Etiological Study Of Short Fetal Limbs In The First And Second Trimester And Noninvasive Prenatal Screening Of Fetal Achondroplasia

Part one Study of genetic etiology in fetuses with severely short limbs in the first and second trimester using whole exome sequencingObjective To investigate pathogenic genes related to the phenotype of fetuses with severely short limbs in the first and second trimester.Methods Thirteen fetuses with severely short limbs detected by ultrasonography in the first and second trimester referred to the first medical center of Chinese PLA General Hospital were collected from September 2016 to June 2018.All the cases were performed induced abortion by intra-amniotic instillation with ethacridine,some of which were carried out karyotyping in the meantime.Under the trio analysis,whole exome sequencing(WES)and copy number variations(CNV)were performed on specimens from fetal tissues after labor induction.The suspected pathogenic mutations were validated by Sanger sequencing reactions.Results No abnormal karyotypes or pathological copy number variations were found.In 11 fetuses,pathogenic or possibly pathogenic mutations were detected in the following genes:COL2A1,FGFR3,COL1A1,COL1A2,DYNC2LI1 and TRIP11,all of which were essential to skeletal development.The diagnostic yield of WES in the fetuses suspected genetic skeletal dysplasias was 11/14.Conclusion In the first and second trimester,most of the fetuses with extremely short limbs suffer from monogenic diseases.WES is likely to be a valuable diagnostic testing option for the fetuses with severe limb shortening.Part Two The research of noninvasive prenatal screening of achondroplasia based on droplet digital PCRObjective To explore the feasibility of clinical application of new noninvasive prenatal screening for achondroplasia(ACH)using droplet digital PCR(ddPCR).Methods The pregnant women with fetus suspected for achondroplasia were enrolled from January 2015 to July 2018 at our hospital.Amniocentesis was performed and 10ml amniotic fluid was collected for Sanger sequencing to detect FGFR3 c.1138 locus.If no mutations were found,SNP-array was further performed to detect chromosomal microdeletions and micro-duplications.Before amniocentesis,we extracted cell-free DNA(cfDNA)from 10 milliliters of peripheral blood of these pregnant women.They were Stored in-80? refrigerator.Besides,after the fragmentations of fetal genomic DNA from the aborted fetus affected by achondroplasia caused by the c.1138 G>A mutation in FGFR3 and normal nonpregnant female genomic DNA,artificial mixtures of DNA were designed at different fetal DNA concentrations(containing 50%,25%,12%,6%,3%,1.5%,1%and 0.4%).Dd-PCR was performed to detect the FGFR3 c.1138 G>A allele in artificial samples to test the efficiency of ddPCR and the stability of probes.To further evaluate the performance of noninvasive prenatal screening(NIPS)of achondroplasia in clinical samples,42 pregnant women with highly suspected achondroplasia were tested for dd-PCR and Sanger's test for amniocentesis fluid.Sensitivity,specificity,positive predictive value and negative predictive value were calculated to evaluate the performance.Results The detection rates were 100%at artificial samples of different fetal concentrations and no positive results were detected in the negative controls.The NIPS results of 42 pregnant women were consistent with invasive prenatal diagnosis and pregnancy outcomes.Eleven affected fetuses were identified as c.1138 G>A pathogenic variants of FGFR3 and fifty-five fetuses were normal.The NIPS of ACH using dd-PCR had a sensitivity of 100%(95%CI:71.5%?100%),as well as a specificity of 100%(95%CI:88.8%?100%).The positive predictive value and negative predictive value were 100%(95%CI:71.5%?100%)and 100%(95%CI:88.8%?100%),respectively.Conclusion The NIPT of ACH using dd-PCR has an extremely high sensitivity and specificity in detecting FGFR3 c.1138 G>A of ACH in a short detectigng period.