Abstract
ObjectiveDevelopment of an accurate and affordable test for the non‐invasive prenatal diagnosis of Duchenne and Becker muscular dystrophies (DMD/BMD) to implement in clinical practice.MethodCell‐free DNA was extracted from maternal blood and prepared for massively parallel sequencing on an Illumina MiSeq by targeted capture enrichment of single nucleotide polymorphisms (SNPs) across the dystrophin gene on chromosome X. Sequencing data were analysed by relative haplotype dosage.ResultsSeven healthy pregnant donors and two pregnant DMD carriers all bearing a male fetus were recruited through the non‐invasive prenatal diagnosis for single gene disorders study. Non‐invasive prenatal diagnosis testing was conducted by relative haplotype dosage analysis for X‐linked disorders where the genomic DNA from the chorionic villus sampling (for healthy pregnant donors) or from the proband (for pregnant DMD carriers) was used to identify the reference haplotype. Results for all patients showed a test accuracy of 100%, when the calculated fetal fraction was >4% and correlated with known outcomes. A recombination event was also detected in a DMD patient.ConclusionOur new test for NIPD of DMD/BMD has been shown to be accurate and reliable during initial stages of validation. It is also feasible for implementation into clinical service. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.
Highlights
Since the discovery of cell-free fetal DNA in maternal plasma during pregnancy,[1] many advances have been made in the research for highly sensitive and reliable non-invasive prenatal diagnostic (NIPD) tests.[2] cffDNA is composed of small fragments of extracellular DNA derived from the shedding of placental trophoblasts[3] and only accounts for around 10%
Blood samples from all patients were taken between 11 and 14 weeks of gestation and extracted cell-free DNA (cfDNA) showed varying fetal fractions ranging between 3–27%
536 [interquartile range (IQR) = 106] informative single nucleotide polymorphisms (SNPs) were identified for each patient, and 385 (IQR = 72) of these were used for relative haplotype dosage (RHDO) analysis after quality filtering
Summary
Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma during pregnancy,[1] many advances have been made in the research for highly sensitive and reliable non-invasive prenatal diagnostic (NIPD) tests.[2] cffDNA is composed of small fragments of extracellular DNA derived from the shedding of placental trophoblasts[3] and only accounts for around 10%of cell-free DNA (cfDNA) circulating in the maternal bloodstream.[4,5] the use of cffDNA in clinical applications has been limited to the detection of paternally inherited sequences[6,7,8,9,10] and de novo mutations.[11]. Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma during pregnancy,[1] many advances have been made in the research for highly sensitive and reliable non-invasive prenatal diagnostic (NIPD) tests.[2] cffDNA is composed of small fragments of extracellular DNA derived from the shedding of placental trophoblasts[3] and only accounts for around 10%.
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