Whole Exome Sequencing For Detecting Etiologies Of Non-immune Hydrops Fetalis

BackgroundHydrops fetal（HF）is a pathological condition of fluid environment disorder,which leads to abnormal accumulation of fluid in the interstitial space of the fetus.The diagnosis of HF depends on ultrasonography,which shows at least 2 abnormal fetal body cavity effusions,including pleural effusion,peritoneal effusion,pericardial effusion and skin edema.Fetal edema mainly includes two types:immune fetal edema and non-immune hydrops fetalis（NIHF）.At present,most cases of fetal edema are NIHF,accounting for 85-90%of fetal edema cases.The incidence of pregnancy in NIHF is between 1/1700-3000,with a high mortality rate,ranging from 55%to 90%,depending on the etiology.Understanding the etiology of NIHF is important for effective management of pregnancy and neonatal periods,as morbidity and mortality depend on inherent genetic causes.At present,the recommending first-line examination of NIHF using karyotype and chromosome microarray（CMA）,fetal echocardiography,and viral polymerase chain reaction（PCR）.However,after the evaluation of the recommended standard tests,the etiology of up to half of cases are still unclear.There are many possible single-gene causes of NIHF,some of which can be treated by early intervention,but the current diagnostic methods are not detailed.Whole exome sequencing（WES）,especially the parent-fetus three-person whole exom sequencing（trio WES）strategy has unique advantages in mutation detection.In this study,trio WES was used to detecte and analyze three families with reduplicated NIHF,to explore the application value of combined trio WES in determining the etiology of NIHF when the result of copy number variation sequencing（CNV-seq）was negative.ObjectivesTo study the feasibility of using trio WES to detect the genetic etiology of 3 families with reduplicated NIHF and negative CNV-seq results.Objects and Methods1.ObjectsThis study collected 3 families,due to the previous pregnancy of one or more NIHF fetus,the fetus of this pregnancy was still diagnosed as NIHF.In family 1,the woman was 23 years old and the gestational age was 30 weeks,and the limb structure of the affected fetus was abnormal at the same time;In family 2,the woman was 29 years old and the gestational age was 19 weeks;In family 3,the woman was 25 years old and 29 weeks of gestational weeks,and bilateral renal cortical echo enhancement was found in the affected fetus at the same time.2.MethodsThe skin tissue of the induced labor fetus of family 1 and 5ml of the peripheral blood of both husband and wife,amniotic fluid of pregnant women from family 2 and family 3 and 5ml of peripheral blood of both husband and wife were collected respectively.The whole genome DNA,was extracted with the kit produced by Tiangen biochemical Technology（Beijing）Co.,Ltd.,and the concentration of DNA was quantified by Qubit 2.0 fluorometer（Thermo Fisher Scientific Company,USA）.CNV-seq detection was carried out in 3 families,except family 1,and the results were verified by CMA.After confirming that the results of CNV-seq ware negative,3 families were tested for trio WES.The DNA from the whole exom region was enriched by Illumina liquid phase chip capture system,and then high-throughput sequencing was carried out on the Illumina Novaseq 6000 sequencing platform.Then dbSNP database,1000dGenome database,SIFT and PolyPhen2 software were used for gene screening.The mutant sites were verified by Sanger sequencing.Results1.Family 2 and family 3 were negative for CNV-seq.2.Family 1,SOX18 c.976G>T heterozygous mutation,RYR1 c.844C>T and RYR1 c.9472G>A compound heterozygous mutation were detected.These three mutations were inherited from fetal parents,which confirmed the etiology of NIHF.3.Family 2,the compound heterozygous mutations of PIEZO1 c.6682C>T and PIEZO1 c.4373₄383del were detected.These two mutations were inherited from fetal parents,which confirmed the etiology of NIHF.4.Family 3,the compound heterozygous mutations of TTN c.29860G>C and TTN c.21107A>T were detected.These two mutations were inherited from fetal parents.Conclusion1.In this study,4 gene mutations,SOX18,RYR1,PIEZO1 and TTN,were detected in 3 families with continuous NIHF by trio WES;2.When the CNV-seq results are negative,trio WES can improve the diagnostic rate of NIHF;3.Trio WES data analysis combined with family analysis and fetal clinical manifestations can improve the accuracy of variation interpretation.