Optimized Droplet Digital PCR Assay on Cell-Free DNA Samples for Non-Invasive Prenatal Diagnosis: Application to Beta-Thalassemia.
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 discove...
| Publicado en: | Clinical Chemistry Vol. 68; no. 8; pp. 1053 - 1064 |
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| Autores principales: | , , , , , , , , , |
| Formato: | Journal Article |
| Publicado: |
Oxford University Press / USA
Aug2022
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=158258536&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 158258536 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 00099147 10CS jtl: Clinical Chemistry issn: 00099147 maglogo: N pubinfo: dt: Aug2022 vid: 68 iid: 8 pid: 622 pub: Oxford University Press / USA artinfo: ui: 158258536 10.1093/clinchem/hvac076 158258536 ppf: 1053 ppct: 11 formats: fmt: @attributes: type: P tig: atl: Optimized Droplet Digital PCR Assay on Cell-Free DNA Samples for Non-Invasive Prenatal Diagnosis: Application to Beta-Thalassemia. aug: au: Constantinou, Constantina G. Karitzi, Eleni Byrou, Stefania Stephanou, Coralea Michailidou, Kyriaki Makariou, Christiana Hadjilambi, Georgia Christofides, Agathoklis Kleanthous, Marina Papasavva, Thessalia affil: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus sug: ab: 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 holdsmajor 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. pubtype: Academic Journal doctype: Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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