title: Detection of paternally inherited fetal point mutations for β-thalassemia in maternal plasma using simple fetal DNA enrichment protocol with or without whole genome amplification: an accuracy assessment.
abstract: OBJECTIVE
  To design and evaluate a noninvasive protocol for prenatal diagnosis (PND) of β-thalassemia, using cell free fetal DNA (cff-DNA) in maternal circulation. Traditional current PND which is mainly based on chorionic villous sampling (CVS), amplification refractory mutation system and sequencing holds as gold standard.
  
  
  METHODS
  Ten thalassemia trait couples with distinct mutations for the husband and wife were included in this study. The mutations in carrier fathers were IVSI-1, IVSI-5, FR8/9 and CD44. After maternal plasma isolation and free DNA extraction, all samples subjected to designed protocol including DNA size separation on agarose gel, elution of DNA from the gel slices using a simple and efficient manual purification method, with or without whole genome amplification and the detection method was allele-specific real-time PCR.
  
  
  RESULTS
  Presence or absence of the paternal mutant allele was correctly determined in all of cases and the accuracy of designed protocol was determined 100%.
  
  
  CONCLUSIONS
  The protocol described here is very simple, inexpensive and easy to perform, but with satisfactory accuracy in detection of paternal mutations in cff-DNA. Due to the risk of fetal loss with current invasive sampling for PND, a noninvasive alternative is highly demanded in clinical setting.
authors.affiliation: a Department of Genetics and Molecular Biology , School of Medicine, Isfahan University of Medical Sciences , Isfahan , Iran and.
authors.forename: Mahboubeh
authors.initials: M
authors.lastname: Ramezanzadeh
authors.affiliation: a Department of Genetics and Molecular Biology , School of Medicine, Isfahan University of Medical Sciences , Isfahan , Iran and.
authors.forename: Mansour
authors.initials: M
authors.lastname: Salehi
authors.affiliation: b Department of Community Medicine , Isfahan University of Medical Sciences , Isfahan , Iran.
authors.forename: Ziba
authors.initials: Z
authors.lastname: Farajzadegan
authors.affiliation: a Department of Genetics and Molecular Biology , School of Medicine, Isfahan University of Medical Sciences , Isfahan , Iran and.
authors.forename: Sara
authors.initials: S
authors.lastname: Kamali
authors.affiliation: a Department of Genetics and Molecular Biology , School of Medicine, Isfahan University of Medical Sciences , Isfahan , Iran and.
authors.forename: Rasoul
authors.initials: R
authors.lastname: Salehi
mesh_terms: D000328:Adult; D004247:DNA / Q000097:blood*; D005260:Female; D005312:Fetal Blood / Q000737:chemistry; D005333:Fetus; D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D008297:Male; D000072740:Paternal Inheritance / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D012680:Sensitivity and Specificity; D017086:beta-Thalassemia / Q000175:diagnosis* / Q000235:genetics*
chemical_list: D004247:DNA
keywords: allele specific PCR; free DNA; prenatal diagnosis; real-time polymerase chain reaction; β-thalassemia