Etiology Exploration And Non-invasive Prenatal Test Of Fetal FGFR3 Mutation For Short Fetal Femur

Part One Etiology exploration of 24 cases with short fetal femur in third trimesterObjective To explore the etiology of short fetal femur in third trimester.Methods From January 2015 to December 2016, 24 singleton pregnancies with short fetal femur detected by ultrasonography in third trimester were referred to Chinese PLA General Hospital. Clinical data were collected, karyotype or SNP array, FGFR3 c.1138 mutation detection were carried out via invasive procedure. The deviation of femur length from the mean value of gestational age in ultrasonography was expressed as Z-score,difference between ACH and ISF (isolated short femur) was then explored.Results Among the 24 fetuses, 12 had abnormal genetic test results (50.0%, 12/24),including 10 ACH, one Ellis-van Creveld Syndrome, and one Pallister-Killian Syndrome.In the 12 ISF fetuses (50.0%, 12/24), 3 cases were FGR, one case was normal small for gestational age and 8 cases were unexplained. The Z-scores [median (interquartile range)]for ACH and ISF in third trimester were -5.19 (-6.31 ～-4.77),-2.84 (-4.67～-1.86),respectively. The short femur in ACH was more severe than in ISF (P=0.001).Conclusions The etiology of short fetal femur is complicated, including skeletal dysplasia, chromosomal abnormality, fetal growth restriction, as well as normal variants during fetal development. Genetic test should be considered during the antenatal consellingPart Two Non-invasive prenatal test for FGFR3-related skeletal dysplasia based on next-generation sequencing : methodology establishmentObjective To perform the methodology establishment of non-invasive prenatal test for FGFR3- related skeletal dysplasia based on NGS (next-generation sequencing).Method Fragmented fetal gDNA (genome DNA) of ACH (n=4) or TD I (thanatophoric dysplasia, Type I, n=2) were diluted by corresponding maternal post-delivery plasma DNA into different concentrations including 10%, 6%,3%, 1% and 0.5% (n=30). Plasma DNA collected from pregnant women (n=13) whose fetuses were confirmed free of FGFR3 mutation served as negative control. Then NGS was carried out to detect the fetal mutant alleles in those samples. Se (sensitivity), Sp (specificity), PPV(positive predictive value),and NPV(negative predictive value) were calculated to evaluate the test performance.Results When the fetal gDNA concentration were 10%、6%、3%、1% and 0.5%,the detected rates were 6/6, 6/6, 6/6, 3/6 and 1/6, respectively. NGS had a Se of 100% (95%CI,81.5%-99.9%) when the fetal gDNA concentration≥3%. There was no positive result in negative controls and the Sp of NGS was 100% (75.3%-100%) . The PPV and NPV were 100% (81.5%-100%) and 100% (75.3%-100%), respectively.Conclusion NGS had a greater test accuracy in detecting fetal mutant alleles when fetal gDNA concentration were equal to or more than 3%.Part Three Non-invasive prenatal test for FGFR3-related skeletal dysplasia based on next-generation sequencing: clinical verificationObjective To carry out the clinical verification of non-invasive prenatal test for FGFR3-related skeletal dysplasia.Method Maternal plasma of ACH (n=6) or TD I fetuses (n=3) were collected as positive cases,while negative control plasma were collected from pregnant women whose fetuses were confirmed free of FGFR3 mutation (n=15). Then NGS was carried out to detect the fetal mutant alleles in those samples, and Se, Sp, PPV, and NPV were calculated.Results Fetal FGFR3 mutant alleles were successfully discriminated from all maternal plasma by NGS, without positive result obtained in negative control samples. The Se,Sp,PPV, and NPV of NGS were 100% (95% CI, 66.4%～100%), 100% (78.2% ～100%), 100%(66.4%～100%) and 100% (78.2%～100%), respectively.Conclusion NGS had a favorable test performance in discriminating fetal mutant alleles from maternal plasma, highlighting its promising value in developing NIPT for for de novo and paternal inherited diseases.