{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T01:19:57Z","timestamp":1792804797278,"version":"4.5.0"},"reference-count":45,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2025,1,10]],"date-time":"2025-01-10T00:00:00Z","timestamp":1736467200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Excellence Hubs Program of The Research and Innovation Foundation (RIF), \u201cRestart 2016\u20132020\u201d","award":["Excellence\/1216\/0484"],"award-info":[{"award-number":["Excellence\/1216\/0484"]}],"id":[{"id":"https:\/\/ror.org\/00en9ce74","id-type":"ROR","asserted-by":"crossref"}]},{"name":"The Cyprus Institute of Neurology &amp; Genetics","award":["Excellence\/1216\/0484"],"award-info":[{"award-number":["Excellence\/1216\/0484"]}],"id":[{"id":"https:\/\/ror.org\/01ggsp920","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Non-invasive prenatal testing (NIPT) has been widely adopted for the screening of chromosomal abnormalities; however, its adoption for monogenic disorders, such as \u03b2-thalassaemia, has proven challenging. Haemoglobinopathies are the most common monogenic disorders globally, with \u03b2-thalassaemia being particularly prevalent in Cyprus. This study introduces a non-invasive prenatal haplotyping (NIPH) assay for \u03b2-thalassaemia, utilizing cell-free DNA (cfDNA) from maternal plasma. The assay determines paternal inheritance by analyzing highly heterozygous single-nucleotide variants (SNVs) in the \u03b2-globin gene cluster. To identify highly heterozygous SNVs in the population, 96 randomly selected samples were processed using Illumina DNA-prep NGS chemistry. A custom, high-density NGS genotyping panel, named HAPLONID, was designed with 169 SNVs, including 15 common pathogenic ones. The AmpliSeq for Illumina assay was then applied to cfDNA to evaluate the panel\u2019s efficiency in performing NIPT for \u03b2-thalassaemia. Analysis revealed 219 highly polymorphic SNVs, and the sequencing of 17 families confirmed successful paternal allele determination. The NIPH assay demonstrated 100% success in diagnostic interpretation. This study achieved the advancement of an integrated NGS-NIPT assay for \u03b2-thalassaemia, bringing it one step closer to being a diagnostic assay and thereby enabling a reduction in the number of risky invasive prenatal sampling procedures in Cyprus and elsewhere.<\/jats:p>","DOI":"10.3390\/ijms26020570","type":"journal-article","created":{"date-parts":[[2025,1,13]],"date-time":"2025-01-13T07:40:08Z","timestamp":1736754008000},"page":"570","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Non-Invasive Determination of the Paternal Inheritance in Pregnancies at Risk for \u03b2-Thalassaemia by Analyzing Cell-Free Fetal DNA Using Targeted Next-Generation Sequencing"],"prefix":"10.3390","volume":"26","author":[{"given":"Stefania","family":"Byrou","sequence":"first","affiliation":[{"name":"Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology & Genetics, Nicosia 2371, Cyprus"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7524-6230","authenticated-orcid":false,"given":"Rutger W. W.","family":"Brouwer","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"},{"name":"Center for Biomics, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0836-5579","authenticated-orcid":false,"given":"Marios","family":"Tomazou","sequence":"additional","affiliation":[{"name":"Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology & Genetics, Nicosia 2371, Cyprus"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3414-4972","authenticated-orcid":false,"given":"Stella","family":"Tamana","sequence":"additional","affiliation":[{"name":"Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology & Genetics, Nicosia 2371, Cyprus"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2681-4355","authenticated-orcid":false,"given":"Petros","family":"Kountouris","sequence":"additional","affiliation":[{"name":"Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology & Genetics, Nicosia 2371, Cyprus"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3920-9584","authenticated-orcid":false,"given":"Carsten W.","family":"Lederer","sequence":"additional","affiliation":[{"name":"Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology & Genetics, Nicosia 2371, Cyprus"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miranda","family":"Petrou","sequence":"additional","affiliation":[{"name":"Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology & Genetics, Nicosia 2371, Cyprus"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zeliha","family":"Ozgur","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"},{"name":"Center for Biomics, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3353-8101","authenticated-orcid":false,"given":"Xander","family":"den Dekker","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"},{"name":"Center for Biomics, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zakia","family":"Azmani","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"},{"name":"Center for Biomics, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Soteroula","family":"Christou","sequence":"additional","affiliation":[{"name":"Nicosia Thalassaemia Clinic, Archbishop Makarios III Hospital, Nicosia 2371, Cyprus"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christiana","family":"Makariou","sequence":"additional","affiliation":[{"name":"Thalassaemia Screening Laboratory, Thalassaemia Center, Archbishop Makarios III Hospital, Nicosia 2371, Cyprus"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0064-7034","authenticated-orcid":false,"given":"Marina","family":"Kleanthous","sequence":"additional","affiliation":[{"name":"Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology & Genetics, Nicosia 2371, Cyprus"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0421-8301","authenticated-orcid":false,"given":"Wilfred F. J. van","family":"IJcken","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"},{"name":"Center for Biomics, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9279-4810","authenticated-orcid":false,"given":"Thessalia","family":"Papasavva","sequence":"additional","affiliation":[{"name":"Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology & Genetics, Nicosia 2371, Cyprus"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,10]]},"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":"768","DOI":"10.1086\/301800","article-title":"Quantitative Analysis of Fetal DNA in Maternal Plasma and Serum: Implications for Noninvasive Prenatal Diagnosis","volume":"62","author":"Lo","year":"1998","journal-title":"Am. 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