Byrou Stefania, Brouwer Rutger W W, Tomazou Marios, Tamana Stella, Kountouris Petros, Lederer Carsten W, Petrou Miranda, Ozgur Zeliha, den Dekker Xander, Azmani Zakia, Christou Soteroula, Makariou Christiana, Kleanthous Marina, IJcken Wilfred F J van, Papasavva Thessalia
Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology & Genetics, Nicosia 2371, Cyprus.
Department of Cell Biology, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.
Int J Mol Sci. 2025 Jan 10;26(2):570. doi: 10.3390/ijms26020570.
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.
无创产前检测（NIPT）已被广泛用于染色体异常的筛查；然而，事实证明，将其应用于单基因疾病（如β地中海贫血）具有挑战性。血红蛋白病是全球最常见的单基因疾病，β地中海贫血在塞浦路斯尤为普遍。本研究介绍了一种用于β地中海贫血的无创产前单倍型分析（NIPH）检测方法，该方法利用母体血浆中的游离DNA（cfDNA）。该检测方法通过分析β珠蛋白基因簇中高度杂合的单核苷酸变异（SNV）来确定父系遗传。为了在人群中识别高度杂合的SNV，使用Illumina DNA制备NGS化学方法对96个随机选择的样本进行了处理。设计了一个名为HAPLONID的定制高密度NGS基因分型面板，其中包含169个SNV，包括15个常见的致病SNV。然后将Illumina的AmpliSeq检测方法应用于cfDNA，以评估该面板在进行β地中海贫血NIPT方面的效率。分析发现了219个高度多态性的SNV，对17个家庭的测序证实了父系等位基因的成功确定。NIPH检测在诊断解读方面显示出100%的成功率。本研究实现了β地中海贫血综合NGS-NIPT检测方法的进展，使其更接近成为一种诊断检测方法，从而减少塞浦路斯和其他地区有风险的侵入性产前采样程序的数量。
