ORCID Profile
0000-0001-5216-8815
Current Organisation
Garvan Institute of Medical Research
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Publisher: BMJ
Date: 06-2001
DOI: 10.1136/JMG.38.6.381
Abstract: Autosomal dominant drusen is of particular interest because of its phenotypic similarity to age related macular degeneration. Currently, mutation R345W of EFEMP1 and, in a single pedigree, linkage to chromosome 6q14 have been causally related to the disease. We proposed to investigate and quantify the roles of EFEMP1 and the 6q14 locus in dominant drusen patients from the UK and USA. Molecular genetic analysis. Ten unrelated families and 17 young drusen patients. Exons 1 and 2 of EFEMP1 were characterised by 5′ rapid lification of cDNA ends and direct sequencing. Exons 1-12 of EFEMP1 were then investigated for mutation by direct sequencing. A Hpa II restriction digest test was constructed to detect the EFEMP1 R345W mutation. Marker loci spanning the two dominant drusen linked loci were used to generate haplotype data. Only seven of the 10 families (70%) and one of the 17 sporadic patients (6%) had the R345W mutation. The Hpa II restriction digest test was found to be a reliable and quick method for detecting this. No other exonic or splice site mutation was identified. Of the three families without EFEMP1 mutation, two were linked to the 2p16 region. EFEMP1 R345W accounts for only a proportion of the dominant drusen phenotype. Importantly, other families linked to chromosome 2p16 raise the possibility of EFEMP1 promoter sequence mutation or a second dominant drusen gene at this locus. Preliminary haplotype data suggest that the disease gene at the 6q14 locus is responsible for only a minority of dominant drusen cases.
Publisher: Elsevier BV
Date: 02-2018
DOI: 10.1016/J.CELS.2017.12.005
Abstract: The human transcriptome is so large, erse, and dynamic that, even after a decade of investigation by RNA sequencing (RNA-seq), we have yet to resolve its true dimensions. RNA-seq suffers from an expression-dependent bias that impedes characterization of low-abundance transcripts. We performed targeted single-molecule and short-read RNA-seq to survey the transcriptional landscape of a single human chromosome (Hsa21) at unprecedented resolution. Our analysis reaches the lower limits of the transcriptome, identifying a fundamental distinction between protein-coding and noncoding gene content: almost every noncoding exon undergoes alternative splicing, producing a seemingly limitless variety of isoforms. Analysis of syntenic regions of the mouse genome shows that few noncoding exons are shared between human and mouse, yet human splicing profiles are recapitulated on Hsa21 in mouse cells, indicative of regulation by a deeply conserved splicing code. We propose that noncoding exons are functionally modular, with alternative splicing generating an enormous repertoire of potential regulatory RNAs and a rich transcriptional reservoir for gene evolution.
Publisher: Elsevier BV
Date: 03-2020
Publisher: Wiley
Date: 15-05-2019
DOI: 10.1002/PROS.23823
Abstract: The androgen‐regulated gene TMPRSS2 to the ETS transcription factor gene ERG fusion is the most common genomic alteration acquired during prostate tumorigenesis and biased toward men of European ancestry. In contrast, African American men present with more advanced disease, yet their tumors are less likely to acquire TMPRSS2‐ERG . Data for Africa is scarce. RNA was made available for genomic analyses from 181 prostate tissue biopsy cores from Black South African men, 94 with and 87 without pathological evidence for prostate cancer. Reverse transcription polymerase chain reaction was used to screen for the TMPRSS2‐ERG fusion, while transcript junction coordinates and isoform frequencies, including novel gene fusions, were determined using targeted RNA sequencing. Here we report a frequency of 13% for TMPRSS2‐ERG in tumors from Black South Africans. Present in 12/94 positive versus 1/87 cancer negative prostate tissue cores, this suggests a 92.62% predictivity for a positive cancer diagnosis ( P = 0.0031). At a frequency of almost half that reported for African Americans and roughly a quarter of that reported for men of European ancestry, acquisition of TMPRSS2‐ERG appears to be inversely associated with aggressive prostate cancer. Further support was provided by linking the presence of TMPRSS2‐ERG to low‐grade disease in younger patients ( P = 0.0466), with higher expressing distal ERG fusion junction coordinates. Only the second study of its kind for the African continent, we support a link between TMPRSS2‐ERG status and prostate cancer racial health disparity beyond the borders of the United States. We call for urgent evaluation of androgen deprivation therapy within Africa.
Publisher: Elsevier BV
Date: 2022
DOI: 10.1016/J.GIM.2021.09.001
Abstract: Genetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy). A total of 74 families with erse monogenic conditions (31% prenatal-congenital onset, 47% early childhood, and 22% teenage-adult onset) were triaged into PCR-based RNA testing, with comparative RNA sequencing for 19 cases. Informative RNA assay data were obtained for 96% of cases, enabling variant reclassification for 75% variants that can be used for genetic counseling (71%), to inform clinical care (32%) and prenatal counseling (41%). Variant-associated mis-splicing was highly reproducible for 28 cases with s les from ≥2 affected in iduals or heterozygotes and 10 cases with ≥2 biospecimens. PCR licons encompassing another segregated heterozygous variant was vital for clinical interpretation of 22 of 79 variants to phase RNA splicing events and discern complete from partial mis-splicing. RNA diagnostics enabled provision of a genetic diagnosis for 64% of recruited cases. PCR-based RNA diagnostics has capacity to analyze 81.3% of clinically significant genes, with long licons providing an advantage over RNA sequencing to phase RNA splicing events. The Australasian Consortium for RNA Diagnostics (SpliceACORD) provide clinically-endorsed, standardized protocols and recommendations for interpreting RNA assay data.
Publisher: Springer Science and Business Media LLC
Date: 16-04-2021
DOI: 10.1186/S13059-021-02315-0
Abstract: Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing. All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5–20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden. This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.
Publisher: Springer Science and Business Media LLC
Date: 08-08-2016
DOI: 10.1038/NMETH.3958
Abstract: RNA sequencing (RNA-seq) can be used to assemble spliced isoforms, quantify expressed genes and provide a global profile of the transcriptome. However, the size and ersity of the transcriptome, the wide dynamic range in gene expression and inherent technical biases confound RNA-seq analysis. We have developed a set of spike-in RNA standards, termed 'sequins' (sequencing spike-ins), that represent full-length spliced mRNA isoforms. Sequins have an entirely artificial sequence with no homology to natural reference genomes, but they align to gene loci encoded on an artificial in silico chromosome. The combination of multiple sequins across a range of concentrations emulates alternative splicing and differential gene expression, and it provides scaling factors for normalization between s les. We demonstrate the use of sequins in RNA-seq experiments to measure s le-specific biases and determine the limits of reliable transcript assembly and quantification in accompanying human RNA s les. In addition, we have designed a complementary set of sequins that represent fusion genes arising from rearrangements of the in silico chromosome to aid in cancer diagnosis. RNA sequins provide a qualitative and quantitative reference with which to navigate the complexity of the human transcriptome.
Publisher: Wiley
Date: 04-02-2019
DOI: 10.1002/DVDY.10
Abstract: The timing, location, and level of gene expression are crucial for normal organ development, because morphogenesis requires strict genetic control. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating gene expression level. Although miRNAs are known to be involved in many biological events, the role of miRNAs in organogenesis is not fully understood. Mammalian eyelids fuse and separate during development and growth. In mice, failure of this process results in the eye-open at birth (EOB) phenotype. It has been shown that conditional deletion of mesenchymal Dicer (an essential protein for miRNA processing Dicer miRNAs control eyelid development through Wnt. Developmental Dynamics 248:201-210, 2019. © 2019 Wiley Periodicals, Inc.
Publisher: Wiley
Date: 13-07-2016
DOI: 10.1002/DVDY.24427
Abstract: Non-gustatory filiform papillae play critical roles in helping to grip food, drawing food to the esophagus, cleaning the mouth, and spreading saliva. The molecular mechanisms of filiform tongue papillae development however are not fully understood. We found Ikkα and Irf6 expression in developing tongue epithelium, and describe here specific tongue abnormalities in mice with mutation of these genes, indicating a role for Ikkα and Irf6 in filiform papillae development. Ikkα and Irf6 mutant tongues showed ectopic vertical epithelium at the midline, while lateral sides of mutant tongues adhered to the oral mucosa. Both the ectopic median vertical epithelium and adhered epithelium exhibited the presence of filiform tongue papillae, whereas epithelium between the median vertical epithelium and adhered tongue showed a loss of filiform tongue papillae. Timing of filiform papillae development was found to be slightly different between the midline and lateral regions of the wild-type tongue. Filiform papillae thus develop through distinct molecular mechanisms between the regions of tongue dorsum in the medio-lateral axis, with some filiform papillae developing under the control of Ikkα and Irf6. Developmental Dynamics 245:937-946, 2016. © 2016 Wiley Periodicals, Inc.
Publisher: Springer Science and Business Media LLC
Date: 19-06-2019
DOI: 10.1038/S41596-019-0175-1
Abstract: Next-generation sequencing (NGS) has been widely adopted to identify genetic variants and investigate their association with disease. However, the analysis of sequencing data remains challenging because of the complexity of human genetic variation and confounding errors introduced during library preparation, sequencing and analysis. We have developed a set of synthetic DNA spike-ins-termed 'sequins' (sequencing spike-ins)-that are directly added to DNA s les before library preparation. Sequins can be used to measure technical biases and to act as internal quantitative and qualitative controls throughout the sequencing workflow. This step-by-step protocol explains the use of sequins for both whole-genome and targeted sequencing of the human genome. This includes instructions regarding the dilution and addition of sequins to human DNA s les, followed by the bioinformatic steps required to separate sequin- and s le-derived sequencing reads and to evaluate the diagnostic performance of the assay. These practical guidelines are accompanied by a broader discussion of the conceptual and statistical principles that underpin the design of sequin standards. This protocol is suitable for users with standard laboratory and bioinformatic experience. The laboratory steps require ~1-4 d and the bioinformatic steps (which can be performed with the provided ex le data files) take an additional day.
Publisher: BMJ
Date: 19-03-2014
Publisher: American Association for the Advancement of Science (AAAS)
Date: 02-06-2017
Abstract: Alternative splicing in chromatin-modifying genes is associated with temperature-dependent sex in ergent reptile lineages.
Publisher: Wiley
Date: 16-12-2015
DOI: 10.1002/MC.22441
Publisher: The Company of Biologists
Date: 15-03-2009
DOI: 10.1242/DEV.027979
Abstract: Primary cilia mediate Hh signalling and mutations in their protein components affect Hh activity. We show that in mice mutant for a cilia intraflagellar transport (IFT) protein, IFT88 olaris, Shh activity is increased in the toothless diastema mesenchyme of the embryonic jaw primordia. This results in the formation of ectopic teeth in the diastema, mesial to the first molars. This phenotype is specific to loss of polaris activity in the mesenchyme since loss of Polaris in the epithelium has no detrimental affect on tooth development. To further confirm that upregulation of Shh activity is responsible for the ectopic tooth formation, we analysed mice mutant for Gas1, a Shh protein antagonist in diastema mesenchyme. Gas1mutants also had ectopic diastema teeth and accompanying increased Shh activity. In this context, therefore, primary cilia exert a specific negative regulatory effect on Shh activity that functions to repress tooth formation and thus determine tooth number. Strikingly, the ectopic teeth adopt a size and shape characteristic of premolars, a tooth type that was lost in mice around 50-100 million years ago.
Publisher: Elsevier BV
Date: 06-2023
Publisher: Springer Science and Business Media LLC
Date: 04-07-0012
DOI: 10.1038/S41467-020-17445-5
Abstract: Standard units of measurement are required for the quantitative description of nature however, few standard units have been established for genomics to date. Here, we have developed a synthetic DNA ladder that defines a quantitative standard unit that can measure DNA sequence abundance within a next-generation sequencing library. The ladder can be spiked into a DNA s le, and act as an internal scale that measures quantitative genetics features. Unlike previous spike-ins, the ladder is encoded within a single molecule, and can be equivalently and independently synthesized by different laboratories. We show how the ladder can measure erse quantitative features, including human genetic variation and microbial abundance, and also estimate uncertainty due to technical variation and improve normalization between libraries. This ladder provides an independent quantitative unit that can be used with any organism, application or technology, thereby providing a common metric by which genomes can be measured.
Publisher: Elsevier BV
Date: 06-2012
Publisher: Elsevier BV
Date: 02-2005
DOI: 10.1016/J.ARCHORALBIO.2004.11.019
Abstract: Hypohidrotic ectodermal dysplasia (HED) is a congenital disorder affecting organs of ectodermal origin including teeth, hair and sweat glands. Defects in Ectodysplasin (tabby), Edar (downless) and Edar associated death domain (Edaradd) (crinkled) cause HED in both humans and mice. Ectodysplasin is a tumour necrosis factor (TNF) superfamily member whose downstream signalling is transduced by the inhibitor of kappaB kinase (IKK) complex and inhibitors of kappaB (IkappaB) to activate the transcription factor NFkappaB. NFkappaB signalling is involved in a wide range of cellular processes and at each stage the different family members must be tightly regulated for each function. Recent data have demonstrated the importance of this signalling pathway in odontogenesis, particularly in the formation of cusps. Here we review recent advances in our understanding of Ectodysplasin/NFkappaB signalling in tooth development and in particular the central role of the IKK complex.
Publisher: Wiley
Date: 19-04-2020
Publisher: Proceedings of the National Academy of Sciences
Date: 29-12-2009
Abstract: Changes in tooth shape have played a major role in vertebrate evolution with modification of dentition allowing an organism to adapt to new feeding strategies. The current view is that molar teeth evolved from simple conical teeth, similar to canines, by progressive addition of extra “cones” to form progressively complex multicuspid crowns. Mammalian incisors, however, are neither conical nor multicuspid, and their evolution is unclear. We show that hypomorphic mutation of a cell surface receptor, Lrp4, which modulates multiple signaling pathways, produces incisors with grooved enamel surfaces that exhibit the same molecular characteristics as the tips of molar cusps. Mice with a null mutation of Lrp4 develop extra cusps on molars and have incisors that exhibit clear molar-like cusp and root morphologies. Molecular analysis identifies misregulation of Shh and Bmp signaling in the mutant incisors and suggests an uncoupling of the processes of tooth shape determination and morphogenesis. Incisors thus possess a developmentally suppressed, cuspid crown-like morphogenesis program similar to that in molars that is revealed by loss of Lrp4 activity. Several mammalian species naturally possess multicuspid incisors, suggesting that mammals have the capacity to form multicuspid teeth regardless of location in the oral jaw. Localized loss of enamel may thus have been an intermediary step in the evolution of cusps, both of which use Lrp4 -mediated signaling.
Publisher: SAGE Publications
Date: 06-11-2014
Abstract: Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKβ, an essential component of the NF-κB pathway, under keratin 5 promoter ( K5-Ikkβ). K5-Ikkβ mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkβ mice. The supernumerary incisors in K5-Ikkβ mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions.
Publisher: Association for Research in Vision and Ophthalmology (ARVO)
Date: 11-2003
DOI: 10.1167/IOVS.03-0112
Abstract: To determine the important transcriptional control elements and sites of expression of fibulin-3 in mammalian retina. Sequencing and 5' rapid lification of cDNA ends (RACE) were undertaken to characterize the genomic sequence upstream of the FBLN3 coding sequence. Reporter deletion-mutation constructs were used in luciferase transfection assays to determine the important regulatory motifs. Fibulin-3 expression in mouse and human retina was studied by in situ hybridization and RT-PCR. The effect of 17beta-estradiol on fibulin-3 production was studied in COS-7 and ARPE-19 cells. Two untranslated exons were fully sequenced completing the characterization of FBLN3 that comprises 12 exons. Reporter assays suggest that the FBLN3 proximal promoter is contained within 425 bp upstream of exon 1. Important regulatory elements include three Sp1-binding sites, a Tant motif (trans-activating) and an estrogen response element (ERE) binding site (trans-repressing). No TATA or CAAT regulatory boxes were identified. RT-PCR suggests that the fibulin-3 gene is expressed in murine and human RPE, and in situ studies confirm that Fbln3 is expressed in the outer and inner nuclear layers, but strikingly not in the ganglion cell layer. Fibulin-3 expression in ARPE-19 cells could be modified by varying the amount of estrogen in the cell culture medium. The 5' end of the FBLN3 gene has been characterized, and the important upstream motifs regulating its transcription have been identified. Fibulin-3 is expressed in adult retina and at early stages in human and mouse development. Estrogen may be an important regulator of fibulin-3 expression, and this highlights a novel mechanism by which circulating estrogen may control the composition of the retinal extracellular matrix.
Publisher: Springer Science and Business Media LLC
Date: 10-08-2022
DOI: 10.1038/S41586-022-05054-9
Abstract: The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago
Publisher: Wiley
Date: 09-07-2020
DOI: 10.1111/ODI.13384
Publisher: Springer Science and Business Media LLC
Date: 20-12-2117
Publisher: Springer Science and Business Media LLC
Date: 22-03-2019
DOI: 10.1038/S41467-019-09272-0
Abstract: Chirality is a property describing any object that is inequivalent to its mirror image. Due to its 5′–3′ directionality, a DNA sequence is distinct from a mirrored sequence arranged in reverse nucleotide-order, and is therefore chiral. A given sequence and its opposing chiral partner sequence share many properties, such as nucleotide composition and sequence entropy. Here we demonstrate that chiral DNA sequence pairs also perform equivalently during molecular and bioinformatic techniques that underpin genetic analysis, including PCR lification, hybridization, whole-genome, target-enriched and nanopore sequencing, sequence alignment and variant detection. Given these shared properties, synthetic DNA sequences mirroring clinically relevant or analytically challenging regions of the human genome are ideal controls for clinical genomics. The addition of synthetic chiral sequences (sequins) to patient tumor s les can prevent false-positive and false-negative mutation detection to improve diagnosis. Accordingly, we propose that sequins can fulfill the need for commutable internal controls in precision medicine.
Publisher: Cold Spring Harbor Laboratory
Date: 04-02-2021
DOI: 10.1101/2021.02.03.429474
Abstract: How temperature determines sex remains unknown. A recent hypothesis proposes that conserved cellular mechanisms (calcium and redox ‘CaRe’ status) sense temperature and identify genes and regulatory pathways likely to be involved in driving sexual development. We take advantage of the unique sex determining system of the model organism, Pogona vitticeps , to assess predictions of this hypothesis. P. vitticeps has ZZ male: ZW female sex chromosomes whose influence can be overridden in genetic males by high temperatures, causing male-to-female sex reversal. We compare a developmental transcriptome series of ZWf females and temperature sex reversed ZZf females. We demonstrate that early developmental cascades differ dramatically between genetically driven and thermally driven females, later converging to produce a common outcome (ovaries). We show that genes proposed as regulators of thermosensitive sex determination play a role in temperature sex reversal. Our study greatly advances the search for the mechanisms by which temperature determines sex. In many reptiles and fish, environment can determine, or influence, the sex of developing embryos. How this happens at a molecular level that has eluded resolution for half a century of intensive research. We studied the bearded dragon, a lizard that has sex chromosomes (ZZ male and ZW female), but in which that temperature can override ZZ sex chromosomes to cause male to female sex reversal. This provides an unparalleled opportunity to disentangle, in the same species, the biochemical pathways required to make a female by these two different routes. We sequenced the transcriptomes of gonads from developing ZZ reversed and normal ZW dragon embryos and discovered that different sets of genes are active in ovary development driven by genotype or temperature. Females whose sex was initiated by temperature showed a transcriptional profile consistent with the recently-proposed Calcium-Redox hypotheses of cellular temperature sensing. These findings are an important for understanding how the environment influences the development of sex, and more generally how the environment can epigenetically modify the action of genes.
Publisher: Elsevier BV
Date: 10-2020
Publisher: Springer Science and Business Media LLC
Date: 08-04-2020
DOI: 10.1038/S41467-020-15697-9
Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Publisher: Elsevier BV
Date: 05-2012
Publisher: MDPI AG
Date: 02-02-2021
DOI: 10.3390/NU13020490
Abstract: Creatine metabolism is an important component of cellular energy homeostasis. Via the creatine kinase circuit, creatine derived from our diet or synthesized endogenously provides spatial and temporal maintenance of intracellular adenosine triphosphate (ATP) production this is particularly important for cells with high or fluctuating energy demands. The use of this circuit by tissues within the female reproductive system, as well as the placenta and the developing fetus during pregnancy is apparent throughout the literature, with some studies linking perturbations in creatine metabolism to reduced fertility and poor pregnancy outcomes. Maternal dietary creatine supplementation during pregnancy as a safeguard against hypoxia-induced perinatal injury, particularly that of the brain, has also been widely studied in pre-clinical in vitro and small animal models. However, there is still no consensus on whether creatine is essential for successful reproduction. This review consolidates the available literature on creatine metabolism in female reproduction, pregnancy and the early neonatal period. Creatine metabolism is discussed in relation to cellular bioenergetics and de novo synthesis, as well as the potential to use dietary creatine in a reproductive setting. We highlight the apparent knowledge gaps and the research “road forward” to understand, and then utilize, creatine to improve reproductive health and perinatal outcomes.
Publisher: Springer Science and Business Media LLC
Date: 27-03-2019
DOI: 10.1038/S41467-019-09374-9
Abstract: Fusion genes are a major cause of cancer. Their rapid and accurate diagnosis can inform clinical action, but current molecular diagnostic assays are restricted in resolution and throughput. Here, we show that targeted RNA sequencing (RNAseq) can overcome these limitations. First, we establish that fusion gene detection with targeted RNAseq is both sensitive and quantitative by optimising laboratory and bioinformatic variables using spike-in standards and cell lines. Next, we analyse a clinical patient cohort and improve the overall fusion gene diagnostic rate from 63% with conventional approaches to 76% with targeted RNAseq while demonstrating high concordance for patient s les with previous diagnoses. Finally, we show that targeted RNAseq offers additional advantages by simultaneously measuring gene expression levels and profiling the immune-receptor repertoire. We anticipate that targeted RNAseq will improve clinical fusion gene detection, and its increasing use will provide a deeper understanding of fusion gene biology.
Publisher: Public Library of Science (PLoS)
Date: 20-09-2018
Publisher: Public Library of Science (PLoS)
Date: 13-04-2023
DOI: 10.1371/JOURNAL.PONE.0284327
Abstract: Intragenic CpG dinucleotides are tightly conserved in evolution yet are also vulnerable to methylation-dependent mutation, raising the question as to why these functionally critical sites have not been deselected by more stable coding sequences. We previously showed in cell lines that altered exonic CpG methylation can modify promoter start sites, and hence protein isoform expression, for the human TP53 tumor suppressor gene. Here we extend this work to the in vivo setting by testing whether synonymous germline modifications of exonic CpG sites affect murine development, fertility, longevity, or cancer incidence. We substituted the DNA-binding exons 5–8 of Trp53 , the mouse ortholog of human TP53 , with variant-CpG (either CpG-depleted or -enriched) sequences predicted to encode the normal p53 amino acid sequence a control construct was also created in which all non-CpG sites were synonymously substituted. Homozygous Trp53 -null mice were the only genotype to develop tumors. Mice with variant-CpG Trp53 sequences remained tumor-free, but were uniquely prone to dental anomalies causing jaw malocclusion (p .0001). Since the latter phenotype also characterises murine Rett syndrome due to dysfunction of the trans-repressive MeCP2 methyl-CpG-binding protein, we hypothesise that CpG sites may exert non-coding phenotypic effects via pre-translational cis-interactions of 5-methylcytosine with methyl-binding proteins which regulate mRNA transcript initiation, expression or splicing, although direct effects on mRNA structure or translation are also possible.
Publisher: Wiley
Date: 08-2006
DOI: 10.1111/J.1601-6343.2006.00365.X
Abstract: Understanding of apoptotic mechanisms involved in tissue shaping is of particular interest because of possible targeted modulation of the development of organ structures such as teeth. Research of CD 95 mediated apoptosis has been focused particularly on cell death in the immune system and related disorders. However, CD 95 mediated apoptosis is also involved in embryogenesis of many organs as the kidney, the lung, the intestine and tissue networks such as the nervous system. Narrative review. This review briefly summarizes the current knowledge of CD 95 mediated apoptosis in embryogenesis with possible implication in tooth development. CD 95 receptor and CD 95 ligand are found at early stages of tooth development. The data suggest some positive correlations with dental apoptosis distribution, particularly in the primary enamel knot where apoptosis occurs during elimination of this structure. CD 95 deficient (lpr) adult mouse tooth phenotype, however, did not show any alterations in final tooth pattern and morphology. To date studies of apoptotic machinery during tooth development show spatial localization of many of the components together with precise and localized timing of cell death. There is still much to be learned about the regulation and importance of apoptosis in tooth development. Nevertheless, the involvement of apoptotic regulatory mechanisms interplaying with other molecules participates to the cellular cross-talk in developing tissues, which opens possible targeted modulations as suggested, e.g. for future molecular dentistry.
Publisher: Springer Science and Business Media LLC
Date: 17-02-2019
DOI: 10.1038/S41467-019-11049-4
Abstract: High-throughput single-cell RNA sequencing is a powerful technique but only generates short reads from one end of a cDNA template, limiting the reconstruction of highly erse sequences such as antigen receptors. To overcome this limitation, we combined targeted capture and long-read sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single-cell libraries generated by droplet-based partitioning. We show that Repertoire and Gene Expression by Sequencing (RAGE-Seq) can generate accurate full-length antigen receptor sequences at nucleotide resolution, infer B-cell clonal evolution and identify alternatively spliced BCR transcripts. We apply RAGE-Seq to 7138 cells s led from the primary tumor and draining lymph node of a breast cancer patient to track transcriptome profiles of expanded lymphocyte clones across tissues. Our results demonstrate that RAGE-Seq is a powerful method for tracking the clonal evolution from large numbers of lymphocytes applicable to the study of immunity, autoimmunity and cancer.
Publisher: Frontiers Media SA
Date: 12-04-2019
Publisher: Public Library of Science (PLoS)
Date: 15-04-2021
DOI: 10.1371/JOURNAL.PGEN.1009465
Abstract: How temperature determines sex remains unknown. A recent hypothesis proposes that conserved cellular mechanisms (calcium and redox ‘CaRe’ status) sense temperature and identify genes and regulatory pathways likely to be involved in driving sexual development. We take advantage of the unique sex determining system of the model organism, Pogona vitticeps , to assess predictions of this hypothesis. P . vitticeps has ZZ male: ZW female sex chromosomes whose influence can be overridden in genetic males by high temperatures, causing male-to-female sex reversal. We compare a developmental transcriptome series of ZWf females and temperature sex reversed ZZf females. We demonstrate that early developmental cascades differ dramatically between genetically driven and thermally driven females, later converging to produce a common outcome (ovaries). We show that genes proposed as regulators of thermosensitive sex determination play a role in temperature sex reversal. Our study greatly advances the search for the mechanisms by which temperature determines sex.
Publisher: Elsevier BV
Date: 07-2016
DOI: 10.1016/J.GEP.2016.05.002
Abstract: Members of the Sox gene family play critical roles in many biological processes including organogenesis. We carried out comparative in situ hybridisation analysis of seventeen Sox genes (Sox1-14, 17, 18 and 21) during murine palatogenesis from initiation to fusion of the palatal shelves above the dorsal side of the tongue. At palatal shelf initiation (E12.5), the localized expression of six Sox genes (Sox2, 5, 6, 9, 12 and 13) was observed in the shelves, whereas Sox4 and Sox11 showed ubiquitious expression. During the down-growth of palatal shelves (E13.5), Sox4, Sox5, and Sox9 exhibited restricted expression to the interior side of the palatal shelves facing the tongue. Following elevation of the palatal shelves (E14.5), Sox2, Sox11 and Sox21 expression was present in the midline epithelial seam. We thus identify dynamic spatio-temporal expression of Sox gene family during the process of palatogenesis.
Publisher: Frontiers Media SA
Date: 05-04-2017
Publisher: American Society of Clinical Oncology (ASCO)
Date: 12-2023
DOI: 10.1200/PO.18.00297
Abstract: Before anaplastic lymphoma kinase (ALK) inhibitors, treatment options for ALK-positive inflammatory myofibroblastic tumors (AP-IMTs) were unsatisfactory. We retrospectively analyzed the outcome of patients with AP-IMT treated with crizotinib to document response, toxicity, survival, and features associated with relapse. The cohort comprised eight patients with AP-IMT treated with crizotinib and surgery. Outcome measures were progression-free and overall survival after commencing crizotinib, treatment-related toxicities, features associated with relapse, outcome after relapse, and outcome after ceasing crizotinib. The median follow-up after commencing crizotinib was 3 years (range, 0.9 to 5.5 years). The major toxicity was neutropenia. All patients responded to crizotinib. Five were able to discontinue therapy without recurrence (median treatment duration, 1 year range, 0.2 to 3.0 years) one continues on crizotinib. Two critically ill patients with initial complete response experienced relapse while on therapy. Both harbored RANBP2-ALK fusions and responded to alternative ALK inhibitors one ultimately died as a result of progressive disease, whereas the other remains alive on treatment. Progression-free and overall survival since commencement of crizotinib is 0.75 ± 0.15% and 0.83 ± 0.15%, respectively. We confirm acceptable toxicity and excellent disease control in patients with AP-IMT treated with crizotinib, which may be ceased without recurrence in most. Relapses occurred in two of three patients with RANBP2-ALK translocated IMT, which suggests that such patients require additional therapy.
Publisher: American Association for Cancer Research (AACR)
Date: 14-08-2017
DOI: 10.1158/0008-5472.CAN-16-2550
Abstract: Despite intensive multimodal treatment of sarcomas, a heterogeneous group of malignant tumors arising from connective tissue, survival remains poor. Candidate-based targeted treatments have demonstrated limited clinical success, urging an unbiased and comprehensive analysis of oncogenic signaling networks to reveal therapeutic targets and personalized treatment strategies. Here we applied mass spectrometry–based phosphoproteomic profiling to the largest and most heterogeneous set of sarcoma cell lines characterized to date and identified novel tyrosine phosphorylation patterns, enhanced tyrosine kinases in specific subtypes, and potential driver kinases. ALK was identified as a novel driver in the Aska-SS synovial sarcoma (SS) cell line via expression of an ALK variant with a large extracellular domain deletion (ALKΔ2–17). Functional ALK dependency was confirmed in vitro and in vivo with selective inhibitors. Importantly, ALK immunopositivity was detected in 6 of 43 (14%) of SS patient specimens, one of which exhibited an ALK rearrangement. High PDGFRα phosphorylation also characterized SS cell lines, which was accompanied by enhanced MET activation in Yamato-SS cells. Although Yamato-SS cells were sensitive to crizotinib (ALK/MET-inhibitor) but not pazopanib (VEGFR/PDGFR-inhibitor) monotherapy in vitro, synergistic effects were observed upon drug combination. In vivo, both drugs were in idually effective, with pazopanib efficacy likely attributable to reduced angiogenesis. MET or PDGFRα expression was detected in 58% and 84% of SS patients, respectively, with coexpression in 56%. Consequently, our integrated approach has led to the identification of ALK and MET as promising therapeutic targets in SS. Cancer Res 77(16) 4279–92. ©2017 AACR.
No related grants have been discovered for James Blackburn.