Research On The Genotype-phenotype Correlation Of Prenatally Detected High-frequency Pathogenic CNVs And Whole Genome Sequencing Of Recurrent Spontaneous Abortion

Background:Women with adverse pregnancy and childbearing history account for approximately 20% in clinical pregnancies,with birth defects and recurrent spontaneous abortion(RSA)being the most common types in clinic.However,the etiology and mechanisms of birth defects and RSA are not fully understood.With the development of molecular biology and genetic detecting techniques,more and more patients with birth defects and RSA are being diagnosed definitively.The incidence rate of birth defects in China is about 6%,and chromosomal abnormalities are main cause.Prenatal screening and prenatal diagnosis are primary methods of reducing birth defects,and chromosomal karyotyping is still the gold standard for prenatal diagnosis.In recent years,with the application of molecular genetic techniques such as chromosomal microarray analysis(CMA)and copy number variation sequencing(CNV-seq),the chromosomal microduplications/microdeletions which cannot be detected by karyotypic analysis have been identified.Although these new technologies have greatly improved the detection rates of chromosomal abnormalities,they also increased the difficulty for prenatal genetic counseling.The prenatally detected copy number variations(CNVs),especially the genotype-phenotype correlations of high-frequency pathogenic CNVs,need to be further investigated in depth.RSA occurs in 2% to 5% of clinical pregnancies,the etiology of which is complex.Genetic factors play a critical role in RSA,and the current detecting approaches commonly used in clinic for RSA include CNVs detection in the products of conception(POC)and parental karyotypic analysis.However,even after a comprehensive etiology screening,the causes of approximately 50% of RSA patients are unknown.In recent years,although whole exome sequencing(WES)technologyhas been used to explore the genetic etiology of RSA,most studies focused on couples or POC alone,lacking of an overall assessment of RSA families.In addition,WES cannot detect non-coding regions of the genome due to its technical limitations.With the development of sequencing technology,whole genome sequencing(WGS)has been used in cancer and precision medicine to explore the etiology of complex diseases,but further research on the genetic etiology of RSA based upon WGS remains to be investigated.Objective:1.To study the detecting discrepancies and the impacts on pregnancy outcomes between CMA and karyotyping analysis in prenatal diagnosis,and evaluate the clinical value of their combined application.2.To investigate the genotype-phenotype correlations of prenatally detected pathogenic CNVs with high frequencies.3.To explore the correlations between risk factors for RSA and CNVs in POC based upon the chromosomal abnormalities detected by high-throughput sequencing.4.To comprehensively evaluate the genetic etiology of RSA based on the results of trios-WGS,discover the candidate genes associated with RSA,and provide a basis for the genetic counseling of RSA couples opting for further pregnancy and functional studies of genes associated with RSA.Methods:This study included a total of 6785 singleton pregnant women who underwent amniocentesis and genetic counseling at the Center for Reproductive Medicine,Center for Prenatal Diagnosis,First Hospital of Jilin University for different prenatal diagnosis indications from October 2018 to June 2022.30 m L of amniotic fluid was extracted for every pregnant woman.20 m L was cultured for 7-10 days,then cell harvesting,chromosome preparation,G-banding and karyotypic analysis were processed.The genomic DNA of the other 10 m L of amniotic fluid was extracted and the whole genomic CNVs were detected based on the Cyto Scan 750 K array system.The consistency of the two tests for chromosomal abnormalities was finally compared by SPSS 21.0 software.A total of 1345 singleton pregnancies with normal karyotypes and chromosomal microduplications/microdeletions detected were selected.The data involving the coordinates of all CNVs was processed through professional gene chips statistical software based upon QT.Combined with literature review,the prenatal phenotypes of high-frequency pathogenic CNVs distributed in all chromosomes were summarized.The scale representation of the pathogenic CNVs was mapped based on DECIPHER database(https://decipher.sanger.ac.uk/browser),then the genotype-phenotype correlation was analyzed and relevant pathogenic genes were screened.A total of 1939 POC in RSA couples were included in this study from October 2016 to September 2022 in our center.High-throughput sequencing was performed based upon the Proton sequencing platform to analyze the correlation between aneuploidy and CNVs detected and different RSA-related risk factors.A total of 105 qualified RSA pedigrees were selected.After extracting genomic DNA from the peripheral blood of couples and POC,PE100 whole genome sequencing was performed using the BGISEQ-500 platform.Bioinformatics analysis was applied based upon the Human Phenotype Ontology(HPO)database,OMIM and Mouse Genome Information(MGI)database to establish a list of candidate genes associated with RSA,and the final RSA candidate genes in our study were validated using Sanger sequencing.To analyze the effect of candidate gene mutations on protein conformation,the structural models of wild and mutant proteins were performed using RCSB PDB database,Alpha Fold Protein Structure database,Py MOL software and so on.Results:1.Among the 6785 pregnant women opting for amniocentesis,karyotypic analysis detected 634 cases(9.34%)of chromosomal abnormalities.1280 cases(18.87%)with genetic abnormalities were detected by CMA.The total detection rate of genetic variants in CMA increased by 9.53% compared with karyotypic analysis.2.With the increase of maternal age,the detection rates of chromosomal numerical abnormalities increased and pathogenic(P)/likely pathogenic(LP)CNVs decreased(P<0.05),while the detection rates of chromosomal structural abnormalities and variants of unknown significance(VOUS)were not statistically significantly different among all age groups(P>0.05).The detection rates of chromosomal numerical abnormalities were higher in the two-indicator group and the three-indicator group than in the single-indicator group,and the differences were statistically significant(P<0.05).However,no statistically significant differences were observed in the detection rates of chromosomal structural abnormalities,P/LP pathogenic CNVs and VOUS among all subgroups(P>0.05).3.CMA and karyotypic analysis had the same detection rate for aneuploidy,but chromosomal mosaicism was underdiagnosed to varying degrees in both techniques,so their combined application was beneficial in improving the detection rate of chromosomal mosaicism.In addition,CMA can detect P/LP CNVs in chromosomal balanced structural rearrangements,polymorphisms and small supernumerary marker chromosomes.4.For the detected P/LP CNVs and VOUS in fetuses,the rate of pregnancy termination was higher in the de novo mutation group than in the parentally inherited group(P<0.001).5.The prenatal phenotypes of 1q21.1 microduplication were mainly associated with cardiovascular abnormalities,nasal bone hypoplasia,and increased nuchal translucency(NT).The cardiovascular system abnormalities may be associated with copy number gains of GJA5 and GJA8 genes.The prenatal phenotypes of1q21.1 microdeletion were mainly correlated with urinary system abnormalities,cardiac malformations,and increased NT.6.The prenatal phenotypes of 16p13.11 microduplication mainly exhibited increased NT.The prenatal phenotypes of 16p11.2 microdeletion were mainly associated with spinal/rib abnormalities,cardiovascular system malformations and abnormal soft markers.Spinal/rib dysplasia may be associated with the TBX6 gene.7.The prenatal phenotypes of 17q12 microduplication were mainly associated with duodenal obstruction,cardiac malformation.Duodenal obstruction may be associated with the HNF1 B gene.8.The prenatal phenotypes of 22q11.2 microduplication mainly exhibited increased NT and fetal cerebral ventriculomegaly.The prenatal phenotypes of 22q11.2microdeletion were mainly correlated with cardiac malformations,foot inversion,polyhydramnios and increased NT.The cardiac malformations may be associated with the TBX1 gene.9.The detection rate of genetic abnormalities in the POC by high-throughput sequencing was 50.03%,which mainly included trisomy 16,monosomy X and trisomy 22.The frequency of chromosome 8 was the highest among the segmental duplications/deletions detected in all POC.10.CNVs at 4p16.2p16.3,8p22p23,8q23q34,15q26 and 17p13 may be associated with RSA.11.The detection rates of chromosome anomalies associated with RSA in the advanced maternal age group and early pregnancy group were higher than that in the non-advanced maternal age and late pregnancy group,with statistically significant differences observed(P<0.05).However,the correlations between chromosomal abnormalities in the POC of RSA and conception modes,number of miscarriages and sex of the embryo need to be further investigated.12.The total detection rate of genetic variants in RSA families by trios-WGS was38.1%,and the detection rate of pathogenic CNVs and SNV was 29.52% and8.57%,respectively.WGS was consistent with high-throughput sequencing for chromosomal aneuploidy,and showed higher detection rates in submicroscopic CNVs and uniparental disomy(UPD).13.Nine candidate genes associated with RSA were screened using trios-WGS.Two genes(TLE3,SLC35A2)were due to embryonic factors,five genes(PPP2R3C,TRIM37,SYCP3,CBX2,ATP7B)were due to maternal factors and two genes(NME8,CFAP65)were due to paternal factors.The protein structure prediction models showed that mutations in SYCP3,ATP7 B and NME8 genes altered the structure of the protein.Conclusion:1.The combination of CMA and karyotyping analysis is more helpful to improve the detection rate of prenatal genetic abnormalities.CMA can detect pathogenic microduplications/microdeletions in fetuses carrying balanced chromosomal rearrangements,polymorphisms and small supernumerary marker chromosomes.2.The cardiovascular malformations in prenatally detected 1q21.1microduplications may be associated with copy number gains of GJA5 and GJA8 genes.The spinal/rib dysplasia in prenatally detected 16p11.2 microdeletions may be associated with TBX6 gene.The duodenal obstruction in prenatally detected17q12 microduplications was probably associated with HNF1 B gene.The cardiac malformations in prenatally detected 22q11.2 microdeletions may be associated with the TBX1 gene.3.The detection rate of genetic abnormalities in the POC by high-throughput sequencing was 50.03%,which mainly included trisomy 16,monosomy X and trisomy 22.Chromosomal abnormalities in POC of RSA were closely correlated with the maternal age,gestational week of miscarriage.4.The detection rate of pathogenic SNV in RSA family by trios-WGS was 8.57%.Nine candidate genes associated with RSA were identified using trios-WGS and Sanger sequencing.Two genes(TLE3,SLC35A2)were due to embryonic factors,five genes(PPP2R3C,TRIM37,SYCP3,CBX2,ATP7B)were due to maternal factors and two genes(NME8,CFAP65)were due to paternal factors.