Imprinted genes as potential genetic and epigenetic toxicologic targets

Genomic imprinting is an epigenetic phenomenon in eutherian mammals that results in the differential expression of the paternally and maternally inherited alleles of a gene. Imprinted genes are necessary for normal mammalian development. This requirement has been proposed to have evolved because of an interparental genetic battle for the utilization of maternal resources during gestation and postnatally. The nonrandom requisite for monoallelic expression of a subset of genes has also resulted in the formation of susceptibility loci for neurobehavioral disorders, developmental disorders, and cancer. Since imprinting involves both cytosine methylation within CpG islands and changes in chromatin structure, imprinted genes are potential targets for dysregulation by epigenetic toxicants that modify DNA methylation and histone acetylation.     

Influence of epigenetic changes during oocyte growth on nuclear reprogramming after nuclear transfer

Genomic imprinting is the epigenetic mechanism that distinguishes whether the loci that are inherited from the maternal or paternal genome lead to parent-specific gene expression. The mechanism also regulates development in mammalian embryos. Genomic imprinting is established after implantation according to the specific markers that are imposed on the genome during gametogenesis; the allele-specific gene expression is then maintained throughout embryogenesis. The genomic imprinting markers are erased and renewed on an own-sex basis only in cells that differentiate into germline cells. This report shows that the epigenetic modifications that occur during oogenesis perform the crucial function of establishing the allele-specific expression of imprinted genes, and also suggests that the epigenetic DNA modification is related to the reprogramming and aberrant development seen in manipulated embryos.     

Genomic imprinting in primate embryos and embryonic stem cells

Embryonic stem (ES) cells hold promise for cell and tissue replacement approaches to treating human diseases. However, long-term in vitro culture and manipulations of ES cells may adversely affect their epigenetic integrity including imprinting. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. We demonstrated aberrant biallelic expression of IGF2 and H19 in several rhesus monkey ES cell lines while SNRPN and NDN were normally imprinted and expressed from the paternal allele. In contrast, expanded blastocyst-stage embryos, from which these ES cells were derived, exhibited normal paternal expression of IGF2 and maternal expression of H19. To test the possibility that aberrant methylation at an imprinting centre (IC) upstream of H19 accounts for the relaxed imprinting of IGF2 and H19, we performed comprehensive methylation analysis by investigating methylation profiles of CpG sites within the IGF2/H19 IC. Our results demonstrate abnormal hypermethylation within the IGF2/H19 IC in all analysed ES cell lines consistent with biallelic expression of these genes. Cellular overproliferation and tumour formation resulting from tissue or cell transplantation are potential problems that must be addressed before clinical trials of ES cell-based therapy are initiated.     

Ex vivo early embryo development and effects on gene expression and imprinting

The environment to which the mammalian embryo is exposed during the preimplantation period of development has a profound effect on the physiology and viability of the conceptus. It has been demonstrated that conditions that alter gene expression, and in some instances the imprinting status of specific genes, have all previously been shown to adversely affect cell physiology. Thus, questions are raised regarding the aetiology of abnormal gene expression and altered imprinting patterns, and whether problems can be averted by using more physiological culture conditions. It is also of note that the sensitivity of the embryo to its surroundings decreases as development proceeds. Post compaction, environmental conditions have a lesser effect on gene function. This, therefore, has implications regarding the conditions used for IVF and the culture of the cleavage stage embryo. The developmental competence of the oocyte also impacts gene expression in the embryo, and therefore superovulation has been implicated in abnormal methylation and imprinting in the resultant embryo. Furthermore, the genetics and dietary status of the mother have a profound impact on embryo development and gene expression. The significance of specific animal models for human assisted reproductive technologies (ART) is questioned, given that most cattle data have been obtained from in vitro-matured oocytes and that genes imprinted in domestic and laboratory animals are not necessarily imprinted in the human. Patients treated with ART have fertility problems, which in turn may predispose their gametes or embryos to greater sensitivities to the process of ART. Whether this is from the drugs involved in the ovulation induction or from the IVF, intracytoplasmic sperm injection or culture procedures themselves remains to be determined. Alternatively, it may be that epigenetic alterations are associated with infertility and symptoms are subsequently revealed through ART. Whatever the aetiology, continued long-term monitoring of the children conceived through ART is warranted.     

A Lack of Contact of Sperm with Accessory Sex Gland Secretions Deregulates DNA Methylation and Imprinted Gene Expression in Rodent Embryos

Increased oxidative DNA damage is observed in sperm devoid of contact with accessory sex gland (ASG) secretion. After fertilization, these sperm may produce abnormal embryos. In this study, we investigated the possibility that the pattern of DNA methylation and imprinted gene expression in these embryos may be perturbed. Epididymal sperm, uterine sperm, and embryonic day 13 (E13) embryos were collected from hamster and mouse. The extent of global DNA methylation was determined with an antibody against methylcytosine using an embryo DNA dot. The sperm and embryo Gtl2 promoter and H19 differential methylated region (DMR) were subject to bisulfite sequencing. Expression of their reciprocally activated genes Dlk1 and Igf2 was quantified by real-time PCR. Genome-wide DNA hypo-methylation in both hamster and mouse embryos sired by males without ASG was observed. The imprinting pattern of fetal mouse Gtl2 promoter and fetal hamster H19 DMR were also disrupted while the expression of Dlk1 and Igf2 was dysregulated in the hamster embryo. This study suggests that a lack of contact of sperm with the ASG secretion disrupts genome-wide DNA methylation and also affects the DNA methylation pattern of imprinted genes in embryos.     

辅助生殖技术中基因印迹分析

人类辅助生殖临床数据已经显示,辅助生殖技术（ART）与自发流产、早产和围生期死亡、低体质量儿以及一些印迹疾病有关。在配子及胚胎早期发育过程中,基因印迹需经历印迹擦除、重建和维持过程,其中任何一个环节出错都可能导致胚胎发育缺陷,甚至死亡。ART恰施于这一表观遗传重编程的关键时期。因此,这些异常结局可能与ART导致的印迹基因的异常表达有关。而ART中主要的治疗手段有促排卵、体外受精、胞浆内单精子注射（ICSI）和体外培养。这些操作通过干扰基因印迹的重建和维持,影响基因表达和表型,进而影响配子和早期胚胎的发育,从而影响子代的生长发育潜能。     

Epigenetic Aspects of Fertilization and Preimplantation Development in Mammals: Lessons from the Mouse

During gametogenesis, the parental genomes are separated and are epigenetically marked by modifications that will direct the expression profile of genes necessary for meiosis as well as for the formation of the oocyte and sperm cell. Immediately after sperm-egg fusion, the parental haploid genomes show great epigenetic asymmetry with differences in the levels of DNA methylation and histone tail modifications. The epigenetic program acquired during oogenesis and spermatogenesis must be reset for the zygote to successfully proceed through preimplantation development and this occurs as the two genomes approach each other in preparation for karyogamy. During preimplantation development, the embryo is vested with the responsibility of maintaining the primary imprints. In addition, female embryos must silence one of the X-chromosomes in order to transcribe equal levels of X-linked genes as their male counterparts. This review is intended as a survey of the epigenetic modifications and mechanisms present in zygotes and preimplantation mouse embryos, namely DNA methylation, histone modifications, dosage compensation, genomic imprinting, and regulation by non-coding RNAs.     

基因印记与精子发生障碍的研究进展

基因印记是特定基因的一对等位基因发生差异性的表达,机体仅表达来自亲本一方的等位基因,而另一方保持沉默。精子发生是一个高度复杂的独特分化过程,包括精原细胞发育为精母细胞、单倍体精细胞的形成和精子成熟。本文通过探讨精子发生障碍与基因印记的关系,分析不育男性表观遗传缺陷的潜在风险,为临床男性不育的预防和治疗提供理论支持。     

Putative imprinted gene expression in uniparental bovine embryo models

Altered patterns of gene expression and the imprinted status of genes have a profound effect on cell physiology and can markedly alter embryonic and fetal development. Failure to maintain correct imprinting patterns can lead to abnormal growth and behavioural problems, or to early pregnancy loss. Recently, it has been reported that the Igf2R and Grb10 genes are biallelically expressed in sheep blastocysts, but monoallelically expressed at Day 21 of development. The present study investigated the imprinting status of 17 genes in in vivo, parthenogenetic and androgenetic bovine blastocysts in order to determine the prevalence of this unique phenomenon. Specifically, the putatively imprinted genes Ata3, Impact, L3Mbtl, Magel2, Mkrn3, Peg3, Snrpn, Ube3a and Zac1 were investigated for the first time in bovine in vitro fertilised embryos. Ata3 was the only gene not detected. The results of the present study revealed that all genes, except Xist, failed to display monoallelic expression patterns in bovine embryos and support recent results reported for ovine embryos. Collectively, the data suggest that monoallelic expression may not be required for most imprinted genes during preimplantation development, especially in ruminants. The research also suggests that monoallelic expression of genes may develop in a gene- and time-dependent manner.     

人卵巢组织冷冻对印迹基因IGF-2/H19甲基化程度及表达的影响

目的：探讨程序化冷冻和固相表面玻璃化冷冻对人卵巢组织中印迹基因胰岛素样生长因子2（IGF-2）/H１９（一种非可读框架RNＡ）差异性甲基化区（DMR）CpG岛甲基化程度及两基因表达的影响。方法：15例因其他疾病而手术的患者,将病理检查剩余的卵巢组织切割成皮质小条后随机分配到新鲜组（F组）、程序化冷冻组 （SF组）和固相表面玻璃化组（SSV组）。通过甲基化聚合酶链反应（MS-PCR）检测各组卵巢组织中IGF-2/H19 CpG岛甲基化程度,并分别采用免疫组化法和原位杂交方法检测各组卵泡中IGF-2蛋白和H19 RNA的表达情况。结果：F组、SF组和SSV组卵巢组织甲基化程度分别为26.46%,41.02%和33.09%,虽然SF组和SSV组的甲基化程度略高于F组,但差异无统计学意义。在新鲜和冻融后的卵巢组织中,各级别卵泡的卵母细胞和颗粒细胞胞质中均有IGF-2蛋白阳性表达,3组之间原始和初级卵泡IGF-2的表达差异无统计学意义（P＞0.05）。原始卵泡的颗粒细胞和卵母细胞均可见到H19 RNA阳性表达,3组之间原始卵泡H19 RNA的表达差异无统计学意义（P＞0.05）。结论：未检测到慢速程序化冷冻和快速固相表面玻璃化冷冻对印迹基因IGF-2/H19 CpG岛甲基化程度的影响,亦未发现2种冷冻方法对原始和初级卵泡中IGF-2蛋白和H19 RNA表达的影响。     

男性焦虑和压力对精子印记基因甲基化的影响

目的 近年来,心理健康问题发病率不断增高,有研究报道,母亲妊娠期间因承受压力通过改变胎儿发育的编程而影响下一代的发育,而父源性的负性心理健康因素对后代的影响则研究甚少。本课题通过对男性负性心理健康因素与精子质量以及印记基因甲基化改变的相关性研究,以探讨心理因素对精子质量和印记基因甲基化水平的影响,为促进生育健康和预防不良出生结局提供依据。方法 采用横断面方法调查山西省太原市2所医院620名男性门诊就诊者。包括相关问卷调查,焦虑自评量表和知觉压力量表,收集精液,并进行精子质量分析和印记基因H19、PEG3和MEG3的甲基化水平分析。结果 被研究人群有压力的占27.4%,焦虑的占7.08%。焦虑组精液量低于非焦虑组(P<0.05)。尽管压力组和非压力组在精子质量各项指标方面未发现差异,但压力组精子印记基因PEG3甲基化区的7个CpG位点的平均甲基化水平低于非压力组(P<0.05)。焦虑和压力量表得分与PEG3甲基化区的7个CpG位点的平均甲基化水平之间存在负相关关系,而H19和MEG3与焦虑和压力量表得分均未发现相关。结论 心理健康影响男性的精子质量,并与精子印记基因甲基化水平的改变相关。     

镉转化细胞DNA异常甲基化对肿瘤相关基因表达的影响

目的 对镉转化细胞DNA异常甲基化及其对肿瘤相关基因表达的影响进行研究，探讨镉的外遗传致癌机制。方法 从CdCl2转化BAIB／c—3T3细胞中提取基因组DNA，经甲基化非敏感性酶(Mse1)单独消化或Mse1和甲基化敏感性酶(BstUl)联合消化，消化产物用甲基化敏感性内切酶指纹法(MSRF)进行分析，差异显示出异常甲基化基因片段，进一步以异常甲基化DNA为探针进行Southern分子杂交加以证实，并进行DNA序列测定，与基因文库中的基因进行类比分析。结果 发现镉转化细胞存在异常甲基化DNA，其中一个甲基化DNA片段为p16抑癌基因。结论 DNA高甲基化会导致基因表达抑制，因此，p16基因高甲基化会导致其抑癌功能减弱或丧失，这可能是镉诱导细胞转化及其致癌作用的一种外遗传机制。     

Co-localization of differentially expressed genes and shared susceptibility loci in human autoimmunity

Autoimmune diseases arise from complex interactions between environmental and genetic factors. Genetic linkage scans show that different autoimmune diseases share overlapping susceptibility loci. Lymphocytes from individuals with different autoimmune diseases, as well as unaffected first-degree relatives, also share a common gene expression profile. We sought to determine if genes within this autoimmune expression profile were nonrandomly distributed in the genome and if their distribution overlapped with shared disease susceptibility loci. We found that differentially expressed genes were distributed in a nonrandom fashion in chromosomal domains within the genome. Furthermore, positions of these domains were not statistically different from a number of shared autoimmune disease susceptibility loci. To our knowledge, this is the first study showing concordance between gene expression and genetic linkage results in common complex multifactorial human diseases.     

The Emerging Importance of Genetics in Epidemiologic Research III. Bioinformatics and statistical genetic methods.

PURPOSE: To outline potential benefits of integrating recent developments in bioinformatics and statistical genetics with traditional epidemiologic studies to localize genes influencing complex phenotypes and examine genetic effects on disease susceptibility. METHODS: An overview of bioinformatic and statistical approaches for localizing disease-susceptibility genes as well as challenges associated with identifying functional DNA variants and context-dependent genetic effects concludes this three-part series on the importance of genetics in epidemiologic research. RESULTS: Rapidly evolving bioinformatic and statistical methods are providing invaluable information on newly-discovered genes and molecular variation influencing human diseases that is readily available to epidemiologic researchers. CONCLUSIONS: Integrating bioinformatics and molecular biotechnology with epidemiologic methods of assessing disease risk is rapidly expanding our ability to identify genetic influences on complex human diseases. These technological advances are likely to have a profound impact on current knowledge of complex disease etiology, and may reveal novel approaches to disease treatment and prevention.     

表观遗传学及其应用研究进展

近年来,表观遗传学方面的研究受到人们的广泛关注,表观遗传学是指不涉及DNA序列改变的基因或者蛋白表达的变化,并可以在发育和细胞增殖过程中稳定传递的遗传学分支学科,主要包括DNA甲基化,组蛋白共价修饰,染色质重塑,基因沉默和RNA编辑等调控机制。随着研究的深入,人们发现表观遗传学与多种疾病的发生发展有关,本文就表观遗传学的主要内容及其在相关疾病中的应用做一综述。     

Genomic approach to allergic diseases: present and future perspectives

The rapid advances in genomic research have a major impact on biomedical sciences. In this review the authors summarize the current results of the genomic investigation of allergic diseases. From a genetic point of view allergy is multifactorial, which means that the susceptibility to the disease is determined by interactions between multiple genes, and involve important nongenetic factors such as the environment for their expression. There are two widespread methods for searching for disease susceptibility genes in allergy: (1) genome-wide search and (2) candidate gene association studies. Until now five groups have completed genome-wide searches for asthma, and almost 500 genetic linkage and association studies have reported more than 500 atopy and asthma loci throughout the genome. In this review we selected those results, which were consistently confirmed by several independent studies, or appeared particularly important, or interesting. On the basis of this, 9 chromosome regions can be associated with the susceptibility to allergic diseases: 2q, 5q31-q33, 6p21.3, 7q31, 11q13, 12q14.3-q24.31, 14q11.2-q13, 16p21, 17q11.2. According to the results of the human genome programs, and association studies, the authors discuss the possible roles of candidate genes found in these loci in the pathomechanism of allergy. Besides, the authors summarize briefly the results of pharmacogenomics, and animal linkage and genetics studies related to allergy.     

Gamete imprinting: setting epigenetic patterns for the next generation

The acquisition of genomic DNA methylation patterns, including those important for development, begins in the germ line. In particular, imprinted genes are differentially marked in the developing male and female germ cells to ensure parent-of-origin-specific expression in the offspring. Abnormalities in imprints are associated with perturbations in growth, placental function, neurobehavioural processes and carcinogenesis. Based, for the most part, on data from the well-characterised mouse model, the present review will describe recent studies on the timing and mechanisms underlying the acquisition and maintenance of DNA methylation patterns in gametes and early embryos, as well as the consequences of altering these patterns.