Preliminary Clinical Application Study Of CSMART In Noninvasive Prenatal Test Of ?-thalassemia & Methylation Markers' Screening And Identification For Fetal ?-thalassemia Major

Background ? thalassemia is a common single-gene disease,mainly distributed in Southeast Asia,the Mediterranean coast,the Middle East and southern China,with a carriage rate of nearly 6.8% in the Guangdong region of China.The current clinical practice is to test fetuses for HBB mutations through the Invasive Prenatal Test?IPT?and to recommend termination of pregnancy in fetuses with thalassemia major.However,this method causes a certain risk of miscarriage,causing great mental stress and anxiety to the pregnant woman.The discovery of plasma cell free fetal DNA in pregnant women provides a safe and reliable pathway for prenatal testing.At present,there are few studies on non-invasive prenatal ?-thalassemia major testing,which needs to be studied in depth to prevent the birth of children with ?-thalassemia major and to reduce social pressure.Chapter ?:Preliminary clinical application study of cSMART in noninvasive prenatal test of ?-thalassemiaObjective To evaluate the effectiveness of the cSMART?circulating Single-Molecule amplification and resequencing technology?method for non-invasive prenatal ?-thalassemia testing,with combination of relative mutation dosage?RMD?analysis.Methods Seven couples were selected as carriers of the HBB gene mutation,including pregnant women with HBB:c.126₁29del CTTT?CD41-42?heterozygous mutation,spouses with HBB:c.126₁29del CTTT?CD41-42?heterozygous mutation,HBB: c.316-197C>T?IVS-II-654?heterozygous mutation,HBB:c.-78A>G?-28?heterozygous mutation and HBB:c.52A>T?CD17?heterozygous mutation.10 mL of pregnant women's peripheral blood was collected,plasma was isolated and free DNA was extracted,and a CVS was performed and further fetal HBB genotyping was performed as a control method.Results Among the 7 cases,the cSMART technique was used for non-invasive prenatal ?-deprivation testing in 2 cases of HBB: c.126₁29delCTTT?CD41-42?homozygous,2 cases of HBB: c.126₁29delCTTT?CD41-42?heterozygous mutation,1 case of HBB: c.52A>T?CD17?heterozygous mutation and 2 cases were normal.1 case of HBB: c.126₁29delCTTT?CD41-42?heterozygous mutation was detected by the invasive method,but the cSMART method was normal.Conclusion The cSMART technology can be used to detect HBB gene point mutations noninvasively,but the detection of the deletion type mutation?HBB: c.126₁29del CTTT?CD41-42??may be inconsistent with the results of invasive prenatal testing,suggesting the potential feasibility of cSMART technology for noninvasive prenatal detection of fetal ?-thalassemia.Chapter ?:Methylation markers' screening and identification for fetal ?-thalassemia majorObjective Preliminary genome-wide screening of significant DNA methylation differential loci present between normal and heavy ?-depleted fetuses to provide candidate fetal DNA methylation markers for noninvasive prenatal detection of ? thalassemia major fetuses.Methods collected chorionic specimens from 16 fetuses with ? thalassemia major and 16 fetuses with normal genotype,which were diagnosed by invasive prenatal diagnosis.Firstly,we used the Illumina Infinium ? Human Methylation 850 K Bead Chip DNA methylation microarray to screen the genome-wide DNA methylation loci in the chorionic villus samples of 5 heavy ?-depleted fetuses and 5 normal genotype fetuses,compared the differential methylation loci,and looked up the database?GO analysis?for bioinformatics analysis to select the loci that were hematopoietically relevant and highly methylated in heavy ?-depleted fetuses but hypomethylated in normal fetuses.Results The Illumina 850 K DNA methylation microarray screening of chorionic villus samples from five ? thalassemia major and five normal genotype fetuses revealed 3394 statistically significant differential methylation loci and 60 genes were involved?Diff Score values less than or greater than-13 and Delta_Beta greater than or less than-0.1 were differentially methylated genes?.By direct sequencing of the destination fragment and cloning,the results indicated that CUBN-cg11334510,PTPRN2-cg 0753 8293,PTPRN2-cg25402818,PTPRN2-cg26859186,PRDM16-cg12096707,PR DM16-cg08110058 total 6 SNP loci were hypermethylated in 11 ? thalassemia major fetuses and hypermethylated in 11 normal fetuses,with statistical differences?P<0.05?.Conclusion The six SNP loci identified in this study,CUBN-cg11334510,PTP RN2-cg 07538293,PTPRN2-cg25402818,PTPRN2-cg26859186,PRDM16-cg12096707,PRD M16-cg08110058,can be used as markers for the detection of fetal ? thal assemia major and provide experimental basis for further non-invasive prenatal dete ction of ? thalassemia.