The MyD88+ phenotype is an adverse prognostic factor in epithelial ovarian cancer

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.

Modeling conditional correlations of asset returns : a smooth transition approach

In this paper we propose a new multivariate GARCH model with time-varying conditional correlation structure. The time-varying conditional correlations change smoothly between two extreme states of constant correlations according to a predetermined or exogenous transition variable. An LM–test is derived to test the constancy of correlations and LM- and Wald tests to test the hypothesis of partially constant correlations. Analytical expressions for the test statistics and the required derivatives are provided to make computations feasible. An empirical example based on daily return series of five frequently traded stocks in the S&P 500 stock index completes the paper.

A high and low fear phenotype mouse model

Fear-related illnesses such as post-traumatic stress disorder (PTSD) impose a tremendous burden on individual quality of life, families, and the national economy. In the military population, 17-20% of services members returning from deployment are diagnosed with PTSD. While treatments have improved for PTSD and are helpful for some, many people continue to suffer despite therapy. The aim of this research is to examine fear memory behaviourally and at the cellular level in the amygdala by using a unique inter-cross strain of high and low fear phenotype mice. An extended outcross C57BL/6J x DBA/2J (F8) are selected for high or low Pavlovian fear memory to context and cue. On presentation of either the original learning context or the cue (tone) mice display high or low levels of freezing as a behavioural measure of fear. In order to identify key aspects of the cellular basis of this difference in fear memory behaviour we are making measurements of protein levels and neuron numbers of a known pathway involved in the consolidation of a long term fear memory (pMAPK). Ongoing studies aim to determine if high fear behaviour is associated with differential signalling in the lateral amygdala compared to low fear behaviour. Additionally, by blocking this pathway in the lateral amygdala (LA), we aim to reduce fear behaviour following Pavlovian fear conditioning. This research will help to unravel the cellular mechanisms underlying high fear behaviour and advance the field toward targeted treatment and improved outcomes, ultimately improving human quality of life.

H2AX phosphorylation screen of cells from radiosensitive cancer patients reveals a novel DNA double-strand break repair cellular phenotype

BACKGROUND: About 1-5% of cancer patients suffer from significant normal tissue reactions as a result of radiotherapy (RT). It is not possible at this time to predict how most patients' normal tissues will respond to RT. DNA repair dysfunction is implicated in sensitivity to RT particularly in genes that mediate the repair of DNA double-strand breaks (DSBs). Phosphorylation of histone H2AX (phosphorylated molecules are known as gammaH2AX) occurs rapidly in response to DNA DSBs, and, among its other roles, contributes to repair protein recruitment to these damaged sites. Mammalian cell lines have also been crucial in facilitating the successful cloning of many DNA DSB repair genes; yet, very few mutant cell lines exist for non-syndromic clinical radiosensitivity (RS). METHODS: Here, we survey DNA DSB induction and repair in whole cells from RS patients, as revealed by gammaH2AX foci assays, as potential predictive markers of clinical radiation response. RESULTS: With one exception, both DNA focus induction and repair in cell lines from RS patients were comparable with controls. Using gammaH2AX foci assays, we identified a RS cancer patient cell line with a novel ionising radiation-induced DNA DSB repair defect; these data were confirmed by an independent DNA DSB repair assay. CONCLUSION: gammaH2AX focus measurement has limited scope as a pre-RT predictive assay in lymphoblast cell lines from RT patients; however, the assay can successfully identify novel DNA DSB repair-defective patient cell lines, thus potentially facilitating the discovery of novel constitutional contributions to clinical RS.

FGF-2 induces the proliferation of human periodontal ligament cells and modulates their osteoblastic phenotype by affecting Runx2 expression in the presence and absence of osteogenic inducers

The exact phenotype of human periodontal ligament cells (hPDLCs) remains a controversial area. Basic fibroblast growth factor (FGF‑2) exhibits various functions and its effect on hPDLCs is also controversial. Therefore, the present study examined the effect of FGF‑2 on the growth and osteoblastic phenotype of hPDLCs with or without osteogenic inducers (dexamethasone and β‑glycerophosphate). FGF‑2 was added to defined growth culture medium and osteogenic inductive culture medium. Cell proliferation, osteogenic differentiation and mineralization were measured. The selected differentiation markers, Runx2, collagen type Ⅰ, α1 (Col1a1), osteocalcin (OCN) and epidermal growth factor receptor (EGFR), were investigated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Runx2 and OCN protein expression was measured by western blotting. FGF‑2 significantly increased the proliferation of hPDLCs, but did not affect alkaline phosphatase activity. RT‑qPCR analysis revealed enhanced mRNA expression of Runx2, OCN and EGFR, but suppressed Col1a1 gene expression in the absence of osteogenic inducers, whereas all these gene levels had no clear trend in their presence. The Runx2 protein expression was clearly increased, but the OCN protein level showed no evident trend. The mineralization assay demonstrated that FGF‑2 inhibited mineralized matrix deposition with osteogenic inducers. These results suggested that FGF‑2 induces the growth of immature hPDLCs, which is a competitive inhibitor of epithelial downgrowth, and suppresses their differentiation into mineralized tissue by affecting Runx2 expression. Therefore, this may lead to the acceleration of periodontal regeneration.

Epigenetic modelling

The field of epigenetics looks at changes in the chromosomal structure that affect gene expression without altering DNA sequence. A large-scale modelling project to better understand these mechanisms is gaining momentum. Early advances in genetics led to the all-genetic paradigm: phenotype (an organism's characteristics/behaviour) is determined by genotype (its genetic make-up). This was later amended and expressed by the well-known formula P = G + E, encompassing the notion that the visible characteristics of a living organism (the phenotype, P) is a combination of hereditary genetic factors (the genotype, G) and environmental factors (E). However, this method fails to explain why in diseases such as schizophrenia we still observe differences between identical twins. Furthermore, the identification of environmental factors (such as smoking and air quality for lung cancer) is relatively rare. The formula also fails to explain cell differentiation from a single fertilized cell. In the wake of early work by Waddington, more recent results have emphasized that the expression of the genotype can be altered without any change in the DNA sequence. This phenomenon has been tagged as epigenetics. To form the chromosome, DNA strands roll over nucleosomes, which are a cluster of nine proteins (histones), as detailed in Figure 1. Epigenetic mechanisms involve inherited alterations in these two structures, eg through attachment of a functional group to the amino acids (methyl, acetyl and phosphate). These 'stable alterations' arise during development and cell proliferation and persist through cell division. While information within the genetic material is not changed, instructions for its assembly and interpretation may be. Modelling this new paradigm, P = G + E + EpiG, is the object of our study.

Variable phenotype in 16p duplication within a family

Background More individuals are now being identified with very rare genetic syndromes. We present a family with an inherited duplication of 16p11.2 to 16q12.1 in ring formation. Three of the four children, (aged 15 months to 10 years), mother, uncle, and grandmother are affected. Our aim was to provide preliminary evidence of possible phenotypic patterns of learning and behaviour associated with this chromosome anomaly. Method Psychometric assessments were undertaken with all four children. The mother and uncle also agreed to participate in the study. Measures of development (Bayley or Mullen), intellectual ability (WISC-IV or WAIS-III), academic achievement (WIAT-II), adaptive behaviour (Vinelands), and other relevant aspects of functioning (e.g., Children’s Memory Scale) were administered. Results. The first-born child is the only one who is unaffected. Her intellectual ability was assessed as being within the superior range. The second child experienced early difficulties with speech and motor skills. Although his intelligence is average, he has learning difficulties and significant auditory memory problems. The third child’s speech and motor milestones were markedly delayed. He has a complex medical history that includes a vitamin B12 deficiency. On the Mullen Scales at age 4 his scores ranged from average to very low. The development of the youngest child (aged 15 months), who also had a B12 deficiency but was treated early, was assessed as being within typical limits. Conclusions There is considerable developmental variability among the three children with this inherited 16p duplication. We discuss the intriguing similarities and differences, considering common features that may reflect phenotypic patterns and speculating about possible explanations for the variable presentations.

SECA: SNP effect concordance analysis using genome-wide association summary results

The genomics era provides opportunities to assess the genetic overlap across phenotypes at the measured genotype level; however, current approaches require individual-level genome-wide association (GWA) single nucleotide polymorphism (SNP) genotype data in one or both of a pair of GWA samples. To facilitate the discovery of pleiotropic effects and examine genetic overlap across two phenotypes, I have developed a user-friendly web-based application called SECA to perform SNP effect concordance analysis using GWA summary results. The method is validated using publicly available summary data from the Psychiatric Genomics Consortium.

Common variants in the CYP2C19 gene are associated with susceptibility to endometriosis

Objective: To follow-up previous studies highlighting a possible role for cytochrome P450, family 2, subfamily C, 19 (CYP2C19) in susceptibility to endometriosis by searching for additional variants in the CYP2C19 gene that may be associated with the disease. Design Case-control study. Setting Academic research. Subject(s) The cases comprised 2,271 women with surgically confirmed endometriosis; the controls comprised 939 women with self-report of no endometriosis and 1,770 unscreened population samples. Intervention(s) Sequencing of the CYP2C19 region and follow-up of 80 single nucleotide polymorphisms (SNPs) in two case-control samples. Main Outcome Measure(s) Allele frequency differences between cases and controls. Result(s) Sequencing of the CYP2C19 gene region resulted in the detection of a large number of known and novel SNPs. Genotyping of 80 polymorphic SNPs in 901 endometriosis cases and 939 controls resulted in study-wide significant association signals for SNPs in moderate or complete linkage disequilibrium with rs4244285, a functional SNP in exon 5 that abrogates CYP2C19 function through the creation of an alternative splice site. Evidence of association was also detected for another functional SNP in the CYP2C19 promoter, rs12248560, which was highlighted in our previous study. Conclusion(s) Functional variants in CYP2C19 may contribute to endometriosis susceptibility in both familial and sporadic cases. © 2014 by American Society for Reproductive Medicine.

Identification of genetic variants contributing to the migraine phenotype in different Australian populations

This project aimed to identify novel genetic risk variants associated with migraine in the Norfolk Island population. Statistical analysis and bioinformatics approaches such as polygenic modeling and gene clustering methods were carried out to explore genotypic and expression data from high-throughput techniques. This project had a particular focus on hormonal genes and other genetic variants and identified a modest effect size on the migraine phenotype.

Genome-Wide Association Studies of Asthma in Population-Based Cohorts Confirm Known and Suggested Loci and Identify an Additional Association near HLA

Rationale: Asthma has substantial morbidity and mortality and a strong genetic component, but identification of genetic risk factors is limited by availability of suitable studies. Objectives: To test if population-based cohorts with self-reported physician-diagnosed asthma and genome-wide association (GWA) data could be used to validate known associations with asthma and identify novel associations. Methods: The APCAT (Analysis in Population-based Cohorts of Asthma Traits) consortium consists of 1,716 individuals with asthma and 16,888 healthy controls from six European-descent population-based cohorts. We examined associations in APCAT of thirteen variants previously reported as genome-wide significant (P<5x10-8) and three variants reported as suggestive (P<5×10-7). We also searched for novel associations in APCAT (Stage 1) and followed-up the most promising variants in 4,035 asthmatics and 11,251 healthy controls (Stage 2). Finally, we conducted the first genome-wide screen for interactions with smoking or hay fever. Main Results: We observed association in the same direction for all thirteen previously reported variants and nominally replicated ten of them. One variant that was previously suggestive, rs11071559 in RORA, now reaches genome-wide significance when combined with our data (P = 2.4×10-9). We also identified two genome-wide significant associations: rs13408661 near IL1RL1/IL18R1 (PStage1+Stage2 = 1.1x10-9), which is correlated with a variant recently shown to be associated with asthma (rs3771180), and rs9268516 in the HLA region (PStage1+Stage2 = 1.1x10-8), which appears to be independent of previously reported associations in this locus. Finally, we found no strong evidence for gene-environment interactions with smoking or hay fever status. Conclusions: Population-based cohorts with simple asthma phenotypes represent a valuable and largely untapped resource for genetic studies of asthma. © 2012 Ramasamy et al.

HLA class I associations of ankylosing spondylitis in the white population in the United Kingdom

Objective - To investigate the HLA class I associations of ankylosing spondylitis (AS) in the white population, with particular reference to HLA-B27 subtypes. Methods - HLA-B27 and -B60 typing was performed in 284 white patients with AS. Allele frequencies of HLA-B27 and HLA-B60 from 5926 white bone marrow donors were used for comparison. HLA-B27 subtyping was performed by single strand conformation polymorphism (SSCP) in all HLA-B27 positive AS patients, and 154 HLA-B27 positive ethnically matched blood donors. Results - The strong association of HLA-B27 and AS was confirmed (odds ratio (OR) 171, 95% confidence interval (CI) 135 to 218; p < 10-99). The association of HLA-B60 with AS was confirmed in HLA-B27 positive cases (OR 3.6, 95% CI 2.1 to 6.3; p < 5 x 10-5), and a similar association was demonstrated in HLA-B27 negative AS (OR 3.5, 95% CI 1.1 to 11.4; p < 0.05). No significant difference was observed in the frequencies of HLA-B27 allelic subtypes in patients and controls (HLA-B*2702, three of 172 patients v five of 154 controls; HLA-B*2705, 169 of 172 patients v 147 of 154 controls; HkA-B*2708, none of 172 patients v two of 154 controls), and no novel HLA-B27 alleles were detected. Conclusion - HLA-B27 and -B60 are associated with susceptibility to AS, but differences in BLA-B27 subtype do not affect susceptibility to AS in this white population.

The F158V polymorphism in FcγRIIIA shows disparate associations with rheumatoid arthritis in two genetically distinct populations

Objectives: To investigate the association of the FcγRIIIA gene with rheumatoid orthritis (RA) in two genetically distinct groups: a white group from the United Kingdom and a northern Indian group. Methods: The distributions of the two alleles of the FcγRIIIA F158V polymorphism were determined in 398 white patients from the United Kingdom and 63 Indian patients with RA and compared with those from 289 United Kingdom and 93 Indian healthy controls, respectively. Results: Among the Indian patients, the frequency of the rare 158V allele and the proportion of 158VV homozygotes were reduced (relative risk (RR)=0.3, 95% confidence interval (95% CI) 0.1 to 1.1, p<0.06), reaching statistical significance for carrying the 158VV phenotype relative to 158FV or FF (RR=0.2, 95% CI 0.05-0.9, p<0.02). Conversely, no significant deviation in allelic frequencies was noted between the patients and controls from the United Kingdom. Conclusions: The 158VV phenotype showed a weak protective effect against developing RA in the Indian group. However, this sample was small (resulting in a low power for statistical analysis) and no independent confirmation was found in the larger white United Kingdom group. Thus the FcγRIIIA locus is unlikely to be of major importance in causing RA.

Investigation of the role of ANKH in ankylosing spondylitis

Objective The ank/ank mouse develops a phenotype similar to ankylosing spondylitis (AS) in humans. ANKH, the human homolog of the mutated gene in the ank/ank mouse, has been implicated in familial autosomal-dominant chondrocalcinosis and autosomal-dominant craniometaphyseal dysplasia. This study was undertaken to investigate the role of ANKH in susceptibility to and clinical manifestations of AS. Methods Sequence variants were identified by genomic sequencing of the 12 ANKH exons and their flanking splice sites in 48 AS patients; variants were then screened in 233 patients and 478 controls. Linkage to the ANKH locus was assessed in 185 affected-sibling-pair families. Results Five single-nucleotide polymorphisms were identified within the coding region and flanking splice sites. No association between either susceptibility to AS or its clinical manifestations and these novel polymorphisms, or between disease susceptibility and 3 known promoter variants, was seen. No linkage between the ANKH locus and AS was observed. Multipoint exclusion mapping rejected the hypothesis of a locus of a magnitude λ≥1.4 (logarithm of odds score <-2) (equivalent to a genetic contribution of >10% to the AS sibling recurrence risk ratio) within this area contributing to AS. Conclusion These findings indicate that ANKH is not significantly involved in susceptibility to or clinical manifestations of AS.

Genome-wide association study for radiographic vertebral fractures: A potential role for the 16q24 BMD locus

Vertebral fracture risk is a heritable complex trait. The aim of this study was to identify genetic susceptibility factors for osteoporotic vertebral fractures applying a genome-wide association study (GWAS) approach. The GWAS discovery was based on the Rotterdam Study, a population-based study of elderly Dutch individuals aged >55years; and comprising 329 cases and 2666 controls with radiographic scoring (McCloskey-Kanis) and genetic data. Replication of one top-associated SNP was pursued by de-novo genotyping of 15 independent studies across Europe, the United States, and Australia and one Asian study. Radiographic vertebral fracture assessment was performed using McCloskey-Kanis or Genant semi-quantitative definitions. SNPs were analyzed in relation to vertebral fracture using logistic regression models corrected for age and sex. Fixed effects inverse variance and Han-Eskin alternative random effects meta-analyses were applied. Genome-wide significance was set at p<5×10-8. In the discovery, a SNP (rs11645938) on chromosome 16q24 was associated with the risk for vertebral fractures at p=4.6×10-8. However, the association was not significant across 5720 cases and 21,791 controls from 14 studies. Fixed-effects meta-analysis summary estimate was 1.06 (95% CI: 0.98-1.14; p=0.17), displaying high degree of heterogeneity (I2=57%; Qhet p=0.0006). Under Han-Eskin alternative random effects model the summary effect was significant (p=0.0005). The SNP maps to a region previously found associated with lumbar spine bone mineral density (LS-BMD) in two large meta-analyses from the GEFOS consortium. A false positive association in the GWAS discovery cannot be excluded, yet, the low-powered setting of the discovery and replication settings (appropriate to identify risk effect size >1.25) may still be consistent with an effect size <1.10, more of the type expected in complex traits. Larger effort in studies with standardized phenotype definitions is needed to confirm or reject the involvement of this locus on the risk for vertebral fractures.