Hypomethylation of the miRNA-34a gene promoter is associated with Severe Preeclampsia

Purpose: PE is a pregnancy-specific complication, which genetic and epigenetic factors play key roles in its pathogenesis. DNA methylation is a main epigenetic alteration with important roles in gene regulation. Micro RNAs (miRNAs) as another member of epigenetic machinery regulate the gene expression and involve in different biological pathways including apoptosis and placental development. Therefore, the present study performed to assess the association between miRNA-34a promoter methylation and PE susceptibility. Methods: The placenta of 104 PE pregnant women and 119 normotensive pregnant women were collected after delivery. The methylation status of the miRNA-34a promoter was assessed using Methylation Specific PCR (MSP). Results: The frequency of the hemi-methylated (MU) miR-34a promoter was significantly lower in PE women compared to the controls (17.3 vs. 29.4%) (OR, 0.45 [95% CI, 0.2–0.9], P = 0.016). The overall methylation rate was 23.1% in PE women and 41.2% in the control group and was significantly lower in PE women (OR, 0.4 [95% CI, 0.2–0.8], P = 0.004). The frequency of hemi-methylated (MU) and overall methylated (MU+MM) promoter of miR-34a gene was significantly lower in severe PE but not in mild PE women compared to the controls [(OR, 0.3 [95% CI, 0.1–0.8], P = 0.02) and (OR, 0.3 [95% CI, 0.1–0.7], P = 0.009), respectively]. There was an association between hemi-methylated (MU) and overall methylated (MU+MM) promoter and late onset PE [(OR, 0.4 [95% CI, 0.2–0.9], P = 0.03) and (OR, 0.4 [95% CI, 0.2–0.8], P = 0.01), respectively]. Conclusions: An association was found between hypo-methylation of the miR-34a promoter and PE and PE severity.     

Hypomethylation of DNA derived from purified human erythroid cells correlates with gene activity of the beta-globin cluster

Analysis of methylation at the beta-globin gene cluster was carried out on DNA derived from nucleated RBCs (orthochromatic normoblasts) isolated from peripheral blood of patients with beta-thalassemia major or other congenital hemolytic anemia after splenectomy. A procedure to separate these normoblasts from the other nucleated cells of the peripheral blood was developed, providing us with a convenient source of DNA for investigating parameters related to human erythroid differentiation. Blood samples were obtained from six adult patients who express their gamma-globin genes at different levels. Inverse correlation between methylation and gene activity was consistently observed for five of the eight sites analyzed. A site 3' to the beta gene was always unmethylated, two sites flanking the epsilon gene were always found to be methylated, and two sites 5' to the two gamma genes, G gamma and A gamma, were hypomethylated in correlation with gamma gene activity of the individual patients. A site 5' to the delta gene was unmethylated in normoblasts as well as in WBC. No apparent relation between hypomethylation and gene activity was observed for two additional sites. The results suggest that methylation at specific chromosomal locations participate in genetic regulation of the beta- like globin genes in humans.     

POMC overexpression in the ventral tegmental area ameliorates dietary obesity

The activation of proopiomelanocortin (POMC) neurons in different regions of the brain, including the arcuate nucleus of the hypothalamus (ARC) and the nucleus of the solitary tract curtails feeding and attenuates body weight. In this study, we compared the effects of delivery of a recombinant adeno-associated viral (rAAV) construct encoding POMC to the ARC with delivery to the ventral tegmental area (VTA). F344×Brown Norway rats were high-fat (HF) fed for 14 days after which self-complementary rAAV constructs expressing either green fluorescent protein or the POMC gene were injected using coordinates targeting either the VTA or the ARC. Corresponding increased POMC levels were found at the predicted injection sites and subsequent α-melanocyte-stimulating hormone levels were observed. Food intake and body weight were measured for 4 months. Although caloric intake was unaltered by POMC overexpression, weight gain was tempered with POMC overexpression in either the VTA or the ARC compared with controls. There were parallel decreases in adipose tissue reserves. In addition, levels of oxygen consumption and brown adipose tissue uncoupling protein 1 were significantly elevated with POMC treatment in the VTA. Interestingly, tyrosine hydroxylase levels were increased in both the ARC and VTA with POMC overexpression in either the ARC or the VTA. In conclusion, these data indicate a role for POMC overexpression within the VTA reward center to combat HF-induced obesity.     

Prospective study of thymic carcinoids in patients with multiple endocrine neoplasia type 1

Little is known of the natural history of thymic carcinoids in multiple endocrine neoplasia type 1 (MEN1). This is important because in 1993 they were identified as a frequent cause of death, yet only small retrospective studies and case reports exist. We report results of a prospective study of 85 patients with MEN1 evaluated for pancreatic endocrine tumors and followed over a mean of 8 yr with serial chest computed tomography, magnetic resonance imaging (MRI), chest x-ray, and, since 1994, octreoscans [somatostatin receptor scintigraphy (SRS)]. Seven patients (8%) developed thymic carcinoids. Patients with and without carcinoids did not differ in clinical, laboratory, or MEN1 tumor features, except for male gender and the presence of a gastric carcinoid. All thymic tumors were hormonally inactive. Four thymic carcinoids lacked 11q loss of heterozygosity, although it was found in three pancreatic endocrine tumors. Computed tomography and/or MRI were more sensitive than SRS or chest x-ray in detecting tumors initially or with recurrence. All patients underwent resection of the thymic carcinoid, and in all patients followed more than 1 yr, the tumor recurred. Bone metastases developed in two patients and were detected early only on MRI, not SRS. This study provides information on early thymic carcinoids and allows modifications of existing guidelines to be recommended for their diagnosis, surveillance, and treatment.     

以基因芯片技术研究胸腺类癌引起异位ACTH综合征的基因表达

目的　研究引起异位ACTH综合征的胸腺类癌组织与正常胸腺组织的基因表达差异,探讨胸腺类癌引起异位ACTH综合征的发病机制。方法　利用基因芯片技术,通过不同荧光(Cy3和Cy5)标记的对照和胸腺类癌组织RNA样本与芯片杂交,观察两种组织间的基因差异表达。结果　研究发现,基因芯片的4 224个基因中403个基因在胸腺类癌组织中表达下调, 394个基因表达上调2倍以上(其中23个与细胞分裂有关), 51个基因上调5倍以上(其中与细胞分裂有关的基因1个,即PAK3)。结论　引起异位ACTH综合征的胸腺类癌组织与正常胸腺组织存在多个差异表达的基因,PAK3可能参与了该肿瘤的发生。     

TNF-alpha promoter region gene polymorphisms in HIV-positive patients with lipodystrophy

OBJECTIVE: The pathogenic mechanisms underlying lipodystrophy in HIV-positive patients are largely unknown. TNF-alpha has many actions that are consistent with the features of lipodystrophy; therefore, an analysis was carried out to determine whether functionally active polymorphisms in the promoter region of the TNF-alpha gene are associated with the development of lipodystrophy. DESIGN: Genetic case-control association study. METHODS: Individuals were genotyped for the -238 and -308 polymorphisms in the TNF-alpha gene using polymerase chain reaction-restriction fragment length polymorphism analysis. The genotype and allele frequencies for 61 HIV-positive patients with lipodystrophy were compared with (a) 35 HIV-positive patients with no evidence of lipodystrophy and (b) 239 healthy HIV-negative individuals. RESULTS: The frequency of the variant rare -238 allele was significantly different (P = 0.01) in HIV-positive patients with lipodystrophy than in those without lipodystrophy. At the genotype level, a trend towards a difference between patients with and without lipodystrophy was observed (chi2 for linear trend 5.2, P = 0.02). For the -308 polymorphism, no difference was found in genotype and allele frequencies between HIV patients with and without lipodystrophy. CONCLUSIONS: The data suggest that the -238 (but not the -308) promoter region TNF-alpha gene polymorphism is a determinant in the development of HIV-related lipodystrophy. However, the results need to be confirmed in larger numbers of patients as well as in an ethnically diverse population.     

Age-related changes in proopiomelanocortin (POMC) gene expression in the periarcuate region of ovariectomized rats

Aging of the female reproductive system results in a decline in estrous cyclicity which is due, in part, to alterations in hypothalamic function. Opioid peptides, especially the proopiomelanocortin (POMC)-derived neuropeptide, beta-endorphin, are thought to play a role in maintaining normal patterns of LH secretion. Previous studies have shown that the level of hypothalamic beta-endorphin and POMC messenger RNA (mRNA) decreases in old animals; however, it is unknown whether opioid peptides are involved in age-related reproductive decline. To determine whether POMC gene expression changes with age and is related to reproductive status, we assessed POMC mRNA levels by in situ hybridization histochemistry in the periarcuate region of young (3-4 months), middle-aged (10-12 months), and old (17-19 months) ovariectomized rats. Two methods of quantitation were used: 1) slides were apposed to x-ray film and POMC mRNA levels were quantitated over the entire periarcuate region, and 2) the same slides were dipped in emulsion and the level of POMC was quantitated in individual cells. POMC mRNA levels decreased 20-30% by the time animals were middle-aged, and no further decline was noted in the old animal groups. The decrease in POMC mRNA levels in the middle-aged and old animals occurred regardless of their reproductive status prior to ovariectomy. In addition, there was a 30-40% decline in the number of cells expressing POMC mRNA in middle-aged and old animals, suggesting an overall age-related decline in POMC gene expression in middle-aged and old animals independent of reproductive status.     

Evaluating the radiation of the POMC gene in teleosts: characterization of American eel POMC

A distinctive feature of the pituitary hormone precursor, proopiomelanocortin (POMC), is the presence of multiple melanocortin core sequences (HFRW), and one copy of the opioid, beta-endorphin. In the older lineages of ray-finned fish (i.e., orders Acipenseriformes and Semionotiformes), certain extant lobe-finned fish (Australian lungfish and African lungfish), and the tetrapods there are three melanocortin regions in POMC: ACTH/alphaMSH, beta-MSH, and gamma-MSH. However, among the teleosts, the most recent radiation of the ray-finned fishes, the gamma-MSH sequence is absent from the POMC genes of euteleosts like the carp, tilapia, chum salmon, sockeye salmon, and rainbow trout. The objective of this study was to determine whether the gamma-MSH sequence still may be present in the POMC gene of a more basal lineage of the teleosts such as a representative from subdivision Elopomorpha. To this end, a POMC cDNA was cloned and sequenced from the pituitary of the American eel, Anguilla rostrata (order Anguilliformes, family Anguillidae). The open reading frame of the eel POMC cDNA was 648 nucleotides in length and encoded 216 amino acids. As predicted, eel POMC contained the deduced amino acid sequences for beta-endorphin, ACTH/alpha-MSH, and beta-MSH. These end-products displayed primary sequence features that are common to ray-finned fish. Eel POMC lacks a gamma-MSH sequence and a large portion of the joining peptide region. In this regard, the eel POMC gene thus displays features very similar to the POMC genes that have been sequenced from euteleosts. Although it is conceivable that the gamma-MSH sequence may be present in representatives from the other basal extant lineages of teleosts (i.e., subdivisions Osteoglossomorpha or Clupeomorpha), it is also possible that the deletion that resulted in the loss of the gamma-MSH sequence occurred in the ancestral neopterygian that gave rise to the teleosts. In this case, the gamma-MSH sequence should be absent in all extant teleosts.     

Suppressed kindling epileptogenesis in mice with ectopic overexpression of galanin

The neuropeptide galanin has been shown to suppress epileptic seizures. In cortical and hippocampal areas, galanin is normally mainly expressed in noradrenergic afferents. We have generated a mouse overexpressing galanin in neurons under the platelet-derived growth factor B promoter. RIA and HPLC analysis revealed up to 8-fold higher levels of galanin in transgenic as compared with wild-type mice. Ectopic galanin overexpression was detected especially in dentate granule cells and hippocampal and cortical pyramidal neurons. Galanin-overexpressing mice showed retardation of seizure generalization during hippocampal kindling, a model for human complex partial epilepsy. The high levels of galanin in mossy fibers found in the transgenic mice were further increased after seizures. Frequency facilitation of field excitatory postsynaptic potentials, a form of short-term synaptic plasticity assessed in hippocampal slices, was reduced in mossy fiber-CA3 cell synapses of galanin-overexpressing mice, indicating suppressed glutamate release. This effect was reversed by application of the putative galanin receptor antagonist M35. These data provide evidence that ectopically overexpressed galanin can be released and dampen the development of epilepsy by means of receptor-mediated action, at least partly by reducing glutamate release from mossy fibers.     

Aberrant localization of beta-catenin correlates with overexpression of its target gene in human papillary thyroid cancer

Alterations of the Wnt/beta-catenin signaling pathway are known to occur in mutations of the component genes such as APC, Axin, and beta-catenin, and play a pathogenetic role in tumorigenesis. Activated Wnt signaling stabilizes beta-catenin, which associates with T cell factor, resulting in transactivation of the downstream target genes including c-myc and cyclin D1. To investigate the involvement of Wnt/beta-catenin signaling pathway in thyroid tumorigenesis, we analyzed its activation and localization in 5 human thyroid cancer cell lines and 132 thyroid tumor tissue samples. Dislocalization of beta-catenin was observed in all cell lines. Constitutive activation of T cell factor in two of four thyroid cancer cell lines was observed using reporter gene assay. Furthermore, high expression levels of c-Myc and cyclin D1 were observed in cell lines that showed cytoplasmic or nuclear accumulation of beta-catenin. In 132 paraffin-embedded thyroid carcinoma tissue samples, cytoplasmic beta-catenin was immunohistochemically observed in 52 out of 78 (67%) papillary thyroid cancers, but only in 3 of 34 (9%) follicular adenomas and 5 of 20 (25%) follicular cancers. Cytoplasmic localization of beta-catenin significantly correlated with overexpression of cyclin D1 in papillary carcinomas. Our results suggest that aberrant activation of Wnt/beta-catenin signaling is strongly involved in thyroid tumorigenesis.     

Hypomethylation of the HTR1A promoter region and high expression of HTR1A in the peripheral blood lymphocytes of patients with systemic lupus erythematosus

The occurrence of systemic lupus erythematosus (SLE) involves a gene-environment interaction and epigenetic regulations, such as DNA methylation, may play important role in the etiology of SLE. Some neurotransmitters, such as serotonin, can regulate T- and B-cell proliferation via the 5-HT1A receptor and are involved in the pathology of SLE. The abnormal methylation of DNA has been reported in SLE, but there has been no study concerning the serotonin system. This study was conducted to explore the DNA methylation status of the promoter region of HTR1A (PR-HTR1A) and the level of HTR1A mRNA in the peripheral blood lymphocytes (PBLC) of SLE patients and healthy controls (HC). In this study, the DNA methylation status of PR-HTR1A and the level of HTR1A mRNA were detected in the PBLC of SLE patients and HC. The results showed significant hypomethylation of PR-HTR1A in SLE patients compared with HC. The patients also showed a significantly higher HTR1A mRNA level than did the controls. Relatively higher percentage of anti-histone antibodies in methylated SLE patients was found compared with unmethylated patients. Our results support the hypothesis that the hypomethylation of PR-HTR1A and overexpression of HTR1A might contribute to SLE. These results also reveal that epigenetic regulation via the serotonin system may contribute to SLE, and reveal the link between the brain and the immune system.     

肝豆状核变性基因启动子区的多态和突变研究

目的　检测肝豆状核变性 (wilsondisease ,WD)基因启动子区的DNA序列 ,发现存在的多态和突变。方法　 2 0 0 1- 0 2～ 2 0 0 4 - 0 2检测 36个WD家系 71名成员 (其中 4 8例WD患者 )及 2 0名正常人的基因组DNA序列 ,进行分析。结果　在正常对照、患者一级亲属和WD患者的启动子区 - 190、- 78和 2 6 0位 (转录起始点为 1)均发现存在单个碱基的不同 ;在 4 8例病人中发现 3例存在 - 183位C→T突变 ,其中 2例为纯合突变 ,另 1例为杂合突变 ,在正常对照、患者一级亲属中未发现此改变。结论　启动子区在调控WD基因转录活性中起重要的作用 ,提示启动子区的突变是WD的分子发病机制之一。