Application QF-PCR Technology Rapid Diagnosis Of Fetal Chromosome Aneuploidy Disease

Objective: Chromosomal diseases including number of chromosomesabnormalities and structures of chromosomes abnormalities. The chromosomeaneuploidy disease belongs to number of chromosomes abnormalities,andincluding haplotypes, polysomy and trisomy and chimeras. In the clinical it iscommon that21-trisomy syndrome (Down Syndrome),18-trisomy (EdwardSyndrome),13-trisomy syndrome (Patau Syndrome),as well as some sexchromosome aberrations, such as Klinefelter syndrome (47,XXY) and Turnersyndrome (45,X). Studies have shown that13,18,21and X/Y-chromosomeabnormalities accounted for95%of neonatal number of chromosomesabnormalities, which put a heavy burden to families and society. At present,there are no effective treatment methods for such chromosome diseases. Theprevention focus is conscientiously carrying out antenatal screening andprenatal diagnosis. Karyotype analysis is the gold diagnosis standard on theclinical. Yet the technology has great limitations. Such as the cultivationmethod of amniotic fluid program trivial, inspection cycle is long；Amnioticfluid cells is not easy to cultivate, the reliability of the test result is dependslargely on the experience and technology of operator. There are not enoughprenatal diagnosis centers that can complete stability, high quality theamniotic fluid cultivation in our country. It is detrimental to large-scaleclinical screening and prenatal diagnosis. The development of quantitativefluorescence polymerase chain reaction (QF-PCR) has opened new avenuesfor the chromosome aneuploidy disease. The technology has a high sensitivityand specificity to the un-mosaic chromosome aneuploidy disease. In this study,we use short tandem repeat of13,18,21and sex chromosomes, applyquantitative fluorescence polymerase chain reaction technology on diagnosispregnant women who has chromosome abnormality at high-risk for diagnosis, and establish the fast prenatal diagnosis technology of the commonchromosome aneuploidy disease.Methods: Laboratory specimen is from the Second Hospital of HebeiMedical University obstetrics clinic, ward and prenatal diagnostic center inMarch2010to December2011.(1) Object of study: the pregnant women who is serological screeninghigh-risk,type-B ultrasonic showed abnormal fetal growth or with pregnancycomplications need significant inducing labor；②Neonatal peripheral bloodwhose postnatal signs suspected chromosomal abnormalities；③Peripheralblood of children who is abnormal development signs.(2) Sample collection: pregnant women are implemented amniocentesisby obstetricians in type-B ultrasonic-guided positioning. Extract amniotic fluidspecimens2-5ml for DNA extraction, and the remaining15-20ml for cellculture karyotype analysis；selected newborns who suspected of chromosomalabnormalities, after birth extract peripheral blood1ml, heparinize it and0.2mlused for DNA extraction,0.8ml for the cell culture karyotype analysis.(3) DNA extracted and quantitative: after specimen collection, weimmediately extract DNA using BloodGen Mini Kit, then-20℃contain DNA.Then batch DNA is miniature gel electrophoresis quantitative and ultravioletspectrophotometer quantitative. Select one highly concentration and purityDNA.(4) STR amplification and analysis results：using the method of QF-PCRamplification the STR of DNA samples in chromosome21,13,18and sex thatD21S11, D18S535, D13S631, X22and AMXY. The PCR product is incapillary electrophoresis, combined with Genescan software analysis andcalculation of the peak area ratio, and compared with the karyotype analysisresults.Result: It is consistent the results of118cases by fluorescencequantitative PCR rapid diagnosis and karyotype analysis.(1) Normal karyotype is113cases. The heterozygote samples show thesame fluorescence intensity1:1twin peaks, and homozygote samples show the single-peak. The Double allele peak area ratio of heterozygote samples STRsites are respectively D21S111.033-1.301, D18S5351.032-1.397, D13S6311.009-1.229, X221.031-1.244, AMXY site has59heterozygote cases and54homozygote cases, namely59cases are male and54cases are female.(2) Abnormal karyotype is5cases.21-trisomy syndrome are3cases；18-trisomy syndrome are1cases；47, XXY (Klinefelter syndrome) are1cases.Three cases of21-trisomy syndrome samples and one case of18-trisomysyndrome samples show fluorescence intensity of1:1:1there-Allele peak. Thismeans the four cases are normalized form.Conclusions: This study demonstrates that the quantitative fluorescencepolymerase chain reaction rapid prenatal diagnostic technique forchromosome aneuploidy disease diagnosis is a simple, rapid, accurateadvantage. The use of five STR sites can diagnose the abnormal number of13,18,21and the X/Y chromosome together. And it can automate the detection ofa large number of samples, suitable for clinical large-scale application. Itapplies to pregnant women age35years or more, higher-risk maternitychromosome abnormal fetus； pregnant women who has the highlychromosomal abnormalities fetal risk after the triple serum screening；pregnant women who was showed chromosomal abnormalities sign byultrasound or who eager to know the results；known one of the parentssuffering from Robertsonian such as translocation13/21of the fetus. Theestablishment of the technology has the great significance for reducing thecommon chromosomal disease, especially the incidence of the commonchromosome aneuploidy disease, improve the quality of the newborn, prenataland postnatal care.