Effects Of Assisted Reproductive Technology On DNA Methylation Patterns Of SNRPN At Imprinting Control Region Of PWS/AS

Background and objectiveIn the past decades,assisted reproductive technology(ART) have developed rapidly and are important treatment for infertile couples.There are more than three millions of children conceived by ART,which now accout for 1-3%of the total annual births in developed countries.So people take more concerns on the safty of ART.ART have been generally considered to be relatively safe medical treatments.But rencent studies have suggested that there may be links between ART and an increased risk of birth defects,imprinting disorders and childhood cancer.Several clinical studies have reported an increased frequency of ART conceptions among children with Prader-Willi syndrome(PWS) and Angelman syndrome(AS).The absolute risk of an imprinting disorder after ART appears to be very small,but animal study have demonstated that in vitro embryo culture is associated with disordered genomic imprinting and alterations in epigenetics.So it is important to do some researches investigating the safty of ART.PWS and AS are two congenital neurobehavioural disturbances syndrome.They are the most typical model of genomic imprinting and have different clinical characters and genetic patterns.A common region involved in these syndromes is the 15q11-13.PWS is characterized by obesity,short stature,muscular hypotonia and mild mental retardation and AS is characterized by poor balance accompanied by jerky movements,severe mental retardation,absence of speech and happy disposition.PWS is caused by a deficiency of chromosome 15q11-13:mainly a paternal deletion(70%),a maternal disomy(25%) or an imprinting mutation(5%).In contrast,AS genotype is charaterised by a maternal deletion(70-80%),a paternal disomy(2%) or an imprinting mutation of UBE3A(20%) in AS gene located on the same chromosomal regions.UBE3A is mainly expressed in brain and encodes a ubiquitin-protease which involved in renew process of protein.Abonormal expression of UBE3A in encephalization is associated with AS.PWS is caused by deletion of paternal 15q11-13 and AS is caused by deletion of maternal 15q11-13,they have a close relationship with regulation of genomic imprinting.There are several imprinting genes on chromosomal 15q11-13,such as SNRPN(small nuclear riboproteinassociated polypeptide N),NDN and ZNF127.Paternal expressed SNRPN takes an important role in PWS and AS.SNRPN gene encodes a polypeptide of a small nuclear ribonucleoprotein N which is indispensable for pre-mRNA processing.Either any parental deletion or UPD lost one of the active parental allele resulting in PWS or AS,which indicates maternal and paternal 15q11-13 has different functions.Although imprinting is involved in these syndromes,the specific contributions of these genes in imprinting defects were unclear.The mechanisms between imprinting gene and imprinting disorders need to be further investigated.The unusual relationship of ART and impinting disorders is poorly understanded.ART put fertilization and early embryo culture in laboratory condition which provide the best model for researching epigenetics of fertilization and early embryo development.Epigenetics is a genomic modification that is not involved in DNA sequence such as DNA methylation,genomic imprinting,histone modification,chromatin remodeling and noncoding RNA.This modifications not only affect the individual development,but also are heritable during cell division.Epigentics is one of the key researches in genomic functions and gene expressions,genomic imprinting is an important part of it.Genomic imprinting or parental imprinting is a non-Mendelian inheritance which was described in 80s twentieth century.Mammals inherit paternal and maternal chromosomes,most of genes express two parental alleles,one is from paternal,another is from maternal.Only a small number of genes express predominantly from one parental allele,accouting for about 0.1%in the mammalian genomes.This epigentics modification phenomenon is called genomic imprinting. Paternal(maternal) imprinting means paternal(maternal) allele is silenced.Many imprinted genes are essential for regulating embryonic and placenta growth,especially for the placenta growth.Correct expression of imprinting genes is associated with normal development of embryo,placenta and behaviors.Disordered imprinting has been implicated in the pathogenesis of genetic diseases and cancer.Disregulation of the expression of genomic imprinting gives rise to a variety of development abnormalities,stillbirths and children tumor.A uniparental disomy, a deletion after hybridisation,a point mutation or an loss of imprinting can be seen on chromosome levels of imprinting associated diseases.It is so clear that genomic imprinting is a new non-Mendelian heredity elucidating the profoud mechanisms underlying human embryonic development.DNA methylation is a crucial epigenetic modification of genome that is involved in regulating differentiation of embryonic development and tumorigenesis.DNA methylation is a enzymatic reaction after DNA duplication which transfer methylium of S-adenosylmethionine(SAM) to cytosine at position C~5 by DNA methyltransferases.The tranfered methylium result in changes of spatial structure what affect DNA binding to protein.This will result in the inactivation of gene transcriptions and silence of gene expression,even lose gene functions.Cytosine at position C~5 in CpG dinucleotides are target sites of methylation.Methylation-specific polymerase chain reaction is a breakthrough in methylation analysis.Its principle is as below:genomic DNA is first subjected to a bisulfite treatment,which serves to deaminate unmethylated cytosines,thus converting them to uracils,whereas methylated cytosines are not converted.Two different PCR are then performed per individual using primers specific for either the unmethylated or the methylated alleles(allele-specific PCR).In this way,we can distinguish methylated alleles from unmethylatd alleles.In normal situations, the human SNRPN gene is expressed from the unmethylated paternal allele,while the silenced maternal copy is methylated.In normal human genomic DNA,We can get PCR specific product of methylated maternal and unmethylated paternal alleles through MS-PCR which aim at SNRPN gene promoter region.We use MS-PCR detecting methylation status on SNRPN imprinting control region in samples which are related to assisted reproductive technologies like early pregnancy villus gotten from mutiplefetal reduction and spontaneous abortion,cord blood of ART births,using nature early pregnancy villus and cord blood of nature birth as control.We want to investigate the correlation between assisted reproductive technology and imprinting diseases to settle preliminary theory foundation in researching the safty of ART.Materials and methodswe have collected early pregnancy villus gotten from mutiplefetal reduction (10 samples) and spontaneous abortion of ART(6 samples),early pregnancy villus(10 samples) and spontaneous abortion villus(6 smaples) of spontaneous pregnancy,cord blood of ART births(12 samples) and spontaneous pregnancy births(10 samples).These collections were finished from January to December 2007 in Nanfang Hospital.We have detected methylation status of SNRPN at imprinting control region in above-mentioned collections.Main results1.MS-PCR on genomic DNA from early pregnancy villus gotten from mutiplefetal pregnancy reduction of ART(10 samples) showed both a 174bp product from the methylated maternal chromosome and a 100bp product from the unmethylated paternal chromosome.2.MS-PCR on genomic DNA from early pregnancy villus of spontaneous pregnancy(10 samples) showed both a 174bp product from the methylated maternal chromosome and a 100bp product from the unmethylated paternal chromosome.3.Four spontaneous abortion villus which got pregnancy by ART only shows a 174bp product from the methylated maternal chromosome;three spontaneous abortion villus of spontaneous pregnancy only shows a 100bp product from the unmethylated paternal chromosome.4.MS-PCR on genomic DNA from cord blood of ART births(12 samples) showed both a 174bp product from the methylated maternal chromosome and a 100bp product from the unmethylated paternal chromosome.Conclusion1.We first use MS-PCR to detect methylation status of SNRPN at imprinting center 15q11-13 in early pregnancy villus and spontaneous abortion villus.2.The MS-PCR of cord blood and mutiplefetal pregnancy reduction villus after ART revealed a methylation status at chromosome 15q11-13 identical to the methylation pattern in the normal control;the results of the present study do not indicate a higher risk of imprinting disorders related to assisted reproductive technologies. 3.The abnormal status of SNRPN in spontaneous abortion villus indicated the abnormality of epigenetics might play an important role in spontaneous abortion.4.Spontaneous abortion villus by ART miss a unmethylated paternal product on imprinting center of PWS/AS,which can be cause by genetics,epigenetics, endocrine,infection,immunization or ART procedures.But there is no evidence to demonstate a direct link between ART and imprinting disorders.