The Study Of Imprinted Genes On Expression In Human Oocytes And Preimplantation Embryos And On Molecular Diagnosis Of Imprinting Disease Prader-Willi Syndrome

Gene imprinting is characterized by expression of only one of the parental alleles according to its inheritance. It's of expression of only one of the alleles from the mother or the father. It's also named parental imprinting. The gene which with the characteristic above is named as imprinted gene. Being different from tranditional Mendelian inheritance which indicates that gene is expressed from both alleles of parents and is not influenced by the parental origin of the allele. Gene imprinting isn't a kind of classic inheritance gene regulation but a appearance genetic modification. DNA methylation is presumed its major modificatory mode. Gene imprinting betides gametogenesis and can affect oocyte maturation, embryogenesis, embryo development, foetus growth and placenta differentiation. Abnormality imprinting in imprinting centre results in series of inherited imprinting diseases, such as abortion, fetal death, malformation, dysnoesia and tumors of children and adult associated with gene imprinting. At present, the problems about expression on oocytes and nonage embryos, formation of imprinting, whether or not cultivation in vitro and micromanipulation etc in assisted reproductive technology(ART) interfere with the status of methylation imprinting, already became hot spots in researches of reproductive medicine and genetics.ART have revolutionized the treatment of infertility around the world from borning of the frist baby in vitro fertilization(IVF) in 1978 in the United Kingdom and blest treating of male sterility by intracytoplasmic sperm injection (ICSI). For the past few years,it has been investigated in the United States, the United Kingdom and Germany etc that culture in vitro and micromanipulation by ART may interfere with methylation imprinting status of imprinted gene of oocytes and early embryos,leading to imprinting diseases, for example, Beckwith-Wiedemann syndrome (BWS) and Angelman syndrome(AS) etc. Comparing incidence between 1316500 naturally born babies and 14894 babies(IVF), Halliday discovered onset risk of BWS was more 9 times in IVF. At the same time,they reported that six cases had existed loss of imprinting of paternal gene KCNQ1OT1 or H19 during the seven cases of BWS patients born by ART. Orstavik and Cox et al reported three children born by ICSI,had got AS for loss of imprinting of maternal imprited gene UBE3A on chromosome 15qll-13.In 70% of BWS and 100% of AS conceived by ART,abnormality methylation of maternal allele induced loss of imprinting. But only 50%-60% of BWS and 5% patients of AS had abnormality of methylation among common patients. Moreover, whether or not human embryonic stem cells cultivation in vitro interfere with the status of gene imprinting ,which needs further researches.Owing to the restriction of human oocytes and embryos' source and related ethics, furthermore, samples of only containing single cell to several cells, extreme low mRNA content and very difficult skills, related researches centralized mostly in animals. In human, only seldom overseas documents reported the expression of several imprinted genes on oocytes and preimplantation embryos, for example, IGF2 and H19 etc. But information about the expression of following imprinted genes on oocytes and preimplantation embryos was seldom reported, for example, imprinted genes SNRPN,NDN related with PWS, UBE3A related with AS, KvLQT,MASH2 related with BWS, PEG1 related with AS and etc. The candidate imprinted genes PEG10, ASB4 related with SRS aren't yet reported at home and abroad now. The research detected mRNA expression of above eight imprinted genes relative to imprinting diseases by using monoplast nested reverse transcription-polymerase chain raction (RT-PCR) technology and the purpose is to understand the expression status of imprinted genes on oocytes and preimplantatione mbryos, to investigate various abnormal expression, imprinting disease and mechanism of gene imprinting regulation, and to provide theoretical and experimental basis for preimplantation genetic diagnosis (PGD) development of genetic imprinting diseases and for research the relation between ART and imprinting diseases. Meanwhile, we may make an exact causal diagnosis to a child who was suspected of PWS at clinic on gene level and on chromosome level by detecting micro-depletion, translocation, uniparental diplont and etc. It may give clinical genetic counseling for his parents another gestation and avoid PWS child born again. It can also provide experimental basis for preventing PWS before delivery, even before embryonic implantation.Chapter One The mRNA Expression of Imprinted Genes in Human Oocytes and Preimplantation EmbryosObjective: To detect the mRNA expression of imprinted genes in human oocytes and preimplantation embryos, to discuss its biological meanings, mechanism of imprinting regulation, relation bewteen ART and imprinting diseases, to provide theoretical and experimental evidence for PGD development.Methods: Choosing disuse oocytes of GV, Mâ… , Mâ…¡times and embryos of 2-cell, 4-cell, 8-cell and blastocysts stages or frozen embryos, voluntary donated by patients who had born normal baby by IVF/ICSI-ET from the ART Medical Center of Peking University Shenzhen Hospital from June to September in 2005.The patients all had no family history of inherited diseases. The study was consented by the patients and by medical ethics association of the hospital. Under inverted microscope pellucid zone was removed. Using cell lysis, cDNA preparation and monoplast nested reverse transcription-polymerase chain raction(RT-PCR) technology, we detected the mRNA expression of eight imprinted genes relative to imprinting disease such as SNRPN, NPN, UBE3A, KvLQT₁, MASH2, PEG1, PEG10, ASB4 etc. Moreover, undertaking purification to PCR products, linkage and translation, shake bacterium for extracting plasmid to sequence and verificate about imprinted genes SNRPN and UBE3A.Results:1. The mRNA expression of SNRPN and PEG1 genes existed in oocytes of GV,M I ,M II and embryos of 2-cell, 4-cell, 8-cell and blastocyst stages.2. UBE3A gene was expressed from M I oocyte to all stages of preimplantation embryos.3. The mRNA of NDN gene was detected in GV, Mâ… , Mâ…¡oocytes and 8-cell embryos and blastocyst.4. KvLQT₁ gene couldn't be detected in all stages from oocyte to blastocyst.5. MASH2 gene was only expressed in Mâ…¡oocyte, 8-cell embryo and blastocyst.6. The mRNA expression of PEG10 and ASB4 genes existed in Mâ…¡oocyte, 4-cell, 8-cell embryos and blastocyst.Conclusion:1. At home it was first confirmed that the paternal imprinted gene SNRPN associated with PWS on chromosomes 15qll-13 were expressed in times of GV,M I ,M II oocytes and stages of 2-cell,4-cell, 8-cell embryos and blastocyst and NDN gene were expressed in times of GV,M I ,M II oocytes and 8-cell embryos and blastocyst. It indicates that the two genes can promote oocyte maturation and early embryo growth and development.2. UBE3A, maternal imprited gene closely correlated with AS on chromosome 15q11-13, was detected in the Mâ… , Mâ…¡oocytes and all stages of preimplantation embryos in domestic. This indicates that the gene imprinting status is possibly disturbed by some elements of ART, which results in raising of incidence of AS.3. Paternal expressive proto-oncogene PEG10 on chromosome 7, was first proved the expression in Mâ…¡oocyte and 4-cell, 8-cell embryos and blastocyst. It suggests its imprinting status is possibly disturbed by some elements of ART and results in activation of PEG10 gene, which generates related diseases such as SRS and chorionic carcinoma etc. Monitoring incidence of SRS and tumor is warned through necessary long-term follow-up crowd born by ART.4. ASB4 as a new paternal imprinted gene on chromosome 7, was first confirmed its expression in Mâ…¡oocyte and all stages of preimplantation embryos except for 2-cell.5. MASH2, maternal imprited gene correlated with placenta development on chromosome 11, was detected its expression in Mâ…¡human oocyte, 8-cell embryo and blastocyst in domestic.6. The study confirmed imprinted genetic expression in the different stages of oocytes and embryos, having horary specificity. It is presumd also horary specificity in the erasion, establishment and maintenance of various genomic imprinting.7. By monoplast nested RT-PCR technology, mRNA expression of eight imprinted genes were detected. The amplification achievement ratio of was 81.0% (64/79) .This indicates that the technology is comparatively accurate and stable,which laid down a foundation for further developing PGD of imprinting diseases.Chapter Two The Study of Molecular Diagnosis of Imprinting Disease PWSObjective: To make a definite diagnosis for PWS and its etiopathogenisis on chromosome level and gene level by detecting imprinted gene SNRPN of suspected PWS child at clinic and his parents, to provide experimental evidence for gestation again, to guide clinical genetic counseling, treatment and prenatal diagnosis.Methods: Using methylation specific PCR (MS-PCR) to investigate the methylation status of parents allele from the 19 CG sizes of the exon alpha domain of the imprinted gene SNRPN locus on chromosome 15q11-13 ,which is close related to PWS. Its function principle according to the next: When the DNA modified by natrium bisulfurosum, the cytosine from the paternal unmethylation turned to uracil ,but the maternal methylation Sequence kept the same. Then primers, the pair of high degree of specificity on methylation and unmethylation, were amplified. According to the different straps after amplification,we could diagnose the cause of a disease. We made a precise diagnosis on chromosome level by fluorescence in situ hybridization (FISH) technique.Results:1. The child's result of MS-PCR was no amplification straps of paternal unmethylation sequence,which confirmed the loss of paternal allele in exon alpha domain of SNRPN gene or maternal uniparental diplont (matUPD). The child was diagnosed as PWS.The microdeletions of SNRPN gene was detected on chromosome 15q11-13 by FISH. Considered the result of MS-PCR, the cause of disease of the child was the paternal microdeletion of SNRPN gene on chromosome 15q11-13 section and the matUPD(15) was excluded.2. The results of parents of the child and the control normal adult were normal.3. The result of PCR based sequencing confirmed that there were 19 CG sizes on exon alpha domain of SNRPN gene. DNA sequence from his mather was of methylation and DNA sequence from his father was of unmethylation.Conclusion:1. The diagnosis of genomic imprinting diseases such as PWS etc can be puted out quickly and exactly at chromosome level by combining FISH with molecular probe technique and at gene level by MS-PCR technique. Both of them are able to as a rout technology of making a definite diagnosis and prenatal screening on genomic imprinting diseases.2. Combining MS-PCR technique and the results of PCR products' sequencing, we can know methylation conditions of some genes' CpG islands. It brings about new idea to block development and cure of imprinting diseases before embryo implantation through kickback of abnormality methylation sites.