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Fusion genes in breast cancer
Fusion genes caused by chromosomal rearrangements are a common and important feature in
haematological malignancies, but have until recently been seen as unimportant in epithelial
cancers. The discovery of recurrent fusion genes in prostate and lung cancer suggests that
fusion genes may play an important role in epithelial carcinogenesis, and that they have been
previously under-reported due to the difficulties of cytogenetic analysis of solid tumours. In
particular, breast cancers often have complex, highly rearranged karyotypes which have proved
difficult to analyse using classical cytogenetic techniques.
The aim of this project was to search for fusion genes in breast cancer by using high-resolution
mapping of chromosome rearrangements in breast cancer cell lines. Mapping the chromosome
rearrangements was initially done using high-resolution DNA microarrays and fluorescence in-
situ hybridisation, but moved to high-throughput sequencing as it became available. Interesting
candidate genes identified from the mapped chromosome rearrangements were investigated
on a larger set of cell lines and primary tumours.
The complete karyotypes of two breast cancer cell lines were constructed using a combination
of microarrays, fluorescence microscopy, and high-throughput sequencing. A number of
potential fusion genes were identified in these two cell lines. Although no expressed fusion
genes were found, the complete karyotypes gave insight into the number and mechanisms of
chromosome rearrangement in breast cancer, and identified interesting candidate genes which
may be of importance in tumourigenesis. Two genes which were fused in other breast cancer
cell lines, BCAS3 and ODZ4, were disrupted by chromosome rearrangements and identified as
interesting candidate genes in tumorigenesis.
A bioinformatic pipeline to process high-throughput sequencing data was set up and validated,
and shown to more accurately predict fusion genes than other methods, and can be used to
investigate further cell lines and tumours for recurrent fusion genes. The pipeline was used to
analyse data from 3 other breast cancer cell lines and predict chromosomal rearrangements
and fusion genes, several of which were found to be expressed. Of the fusions predicted in the
cell line ZR-75-30, 7 expressed fusion genes were identified, and may have functional
significance in breast cancer.This work was supported by a grant from Breast Cancer Campaign
PML nuclear bodies and the spatial analysis of interphase mammalian cell nuclear architecture
Promyelocytic leukaemia nuclear bodies (PML NBs) are found within the nucleus of
mammalian cells. Numbering between 10 and 30 per nucleus, they are an obvious
feature of the nuclear landscape, yet their functions have still to be unambiguously
defined. In the mammalian nucleus, compartmentalization of functions is apparent, as
reflected in the wide-range of other nuclear compartments that can be identified.
Quantification of relationships between PML NBs and other nuclear functional
compartments is essential for a complete understanding of PML NB function. Initially,
PML size, number, distance relationships, and spatial organisation in relation to each
other, and the nuclear boundary and centroid, under the spatial point pattern theory
hypothesis of Complete Spatial Randomness (CSR), were investigated in both normal
and SV40 transformed MRC5 and WI38 human foetal lung fibroblasts. This was also
completed in normal MRC5 cells treated with heat shock, and interferon β (both of
which alter PML NB morphometrics), and also serum starvation. PML NBs appeared to
locate according to CSR with respect to each other, and inter – PML distances were
dependent upon median PML NB number per nucleus. PML NBs did not tend to
associate with the nuclear centroid, and were repelled from the nuclear boundary in all
cell lines and conditions. The distance and spatial organisation relationships between
PML NBs and eleven different nuclear compartments were also compared and
contrasted in the cell lines and conditions mentioned previously. PML NBs were shown
to share strong distance and spatial organisation relationships with the 11S
immunoproteasome regulator, SC35 domains,
and transcriptional compartments in
normal asynchronous nuclei, and with telomeres in transformed cells, highlighting likely
functions for the bodies. Lastly, the three dimensional spatial preference of functional
compartments in the nucleus was determined using an aggregate map, which provided a
novel means to visualise the nuclear location of functional compartments in relation to
each other, and under different cellular conditions. Spatial preference fell into four
categories: 1) diffuse, 2) annular, 3) core, and 4) polar. Nucleoli and RNA polymerase
maintained their spatial preference across cell lines and conditions, whereas other
compartments showed altered spatial preferences. Interestingly, viral transformation led
to global disorganisation of the nucleus, where most compartments (including PML
NBs) reverted to a diffuse spatial preference
Harnessing genomics in the battle against antimicrobial resistance and neglected tropical diseases.
Task-related default mode network modulation and inhibitory control in ADHD: effects of motivation and methylphenidate
Background: Deficits characteristic of Attention Deficit/Hyperactivity Disorder (ADHD), including poor attention and inhibitory control, are at least partially alleviated by factors that increase engagement of attention, suggesting a hypodopaminergic reward deficit. Lapses of attention are associated with attenuated deactivation of the Default Mode Network (DMN), a distributed brain system normally deactivated during tasks requiring attention to the external world. Task-related DMN deactivation has been shown to be attenuated in ADHD relative to controls. We hypothesised that motivational incentives to balance speed against restraint would increase task engagement during an inhibitory control task, enhancing DMN deactivation in ADHD. We also hypothesised that methylphenidate, an indirect dopamine agonist, would tend to normalise abnormal patterns of DMN deactivation.
Method: We obtained functional magnetic resonance images from eighteen methylphenidate-responsive children with ADHD (DSM-IV combined subtype) and 18 pairwise-matched typically developing children aged 9-15 years while they performed a paced Go/No-go task. We manipulated motivational incentive to balance response speed against inhibitory control, and tested children with ADHD both on and off methylphenidate.
Results: When children with ADHD were off-methylphenidate and task incentive was low, event-related DMN deactivation was significantly attenuated compared to controls, but the two groups did not differ under high motivational incentives. The modulation of DMN deactivation by incentive in the children with ADHD, off- methylphenidate, was statistically significant, and significantly greater than in typically developing children. When children with ADHD were on-methylphenidate, motivational modulation of event-related DMN deactivation was abolished, and no attenuation relative to their typically developing peers was apparent in either motivational condition.
Conclusions: During an inhibitory control task, children with ADHD exhibit a raised motivational threshold at which task-relevant stimuli become sufficiently salient to deactivate the DMN. Treatment with methylphenidate normalises this threshold, rendering their pattern of task-related DMN deactivation indistinguishable from that of typically developing children
Insights into Platypus Population Structure and History from Whole-Genome Sequencing
The platypus is an egg-laying mammal which, alongside the echidna, occupies a unique place in the mammalian phylogenetic tree. Despite widespread interest in its unusual biology, little is known about its population structure or recent evolutionary history. To provide new insights into the dispersal and demographic history of this iconic species, we sequenced the genomes of 57 platypuses from across the whole species range in eastern mainland Australia and Tasmania. Using a highly improved reference genome, we called over 6.7 M SNPs, providing an informative genetic data set for population analyses. Our results show very strong population structure in the platypus, with our sampling locations corresponding to discrete groupings between which there is no evidence for recent gene flow. Genome-wide data allowed us to establish that 28 of the 57 sampled individuals had at least a third-degree relative among other samples from the same river, often taken at different times. Taking advantage of a sampled family quartet, we estimated the de novo mutation rate in the platypus at 7.0 × 10−9/bp/generation (95% CI 4.1 × 10−9–1.2 × 10−8/bp/generation). We estimated effective population sizes of ancestral populations and haplotype sharing between current groupings, and found evidence for bottlenecks and long-term population decline in multiple regions, and early divergence between populations in different regions. This study demonstrates the power of whole-genome sequencing for studying natural populations of an evolutionarily important species.We thank the
High-Throughput Genomics Group at the Wellcome Centre
for Human Genetics (funded by Wellcome Trust grant reference
090532/Z/09/Z) for the generation of sequencing data.
This work was supported by a Wellcome Trust Core Award
(090532/Z/09/Z) to P.D. and by a University of Sydney StartUp
Research grant to J.G
Reduced Prefrontal Gyrification in Carriers of the Dopamine D4 Receptor 7-Repeat Allele With Attention Deficit/Hyperactivity Disorder: A Preliminary Report
Objective: Structural and functional abnormalities have been noted in the prefrontal cortex of individuals with neurodevelopmental disorders such as attention deficit/hyperactivity disorder (ADHD). Cortical thickness and gyrification, both of which have been reported as abnormal in the prefrontal cortex in ADHD, are thought to be modulated by genetic influences during neural development. This study aimed to investigate the effects of a polymorphism of the dopamine DRD4 gene (the 7-repeat (7R) “risk” allele) on thickness and gyrification as distinct parameters of prefrontal cortical structure in children with ADHD.Method: Structural images and genetic samples were obtained from 49 children aged 9–15 years (25 with ADHD and 24 matched controls), and measures of cortical thickness and gyrification for inferior, middle, and superior frontal cortex were calculated.Results: A significant interaction between diagnosis and genotype on prefrontal gyrification was observed, largely driven by reduced inferior frontal gyrification in patients who carried the DRD4 7R allele. Furthermore, inferior frontal gyrification—but not thickness—related to everyday executive functioning in 7R allele carriers across groups.Conclusions: Prefrontal gyrification is reduced in children with ADHD who also carry the DRD4 7R allele, and it relates to critical functional skills in the executive domain in carriers of the risk allele. More broadly, these effects highlight the importance of considering precise neurodevelopmental mechanisms through which risk alleles influence cortical neurogenesis and migration
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