Functional evaluation of genetic variants associated with endometriosis near GREB1

STUDY QUESTION: Do DNA variants in the growth regulation by estrogen in breast cancer 1 (GREB1) region regulate endometrial GREB1 expression and increase the risk of developing endometriosis in women? SUMMARY ANSWER: We identified new single nucleotide polymorphisms (SNPs) with strong association with endometriosis at the GREB1 locus although we did not detect altered GREB1 expression in endometriosis patients with defined genotypes. WHAT IS ALREADY KNOWN: Genome-wide association studies have identified the GREB1 region on chromosome 2p25.1 for increasing endometriosis risk. The differential expression of GREB1 has also been reported by others in association with endometriosis disease phenotype. STUDY DESIGN, SIZE, DURATION: Fine mapping studies comprehensively evaluated SNPs within the GREB1 region in a large-scale data set (>2500 cases and >4000 controls). Publicly available bioinformatics tools were employed to functionally annotate SNPs showing the strongest association signal with endometriosis risk. Endometrial GREB1 mRNA and protein expression was studied with respect to phases of the menstrual cycle (n = 2-45 per cycle stage) and expression quantitative trait loci (eQTL) analysis for significant SNPs were undertaken for GREB1 [mRNA (n = 94) and protein (n = 44) in endometrium]. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants in this study are females who provided blood and/or endometrial tissue samples in a hospital setting. The key SNPs were genotyped using Sequenom MassARRAY. The functional roles and regulatory annotations for identified SNPs are predicted by various publicly available bioinformatics tools. Endometrial GREB1 expression work employed qRT-PCR, western blotting and immunohistochemistry studies. MAIN RESULTS AND THE ROLE OF CHANCE: Fine mapping results identified a number of SNPs showing stronger association (0.004 < P < 0.032) with endometriosis risk than the original GWAS SNP (rs13394619) (P = 0.034). Some of these SNPs were predicted to have functional roles, for example, interaction with transcription factor motifs. The haplotype (a combination of alleles) formed by the risk alleles from two common SNPs showed significant association (P = 0.026) with endometriosis and epistasis analysis showed no evidence for interaction between the two SNPs, suggesting an additive effect of SNPs on endometriosis risk. In normal human endometrium, GREB1 protein expression was altered depending on the cycle stage (significantly different in late proliferative versus late secretory, P < 0.05) and cell type (glandular epithelium, not stromal cells). However, GREB1 expression in endometriosis cases versus controls and eQTL analyses did not reveal any significant changes. LIMITATIONS, REASONS FOR CAUTION: In silico prediction tools are generally based on cell lines different to our tissue and disease of interest. Functional annotations drawn from these analyses should be considered with this limitation in mind. We identified cell-specific and hormone-specific changes in GREB1 protein expression. The lack of a significant difference observed following our GREB1 expression studies may be the result of moderate power on mixed cell populations in the endometrial tissue samples. WIDER IMPLICATIONS OF THE FINDINGS: This study further implicates the GREB1 region on chromosome 2p25.1 and the GREB1 gene with involvement in endometriosis risk. More detailed functional studies are required to determine the role of the novel GREB1 transcripts in endometriosis pathophysiology. STUDY FUNDING/COMPETING INTERESTS: Funding for this work was provided by NHMRC Project Grants APP1012245, APP1026033, APP1049472 and APP1046880. There are no competing interests.

Variants in EMX2 and PTEN do not contribute to risk of endometriosis

Endometriosis has a genetic component, and significant linkage has been found to a region on chromosome 10q. Two candidate genes, EMX2 and PTEN, implicated in both endometriosis and endometrial cancer, lie on chromosome 10q. We hypothesized that variation in EMX2 and/or PTEN could contribute to the risk of endometriosis and may account for some of the linkage signal on 10q. We genotyped single nucleotide polymorphisms (SNPs) in a case-control design to evaluate association between endometriosis and common variations in these two genes. The genotyping and statistical analysis were based on samples collected from Australian volunteers. The cases were 768 unrelated women with surgically confirmed endometriosis selected from affected sister pair (ASP) families participating in the Australian Genes behind Endometriosis Study. The controls were 768 female participants in twin studies who, based on screening questions, did not have a diagnosis of endometriosis. Genotypes of 22 SNPs in the EMX2 gene and 15 SNPs in the PTEN gene were the main outcome measures. Statistical analysis provided measures of linkage disequilibrium and association. Permutation testing showed no globally significant association between any SNPs or haplotypes and endometriosis for either gene. It is unlikely that the EMX2 or PTEN gene variants investigated contribute to risk for initiation and/or development of endometriosis.

Mice lacking the serotonin Htr2B receptor gene present an antipsychotic-sensitive schizophrenic-like phenotype

Impulsivity and hyperactivity share common ground with numerous mental disorders, including schizophrenia. Recently, a population-specific serotonin 2B (5-HT2B) receptor stop codon (ie, HTR2B Q20*) was reported to segregate with severely impulsive individuals, whereas 5-HT2B mutant (Htr2B−/−) mice also showed high impulsivity. Interestingly, in the same cohort, early-onset schizophrenia was more prevalent in HTR2B Q*20 carriers. However, the putative role of 5-HT2B receptor in the neurobiology of schizophrenia has never been investigated. We assessed the effects of the genetic and the pharmacological ablation of 5-HT2B receptors in mice subjected to a comprehensive series of behavioral test screenings for schizophrenic-like symptoms and investigated relevant dopaminergic and glutamatergic neurochemical alterations in the cortex and the striatum. Domains related to the positive, negative, and cognitive symptom clusters of schizophrenia were affected in Htr2B−/− mice, as shown by deficits in sensorimotor gating, in selective attention, in social interactions, and in learning and memory processes. In addition, Htr2B−/− mice presented with enhanced locomotor response to the psychostimulants dizocilpine and amphetamine, and with robust alterations in sleep architecture. Moreover, ablation of 5-HT2B receptors induced a region-selective decrease of dopamine and glutamate concentrations in the dorsal striatum. Importantly, selected schizophrenic-like phenotypes and endophenotypes were rescued by chronic haloperidol treatment. We report herein that 5-HT2B receptor deficiency confers a wide spectrum of antipsychotic-sensitive schizophrenic-like behavioral and psychopharmacological phenotypes in mice and provide first evidence for a role of 5-HT2B receptors in the neurobiology of psychotic disorders

Peritoneal washing cytology

Peritoneal washing cytology (PWC) is a useful indicator of ovarian surface involvement and peritoneal dissemination by ovarian tumours. It may identify subclinical peritoneal spread and thus provide valuable staging and prognostic information, particularly for non-serous ovarian tumours. The role of PWC as a prognostic indicator for endometrial carcinoma is less clear, due in part to the questionable significance of identifying endometrial tumour cells in the peritoneum. Detection of metastatic carcinoma in PWC is based on recognition of non-mesothelial cell characteristics, however a number of conditions such as reactive mesothelial cells, endometriosis and endosalpingiosis may mimic this appearance. Cells from these conditions may have a similar presentation in PWC to that of serous borderline tumours and low grade serous carcinoma. The presence of cilia, lack of single atypical cells, prominent cytoplasmic vacuolation, marked nuclear atypia or two distinct cell populations are features favouring a benign process. Attention to these features along with close correlation with clinical history and the results of surgical pathology should help avoid errors. Additional assistance may be provided by the use of cell blocks and special stains.

Induced higher-order aberrations after laser in situ keratomileusis (LASIK) performed with wavefront-guided IntraLase femtosecond laser in moderate to high astigmatism

Background Wavefront-guided Laser-assisted in situ keratomileusis (LASIK) is a widespread and effective surgical treatment for myopia and astigmatic correction but whether it induces higher-order aberrations remains controversial. The study was designed to evaluate the changes in higher-order aberrations after wavefront-guided ablation with IntraLase femtosecond laser in moderate to high astigmatism. Methods Twenty-three eyes of 15 patients with moderate to high astigmatism (mean cylinder, −3.22 ± 0.59 dioptres) aged between 19 and 35 years (mean age, 25.6 ± 4.9 years) were included in this prospective study. Subjects with cylinder ≥ 1.5 and ≤2.75 D were classified as moderate astigmatism while high astigmatism was ≥3.00 D. All patients underwent a femtosecond laser–enabled (150-kHz IntraLase iFS; Abbott Medical Optics Inc) wavefront-guided ablation. Uncorrected (UDVA), corrected (CDVA) distance visual acuity in logMAR, keratometry, central corneal thickness (CCT) and higher-order aberrations (HOAs) over a 6 mm pupil, were assessed before and 6 months, postoperatively. The relationship between postoperative change in HOA and preoperative mean spherical equivalent refraction, mean astigmatism, and postoperative CCT were tested. Results At the last follow-up, the mean UDVA was increased (P < 0.0001) but CDVA remained unchanged (P = 0.48) and no eyes lost ≥2 lines of CDVA. Mean spherical equivalent refraction was reduced (P < 0.0001) and was within ±0.50 D range in 61 % of eyes. The average corneal curvature was flatter by 4 D and CCT was reduced by 83 μm (P < 0.0001, for all), postoperatively. Coma aberrations remained unchanged (P = 0.07) while the change in trefoil (P = 0.047) postoperatively, was not clinically significant. The 4th order HOAs (spherical aberration and secondary astigmatism) and the HOA root mean square (RMS) increased from −0.18 ± 0.07 μm, 0.04 ± 0.03 μm and 0.47 ± 0.11 μm, preoperatively, to 0.33 ± 0.19 μm (P = 0.004), 0.21 ± 0.09 μm (P < 0.0001) and 0.77 ± 0.27 μm (P < 0.0001), six months postoperatively. The change in spherical aberration after the procedure increased with an increase in the degree of preoperative myopia. Conclusions Wavefront-guided IntraLASIK offers a safe and effective option for vision and visual function improvement in astigmatism. Although, reduction of HOA is possible in a few eyes, spherical-like aberrations are increased in majority of the treated eyes.

Polysaccharopeptide enhanced the anti-cancer effect of gammatocotrienol through activation of AMPK

Prostate cancer (PCa) frequently relapses after hormone ablation therapy. Unfortunately, once progressed to the castration resistant stage, the disease is regarded as incurable as prostate tumours are highly resistant to conventional chemotherapy. Therefore, an effective treatment strategy is urgently needed for improving the treatment outcome of the patients.

Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells

Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.