title: COLD-PCR and microarray: two independent highly sensitive approaches allowing the identification of fetal paternally inherited mutations in maternal plasma.
abstract: Until now, non-invasive prenatal diagnosis of genetic diseases found only limited routine applications. In autosomal recessive diseases, it can be used to determine the carrier status of the fetus through the detection of a paternally inherited disease allele in cases where maternal and paternal mutated alleles differ.
  
  
  
  Conditions for non-invasive identification of fetal paternally inherited mutations in maternal plasma were developed by two independent approaches: coamplification at lower denaturation temperature-PCR (COLD-PCR) and highly sensitive microarrays. Assays were designed for identifying 14 mutations, 7 causing β-thalassaemia and 7 cystic fibrosis.
  
  
  
  In total, 87 non-invasive prenatal diagnoses were performed by COLD-PCR in 75 couples at risk for β-thalassaemia and 12 for cystic fibrosis. First, to identify the more appropriate methodology for the analysis of minority mutated fetal alleles in maternal plasma, both fast and full COLD-PCR protocols were developed for the most common Italian β-thalassaemia Cd39 and IVSI.110 mutations. In 5 out of 31 samples, no enrichment was obtained with the fast protocol, while full COLD-PCR provided the correct fetal genotypes. Thus, full COLD-PCR protocols were developed for all the remaining mutations and all analyses confirmed the fetal genotypes obtained by invasive prenatal diagnosis. Microarray analysis was performed on 40 samples from 28 couples at risk for β-thalassaemia and 12 for cystic fibrosis. Results were in complete concordance with those obtained by both COLD-PCR and invasive procedures.
  
  
  
  COLD-PCR and microarray approaches are not expensive, simple to handle, fast and can be easily set up in specialised clinical laboratories where prenatal diagnosis is routinely performed.
authors.affiliation: Unit of Genomic for the Diagnosis of Human Pathologies, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Galbiati
authors.affiliation: Unit of Genomic for the Diagnosis of Human Pathologies, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Monguzzi
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Milano, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Damin
authors.affiliation: Unit of Genomic for the Diagnosis of Human Pathologies, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Nadia
authors.initials: N
authors.lastname: Soriani
authors.affiliation: Cystic Fibrosis Centre, Azienda Ospedaliera Universitaria Integrata, Verona, Italy.
authors.forename: Marianna
authors.initials: M
authors.lastname: Passiu
authors.affiliation: Cystic Fibrosis Centre, Azienda Ospedaliera Universitaria Integrata, Verona, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Castellani
authors.affiliation: Clinical Genetics Unit, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Natacci
authors.affiliation: Clinical Genetics Unit, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Curcio
authors.affiliation: Clinical Genetics Unit, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Seia
authors.affiliation: Clinical Genetics Unit, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy.
authors.forename: Faustina
authors.initials: F
authors.lastname: Lalatta
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Milano, Italy.
authors.forename: Marcella
authors.initials: M
authors.lastname: Chiari
authors.affiliation: Unit of Genomic for the Diagnosis of Human Pathologies, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Laboratory of Clinical Molecular Biology, IRCCS Ospedale San Raffaele, Milan, Italy Università Vita-Salute San Raffaele, Milano, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Ferrari
authors.affiliation: Unit of Genomic for the Diagnosis of Human Pathologies, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Cremonesi
mesh_terms: D000483:Alleles; D003080:Cold Temperature; D003550:Cystic Fibrosis / Q000235:genetics; D004247:DNA; D005333:Fetus; D005838:Genotype; D006801:Humans; D046228:Microarray Analysis / Q000379:methods; D009154:Mutation / Q000235:genetics*; D000072740:Paternal Inheritance / Q000235:genetics*; D010949:Plasma / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods; D011296:Prenatal Diagnosis / Q000379:methods*; D017086:beta-Thalassemia / Q000235:genetics
chemical_list: D004247:DNA
keywords: Clinical genetics; Cystic fibrosis; Diagnosis