title: Development of a quantitative real-time PCR assay for detection of unknown alpha-globin gene deletions.
abstract: BACKGROUND
  Alpha-Thalassemia is the most common inherited disorder of hemoglobin (Hb) synthesis in the world. Unlike beta-thalassemia, in which non-deletional mutations predominate, most of recognized alpha-thalassemia mutations include deletion of one or both alpha-globin genes. The importance of alpha-thalassemia detection is mainly due to its shared blood parameters with beta-thalassemia and its impact on discrimination between unknown alpha-thalassemia and normal HbA2 beta-thalassemia during thalassemia prevention program.
  
  
  METHODS
  Cases with hematologic profile of low MCV, MCH, and normal HbA2 were enrolled in this study. Common alpha-globin deletional mutations including alpha(3.7)kb, alpha(4.2)kb, alpha(20.5)kb, and alpha(MED) and point mutation including 5 nt, Constant Spring (CS), and C19 were checked using either GAP-PCR or ARMS-PCR. Cases with unknown molecular defects were investigated further by direct gene sequencing. Finally, further study was done for probable unknown deletions by gene dosage analysis using real-time PCR. For this, five pairs of primers were used spanning from theta-globin gene up to the 3' upstream of alpha(2) gene.
  
  
  RESULTS
  After validation of primers specificity and performing serial dilution analysis in order to calculate PCR efficiency, the assay was performed on normal samples and cases with known alpha-globin gene deletions as positive and negative controls, respectively. The assay was able to diagnose the control groups successfully. In 21 out of 29 unknown cases (72.4%), the assay showed various patterns of deletions in at 2 to 5 screened regions (theta gene up to the upstream of alpha2 gene). In 8 (27.6%) cases, deletions were seen in all regions.
  
  
  CONCLUSIONS
  Gene dosage study by quantitative real-time PCR can be suggested as a rapid and reliable assay to screen probable carrier of alpha-thalassemia for unknown alpha-globin gene deletions.
authors.affiliation: National Institute for Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Mohammad-Sadegh
authors.initials: MS
authors.lastname: Fallah
authors.forename: Reza
authors.initials: R
authors.lastname: Mahdian
authors.forename: Seyyed-Ahmad
authors.initials: SA
authors.lastname: Aleyasin
authors.forename: Somayeh
authors.initials: S
authors.lastname: Jamali
authors.forename: Mina
authors.initials: M
authors.lastname: Hayat-Nosaeid
authors.forename: Moretza
authors.initials: M
authors.lastname: Karimipour
authors.forename: Marzieh
authors.initials: M
authors.lastname: Raeisi
authors.forename: Sirous
authors.initials: S
authors.lastname: Zeinali
mesh_terms: D005260:Female; D017353:Gene Deletion; D006801:Humans; D007492:Iran; D008297:Male; D005810:Multigene Family; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D055542:alpha-Globins / Q000235:genetics*; D017085:alpha-Thalassemia / Q000175:diagnosis / Q000235:genetics*
chemical_list: D055542:alpha-Globins