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Effects of Assisted Reproductive Technology on DNA Methylation Patterns of SNRPN at Imprinting Control Region of PWS/AS

Author: XiongXiaSi
Tutor: ChenShiLing
School: Southern Medical University,
Course: Obstetrics and Gynecology
Keywords: Assisted Reproductive Technology S \u0026 P - Wei syndrome and Angelina Germain syndrome The small nuclear Kernel ribonucleoprotein Methylation-specific polymerase chain reaction
CLC: R714.8
Type: Master's thesis
Year: 2008
Downloads: 180
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Abstract

Research background and purpose of assisted reproductive technology through the development of recent decades, and has become an important means to treat a variety of reasons for infertility. Around the world there are more than three million children born through assisted reproductive technology, accounted for 1-3% of the proportion of all births in developed countries. For assisted reproductive technology security issues become the focus of research and hotspots. Operation of assisted reproductive technology is generally believed to be relatively safe, but recent studies have shown that increased birth defects after assisted reproductive technology, the risk of imprinting disorders and the incidence of childhood cancer. There are several small-scale clinical trials reported an increased risk of assisted reproductive technology born child \u0026 P - Wei syndrome (Prader-Willi syndrome, PWS) and Angelina Pullman syndrome (Angelman syndrome, AS). Born children through assisted reproductive technology imprinted gene disease absolute risk is small, but the animal study found that abnormal embryos cultured in vitro expression of imprinted genes, epigenetic change, leading to imprint expression disorder. It is necessary to investigate the safety of assisted reproductive technology through experimental studies of assisted reproductive technology birth children and ART specimens. General - Wei syndrome (PWS) and Angelina Germain syndrome (AS) is the two congenital neurobehavioral development of abnormal syndrome, genomic imprinting of the most typical examples. Both have different clinical symptoms and genetic forms, but the pathogenic factor in the long arm of chromosome 15 11-13 zone (15q11-13) of imprinted genes related to the enclave. The PWS performance obesity, short stature, hypotonia and mild mental retardation. AS ataxia, severe mental disorders, few words, facial expressions pleasure. About 70% of the PWS patients causes deletion of chromosome 15q11-13, and deletion occurs mainly in the parent source chromosome. 25% of patients with karyotype visible of maternal uniparental Body Type (uniparental disomy UPD) about 5% of patients imprint mutation (the imprinting a mutation). ,70-80% of AS patients with PWS maternal chromosome deletion paternally Body Type 2% in AS patients with no chromosomal deletions about 20% of this area maternal expression of UBE3A truncating mutations. UBE3A expression mainly in the brain tissue, encodes a ubiquitin-proteasome involved in the update process protein UBE3A expression of abnormal brain formation process with AS. Thus, PWS 15q11-13 parent source caused by the lack of gene expression, and AS is caused by maternal gene expression defects, both genomic imprinting regulation. 15q11-13 region there are several imprinted genes (such as SNRPN, NDN, ZNF127, etc.), which paternally expressed SNRPN (smallnuclear riboprotein-associated polypeptide N) has played a key role in the PWS. SNR-PN encodes a nuclear protein related small nucleoprotein N, play an important role in the regulation of mRNA splicing process. Whether it is the lack of a parent or single-parent body results are lost a parent on active allele PWS and AS, the parent source maternal 15q11-q13 function. Mark on the participation of two types of this syndrome obvious, but so far it is not clear enough on the specific role of these genes in disease, the relationship between these imprinted genes associated with the mark disease pathogenesis needs further study. Unusual correlation between assisted reproductive technology and imprinted gene disorders is a yet to unlock the mysteries. Assisted reproductive technology to human sperm-egg fertilization and early embryo fertility process placed under laboratory conditions, and to provide the best model for fertilization and early embryonic development, epigenetics research. Epigenetics (epigenetics) is a major does not involve DNA sequence changes the modification of the genome, such as DNA methylation, genomic imprinting, histone modification, chromatin remodeling and non-coding RNA. Such modifications can affect not only the development of the individual, but also in the process of cell division genetic continues. Epigenetics is one of the key research areas elaborated genome function and gene expression. Genomic imprinting is one important part. Genomic imprinting (genomic imprinting), also known as parental imprinting (parentalimprinting) is proposed in the 1980s, a non-Mendelian mode of inheritance. Group of placental mammals inherited from father and maternal sets of chromosomes, which most genes biallelic expression, but part only derived from a single allele of the parental specific expression, accounting for 0.1% of the total genes, this epigenetic phenomenon known as genomic imprinting. Parent (mother) source allele did not express, said the father (mother) source imprint. The effect produced by the imprinting gene plays an important role in the process of the development of the embryo and placenta, particularly placental development is extremely important, the correct expression is associated with the normal development of the embryo, placenta, and behavior. Age imprint will affect the occurrence of certain genetic diseases and some tumors, and its incidence, penetrance, expressivity. Disorders of genomic imprinting function will cause a variety of developmental abnormalities, stillbirth and childhood tumors. Uniparental disomy chromosome lost after hybridization and gene imprinting loss phenomenon can be seen with the imprint-related diseases in the chromosome level. Shows that genomic imprinting is a new genetic mechanism, the occurrence of such non-Mendelian inheritance, to reveal the mechanism of human embryos to develop deeper. DNA methylation is an important mechanism of gene epigenetics, now known to differentiation and the development of tumor cells with human embryonic development. It is a DNA replication after the enzymatic reaction process. After DNA replication, in the role of DNA methylation enzyme, the S-adenosyl-acyloxy methionine (SAM) on the molecule methyl transferred to the 5 carbon atom of the cytosine residues in the DNA molecule. DNA molecules structural alterations can be the introduction of a methyl group, affecting the binding of DNA and protein molecules, resulting in certain gene transcription inactivation, the expression was inhibited, and even loss of function. 5 carbon atoms target site is methylated cytosine in the CpG dinucleotide. Methylation-specific polymerase chain reaction (methylation-specific PCR, MS-PCR) analysis of gene methylation breakthrough in speed and sensitivity. The principle is to first use of DNA bisulfite treatment, so that the unmethylated cytosine into uracil, and the methylation of the unchanged, designed based on this difference, the two pairs of specific primers: a pair of methylation-specific primer and a pair of non-methylation specific primers can be methylated allele chemical modification of the DNA sequence with the unmethylated allele distinction. The SNRPN gene of CpG islands in the chromosome of paternal Under normal circumstances is not methylated, and the maternal chromosomes occurs methylated. SNRPN gene promoter region of the MS-PCR in the detection of a normal human genomic DNA can be paternal SNRPN the unmethylated locus specific PCR product as a template, but also able to maternal SNRPN methylation locus specific PCR product as a template, and to help determine the SNRPN gene whether the presence of abnormal methylation sites. Study the contents of this article using MS-PCR to detect the related specimens ART by ART pregnancy chorionic villi and early spontaneous abortion, ART born children of umbilical cord blood genomic DNA of the PWS / AS imprinting control region SNRPN methylation status and nature pregnancy fluff and birth children cord blood compared to find a correlation may exist between assisted reproductive technology and imprinted gene disease PWS / AS, to explore epigenetics assisted reproductive technology and imprinted gene disorders that may exist between lay a preliminary theoretical basis for the relationship, in order to further verify the security of ART. Materials and methods objects in Nanfang Hospital from January 2007 to December patient flow collection, ART pregnancy chorionic villi (10 cases) and spontaneous abortion (6 cases); natural pregnancy chorionic villi (10 cases) and spontaneous abortion (6 cases) and by in vitro fertilization - embryo transfer technology born children of cord blood specimens (12 cases) and normal natural pregnancy cord blood specimens (10 cases). In this paper, by using methylation-specific polymerase chain reaction (MSP) to detect methylation of the regulatory region of the villi and umbilical cord blood genomic DNA imprint SNRPN gene expression status. The main results of 1.10 cases of pregnancy through ART the chorionic villi genomic DNA after MS-PCR showed maternally methylated 174bp positive products and paternal unmethylated 100bp positive products bands. 2.10 cases natural pregnancy chorionic villi of genomic DNA are maternally methylated 174bp positive products and paternal unmethylated 100bp positive products bands after MS-PCR electrophoresis. 3 ART pregnancy spontaneous abortion fluff genomic DNA line MSP After electrophoresis four cases appear only Dam methylation 174bp positive product band occurs the Sire unmethylated product lost; natural pregnancy spontaneous abortion of fluff by MSP After electrophoresis three cases appears only Sire unmethylated 100bp positive product strip, the a Dam methylation product loss occurs. 4.12 cases the ART technology born child cord blood genomic DNA after MSP electrophoresis maternal methylation a 174bp positive product and paternal unmethylated 100bp positive product. The main conclusions of this paper in the country for the first time using methylation-specific PCR to detect chorionic villi and spontaneous abortion PWS / AS imprinting control region 15q11-13 SNRPN gene methylation status. After assisted reproductive technology born children of cord blood, and the chorionic villi genomic DNA of the PWS / AS imprinting control region SNRPN gene methylation status of the same as the natural pregnancy; assisted reproductive technology not found the risk of an increase in the incidence of the disease of imprinted genes. In spontaneous abortion regardless of assisted reproductive technology pregnancy or natural pregnancy, the PWS / AS imprinting control region SNRPN gene methylation abnormal expression of genomic DNA, prompt exception epigenetics may lead to spontaneous abortion of a important factor. 4 assisted reproductive technology pregnancy occurring after spontaneous abortion part villi samples of genomic DNA PWS / AS imprinting control region SNRPN gene occurred the paternal unmethylated product expression is lost, may be genetic, epigenetic, endocrine, infectious, immune and various operational aspects of assisted reproductive technology related, but no clear evidence that that is a direct result of related operations by the ART.

Effects of Assisted Reproductive Technology on DNA Methylation Patterns of SNRPN at Imprinting Control Region of PWS/AS

Abstract

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