Development of a quantitative real-time PCR assay for detection of unknown α-globin gene deletions

Fallah, Mohammad‐Sadegh; Mahdian, Reza; Aleyasin, Seyyed-Ahmad; Jamali, Salar; Hayat-Nosaeid, Mina; Karimipour, Morteza; Raeisi, Marzieh; Zeinali, Sirous

doi:10.1016/j.bcmd.2010.03.001

Cited by 15 publications

(20 citation statements)

References 27 publications

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“…SYBR Green real-time qPCR has been used for analysis of copy number variation in both the α- and β-globin gene clusters [13,19]. Fallah et al [13] reported one of the first assays to detect unknown α-thalassemia deletions based on the relative, quantitative PCR method using SYBR Green chemistry.…”

Section: Discussionmentioning

confidence: 99%

“…Several methods have been developed to identify unknown deletions and duplications in the α-globin genes, using quantitative real-time PCR (real-time qPCR) assays [13-15] or multiplex ligation-dependent probe amplification (MLPA) assays [16,17]. Here, we describe a fast, robust and specific method, designated HBA-CNV, that measures copy number variation using real-time qPCR.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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BackgroundAlpha-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).ResultsQuantitative real-time PCR was performed using four TaqMan® assays which specifically amplify target sequences representing both the α-globin genes, the –α3.7 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.ConclusionsHBA-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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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“…Primer panels targeted to the most common deletion found in the area of the geographic origin of the proband can be used Chong et al 2000;Old et al 2001). MLPA (Harteveld et al 2005) or quantitative real-time PCR assay may also be used to detect less common mutations or novel deletion (Fallah et al 2010). a-Thalassemia mental retardation 16 (ATR16), a contiguous gene deletion, may also be detected by MLPA or array comparative genome hybridization (CGH) (Gibson et al 2008).…”

Section: Molecular Diagnosismentioning

confidence: 99%

The Prevention of Thalassemia

Cao

Kan

2013

Cold Spring Harbor Perspectives in Medicine

200

157

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The thalassemias are among the most common inherited diseases worldwide, affecting individuals originating from the Mediterranean area, Middle East, Transcaucasia, Central Asia, Indian subcontinent, and Southeast Asia. As the diseases require long-term care, prevention of the homozygous state constitutes a major armament in the management. This article discusses the major prevention programs that are set up in many countries in Europe, Asia, and Australia, often drawing from the experience in Sardinia. These comprehensive programs involve carrier detections, molecular diagnostics, genetic counseling, and prenatal diagnosis. Variability of clinical severity can be attributable to interactions with a-thalassemia and mutations that increase fetal productions. Special methods taht are currently quite expensive and not widely applicable are preimplantation and preconception diagnosis. The recent successful studies of fetal DNA in maternal plasma may allow future prenatal diagnosis that is noninvasive for the fetus.

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“…Primers were designed and verified using Beacon Designer 7 (Premier Biosoft International, Palo Alto, CA, USA). Data analysis was carried out as described previously (25,26). To characterize the suspected deletion, gap-PCR with forward primer P1 (5 0 -ATC TAC TTT CTG CTG GGA TTT GCC C-3 0 ) and reverse primer P2 (5 0 -GGC GGC TCC AGA TTC AGA CTC CT-3 0 ) was used to amplify across the breakpoints and the PCR product was sequenced directly.…”

Section: Molecular Diagnosismentioning

confidence: 99%

AnAluElement-Mediated 28.5 kb α-Thalassemia Deletion Found in a Chinese Family

Xie

Luo

et al. 2014

Hemoglobin

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Over 95.0% of the α-thalassemia (α-thal) cases in southern China are caused by large deletions involving the α-globin gene. Here, we describe the molecular characterization of a novel 28.5 kb deletion that eliminated one of the duplicated α-globin genes in a Chinese family. The deletion breakpoint fragment involved Alu repeat sequences, suggesting a homologous recombination event. Phenotypic analysis on the heterozygous carrier of this deletion revealed that it leads to a very mild phenotype. Because of a 25.0% risk of Hb H (β4) disease in the offspring when in combination with another α(0)-thal allele, we should not ignore screening the deletion in prenatal diagnosis in order to decrease reproductive risk.

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Development of a quantitative real-time PCR assay for detection of unknown α-globin gene deletions

Cited by 15 publications

References 27 publications

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

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

The Prevention of Thalassemia

AnAluElement-Mediated 28.5 kb α-Thalassemia Deletion Found in a Chinese Family

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