{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T14:42:11Z","timestamp":1790088131927,"version":"4.0.1"},"reference-count":42,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2022,3,4]],"date-time":"2022-03-04T00:00:00Z","timestamp":1646352000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"FP7 HEALTH 2012 INNOVATION-1","award":["306201"],"award-info":[{"award-number":["306201"]}]},{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["Innovator Award 208872\/Z\/17\/Z"],"award-info":[{"award-number":["Innovator Award 208872\/Z\/17\/Z"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]},{"name":"AIFA, IT","award":["AIFA-2016-02364887"],"award-info":[{"award-number":["AIFA-2016-02364887"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Non-invasive prenatal testing (NIPT) is based on the detection and characterization of circulating cell-free fetal DNA (ccffDNA) in maternal plasma and aims to identify genetic abnormalities. At present, commercial NIPT kits can detect only aneuploidies, small deletions and insertions and some paternally inherited single-gene point mutations causing genetic diseases, but not maternally inherited ones. In this work, we have developed two NIPT assays, based on the innovative and sensitive droplet digital PCR (ddPCR) technology, to identify the two most common \u03b2 thalassemia mutations in the Mediterranean area (\u03b2+IVSI-110 and \u03b2039), maternally and\/or paternally inherited, by fetal genotyping. The assays were optimized in terms of amplification efficiency and hybridization specificity, using mixtures of two genomic DNAs with different genotypes and percentages to simulate fetal and maternal circulating cell-free DNA (ccfDNA) at various gestational weeks. The two ddPCR assays were then applied to determine the fetal genotype from 52 maternal plasma samples at different gestational ages. The diagnostic outcomes were confirmed for all the samples by DNA sequencing. In the case of mutations inherited from the mother or from both parents, a precise dosage of normal and mutated alleles was required to determine the fetal genotype. In particular, we identified two diagnostic ranges for allelic ratio values statistically distinct and not overlapping, allowing correct fetal genotype determinations for almost all the analyzed samples. In conclusion, we have developed a simple and sensitive diagnostic tool, based on ddPCR, for the NIPT of \u03b2+IVSI-110 and \u03b2039 mutations paternally and, for the first time, maternally inherited, a tool, which may be applied to other single point mutations causing monogenic diseases.<\/jats:p>","DOI":"10.3390\/ijms23052819","type":"journal-article","created":{"date-parts":[[2022,3,6]],"date-time":"2022-03-06T20:38:16Z","timestamp":1646599096000},"page":"2819","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":26,"title":["Droplet Digital PCR for Non-Invasive Prenatal Detection of Fetal Single-Gene Point Mutations in Maternal Plasma"],"prefix":"10.3390","volume":"23","author":[{"given":"Elisabetta","family":"D\u2019Aversa","sequence":"first","affiliation":[{"name":"Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giulia","family":"Breveglieri","sequence":"additional","affiliation":[{"name":"Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Effrossyni","family":"Boutou","sequence":"additional","affiliation":[{"name":"Molecular Genetics Laboratory, Thalassemia and Hemoglobinopathies Center, Laiko General Hospital, 11526 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angeliki","family":"Balassopoulou","sequence":"additional","affiliation":[{"name":"Molecular Genetics Laboratory, Thalassemia and Hemoglobinopathies Center, Laiko General Hospital, 11526 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ersi","family":"Voskaridou","sequence":"additional","affiliation":[{"name":"Thalassemia and Hemoglobinopathies Center, Laiko General Hospital, 11526 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrizia","family":"Pellegatti","sequence":"additional","affiliation":[{"name":"Operative Unit of Laboratory Analysis, University Hospital S. Anna, 44121 Ferrara, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giovanni","family":"Guerra","sequence":"additional","affiliation":[{"name":"Operative Unit of Laboratory Analysis, University Hospital S. Anna, 44121 Ferrara, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4058-4787","authenticated-orcid":false,"given":"Chiara","family":"Scapoli","sequence":"additional","affiliation":[{"name":"Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9205-6033","authenticated-orcid":false,"given":"Roberto","family":"Gambari","sequence":"additional","affiliation":[{"name":"Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy"},{"name":"Thal-LAB, Research Laboratory \u201cElio Zago\u201d on the Pharmacologic and Pharmacogenomic Therapy of Thalassemia, University of Ferrara, 44121 Ferrara, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8470-2496","authenticated-orcid":false,"given":"Monica","family":"Borgatti","sequence":"additional","affiliation":[{"name":"Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy"},{"name":"Biotechnology Center, University of Ferrara, 44121 Ferrara, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,3,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1016\/S0140-6736(97)02174-0","article-title":"Presence of fetal DNA in maternal plasma and serum","volume":"350","author":"Lo","year":"1997","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"19920","DOI":"10.1073\/pnas.0810373105","article-title":"Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma","volume":"105","author":"Lun","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1016\/j.earlhumdev.2006.11.001","article-title":"Early detection of cell-free fetal DNA in maternal plasma","volume":"83","author":"Illanes","year":"2007","journal-title":"Early Hum. Dev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/bs.acc.2016.05.004","article-title":"Cell-Free Fetal DNA Testing for Prenatal Diagnosis","volume":"76","author":"Drury","year":"2016","journal-title":"Adv. Clin. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1429","DOI":"10.1177\/1933719115584445","article-title":"Effects of maternal and fetal characteristics on cell-free fetal DNA fraction in maternal plasma","volume":"22","author":"Zhou","year":"2015","journal-title":"Reprod. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1159\/000444524","article-title":"Early Prediction of Hypertensive Disorders of Pregnancy Using Cell-Free Fetal DNA, Cell-Free Total DNA, and Biochemical Markers","volume":"40","author":"Kim","year":"2016","journal-title":"Fetal Diagn. Ther."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"912","DOI":"10.1002\/pd.3919","article-title":"A multifactorial relationship exists between total circulating cell-free DNA levels and maternal BMI","volume":"32","author":"Vora","year":"2012","journal-title":"Prenat. Diagn."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"967","DOI":"10.1002\/pd.2814","article-title":"Quantification of free fetal DNA in multiple pregnancies and relationship with chorionicity","volume":"31","author":"Attilakos","year":"2011","journal-title":"Prenat. Diagn."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Wright, C.F., Wei, Y., Higgins, J.P., and Sagoo, G.S. (2012). Non-invasive prenatal diagnostic test accuracy for fetal sex using cell-free DNA a review and meta-analysis. BMC Res. Notes, 5.","DOI":"10.1186\/1756-0500-5-476"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"283","DOI":"10.3390\/diagnostics3020283","article-title":"Overview of Five-Years of Experience Performing Non-Invasive Fetal Sex Assessment in Maternal Blood","volume":"3","author":"Horcajada","year":"2013","journal-title":"Diagnostics"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"154","DOI":"10.1016\/j.transci.2014.02.008","article-title":"Noninvasive fetal RhD genotyping","volume":"50","author":"Clausen","year":"2014","journal-title":"Transfus. Apher. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1016\/j.siny.2015.10.006","article-title":"Hemolytic disease of the fetus and newborn in the molecular era","volume":"21","author":"Fasano","year":"2016","journal-title":"Semin. Fetal Neonatal. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1007\/s40291-016-0245-9","article-title":"Cell-Free Fetal DNA for the Prediction of Pre-Eclampsia at the First and Second Trimesters: A Systematic Review and Meta-Analysis","volume":"21","author":"Contro","year":"2017","journal-title":"Mol. Diagn. Ther."},{"key":"ref_14","first-page":"R137","article-title":"Cell-free Fetal DNA and Spontaneous Preterm Birth","volume":"155","author":"Davidson","year":"2017","journal-title":"Reproduction"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"722","DOI":"10.1002\/uog.18993","article-title":"Association between fetal fraction on cell-free DNA testing and first trimester markers for pre-eclampsia","volume":"52","author":"Rolnik","year":"2018","journal-title":"Ultrasound Obstet. Gynecol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.bpobgyn.2017.02.007","article-title":"Noninvasive prenatal testing for fetal aneuploidy and single gene disorders","volume":"42","author":"Skrzypek","year":"2017","journal-title":"Best Pract. Res. Clin. Obstet. Gynaecol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"980","DOI":"10.1002\/pd.4639","article-title":"Cell-free DNA screening and sex chromosome aneuploidies","volume":"35","author":"Mennuti","year":"2015","journal-title":"Prenat. Diagn."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1093\/humupd\/dmn047","article-title":"The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis","volume":"15","author":"Wright","year":"2009","journal-title":"Hum. Reprod. Update"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1016\/j.gene.2012.04.045","article-title":"Non-invasive prenatal diagnosis of single-gene disorders from maternal blood","volume":"504","author":"Perlado","year":"2012","journal-title":"Gene"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Perlado, S., Bustamante-Aragon\u00e9s, A., Donas, M., Lorda-S\u00e1nchez, I., Plaza, J., and Rodr\u00edguez de Alba, M. (2016). Fetal Genotyping in Maternal Blood by Digital PCR: Towards NIPD of Monogenic Disorders Independently of Parental Origin. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0153258"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"948","DOI":"10.1016\/j.clinbiochem.2015.03.015","article-title":"Digital PCR analysis of circulating nucleic acids","volume":"48","author":"Hudecova","year":"2015","journal-title":"Clin. Biochem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1373\/clinchem.2016.268375","article-title":"Universal Haplotype-Based Noninvasive Prenatal Testing for Single Gene Diseases","volume":"63","author":"Hui","year":"2017","journal-title":"Clin. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"645","DOI":"10.1007\/s00404-017-4459-9","article-title":"Principles of first trimester screening in the age of non-invasive prenatal diagnosis: Screening for chromosomal abnormalities","volume":"296","author":"Kagan","year":"2017","journal-title":"Arch. Gynecol. Obstet."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1002\/pd.5207","article-title":"cfDNA screening and diagnosis of monogenic disorders-where are we heading?","volume":"38","author":"Chiu","year":"2018","journal-title":"Prenat. Diagn."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1002\/uog.17228","article-title":"Clinical utility of non-invasive prenatal testing in pregnancies with ultrasound anomalies","volume":"49","author":"Beulen","year":"2017","journal-title":"Ultrasound Obstet. Gynecol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1016\/j.siny.2017.12.002","article-title":"Beyond screening for chromosomal abnormalities: Advances in non-invasive diagnosis of single gene disorders and fetal exome sequencing","volume":"23","author":"Hayward","year":"2018","journal-title":"Semin. Fetal Neonatal. Med."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.cca.2017.11.015","article-title":"A new approach of digital PCR system for non-invasive prenatal screening of trisomy 21","volume":"476","author":"Lee","year":"2018","journal-title":"Clin. Chim. Acta"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"82","DOI":"10.3892\/br.2021.1458","article-title":"Droplet-based digital PCR for non-invasive prenatal genetic diagnosis of \u03b1 and \u03b2-thalassemia","volume":"15","author":"Sawakwongpra","year":"2021","journal-title":"Biomed. Rep."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1186\/s10020-018-0016-7","article-title":"Non-invasive fetal sex diagnosis in plasma of early weeks pregnants using droplet digital PCR","volume":"24","author":"Breveglieri","year":"2018","journal-title":"Mol. Med."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1003","DOI":"10.1021\/ac202578x","article-title":"Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification","volume":"84","author":"Pinheiro","year":"2012","journal-title":"Anal. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"8604","DOI":"10.1021\/ac202028g","article-title":"High-throughput droplet digital PCR system for absolute quantitation of DNA copy number","volume":"83","author":"Hindson","year":"2011","journal-title":"Anal. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1101\/cshperspect.a011700","article-title":"The molecular basis of \u03b2-thalassemia","volume":"3","author":"Thein","year":"2013","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.hoc.2017.11.006","article-title":"Clinical Classification, Screening and Diagnosis for Thalassemia","volume":"32","author":"Viprakasit","year":"2018","journal-title":"Hematol. Oncol. Clin. N. Am."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"5463","DOI":"10.1073\/pnas.74.12.5463","article-title":"DNA sequencing with chain-terminating inhibitors","volume":"74","author":"Sanger","year":"1977","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2239","DOI":"10.1039\/C8AN02018C","article-title":"A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies","volume":"144","author":"Tan","year":"2019","journal-title":"Analyst"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1093\/humupd\/dmh053","article-title":"Cell-free fetal DNA in maternal blood: Kinetics, source and structure","volume":"11","author":"Bischoff","year":"2005","journal-title":"Hum. Reprod. Update"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1373\/clinchem.2003.024893","article-title":"Size distributions of maternal and fetal DNA in maternal plasma","volume":"50","author":"Chan","year":"2004","journal-title":"Clin. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1002","DOI":"10.1373\/clinchem.2003.029835","article-title":"Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms","volume":"50","author":"Li","year":"2004","journal-title":"Clin. Chem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"17675","DOI":"10.1038\/s41598-018-35738-0","article-title":"Enrichment of the fetal fraction in non-invasive prenatal screening reduces maternal background interference","volume":"8","author":"Liang","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1007\/s40291-019-00385-2","article-title":"Non-invasive Prenatal Testing Using Fetal DNA","volume":"23","author":"Breveglieri","year":"2019","journal-title":"Mol. Diagn. Ther."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"728","DOI":"10.1515\/cclm-2017-0689","article-title":"Non-invasive prenatal diagnosis of paternally inherited disorders from maternal plasma: Detection of NF1 and CFTR mutations using droplet digital PCR","volume":"56","author":"Gruber","year":"2018","journal-title":"Clin. Chem. Lab. Med."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1802332","DOI":"10.1002\/advs.201802332","article-title":"Cell-free DNA Barcode-Enabled Single-Molecule Test for Non invasive Prenatal Diagnosis of Monogenic Disorders: Application to \u03b2-Thalassemia","volume":"6","author":"Yang","year":"2019","journal-title":"Adv. Sci."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/5\/2819\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:32:09Z","timestamp":1760135529000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/5\/2819"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,3,4]]},"references-count":42,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2022,3]]}},"alternative-id":["ijms23052819"],"URL":"https:\/\/doi.org\/10.3390\/ijms23052819","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,3,4]]}}}