Efficiency Of Whole Genome Sequencing Applied For Detecting Chromosomal Anomalies In Non-Invasive Prenatal/Preimplantation Diagnosis

Part I:Prevalence survey of chromosomal anomalies detecting by non-invasive prenatal tests,a five years retrospective study compared with cytogenetic karyotypeBackground:Since 2013 NIPT has been not only applied for the screening of chromosome 21 trisomy but also used as an alternative to amniocenteses in clinic.Although the current low-coverage whole-genome sequencing(3.5 Mb useable reads)is powerful to detect CNVs greater than 5 Mb,the efficiency of NIPT is limited by sequencing depth and cff-DNA concentration,and the false positive/negative results would also resulted from placenta mosaic.Whether the current low-coverage whole-genome sequencing(3.5 Mb useable reads)can represent the rountine cytogenetic analysis in practice remains to be elucidated.And this case-report study would also to elucidate the epidemiologic distribution of imbalance occurred in each chromosome.Aim:In this study,we conducted a large sample retrospective survey to investigate the practical detecting rates of NIPT for diagnosing the chromosomal anomalies compared with the golden standard cytogenetic karyotyping.Moreover,the distribution of anomalies in individual chromosome was observed.Materials and Methods:(1)This retrospective case control survey were carried out to compare the prevalence of chromosomal anomalies between the NIPT and karyotype groups(2)The study population was composed by 22170 and 20168 pregnant women initially selected the NIPT and invasively karyotype tests during the period from June,2013 to June 2018,at Prenatal Diagnosis Center of Taizhou City.(3)Among the NIPT group,7550 cases with the indication of women aged over than 35,other 10011 cases with risky of serum screening(risk value less than 1/1000).(4)NIPT was performed by the same semiconductor platform using Ion Proton Sequencer at 400 flows in the sequencing depth at 3.5 Mb usable reads.Samples from amniocenteses were cultured and harvested after day 12.Cytogenetic karyotyping was carried by standard chromosome analysis at 350-bands level.(5)Positive results of NIPT were confirmed by invasive diagnoses or chromosome examination of the fetal sample after birth.Aneuploids were confirmed by karyotype or CMA,whereas the micro-deletion/duplication results were determined by CMA.Only true positive results were calculated.(6)Main outcomes are the standardized detecting rates of chromosomal imbalance detecting initially by NIPT and karyotype.And the imbalances include the aneuplo ids and the structural imbalance.The indication stratum-specific standardized rates were calculated for comparing the detecting rates of two techniques.Subgroup analyses were conducted between aneuploid and duplication/deletion.Results:(1)Standardized detecting rate of chromosomal anomalies between NIPT and karyotypeIn standardized population,NIPT presents the similar detecting rates for aneuploids,compared with the golden standard karyotyping.the age standardized rates of NIPT and karyotype groups were 1.49%and 1.54%(?2=0.319,p=0.572),and risky value standardized rates were 1.2%and 1.3%(?2=0.900,p=0.343),respectively.Also,no significant differences have been found in standardized rates for detecting structural imbalance between two groups.The adjusted age standardized rates of two techniques for detecting structural imbalance were 0.15%and 0.1%(?2=0.276 p=0.131),and risky value standardized rates were 0.14%and 0.18%in NIPT and karyotype groups,respectively(?2=0.861,p=0.353).(2)Distribution of chromosomal anomaliesPrevalence of aneuploids was significant associated with the age(P=0.785,p=0.007 and ?=0.862,p=0.001 for karyotype and NIPT,respectively)and risky value of serum screening(?=-0.415 p=0.013 and ?=-0.455,p=0.038 for karyotype and NIPT,respectively),but chromosomal deletions and duplications were not,both in NIPT and karyotype groups.Distribution of aneuploids were inhomogenized among the chromosomes,T21,T18,and X chromosome trisomy/monosomy take up more than 90%of aneuploids,whereas deletions and duplications were homogenized,both in NIPT and karyotype.Conclusions:(1)In standardized population,NIPT present the similar detecting rates for aneuploids and structural imbalance compared with the cytogenetic karyotyping.The current low-coverage whole-genome sequencing can overall represent the cytogenetic analysis in practice.(2)Prevalence of aneuploids associated with the age and distributed centralized among the chromosomes,whereas distribution of structural imbalance was discrete contrary.These prompt that the aneuploids and structural anomalies were different embryo logic origin.Part ?:Comparison of efficiencies of non-invasive prenatal testing,karyotyping,and chromosomal micro-array for diagnosing fetal chromosomal anomaliesBackground:Our cases-control study prompts that the adjusted detecting rates of chromosomal anomalies(aneuploid and structural imbalance)were similar between the standardized NIPT and karyotype population.And the distribution of the imbalance in NIPT group was also correspondent with the karyotype results.However,due to the difficulty in t getting the total false negative cases,the efficacy evaluate of NIPT remains unclear.Furthermore,the resolutions of diagnostic tests were unable to assess in this retrospective study.Therefore,a diagnostic test was designed to evaluate the efficacy between the NIPT and karyotype,compared with the golden standard CMA results.And we were further determining the resolution cutoffs of two techniques according the ROC curve.Aim:In this study,we aimed to compare the efficiency of non-invasive prenatal testing(NIPT),karyotyping,and chromosomal micro-array(CMA)for the diagnosis of fetal chromosomal anomalies.Material and Methods:(1)A prospective self-controlled diagnostic trial was carried out to evaluate the efficiency of the NIPT,karyotyping and CMA for detection of fetal chromosomal abnormality.(2)The clinical study participants were recruited from the Prenatal Clinic of Taizhou Hospital,Zhejiang,China between January,2016 and July,2017.Women with single pregnancy who needed middle or late trimester amniocenteses for CMA testing with indications of fetal ultra-sonographic structural anomalies or positive maternal serum biochemical screening were recruited.(3)Women with multiple pregnancies,a history of abortion risks,acute infectious disease or allergenic blood cell transfusion were excluded.We obtained 802 cases for complete analysis.(4)Testing:NIPT procedure was performed using the Ion Proton Sequencer(Life Technologies)in a semiconductor platform at 400 flows,the amniotic fluid cells were cultured following the local prenatal diagnostic standards.And the CMA was detected using Affymetrix CytoScan 750K chip with the resolution of 0.1Mb.(5)Results interpretation:CNVs were categorized as pathogenic,likely pathogenic,of uncertain significance,or benign based on the region,size,gene content,and inheritance pattern.All CNVs of uncertain significance were compared to the parents' genome,CNVs inherited from normal phenotype parents were regarded as benign,whereas fetal new mutation were regarded as likely pathogenic.(6)Main outcome:Only the chromosome imbalance abnormal were calculated in this study,and the balanced translocations,inversions and loss of heterozygosity,LOH were rule out.Reality outcome include sensitivity(Sen)and specificity(Spe),and efficacy outcome include positive predictive value(PPV)and negative predictive value(NPV)?The equations as following:Sen=NIPT/karyotype ture positive/CMA positive?Spe=NIPT/karyotype ture negative/CMA negative?PPV=ture positive/NIPT/karyotype positive?NPV=ture negaive/NIPT/karyotype negative(7)Data processing and statistical calculations were performed by Microsoft Excel and SPSS 22 respectively.CNVs inherited from normal phenotype parents were regarded as benign,whereas fetal new mutation was regarded as likely pathogenic.A receiver operating characteristic(ROC)curve,which was created by plotting the sensitivity and 1-specificity,was used to illustrate the diagnostic ability of NIPT and karyotyping at various CNV sizes.The sample size is calculated according to expected sensitivity of NIPT for detecting CNVs to be 80%and specificity 90%,whereas for karyotyping this rate is 79.6%and 99%,respectively.A usable sample size of 61 patients is required in anomalies group with an allowable error of 0.1 in a two-tailed test with p<0.05.Results:(1)Demographic characteristics of samplesWe confirmed 69 imbalanced chromosomal anomalies with 27 instances of aneuploidy.Thirteen of 42 deletion/duplications were categorized to be benign or inherited from normal phenotype parents.We found 1 case of sex chromosome monosomy being misdiagnosis by the NIPT technique that was confirmed to be a mosaic 45,X[48]/46,XX[62]both by the karyotyping and CMA methods.There were 29 pathogenic CNVs,9 of which were detected by NIPT whereas karyotype detected only 7(table 2-3-1-4)?(2)The accuracy and efficiency of NIPT and karyotype compared to CMA.NIPT showed similar accuracy,sensitivity and specificity as the karyotyping,whatever in detecting(table 2-3-2-1,p>0.05).(3)Compared the diagnostic resolution of NIPT and karyotype for detecting chromosomal anomalies.NIPT presented a slightly higher sensitivity than karyotyping for detecting CNVs with size ranged from 5 to 20M(table 2-3-2-2,p<0.05).The AUC of the two methods was also similar(Z=0.314,p=0.363),whereas the resolution cutoff of NIPT for detecting CNVs was 5.4 Mb(sensitivity=0.973,specificity=0.682),and this cutoff was 13.2 Mb for karyotyping(sensitivity=0.914,specificity=0.761).These results suggested that although NIPT present a higher resolution than karyotyping.Conclusions(1)NIPT sequencing with low-coverage whole-genome library sequencing displays similar accuracy levels compared to the 350-band karyotyping method for detection of chromosomal anomalies.(2)We suggest that NIPT might be more sensitive to micro-duplication/deletion events that can be confirmed downstream by more precise techniques such as CMA or CNV-sequencing to verify the positive results.Part III:WGS applied for noninvasive preimplantation genetic screeningBackground:PGS in clinic practice is archived by embryo biopsy,it needs complex manipulations and probably lead to embryo injure,which limited the extensive application.Recently NIPT performed by sequencing the cffDNA isolated from maternal peripheral blood has revolutionized the field of PGD:the non invasive preimplantation genetic testing,NI-PGT?CfDNA detected from spent culture media(SCM),blastocoel fluid(BF)were sufficient for whole genome sequence.However the spent culture media is a mixed DNA sourced from spermatozoa and granule cell,whereas the blastocoel cfDNA might mixed with the genome of mosaic rescue.Whether the accordance of cfDNA compared to the blastocysts is sufficient to detecting the chromosomal anomalies was undetermined.This study try to analyze the cfDNA extracted from spent culture media and blastocoel fluid,and compared the accordance to the blastocysts.Aim:(1)Whole genome amplified the cfDNA from spent culture media,blastocoel fluid,to determine the content and sequence.(2)Survey the chromosomal concordance of spent culture media,blastocoel fluid with TE.Material and Methods:(1)Sample collecting:Continue culture the spent D3 cleavage embryos obtained from IVF/ICSI till the D5 blastocysts.The blastocoel fluid and TE were collected by micromanipulation for whole genome amplification and sequence.And spent culture media from ICSI embryos were collected synchronously.(2)Same whole genome amplification(GenomePlex single cell WGS amplification kit,Sigma)and sequence platform(Ion Proton sequencer,Life Technologies)applied for samples,with a same sequence deep compared to NIPT.Results:(1)Seven of 14 blastocystes were originated from ICSI,in which the spent culture media and blastocoel fluid analyzed simultaneously;one blastocoel fluid was lost during operation.Others 7 cases originated from IVF were compared the blastocoel fluid with TE only.Totally 34 sample amplicated,the success rate of TE,blastocoel fluid,and culture media were 93%,76.9%and 85.7%,respectively.And WGS found 8 in 11 TE were normal karyotype,one case of aneuploid and 2 cases of dup/del.(2)The concordance of spent culture media and blastocoel fluid for detecting aneupliods were 90%and 66%,respectively.Sensitivity of blastocoel fluid for detecting aneuploids is 100%,whereas specifitivity of two materials is 75%and 80%for blastocoel fluid and spent culture media,respectively.None of dup/del confirmed by two materials.Conclusions:The cfDNA amplified from blastocoel fluid and spent culture media cover the whole genome information and both can practice for detecting aneuploid but not dup/del.