title: Detection of α-globin gene deletion and duplication using quantitative multiplex PCR of short fluorescent fragments.
abstract: BACKGROUND
  The aim of this study was to establish a sensitive method that can detect the presence of not only the common but also the unusual or unknown α-globin gene deletions for screening of α-thalassemia. We used quantitative multiplex PCR of short fluorescent fragments (QMPSF) for the α-globin genes (HBA) to screen α-thalassemia deletions.
  
  
  METHODS
  We set up and validated HBA-QMPSF using 50 negative and 100 positive controls of deletional α-thalassemia. To evaluate its ability to detect the presence of the common and unusual or unknown α-globin gene deletions, 579 unrelated samples were simultaneously analyzed using this assay and multiplex Gap polymerase chain reaction (Gap-PCR). The inconsistent results were further confirmed by multiplex ligation-dependent probe amplification (MLPA).
  
  
  RESULTS
  HBA-QMPSF was capable of detecting α-globin gene deletions with an acceptable variability as shown by mean values (SD) of allele dosage for the heterozygous deleted control obtained from intra- and inter-experimental replicates [0.63 (0.01) and 0.61 (0.03)]. In 572 out of the 579 unrelated subjects, HBA-QMPSF and multiplex Gap-PCR gave consistent results. In seven cases which were finally proved to be composed of one rare deletion--Thai/-α3.7, one novel deletion--SEA/-α2.8, four αααanti3.7/αα and one αααanti4.2/αα triplications, HBA-QMPSF showed deletion or duplication in the α-globin gene while multiplex Gap-PCR failed to give the correct diagnosis.
  
  
  CONCLUSIONS
  HBA-QMPSF is able to detect the presence of the common and unusual or unknown α-thalassemia deletions and duplications. It can be used as an initial screening test for α-thalassemia caused by HBA gene copy number alteration.
authors.affiliation: Chongqing Medical University, Chongqing, PR China.
authors.forename: Zong-Ping
authors.initials: ZP
authors.lastname: Mo
authors.forename: Chang-Shun
authors.initials: CS
authors.lastname: Yu
authors.forename: Ya-Juan
authors.initials: YJ
authors.lastname: Li
authors.forename: Wei-xi
authors.initials: WX
authors.lastname: Cao
authors.forename: Zheng-Yu
authors.initials: ZY
authors.lastname: Zeng
authors.forename: Yi-Xin
authors.initials: YX
authors.lastname: Zhan
authors.forename: Gui-ling
authors.initials: GL
authors.lastname: Mo
authors.forename: Chaohui
authors.initials: C
authors.lastname: Hu
authors.forename: Wenli
authors.initials: W
authors.lastname: Feng
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D017353:Gene Deletion; D020440:Gene Duplication; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D055542:alpha-Globins / Q000235:genetics*; D017085:alpha-Thalassemia / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; D055542:alpha-Globins