Study On Fetal Chromosome Abnormality And Appropriate Prenatal Diagnostic Technique

Chapter1Study on the distribution of fetal abnormal chromosome in the high-risk mid-pregnancyPart1Study on the distribution of fetal abnormal chromosome in the high-risk mid-pregnancy in Zhejiang province from2001to2010Objective:To evaluate the distribution of fetal abnormal chromosome in the high-risk mid-pregnancy in Zhejiang province.Method:12841cases of fetal karyotype achieved between2001-2010were analyzed. The indications for prenatal diagnosis were grouped as follows:positive screening for trisomy21, positive screening for trisomy18, advanced maternal age, abnormal history of pregnancies, abnormal family history, fetal structural abnormalities and others. Chromosome abnormality (CA) were analyzed in two groups:high-risk CA and Low-risk CA. Results:There were462cases out of12481with a chromosomal abnormality (3.60%), with215cases of high risk (detection rate1.67%) and247cases of low risk (detection rate1.92%). Among the total high-risk CA, the combination of trisomy21, trisomy18and trisomy13(T21/18/13) accounted for72.56%, while the aneuploidies of chromosome21,18,13, X, Y (21/18/13/X/Y Group) accounted for94.88%. For the common indication of positive screening for trisomy21, positive screening for trisomy18, advanced maternal age, and fetal structural abnormalities, the T21/18/13cases accounted for78.13%,92.59%,66.07%and54.17%respectively in the high risk group of chromosomal abnormality, while the21/18/13/X/Y aneuploidies accounted for95.83%、96.30%、94.64%and95.83%respectively in the high risk group of chromosomal abnormality.Conclusion:The21/18/13/X/Y Group were the most common high-risk CA, which accounted for about95%in total high-risk CA. The detection rate was various under different referral indications. It is suggested to select appropriate technique according to specific indication in clinical prenatal diagnosis. Chapter1Part2:A multicenter study of fetal chromosomal abnormalities in Chinese women of advanced maternal age from2001to2010Objective:To Understand the trend of advanced maternal age (AMA) in the indication of prenatal diagnosis and to evaluate the distribution of fetal abnormal chromosome in the high-risk mid-pregnancy in Chinese women.Method:The results of fetal karyotype (January2001to December2010) from a total of46,258women at13prenatal diagnosis centers in China were collected. The women were divided into seven groups based on their age at their due date:35,36,37,38,39, and40years of age and over40years of age. The high-risk Chromosome abnormality (CA) was analyzed according to different group.Results:From2001to2010, the proportion of AMA women undergoing prenatal diagnosis increased from20%to46%. Among the46,258women, there were708cases of high-risk C A with rate of1.53%.The combination of trisomy21, trisomy18and trisomy13(T21/18/13) accounted for73.59%, while the aneuploidies of chromosome21,18,13, X, Y (21/18/13/X/Y Group) accounted for93.93%. The rare high-risk CA, such as partial chromosome deletions and additions, and unbalanced translocations, account for6.07%of all high-risk CA. There were no significant differences in the incidence of T21/18/13and21/18/13/X/Y Group among women35,36,37, and38years of age (P=0.133). However, the incidences of T21/18/13and21/18/13/X/Y Group in women39, 40, and more than40years of age was significantly different from that of the35-38year age group (P<0.001), which accounted for77.35%and96.86%respectively in high-risk C A. The incidence of t rare high-risk CA did not increase with age, and their relative proportion of clinically significant chromosomal abnormalities tended to decrease with age.Conclusion:The need for fetal diagnosis due to AMA is increasing in China, placing additional burden on cytogenetic testing resources. In AMA women in China, the21/18/13/X/Y Group were the most common high-risk CA, which accounted for93.93%of all high-risk CA. The incidence of21/18/13/X/Y Group increased significantly in those39years of age and older. These results provide a basis for the selection of prenatal diagnostic technologies for prenatal diagnosis. Chapter2Study on STR population genetics in Chinese han population and to establish high thoughput STR testing assay in a Han population from ChinaObjective:To establish high throughput Short tandem repeats (STR) detection assay and to establish STR genetic data in a Han population from China. Subject and Sample:44candidate STR on chromosome21,18,13,X,Y and TAF9b, Amelogenin (AMEL), SRY were analyzed; Samples include blood from500unrelated Han individuals,558cases of fetal cell collected from amniotic fluid, and42cases of villi.Method:1) The preparation for the establishment of high throughput STR detection assay: All primers (44candidate STR and TAF9b, AMEL, SRY)were designed with PRIMER3; The preferred primers were separately labeled with5’ROX、5’TAMRA、5’HEX、6’FAM; To optimize the PCR reaction assay; To extract sample DNA with Chelex method; All locus were grouped in3multiplex STR-PCR (D21、1318XY and1318XYplus) and were applied to analyze500unrelated Han individuals:PCR on ABI9700and electrophoresis on the Genetic Analyzer (ABI3130). Based on the genotype analysis, the41STR genetic population data were initial set up.2) To establish D21high throughput STR detection assay (D21assay) and genetic analysis of11STR markers on chromosome21in Han population:11STR markers on chromosome21(D2181432、D21811、D2181246、D2181412、D2181437、D2181413、 D21S2039、D21S1270、D21S1435、D21S1446、D21S1409) and Amel were adopted to establish D21assay;To constitute D21allele standards (Allelic Ladder); To program D21genotyping software; Based on the analysis of the genotype and sequence of500unrelated Han individuals, the genetic population data of11STR markers on chromosome21in Han population were established.558cases of fetal cell collected from amniotic fluid and42cases of villi were detected with D21assay, to confirm the reliability of D21assay on detecting trisomy21according to the karyotype.3) To establish D1318XY high throughput STR detection assay (D1318XY assay): 19STR markers on chromosome13,18,X,Y (D18S535、D13S797、DXS1187、 DXYS267、D13S305、DXS6803、D13S628、DXS981、XHPRT、D18S386、D13S634、 D18S390、D18S1002、D13S258、DXS6809、D13S742、D18S391、X22、D18S499) and TAF9b、Amel、SRY were adopted to establish D1318XY assay;To constitute D1318XY allele standards (Allelic Ladder); To program D1318XY genotyping software;558cases of fetal cell collected from amniotic fluid and42cases of villi were detected with D1318XY assay, to confirm the reliability of D1318XY assay on detecting aneuploidies of chromosome13,18,X,Y according to the karyotype.Results:Two high thoughput STR testing assay (D21assay and D1318XY assay) were successfully established, which could detect all the target chromosome abnormality (Trisomy21,13,18and aneuploidy of X, Y) in558cases of fetal cell collected from amniotic fluid and42cases of villi. Based on the analysis on the genotype of30STR (D21assay and D1318XY assay) of500unrelated Han individuals,19STR marker were found with high polymorphism and heterogeneity, which preferred for QF-PCR establishment.Conclusion:The established high thoughput STR testing assay (D21assay and D1318XY assay) laid the foundation for QF-PCR; The genetic population data of41STR on chromosome21,13,18,X,Y provide the basis for selection of appropriate STR markers for QF-PCR assay. Chapter3The establishment of the QF-PCR detection assay for Chinese peopleObjective:To establish the QF-PCR detection assay for Chinese people.Samples:558cases of fetal cell collected from amniotic fluid (with known kayrotype);15cases of villi (with known FISH result); Several blood from abnormal fetus’s parents; a small amount of different kind of sample:blood stains, hair follicle, saliva stain, muscle, etal.Method:23gene loci including21STR spread over chromosome21,18,13,X,Y (D21S1432、D21S1270、D21S11、D21S1412、D21S1442、D21S1435、D18S535、 D18S386、D18S1002、D18S977、D18S499、D13S305、D13S634、D13S258、D13S742、 DXS1187、DXYS218、DXYS267、DXS981、DXS6809、X22)and AMEL、SRY were adopted to establish a new high throughput STR detection assay (QF-PCR):All primers were designed with PRIMER3again according to new assignment; The preferred primers were separately labeled with5’ROX、5’TAMRA、5’HEX、6’FAM;To optimize the PCR reaction assay; To constitute QF-PCR allele standards (Allelic Ladder); To program QF-PCR genotyping software; To evaluate the testing sensibility of QF-PCR with DNA standard9947A; To evaluate the discrimination ability of QF-PCR on mixed samples with9947A and9948(with known genotype); To evaluate the detection capability of QF-PCR on different kind of sample of blood stains, hair follicle, saliva stain and muscle; To evaluate QF-PCR on test turnaround time and test throughput; By testing558cases of fetal cell collected from amniotic fluid and15cases of villi, verify the diagnosis standard of QF-PCR and the diagnostic ability of QF-PCR on target abnormal chromosome, according to karyotype or FISH results; To identify the paternity origin of additional or deletion chromosome, several parents’sample were analyzed with the abnormal fetus’s.Results:The QF-PCR was established with23gene locus, which could detect all the target chromosome abnormality (Trisomy21,13,18and aneuploidy of X, Y) in a single assay. The established QF-PCR were test good in performance:high sensibility, accuracy, fast, high throughput, suitable for various kind of sample, able to detect the second karyotype presenting in more than20%of cells; able to identify the paternity origin of additional or deletion chromosome. However, there exsit some gray area in two allele peaks in some STR locus and some single abnormal STR peaks in particular STR locus, which allowed further analysis with more tests.Conclusion:QF-PCR detection assay was successfully established for Chinese Han people. Chapter4Prospective study on the clinical application of QF-PCR for Chinese peopleObjective:To evaluate the Suitability of QF-PCR in clinical applicationMethod:From Jan.2012to June2014,925cases of residual amniotic fluid2-3ml were collected from the high risk pregnant women who perform prenatal diagnosis (karyotyping) in our center. For each case,1ml amniotic fluid was used to extract DNA with Chelex method. For each batch,90samples were processed with QF-PCR kit, amplifying on common PCR machine and electrophoresis on ABI3130genetic analyzer. To evaluate the test accuracy, turnaround time, throughput and accessibility.Results:925amniotic fluid samples were totally successful tested. Comparing with karyotyping, the QF-PCR detected15out of16cases of target chromosome abnormality:8cases of trisomy21,3cases of trisomy18,1case of trisomy13,1case of45X,1case of47XXY, and1case of47XXX.1case of45X mosaic (46,XX[46]/45, X [4])was missed. On the detection of5target chromosomes, QF-PCR showed high consistency with karyotyping:the total consistency was99.89%(95%CI99.78-100%), the positive testing consistency was93.75%(95%CI92.95-94.55%), and the negative testing consistency was100%. Samples could be performed in batch, for each sample, the detection turnaround time could be16-17min; QF-PCR could be conveniently performed in PCR lab.Conclusion:The established QF-PCR detection assay is fast, accurate, high throughput, convenient, cost effective, and easily quality control, which is suitable for performing clinical prenatal diagnosis in Chinese population and has a promising prospect.