Rapid and reliable detection of α-globin copy number variations by quantitative real-time PCR

BMC Hematology

Armin P Piehler¹  Olav Klingenberg³  Petter Urdal²  Runa M Grimholt² 

[1] Fürst Medical Laboratory, 1051 Oslo, Norway;Department of Medical Biochemistry, Oslo University Hospital, Ullevaal, 0424 Oslo, Norway;Department of Medical Biochemistry, Oslo University Hospital, Rikshospitalet, 0424 Oslo, Norway

关键词: Unknown deletions;    HS-40;    α-globin gene cluster;    Quantitative real-time PCR;    Copy number variation;    α-thalassemia;   

Others  :  865375
DOI  :  10.1186/2052-1839-14-4

 received in 2013-07-26, accepted in 2014-01-22,  发布年份 2014

【 摘 要 】

Background

Alpha-thalassemia is the most common human genetic disease worldwide. Copy number variations in the form of deletions of α-globin genes lead to α-thalassemia while duplications of α-globin genes can cause a severe phenotype in β-thalassemia carriers due to accentuation of globin chain imbalance. It is important to have simple and reliable methods to identify unknown or rare deletions and duplications in cases in which thalassemia is suspected but cannot be confirmed by multiplex gap-PCR. Here we describe a copy number variation assay to detect deletions and duplications in the α-globin gene cluster (HBA-CNV).

Results

Quantitative real-time PCR was performed using four TaqMan® assays which specifically amplify target sequences representing both the α-globin genes, the –α^3.7 deletion and the HS-40 region. The copy number for each target was determined by the 2^-ΔΔCq method. To validate our method, we compared the HBA-CNV method with traditional gap-PCR in 108 samples from patients referred to our laboratory for hemoglobinopathy evaluation. To determine the robustness of the four assays, we analyzed samples with and without deletions diluted to obtain different DNA concentrations. The HBA-CNV method identified the correct copy numbers in all 108 samples. All four assays showed the correct copy number within a wide range of DNA concentrations (3.2-100 ng/μL), showing that it is a robust and reliable method. By using the method in routine diagnostics of hemoglobinopathies we have also identified several deletions and duplications that are not detected with conventional gap-PCR.

Conclusions

HBA-CNV is able to detect all known large deletions and duplications affecting the α-globin genes, providing a flexible and simple workflow with rapid and reliable results.

【 授权许可】

   
2014 Grimholt et al.; licensee BioMed Central Ltd.

【 图 表 】

【 参考文献 】

• [1]Almal SH, Padh H: Implications of gene copy-number variation in health and diseases. J Hum Genet 2012, 57:6-13.
• [2]Perry GH: The evolutionary significance of copy number variation in the human genome. Cytogenet Genome Res 2008, 123:283-287.
• [3]Higgs DR, Engel JD, Stamatoyannopoulos G: Thalassaemia. Lancet 2011, 379:373-383.
• [4]Harteveld CL, Higgs DR: Alpha-thalassaemia. Orphanet J Rare Dis 2010, 5:13. BioMed Central Full Text
• [5]Steinberg MH, Forget BG, Higgs DR, Weatherall DJ: Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. 2nd edition. 2012.
• [6]Weatherall DJ: Phenotype-genotype relationships in monogenic disease: lessons from the thalassaemias. Nat Rev Genet 2001, 2:245-255.
• [7]Harteveld CL, Refaldi C, Cassinerio E, Cappellini MD, Giordano PC: Segmental duplications involving the alpha-globin gene cluster are causing beta-thalassemia intermedia phenotypes in beta-thalassemia heterozygous patients. Blood Cells Mol Dis 2008, 40:312-316.
• [8]Sollaino MC, Paglietti ME, Perseu L, Giagu N, Loi D, Galanello R: Association of alpha globin gene quadruplication and heterozygous beta thalassemia in patients with thalassemia intermedia. Haematologica 2009, 94:1445-1448.
• [9]Ribeiro DM, Sonati MF: Regulation of human alpha-globin gene expression and alpha-thalassemia. Genet Mol Res 2008, 7:1045-1053.
• [10]Chong SS, Boehm CD, Higgs DR, Cutting GR: Single-tube multiplex-PCR screen for common deletional determinants of alpha-thalassemia. Blood 2000, 95:360-362.
• [11]Tan AS, Quah TC, Low PS, Chong SS: A rapid and reliable 7-deletion multiplex polymerase chain reaction assay for alpha-thalassemia. Blood 2001, 98:250-251.
• [12]Hb V: A Database of Human Haemoglobin Variants and Thalassemias. 25-1 edition. 2012. Available at http://globin.cse.psu.edu/menu.html webcite
• [13]Fallah MS, Mahdian R, Aleyasin SA, Jamali S, Hayat-Nosaeid M, Karimipour M, et al.: Development of a quantitative real-time PCR assay for detection of unknown alpha-globin gene deletions. Blood Cells Mol Dis 2010, 45:58-64.
• [14]Mo ZP, Yu CS, Li YJ, Cao WX, Zeng ZY, Zhan YX, et al.: Detection of alpha-globin gene deletion and duplication using quantitative multiplex PCR of short fluorescent fragments. Clin Chem Lab Med 2012, 50:649-654.
• [15]Zhou WJ, Liu WY, Liu D, Jiang JH, Zhou DM, Zhong ZY, et al.: A quantitative assay to detect alpha-thalassemia deletions and triplications using multiplex nested real-time quantitative polymerase chain reaction. Anal Biochem 2012, 427:144-150.
• [16]Colosimo A, Gatta V, Guida V, Leodori E, Foglietta E, Rinaldi S, et al.: Application of MLPA assay to characterize unsolved alpha-globin gene rearrangements. Blood Cells Mol Dis 2011, 46:139-144.
• [17]Kipp BR, Roellinger SE, Lundquist PA, Highsmith WE, Dawson DB: Development and clinical implementation of a combination deletion PCR and multiplex ligation-dependent probe amplification assay for detecting deletions involving the human alpha-globin gene cluster. J Mol Diagn 2011, 13:549-557.
• [18]Ryan K, Bain BJ, Worthington D, James J, Plews D, Mason A, et al.: Significant haemoglobinopathies: guidelines for screening and diagnosis. Br J Haematol 2010, 149:35-49.
• [19]Babashah S, Jamali S, Mahdian R, Nosaeid MH, Karimipoor M, Alimohammadi R, et al.: Detection of unknown deletions in beta-globin gene cluster using relative quantitative PCR methods. Eur J Haematol 2009, 83:261-269.
• [20]Wang W, Ma ES, Chan AY, Prior J, Erber WN, Chan LC, et al.: Single-tube multiplex-PCR screen for anti-3.7 and anti-4.2 alpha-globin gene triplications. Clin Chem 2003, 49:1679-1682.
• [21]Meyer LR, Zweig AS, Hinrichs AS, Karolchik D, Kuhn RM, Wong M, et al.: The UCSC genome browser database: extensions and updates 2013. Nucleic Acids Res 2013, 41:D64-D69.
• [22]Mayo P, Hartshorne T, Li K, McMunn-Gibson C, Spencer K, Schnetz-Boutaud N: CNV analysis using TaqMan copy number assays. Curr Protoc Hum Genet 2010, 67:2.13.1. 2.13.10