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Objective: The aim of this study is to explore the utility of rapid medical trio exome sequencing (ES) for prenatal diagnosis using the skeletal dysplasia as an exemplar., Method: Pregnant women who were referred for genetic testing because of ultrasound detection of fetal abnormalities suggestive of a skeletal dysplasia were identified prospectively. Fetal samples (amniocytes or cord blood), along with parental blood, were send for rapid copy number variations testing and medical trio ES in parallel., Results: Definitive molecular diagnosis was made in 24/27 (88.9%) cases. Chromosomal abnormality (partial trisomy 18) was detected in one case. Sequencing results had explained the prenatal phenotype enabling definitive diagnoses to be made in 23 cases. There were 16 de novo dominant pathogenic variants, four dominant pathogenic variants inherited maternally or paternally, two recessive conditions with pathogenic variants inherited from unaffected parents, and one X-linked condition. The turnaround time from receipt of samples in the laboratory to reporting sequencing results was within 2 weeks., Conclusion: Medical trio ES can yield very timely and high diagnostic rates in fetuses presenting with suspected skeletal dysplasia. These definite diagnoses aided parental counseling and decision making in most of cases., (© 2020 John Wiley & Sons, Ltd.)

Objective: To explore the feasibility and accuracy of a noninvasive prenatal test for fibroblast growth factor receptor 3 (FGFR3)-related skeletal dysplasia based on next-generation sequencing (NGS) of plasma cell-free DNA., Method: Fragmented genome DNA (gDNA) of fetuses with achondroplasia (ACH) and thanatophoric dysplasia type I (TD I) was mixed with postdelivery maternal plasma cell-free DNA to generate spiked samples of different modeled fetal fractions. Multiplex polymerase chain reaction was used to amplify the 19 FGFR3 loci, and the amplification products were then sequenced by NGS to detect the fetal mutant alleles. Then, maternal plasma samples of pregnant women carrying ACH (n = 4) and TD I fetuses (n = 2), as well as healthy controls (n = 15), were tested by NGS, and the test performance was evaluated., Results: Fetal FGFR3 mutations were detected in all artificial mixtures with fetal gDNA concentrations above 3%. In clinical validation, our method identified all fetal FGFR3 mutant alleles from maternal plasma, with no false positive results. The sensitivity and specificity of our method were 100% (95% CI, 54.1%-100%) and 100% (78.2%-100%), respectively., Conclusion: Our method had a favorable performance for noninvasively detecting fetal FGFR3 mutations in maternal plasma, highlighting its promising value in developing a noninvasive prenatal test for de novo and paternally inherited disorders., (© 2018 John Wiley & Sons, Ltd.)

Objective: Evaluate the costs of offering non-invasive prenatal diagnosis (NIPD) for single gene disorders compared to traditional invasive testing to inform NIPD implementation into clinical practice., Method: Total costs of diagnosis using NIPD or invasive testing pathways were compared for a representative set of single gene disorders., Results: For autosomal dominant conditions, where NIPD molecular techniques are straightforward, NIPD cost £314 less than invasive testing. NIPD for autosomal recessive and X-linked conditions requires more complicated technical approaches and total costs were more than invasive testing, e.g. NIPD for spinal muscular atrophy was £1090 more than invasive testing. Impact of test uptake on costs was assessed using sickle cell disorder as an example. Anticipated high uptake of NIPD resulted in an incremental cost of NIPD over invasive testing of £48 635 per 100 pregnancies at risk of sickle cell disorder., Conclusion: Total costs of NIPD are dependent upon the complexity of the testing technique required. Anticipated increased demand for testing may have economic implications for prenatal diagnostic services. Ethical issues requiring further consideration are highlighted including directing resources to NIPD when used for information only and restricting access to safe tests if it is not cost-effective to develop NIPD for rare conditions. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd., (© 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.)

Objective: Accurate prenatal diagnosis of genetic conditions can be challenging and usually requires invasive testing. Here, we demonstrate the potential of next-generation sequencing (NGS) for the analysis of cell-free DNA in maternal blood to transform prenatal diagnosis of monogenic disorders., Methods: Analysis of cell-free DNA using a PCR and restriction enzyme digest (PCR-RED) was compared with a novel NGS assay in pregnancies at risk of achondroplasia and thanatophoric dysplasia., Results: PCR-RED was performed in 72 cases and was correct in 88.6%, inconclusive in 7% with one false negative. NGS was performed in 47 cases and was accurate in 96.2% with no inconclusives. Both approaches were used in 27 cases, with NGS giving the correct result in the two cases inconclusive with PCR-RED., Conclusion: NGS provides an accurate, flexible approach to non-invasive prenatal diagnosis of de novo and paternally inherited mutations. It is more sensitive than PCR-RED and is ideal when screening a gene with multiple potential pathogenic mutations. These findings highlight the value of NGS in the development of non-invasive prenatal diagnosis for other monogenic disorders., (© 2015 John Wiley & Sons, Ltd.)

Thanatophoric dysplasia (TD) is one of the most common neonatal lethal skeletal dysplasias. Prenatal sonographic and molecular genetic diagnoses of three cases of TD type I (TD1) and one case of TD type II (TD2) are presented here. Two fetuses of TD1 were characterized by polyhydramnios, macrocephaly, short limbs, a narrow thoracic cage and curved short femora, but without a cloverleaf skull at 27 and 31 weeks' gestation, respectively. The third fetus with TD1 was, however, not associated with macrocephaly, polyhydramnios, chest narrowing and severe femoral bowing on prenatal ultrasound at 18 weeks' gestation. The TD2 fetus was characterized by polyhydramnios, short limbs, a narrow thoracic cage, straight short femora, hydrocephalus and a cloverleaf skull at 24 weeks' gestation. Three-dimensional ultrasound was able to enhance the visualization of thickened, redundant skin folds and craniofacial and limb deformities associated with TD. Molecular analysis of the fibroblast growth factor receptor 3 (FGFR3) gene by restriction enzyme digestion analysis and direct sequencing using cultured amniotic fluid cells or cord blood cells revealed a missense mutation of 742C-->T (Arg248Cys) in all cases with TD1 and a missense mutation of 1948A-->G (Lys650Glu) in the case with TD2. The present report shows that adjunctive applications of molecular genetic analysis of the FGFR3 gene and three-dimensional ultrasound are useful for prenatal diagnosis of TD., (Copyright 2001 John Wiley & Sons, Ltd.)

Thanatophoric dysplasia (TD) is the most frequent form of neonatal lethal skeletal dysplasia. Recently. mutations in the fibroblast growth factor receptor 3 (FGFR3) gene that cause two subtypes of this disorder, type I (TDI) and type II (TDII), have been identified. This discovery has now made it possible to make a definite diagnosis of TD by molecular methods. To date, prenatal diagnosis of TD has been accomplished by ultrasonography in the second trimester. However, it is not always possible to distinguish TD fetuses it utero from the other osteochondrodysplasias by ultrasonography or radiography. We report on the prenatal diagnosis of a TD fetus, showing severe shortness of limbs and polyhydramnios, by identification of a mutation in the FGFR3 gene. Genomic DNA was isolated from the amniotic fluid and then subjected to PCR amplification. The common TDI mutation, C-->T transition at nucleotide 742 in the FGFR3 gene, was identified using restriction enzyme analysis. This information was critical in obstetric management decisions later in pregnancy. However, although the mutation responsible for TDI was detected previously, we noticed some inconsistencies in the published PCR results and have proposed a correction.