C Guissart1, C Dubucs2, C Raynal1, A Girardet1, F Tran Mau Them2, V Debant2, C Rouzier3, A Boureau-Wirth3, E Haquet4, J Puechberty4, E Bieth5, D Dupin Deguine5, P Khau Van Kien6, M P Brechard7, V Pritchard8, M Koenig1, M Claustres1, M C Vincent9. 1. Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France; Equipe Accueil EA7402, Université Montpellier, Montpellier, France. 2. Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France. 3. CHU de Nice, Service de Génétique Médicale, Nice, France. 4. CHU de Montpellier, Service de Génétique Médicale, Montpellier, France. 5. CHU de Toulouse, Service de Génétique Médicale, Toulouse, France. 6. CHU de Nîmes, Laboratoire de Cytologie Clinique et Cytogénétique, Nîmes, France. 7. Hôpital Saint-Joseph, Service de Diagnostic Prénatal, Marseille, France. 8. Brammer Bio, Alachua, FL, USA. 9. Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France; Equipe Accueil EA7402, Université Montpellier, Montpellier, France. Electronic address: marie-claire.vincent@inserm.fr.
Abstract
BACKGROUND: Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches. METHODS: To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence. RESULTS: We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses. CONCLUSIONS: This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF.
BACKGROUND: Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches. METHODS: To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence. RESULTS: We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses. CONCLUSIONS: This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF.
Authors: Scott C Bell; Marcus A Mall; Hector Gutierrez; Milan Macek; Susan Madge; Jane C Davies; Pierre-Régis Burgel; Elizabeth Tullis; Claudio Castaños; Carlo Castellani; Catherine A Byrnes; Fiona Cathcart; Sanjay H Chotirmall; Rebecca Cosgriff; Irmgard Eichler; Isabelle Fajac; Christopher H Goss; Pavel Drevinek; Philip M Farrell; Anna M Gravelle; Trudy Havermans; Nicole Mayer-Hamblett; Nataliya Kashirskaya; Eitan Kerem; Joseph L Mathew; Edward F McKone; Lutz Naehrlich; Samya Z Nasr; Gabriela R Oates; Ciaran O'Neill; Ulrike Pypops; Karen S Raraigh; Steven M Rowe; Kevin W Southern; Sheila Sivam; Anne L Stephenson; Marco Zampoli; Felix Ratjen Journal: Lancet Respir Med Date: 2019-09-27 Impact factor: 30.700
Authors: Justyna Domaradzka; Marta Deperas; Ewa Obersztyn; Anna Kucińska-Chahwan; Nathalie Brison; Kris Van Den Bogaert; Tomasz Roszkowski; Marta Kędzior; Magdalena Bartnik-Głaska; Alicja Łuszczek; Krystyna Jakubów-Durska; Joris Robert Vermeesch; Beata Anna Nowakowska Journal: Mol Cytogenet Date: 2021-03-15 Impact factor: 2.009