Rapid detection of non-deletional mutations causing α-thalassemia by multicolor melting curve analysisClinical Chemistry and Laboratory Medicine (CCLM)

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Authors
Qiuying Huang, Xudong Wang, Ning Tang, Chunjiang Zhu, Tizhen Yan, Ping Chen, Qingge Li
Year
2015
DOI
10.1515/cclm-2015-0173
Subject
Clinical Biochemistry / Biochemistry, medical

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Clin Chem Lab Med 2015; aop aThese authors contributed equally to this work. *Corresponding authors: Ping Chen, Guangxi Key Laboratory of Thalassemia Research, Guangxi Zhuang Autonomous Region,

Nanning 530021, China, E-mail: cping62@hotmail.com; and

Hemoglobin Laboratory, the First Affiliated Hospital of Guangxi

Medical University, Guangxi Key Laboratory of Thalassemia

Research, Guangxi Zhuang Autonomous Region, Nanning,

China; and Qingge Li, Engineering Research Center of Molecular

Diagnostics, Ministry of Education, School of Life Sciences, Xiamen

University, Xiamen 361005, Fujian, China, E-mail: qgli@xmu.edu.cn; and State Key Laboratory of Cellular Stress Biology, State Key

Laboratory of Molecular Vaccinology and Molecular Diagnostics,

Engineering Research Centre of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen,

Fujian, China

Qiuying Huang and Xudong Wang: State Key Laboratory of Cellular

Stress Biology, State Key Laboratory of Molecular Vaccinology and

Molecular Diagnostics, Engineering Research Centre of Molecular

Diagnostics, Ministry of Education, School of Life Sciences, Xiamen

University, Xiamen, Fujian, China

Ning Tang and Tizhen Yan: Department of Clinical Laboratory,

Liuzhou Key Laboratory of Birth Defects Prevention and Control,

Liuzhou Maternal and Child Health Hospital, Liuzhou, Guangxi, China

Chunjiang Zhu: Department of Genetics, Affiliated Hospital of Guilin

Medical University, Guilin, Guangxi, China

Qiuying Huanga, Xudong Wanga, Ning Tang, Chunjiang Zhu, Tizhen Yan, Ping Chen* and Qingge Li*

Rapid detection of non-deletional mutations causing α-thalassemia by multicolor melting curve analysis

DOI 10.1515/cclm-2015-0173

Received February 19, 2015; accepted July 22, 2015

Abstract

Background: α-Thalassemia, caused by mutations in the α-globin genes, is one of the most common monogenic inherited disorders in the world. However, non-deletional α-thalassemia mutations remain undetected in routine clinical testing due to the lack of a suitable method. In this study, a closed- and single-tube assay for the detection of six common non-deletional α-thalassemia mutations in the HBA2 gene was developed based on multicolor melting curve analysis.

Methods: The assay consisted of one pair of primers specific for the HBA2 gene and four dual-labeled, self-quenched probes targeting six non-deletional α-thalassemia mutations. The sensitivity, reproducibility, and accuracy of the method were validated via 700 genomic DNA samples.

Results: The assay had a reproducibility of 100%, could detect gDNA of different genotype as low as 1 ng per reaction, and had an overall accuracy of 100% when compared with RDB analysis and Sanger sequencing.

Conclusions: The developed assay is rapid, robust, and cost-effective while maintaining high sensitivity, specificity, and throughput.

Keywords: α-thalassemia; hemoglobin H disease; melting curve analysis; non-deletional mutations; real-time PCR.

Introduction α-Thalassemia, caused by mutations in the α-globin gene cluster on chromosome 16p13.3, is one of the most common recessively inherited hemoglobin (Hb) disorders in the world [1]. In southern China, the prevalence rates can be up to 17% [2]. Normally, there are four α-globin genes with two α-globin genes (HBA2 and HBA1) located sequentially on each chromosome 16. Loss of all four α-globin genes results in severe anemia in utero (Hb

Bart’s hydrops fetalis) and the deletion or dysfunction of three alleles results in thalassemia intermedia (Hb

H disease). More than 95% of α-thalassemia mutations involve deletions of one (-α) or both (--) α-globin genes, and the rest are caused by non-deletional mutations [3].

The importance of the non-deletional α-thalassemia mutations is recognized by the observations that nondeletional Hb H (αTα/--) has more severe symptoms than patients with deletional Hb H (-α/--) [4, 5]. Furthermore,

Hb H disease could be caused by compound heterozygosity or homozygosity of non-deletional mutations (αTα/αTα) [4]. The overall carrier rate of non-deletional mutations can be as high as 2%–3% in southern China and non-deletional Hb H accounts for up to 50% of all Hb

H patients [4, 5]. It is thus important to have a rapid and

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Download Date | 8/31/15 3:24 PM 2      Huang et al.: MMCA detecting non-deletional α-thalassemia mutations inexpensive screening method for those common nondeletional α-thalassemia mutations in the α-thalassemia endemic areas.

There are in total 12 non-deletional α-thalassemia mutations reported in the Chinese population. Among them, six mutations, i.e. the codon 142 TAA→CAA (Hb Constant Spring, Hb CS, c.427T > C), the codon 125

CTG→CCG (Hb Quong Sze, Hb QS, c.377T > C), the codon 122 CAC→CAG (Hb Westmead, c.369T > C), the codon 30 (-GAG), the codon 31 AGG→AAG, and the codon 59

GGC→GAC) in HBA2 gene (NCBI Reference Sequence:

NM_000517.4) account for more than 98% of the Chinese non-deletional α-thalassemia population, and are often listed as the screening targets. Many methods have been reported for the detection of the non-deletional α-thalassemia mutations, including amplification refractory mutation system (ARMS) [6], PCR-restriction fragment length polymorphism (PCR-RFLP) [7], PCR-reverse dot blot hybridization (PCR-RDB) [8, 9], denaturing HPLC-based assay [3, 10], multiple minisequencing assay [5], pyrosequencing [11], and high resolution melting curve (HRM) [2, 12, 13]. A common shortcoming of these methods is that they either involve complex post-PCR manipulations that are both time consuming and labor intensive or require dedicated instruments that are not often used in a diagnostic laboratory.

Multicolor melting curve analysis (MMCA) is a realtime PCR-based method designed to detect multiple mutations in a single reaction by using a mixture of dual-labeled, self-quenched probes [14]. High accuracy, reproducibility, throughput, and cross-platform compatibility have been demonstrated in the detection of 24 β-thalassemia mutations [15] and 30 isoniazid-resistant mutations in Mycobacterium tuberculosis [16]. In this study, we aimed to develop an MMCA assay to rapidly and reliably detect the six Chinese non-deletional α-thalassemia mutations. We systematically studied its analytical performance and also evaluated its clinical performance by using 700 genomic DNA (gDNA) samples collected from three hospitals located in southern China.