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Abstract
Non-invasive prenatal testing (NIPT) is based on the detection and characterization of circulating cell-free fetal DNA (ccffDNA) in maternal plasma and aims to identify genetic abnormalities. At present, commercial NIPT kits can detect only aneuploidies, small deletions and insertions and some paternally inherited single-gene point mutations causing genetic diseases, but not maternally inherited ones. In this work, we have developed two NIPT assays, based on the innovative and sensitive droplet digital PCR (ddPCR) technology, to identify the two most common β thalassemia mutations in the Mediterranean area (β+IVSI-110 and β039), maternally and/or paternally inherited, by fetal genotyping. The assays were optimized in terms of amplification efficiency and hybridization specificity, using mixtures of two genomic DNAs with different genotypes and percentages to simulate fetal and maternal circulating cell-free DNA (ccfDNA) at various gestational weeks. The two ddPCR assays were then applied to determine the fetal genotype from 52 maternal plasma samples at different gestational ages. The diagnostic outcomes were confirmed for all the samples by DNA sequencing. In the case of mutations inherited from the mother or from both parents, a precise dosage of normal and mutated alleles was required to determine the fetal genotype. In particular, we identified two diagnostic ranges for allelic ratio values statistically distinct and not overlapping, allowing correct fetal genotype determinations for almost all the analyzed samples. In conclusion, we have developed a simple and sensitive diagnostic tool, based on ddPCR, for the NIPT of β+IVSI-110 and β039 mutations paternally and, for the first time, maternally inherited, a tool, which may be applied to other single point mutations causing monogenic diseases.
Highlights
Non-invasive prenatal testing (NIPT) is based on the detection and characterization of circulating cell-free fetal DNA in maternal plasma [1], using a simple maternal peripheral blood sampling technique and avoiding risks associated with conventional invasive procedures, such as amniocentesis and chorionic villus sampling. 4.0/).ccffDNA comprises only a small portion (10–20%) of total circulating cell-free DNA in the last gestational weeks [2] and is detectable from the 4th week of gestation until delivery [3], increasing by 0.1% every week between the 10th and 21st gestational weeks and by almost 1% increment/week after the 21st week [4,5].In addition, the amount of ccffDNA depends, besides the gestation period, on several other factors, such as maternal characteristics and body weight [5–7], aneuploidies [5] and twin pregnancies [8]
After the optimization of experimental conditions for β+ IVSI-110 (Figure S1, Supplementary Materials) and β0 39 (Figure S2, Supplementary Materials) thalassemia droplet digital polymerase chain reactions (PCR) (ddPCR) assays, both assays were validated for use in NIPT of β thalassemia
In order to simulate the fragmentation of circulating cell-free DNA (ccfDNA), genomic DNA was digested using SspI restriction enzymes before the ddPCR experiment
Summary
Non-invasive prenatal testing (NIPT) is based on the detection and characterization of circulating cell-free fetal DNA (ccffDNA) in maternal plasma [1], using a simple maternal peripheral blood sampling technique and avoiding risks associated with conventional invasive procedures, such as amniocentesis and chorionic villus sampling. 4.0/).ccffDNA comprises only a small portion (10–20%) of total circulating cell-free DNA (ccfDNA) in the last gestational weeks [2] and is detectable from the 4th week of gestation until delivery [3], increasing by 0.1% every week between the 10th and 21st gestational weeks and by almost 1% increment/week after the 21st week [4,5].In addition, the amount of ccffDNA depends, besides the gestation period, on several other factors, such as maternal characteristics and body weight [5–7], aneuploidies [5] and twin pregnancies [8]. Non-invasive prenatal testing (NIPT) is based on the detection and characterization of circulating cell-free fetal DNA (ccffDNA) in maternal plasma [1], using a simple maternal peripheral blood sampling technique and avoiding risks associated with conventional invasive procedures, such as amniocentesis and chorionic villus sampling. CcffDNA comprises only a small portion (10–20%) of total circulating cell-free DNA (ccfDNA) in the last gestational weeks [2] and is detectable from the 4th week of gestation until delivery [3], increasing by 0.1% every week between the 10th and 21st gestational weeks and by almost 1% increment/week after the 21st week [4,5]. Commercial NIPT assays can detect only aneuploidies, fetal sex and small deletions or insertions, but not maternally inherited single point mutations [23–25] NIPT has been applied to determine fetal sex [9,10], fetal rhesus D (RhD) genotyping [11,12], some pregnancy-associated conditions, including preeclampsia [13–15], aneuploidies [16,17] and the identification of paternally inherited monogenic disorders [16,18,19].
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