| Part ? Genetic polymorphism of 15 STR Loci in ?-globin gene of Guangxi populationObjective: To study the genetic polymorphism of 15 short tandem repeat(STR)loci closely linked to ?-globin(HBB)gene in Guangxi population,thus can provide experimental evidence for the establishment of preimplantation genetic haplotyping(PGH)of preimplantation genetic diagnosis(PGD).Methods: Peripheral venous blood from 150 unrelated individuals in Guangxi was collected and the genomic DNA of peripheral blood was extracted.Fifteen STR loci(HBB4506?D11S988?HBB4677?D11S2362?HBB5089?D11S1243?HBB5138?HBB5178?HBB5205?D11S1760?HBB5576?HBB5655?HBB5820?HBB5859?D11S1338)closely linked to beta-globin(HBB)gene(from HBB gene<1 Mb)were amplified by fluorescence PCR.The amplified products were analyzed by capillary electrophoresis using an ABI3500 Dx genetic analyzer.The results were analyzed using Genemaper 4.1 software.Finally,the allele frequency distribution and polymorphism information content(PIC)of 15HBB-STR loci were calculated by using Microsoft Excel software.Results: 18,24,22,11,18,27,13,13,16,23,22,16,17,18 and 7 alleles were detected on the 15 HBB-STR loci(HBB4506?D11S988?HBB4677?D11S2362?HBB5089?D11S1243?HBB5138?HBB5178?HBB5205?D11S1760?HBB5576?HBB5655?HBB5820?HBB5859?D11S1338).The allele frequency ranged from0.003333 to 0.383333.The polymorphism information content(PIC)of these 15HBB-STR loci were0.8457?0.8983?0.8931?0.7592?0.8698?0.9046?0.7940?0.7925?0.7708?0.9102?0.8583?0.8467?0.7438?0.8366 and 0.7704?The PIC of these 15 loci was> 0.7000,with an average of 0.8329.There were 6HBB-STR loci in PIC> 0.8500.Conclusion: The polymorphism information content(PIC)of these 15HBB-STR loci detected in this study was higher.They have rich genetic polymorphism.It has a high value in the preimplantation genetic haplotyping(PGH)of preimplantation genetic diagnosis(PGD)of ?-thalassemia,which provided the experimental basis for the follow-up work.Part ? Comparison of different diagnostic methods in preimplantation genetic diagnosis of ? – thalassemiaObjective: The reverse dot blot(RDB)combined with STR haploid analysis and Next generation sequencing(NGS)were used in the diagnosis of ?-thalassemia PGD.The diagnostic time,diagnosis rate,reliability,cost and so on of the two methods were compared.We strive to establish the most suitable genetic diagnostic techniques for ?-thalassemia PGD in Guangxi.Methods: Choose both couples who are ?-thalassemia gene mutations carriers.They have PGD indications and agreed to participate in this study.Patient couples and three other family members(woman mother,man mother and man father)were treated with peripheral venous blood for 15 STR loci linkage analysis.The woman was subjected to conventional ovarian stimulation,oocyte retieval,intracytoplasmic sperm injection(ICSI),embryo culture and so on.The trophectoderm cells were biopsied on Day 5 after oocyte retieval.And then all the blastocysts will be vitrified frozen.The whole genome amplification(WGA)of trophectoderm cells was performed by multiple displacement amplification(MDA).A portion of the MDA amplification product was genetically diagnosed using reverse dot blot(RDB)in combination with 15 short tandem repeat(STR)loci of PGH technique(A group);the other part of MDA amplification products were diagnosed by NGS method(group B).The detection rate,the diagnostic coincidence rate,the required time and cost of the two methods are compared.Finally,choose the most suitable blastocysts for thawing transplantation.Results: The patient in this study received 24 oocytes and 13 embryos were formed.Six blastocysts were formed after blastocyst culture.The blastocyst scores were 4BB,4BC,3CC,3CC,3BC and 3CC,respectively.Cell biopsy,6 samples of MDA amplification were amplified successfully,the amplification success rate of 100%.In group A,the diagnosis result of blastocyst 1 was ?-28 /?CD41-42;the diagnosis result of blastocyst 2 was ?CD41-42 / ?N;the diagnosis result of blastocyst 3 was ?N / ?N;the diagnosis result of blastocyst 4was?-28 / ?N / ?N;the diagnosis of thalassemia genotypes of blastocysts 5 and6 were ?-28 / ?N.The results of genotype diagnosis of thalassemia in group B were identical with those in group A,and group B was also tested for chromosome copy number of blastocysts 2-6.The blastocyst 2 was euploid and blastocysts 3-6 were aneuploid.A group of diagnostic time required for 2-3 days,the results of the analysis time is about 1 working day;Group B required diagnostic time of 2-3 days,the results were sent to Jia bao ren he company analysis,the analysis time required for 7 working days.Group A costs 2,000 yuan / blastocyst,and Group B costs 3,500 yuan / blastocyst.Conclusion: Compared with the second generation sequencing(NGS)technique,reverse dot hybridization combined with STR haplotype analysis can also effectively and accurately diagnose the thalassemia genotypes of preimplantation blastocysts in ?-thalassemia PGD,which can be applied to ?Thalassemia PGD clinical.Although the current second-generation sequencing is expensive,the requirements for results of the analyst are high,clinical application is limited,but NGS is PGD genetic diagnostic technology development direction and future trends.However,taking into account the current restrictions on various aspects of the conditions and in Guangxi to carry out the urgent needs of ?-thalassemia PGD,RDB combined with 15 STR loci haplotype analysis of the diagnostic method is more suitable for the current Guangxi ?-thalassemia PGD,is worthy of popularization and application.Part ? Clinical application of MDA combined with RDB and STR haplotype analysis in ?-thalassemia PGDObjective: To investigate the clinical application of multiple displacement amplification(MDA)combined with reverse dot blot(RDB)and short tandem repeat(STR)haplotype analysis in ?-thalassemia PGD.Methods: There are eight couples on both sides of the ?-thalassemia gene mutation carriers who required beta-thalassemia PGD treatment.Carrier couples and their parents(each family of a total of six members)were taken peripheral venous blood for 15 STR loci linkage analysis.The patients were subjected to conventional ovarian stimulation,oocyte retieval,intracytoplasmic sperm injection(ICSI),embryo culture and so on.The trophectoderm cells were biopsied and then all the blastocysts will be vitrified frozen.The whole genome amplification(WGA)of trophectoderm cells was performed by multiple displacement amplification(MDA).The MDA amplification products were genetically diagnosed using reverse dot blot(RDB)in combination with 15 short tandem repeat(STR)loci.Finally,choose the most suitable blastocysts for thawing transplantation.The diagnosis of PGD was further confirmed by prenatal diagnosis of amniotic fluid from 18-22 weeks of gestation.Results: The mutation types of these 8 couples were CD41-42 / N,CD17 / N,-28 / N or IVS-II-654 / N respectively.The results showed that the STR loci of each family had 7 or more(HBB gene upstream or downstream have more than two STRs)could provide diagnostic information,and the haplotype was successful.The total of 147 embryos were obtained and 86 blastocysts were formed.Among them,82 blastocysts were successfully amplified and 95.35% successful.80 blastocysts were clearly diagnosed,of which 24 blastocysts were normal homozygous;38 blastocysts were diagnosed as heterozygous;18blastocysts could not be transplanted with abnormal homozygous or double heterozygotes.A total of 4 patients in the 8 patients underwent thawed embryo transfer.Three cases received pregnancy,otherwise 1 case without pregnancy.The pregnancy rate was 75%(3/4).Family 1 and family 2 patients were transplanted normal homozygous blastocyst(genotype ? N / ? N),prenatal diagnosis of amniotic fluid and PGD diagnosis results are consistent.Family 3patient was transplanted heterozygous blastocyst(genotype ?CD41-42 / ?N),currently in pregnancy,but there is no prenatal diagnosis to verify the results of PGD diagnosis.Conclusin: In this study,we established the ?-thalassemia PGD method,that is,after multiple displacement amplification(MDA),reverse dot hybridization(RDB)combined with short tandem repeat(STR)haplotype linkage analysis.This method has proven to be effective and accurate,can be popularized in the clinical practice of ?-thalassemia PGD in Guangxi.We look forward to a larger sample size of the study confirmed. |
PDF Full Text Request