title: Identification of an 18 bp deletion in the TWIST1 gene by CO-amplification at lower denaturation temperature-PCR (COLD-PCR) for non-invasive prenatal diagnosis of craniosynostosis: first case report.
abstract: BACKGROUND
  Non-invasive prenatal diagnosis has found application in a limited number of genetic diseases due to the difficulty in detecting a few copies of fetal mutated sequences in the presence of a large excess of wild-type maternal alleles, even in the case of single-base mutations.
  
  
  METHODS
  We developed conditions for the enrichment of fetal mutated alleles in maternal plasma based on CO-amplification at lower denaturation temperature-PCR (COLD-PCR). In particular, we applied a full COLD-PCR protocol to the identification of a p.A87_G92del mutation in the TWIST1 gene causing craniosynostosis in a couple at risk for the disease.
  
  
  RESULTS
  The use of the COLD-PCR protocol coupled with direct sequencing enabled correct identification of the fetal paternally inherited mutated allele, in accordance with the result obtained on DNA extracted from chorionic villi.
  
  
  CONCLUSIONS
  COLD-PCR proved to be a simple and powerful tool for the identification of minority mutated alleles even in the case of a moderately large deletion (18 bp) and confirmed to be very suitable for non-invasive prenatal diagnosis of a variety of genetic diseases.
authors.forename: Silvia
authors.initials: S
authors.lastname: Galbiati
authors.forename: Stefania
authors.initials: S
authors.lastname: Stenirri
authors.forename: Luca
authors.initials: L
authors.lastname: Sbaiz
authors.forename: Marco
authors.initials: M
authors.lastname: Barberis
authors.forename: Laura
authors.initials: L
authors.lastname: Cremonesi
authors.forename: Gabriella
authors.initials: G
authors.lastname: Restagno
authors.forename: Maurizio
authors.initials: M
authors.lastname: Ferrari
mesh_terms: D000483:Alleles; D001483:Base Sequence; D003080:Cold Temperature; D003398:Craniosynostoses / Q000175:diagnosis* / Q000235:genetics; D004252:DNA Mutational Analysis; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011296:Prenatal Diagnosis; D011489:Protein Denaturation; D017384:Sequence Deletion / Q000235:genetics*; D051879:Twist-Related Protein 1 / Q000235:genetics*
chemical_list: D009687:Nuclear Proteins; C103173:TWIST1 protein, human; D051879:Twist-Related Protein 1