Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by symptoms related to altered social interactions/communication and restricted and repetitive behaviors. In addition to genetic risk, epigenetic mechanisms (which include DNA methylation/demethylation) are thought to be important in the etiopathogenesis of ASD. We studied epigenetic mechanisms underlying the transcriptional regulation of candidate genes in cerebella of ASD patients, including the binding of MeCP2 (methyl CpG binding protein-2) to the glutamic acid decarboxylase 67 (GAD1), glutamic acid decarboxylase 65 (GAD2), and Reelin (RELN) promoters and gene bodies. Moreover, we performed methyl DNA immunoprecipitation (MeDIP) and hydroxymethyl DNA immunoprecipitation (hMeDIP) to measure total 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in the same regions of these genes. The enrichment of 5-hmC and decrease in 5-mC at the GAD1 or RELN promoters detected by 5-hmC and 5-mC antibodies was confirmed by Tet-assisted bisulfite (TAB) pyrosequencing. The results showed a marked and significant increase in MeCP2 binding to the promoter regions of GAD1 and RELN, but not to the corresponding gene body regions in cerebellar cortex of ASD patients. Moreover, we detected a significant increase in TET1 expression and an enrichment in the level of 5-hmC, but not 5-mC, at the promoters of GAD1 and RELN in ASD when compared with CON. Moreover, there was increased TET1 binding to these promoter regions. These data are consistent with the hypothesis that an increase of 5-hmC (relative to 5-mC) at specific gene domains enhances the binding of MeCP2 to 5-hmC and reduces expression of the corresponding target genes in ASD cerebella.

Estrogen replacement therapy has been correlated with an increased risk of developing breast or endometrial cancer. 4-Hydroxyequilenin (4-OHEN) is a catechol metabolite of equilenin which is a minor component of the estrogen replacement formulation marketed under the name of Premarin (Wyeth-Ayerst). Previously, we showed that 4-OHEN autoxidizes to quinoids which can consume reducing equivalents and molecular oxygen, are potent cytotoxins, and cause a variety of damage to DNA, including formation of bulky stable adducts, apurinic sites, and oxidation of the phosphate-sugar backbone and purine/pyrimidine bases [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1127]. All of these deleterious effects could contribute to the cytotoxic and genotoxic effects of equilenin in vivo. In the study presented here, we examined the relative toxicity of 4-OHEN in estrogen receptor (ER) positive cells (MCF-7 and S30) compared to that in breast cancer cells without the estrogen receptor (MDA-MB-231). The data showed that 4-OHEN was 4-fold more toxic to MCF-7 cells (LC(50) = 6.0 +/- 0. 2 microM) and 6-fold more toxic to S30 cells (LC(50) = 4.0 +/- 0.1 microM) than to MDA-MB-231 cells (LC(50) = 24 +/- 0.3 microM). Using the single-cell gel electrophoresis assay (comet assay) to assess DNA damage, we found that 4-OHEN causes concentration-dependent DNA single-strand cleavage in all three cell lines, and this effect could be enhanced by agents which catalyze redox cycling (NADH) or deplete cellular GSH (diethyl maleate). In addition, the ER(+) cell lines (MCF-7 and S30) were considerably more sensitive to induction of DNA damage by 4-OHEN than the ER(-) cells (MDA-MB-231). 4-OHEN also caused a concentration-dependent increase in the amount of mutagenic lesion 8-oxo-dG in the S30 cells as determined by LC/MS-MS. Cell morphology assays showed that 4-OHEN induces apoptosis in these cell lines. As observed with the toxicity assay and the comet assay, the ER(+) cells were more sensitive to induction of apoptosis by 4-OHEN than MDA-MB-231 cells. Finally, the endogenous catechol estrogen metabolite 4-hydroxyestrone (4-OHE) was considerably less effective at inducing DNA damage and apoptosis in breast cancer cell lines than 4-OHEN. Our data suggest that the cytotoxic effects of 4-OHEN may be related to its ability to induce DNA damage and apoptosis in hormone sensitive cells in vivo, and these effects may be potentiated by the estrogen receptor.

Design and synthesis of nonpeptidal bis-tetrahydrofuran ligands based upon the X-ray crystal structure of the HIV-1 protease-inhibitor complex 1 led to replacement of two amide bonds and a 10 pi-aromatic system of Ro 31-8959 class of HIV protease inhibitors. Detailed structure-activity studies have now established that the position of ring oxygens, ring size, and stereochemistry are all crucial to potency. Of particular interest, compound 49 with (3S,3aS,6aS)-bis-Thf is the most potent inhibitor (IC50 value 1.8 +/- 0.2 nM; CIC95 value 46 +/- 4 nM) in this series. The X-ray structure of protein-inhibitor complex 49 has provided insight into the ligand-binding site interactions. As it turned out, both oxygens in the bis-Thf ligands are involved in hydrogen-bonding interactions with Asp 29 and Asp 30 NH present in the S2 subsite of HIV-1 protease. Stereoselective routes have been developed to obtain these novel ligands in optically pure form.

Introduction Mutations in cardiac ion channels, structural proteins and signaling molecules have been identified in European whites with early-onset AF (EOAF). However, it remains unclear if genetic variation also contributes to the etiology of EOAF in ethnic minorities. Purpose To determine the prevalence of disease causing variants in candidate AF genes in African American and Hispanic/Latino probands with EOAF. Method In this family-based study, probands of African and Hispanic descent with EOAF (defined as AF ≤65 years) were prospectively enrolled in a clinical-DNA biorepository and underwent targeted sequencing for 60 AF candidate genes. Variants were filtered at 20X read depth and clinically evaluated with American College of Medical Genetics and Genomics and Association for Molecular Pathology (ACMG/AMP) as well as the Association for Clinical Genomic Science (ACGS) criteria for disease-causing mutations. Results Among 227 EOAF probands with mean (SD) age of AF 51.0 (9.9) years, 132 (58.0%) were men and 148 (65.0%) African American and 79 (35.0%) Hispanic/Latino. Sequencing 60 candidate AF genes revealed 90 variants that met filtering criteria and underwent clinical evaluation. We identified 16 (7.0%) EOAF probands with a likely pathogenic or pathogenic variant with the majority being loss of function (62.5%) and located in the TTN gene (50.0%). We confirmed a family history of AF in 24 probands (10.6%) and 6 families with >1 affected member a variant of unknown significance (VUS) in genes encoding for a sodium channel (SCN10A), potassium channel (KCNE5), sarcomeric proteins (MYH6, TTN) and atrial natriuretic peptide (NPPA) co-segregated with AF. Conclusion Gene sequencing in African American and Hispanic/Latinos probands with EOAF identified a small percentage of disease causing variants in patients with EOAF. Our findings not only represent important progress toward molecular phenotyping of EOAF, but also provides insight into the underlying pathophysiology toward targeted mechanism-based therapies for AF in ethnic minorities. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): American Heart Association

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