The Research On SMN Gene Carrier Screening Among Pregnant Women With FQ-PCR And Application On Prenatal Diagnosis

OBJECTIVEThe experiment purpose is to establish an accurate,high-throughput,rapid and simple screening and diagnosis method by detecting the deletion of SMN1 gene exon7 and/or exon 8 in pregnant women.It is helpful to improve the quality of the birth population by preventing the birth of children with spinal muscular dystrophy(SMA).METHODIn this study,we used FQ-PCR to diagnose the genotypes of 162 pregnant women in the Center Hospital of Obstetrics and Gynecology of Tianjin during December2014 to October 2016.The experiment was divided into two parts.Experiment 1 was used to detect the deletion of exon 7 and 8 in SMN1 gene by FQ-PCR in 5 families with SMA cases.The Sequencing and MLPA were used to confirm the FQ-PCR to detect the feasibility of this genetic change.Experiment 2 was the main part of the experiment,162 pregnant women were diagnosed by FQ-PCR.If one's genotypes was SMN1 heterozygous deletion,she would be identified as a SMA carrier.Peripheral blood of the pregnant women with SMN1 heterozygous detection were collected for DNA detection.When the couples were SMN1 heterozygous deletion,the amniotic fluid samples of their fetus were collected for prenatal diagnosis.RESULTSExperiment 1(1)Quality of 5 SMA familiy members' extracted DNA: DNA of peripheral blood was extracted by centrifugal method.DNA concentration ranged from 22.7ng/?l~61.4 ng/?l(36.8±12.1 ng/?l),OD260/OD280 ranged from 1.75 to1.89.(2)The results of FQ-PCR and Sequencing and MLPA for the members of the 5SMA familiyThe exon 7 in SMN1 was homozygous deletion in all SMA,however,1 case of them also had SMN1 exon 8 heterozygous deletion.In the 4 families who had SMA cases with SMN1 exon 7 and exon 8 homozygous deletion,the parents' genotypes were SMN1 exon 7 and exon 8 heterozygous deletion,while the parents' genotypes of the case with SMN1 exon 7 homozygous deletion and exon 8 heterozygous weredifferent.The mother's genotype was SMN1 exon 7 and exon 8 heterozygous deletion,the father showed that only exon 7 was heterozygous deletion,exon 8 was no deletion change.The results were consistent with Sequencing and MLPA.Experiment 2(1)Quality of extracted DNA:DNA of peripheral blood and amniotic fluid was also extracted by centrifugal method.DNA concentration of peripheral blood ranged from 6.8 ng/?l~194.1 ng/?l(67.3±33.1 ng/?l),OD260/OD280 ranged from 1.75 to1.89.In the 2 high-risk families,the DNA concentration of amniotic fluid were 22.9ng/?l and 27.1 ng/?l,OD260/OD28 were 1.81 and 1.83.(2)The results of FQ-PCR for pregnant women There were 6 carriers with SMN1 exon 7 heterozygous deletion in 162 pregnant women.(3)The results of FQ-PCR for the carriers' spouses Among the 6 cases,4 cases showed that exon 7 and exon 8 were both normal.2 cases showed exon 7 and exon 8were heterozygous deletion.(4)The results of FQ-PCR and Sequencing and MLPA for carriers Excluding the effect of maternal blood contamination,1 fetus had exon 7 and exon 8homozygous deletion,and the other one had exon 7 and exon 8 heterozygous deletions.It was same with the result of Sequencing with MLPA.(5)Prenatal diagnosis and genetic counseling were made for the carriers.CONCLUSIONS(1)The exon 7 of SMN1 gene is closely related to SMA.Distinction of SMA patients,carriers and normal population can be made by exon 7 deletion in SMN1.(2)FQ-PCR can be applied to the initial diagnosis of SMA and carriers in population screening.Compareing with other detection methods,FQ-PCR has advantages of simple,rapid,low cost,accurate and reliable detection result.(3)SMA is seriously harmful,and there is a high carrying rate in population.Screening for SMA carriers in pregnant women and making prenatal diagnosis is meaningful.