Basic And Applied Research Of High-throughput Sequencing Techniques In Preimplantation Genetic Diagnosis

Clinical application of preimplantation genetic diagnosis in single gene disordersObjective: The study aimed to explore the application of multiple displacement amplification(MDA)combined with next generation sequencing(NGS)-haplotype analysis in PGD for single gene disorders.Materials and methods: Whole cell genome was amplified by single cell biopsy with MDA method.The gene mutation was taken as the target region.A number of single nucleotide polymorphisms(SNPs)were selected as genetic markers in the upstream and downstream of the mutation by NGS based on preimplantation genetic haplotyping(PGH)and aneuploidy screening.The results of all embryo haplotype analysis were validated by Sanger sequencing.Results: The 14 patients with their pedigrees included 11 single gene disorders,with a mean age of 31.7 ± 5.5 years old.A total of 193 oocytes were obtained by control ovarian stimulation,of which oocytes maturation rate was 84.5%(163/193),fertilization rate was 83.4%(136/163)and cleavage rate was 98.5%(134/136).A total of 984 SNPs were validated by haplotype pre-testing in 14 pedigrees.A total of 72 embryos were biopsied.The percentage of normal embryos was 22.2%(16/72)by PGH and PGS.Of 72 embryos,6.9%(5/72)were unidentified embryos and recombined events occurred in 4 of 72 embryos(5.6%).The results of PGH were validated by Sanger sequencing which showed 100% consistency.3 of the 14 patients had not yet been transferred,5 patients got no normal embryos,and 6 patients had been transferred.A total of 4 patients got successful pregnancies(congenital hereditary deafness,infantile axonal dystrophy,phenylketonuria and adrenoleukodystrophy),resulting in 2 successful term deliveries and 2 ongoing pregnancies.Conclusion: MDA combined with NGS-PGH method can help families of high genetic risk of single gene disorders to obtain healthy offsprings.NGS-PGH can simultaneously analyze embryo genotypes and haplotypes and is a highly reliable method.The Value and Risk of Transferring Mosaic EmbryoObjective: Mosaic embryos are common in early embryos of human assisted reproductive technology.This study explored the clinical value of mosaic embryos.Materials and methods: The chromosomal composition,clinical pregnancy outcome and amniocentesis results were analyzed in 28 PGD cycle of transferring mosaic embryos.The four mosaic blastocysts donated by the patients were divided into inner cell mass and trophectoderm part for NGS reanalysis.Results: 28 PGD cycles of transferring mosaic blastocysts resulted in 10 clinical pregnancies(35.7%).Among these 10 pregnant cases,there were 4 live births and the infants were healthy,one patient had an ongoing pregnancy,5 patients had early miscarriage.Among 5 successful clinical pregnancies,3 cases of the amniotic fluid karyotype were normal and 1 case showed chromosomal translocation,one patient refused amniocentesis.NGS was applied to reanalyze 4 mosaic blastocysts.3 blastocysts were normal based on trophectoderm and internal cell mass diagnosis,trophectoderm diagnosis of one blastocyst left was normal,however,the inner cell mass diagnosed was mosaic: microdeletion of 12.46Mb(7p21.3-p15.3).Conclusion: Mosaic embryos can be attempt to transferred when there are no normal embryos in the PGD and PGS cycles,but the risk of transferring a mosaic embryo must be fully understood by patients and a subsequent invasive prenatal diagnosis must be followed.Molecular mechanism of KDM5 C mutation in X-linked mental retardation syndromeObjective: To investigate the function of the KDM5 C novel mutation c.673 del C at both transcriptional and posttranscriptional levels in an intellectual disability pedigree.Materials and methods: The wild type and mutant plasmids of KDM5 C gene were transiently transfected into human SH-SY5 Y cell line,and si RNA was also transfected.Real-time fluorescence quantitative PCR(QPCR)and Western blot(WB)were used to detect the expression of messenger RNA(m RNA)and protein respectively.The H3K4 trimethylation levels and apoptosis were detected by flow cytometry.Results: Compared with the SH-SY5 Y cell line transfected with wild type plasmid,the expression of KDM5 C in transfected mutant plasmid was significantly decreased(P<0.05),while there was no significant difference in the H3K4 trimethylated levels(P>0.05).The rate of H3K4 trimethylated levels in group of SH-SY5 Y cells transfected with si RNA and negative control group was comparable(P>0.05).Conclusion: The c.673 del C mutation of KDM5 C gene reduced the expression of KDM5 C,but did not affect the overall H3K4 trimethylation levels.Interfering with the expression of KDM5 C gene could not affect the overall H3K4 trimethylation levels in SH-SY5 Y cells.