A Multiplex qPCR Gene Dosage Assay for Rapid Genotyping and Large-Scale Population Screening for Deletional α-Thalassemia

Abstract

The predominant determinants of α-thalassemia are deletions in the human α-globin gene cluster. A rapid DNA-based assay is needed for mass screening in thalassemia-prevention programs. Herein, we established a novel quadruplex TaqMan qPCR gene dosage assay with two separate combination reactions. The assay directly determined the copy number of human α-globin genes based on relative quantitation of three target genes (HBA2, HBA1, and HBZ or HBPA1) versus a control gene (CREBBP). The assay showed good accuracy, with mean intra-assay and interassay variations of 3.31% ± 1.02% and 5.49% ± 0.32%, respectively. The assay was evaluated using 678 pretyped clinical DNA samples containing six α-thalassemia deletions in 13 genotypes and 186 normal samples previously screened by multiplex ligation-dependent probe amplification or gap PCR. As determined by the 2(-ΔΔCq) method, deleted gene dosage ratios were 0.46 to 0.60 in heterozygotes, 0.0 in homozygotes, and 0.97 to 1.07 in nondeleted samples. We found 99.3% concordance between the quantitative PCR and multiplex ligation-dependent probe amplification or gap-PCR results. Furthermore, routine screening for α-thalassemia deletions was performed on 3000 random samples in a blind analysis. Results for all 279 positives, which had different deletions, were fully coincident with results from standard methods. We also identified two novel deletions confirmed by multiplex ligation-dependent probe amplification. Assays using the novel method are simple and suitable for rapid genotyping and mass screening.

Mammalian target of rapamycin/p70 ribosomal S6 protein kinase signaling is altered by sevoflurane and/or surgery in aged rats

“…All PCR reactions used the following conditions: 94˚C for 3 min followed by 40 cycles at 94˚C for 30 sec and 72˚C for 45 sec. The data were analyzed using the ΔΔCq method (21), with β-actin as the internal control. The primer sequences, the expected PCR product sizes (base pairs; bp), and the annealing temperatures were as follows: mTOR, sense 5'-TGT CGA GAG GCA ACA ACC TC-3' and antisense 5'-TTG TTG GCT GCA TTG TGA CG-3' (298 bp, 60˚C); p70S6K, sense 5'-GGA TTT CTG GGG ACG AGG TG-3' and antisense 5'-AGA GTT GGG CTG TCG GAT TG-3' (148 bp, 58˚C); tau, sense 5'-CTG AAG AAG CAG GCA TCG GA-3' and antisense 5'-GGT GGT TCA CCT GAT CCT GG-3' (265 bp, 59˚C); caspase-3, sense 5'-CGG ACC TGT GGA CCT GAA AA-3' and antisense 5'-TAA CCG GGT GCG GTA GAG TA-3' (216 bp, 60˚C); β-actin, Statistical analysis.…”

A combination of the (αα)GZ and ‐‐SEA deletions causing a severe form of hemoglobin H disease

“…To conduct this study, multiplex ligation-dependent probe amplification (MLPA) assays were performed to identify a possible unknown deletion in the α-globin gene cluster. 7 Interestingly, we found that the proband had both the --SEA deletion and a novel deletion upstream of the α-globin gene ( Figure 2C), which was further tested by next-generation sequencing (NGS) for precisely defining the location of this novel deletion ( Figure 2D). The breakpoint of this deletion was confirmed by direct PCR amplification of about 800-bp products ( Figure 2E LETTER TO THE EDITOR of this novel deletion encompassed in the α-globin gene expression enhancer HS-40 that is an important binding region for numerous transcription factors.…”

A novel 223 kb deletion in the beta‐globin gene cluster was identified in a Chinese thalassemia major patient

“…Deletions in the human α-globin gene cluster which have a high incidence in south China were investigated by a quadruplex TaqMan qPCR gene dosage assay. 6 Sanger sequencing was performed to detect mutations in HBB gene (PCR primers were as follow: forward primer 5′-AACTCCTAAGCCAGTGCCAGTGCCAGAAGAGC-3′; reverse primer 5′-ATGCACTGACCTCCCACATTCCCT-3′). MLPA kits (MRC-Holland, Amsterdam, the Netherlands) were used to identify rearrangements in the beta-globin gene cluster (SALSA MLPA KIT P102 HBB).…”