Simultaneous Genotyping of α-Thalassemia Deletional and Nondeletional Mutations by Real-Time PCR–Based Multicolor Melting Curve Analysis
2017; Elsevier BV; Volume: 19; Issue: 4 Linguagem: Inglês
10.1016/j.jmoldx.2017.04.003
ISSN1943-7811
AutoresQiuying Huang, Xudong Wang, Ning Tang, Tizhen Yan, Ping Chen, Qingge Li,
Tópico(s)Forensic and Genetic Research
Resumoα-Thalassemia, which is caused by defective synthesis of the hemoglobin α-globin chains, is the most commonly inherited recessive hemoglobin abnormality. Genetic detection of a defective α-globin gene is challenging because of a variety of large deletions of the α-globin gene cluster and nondeletional mutations. Separate detections of them are often required using complex and error-prone open-tube methods. We report a novel real-time PCR–based assay that can simultaneously genotype four major deletional and three common nondeletional mutations in two parallel reactions by using multicolor melting curve analysis. The turnaround time of this closed-tube assay was within 3.5 hours, the limit of detection was 5 ng of human genomic DNA per reaction, and as low as 5% mutant DNA could be detected in the mosaic samples. The assay was evaluated using 1213 precharacterized genomic DNA samples in a double-blind manner. All seven α-thalassemia mutations were accurately genotyped, yielding a 99.3% concordance with the comparison assays. The 14 discordant samples contained the HKαα allele that was undetected by the traditional methods. Considering its rapidity, ease of use, and accuracy, we concluded that our real-time PCR assay may be recommended as an alternative screening and diagnostic tool for α-thalassemia. α-Thalassemia, which is caused by defective synthesis of the hemoglobin α-globin chains, is the most commonly inherited recessive hemoglobin abnormality. Genetic detection of a defective α-globin gene is challenging because of a variety of large deletions of the α-globin gene cluster and nondeletional mutations. Separate detections of them are often required using complex and error-prone open-tube methods. We report a novel real-time PCR–based assay that can simultaneously genotype four major deletional and three common nondeletional mutations in two parallel reactions by using multicolor melting curve analysis. The turnaround time of this closed-tube assay was within 3.5 hours, the limit of detection was 5 ng of human genomic DNA per reaction, and as low as 5% mutant DNA could be detected in the mosaic samples. The assay was evaluated using 1213 precharacterized genomic DNA samples in a double-blind manner. All seven α-thalassemia mutations were accurately genotyped, yielding a 99.3% concordance with the comparison assays. The 14 discordant samples contained the HKαα allele that was undetected by the traditional methods. Considering its rapidity, ease of use, and accuracy, we concluded that our real-time PCR assay may be recommended as an alternative screening and diagnostic tool for α-thalassemia. α-Thalassemia is the most commonly inherited recessive hemoglobin (Hb) abnormality in the tropical and subtropical regions.1Frédéric B.P. David J.W. The α-thalassemias.N Engl J Med. 2014; 371: 1908-1916Crossref PubMed Scopus (184) Google Scholar It is characterized by a reduction or complete absence of the Hb α-chains, resulting mainly from large deletions of the α-globin gene cluster and occasionally from nondeletional mutations. The α-globin gene cluster is located on chromosome 16p13.3, which contains two nearly identical α-globin genes (HBA2 and HBA1) with high guanine-cytosine (GC) content. The most common causes of α-thalassemia are the loss of one (-α) or two (--) of the α-globin genes, which usually do not manifest in any clinical symptoms. The deletion of three α-globin genes (--/-α) results in Hb H disease, and the complete deletion of four α-globin genes leads to severe anemia in utero (Hb Bart's hydrops fetalis).2Chui D.H. Alpha-thalassemia: Hb H disease and Hb Barts hydrops fetalis.Ann N Y Acad Sci. 2005; 1054: 25-32Crossref PubMed Scopus (80) Google Scholar Moreover, the nondeletional mutations are noteworthy because they can cause a more severe Hb H disease than the pure deletional mutations when combined with deletions of two α-globin genes.3Farashi S. Bayat N. Vakili S. Faramarzi G.N. Ashki M. Imanian H. Najmabadi H. Azarkeivan A. Point mutations which should not be overlooked in Hb H disease.Expert Rev Hematol. 2015; 9: 1-7PubMed Google Scholar The most common deletional α-thalassemia mutations are the -α3.7, -α4.2, --SEA, and --THAI in Southeast Asia and China, whereas the most prevalent nondeletional mutations are the codon 142 TAA→CAA (Hb Constant Spring, c.427T>C), the codon 125 CTG→CCG (Hb Quong Sze, Hb QS, c.377T>C), and the codon 122 CAC→CAG (Hb Westmead, c.369T>C) in the HBA2 gene [National Center for Biotechnology Information (NCBI) reference sequence: NG_000006.1] in China.4Harteveld C.L. Higgs D.R. Alpha-thalassaemia.Orphanet J Rare Dis. 2010; 5: 53-90Crossref Scopus (387) Google Scholar, 5Traeger-Synodinos J. Molecular basis of a-thalassaemia.Thalassemia Reports. 2012; 3: a011718Google Scholar Therefore, genotyping of both deletional and nondeletional mutations is essential for carrier screening, prenatal diagnosis, and newborn screening for α-thalassemia in these geographic regions. A variety of methods have been developed for the diagnosis of α-thalassemia mutations, including multiplex gap–PCR,6Chong S.S. Boehm C.D. Cutting G.R. Higgs D.R. Simplified multiplex-PCR diagnosis of common Southeast Asian deletional determinants of alpha-thalassemia.Clin Chem. 2000; 46: 1692-1695PubMed Google Scholar, 7Liu Y.T. Old J.M. Miles K. Fisher C.A. Weatherall D.J. Clegg J.B. Rapid detection of α-thalassaemia deletions and α-globin gene triplication by multiplex polymerase chain reactions.Br J Haematol. 2000; 108: 295-299Crossref PubMed Scopus (314) Google Scholar reverse dot blotting (RDB) assay,8Chan V. Yam I. Chen F.E. Chan T.K. A reverse dot-blot method for rapid detection of non-deletion α thalassaemia.Br J Haematol. 1999; 104: 513-515Crossref PubMed Scopus (58) Google Scholar, 9Lin M. Zhu J.J. Wang Q. Xie L.X. Lu M. Wang J.L. Wang C.F. Zhong T.Y. Zheng L. Pan M.C. Development and evaluation of a reverse dot blot assay for the simultaneous detection of common alpha and beta thalassemia in Chinese.Blood Cells Mol Dis. 2011; 48: 86-90Crossref PubMed Scopus (36) Google Scholar amplification refractory mutation system,10Newton C.R. Graham A. Heptinstall L.E. Powell S.J. Summers C. Kalsheker N. Smith J.C. Markham A.F. Analysis of any point mutation in DNA: the amplification refractory mutation system (ARMS).Nucleic Acids Res. 1989; 17: 2503-2516Crossref PubMed Scopus (2089) Google Scholar denaturing high-pressure liquid chromatography,11Lacerra G. Fiorito M. Musollino G. Di N.F. Esposito M. Nigro V. Gaudiano C. Carestia C. Sequence variations of the alpha-globin genes: scanning of high CG content genes with DHPLC and DG-DGGE.Hum Mutat. 2004; 24: 338-349Crossref PubMed Scopus (32) Google Scholar, 12Liu J. Jia X. Tang N. Zhang X. Wu X. Cai R. Wang L. Liu Q. Xiao B. Zhu J. Novel technique for rapid detection of alpha-globin gene mutations and deletions.Transl Res. 2010; 155: 148-155Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar capillary electrophoresis analysis,13Liao Y.M. Lin S.K. Liu T.C. Chiou S.S. Lu H.C. Kao C.F. Chang J.G. Rapid identification of the copy number of α-globin genes by capillary electrophoresis analysis.Clin Biochem. 2012; 45: 798-805Crossref PubMed Scopus (5) Google Scholar and sequencing. A common drawback of these methods is that they involve multiple post-PCR manipulation steps that are lengthy, labor intensive, and amenable to amplicon contamination. Moreover, separate reactions are often required to detect both deletional and nondeletional mutations, further increasing the complexity of the detection procedure. Real-time PCR–based methods for the diagnosis of α-thalassemia mutations have also been described, including dye-based melting curve analysis,14Liu J. Yan M. Wang Z. Wang L. Zhou Y. Xiao B. Molecular diagnosis of α-thalassemia by combining real-time PCR with SYBR Green1 and dissociation curve analysis.Transl Res. 2006; 148: 6-12Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 15Munkongdee T. Vattanaviboon P. Thummarati P. Sewamart P. Winichagoon P. Fucharoen S. Svasti S. Rapid diagnosis of α-thalassemia by melting curve analysis.J Mol Diagn. 2010; 12: 354-358Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar multiplex real-time quantitative PCR assay,16Zhou W. Ge W. Zhao X. Fu X. Zhou S. Peng J. Cheng Y. Xu S. Xu X. A multiplex qPCR gene dosage assay for rapid genotyping and large-scale population screening for deletional α-thalassemia.J Mol Diagn. 2013; 15: 642-651Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar and probe-based melting curve analysis.17Lan G. Liu Y. Sun M. Ying Z. Xie R. Yi H. Xu W. Liu J. Lin Y. Lou J. Rapid detection of α-thalassaemia alleles of -- SEA/, -α 3.7/ and -α 4.2/ using a dual labelling, self-quenching hybridization probe/melting curve analysis.Mol Cell Probes. 2015; 29: 438-441Crossref PubMed Scopus (4) Google Scholar, 18Timmann C. Moenkemeyer F. Evans J.A. Foerster B. Tannich E. Haase S. Sievertsen J. Kohne E. Horstmann R.D. Diagnosis of alpha+-thalassemias by determining the ratio of the two alpha-globin gene copies by oligonucleotide hybridization and melting curve analysis.Clin Chem. 2005; 51: 1711-1713Crossref PubMed Scopus (7) Google Scholar, 19Huang Q. Wang X. Tang N. Zhu C. Yan T. Chen P. Li Q. Rapid detection of non-deletional mutations causing α-thalassemia by multicolor melting curve analysis.Clin Chem Lab Med. 2016; 54: 397-402Crossref PubMed Scopus (10) Google Scholar Despite their closed-tube nature and ease of use, current real-time PCR–based methods can neither distinguish between different genotypes nor simutaneously detect the deletional and nondeletional mutations. An improved method that allows for complete genotyping of the deletional and nondeletional mutations in one assay is thus warranted. In this report, we describe a novel real-time PCR–based multicolor melting curve analysis (MMCA) that allows simultaneous genotyping of four deletional (-α3.7, -α4.2, --SEA, and --THAI) and three nondeletional α-thalassemia mutations (c.369 C>G, c.377 T>C, and c.427 T>C). We systematically studied its analytical performance and evaluated its clinical performance by using 1213 human genomic DNA (gDNA) samples from two different hospitals in southern China. Twenty-four whole-blood samples of known α-globin gene genotypes, including four αα/αα, two --SEA/αα, two -α4.2/αα, two -α3.7/αα, one --THAI/αα, one --THAI/--SEA, one -α3.7/-α3.7, one -α4.2/-α3.7, one -α4.2/-α4.2, one --SEA/-α3.7, one --SEA/-α4.2, one --SEA/-αCSα, one --SEA/-αWSα, two αCSα/αα, one αQSα/αα, one αWSα/αα, and one αCSα/αWSα, were used for assay development. The gDNA was extracted using Lab-Aid 820 Nucleic Acid Extraction System (Zeesan Biotech, Xiamen, China) according to the manufacturer's instructions. Concentrations were determined by ND-1000 UV-VIS spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). The extracted gDNA was stored at −20°C before use. For clinical evaluation, 1213 gDNA samples (including 936 gDNA samples extracted from whole blood and 277 gDNA samples extracted from amniotic fluid) previously genotyped by multiple gap–PCR/RDB analysis were collected from the First Affiliated Hospital of Guangxi Medical University and Liuzhou Maternal and Child Health Care Hospital. The identities of the gDNA samples and their respective genotypes were not disclosed to the individuals who performed the testing. The Research Ethics Committee of each hospital approved this study, and informed consent was obtained from the individual patients or their guardians. DNA sequences of the α-globin gene cluster (NCBI reference sequence: NG_000006.1) were retrieved from the NCBI website (http://www.ncbi.nlm.nih.gov). According to the principles of gap-PCR and MMCA, four sets of primers specific for the α-globin gene cluster, five probes targeting the seven α-thalassemia mutations, and one set of primers and one probe specific for the ABL gene (internal positive control) (Table 1) were designed using the TM Utility version 1.3 (Idaho Technologies Inc., Salt Lake City, UT) and Oligo 6 (AVG Technologies Inc., Chelmsford, MA). All the primers and probes were synthesized and purified by Sangon (Shanghai, China).Table 1Sequence Information of Primers and Probes Used in the Multicolor Melting Curve Analysis AssayReactionNameSequenceGenBank accession no.: nucleotidesAmplicon sizeAABL-F5′-GAACGAAGCTGGTTTCCAAAG-3′NC_000009.12: 140867 → 140887164 bpABL-R5′-CTGTTGACTGGCGTGATGTAG-3′NC_000009.12: 141029 → 141009P15′-FAM-CCCGGAGCTTTTCACCTGAG-BHQ1-3′NC_000009.12: 140935 → 1409163.7-F5′-CCCCTCACCTACATTCTGCAAC-3′NG_000006.1: 32785 → 328063.7-R5′-GCCCACTCRGACTTTATTCAAAGA-3′NG_000006.1: 34569 → 345461785 bp∗The amplicon size of wild-type allele.NG_000006.1: 38381 → 38358∼1.8 kb†The estimated amplicon size of deletional allele.P25′-ROX-CGTCAAGCTGGAGCCTCGGTAGCCG-BHQ2-3′NG_000006.1: 34458 → 34482NG_000006.1: 38269 → 38292P35′-CY5-CGGTGCACGCCTCCCCGGACAAGTTCC-BHQ2-3′NG_000006.1: 34396 → 34422BSEA-F1‡The two forward primers amplified with the universal reverse primer.5′-AGAGGCTGAGGTGGGAAG-3′NT_037887.4: 174570 →174587210 bpSEA-F2‡The two forward primers amplified with the universal reverse primer.5′-GATCTGGGCTCTGTGTTCTC-3′NG_000006.1: 26121 → 26140221 bpSEA-R5′-GGAGTGCAGTGTTGTAGTCA-3′NT_037887.4: 174779 → 174760P45′-FAM-CCAGTTACTTGGAGGCGGGCAGGA-BHQ1-3′NT_037887.4: 174694 → 1747194.2-F5′-GCACTTCCTGATCTTTGAATGA-3′NG_000006.1: 30277 → 302994.2-R5′-CCGTGCCTGTCGGATTTTAG-3′NG_000006.1: 31374 → 313551098 bp∗The amplicon size of wild-type allele.NG_000006.1: 35630 → 35610∼1.1 kb†The estimated amplicon size of deletional allele.P55′-ROX-CCAGAGCATTGTTATTTCAGCAGAAACACT-BHQ2-3′NG_000006.1: 31313 → 31342NG_000006.1: 35568 → 35597THAI-F1‡The two forward primers amplified with the universal reverse primer.5′-GCTCTCAGACTCCTGTAACT-3′NT_037887.4: 173187 → 173206238 bpTHAI-F2‡The two forward primers amplified with the universal reverse primer.5′-CATTTGCCTTTGACTGCATC-3′NG_000006.1: 10494 → 10513347 bpTHAI-R5′-CTTGAGTGGGCATGAGTC-3′NT_037887.4: 173424 → 173407P65′-CY5-CCCTGGCTCAAGGGCTCAGCCCA-BHQ2-3′NT_037887.4: 173306 → 173328GenBank can be found at: https://www.ncbi.nlm.nih.gov/genbank∗ The amplicon size of wild-type allele.† The estimated amplicon size of deletional allele.‡ The two forward primers amplified with the universal reverse primer. Open table in a new tab GenBank can be found at: https://www.ncbi.nlm.nih.gov/genbank PCR and melting curve analysis were performed on a SLAN-96P real-time system (Hongshi Medical Technology Co., Ltd., Shanghai, China) in a 25-μL reaction that contained 1× PCR buffer [67 mmol/L Tris-HCl (pH 8.8), 16 mmol/L (NH4)2SO4, and 0.01% Tween-20 (w/v)], 5.0 mmol/L MgCl2, 0.4 mmol/L dNTPs, 7% dimethyl sulfoxide, 3.0 U of hot-start Taq polymerase (TaKaRa, Dalian, China), 0.02 to 0.4 μmol/L limiting primers, 0.4 to 2.0 μmol/L excess primers, 0.3 to 0.4 μmol/L probes, and 5 μL of extracted gDNA. The reaction started with a contamination control procedure of 2 minutes at 50°C to prevent carryover of DNA amplicons by uracil-N-glycosylase. After a denaturation step at 95°C for 10 minutes, a touchdown program was performed with 10 cycles at 95°C for 30 seconds, 70°C for 30 seconds (−1°C per cycle), and 76°C for 45 seconds, followed by 50 cycles at 95°C for 30 seconds, 60°C for 30 seconds, and 76°C for 45 seconds. Melting curve analysis started with a denaturation step of 1 minute at 95°C, a hybridization step of 3 minutes at 37°C, and a continuous temperature increase from 45°C to 85°C at a ramp rate of 0.04°C/s. Fluorescence data from the FAM, ROX, and CY5 channels were recorded at the annealing step during the second 50 cycles and at each step of continuous temperature increase during the melting curve analysis procedure. Melting curves were obtained by plotting the negative derivative of fluorescence with respect to temperature versus temperature (−dF/dT), and the Tm values were obtained automatically from the melting curves through the software of the SLAN-96P real-time PCR system (Hongshi Medical Technology Co., Ltd.). To study the reproducibility of Tm measurement, 13 gDNA samples of known genotypes (four αα/αα, one -α3.7/αα, one -α3.7/-α3.7, one αCSα/αα, one αWSα/αα, one αQSα/αα, one --SEA/αα, one -α4.2/αα, one -α4.2/-α4.2, and one --THAI/αα) were analyzed by two technicians. Each technician detected all the above gDNA samples in duplicate on five consecutive days. The Tm value and the Tm difference (ΔTm) between the wild-type and mutant peaks were measured. To study the analytical sensitivity of the MMCA assay, eight gDNA samples of known genotypes (one αα/αα, one -α3.7/αα, one αCSα/αα, one αWSα/αα, one αQSα/αα, one --SEA/αα, one -α4.2/αα, and one --THAI/αα) were serially diluted with 10 mmol/L Tris-HCl that contained 1 mmol/L EDTA (pH 8.5), yielding gDNA concentrations of 20, 2, 1, and 0.2 ng/μL, respectively. The limit of detection was defined as the lowest concentration that gave no more than one negative result in 20 replicates (ie, positive rate ≥95%) for all the above genotypes. To determine the lowest variant allele frequency for the detection of mosaic samples, mutant plasmids (2 × 104 copies/μL) of -α3.7, c.369 C>G, --SEA, and -α4.2 were mixed with corresponding wild-type plasmids, respectively, to generate mixed samples containing 0%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% mutant DNA, respectively, and each sample was analyzed in triplicate. A double-blind analysis (X.W. and Q.H.) was performed for the 1213 gDNA samples by the MMCA assay. The results were compared with the original results that were previously determined using a commercial gap–PCR kit (Yishengtang Biological Products Co. Ltd., Shenzhen, China), which could detect the four deletional mutations -α3.7, -α4.2, --SEA, and --THAI, and a commercial RDB kit (Yaneng Bio, Shenzhen, China), which could detect the three nondeletional mutations Hb Constant Spring, Hb QS, and Hb Westmead. The means ± 3 SDs were used to estimate the reproducibility of the MMCA assay. Statistical calculations were performed with the SPSS software version 14.0 (SPSS Inc., Chicago, IL). The working principle of the MMCA assay is illustrated in Figure 1. To genotype deletions of known breakpoints (--SEA and --THAI), three primers were used and a probe was designed for each deletion to span the breakpoint juncture. The presence of a deletion would result in a lower Tm value than that of the wild-type allele (Figure 2). To genotype deletions of unknown breakpoints (-α3.7 and -α4.2), the forward primer was designed upstream of the homologous region, whereas the reverse primer and the probe were inside the homologous region (-α4.2 in X sequence homology box, -α3.7 in Z sequence homology box). The probe-binding region contained variant sites so that the presence of the deletion would generate a Tm value different from that of the wild type (Figure 2). The three nondeletional mutations were genotyped using one primer pair (shared with -α3.7) and two probes. In addition, the PCR product from the ABL gene served as an internal positive control for general PCR amplification success. To avoid cross-amplification, the primers of -α3.7 and -α4.2 were placed in separate reactions. The formed assay had two reactions. Reaction A contained four primers (ABL-F, ABL-R, 3.7-F, and 3.7-R) and three differently labeled self-quenched probes (P1, P2, and P3) targeting the ABL gene, -α3.7, c.369 C>G, c.377 T>C, c.427 T>C. Reaction B contained eight primers (SEA-F1, SEA-F2, SEA-R, 4.2-F, 4.2-R, THAI-F1, THAI-F2, and THAI-R) and three differently labeled self-quenched probes (P4, P5, and P6) targeting --SEA, -α4.2, and --THAI, respectively.Figure 2Melting curve results from genomic DNA samples with various α-globin genotypes. Melting curves and corresponding genotypes of the four deletions and three nondeletional mutations and the wild-type (WT) allele are shown by their corresponding probes. Gray lines indicate the no template control. −dF/dT, negative derivative of fluorescence with respect to temperature versus temperature.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Using the established assay, we successfully determined the genotypes of all the mutations from the reference samples with a resolution of ΔTm > 4°C, where ΔTm represents the difference between Tm of the wild-type and mutant alleles. The reproducibility study found that the SDs of Tm and ΔTm were less than 1°C in the replicate analysis (Table 2). These results indicate unequivocal distinction between the wild-type and mutant alleles. The analytical sensitivity study found that the assay could repeatedly obtain the genotypes from the reference samples ranged from 5 to 100 ng of gDNA per reaction. The limit of detection of the MMCA assay was determined to be 5 ng of gDNA per reaction, which was low enough to allow for clinical samples from various sources. The lowest variant allele frequency study found that as low as 5% mutant DNA could be repeatedly detected for all kinds of mutations (Supplemental Figure S1).Table 2The Tm of Four Deletions and Three Nondeletional Mutations in the Multicolor Melting Curve Analysis AssayProbeGenotypeTm ± 3 SDs, °CΔTm ± 3 SDs, °CP1Control60.08 ± 0.69NAP2Wild-type66.50 ± 0.70NA-α3.757.97 ± 0.718.63 ± 0.18c.427T>C (Hb Constant Spring)71.46 ± 0.46−5.50 ± 0.13P3Wild-type69.04 ± 0.59NAc.369C>G (Hb WS)60.75 ± 0.508.35 ± 0.33c.377T>C (Hb QS)74.38 ± 0.51−5.70 ± 0.29P4Wild-type64.97 ± 0.68NA--SEA56.90 ± 0.788.29 ± 0.12P5Wild-type63.46 ± 0.81NA-α4.268.03 ± 0.58−4.84 ± 0.18P6Wild-type67.91 ± 0.65NA--THAI61.44 ± 0.276.62 ± 0.31ΔTm, Tm (wild-type) − Tm (mutant).Hb, hemoglobin; NA, not applicable; QS, Quong Sze; WS, Westmead. Open table in a new tab ΔTm, Tm (wild-type) − Tm (mutant). Hb, hemoglobin; NA, not applicable; QS, Quong Sze; WS, Westmead. To validate the clinical performance of the assay, we performed a double-blind analysis (N.T., T.Y., and P.C.) of 1213 gDNA samples that were precharacterized using the gap-PCR/RDB analysis. All seven targeted mutant alleles were detected, and together with the wild-type allele, they formed 31 genotypes that represented 1199 samples (Table 3). By referring to the recorded genotypes, all these samples were correctly genotyped by our assay.Table 3Genotypes of α-Thalassemia Detected in 1213 Clinical SamplesGenotypeMMCAGap-PCR/RDBGenotypeMMCAGap-PCR/RDBαα/αα112112--SEA/-α3.75555-α3.7/-α3.72222--SEA/-α4.22626-α4.2/-α3.71616--SEA/αWSα1919αCSα/αCSα66--SEA/αCSα1212-α3.7/αCSα66--SEA/αQSα66-α4.2/αCSα55--SEA/HKαα33-α4.2/-α4.255--THAI/αWSα11-α3.7/αWSα55--THAI/αCSα11αCSα/αWSα44--THAI/-α3.711αWSα/αWSα22-α3.7/αα340348-α3.7/αQSα22-α4.2/αα177177HKαα/-α4.222αCSα/αα2525αQSα/αWSα11αWSα/αα1818-α4.2/αWSα11αQSα/αα55-α4.2/αQSα11--SEA/αα290290HKαα/αCSα11--THAI/αα66HKαα/-α3.71ND--SEA/--SEA2828HKαα/αα7ND--SEA/--THAI11MMCA, multicolor melting curve analysis; ND, not detected; RDB, reverse dot blotting. Open table in a new tab MMCA, multicolor melting curve analysis; ND, not detected; RDB, reverse dot blotting. Unexpectedly, we detected a ninth allele, HKαα, which contains -α3.7 and αααanti 4.2, in the remaining 14 samples. Five additional genotypes were formed by HKαα, of which HKαα/--SEA, HKαα/-α4.2, and HKαα/αCSα were discerned by the characteristic melting peaks of the corresponding alleles (Figure 3). The other two genotypes, HKαα/αα and HKαα/-α3.7, were determined through the height ratio of the melting peaks of -α3.7 to the wild type (Figure 3). These two genotypes were, however, mistakenly recorded as -α3.7/αα because of their identical patterns in the gap-PCR/RDB assay. These HKαα-derived genotypes obtained by the MMCA assay were confirmed by a two-round PCR assay (Supplemental Figure S2).20Wang W. Chan A.Y. Chan L.C. Ma E.S. Chong S.S. Unusual rearrangement of the alpha-globin gene cluster containing both the -alpha3.7 and alphaalphaalphaanti-4.2 crossover junctions: clinical diagnostic implications and possible mechanisms.Clin Chem. 2005; 51: 2167-2170Crossref PubMed Scopus (26) Google Scholar Collectively, the MMCA assay accurately detected the genotypes of all 1213 samples. In this report, we successfully established an MMCA assay for the complete genotyping of four major deletions (-α3.7, -α4.2, --SEA, and --THAI) and three common nondeletional mutations (c.369 C>G, c.377 T>C, and c.427 T>C) in two reactions. The reproducibility study found that the assay was highly reproducible as indicated by the Tm measurements and accurate genotypes. The analytical sensitivity study found that as little as 5 ng of gDNA per reaction could be detected; thus, clinical samples from different sources should yield sufficient gDNA for the MMCA assay. It is a challenging task to establish a real-time PCR–based genotyping assay for multiple mutations of the α-globin gene because of the extremely high sequence homology between α1 and α2 (>96%) and the requirement of amplifying long (>1.5 kb) and GC-rich regions (average of >60%).11Lacerra G. Fiorito M. Musollino G. Di N.F. Esposito M. Nigro V. Gaudiano C. Carestia C. Sequence variations of the alpha-globin genes: scanning of high CG content genes with DHPLC and DG-DGGE.Hum Mutat. 2004; 24: 338-349Crossref PubMed Scopus (32) Google Scholar Previously, an MCA assay was proposed for the diagnosis of α+-thalassemia by determining the ratio of the two α-globin alleles without differentiating between -α3.7 and -α4.2.18Timmann C. Moenkemeyer F. Evans J.A. Foerster B. Tannich E. Haase S. Sievertsen J. Kohne E. Horstmann R.D. Diagnosis of alpha+-thalassemias by determining the ratio of the two alpha-globin gene copies by oligonucleotide hybridization and melting curve analysis.Clin Chem. 2005; 51: 1711-1713Crossref PubMed Scopus (7) Google Scholar A dye-based real-time PCR MCA assay was developed for genotyping the -α3.7, -α4.2, --SEA, and c.427 T>C (αCSα) alleles individually in different reactions.14Liu J. Yan M. Wang Z. Wang L. Zhou Y. Xiao B. Molecular diagnosis of α-thalassemia by combining real-time PCR with SYBR Green1 and dissociation curve analysis.Transl Res. 2006; 148: 6-12Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar An improved dye-based real-time PCR MCA assay was developed that allowed for simultaneous genotyping of -α3.7, -α4.2, --SEA, and --THAI alleles in one reaction. However, this assay was unable to identify the -α3.7/αα, -α4.2/αα, -α3.7/-α4.2, and αα/αα alleles.15Munkongdee T. Vattanaviboon P. Thummarati P. Sewamart P. Winichagoon P. Fucharoen S. Svasti S. Rapid diagnosis of α-thalassemia by melting curve analysis.J Mol Diagn. 2010; 12: 354-358Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar Recently, a real-time PCR assay was proposed to detect the relative gene dosage of α1 and α2 without genotyping.16Zhou W. Ge W. Zhao X. Fu X. Zhou S. Peng J. Cheng Y. Xu S. Xu X. A multiplex qPCR gene dosage assay for rapid genotyping and large-scale population screening for deletional α-thalassemia.J Mol Diagn. 2013; 15: 642-651Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar Recently, an MCA assay was developed for simultaneous genotyping of --SEA, -α3.7, and -α4.2 but was unable to differentiate among the -α3.7/αα, -α4.2/αα, -α3.7/-α4.2, and αα/αα alleles.17Lan G. Liu Y. Sun M. Ying Z. Xie R. Yi H. Xu W. Liu J. Lin Y. Lou J. Rapid detection of α-thalassaemia alleles of -- SEA/, -α 3.7/ and -α 4.2/ using a dual labelling, self-quenching hybridization probe/melting curve analysis.Mol Cell Probes. 2015; 29: 438-441Crossref PubMed Scopus (4) Google Scholar Consequently, all these real-time PCR assays failed to compete with the traditional gap-PCR/RDB assay with regard to genotyping coverage. We previously found that MMCA could detect multiple mutations in a single reaction by using a mixture of dual-labeled, self-quenched probes.21Huang Q. Liu Z. Liao Y. Chen X. Zhang Y. Li Q. Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes.PLoS One. 2011; 6: e19206Crossref PubMed Scopus (75) Google Scholar It has been successfully used to detect point mutations and small indels.19Huang Q. Wang X. Tang N. Zhu C. Yan T. Chen P. Li Q. Rapid detection of non-deletional mutations causing α-thalassemia by multicolor melting curve analysis.Clin Chem Lab Med. 2016; 54: 397-402Crossref PubMed Scopus (10) Google Scholar, 22Fu X. Huang Q. Chen X. Zhou Y. Zhang X. Ren C. Chen Y. Xie J. Feng S. Wei X. A melting curve analysis–based PCR assay for one-step genotyping of β-thalassemia mutations: a multicenter validation.J Mol Diagn. 2011; 13: 427-435Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 23Xia Z. Chen P. Tang N. Yan T. Zhou Y. Xiao Q. Huang Q. Li Q. Rapid detection of G6PD mutations by multicolor melting curve analysis.Mol Genet Metab. 2016; 119: 168-173Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 24Hu S. Li G. Li H. Liu X. Niu J. Quan S. Wang F. Wen H. Xu Y. Li Q. Rapid detection of isoniazid resistance in mycobacterium tuberculosis isolates by use of real-time-PCR-based melting curve analysis.J Clin Microbiol. 2014; 52: 1644-1652Crossref PubMed Scopus (28) Google Scholar However, no large deletions have been detected. To address this problem, our strategy was to convert deletions into mutations by taking advantage of gap-PCR and the homologous sequences in the α-globin gene cluster. There are two types of large deletions in this study: one with known breakpoints (ie, --SEA or --THAI) and the other with unknown breakpoints (ie, -α3.7 or -α4.2). To detect the former deletions, we designed a probe spanning the breakpoint juncture together with one reverse primer and two forward primers, one inside and the other outside the deletion region. To detect the latter deletions, a probe spanning the variant sites of the homologous region was designed. The forward primer was located upstream of the homologous region, and the reverse primer was inside the homologous region (-α4.2 in X sequence homology box, -α3.7 in Z sequence homology box). The above designs allow all the large deletions to be distinguished from their wild-type counterparts by the differences in Tm value, which is similar to how single-nucleotide mutations are detected. Notably, by using the peak height ratio, our MMCA assay could further identify those HKαα-derived genotypes that were undetected by the traditional methods. Another difficulty lies in the amplification of long and GC-rich regions of the α-globin gene cluster by probe-based real-time PCR. To overcome this problem, dimethyl sulfoxide was added into the PCR reaction to destabilize the secondary structure of the amplicon.25Musso M. Bocciardi R. Parodi S. Ravazzolo R. Ceccherini I. Betaine, dimethyl sulfoxide, and 7-deaza-dGTP, a powerful mixture for amplification of GC-rich DNA sequences.J Mol Diagn. 2006; 8: 544-550Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar A hot-start Taq polymerase was used to alleviate nonspecific amplification. Moreover, extension at a high temperature up to 76°C during PCR was used to eliminate hindrance of the probe on primer extension.19Huang Q. Wang X. Tang N. Zhu C. Yan T. Chen P. Li Q. Rapid detection of non-deletional mutations causing α-thalassemia by multicolor melting curve analysis.Clin Chem Lab Med. 2016; 54: 397-402Crossref PubMed Scopus (10) Google Scholar Overall, our MMCA assay could work efficiently even if the amplicon is approximately 1.8 kb in length and has approximately 67.8% GC content. To our knowledge, this is the first time that all the major α-thalassemia alleles were successfully genotyped by a real-time PCR-based method. As a closed-tube method, the MMCA assay has many advantages over the widely used open-tube methods, such as multiplex gap–PCR analysis and RDB assay. First, it requires no complex post-PCR manipulations and largely avoids the risk of PCR contamination, both of which are often troublesome in open-tube methods. Second, the MMCA assay has significantly higher throughput. The entire procedure involves only a simple step of adding gDNA into two reaction tubes when the gDNA is available. The MMCA assay can be automatically accomplished within 3.5 hours. Third, the SLAN-96 system used in this study has been developed and systematically calibrated by reference gDNA samples (Table 2), enabling accurate identification of the wild-type and mutation peaks at predefined Tm positions and automatic output of the genotyping results with no manual interpretation. Fourth, the assay is more cost-effective because no additional materials are required except for the PCR in MMCA, whereas extra materials for post-PCR manipulations are needed in traditional methods. By our calculations, the material cost is approximately $3 USD per sample for MMCA analysis. In comparison, the cost is approximately $6 USD per sample for gap-PCR/RDB analysis. Although real-time PCR machines are required for MMCA, they are now commonly available in clinical laboratories; thus, introduction of this assay may not add economic burden. In conclusion, we developed a novel real-time PCR-based MMCA assay that achieves the full genotyping of four major deletional and three common nondeletional mutations in one assay. This assay is rapid, accurate, and cost-effective and could be recommended as an alternative screening and diagnostic tool for α-thalassemia in Southeast Asia and Southern China. Download .pdf (.32 MB) Help with pdf files Supplemental Figure S1Melting curves for different percentages of mosaic samples. Melting curves were from mimic mosaic samples with different percentages (0%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%) of -α3.7 (A), c.369 C>G (B), --SEA (C), and -α4.2 (D). Both wild-type and mutant-type templates were plasmid DNAs, and the concentration was set at 2 × 104 copies/μL. Gray lines represent no template control. Download .pdf (3.12 MB) Help with pdf files Supplemental Figure S2Determination of the HKαα allele by a two-round PCR assays. After the first-round PCR, large fragments of approximately 4 kb were observed in all samples (top panel). In the nested -α3.7 PCR, an approximately 2.0-kb -α3.7 junction fragment was observed only in cases 1 to 14 (middle panel). A similar result is also observed in the nested αααanti4.2 PCR, where an approximately 1.7-kb αααanti4.2 junction fragment is observed in the same 14 samples (bottom panel). The genotypes of cases 1, 13, and 14; case 2; cases 3 to 9 and 10; and cases 11 and 12 are HKαα/--SEA, HKαα/-α3.7, HKαα/αα, HKαα/-αCSα, and HKαα/-α4.2, respectively.
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