| Background and ObjectiveChina is one of the countries with high incidence of birth defects.Chromosomal abnormalities account for more than 80%of the genetic causes of birth defects,including chromosome number and structural abnormalities,the most common of which are chromosome aneuploidy and pathogenic chromosome copy number variation(CNV).Prenatal diagnosis is the most effective means of prevention and treatment of chromosomal abnormalities.Noninvasive prenatal testing(NIPT)based on next generation sequencing(NGS)technology has been widely used in clinical practice.However,NIPT is only used in the prenatal diagnosis of trisomy 21,18 and 13,and it still does not meet the performance requirements of pathogenic CNV detection.The insufficient proportion of fetal free fetal DNA(cffDNA)in maternal peripheral blood is the main influencing factor of NIPT false positive and the main limiting factor of noninvasive detection of fetal CNV.Therefore,optimizing the existing NIPT process,enriching cffDNA fragments and accurately quantifying fetal concentration can reduce NIPT false-positive to a certain extent and developing noninvasive detection method of fetal CNV based on cffDNA enrichment can expand the clinical application of NIPT.Methods1.Based on the short length of cffDNA in pregnant women’s peripheral blood,we enriched cfdDNA,and used 94 pregnant male fetal samples to verify the performance based on fetal DNA fraction quantitative method FFy and larger sample size to verify the enrichment effect based on fetal DNA fraction quantitative method FFy and SeqFF.2.The sequencing data of 303 clinical samples with positive results of NIPT trisomy and puncture test results were retrospectively analyzed to establish a method to distinguish true positive and false positive of NIPT.3.On the basis of cffDNA enrichment and NIPT,a non-invasive CNV detection method was established.The performance of the method was verified by using artificial fetal CNV samples and 40 pregnant women plasma samples confirmed by CGH after puncture.Results1.A method based on gel electrophoresis for the recovery and enrichment of cffDNA fragments was established.The method can increase the fetal concentration by an average of 2.3 times,with an average of 26.5%.In the validation of 94 male fetus samples,1 false negative samples were successfully detected.2.Based on the quantitative method of fetal fraction,the fitting proportion model of fetal chromosomal ploidy abnormality was established.Using 303 cases(200 true positive and 103 false positive)of original NIPT to determine trisomy,the threshold function was obtained to distinguish true positive and false positive.After the threshold function was used to determine,the false positive rates of trisomy 21,18 and 13 in 303 samples were reduced by 82.6%,80.0%and 86.7%respectively.3.On the basis of cffDNA enrichment and existing NIPT,the reference data set was established based on the sequencing data of 500 male fetuses and 500 female fetuses,and the non-invasive detection method and CNV analysis process of fetal CNV based on high-throughput sequencing of maternal plasma free DNA were established.In the validation of artificial simulation samples,3-5 MB CNV can be detected at 20%fetal concentration;In 40 clinical samples,the sensitivity was 87.5%and the positive predictive value was 87.5%for CNV larger than 5MB;For 1-5mb CNV,the sensitivity was 93.8%,and the positive predictive value was 93.8%;For CNV less than 1MB,the sensitivity was 56.3%,and the positive predictive value was 75%.ConclusionOn the basis of the existing NIPT technology process,the cffDNA enrichment method was established.Based on the fetal fraction,the ploidy abnormality proportion function was developed quantitatively,which reduced the false positive of NIPT detection results,and expanded the noninvasive CNV detection applicable to clinical. |
