Jenny N. Fung

Dysregulation of the complement cascade in the hSOD1G93A transgenic mouse model of amyotrophic lateral sclerosis.

BackgroundComponents of the innate immune complement system have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS); however, a comprehensive examination of complement expression in this disease has not been performed. This study therefore aimed to determine the expression of complement components (C1qB, C4, factor B, C3/C3b, C5 and CD88) and regulators (CD55 and CD59a) in the lumbar spinal cord of hSOD1G93A mice during defined disease stages.MethodshSOD1G93A and wild-type mice were examined at four different ages of disease progression. mRNA and protein expression of complement components and regulators were examined using quantitative PCR, western blotting and ELISA. Localisation of complement components within lumbar spinal cord was investigated using immunohistochemistry. Statistical differences between hSOD1G93A and wild-type mice were analysed using a two-tailed t-test at each stage of disease progression.ResultsWe found several early complement factors increased as disease progressed, whilst complement regulators decreased; suggesting overall increased complement activation through the classical or alternative pathways in hSOD1G93A mice. CD88 was also increased during disease progression, with immunolocalisation demonstrating expression on motor neurons and increasing expression on microglia surrounding the regions of motor neuron death.ConclusionsThese results indicate that local complement activation and increased expression of CD88 may contribute to motor neuron death and ALS pathology in the hSOD1G93A mouse. Hence, reducing complement-induced inflammation could be an important therapeutic strategy to treat ALS.

Identifying the Biological Basis of GWAS Hits for Endometriosis

ABSTRACT Endometriosis is a common estrogen-dependent gynecological disease influenced by multiple genetic and environmental factors. Genome-wide association studies (GWAS) have identified eight genomic regions with strong evidence for association with endometriosis risk and excellent replication in multiple studies. The results represent a significant breakthrough toward understanding endometriosis. However, the significance can be realized only when the associated DNA sequence variation is linked to the altered regulation and/or function of specific genes and pathways modifying endometriosis risk. This review sets out the multiple steps required to interpret the genetic association results, identify the specific genes likely to be responsible for the altered risk within each region, and obtain the necessary genomic evidence connecting the genetic results to the target genes. Strategies include fine mapping, functional annotation, genomics, and target gene identification through gene expression, epigenetics, and cell-based studies to define direct interactions between causal single nucleotide polymorphisms (SNPs) and target genes. To help decode GWAS “hits” affecting endometriosis from multiple regions, there is an urgent need for well-powered genome-wide studies of the regulation of gene expression and epigenetic mechanisms in the endometrium and other reproductive tissues. The system genetics and genomic studies needed to follow-up GWAS signals will also provide insights into gene regulation influencing other reproductive functions. These studies require multidisciplinary research combining genetics, genomics, functional biology, and clinical research to determine the biological pathways responsible and translate the new knowledge into better outcomes for patients.

TWEAK/Fn14 promotes apoptosis of human endometrial cancer cells via caspase pathway

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible immediate-early response protein 14 (Fn14) have been detected in several human tumors, and demonstrated to regulate multiple cellular responses, including proliferation, survival, migration, apoptosis and differentiation, suggesting roles in cancer. The objective of this study was to clarify the role of TWEAK/Fn14 in the development of human endometrial cancer. We found that TWEAK gene expression was down-regulated and Fn14 gene expression was up-regulated in human endometrial cancer specimens compared with that in normal endometrial specimens; TWEAK acting on Fn14 decreased cell viability by inducing apoptosis through caspase pathways in endometrial cancer cells. Our results suggest that Fn14 expression is high in endometrial cancers whereas local produced TWEAK may be low. TWEAK/Fn14 pathway activation may promote cancer cell apoptosis, which provides a new therapeutic target for human endometrial cancer treatment.

Expression and In Vitro Functions of the Ghrelin Axis in Endometrial Cancer

Ghrelin is a peptide hormone produced in the stomach and a range of other tissues, where it has endocrine, paracrine and autocrine roles in both normal and disease states. Ghrelin has been shown to be an important growth factor for a number of tumours, including prostate and breast cancers. In this study, we examined the expression of the ghrelin axis (ghrelin and its receptor, the growth hormone secretagogue receptor, GHSR) in endometrial cancer. Ghrelin is expressed in a range of endometrial cancer tissues, while its cognate receptor, GHSR1a, is expressed in a small subset of normal and cancer tissues. Low to moderately invasive endometrial cancer cell lines were examined by RT–PCR and immunoblotting, demonstrating that ghrelin axis mRNA and protein expression correlate with differentiation status of Ishikawa, HEC1B and KLE endometrial cancer cell lines. Moreover, treatment with ghrelin potently stimulated cell proliferation and inhibited cell death. Taken together, these data indicate that ghrelin promotes the progression of endometrial cancer cells in vitro, and may contribute to endometrial cancer pathogenesis and represent a novel treatment target.

Pharmacological inhibition of complement C5a-C5a1 receptor signalling ameliorates disease pathology in the hSOD1G93A mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing motor neuron disease without effective treatment. The complement system is up‐regulated in ALS, with recent studies indicating that the activation product C5a accelerates disease progression via the C5a1 receptor (C5aR1). We therefore examined the therapeutic effect of C5a1 receptor antagonism in hSOD1G93A mice, the most widely used preclinical model of ALS.

Endometriosis risk alleles at 1p36.12 act through inverse regulation of CDC42 and LINC00339

Genome-wide association studies (GWAS) have identified markers within the WNT4 region on chromosome 1p36.12 showing consistent and strong association with increasing endometriosis risk. Fine mapping using sequence and imputed genotype data has revealed strong candidates for the causal SNPs within these critical regions; however, the molecular pathogenesis of these SNPs is currently unknown. We used gene expression data collected from whole blood from 862 individuals and endometrial tissue from 136 individuals from independent populations of European descent to examine the mechanism underlying endometriosis susceptibility. Association mapping results from 7,090 individuals (2,594 cases and 4,496 controls) supported rs3820282 as the SNP with the strongest association for endometriosis risk (P = 1.84 × 10−5, OR = 1.244 (1.126-1.375)). SNP rs3820282 is a significant eQTL in whole blood decreasing expression of LINC00339 (also known as HSPC157) and increasing expression of CDC42 (P = 2.0 ×10−54 and 4.5x10−4 respectively). The largest effects were for two LINC00339 probes (P = 2.0 ×10−54; 1.0 × 10−34). The eQTL for LINC00339 was also observed in endometrial tissue (P = 2.4 ×10−8) with the same direction of effect for both whole blood and endometrial tissue. There was no evidence for eQTL effects for WNT4. Chromatin conformation capture provides evidence for risk SNPs interacting with the promoters of both LINC00339 and CDC4 and luciferase reporter assays suggest the risk SNP rs12038474 is located in a transcriptional silencer for CDC42 and the risk allele increases expression of CDC42. However, no effect of rs3820282 was observed in the LINC00339 expression in Ishikawa cells. Taken together, our results suggest that SNPs increasing endometriosis risk in this region act through CDC42, but further functional studies are required to rule out inverse regulation of both LINC00339 and CDC42.

Endometrial vezatin and its association with endometriosis risk

STUDY QUESTION Do endometriosis risk-associated single nucleotide polymorphisms (SNPs) found at the 12q22 locus have effects on vezatin ( ITALIC! VEZT) expression? SUMMARY ANSWER The original genome-wide association study (GWAS) SNP (rs10859871), and other newly identified association signals, demonstrate strong evidence for ITALIC! cis-expression quantitative trait loci (eQTL) effects on ITALIC! VEZT expression. WHAT IS KNOWN ALREADY GWAS have identified several disease-risk loci (SNPs) associated with endometriosis. The SNP rs10859871 is located within the ITALIC! VEZT gene. ITALIC! VEZT expression is altered in the endometrium of endometriosis patients and is an excellent candidate for having a causal role in endometriosis. Most of the SNPs identified from GWAS are not located within the coding region of the genome. However, they are likely to have an effect on the regulation of gene expression. Genetic variants that affect levels of gene expression are called expression quantitative trait loci (eQTL). STUDY DESIGN, SIZE, DURATION Samples for genotyping and ITALIC! VEZT variant screening were drawn from women recruited for genetic studies in Australia/New Zealand and women undergoing surgery in a tertiary care centre. Coding variants for ITALIC! VEZT were screened in blood from 100 unrelated individuals (endometriosis-dense families) from the QIMR Berghofer Medical Research Institute dataset. SNPs at the 12q22 locus were imputed and reanalysed for their association with endometriosis. Reanalysis of endometriosis risk-association was performed on a final combined Australian dataset of 2594 cases and 4496 controls. Gene expression was performed on 136 endometrial samples. eQTL analysis in whole blood was performed on 862 individuals from the Brisbane Systems Genetics Study. Endometrial tissue-specific eQTL analysis was performed on 122 samples (eutopic endometrium) collected following laparoscopic surgery. VEZT protein expression studies employed ITALIC! n = 56 (western blotting) and ITALIC! n = 42 (immunohistochemistry) endometrial samples. PARTICIPANTS/MATERIALS, SETTING, METHODS The women recruited for this study provided blood and/or endometrial tissue samples in a hospital setting. Genomic DNA was screened for common and coding variants. SNPs of interest in the 12q22 region were genotyped using Agena MassARRAY technology or Taqman SNP genotyping assay. Gene expression profiles from RNA extracted from blood and endometrial tissue samples were generated using Illumina whole-genome expression chips (Human HT-12 v4.0). Whole protein extracted from endometrium was used for VEZT western blots, and paraffin sections of endometrium were employed for VEZT immunohistochemistry semi-quantitative analysis. MAIN RESULTS AND THE ROLE OF CHANCE A total of 11 coding variants of ITALIC! VEZT (including one novel variant) were identified from an endometriosis-dense cohort. Polymorphic coding and imputed SNPs were combined with previous GWAS data to reanalyse the endometriosis risk association of the 12q22 region. The disease association signal at 12q22 was due to coding variants in ITALIC! VEZT or ITALIC! FGD6 (FYVE, RhoGEF and PH domain-containing 6) and SNPs with the strongest signals were either intronic or intergenic. We found strong evidence for ITALIC! VEZT cis-eQTLs with the sentinel SNP (rs10859871) in blood and endometrium, where the endometriosis risk allele (C) was associated with an increase in ITALIC! VEZT expression. We could not demonstrate this genotype-specific effect on VEZT protein expression in endometrium. However, we did observe a menstrual cycle stage specific increase in VEZT protein expression in endometrial glands, specific to the secretory phase ( ITALIC! P = 2.0 × 10(-4)). LIMITATIONS, REASONS FOR CAUTION In comparison to the blood sample datasets, the study numbers of endometrial tissues were substantially reduced. Protein studies failed to complement RNA results, also likely a reflection of the low study numbers in these experiments. ITALIC! In silico prediction tools used in this investigation are typically based on cell lines different to our tissues of interest, thus any functional annotations drawn from these approaches should be considered carefully. Therefore, functional studies on VEZT and related pathway components are still warranted to unequivocally implicate a causal role for VEZT in endometriosis pathophysiology. WIDER IMPLICATIONS OF THE FINDINGS GWAS have proven to be very valuable tools for deciphering complex diseases. Endometriosis is a text-book example of a complex disease, involving genetic, lifestyle and environmental influences. Our focused investigation of the 12q22 region validates an association with increased endometriosis risk. Endometriosis risk SNPs (including rs10859871) located within this locus demonstrated evidence for ITALIC! cis-eQTLs on ITALIC! VEZT expression. By examining women who possess an enhanced genetic risk of developing endometriosis, we have identified an effect on ITALIC! VEZT expression and therefore a potential gene/gene pathway in endometriosis disease establishment and development. STUDY FUNDING/COMPETING INTERESTS Funding for this work was provided by NHMRC Project Grants GNT1012245, GNT1026033, GNT1049472 and GNT1046880. G.W.M. is supported by the NHMRC Fellowship scheme (GNT1078399). S.J.H.-C. is supported by the J.N. Peters Bequest Fellowship. The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

The genetic regulation of transcription in human endometrial tissue

Study question Do genetic effects regulate gene expression in human endometrium? Summary answer This study demonstrated strong genetic effects on endometrial gene expression and some evidence for genetic regulation of gene expression in a menstrual cycle stage-specific manner. What is known already Genetic effects on expression levels for many genes are tissue specific. Endometrial gene expression varies across menstrual cycle stages and between individuals, but there are limited data on genetic control of expression in endometrium. Study design, size, duration We analysed genome-wide genotype and gene expression data to map cis expression quantitative trait loci (eQTL) in endometrium. Participants/materials, setting, methods We recruited 123 women of European ancestry. DNA samples from blood were genotyped on Illumina HumanCoreExome chips. Total RNA was extracted from endometrial tissues. Whole-transcriptome profiles were characterized using Illumina Human HT-12 v4.0 Expression Beadchips. We performed eQTL mapping with ~8 000 000 genotyped and imputed single nucleotide polymorphisms (SNPs) and 12 329 genes. Main results and the role of chance We identified a total of 18 595 cis SNP-probe associations at a study-wide level of significance (P < 1 × 10-7), which correspond to independent eQTLs for 198 unique genes. The eQTLs with the largest effect in endometrial tissue were rs4902335 for CHURC1 (P = 1.05 × 10-32) and rs147253019 for ZP3 (P = 8.22 × 10-30). We further performed a context-specific eQTL analysis to investigate if genetic effects on gene expression regulation act in a menstrual cycle-specific manner. Interestingly, five cis-eQTLs were identified with a significant stage-by-genotype interaction. The strongest stage interaction was the eQTL for C10ORF33 (PYROXD2) with SNP rs2296438 (P = 2.0 × 10-4), where we observe a 2-fold difference in the average expression levels of heterozygous samples depending on the stage of the menstrual cycle. Large scale data The summary eQTL results are publicly available to browse or download. Limitations, reasons for caution A limitation of the present study was the relatively modest sample size. It was not powered to identify trans-eQTLs and larger sample sizes will also be needed to provide better power to detect cis-eQTLs and cycle stage-specific effects, given the substantial changes in expression across the menstrual cycle for many genes. Wider implications of the findings Identification of endometrial eQTLs provides a platform for better understanding genetic effects on endometriosis risk and other endometrial-related pathologies. Study funding/competing interest(s) Funding for this work was provided by NHMRC Project Grants GNT1026033, GNT1049472, GNT1046880, GNT1050208, GNT1105321 and APP1083405. There are no competing interests.

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

Genetics of endometriosis: State of the art on genetic risk factors for endometriosis

Developments in high-throughput genotyping technology have driven discovery of genomic regions associated with an increased risk of endometriosis. In all, 16 genomic regions have been associated with risk of endometriosis in one or more populations. The latest meta-analysis including 17,045 endometriosis cases identified 14 genomic regions supported by results from multiple studies. No independent associations were identified from direct genotyping of common and low-frequency protein-coding variants. This suggests that the most common genetic factors that contribute to endometriosis risk are located in regulatory DNA sequences and alter the regulation of gene transcription. Evidence from different methods is essential to identify the target genes and transcripts that contribute to altered disease risk. Potential target genes in three chromosome regions showing altered gene regulation include LINC00339 and CDC42 on chromosome 1, CDKN2A-AS1 on chromosome 9, and VEZT on chromosome 12. Further functional studies are needed to confirm the causal genes in these and other regions to understand pathways that increase endometriosis risk and help identify novel targets for interventions to improve diagnosis and treatment.