetics 8, 271–278 (2007). https://doi.org/10.1007/s10048-007-0093-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10048-007-0093-1

Keywords

Search

Navigation