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COLD-PCR and microarray: two independent highly sensitive approaches allowing the identification of fetal paternally inherited mutations in maternal plasma
  1. Silvia Galbiati1,
  2. Alessandra Monguzzi1,
  3. Francesco Damin2,
  4. Nadia Soriani1,
  5. Marianna Passiu3,
  6. Carlo Castellani3,
  7. Federica Natacci4,
  8. Cristina Curcio4,
  9. Manuela Seia4,
  10. Faustina Lalatta4,
  11. Marcella Chiari2,
  12. Maurizio Ferrari1,5,6,
  13. Laura Cremonesi1
  1. 1Unit of Genomic for the Diagnosis of Human Pathologies, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy
  2. 2Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Milano, Italy
  3. 3Cystic Fibrosis Centre, Azienda Ospedaliera Universitaria Integrata, Verona, Italy
  4. 4Clinical Genetics Unit, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy
  5. 5Laboratory of Clinical Molecular Biology, IRCCS Ospedale San Raffaele, Milan, Italy
  6. 6Università Vita-Salute San Raffaele, Milano, Italy
  1. Correspondence to Dr Silvia Galbiati, Unit of Genomic for the Diagnosis of Human Pathologies, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Via Olgettina 60, Milan 20132, Italy; galbiati.silvia{at}hsr.it

Abstract

Background 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.

Methods 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.

Results 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.

Conclusions 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.

  • Diagnosis
  • Clinical genetics
  • Cystic fibrosis

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