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Multiplex-PCR Assay for the Deletions Causing Hereditary Persistence of Fetal Hemoglobin

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Abstract

Introduction: Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition caused by the failure of normal switching from the fetal to the adult β-globin gene, resulting in continuous production of fetal hemoglobin beyond the perinatal period. To date, eight deletions of variable size and position have been reported for HPFH. Southern hybridization and PCR are the most common methods used to detect each deletion.

Aim: Our aim was to develop a multiplex-PCR assay to detect these deletions in a single tube in order to facilitate rapid and accurate molecular diagnosis.

Methods and results: This report is the first application of multiplex-gap-PCR to detect all HPFH deletions simultaneously to expedite diagnosis. The deletion breakpoints were precisely identified for each deletion and primers were designed in the unique regions across the breakpoints of HPFH-1 (Black), HPFH-2 (Ghanaian), HPFH-3 (Asian Indian), HPFH-4 (Italian), HPFH-5 (Italian), HPFH-6 (Vietnamese), HPFH-7 (Kenyan), and SEA-HPFH (Southeast Asian). As many as 16 primers were used in a single amplification reaction by adjusting the relative primer concentrations. The multiplex-PCR approach was standardized on known positive control samples. We identified unique deletion-specific products for each deletion. The results were confirmed by sequence analysis.

Conclusions: We conclude that our multiplex-gap PCR strategy provides the most rapid and accurate diagnosis for the deletions in the β-globin gene cluster causing HPFH.

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Acknowledgements

We thank Professor Anagnou and Dr Theodoros Kosteas, Department of Basic Sciences, Institute of Molecular Biology and Biotechnology, Heraklion, Greece, for providing positive controls for HPFH-4, -5, and -6, and Dr Xiang-Min Xu, Department of Cellular Biology and Medical Genetics, First Military Medical University, Guangzhou, People’s Republic of China, for providing a DNA sample for SEA-HPFH deletion.

No federal sources of funding were used to assist in the preparation of this study. The authors have no conflicts of interest that are directly relevant to the content of this study.

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Authors and Affiliations

  1. Department of Pediatrics, David Geffen School of Medicine at University of California, Los Angeles (UCLA), 10833 Le Conte Avenue, Los Angeles, 22-412 MDCC, California, 90095-1752, USA

    Urvashi Bhardwaj &  Edward R. B. McCabe

  2. Mattel Children’s Hospital at UCLA, Los Angeles, California, USA

    Urvashi Bhardwaj &  Edward R. B. McCabe

  3. Department of Human Genetics, David Geffen School of Medicine at University of California, Los Angeles (UCLA), Los Angeles, California, USA

    Edward R. B. McCabe

  4. Molecular Biology Institute, University of California, Los Angeles (UCLA), Los Angeles, California, USA

    Edward R. B. McCabe

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  1. Urvashi Bhardwaj
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  2. Edward R. B. McCabe
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Correspondence to Edward R. B. McCabe.

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Bhardwaj, U., McCabe, E.R.B. Multiplex-PCR Assay for the Deletions Causing Hereditary Persistence of Fetal Hemoglobin. CNS Drugs 9, 151–156 (2005). https://doi.org/10.1007/BF03260083

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