| BackgroundWith the development of cytogenetics and molecular genetics, the technology and method of prenatal screening and prenatal diagnosis are diversified, at present the main screening method commonly used in domestic pregnancy prenatal screening is the maternal plasma screening, which could predict the incidence of high risk of 13,18, trisomy 21 and neural tube fetal screening. In addition, early pregnancy serum screening, early pregnancy fetal ultrasound NT index value screening or early in the pregnancy combined screening program, have been used in some regions. For the above screening high-risk pregnant women, can in the early stage, or late pregnancy by collecting fetal cells, tissue samples for detection of fetal genetic material, main technique is commonly used by placental villi sampling (CVS), in the second trimester amniotic sampling, late pregnancy fetal umbilical cord blood sampling or in some severe malformation cases that can not survive the pregnancy of heart puncture and so have the operation record to obtain fetal cell samples for testing. The accuracy is high, but still has certain limitation, prenatal diagnosis way is not suitable for a screening tool for a large scale use. So, for the genetic disease screening in the population, noninvasive or minimally invasive, effective screening method is needed to be explored in the future.Studies had shown that genome CNVs play important roles in some human diseases. Some famous syndrome, such as:Williams-Beuren syndrome, Angelman/Prader-Willi syndrome, Charcot-Marie-Tooth syndrome, are composed of copy number variation or chromosome fragment deletions or duplications result. Such as single nucleotide polymorphisms, the majority of copy number variation exists in normal polymorphic forms, only a few of the copy number variation is associated with disease. Chromosomal microarray analysis (CMA):the emergence of microarray comparative genomic hybridization and single nucleotide polymorphism microarray, which greatly improves the copy number variation in the human genome found. These chromosomal microarray technology because of its advantages of high flux, high resolution found that prenatal diagnosis in the world widely used.Free DNA free DNA containing a certain proportion of the fetal maternal blood plasma, the DNA fragment length is smaller, the absolute content increased with gestational age, and soon disappeared in the degradation can speed characteristics of pregnancy after the end, makes the extraction and detection of maternal plasma DNA as a non-invasive detection to obtain fetal the genetic information became possible.A new generation of high-throughput sequencing technology can simultaneously analyze millions of nucleotides in single molecule sequencing of the technology, the basic principle is:first of all, the genomic DNA fragmentation, then joined the joint and sequencing primers generated cluster after amplified fragment bridge, will be connected to the flowcell, each time adding reaction a reversible termination, fluorescence scanning. This high-throughput, high accuracy of the emergence of sequencing technology, foundation with free fetal DNA in maternal plasma is relatively stable proportion of, greatly promoting the application of noninvasive prenatal testing (NIPT). An aneuploidy chromosome is on the clinical most common chromosomal abnormalities, and most major disabling the foolishness of hereditary disease, chromosome aneuploidy is somatic diploid chromosome is missing or extra one or more chromosomes, can be a homozygous or heterozygous for. Cell free fetal DNA in maternal plasma was found and based on the application of high-throughput sequencing technology to NIPT, a new era of NIPT pioneered the use of high-throughput sequencing of fetal chromosomal aneuploidy and other genetic diseases around the world, with many research group the clinical applicability of a lot of in this method, the sensitivity and specificity of the currently most common trisomy 21-noninvasive prenatal detection have reached more than 99%. Based on this, studies have shown that from maternal plasma free split a whole genome information of fetal and maternal DNA, the results imply that the maternal plasma DNA fetal copy number variation provides the research foundation detection even single gene diseases as study materialsObjectiveAccording to the clinical non-invasive prenatal detection with high risk pregnant women syndrome refers to the application of a new generation of high-throughput sequencing for prenatal testing, screening out the common fetal autosomal aneuploidies. For abnormal signal NIPT process samples, copy number variation analysis of the signal detected by the possible pathogenic information, carries on the prenatal invasive diagnostic techniques, analysis and verification of NIPT of pathogenic findings may copy number variants using chromosome microarray technology. Through test and verification, copy number variation and clinical consultation and diagnosis in patients with pathogenicity, and to establish a for NIPT technology platform of copy number variation, the results of the study can also provide scientific data copy number variation database.MethodsPregnant women who have NIPT of indications of routinely informed consent and NIPT, through the analysis of high-throughput sequencing we got the fetal aneuploidy screening results. On the common 13,18,21 and sex chromosome aneuploidy non detection results of noninvasive prenatal diagnosis, chromosome karyotype analysis was performed on fetal amniotic fluid or umbilical cord blood cells, for noninvasive prenatal test to verify the results. The results of the data analysis in the reaction of copy number variation related to abnormal signals were pathogenic copy number variation analysis, if according to the discovery of the chromosome copy number variation may be pathogenic to retrieve an existing database, this pregnant women were informed, and prenatal diagnosis of traumatic technique after informed consent, according to the different gestational weeks, analysis of karyotype and CMA on fetal amniotic fluid or blood specimens. If the embryo staining verified exists abnormal copy number variation, we will carry on the analysis of the peripheral blood samples of fetal parents karyotype analysis and chromosome microarray, with genetic sources clear this exception. According to the karyotype and chromosome microarray detection results, the common chromosome aneuploidies and chromosomal microdeletion or microduplication patients were offered with genetic counseling.ResultsIn the fetal chromosomal aneuploidy without the basis of a production techniques, the NIPT of clinical indications of pregnant women were conducted with a sample of fetal chromosomal aneuploidy, on 21,18 and 13 chromosome and sex chromosome aneuploidy situation there have been screening accurately. We found 16 T-21,3 T-18. All screening aneuploidy samples were put to the test by invasive prenatal diagnosis of fetal chromosomal karyotypes,15 in 16 T21 were confirmed by CMA and karyotype and another one is confirmed as an NIPT false positive result. Once again confirms the previous studies in this technique for fetal chromosome aneuploidy detection.There are 9 cases of sex chromosome aneuploidy detected in the noninvasive testing. Among which there are 2 47,XXX,1 47,XXY,1 47,XYY,1 mosaic 47,XYY/46,XY, the left 4 cases were fals positive cases and were not detected by the chromosomal analysis.More over, NIPT showed a 13 microdeletion, a 18 microdupllication, and a 18 deletion. In the invasive prenatal detection of these CNVs, match the results of the CMA analysis. A method was established for NIPT of fetal genome CNVs and we have proved the feasibility of NIPT technique in the detection of fetal genomic CNVs.ConclusionsIt is reliable for the NIPT combined with CMA to find the microdeletion or microduplication in the fetus through analysis of CNVs associate abnormal signals of NIPT results. By using this method, we found two cases of human CNV polymorphism that has never been reported before. The detection results also provided research data for the study of human genome and provided research data for the prenatal screening and prenatal diagnosis methods research and development. |
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