Joshua Atkins

Differential effect of disease-associated ST8SIA2 haplotype on cerebral white matter diffusion properties in schizophrenia and healthy controls

Brain white matter abnormalities are evident in individuals with schizophrenia, and also their first-degree relatives, suggesting that some alterations may relate to underlying genetic risk. The ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 (ST8SIA2) gene, which encodes the alpha-2,8-sialyltransferase 8B enzyme that aids neuronal migration and synaptic plasticity, was previously implicated as a schizophrenia susceptibility gene. This study examined the extent to which specific haplotypes in ST8SIA2 influence white matter microstructure using diffusion-weighted imaging of individuals with schizophrenia (n = 281) and healthy controls (n = 172), recruited across five Australian sites. Interactions between diagnostic status and the number of haplotype copies (0 or ≥1) were tested across all white matter voxels with cluster-based statistics. Fractional anisotropy (FA) in the right parietal lobe was found to show a significant interaction between diagnosis and ST8SIA2 protective haplotype (p < 0.05, family-wise error rate (FWER) cluster-corrected). The protective haplotype was associated with increased FA in controls, but this effect was reversed in people with schizophrenia. White matter fiber tracking revealed that the region-of-interest was traversed by portions of the superior longitudinal fasciculus, corona radiata, and posterior limb of internal capsule. Post hoc analysis revealed that reduced FA in this regional juncture correlated with reduced IQ in people with schizophrenia. The ST8SIA2 risk haplotype copy number did not show any differential effects on white matter. This study provides a link between a common disease-associated haplotype and specific changes in white matter microstructure, which may relate to resilience or risk for mental illness, providing further compelling evidence for involvement of ST8SIA2 in the pathophysiology of schizophrenia.

Germ cell-specific sustained activation of Wnt signalling perturbs spermatogenesis in aged mice, possibly through non-coding RNAs

Dysregulated Wnt signalling is associated with human infertility and testicular cancer. However, the role of Wnt signalling in male germ cells remains poorly understood. In this study, we first confirmed the activity of Wnt signalling in mouse, dog and human testes. To determine the physiological importance of the Wnt pathway, we developed a mouse model with germ cell-specific constitutive activation of βcatenin. In young mutants, similar to controls, germ cell development was normal. However, with age, mutant testes showed defective spermatogenesis, progressive germ cell loss, and flawed meiotic entry of spermatogonial cells. Flow sorting confirmed reduced germ cell populations at the leptotene/zygotene stages of meiosis in mutant group. Using thymidine analogues-based DNA double labelling technique, we further established decline in germ cell proliferation and differentiation. Overactivation of Wnt/βcatenin signalling in a spermatogonial cell line resulted in reduced cell proliferation, viability and colony formation. RNA sequencing analysis of testes revealed significant alterations in the non-coding regions of mutant mouse genome. One of the novel non-coding RNAs was switched on in mutant testes compared to controls. QPCR analysis confirmed upregulation of this unique non-coding RNA in mutant testis. In summary, our results highlight the significance of Wnt signalling in male germ cells.

Advantages of Array-Based Technologies for Pre-Emptive Pharmacogenomics Testing.

As recognised by the National Institutes of Health (NIH) Precision Medicine Initiative (PMI), microarray technology currently provides a rapid, inexpensive means of identifying large numbers of known genomic variants or gene transcripts in experimental and clinical settings. However new generation sequencing techniques are now being introduced in many clinical genetic contexts, particularly where novel mutations are involved. While these methods can be valuable for screening a restricted set of genes for known or novel mutations, implementation of whole genome sequencing in clinical practice continues to present challenges. Even very accurate high-throughput methods with small error rates can generate large numbers of false negative or false positive errors due to the high numbers of simultaneous readings. Additional validation is likely to be required for safe use of any such methods in clinical settings. Custom-designed arrays can offer advantages for screening for common, known mutations and, in this context, may currently be better suited for accredited, quality-controlled clinical genetic screening services, as illustrated by their successful application in several large-scale pre-emptive pharmacogenomics programs now underway. Excessive, inappropriate use of next-generation sequencing may waste scarce research funds and other resources. Microarrays presently remain the technology of choice in applications that require fast, cost-effective genome-wide screening of variants of known importance, particularly for large sample sizes. This commentary considers some of the applications where microarrays continue to offer advantages over next-generation sequencing technologies.

F193. DYSREGULATION OF RETINOID SIGNALLING IN SCHIZOPHRENIA OBSERVED IN WHOLE GENOME SEQUENCE ANALYSIS

Abstract Background Retinoic acid (RA) is intrinsically linked to neurodevelopment and has been implicated in schizophrenia (SZ). This is supported by preliminary trials of a retinoid receptor agonist, Bexarotene, as an adjuvant and the association of five common variants in proximity to members of this pathway at genome wide significance. In addition to these high frequency variants with small effect size, we suspect that this pathway is also affected by rare variants with much higher impact. We aimed to examine the burden of variants in retinoid loci in schizophrenia along with its potential consequences for clinical practice. Methods Whole genome sequencing was performed on SZ cases (N=331) and non-psychiatric controls (N=167). Cases were further clustered by cognitive measures to derive the cognitive deficit (CD; N=166) and cognitively spared (CS; N=165) subtypes. Disease and subtype associated genomic variation was then analysed in a panel of 129 genes selected for involvement in RA biology (Molecular Signatures Database). Rare variation was aggregated at the gene level using the optimal unified sequence kernel association test (SKAT-O). Clinical metadata was further examined for each case with a rare putative high impact loss of function variant in a RA panel gene predicted using SnpEff. The rare variant burden on target genes of RA receptor binding in SZ was investigated by logistic regression of variants mapped to consensus 5 base pair spaced direct repeat (DR5) retinoic acid response element (RARE) motifs. Results Gene level rare variant association uncovered suggestive associations with SZ (P < 0.01) for three retinoid genes – RBP3, ADH1C and RPE65. In addition, a stronger signal was detected overall for CD cases implicating four additional genes including the RA receptors RARB (P = 1.1 x 10–3) and RARG (P = 9.2 x 10–3). SZ patients with a rare high impact genotype predicted in a RA panel gene were more likely to have serious symptomology as defined by a global assessment of functioning (GAF) score below 50, P = 7.1 x 10–3 (Two-Tailed Fisher’s Exact Test). We also found evidence of an increased burden of rare variants within predicted DR5-RARE in SZ (P = 0.017, odds ratio [OR] = 1.094, 95% confidence interval [CI] = 1.023- 1.186), however, there was no significant difference between the cognitive subtypes (CD/CS, P = 0.8, OR = 1.002, 95% CI = 0.961–1.045). Discussion Our findings suggest that RA mediated control of gene expression is heterogeneously disrupted in SZ by rare variants in DR5-RARE motifs. Strong signals in the context of our sample size further support the possibility of enrichment of rare loci in genes involved with RA biology, particularly in CD cases with impaired cognition. Moreover, we identified a subset of patients with likely high effect size genotypes in the RA pathway. Future work will examine whether these high-risk patients would benefit from retinoid based pharmacological intervention.

Common variation in ZNF804A (rs1344706) is not associated with brain morphometry in schizophrenia or healthy participants

Background: The single nucleotide polymorphism (SNP) rs1344706 [A > C] within intron 2 of the zinc finger protein 804A gene (ZNF804A) is associated with schizophrenia at the genome‐wide level, but its function in relation to the development of psychotic disorders, including its influence on brain morphology remains unclear. Methods: Using both univariate (voxel‐based morphometry, VBM; cortical thickness) and multivariate (source‐based morphometry, SBM) approaches, we examined the effects of variation of the rs1344706 polymorphism on grey matter integrity in 214 Caucasian schizophrenia cases and 94 Caucasian healthy individuals selected from the Australian Schizophrenia Research Bank. Results: Neither univariate nor multivariate analyses showed any associations between indices of grey matter and rs1344706 variation in schizophrenia or healthy participants. This was revealed in the context of the typical pattern of decreased grey matter integrity in schizophrenia compared to healthy individuals, including: (1) large grey matter volume reductions in the orbitofrontal and anterior cingulate cortices and the left fusiform/inferior temporal gyri; (2) decreased cortical thickness in the left inferior temporal and fusiform gyri, the left orbitofrontal gyrus, as well as in the right pars opercularis/precentral gyrus; and (3) decreased covariation of grey matter concentration in frontal and limbic brain regions emerging from the SBM analyses. Conclusions: Contrary to some – but not all – previous findings, this study of a large sample of schizophrenia cases and healthy controls reveals no evidence for association between grey matter alterations and variation in rs1344706 (ZNF804A). Differences in sample sizes and ethnicities may account for discrepant findings between the present and previous studies. HighlightsZNF804A rs1344706 [A > C] polymorphism is associated with schizophrenia.Previous studies suggest A‐carriers show alterations of brain structure and function.In a larger sample, rs1344706 polymorphism was not associated with grey matter.

Inhibition of extracellular matrix mediated TGF-β signalling suppresses endometrial cancer metastasis

Although aggressive invasion and distant metastases are an important cause of morbidity and mortality in patients with endometrial cancer (EC), the requisite events determining this propensity are currently unknown. Using organotypic three-dimensional culture of endometrial cancer cell lines, we demonstrated anti-correlated TGF-β signalling gene expression patterns that arise among extracellular matrix (ECM)-attached cells. TGF-β pathway seemed to be active in EC cells forming non-glandular colonies in 3D-matrix but weaker in glandular colonies. Functionally we found that out of several ECM proteins, fibronectin relatively promotes Smad phosphorylation suggesting a potential role in regulating TGF-β signalling in non-glandular colonies. Importantly, alteration of TGF-β pathway induced EMT and MET in both type of colonies through slug protein. The results exemplify a crucial role of TGF-β pathway during EC metastasis in human patients and inhibition of the pathway in a murine model impaired tumour cell invasion and metastasis depicting an attractive target for therapeutic intervention of malignant tumour progression. These findings provide key insights into the role of ECM-derived TGF-β signalling to promote endometrial cancer metastasis and offer an avenue for therapeutic targeting of microenvironment derived signals along with tumour cells.Although aggressive invasion and distant metastases are an important cause of morbidity and mortality in patients with endometrial cancer (EC), the requisite events determining this propensity are currently unknown. Using organotypic three-dimensional culture of endometrial cancer cell lines, we demonstrated anti-correlated TGF-β signalling gene expression patterns that arise among extracellular matrix (ECM)-attached cells. TGF-β pathway seemed to be active in EC cells forming non-glandular colonies in 3D-matrix but weaker in glandular colonies. Functionally we found that out of several ECM proteins, fibronectin relatively promotes Smad phosphorylation suggesting a potential role in regulating TGF-β signalling in non-glandular colonies. Importantly, alteration of TGF-β pathway induced EMT and MET in both type of colonies through slug protein. The results exemplify a crucial role of TGF-β pathway during EC metastasis in human patients and inhibition of the pathway in a murine model impaired tumour cell invasion and metastasis depicting an attractive target for therapeutic intervention of malignant tumour progression. These findings provide key insights into the role of ECM-derived TGF-β signalling to promote endometrial cancer metastasis and offer an avenue for therapeutic targeting of microenvironment derived signals along with tumour cells.

miRNA Enriched in Human Neuroblast Nuclei Bind the MAZ Transcription Factor and Their Precursors Contain the MAZ Consensus Motif

While the cytoplasmic function of microRNA (miRNA) as post-transcriptional regulators of mRNA has been the subject of significant research effort, their activity in the nucleus is less well characterized. Here we use a human neuronal cell model to show that some mature miRNA are preferentially enriched in the nucleus. These molecules were predominantly primate-specific and contained a sequence motif with homology to the consensus MAZ transcription factor binding element. Precursor miRNA containing this motif were shown to have affinity for MAZ protein in nuclear extract. We then used Ago1/2 RIP-Seq to explore nuclear miRNA-associated mRNA targets. Interestingly, the genes for Ago2-associated transcripts were also significantly enriched with MAZ binding sites and neural function, whereas Ago1-transcripts were associated with general metabolic processes and localized with SC35 spliceosomes. These findings suggest the MAZ transcription factor is associated with miRNA in the nucleus and may influence the regulation of neuronal development through Ago2-associated miRNA induced silencing complexes. The MAZ transcription factor may therefore be important for organizing higher order integration of transcriptional and post-transcriptional processes in primate neurons.