Optimized Droplet Digital PCR Assay on Cell-Free DNA Samples for Non-Invasive Prenatal Diagnosis: Application to Beta-Thalassemia. Constantinou, C. G., Karitzi, E., Byrou, S., Stephanou, C., Michailidou, K., Makariou, C., Hadjilambi, G., Christofides, A., Kleanthous, M., & Papasavva, T. Clinical chemistry, 68(8):1053–1063, July, 2022. Place: Englanddoi abstract bibtex 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\textgreaterA (HBB:c.93-21G\textgreaterA) 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\textgreaterA 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\textgreaterA beta-thalassemia, bringing NIPD of monogenic diseases closer to the diagnostic setting.
@article{constantinou_optimized_2022,
title = {Optimized {Droplet} {Digital} {PCR} {Assay} on {Cell}-{Free} {DNA} {Samples} for {Non}-{Invasive} {Prenatal} {Diagnosis}: {Application} to {Beta}-{Thalassemia}.},
volume = {68},
copyright = {American Association for Clinical Chemistry 2022.},
issn = {1530-8561 0009-9147},
doi = {10.1093/clinchem/hvac076},
abstract = {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{\textgreater}A (HBB:c.93-21G{\textgreater}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{\textgreater}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{\textgreater}A beta-thalassemia, bringing NIPD of monogenic diseases closer to the diagnostic setting.},
language = {eng},
number = {8},
journal = {Clinical chemistry},
author = {Constantinou, Constantina G. and Karitzi, Eleni and Byrou, Stefania and Stephanou, Coralea and Michailidou, Kyriaki and Makariou, Christiana and Hadjilambi, Georgia and Christofides, Agathoklis and Kleanthous, Marina and Papasavva, Thessalia},
month = jul,
year = {2022},
pmid = {35652459},
note = {Place: England},
keywords = {*Cell-Free Nucleic Acids/genetics, *Prenatal Diagnosis/methods, *beta-Thalassemia/diagnosis/genetics, Alleles, Cell-Free Nucleic Acids, Female, Humans, Polymerase Chain Reaction, Polymerase Chain Reaction/methods, Pregnancy, Prenatal Diagnosis, beta-Thalassemia},
pages = {1053--1063},
}
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Beta-thalassemia has a particularly high prevalence in Cyprus, with the IVSI-110 G\\textgreaterA (HBB:c.93-21G\\textgreaterA) 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\\textgreaterA 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\\textgreaterA beta-thalassemia, bringing NIPD of monogenic diseases closer to the diagnostic setting.","language":"eng","number":"8","journal":"Clinical chemistry","author":[{"propositions":[],"lastnames":["Constantinou"],"firstnames":["Constantina","G."],"suffixes":[]},{"propositions":[],"lastnames":["Karitzi"],"firstnames":["Eleni"],"suffixes":[]},{"propositions":[],"lastnames":["Byrou"],"firstnames":["Stefania"],"suffixes":[]},{"propositions":[],"lastnames":["Stephanou"],"firstnames":["Coralea"],"suffixes":[]},{"propositions":[],"lastnames":["Michailidou"],"firstnames":["Kyriaki"],"suffixes":[]},{"propositions":[],"lastnames":["Makariou"],"firstnames":["Christiana"],"suffixes":[]},{"propositions":[],"lastnames":["Hadjilambi"],"firstnames":["Georgia"],"suffixes":[]},{"propositions":[],"lastnames":["Christofides"],"firstnames":["Agathoklis"],"suffixes":[]},{"propositions":[],"lastnames":["Kleanthous"],"firstnames":["Marina"],"suffixes":[]},{"propositions":[],"lastnames":["Papasavva"],"firstnames":["Thessalia"],"suffixes":[]}],"month":"July","year":"2022","pmid":"35652459","note":"Place: England","keywords":"*Cell-Free Nucleic Acids/genetics, *Prenatal Diagnosis/methods, *beta-Thalassemia/diagnosis/genetics, Alleles, Cell-Free Nucleic Acids, Female, Humans, Polymerase Chain Reaction, Polymerase Chain Reaction/methods, Pregnancy, Prenatal Diagnosis, beta-Thalassemia","pages":"1053–1063","bibtex":"@article{constantinou_optimized_2022,\n\ttitle = {Optimized {Droplet} {Digital} {PCR} {Assay} on {Cell}-{Free} {DNA} {Samples} for {Non}-{Invasive} {Prenatal} {Diagnosis}: {Application} to {Beta}-{Thalassemia}.},\n\tvolume = {68},\n\tcopyright = {American Association for Clinical Chemistry 2022.},\n\tissn = {1530-8561 0009-9147},\n\tdoi = {10.1093/clinchem/hvac076},\n\tabstract = {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{\\textgreater}A (HBB:c.93-21G{\\textgreater}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{\\textgreater}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{\\textgreater}A beta-thalassemia, bringing NIPD of monogenic diseases closer to the diagnostic setting.},\n\tlanguage = {eng},\n\tnumber = {8},\n\tjournal = {Clinical chemistry},\n\tauthor = {Constantinou, Constantina G. and Karitzi, Eleni and Byrou, Stefania and Stephanou, Coralea and Michailidou, Kyriaki and Makariou, Christiana and Hadjilambi, Georgia and Christofides, Agathoklis and Kleanthous, Marina and Papasavva, Thessalia},\n\tmonth = jul,\n\tyear = {2022},\n\tpmid = {35652459},\n\tnote = {Place: England},\n\tkeywords = {*Cell-Free Nucleic Acids/genetics, *Prenatal Diagnosis/methods, *beta-Thalassemia/diagnosis/genetics, Alleles, Cell-Free Nucleic Acids, Female, Humans, Polymerase Chain Reaction, Polymerase Chain Reaction/methods, Pregnancy, Prenatal Diagnosis, beta-Thalassemia},\n\tpages = {1053--1063},\n}\n\n","author_short":["Constantinou, C. 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