Multiplex-PCR assay for the deletions causing hereditary persistence of fetal hemoglobin.
BACKGROUND
Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition caused by the failure of normal switching from the fetal to the adult beta-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.


OBJECTIVE
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.


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 beta-globin gene cluster causing HPFH.
Bhardwaj
Urvashi
U
Department of Pediatrics, David Geffen School of Medicine at University of California, Los Angeles, UCLA, and Mattel Children's Hospital at UCLA, USA.
McCabe
Edward R B
ER
D005319:Fetal Hemoglobin / Q000235:genetics*; D015894:Genome, Human; D006453:Hemoglobinopathies / Q000235:genetics*; D006801:Humans; D005810:Multigene Family; D016133:Polymerase Chain Reaction / Q000379:methods*; D017384:Sequence Deletion / Q000235:genetics*
D005319:Fetal Hemoglobin