BACKGROUND Thalassemias are inherited blood disorders and by far one of the most common monogenic diseases globally. Beta-thalassemia has a particularly high prevalence in Cyprus, with the IVSI-110 G>A (HBB:c.93-21G>A) pathogenic variation representing almost 79% of the total carriers. The discovery that 3% to 20% of cell-free fetal DNA (cffDNA) is present in the maternal plasma allowed the development of non-invasive prenatal diagnosis (NIPD) of monogenic diseases, like beta-thalassemia, avoiding the risks of invasive procedures. However, the development of NIPD holds major technical challenges and has not yet reached the clinical setting. METHODS In this study, we apply droplet digital PCR (ddPCR) coupled with the relative variant dosage approach to develop a NIPD assay for IVSI-110 G>A beta-thalassemia. We have implemented an optimization process for ddPCR to address the challenges of ddPCR assays such as inconclusive rain droplets and thus increase the sensitivity and specificity of the assay. The established protocol was evaluated on 40 maternal plasma samples with a median gestational age of 10 weeks where both parents carried the same pathogenic variation. RESULTS Thirty-three samples were correctly classified, 6 remained inconclusive, and 1 was misclassified. Our assay exhibited 97.06% accuracy (95% CI, 82.46-99.68), 100% sensitivity (95% CI, 76.84-100), and 95% specificity (95% CI, 75.13-99.87), demonstrating its efficiency for the non-invasive detection of both maternal and paternal alleles. CONCLUSIONS We have developed an efficient, simple, and cost-effective ddPCR assay for the non-invasive determination of fetal genotype in couples at risk of IVSI-110 G>A beta-thalassemia, bringing NIPD of monogenic diseases closer to the diagnostic setting.

中文翻译：

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用于无创产前诊断的无细胞 DNA 样本的优化液滴数字 PCR 检测：在 β-地中海贫血中的应用。

背景地中海贫血是遗传性血液疾病，并且是迄今为止全球最常见的单基因疾病之一。β-地中海贫血在塞浦路斯的患病率特别高，IVSI-110 G>A (HBB:c.93-21G>A) 致病变异占总携带者的近 79%。3% 至 20% 的无细胞胎儿 DNA (cffDNA) 存在于母体血浆中的发现使得对单基因疾病（如 β-地中海贫血）的非侵入性产前诊断 (NIPD) 得以发展，从而避免了侵入性操作的风险. 然而，NIPD的发展面临着重大的技术挑战，尚未达到临床应用。方法 在这项研究中，我们应用液滴数字 PCR (ddPCR) 以及相对变体剂量方法来开发 IVSI-110 G>A β-地中海贫血的 NIPD 检测方法。我们为 ddPCR 实施了优化过程，以解决 ddPCR 检测的挑战，例如不确定的雨滴，从而提高检测的灵敏度和特异性。对 40 个中位胎龄为 10 周的母体血浆样本评估了既定方案，其中父母双方携带相同的致病变异。结果 33 个样本被正确分类，6 个仍然不确定，1 个被错误分类。我们的测定显示出 97.06% 的准确度（95% CI，82.46-99.68）、100% 的灵敏度（95% CI，76.84-100）和 95% 的特异性（95% CI，75.13-99.87），证明了其对非母亲和父亲等位基因的侵入性检测。结论 我们开发了一种高效、简单、