The Application Research Of NIPT For Fetal Chromosomal Abnormalities And Functional Analysis Of GUCY2D Gene Mutations

Part I The application research of NIPT for fetal chromosomal abnormalitiesObjective:The aim of this study was to assess the performance of non-invasive prenatal testing（NIPT）for fetal chromosome number and structural abnormalities in 62,780 pregnant women,to evaluate its clinical value on fetal chromosome number and structure abnormality.Materials and methods:NIPT was offered to screen 60,961 single and 1819 twin pregnant women.Sequencing data was analyzed to identify the 13,18,21 aneuploidy,sex chromosome aneuploidy,rare autosomal trisomies,fetal chromosome structure abnormality and genome copy number variations.Written informed consent were obtained from all participants in this study.Karyotype analysis was used to confirm chromosome aneuploidy and large fragment abnormality,while chromosome microarray chip（CMA）was used for genome copy number variation（CNV）.Results:1.NIPT was performed on a total of 60,961 pregnant women.482 cases were at risk of trisomy 13,18 and 21 among which karyotype analysis was carried out on 368 cases.311 of them were confirmed and the other 57 cases were false positive.270 cases were at high risk of sex chromosome aneuploidy among which karyotype analysis was carried out on 138 cases.79 cases of them were confirmed and the other 59 cases were false positive.158 cases were at high risk of rare autosomal trisomy,among which karyotype analysis was carried out on 94 cases.8 cases of them were confirmed and the other 86 cases were false positive.Two trisomy 21 cases and one trisomy 18 case tured out to be false negative.2.A total of 301 cases of pregnant women were at high risk of T21 and 251 cases were verified by karyotype.Among them,236 cases were confirmed（including 6 cases of mosaic trisomy 21,3 cases of trisomy 21 translocation type）,and the other 15 cases were false positive.In this study,the sensitivity（SEV）,specificity（SPC）,positive predictive value（PPV）and negative predictive value（NPV）of T21 were 99.16%（97.00-99.90%）,99.98%（99.96-99.99%）,94.02%（90.46-96.31%）and 100.00%（99.99-100.00%）,respectively.A total of 127 cases of pregnant women were at high risk of T18 and 82 cases were verified by karyotype.Among them,61 cases were confirmed（including 2 cases of mosaic trisomy 18）,and the other 21 cases were false positive.The SEV,SPC,PPV,NPV of T18 were 98.41%（91.47-99.96%）,99.97%（99.95-99.98%）,74.70%（65.79-81.93%）and 100.000%（99.99-100.00%）,respectively.A total of 54 cases of pregnant women were at high risk of T13 and 35 cases were verified by karyotype.Among them,14 cases were confirmed,and the other 21 cases were false positive.The SEV,SPC,PPV,NPV was 100%（76.84-100.00%）,99.97%（99.95-99.98%）,40.00%（65.79-81.93%）,and 100.000%,respectively.3.120 pregnant women were found to have 45,X by NIPT,and karyotype was performed on 58 cases.18 cases were confirmed to be 45,X,the other 40 cases were false positive.The SEV,SPC,PPV,NPV was 100%,99.93%,31.03%,and 100.00%,respectively.There were 62 cases at risk of 47,XXX,among which karyotype analysis was carried out on 35 cases.22 cases of them were confirmed,and the other 13 cases were false positive.The SEV,SPC,PPV,NPV was 100.00%,99.98%,62.86%,and 100.00%,respectively.48 pregnant women were found to have 47,XXY by NIPT,and karyotype was performed on 28 cases.24 cases were confirmed to be 47,XXY,and the other 4 cases were false positive.The SEV,SPC,PPV5 NPV was 100.00%,99.99%,85.71%,and 100.00%,respectively.47 pregnant women were found to have 47,XYY by NIPT,and karyotype was performed on 17 cases.15 cases were confirmed,and the other 2 cases were false positive.The SEV,SPC,PPV,NPV was 100.00%,100%,85.24%,and 100.00%,respectively.4.There were 158 cases of pregnant women at high risk of rare autosomal trisomies,and 95 cases were verified by karyotype.8 cases were confirmed,and the other 87 cases were false positive.The 8 cases of rare autosomal trisomies were:47,XN,+2[7]/46,XN[43];47,XN,+5[25]/46,XN[25];47,XN,+15[4]/46,XN[46];47,XN,+22[10]/46,XN[40]:47,XN,+16[2]/46,XN[74];47,XN,+16[2]/46,XN[98]:47,XN,+9[10]/46,XN[40];47,XN,+9[11]/46,XN[39].5.There were 133 cases were at high risk of abnormal chromosomal structure and genome copy number variation（CNV）,among which karyotype analysis was carried out on 72 cases.34 cases were confirmed,and the other 38 cases were false positive.One case of unbalanced translocation with chromosome deletion and duplication,one case of chromosome partial trisomy 16,two cases of Emanuel syndrome,etc.,false negative diagnosis of two fetuses,one case of Phelan--McDermid syndrome（Phelan--McDermid syndrome,PMDS,OMIM 606232）with 4.03mb fragment missing in 22q 13.3 region,and the other case of Xp22.33 pseudoautosomal region with 857kb heterozygotic deletion.6.A total of 1819 cases of twin pregnant women were detected,and 13 cases of high-risk pregnant women were detected.Karyotype analysis was carried out in 10 cases,5 cases with high risk of trisomy 21 were trisomy 21 of one twin,3 cases with high risk of trisomy 18,2 cases were confirmed as trisomy 18 of one twin.1 case with high risk of XXX was confirmed as 47,XXX of one twin.The positive result was 6.19Mb deletion on chromosome 13ql4.3-q21.1 by NIPT,and 3.06Mb deletion of maternal CNV was found to be the explanation for false positive result.Conclusion:1.A total of 62,780 pregnant women were tested by NTPT,including 60,961 single pregnant women and 1819 twin pregnant women.The results of our study showed that NIPT enabled 98.3%（61724/62780）pregnant women to avoid unnecessary invasive prenatal diagnostic procedures,and reduce the financial burden and unnecessary psychological pressure of the pregnant women.2.The SEV and SPC of NIPT for T13,T18 and T21 are all higher than 98%,but there are still limitations that may lead to false positive and false negative results.The positive results still need to be confirmed by invasive prenatal diagnosis3.Although NIPT technology can detect the abnormality of sex chromosome,the predictive value of 45,X positive is still low.NIPT screening can be expanded to sex chromosome aneuploidy,the incidence of false positive was higher than that of T13,T18 and T21.The positive results especially for Turner syndrome need to be confirmed by invasive prenatal diagnosis.4.Among the 60,961 cases,only 8 cases of rare mosaicism trisomy were detected.Due.to the different karyotype,degree and range of some rare mosaicism trisomy chromosomes,there was no fixed phenotype to follow.NIPT is generally not recommended for screening rare autosomal aneuploidy other than chromosome 13,18 and 21.When NIPT is expanded to detect rare aneuploidy,the possibility of false positives should be explained to patients,and ultrasonography and invasive prenatal diagnosis should be performed for further diagnosis.5.NIPT are feasible for prenatal screening of chromosomal microdeletions and microduplications,and in some cases may hint the parents are translocation carriers.The result can provide clear prenatal diagnosis and genetic counseling for pregnant couples.For pregnant women with abnormal clinical phenotype,when the NIPT test shows positive results,the influence of the maternal genetic background should be excluded first.If the false positive is caused by maternal source element,we also should not give up to its deep analysis.The in-depth analysis of these genetic factors is helpful for the analysis of the results of NIPT detection of chromosomal microstructural abnormalities.6.NIPT has a good detection efficiency in twin pregnancies,and the detection rate and SPC of trisomy 21 are 100%.Although the number and location of abnormal fetuses in twins cannot be determined by NIPT,the pain and abortion risk caused by amniocentesis of more than 98%of twin pregnant women can be avoided.The number of reports on twin NIPT studies is small,and the sample size is limited.Therefore,more studies of the detection of twin NIPT is still needed.7.Despite the high SEV and SPC of NIPT test,this technique should only be used for screening rather than diagnosis and confirmation of fetal karyotype.The risk of false positive results of NIPT should still be avoided by invasive prenatal diagnosis.Part Ⅱ Functional analysis of GUCY2D gene mutationsObjective:Leber’s congenital amaurosis is an autosomal recessive inherited disease,which can result in complete loss of vision within months of birth and a series of serious complications.Through the NGS and Sanger sequencing technology,the GUCY2D gene was detected in the proband families and three variant of undetermined significance mutations were found[c.139₁39delC（Ala49Profs*36）、c.2783G>A（Gly928Glu）and c.835G>A（Asp279Asn）].None of the three mutations has been reported so far,which is a newly discovered mutation after consulting literatures and databases.The researcher used SIFT,Polyphen-2 and Mutation Tasting to predict and analyze the mutation,whose results were all harmful and pathogenic mutations.Function analysis in these mutations will provide pathogenic mechanism of GUCY2D gene.Materials and Results:UCSF Chimera software was used to analyze the tertiary structure of Gly928 of wildtype and mutant proteins,and the result indicated that Gly928 mutation reduced the catalytic function.Widetype and mutant Ala49Profs*36,Asp279Asn and Gly928Glu were constructed to fuse expression vectors of green fluorescent protein EGFR and observed the weak fluorescence signal of Ala49Profs*36 by fluorescence confocal microscope,indicating the protein degradation.Asp279Asn and Gly928Glu were found to have lower cGMP by HPLC-MS/MS analysis,suggesting that the mutation changed the enzyme activity.Conclusion:Fluorescence confocal microscopy and HPLC-MS/MS analysis confirmed that Ala49Profs*36,Asp279Asn and Gly928Glu were pathogenic mutations of GUCY2D gene,respectively.Based on the NGS,this study conducted functional studies on three new mutation sites found in GUCY2D gene,which can not only clarify the pathogenic genes,pathogenic mutations and their functional effects of patients,but also enrich the disease and mutation spectrum,providing theoretical basis for the study of the pathogenic mechanism of GUCY2D gene,which has important application value for clinical genetic services such as genetic counseling,gene diagnosis,prenatal diagnosis and preimplantation genetic diagnosis.The studies have confirmed that the NGS has great application value in the diagnosis of genetic diseases,and the study of pathogenic mutation function provides theoretical basis for the exploration of disease mechanism.