Detection of unknown deletions in β‐globin gene cluster using relative quantitative PCR methods

Babashah, Sadegh; Jamali, Salar; Mahdian, Reza; Nosaeid, Mina Hayat; Karimipoor, Morteza; Alimohammadi, R; Raeisi, Marzieh; Maryami, Fereshteh; Masoudifar, Mahboubeh; Zeinali, Sirous

doi:10.1111/j.1600-0609.2009.01264.x

Cited by 17 publications

(12 citation statements)

References 24 publications

(35 reference statements)

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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%

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%

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

et al. 2014

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“…Glucose-6-Phosphate Dehydrogenase ( G6PDH ), Hydroxymethylbilane Synthase ( HEM3 ) and Chloride channel 7 ( CLCN7 ) genes were selected as internal controls for varying input DNA amounts as recommended by prior published guidelines [9], [10]. Thus, any difference in the real time PCR obtained for test primers/markers would correspond to differences in the amount of the target sequence primers.…”

Section: Methodsmentioning

confidence: 99%

Integration of SNP and mRNA Arrays with MicroRNA Profiling Reveals That MiR-370 Is Upregulated and Targets NF1 in Acute Myeloid Leukemia

et al. 2012

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BackgroundDeregulated miRNA expression plays a crucial role in carcinogenesis. Recent studies show different mechanisms leading to miRNA deregulation in cancer; however, alterations affecting miRNAs by DNA copy number variations (CNV) remain poorly studied.ResultsOur integrative analysis including data from high resolution SNPs arrays, mRNA expression arrays, and miRNAs expression profiles in 16 myeloid cell lines highlights that CNV are alternative mechanisms to deregulate the expression of miRNAs in acute myeloid leukemia (AML), and represent a novel approach to identify novel candidate genes involved in AML. We found association between the expression levels of 19 miRNAs and CNVs affecting their loci. Functional analysis showed that NF1 is a direct target of miR-370, and that overexpression of miR-370 has similar effects that NF1 inactivation, increasing proliferation and colony formation in AML cells. Moreover, real time RT-PCR showed that NF1 downregulation is a recurrent event in AML (30.8%), and western blot analysis confirmed this result. MiR-370 overexpression and deletions affecting the NF1 locus were identified as alternative mechanisms to downregulate NF1.Conclusions NF1 downregulation is a common event in AML, and both deletions in the NF1 locus and overexpression of miR-370 are alternative mechanisms to downregulate NF1 in this disease. Our results suggest a leukemogenic role of miR-370 through NF1 downregulation in AML cells. Since NF1 deficiency leads to RAS activation, patients with AML and overexpression of miR-370 may potentially benefit from additional treatment with either RAS or mTOR inhibitors.

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“…The Turkish type of inv/del (δβ)0 thalassemia was first characterized at the molecular level by Kulozik et al in a Turkish patient living in Germany with normal HbA2 and elevated HbF levels in 1992 [21]. It was also associated with elevated HbF and normal HbA2 levels in another later study [22]. Our study revealed that 7 out of 9 patients carrying the Turkish inv/del (δβ)0 had elevated HbF levels, while the remaining 2 had normal HbF levels.…”

Section: Discussionmentioning

confidence: 99%

Gap-PCR Screening for Common Large Deletional Mutations of β-Globin Gene Cluster Revealed a Higher Prevalence of the Turkish Inversion/Deletion (δβ)0 Mutation in Antalya

Bilgen¹,

Clark²,

Öztürk³

et al. 2016

Tjh

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Objective:Although the calculated carrier frequency for point mutations of the β-globin gene is around 10% for Antalya Province, nothing is known about the profile of large deletional mutations involving the β-globin gene. In this study, we aimed to screen common deletional mutations in the β-globin gene cluster in patients for whom direct DNA sequencing was not able to demonstrate the mutation(s) responsible for the disease phenotype.Materials and Methods:Thirty-one index cases selected with a series of selection events among 60 cases without detected β-globin gene mutation from 580 thalassemia-related cases tested by direct sequencing over the last 4 years in our diagnostic center were screened for the most common 8 different large deletional mutations of the β-globin gene cluster by gap-PCR.Results:We detected 1 homozygous and 9 heterozygous novel unrelated cases for the Turkish inversion/deletion (δβ)0 mutation in our series of 31 cases. Our study showed that the Turkish inversion/deletion (δβ)0 mutation per se accounts for 16.6% of the unidentified causative alleles and also accounts for 1.5% of all detected mutations over the last 4 years in our laboratory.Conclusion:Since molecular diagnosis of deletional mutations in the β-globin gene cluster warrants different approaches, it deserves special attention in order to provide prenatal diagnosis and prevention opportunities to the families involved. We conclude that the Turkish inversion/deletion (δβ)0, as the most prevalent deletional mutation detected so far, has to be routinely tested for in Antalya, and the gap-PCR approach has valuable diagnostic potential in the patients at risk.

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Detection of unknown deletions in β‐globin gene cluster using relative quantitative PCR methods

Cited by 17 publications

References 24 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

Integration of SNP and mRNA Arrays with MicroRNA Profiling Reveals That MiR-370 Is Upregulated and Targets NF1 in Acute Myeloid Leukemia

Gap-PCR Screening for Common Large Deletional Mutations of β-Globin Gene Cluster Revealed a Higher Prevalence of the Turkish Inversion/Deletion (δβ)0 Mutation in Antalya

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