title: Detection of fetal mutations causing hemoglobinopathies by non-invasive prenatal diagnosis from maternal plasma.
abstract: BACKGROUND
  Prenatal diagnosis of hemoglobinopathies enables couples at risk to have a healthy child. Currently used fetal sampling procedures are invasive with some risk of miscarriage. A non-invasive approach to obtain fetal deoxyribonucleic acid (DNA) for diagnosis would eliminate this risk.
  
  
  OBJECTIVE
  To develop and evaluate a non-invasive prenatal diagnostic approach for hemoglobinopathies using cell-free fetal DNA circulating in the maternal plasma.
  
  
  METHODS
  Couples referred to us for prenatal diagnosis of hemoglobinopathies where the maternal and paternal mutations were different were included in the study.
  
  
  METHODS
  Maternal peripheral blood was collected at different periods of gestation before the invasive fetal sampling procedure was done. The blood was centrifuged to isolate the plasma and prepare DNA. A size separation approach was used to isolate fetal DNA. Nested polymerase chain reaction (PCR)-based protocols were developed for detection of the presence or absence of the paternal mutation.
  
  
  CONCLUSIONS
  There were 30 couples where the parental mutations were different. Of these, in 14 cases the paternal mutation was absent and in 16 cases it was present in the fetus. Using cell-free fetal DNA from maternal plasma, the absence of the paternal mutation was accurately determined in 12 of the 14 cases and the presence of the paternal mutation was correctly identified in 12 of the 16 cases. Thus, this non-invasive approach gave comparable results to those obtained by the conventional invasive fetal sampling methods in 24 cases giving an accuracy of 80.0%. Although the nested PCR approach enabled amplification of small quantities of cell-free DNA from maternal plasma at different periods of gestation after size separation to eliminate the more abundant maternal DNA, an accurate diagnosis of the presence or absence of the paternal mutation in the fetus was not possible in all cases to make it clinically applicable.
authors.affiliation: Department of Haematogenetics, National Institute of Immunohaematology (ICMR), K.E.M. Hospital Campus, Parel, Mumbai, Maharashtra, India.
authors.forename: E
authors.initials: E
authors.lastname: D'Souza
authors.forename: P M
authors.initials: PM
authors.lastname: Sawant
authors.forename: A H
authors.initials: AH
authors.lastname: Nadkarni
authors.forename: A
authors.initials: A
authors.lastname: Gorakshakar
authors.forename: K
authors.initials: K
authors.lastname: Ghosh
authors.forename: R B
authors.initials: RB
authors.lastname: Colah
mesh_terms: D000653:Amniotic Fluid / Q000737:chemistry; D002648:Child; D005260:Female; D005315:Fetal Diseases / Q000097:blood / Q000175:diagnosis* / Q000235:genetics*; D005333:Fetus / Q000166:cytology*; D006453:Hemoglobinopathies / Q000175:diagnosis* / Q000453:epidemiology / Q000235:genetics; D006801:Humans; D008431:Maternal-Fetal Exchange / Q000235:genetics; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D055542:alpha-Globins / Q000235:genetics*
chemical_list: D055542:alpha-Globins