Non-Invasive Determination of the Paternal Inheritance in Pregnancies at Risk for β-Thalassaemia by Analyzing Cell-Free Fetal DNA Using Targeted Next-Generation Sequencing
Authors:
- Stefania ByrouSBStefania Byrou
- Rutger W. W. BrouwerRBRutger W. W. Brouwer
- Marios TomazouMTMarios Tomazou
- Stella TamanaSTStella Tamana
- Petros KountourisPetros Kountouris
- Carsten W. LedererCarsten W. Lederer
- Miranda PetrouMPMiranda Petrou
- Zeliha OzgurZOZeliha Ozgur
- Xander Den DekkerXDXander Den Dekker
- Zakia AzmaniZAZakia Azmani
- Soteroula ChristouSCSoteroula Christou
- Christiana MakariouCMChristiana Makariou
Open Access
- 10 January 2025
- journal article
- research article
- Published by MDPI AG in International Journal of Molecular Sciences
- Vol. 26 (2)
Abstract
Non-invasive prenatal testing (NIPT) has been widely adopted for the screening of chromosomal abnormalities; however, its adoption for monogenic disorders, such as β-thalassaemia, has proven challenging. Haemoglobinopathies are the most common monogenic disorders globally, with β-thalassaemia being particularly prevalent in Cyprus. This study introduces a non-invasive prenatal haplotyping (NIPH) assay for β-thalassaemia, utilizing cell-free DNA (cfDNA) from maternal plasma. The assay determines paternal inheritance by analyzing highly heterozygous single-nucleotide variants (SNVs) in the β-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’s efficiency in performing NIPT for β-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 β-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.Keywords
Funding Information
- Excellence Hubs Program of The Research and Innovation Foundation (RIF), “Restart 2016–2020” (Excellence/1216/0484)
- the Cyprus Institute of Neurology & Genetics (Excellence/1216/0484)
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