ABSTRACT The most integrated approach toward understanding the multiple molecular events and mech... more ABSTRACT The most integrated approach toward understanding the multiple molecular events and mechanisms by which cancer may develop is the application of gene expression profiling using microarray technologies. As molecular alterations in breast cancer are complex and involve cross-talk between multiple cellular signalling pathways, microarray technology provides a means of capturing and comparing the expression patterns of the entire genome across multiple samples in a high throughput manner. Since the development of microarray technologies, together with the advances in RNA extraction methodologies, gene expression studies have revolutionised the means by which genes suitable as targets for drug development and individualised cancer treatment can be identified. As of the mid-1990s, expression microarrays have been extensively applied to the study of cancer and no cancer type has seen as much genomic attention as breast cancer. The most abundant area of breast cancer genomics has been the clarification and interpretation of gene expression patterns that unite both biological and clinical aspects of tumours. It is hoped that one day molecular profiling will transform diagnosis and therapeutic selection in human breast cancer toward more individualised regimes. Here, we review a number of prominent microarray profiling studies focussed on human breast cancer and examine their strengths, their limitations, clinical implications including prognostic relevance and gene signature significance along with potential improvements for the next generation of microarray studies. Yes Yes
The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studi... more The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) 4 in culture and detail the accompanying genetic alterations that have been observed. 4 Abbreviations: human mammary epithelial cells (HMEC); human mammary fibroblasts (HMF); the catalytic subunit of telomerase (hTERT); senesence associated-β-galactosidase (SA-βgal); human papilloma virus (HPV).
The most integrated approach toward understanding the multiple molecular events and mechanisms by... more The most integrated approach toward understanding the multiple molecular events and mechanisms by which cancer may develop is the application of gene expression profiling using microarray technologies. As molecular alterations in breast cancer are complex and involve cross-talk between multiple cellular signalling pathways, microarray technology provides a means of capturing and comparing the expression patterns of the entire genome across multiple samples in a high throughput manner. Since the development of microarray technologies, together with the advances in RNA extraction methodologies, gene expression studies have revolutionised the means by which genes suitable as targets for drug development and individualised cancer treatment can be identified. As of the mid-1990s, expression microarrays have been extensively applied to the study of cancer and no cancer type has seen as much genomic attention as breast cancer. The most abundant area of breast cancer genomics has been the c...
A new strategy has emerged to improve healing of bone defects using exogenous glycosaminoglycans ... more A new strategy has emerged to improve healing of bone defects using exogenous glycosaminoglycans by increasing the effectiveness of bone-anabolic growth factors. Wnt ligands play an important role in bone formation. However, their functional interactions with heparan sulfate/heparin have only been investigated in non-osseous tissues. Our study now shows that the osteogenic activity of Wnt3a is cooperatively stimulated through physical interactions with exogenous heparin. N-Sulfation and to a lesser extent O-sulfation of heparin contribute to the physical binding and optimal co-stimulation of Wnt3a. Wnt3a-heparin signaling synergistically increases osteoblast differentiation with minimal effects on cell proliferation. Thus, heparin selectively reduces the effective dose of Wnt3a needed to elicit osteogenic, but not mitogenic responses. Mechanistically, Wnt3a-heparin signaling strongly activates the phosphoinositide 3-kinase/Akt pathway and requires the bone-related transcription fact...
Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradatio... more Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origen is difficult. We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell ...
Journal of mammary gland biology and neoplasia, 2001
The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studi... more The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed.
Senescence and genomic integrity are thought to be important barriers in the development of malig... more Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of...
Since microRNAs (miRNAs) act as translational regulators of multiple genes, single nucleotide pol... more Since microRNAs (miRNAs) act as translational regulators of multiple genes, single nucleotide polymorphisms (SNP) in them can have potentially wide-ranging effects. Using an association approach, this research examined the effects of the rs6505162 SNP, an A > C polymorphism located in the pre-miRNA region of miR-423, on breast cancer development. Caucasian Australian women with breast cancer and controls matched for age and ethnicity were genotyped for rs6505162 and their genotypic and allelic frequencies analysed for significant differences. Analysis indicated that there were significant differences between the case and control populations (χ2 = 6.70, p = 0.035), with the CC genotype conferring reduced risk of breast cancer development (odds ratio = 0.50 95% confidence interval = 0.27-0.92, p = 0.03). Further functional research is required to determine the mechanism of action of this SNP on miRNA function.
Breast cancer is the second most common cancer worldwide and the most common cancer reported in w... more Breast cancer is the second most common cancer worldwide and the most common cancer reported in women. This malignant tumour is characterised by a number of specific features including uncontrolled cell proliferation. It ranks fifth in the world as a cause of cancer death overall in developed countries and is the second most frequent cause of cancer death in women. Early diagnosis increases 5-year survival rates up to 95%. Heparan sulfate proteoglycans (HSPGs) are complex proteins composed of a core protein to which a number of highly sulfated side chains attach, ubiquitous to the cell surface and within the extracellular matrix. HSPG side chains are synthesised by a highly co-ordinated process resulting in distinct sulfation patterns, which determine specific interactions with cell-signalling partners including growth factors, their receptors, ligands and morphogens. The enzymes responsible for chain initiation, elongation and sulfation are critical for creating HS chain variability conferring biological functionality. This study investigated a single nucleotide polymorphism in SULF1, the enzyme responsible for the 6-O desulfation of heparan sulfate side chains. We investigated this SNP in an Australian Caucasian case-control breast cancer population and found a significant association between SULF1 and breast cancer at both the allelic and genotypic levels (allele, p=0.016; genotype, p=0.032). Our results suggest that the rs2623047 SNP in SULF1 may impact breast cancer susceptibility. Specifically, the T allele of rs2623047 in SULF1 is associated with an increased risk of developing breast cancer in our cohort. The identification of markers including SULF1 may improve detection of this disease at its earliest stages improving patient treatment and prognosis.
There is evidence that folate metabolism has a role in migraine pathophysiology, particularly in ... more There is evidence that folate metabolism has a role in migraine pathophysiology, particularly in the migraine with aura (MA) subtype. In this study, we investigate whether two non-synonymous single nucleotide polymorphisms (SNPs), rs1950902 (C401T; R134K) and rs2236225 (G1958A; R653Q), in MTHF dehydrogenase (MTHFD1) are associated with migraine in an Australian case-control population. Increased plasma levels of homocysteine, one of the metabolites produced in the folate pathway, has been found to be a risk factor for migraine. There is also a genetic link: a common polymorphism (rs1801133, C667T) that reduces the catalytic activity of the enzyme that catalyzes the formation of homocysteine, methylenetetrahydrofolate reductase (MTHFR), is associated with an increase in risk of MA. MTHFD1 is a crucial multifunctional enzyme that catalyzes three separate reactions of the folate pathway and therefore variants in MTHFD1 may also influence migraine susceptibility. The R134K and R653Q variants in MTHFD1 were genotyped in an Australian cohort of 520 unrelated migraineurs (162 were diagnosed with migraine without aura [MO] and 358 with MA) and 520 matched controls. Data were analyzed for association with migraine and for interaction with the MTHFR C667T polymorphism. We find no significant differences in genotype or allele frequencies for either SNP between migraineurs and controls, or when either MO or MA cases were compared with controls. In addition, these MTHFD1 polymorphisms did not appear to influence the risk of MA conferred by the MTHFR 667T allele. We find no evidence for association of the MTHFD1 R134K and R653Q polymorphisms with migraine in our Australian case-control population. However, as folate metabolism appears to be important in migraine, particularly with respect to the aura component, future studies using high throughput methods to expand the number of SNPs in folate-related genes genotyped and investigation of interactions between SNPs may be justified.
Computational epigenetics is a new area of research focused on exploring how DNA methylation patt... more Computational epigenetics is a new area of research focused on exploring how DNA methylation patterns affect transcription factor binding that affect gene expression patterns. The aim of this study was to produce a new protocol for the detection of DNA methylation patterns using computational analysis which can be further confirmed by bisulfite PCR with serial pyrosequencing. The upstream regulatory element and preinitiation complex relative to CpG islets within the methylenetetrahydrofolate reductase gene were determined via computational analysis and online databases. The 1,104 bp long CpG island located near to or at the alternative promoter site of methylenetetrahydrofolate reductase gene was identified. The CpG plot indicated that CpG islets A and B, within the island, contained 62 and 75 % GC content CpG ratios of 0.70 and 0.80-0.95, respectively. Further exploration of the CpG islets A and B indicates that the transcription start sites were GGC which were absent from the TATA boxes. In addition, although six PROSITE motifs were identified in CpG B, no motifs were detected in CpG A. A number of cis-regulatory elements were found in different regions within the CpGs A and B. Transcription factors were predicted to bind to CpGs A and B with varying affinities depending on the DNA methylation status. In addition, transcription factor binding may influence the expression patterns of the methylenetetrahydrofolate reductase gene by recruiting chromatin condensation inducing factors. These results have significant implications for the understanding of the architecture of transcription factor binding at CpG islets as well as DNA methylation patterns that affect chromatin structure.
Migraine is a painful and debilitating, neurovascular disease. Current migraine head pain treatme... more Migraine is a painful and debilitating, neurovascular disease. Current migraine head pain treatments work with differing efficacies in migraineurs. The opioid system plays an important role in diverse biological functions including analgesia, drug response and pain reduction. The A118G single nucleotide polymorphism (SNP) in exon 1 of the l-opioid receptor gene (OPRM1) has been associated with elevated pain responses and decreased pain threshold in a variety of populations. The aim of the current preliminary study was to test whether genotypes of the OPRM1 A118G SNP are associated with head pain severity in a clinical cohort of female migraineurs. This was a preliminary study to determine whether genotypes of the OPRM1 A118G SNP are associated with head pain severity in a clinical cohort of female migraineurs. A total of 153 chronic migraine with aura sufferers were assessed for migraine head pain using the Migraine Disability Assessment Score instrument and classified into high and low pain severity groups. DNA was extracted and genotypes obtained for the A118G SNP. Logistic regression analysis adjusting for age effects showed the A118G SNP of the OPRM1 gene to be significantly associated with migraine pain severity in the test population (P = 0.0037). In particular, G118 allele carriers were more likely to be high pain sufferers compared to homozygous carriers of the A118 allele (OR = 3.125, 95 % CI = 1.41, 6.93, P = 0.0037). These findings suggest that A118G genotypes of the OPRM1 gene may influence migraineassociated head pain in females. Further investigations are required to fully understand the effect of this gene variant on migraine head pain including studies in males and in different migraine subtypes, as well as in response to head pain medication.
In a human intervention study, coffee combining natural green coffee bean constituents and dark r... more In a human intervention study, coffee combining natural green coffee bean constituents and dark roast products was identified as a genotype-dependent inducer of the Nrf2/ARE pathway, significantly affecting Nrf2 gene expression and downstream GST1A1 and UGT1A1 gene transcription. The observed transcriptional changes correlated with the presence of specific Nrf2 genotypes suggesting their influence on both Nrf2 and subsequent ARE-dependent GST1A1 and UGT1A1 transcription. While the presence of the -Â 653 SNP seems to be advantageous, resulting in higher Nrf2, GST1A1 and UGT1A1 gene transcription following coffee consumption, in contrast, the presence of the -Â 651 SNP significantly down-regulated the response to the study coffee. Furthermore, the presence of the B/B genotype in GST1A1 along with the frequency of the [TA]6/6 and [TA]7/7 polymorphisms in UGT1A1 appeared to significantly increase sensitivity toward coffee-induced gene transcription. This data suggests that when examining the role of the Nrf2/ARE pathway in the regulation of antioxidative and chemopreventive phase II efficacy, individual genotypes should be included when considering the potency of bioactive food/food constituents and their therapeutic potential.
Breast cancer is the cancer that most commonly affects women worldwide. This type of cancer is ge... more Breast cancer is the cancer that most commonly affects women worldwide. This type of cancer is genetically complex, but is strongly linked to steroid hormone signaling systems. Because microRNAs act as translational regulators of multiple genes, including the steroid nuclear receptors, single nucleotide polymorphisms (SNPs) in microRNA genes can have potentially wide-ranging influences on breast cancer development. Thus, this study was conducted to investigate the relationships between six SNPs (rs6977848, rs199981120, rs185641358, rs113054794, rs66461782, and rs12940701) located in four miRNA genes predicted to target the estrogen receptor (miR-148a, miR-221, miR-186, and miR-152) and breast cancer risk in Caucasian Australian women. By using high resolution melt analysis (HRM) and polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP), 487 samples including 225 controls and 262 cases were genotyped. Analysis of their genotype and allele frequencies indicate...
Background.-Migraine is a highly disabling disease affecting a significant proportion of the Aust... more Background.-Migraine is a highly disabling disease affecting a significant proportion of the Australian population. The methylenetetrahydrofolate reductase (MTHFR) C677T variant has been associated with increased levels of homocysteine and risk of migraine with aura (MA). Folic acid (FA), vitamin B6, and B12 supplementation has been previously shown to reduce increased levels of homocysteine and decrease migraine symptoms. However, the influence of dietary folate intake on migraine has been unclear. The aim of the current study was to analyze the association of dietary folate intake in the form of dietary folate equivalent, FA, and total food folate (TFF) on migraine frequency, severity, and disability.
The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be ... more The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be fully elucidated. However, it has become clear that the family of fibroblast growth factors (FGFs) are centrally involved. In this study we examined the role of the FGF receptors (FGFRs 1-4) during osteogenesis in murine ES cells. Single cells were obtained after the formation of embryoid bodies, cultured on gelatin-coated plates, and coaxed to differentiate along the osteogenic lineage. Upregulation of genes was analyzed at both the transcript and protein levels using gene array, relative-quantitative PCR (RQ-PCR), and Western blotting. Deposition of a mineralized matrix was evaluated with Alizarin Red staining. An FGFR1-specific antibody was generated and used to block FGFR1 activity in mES cells during osteogenic differentiation. Upon induction of osteogenic differentiation in mES cells, all four FGFRs were clearly upregulated at both the transcript and protein levels with a number of genes known to be involved in osteogenic differentiation including bone morphogenetic proteins (BMPs), collagen I, and Runx2. Cells were also capable of depositing a mineralized matrix, confirming the commitment of these cells to the osteogenic lineage. When FGFR1 activity was blocked, a reduction in cell proliferation and a coincident upregulation of Runx2 with enhanced mineralization of cultures was observed. These results indicate that FGFRs play critical roles in cell recruitment and differentiation during the process of osteogenesis in mES cells. In particular, the data indicate that FGFR1 plays a pivotal role in osteoblast lineage determination.
Migraine is a debilitating neurovascular disease that is associated with pulsating head pain acco... more Migraine is a debilitating neurovascular disease that is associated with pulsating head pain accompanied by nausea, vomiting, photophobia, phonophobia and sometimes visual sensory disturbances. Because of its role in nitric oxide regulation and interleukin release, apolipoprotein E (APOE) has been suggested to play a role in the migraine pathogenesis pathway. This study evaluated the potential role of three APOE variants in an Australian population and the role that they may play in susceptibility to migraine. The study found no significant association between the tested variants and migraine for any of the APOE variants investigated.
ABSTRACT The most integrated approach toward understanding the multiple molecular events and mech... more ABSTRACT The most integrated approach toward understanding the multiple molecular events and mechanisms by which cancer may develop is the application of gene expression profiling using microarray technologies. As molecular alterations in breast cancer are complex and involve cross-talk between multiple cellular signalling pathways, microarray technology provides a means of capturing and comparing the expression patterns of the entire genome across multiple samples in a high throughput manner. Since the development of microarray technologies, together with the advances in RNA extraction methodologies, gene expression studies have revolutionised the means by which genes suitable as targets for drug development and individualised cancer treatment can be identified. As of the mid-1990s, expression microarrays have been extensively applied to the study of cancer and no cancer type has seen as much genomic attention as breast cancer. The most abundant area of breast cancer genomics has been the clarification and interpretation of gene expression patterns that unite both biological and clinical aspects of tumours. It is hoped that one day molecular profiling will transform diagnosis and therapeutic selection in human breast cancer toward more individualised regimes. Here, we review a number of prominent microarray profiling studies focussed on human breast cancer and examine their strengths, their limitations, clinical implications including prognostic relevance and gene signature significance along with potential improvements for the next generation of microarray studies. Yes Yes
The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studi... more The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) 4 in culture and detail the accompanying genetic alterations that have been observed. 4 Abbreviations: human mammary epithelial cells (HMEC); human mammary fibroblasts (HMF); the catalytic subunit of telomerase (hTERT); senesence associated-β-galactosidase (SA-βgal); human papilloma virus (HPV).
The most integrated approach toward understanding the multiple molecular events and mechanisms by... more The most integrated approach toward understanding the multiple molecular events and mechanisms by which cancer may develop is the application of gene expression profiling using microarray technologies. As molecular alterations in breast cancer are complex and involve cross-talk between multiple cellular signalling pathways, microarray technology provides a means of capturing and comparing the expression patterns of the entire genome across multiple samples in a high throughput manner. Since the development of microarray technologies, together with the advances in RNA extraction methodologies, gene expression studies have revolutionised the means by which genes suitable as targets for drug development and individualised cancer treatment can be identified. As of the mid-1990s, expression microarrays have been extensively applied to the study of cancer and no cancer type has seen as much genomic attention as breast cancer. The most abundant area of breast cancer genomics has been the c...
A new strategy has emerged to improve healing of bone defects using exogenous glycosaminoglycans ... more A new strategy has emerged to improve healing of bone defects using exogenous glycosaminoglycans by increasing the effectiveness of bone-anabolic growth factors. Wnt ligands play an important role in bone formation. However, their functional interactions with heparan sulfate/heparin have only been investigated in non-osseous tissues. Our study now shows that the osteogenic activity of Wnt3a is cooperatively stimulated through physical interactions with exogenous heparin. N-Sulfation and to a lesser extent O-sulfation of heparin contribute to the physical binding and optimal co-stimulation of Wnt3a. Wnt3a-heparin signaling synergistically increases osteoblast differentiation with minimal effects on cell proliferation. Thus, heparin selectively reduces the effective dose of Wnt3a needed to elicit osteogenic, but not mitogenic responses. Mechanistically, Wnt3a-heparin signaling strongly activates the phosphoinositide 3-kinase/Akt pathway and requires the bone-related transcription fact...
Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradatio... more Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origen is difficult. We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell ...
Journal of mammary gland biology and neoplasia, 2001
The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studi... more The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed.
Senescence and genomic integrity are thought to be important barriers in the development of malig... more Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of...
Since microRNAs (miRNAs) act as translational regulators of multiple genes, single nucleotide pol... more Since microRNAs (miRNAs) act as translational regulators of multiple genes, single nucleotide polymorphisms (SNP) in them can have potentially wide-ranging effects. Using an association approach, this research examined the effects of the rs6505162 SNP, an A > C polymorphism located in the pre-miRNA region of miR-423, on breast cancer development. Caucasian Australian women with breast cancer and controls matched for age and ethnicity were genotyped for rs6505162 and their genotypic and allelic frequencies analysed for significant differences. Analysis indicated that there were significant differences between the case and control populations (χ2 = 6.70, p = 0.035), with the CC genotype conferring reduced risk of breast cancer development (odds ratio = 0.50 95% confidence interval = 0.27-0.92, p = 0.03). Further functional research is required to determine the mechanism of action of this SNP on miRNA function.
Breast cancer is the second most common cancer worldwide and the most common cancer reported in w... more Breast cancer is the second most common cancer worldwide and the most common cancer reported in women. This malignant tumour is characterised by a number of specific features including uncontrolled cell proliferation. It ranks fifth in the world as a cause of cancer death overall in developed countries and is the second most frequent cause of cancer death in women. Early diagnosis increases 5-year survival rates up to 95%. Heparan sulfate proteoglycans (HSPGs) are complex proteins composed of a core protein to which a number of highly sulfated side chains attach, ubiquitous to the cell surface and within the extracellular matrix. HSPG side chains are synthesised by a highly co-ordinated process resulting in distinct sulfation patterns, which determine specific interactions with cell-signalling partners including growth factors, their receptors, ligands and morphogens. The enzymes responsible for chain initiation, elongation and sulfation are critical for creating HS chain variability conferring biological functionality. This study investigated a single nucleotide polymorphism in SULF1, the enzyme responsible for the 6-O desulfation of heparan sulfate side chains. We investigated this SNP in an Australian Caucasian case-control breast cancer population and found a significant association between SULF1 and breast cancer at both the allelic and genotypic levels (allele, p=0.016; genotype, p=0.032). Our results suggest that the rs2623047 SNP in SULF1 may impact breast cancer susceptibility. Specifically, the T allele of rs2623047 in SULF1 is associated with an increased risk of developing breast cancer in our cohort. The identification of markers including SULF1 may improve detection of this disease at its earliest stages improving patient treatment and prognosis.
There is evidence that folate metabolism has a role in migraine pathophysiology, particularly in ... more There is evidence that folate metabolism has a role in migraine pathophysiology, particularly in the migraine with aura (MA) subtype. In this study, we investigate whether two non-synonymous single nucleotide polymorphisms (SNPs), rs1950902 (C401T; R134K) and rs2236225 (G1958A; R653Q), in MTHF dehydrogenase (MTHFD1) are associated with migraine in an Australian case-control population. Increased plasma levels of homocysteine, one of the metabolites produced in the folate pathway, has been found to be a risk factor for migraine. There is also a genetic link: a common polymorphism (rs1801133, C667T) that reduces the catalytic activity of the enzyme that catalyzes the formation of homocysteine, methylenetetrahydrofolate reductase (MTHFR), is associated with an increase in risk of MA. MTHFD1 is a crucial multifunctional enzyme that catalyzes three separate reactions of the folate pathway and therefore variants in MTHFD1 may also influence migraine susceptibility. The R134K and R653Q variants in MTHFD1 were genotyped in an Australian cohort of 520 unrelated migraineurs (162 were diagnosed with migraine without aura [MO] and 358 with MA) and 520 matched controls. Data were analyzed for association with migraine and for interaction with the MTHFR C667T polymorphism. We find no significant differences in genotype or allele frequencies for either SNP between migraineurs and controls, or when either MO or MA cases were compared with controls. In addition, these MTHFD1 polymorphisms did not appear to influence the risk of MA conferred by the MTHFR 667T allele. We find no evidence for association of the MTHFD1 R134K and R653Q polymorphisms with migraine in our Australian case-control population. However, as folate metabolism appears to be important in migraine, particularly with respect to the aura component, future studies using high throughput methods to expand the number of SNPs in folate-related genes genotyped and investigation of interactions between SNPs may be justified.
Computational epigenetics is a new area of research focused on exploring how DNA methylation patt... more Computational epigenetics is a new area of research focused on exploring how DNA methylation patterns affect transcription factor binding that affect gene expression patterns. The aim of this study was to produce a new protocol for the detection of DNA methylation patterns using computational analysis which can be further confirmed by bisulfite PCR with serial pyrosequencing. The upstream regulatory element and preinitiation complex relative to CpG islets within the methylenetetrahydrofolate reductase gene were determined via computational analysis and online databases. The 1,104 bp long CpG island located near to or at the alternative promoter site of methylenetetrahydrofolate reductase gene was identified. The CpG plot indicated that CpG islets A and B, within the island, contained 62 and 75 % GC content CpG ratios of 0.70 and 0.80-0.95, respectively. Further exploration of the CpG islets A and B indicates that the transcription start sites were GGC which were absent from the TATA boxes. In addition, although six PROSITE motifs were identified in CpG B, no motifs were detected in CpG A. A number of cis-regulatory elements were found in different regions within the CpGs A and B. Transcription factors were predicted to bind to CpGs A and B with varying affinities depending on the DNA methylation status. In addition, transcription factor binding may influence the expression patterns of the methylenetetrahydrofolate reductase gene by recruiting chromatin condensation inducing factors. These results have significant implications for the understanding of the architecture of transcription factor binding at CpG islets as well as DNA methylation patterns that affect chromatin structure.
Migraine is a painful and debilitating, neurovascular disease. Current migraine head pain treatme... more Migraine is a painful and debilitating, neurovascular disease. Current migraine head pain treatments work with differing efficacies in migraineurs. The opioid system plays an important role in diverse biological functions including analgesia, drug response and pain reduction. The A118G single nucleotide polymorphism (SNP) in exon 1 of the l-opioid receptor gene (OPRM1) has been associated with elevated pain responses and decreased pain threshold in a variety of populations. The aim of the current preliminary study was to test whether genotypes of the OPRM1 A118G SNP are associated with head pain severity in a clinical cohort of female migraineurs. This was a preliminary study to determine whether genotypes of the OPRM1 A118G SNP are associated with head pain severity in a clinical cohort of female migraineurs. A total of 153 chronic migraine with aura sufferers were assessed for migraine head pain using the Migraine Disability Assessment Score instrument and classified into high and low pain severity groups. DNA was extracted and genotypes obtained for the A118G SNP. Logistic regression analysis adjusting for age effects showed the A118G SNP of the OPRM1 gene to be significantly associated with migraine pain severity in the test population (P = 0.0037). In particular, G118 allele carriers were more likely to be high pain sufferers compared to homozygous carriers of the A118 allele (OR = 3.125, 95 % CI = 1.41, 6.93, P = 0.0037). These findings suggest that A118G genotypes of the OPRM1 gene may influence migraineassociated head pain in females. Further investigations are required to fully understand the effect of this gene variant on migraine head pain including studies in males and in different migraine subtypes, as well as in response to head pain medication.
In a human intervention study, coffee combining natural green coffee bean constituents and dark r... more In a human intervention study, coffee combining natural green coffee bean constituents and dark roast products was identified as a genotype-dependent inducer of the Nrf2/ARE pathway, significantly affecting Nrf2 gene expression and downstream GST1A1 and UGT1A1 gene transcription. The observed transcriptional changes correlated with the presence of specific Nrf2 genotypes suggesting their influence on both Nrf2 and subsequent ARE-dependent GST1A1 and UGT1A1 transcription. While the presence of the -Â 653 SNP seems to be advantageous, resulting in higher Nrf2, GST1A1 and UGT1A1 gene transcription following coffee consumption, in contrast, the presence of the -Â 651 SNP significantly down-regulated the response to the study coffee. Furthermore, the presence of the B/B genotype in GST1A1 along with the frequency of the [TA]6/6 and [TA]7/7 polymorphisms in UGT1A1 appeared to significantly increase sensitivity toward coffee-induced gene transcription. This data suggests that when examining the role of the Nrf2/ARE pathway in the regulation of antioxidative and chemopreventive phase II efficacy, individual genotypes should be included when considering the potency of bioactive food/food constituents and their therapeutic potential.
Breast cancer is the cancer that most commonly affects women worldwide. This type of cancer is ge... more Breast cancer is the cancer that most commonly affects women worldwide. This type of cancer is genetically complex, but is strongly linked to steroid hormone signaling systems. Because microRNAs act as translational regulators of multiple genes, including the steroid nuclear receptors, single nucleotide polymorphisms (SNPs) in microRNA genes can have potentially wide-ranging influences on breast cancer development. Thus, this study was conducted to investigate the relationships between six SNPs (rs6977848, rs199981120, rs185641358, rs113054794, rs66461782, and rs12940701) located in four miRNA genes predicted to target the estrogen receptor (miR-148a, miR-221, miR-186, and miR-152) and breast cancer risk in Caucasian Australian women. By using high resolution melt analysis (HRM) and polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP), 487 samples including 225 controls and 262 cases were genotyped. Analysis of their genotype and allele frequencies indicate...
Background.-Migraine is a highly disabling disease affecting a significant proportion of the Aust... more Background.-Migraine is a highly disabling disease affecting a significant proportion of the Australian population. The methylenetetrahydrofolate reductase (MTHFR) C677T variant has been associated with increased levels of homocysteine and risk of migraine with aura (MA). Folic acid (FA), vitamin B6, and B12 supplementation has been previously shown to reduce increased levels of homocysteine and decrease migraine symptoms. However, the influence of dietary folate intake on migraine has been unclear. The aim of the current study was to analyze the association of dietary folate intake in the form of dietary folate equivalent, FA, and total food folate (TFF) on migraine frequency, severity, and disability.
The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be ... more The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be fully elucidated. However, it has become clear that the family of fibroblast growth factors (FGFs) are centrally involved. In this study we examined the role of the FGF receptors (FGFRs 1-4) during osteogenesis in murine ES cells. Single cells were obtained after the formation of embryoid bodies, cultured on gelatin-coated plates, and coaxed to differentiate along the osteogenic lineage. Upregulation of genes was analyzed at both the transcript and protein levels using gene array, relative-quantitative PCR (RQ-PCR), and Western blotting. Deposition of a mineralized matrix was evaluated with Alizarin Red staining. An FGFR1-specific antibody was generated and used to block FGFR1 activity in mES cells during osteogenic differentiation. Upon induction of osteogenic differentiation in mES cells, all four FGFRs were clearly upregulated at both the transcript and protein levels with a number of genes known to be involved in osteogenic differentiation including bone morphogenetic proteins (BMPs), collagen I, and Runx2. Cells were also capable of depositing a mineralized matrix, confirming the commitment of these cells to the osteogenic lineage. When FGFR1 activity was blocked, a reduction in cell proliferation and a coincident upregulation of Runx2 with enhanced mineralization of cultures was observed. These results indicate that FGFRs play critical roles in cell recruitment and differentiation during the process of osteogenesis in mES cells. In particular, the data indicate that FGFR1 plays a pivotal role in osteoblast lineage determination.
Migraine is a debilitating neurovascular disease that is associated with pulsating head pain acco... more Migraine is a debilitating neurovascular disease that is associated with pulsating head pain accompanied by nausea, vomiting, photophobia, phonophobia and sometimes visual sensory disturbances. Because of its role in nitric oxide regulation and interleukin release, apolipoprotein E (APOE) has been suggested to play a role in the migraine pathogenesis pathway. This study evaluated the potential role of three APOE variants in an Australian population and the role that they may play in susceptibility to migraine. The study found no significant association between the tested variants and migraine for any of the APOE variants investigated.
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Papers by Larisa Haupt