Systematic chemical and molecular profiling of MLL-rearranged infant acute lymphoblastic leukemia reveals efficacy of romidepsin.

To address the poor prognosis of mixed lineage leukemia (MLL)-rearranged infant acute lymphoblastic leukemia (iALL), we generated a panel of cell lines from primary patient samples and investigated cytotoxic responses to contemporary and novel Food and Drug Administration-approved chemotherapeutics. To characterize representation of primary disease within cell lines, molecular features were compared using RNA-sequencing and cytogenetics. High-throughput screening revealed variable efficacy of currently used drugs, however identified consistent efficacy of three novel drug classes: proteasome inhibitors, histone deacetylase inhibitors and cyclin-dependent kinase inhibitors. Gene expression of drug targets was highly reproducible comparing iALL cell lines to matched primary specimens. Histone deacetylase inhibitors, including romidepsin (ROM), enhanced the activity of a key component of iALL therapy, cytarabine (ARAC) in vitro and combined administration of ROM and ARAC to xenografted mice further reduced leukemia burden. Molecular studies showed that ROM reduces expression of cytidine deaminase, an enzyme involved in ARAC deactivation, and enhances the DNA damage-response to ARAC. In conclusion, we present a valuable resource for drug discovery, including the first systematic analysis of transcriptome reproducibility in vitro, and have identified ROM as a promising therapeutic for MLL-rearranged iALL.

ViPAR: a software platform for the Virtual Pooling and Analysis of Research Data.

BACKGROUND: Research studies exploring the determinants of disease require sufficient statistical power to detect meaningful effects. Sample size is often increased through centralized pooling of disparately located datasets, though ethical, privacy and data ownership issues can often hamper this process. Methods that facilitate the sharing of research data that are sympathetic with these issues and which allow flexible and detailed statistical analyses are therefore in critical need. We have created a software platform for the Virtual Pooling and Analysis of Research data (ViPAR), which employs free and open source methods to provide researchers with a web-based platform to analyse datasets housed in disparate locations. METHODS: Database federation permits controlled access to remotely located datasets from a central location. The Secure Shell protocol allows data to be securely exchanged between devices over an insecure network. ViPAR combines these free technologies into a solution that facilitates 'virtual pooling' where data can be temporarily pooled into computer memory and made available for analysis without the need for permanent central storage. RESULTS: Within the ViPAR infrastructure, remote sites manage their own harmonized research dataset in a database hosted at their site, while a central server hosts the data federation component and a secure analysis portal. When an analysis is initiated, requested data are retrieved from each remote site and virtually pooled at the central site. The data are then analysed by statistical software and, on completion, results of the analysis are returned to the user and the virtually pooled data are removed from memory. CONCLUSIONS: ViPAR is a secure, flexible and powerful analysis platform built on open source technology that is currently in use by large international consortia, and is made publicly available at [http://bioinformatics.childhealthresearch.org.au/software/vipar/].

Novel BRD4-NUT fusion isoforms increase the pathogenic complexity in NUT midline carcinoma.

Nuclear protein in testis (NUT)-midline carcinoma (NMC) is a rare, aggressive disease typically presenting with a single t(15;19) translocation that results in the generation of a bromodomain-containing protein 4 (BRD4)-NUT fusion. PER-624 is a cell line generated from an NMC patient with an unusually complex karyotype that gave no initial indication of the involvement of the NUT locus. Analysis of PER-624 next-generation transcriptome sequencing (RNA-Seq) using the algorithm FusionFinder identified a novel transcript in which Exon 15 of BRD4 was fused to Exon 2 of NUT, therefore differing from all published NMC fusion transcripts. The three additional exons contained in the PER-624 fusion encode a series of polyproline repeats, with one predicted to form a helix. In the NMC cell line PER-403, we identified the 'standard' NMC fusion and two novel isoforms. Knockdown by small interfering RNA in either cell line resulted in decreased proliferation, increased cell size and expression of cytokeratins consistent with epithelial differentiation. These data demonstrate that the novel BRD4-NUT fusion in PER-624 encodes a functional protein that is central to the oncogenic mechanism in these cells. Genomic PCR indicated that in both PER-624 and PER-403, the translocation fuses an intron of BRD4 to a region upstream of the NUT coding sequence. Thus, the generation of BRD4-NUT fusion transcripts through post-translocation RNA-splicing appears to be a common feature of these carcinomas that has not previously been appreciated, with the mechanism facilitating the expression of alternative isoforms of the fusion. Finally, ectopic expression of wild-type NUT, a protein normally restricted to the testis, could be demonstrated in PER-403, indicating additional pathways for aberrant cell signaling in NMC. This study contributes to our understanding of the genetic diversity of NMC, an important step towards finding therapeutic targets for a disease that is refractory to current treatments.

Promoter polymorphisms in two overlapping 6p25 genes implicate mitochondrial proteins in cognitive deficit in schizophrenia.

In a previous study, we detected a 6p25-p24 region linked to schizophrenia in families with high composite cognitive deficit (CD) scores, a quantitative trait integrating multiple cognitive measures. Association mapping of a 10 Mb interval identified a 260 kb region with a cluster of single-nucleotide polymorphisms (SNPs) significantly associated with CD scores and memory performance. The region contains two colocalising genes, LYRM4 and FARS2, both encoding mitochondrial proteins. The two tagging SNPs with strongest evidence of association were located around the overlapping putative promoters, with rs2224391 predicted to alter a transcription factor binding site (TFBS). Sequencing the promoter region identified 22 SNPs, many predicted to affect TFBSs, in a tight linkage disequilibrium block. Luciferase reporter assays confirmed promoter activity in the predicted promoter region, and demonstrated marked downregulation of expression in the LYRM4 direction under the haplotype comprising the minor alleles of promoter SNPs, which however is not driven by rs2224391. Experimental evidence from LYRM4 expression in lymphoblasts, gel-shift assays and modelling of DNA breathing dynamics pointed to two adjacent promoter SNPs, rs7752203-rs4141761, as the functional variants affecting expression. Their C-G alleles were associated with higher transcriptional activity and preferential binding of nuclear proteins, whereas the G-A combination had opposite effects and was associated with poor memory and high CD scores. LYRM4 is a eukaryote-specific component of the mitochondrial biogenesis of Fe-S clusters, essential cofactors in multiple processes, including oxidative phosphorylation. LYRM4 downregulation may be one of the mechanisms involved in inefficient oxidative phosphorylation and oxidative stress, increasingly recognised as contributors to schizophrenia pathogenesis.

Significant association between common polymorphisms in the aromatase gene CYP19A1 and bone mineral density in postmenopausal women.

17ß-Estradiol is important in maintaining bone structure, and regulation of its synthesis plays an important role in the development of postmenopausal osteoporosis. We and others have demonstrated associations between variation in the CYP19A1 gene (encoding aromatase) and areal bone mineral density (aBMD) phenotypes in women. In the present study 33 tag polymorphisms were genotyped across the CYP19A1 gene in a population of 1,185 Caucasian postmenopausal women to test the association between sequence variations, total DXA hip aBMD, and circulating 17ß-estradiol levels. An in silico bioinformatics analysis was performed for single nucleotide polymorphisms (SNPs) associated with aBMD to identify putative functional effects, while linkage disequilibrium analysis of these SNPs was undertaken with previously published sequence variants. Five SNPs located in the central third of the gene were strongly associated with total-hip aBMD after adjustment for age (P = 0.006-0.013). A haplotype analysis of these five SNPs revealed an association between the haplotype C-G-G-G-C and increased aBMD (P = 0.008) and the haplotype A-A-A-A-A and a decreased aBMD (P = 0.021). The haplotype frequency was 9.0% for C-G-G-G-C and 15.4% for A-A-A-A-A, with the variation in mean total-hip aBMD explained by the haplotype analyses being 5% and 7%, respectively. None of these polymorphisms was significantly associated with circulating 17ß-estradiol levels. In conclusion, common genetic variations within the CYP19A1 gene are significantly associated with aBMD in postmenopausal Caucasian women.

Combined analysis of three whole genome linkage scans for Ankylosing Spondylitis.

OBJECTIVE: Ankylosing spondylitis (AS) is a debilitating chronic inflammatory condition with a high degree of familiality (lambda(s) = 82) and heritability (>90%) that primarily affects spinal and sacroiliac joints. Whole genome scans for linkage to AS phenotypes have been conducted, although results have been inconsistent between studies and all have had modest sample sizes. One potential solution to these issues is to combine data from multiple studies in a retrospective meta-analysis. METHODS: The International Genetics of Ankylosing Spondylitis Consortium combined data from three whole genome linkage scans for AS (n = 3744 subjects) to determine chromosomal markers that show evidence of linkage with disease. Linkage markers typed in different centres were integrated into a consensus map to facilitate effective data pooling. We performed a weighted meta-analysis to combine the linkage results, and compared them with the three individual scans and a combined pooled scan. RESULTS: In addition to the expected region surrounding the HLA-B27 gene on chromosome 6, we determined that several marker regions showed significant evidence of linkage with disease status. Regions on chromosome 10q and 16q achieved 'suggestive' evidence of linkage, and regions on chromosomes 1q, 3q, 5q, 6q, 9q, 17q and 19q showed at least nominal linkage in two or more scans and in the weighted meta-analysis. Regions previously associated with AS on chromosome 2q (the IL-1 gene cluster) and 22q (CYP2D6) exhibited nominal linkage in the meta-analysis, providing further statistical support for their involvement in susceptibility to AS. CONCLUSION: These findings provide a useful guide for future studies aiming to identify the genes involved in this highly heritable condition.

The C-480T hepatic lipase polymorphism is associated with HDL-C but not with risk of coronary heart disease.

High-density lipoprotein cholesterol (HDL-C) is a known predictor of coronary heart disease (CHD). Studies have shown that the C-480T polymorphism of the hepatic lipase (HL) gene is predictive of HDL-C; however, its observed relationship with the risk of CHD has been inconsistent. We analysed four biallelic polymorphisms in the HL gene in participants from three independent Western Australian populations. Samples were collected from two cross-sectional studies of 1111 and 4822 community-based subjects assessed for cardiovascular risk factors, and a third sample of 556 subjects with physician-diagnosed CHD. Genotypes were tested for association with plasma lipids and the risk of CHD. All polymorphisms were highly correlated (D' > 0.97, r(2) > 0.90); therefore, only C-480T was analysed. The -480T allele was significantly associated with an increase in HDL-C of between 0.08 and 0.16 mmol/l in all three populations (p < 0.001). No associations with other lipids were observed, nor was an association with CHD in a case-control study of males. The TT genotype was however associated with decreased risk of myocardial infarction among cases (odds ratio = 0.39, 95% confidence interval = 0.19-0.78, p = 0.008). These findings replicate those of previous studies in three independent populations and suggest that the genetic determinants of CHD are complex and cannot be entirely explained through intermediate phenotypes.