Multiple analysis of three common genetic alterations associated with thrombophilia

We have previously reported the use of a novel mini-sequencing protocol for detection of the factor V Leiden variant, the first nucleotide change (FNC) technology. This technology is based on a single nucleotide extension of a primer, which is hybridized immediately adjacent to the site of mutation. The extended nucleotide that carries a reporter molecule (fluorescein) has the power to discriminate the genotype at the site of mutation. More recently, the prothrombin 20210 and thermolabile methylene tetrahydrofolate reductase (MTHFR) 677 variants have been identified as possible risk factors associated with thrombophilia. This study describes the use of the FNC technology in a combined assay to detect factor V, prothrombin and MTHFR variants in a population of Australian blood donors, and describes the objective numerical methodology used to determine genotype cut-off values for each genetic variation. Using FNC to test 500 normal blood donors, the incidence of Factor V Leiden was 3.6% (all heterozygous), that of prothrombin 20210 was 2.8% (all heterozygous) and that of MTHFR was 10% (homozygous). The combined FNC technology offers a simple, rapid, automatable DNA-based test for the detection of these three important mutations that are associated with familial thrombophilia. (C) 2000 Lippincott Williams and Wilkins.

Characterisation of the TaNF-Y family of transcription factors in wheat

Light plays a unique role for plants as it is both a source of energy for growth and a signal for development. Light captured by the pigments in the light harvesting complexes is used to drive the synthesis of the chemical energy required for carbon assimilation. The light perceived by photoreceptors activates effectors, such as transcription factors (TFs), which modulate the expression of light-responsive genes. Recently, it has been speculated that increasing the photosynthetic rate could further improve the yield potential of three carbon (C3) crops such as wheat. However, little is currently known about the transcriptional regulation of photosynthesis genes, particularly in crop species. Nuclear factor Y (NF-Y) TF is a functionally diverse regulator of growth and development in the model plant species, with demonstrated roles in embryo development, stress response, flowering time and chloroplast biogenesis. Furthermore, a light-responsive NF-Y binding site (CCAAT-box) is present in the promoter of a spinach photosynthesis gene. As photosynthesis genes are co-regulated by light and co-regulated genes typically have similar regulatory elements in their promoters, it seems likely that other photosynthesis genes would also have light-responsive CCAAT-boxes. This provided the impetus to investigate the NF-Y TF in bread wheat. This thesis is focussed on wheat NF-Y members that have roles in light-mediated gene regulation with an emphasis on their involvement in the regulation of photosynthesis genes. NF-Y is a heterotrimeric complex, comprised of the three subunits NF-YA, NF-YB and NF-YC. Unlike the mammalian and yeast counterparts, each of the three subunits is encoded by multiple genes in Arabidopsis. The initial step taken in this study was the identification of the wheat NF-Y family (Chapter 3). A search of the current wheat nucleotide sequence databases identified 37 NF-Y genes (10 NF-YA, 11 NF-YB, 14 NF-YC & 2 Dr1). Phylogenetic analysis revealed that each of the three wheat NF-Y (TaNF-Y) subunit families could be divided into 4-5 clades based on their conserved core regions. Outside of the core regions, eleven motifs were identified to be conserved between Arabidopsis, rice and wheat NF-Y subunit members. The expression profiles of TaNF-Y genes were constructed using quantitative real-time polymerase chain reaction (RT-PCR). Some TaNF-Y subunit members had little variation in their transcript levels among the organs, while others displayed organ-predominant expression profiles, including those expressed mainly in the photosynthetic organs. To investigate their potential role in light-mediated gene regulation, the light responsiveness of the TaNF-Y genes were examined (Chapters 4 and 5). Two TaNF-YB and five TaNF-YC members were markedly upregulated by light in both the wheat leaves and seedling shoots. To identify the potential target genes of the light-upregulated NF-Y subunit members, a gene expression correlation analysis was conducted using publically available Affymetrix Wheat Genome Array datasets. This analysis revealed that the transcript expression levels of TaNF-YB3 and TaNF-YC11 were significantly correlated with those of photosynthesis genes. These correlated express profiles were also observed in the quantitative RT-PCR dataset from wheat plants grown under light and dark conditions. Sequence analysis of the promoters of these wheat photosynthesis genes revealed that they were enriched with potential NF-Y binding sites (CCAAT-box). The potential role of TaNF-YB3 in the regulation of photosynthetic genes was further investigated using a transgenic approach (Chapter 5). Transgenic wheat lines constitutively expressing TaNF-YB3 were found to have significantly increased expression levels of photosynthesis genes, including those encoding light harvesting chlorophyll a/b-binding proteins, photosystem I reaction centre subunits, a chloroplast ATP synthase subunit and glutamyl-tRNA reductase (GluTR). GluTR is a rate-limiting enzyme in the chlorophyll biosynthesis pathway. In association with the increased expression of the photosynthesis genes, the transgenic lines had a higher leaf chlorophyll content, increased photosynthetic rate and had a more rapid early growth rate compared to the wild-type wheat. In addition to its role in the regulation of photosynthesis genes, TaNF-YB3 overexpression lines flower on average 2-days earlier than the wild-type (Chapter 6). Quantitative RT-PCR analysis showed that there was a 13-fold increase in the expression level of the floral integrator, TaFT. The transcript levels of other downstream genes (TaFT2 and TaVRN1) were also increased in the transgenic lines. Furthermore, the transcript levels of TaNF-YB3 were significantly correlated with those of constans (CO), constans-like (COL) and timing of chlorophyll a/b-binding (CAB) expression 1 [TOC1; (CCT)] domain-containing proteins known to be involved in the regulation of flowering time. To summarise the key findings of this study, 37 NF-Y genes were identified in the crop species wheat. An in depth analysis of TaNF-Y gene expression profiles revealed that the potential role of some light-upregulated members was in the regulation of photosynthetic genes. The involvement of TaNF-YB3 in the regulation of photosynthesis genes was supported by data obtained from transgenic wheat lines with increased constitutive expression of TaNF-YB3. The overexpression of TaNF-YB3 in the transgenic lines revealed this NF-YB member is also involved in the fine-tuning of flowering time. These data suggest that the NF-Y TF plays an important role in light-mediated gene regulation in wheat.

Development of novel vaccine strategies to prevent genital tract chlamydial infections

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection in the developed world and the leading cause of preventable blindness worldwide. As reported by the World Health Organization in 2001, there are approximately 92 million new infections detected annually, costing health systems billions of dollars to treat not only the acute infection, but also to treat infection-associated sequelae. The majority of genital infections are asymptomatic, with 50-70% going undetected. Genital tract infections can be easily treated with antibiotics when detected. Lack of treatment can lead to the development of pelvic inflammatory disease, ectopic pregnancies and tubal factor infertility in women and epididymitis and prostatitis in men. With infection rates on the continual rise and the large number of infections going undetected, there is a need to develop an efficacious vaccine which prevents not only infection, but also the development of infection-associated pathology. Before a vaccine can be developed and administered, the pathogenesis of chlamydial infections needs to be fully understood. This includes the kinetics of ascending infection and the effects of inoculating dose on ascension and development of pathology. The first aim in this study was to examine these factors in a murine model. Female BALB/c mice were infected intravaginally with varying doses of C. muridarum, the mouse variant of human C. trachomatis, and the ascension of infection along the reproductive tract and the time-course of infection-associated pathology development, including inflammatory cell infiltration, pyosalpinx and hydrosalpinx, were determined. It was found that while the inoculating dose did affect the rate and degree of infection, it did not affect any of the pathological parameters examined. This highlighted that the sexual transmission dose may have minimal effect on the development of reproductive sequelae. The results of the first section enabled further studies presented here to use an optimal inoculating dose that would ascend the reproductive tract and cause pathology development, so that vaccine efficacy could be determined. There has been a large amount of research into the development of an efficacious vaccine against genital tract chlamydial infections, with little success. However, there have been no studies examining the effects of the timing of vaccination, including the effects of vaccination during an active genital infection, or after clearance of a previous infection. These are important factors that need to be examined, as it is not yet known whether immunization will enhance not only the individual's immune response, but also pathology development. It is also unknown whether any enhancement of the immune responses will cause the Chlamydia to enter a dormant, persistent state, and possibly further enhance any pathology development. The second section of this study aimed to determine if vaccination during an active genital tract infection, or after clearance of a primary infection, enhanced the murine immune responses and whether any enhanced or reduced pathology occurred. Naïve, actively infected, or previously infected animals were immunized intranasally or transcutaneously with the adjuvants cholera toxin and CpG-ODN in combination with either the major outer membrane protein (MOMP) of C. muridarum, or MOMP and ribonucleotide reductase small chain protein (NrdB) of C. muridarum. It was found that the systemic immune responses in actively or previously infected mice were altered in comparison to animals immunized naïve with the same combinations, however mucosal antibodies were not enhanced. It was also found that there was no difference in pathology development between any of the groups. This suggests that immunization of individuals who may have an asymptomatic infection, or may have been previously exposed to a genital infection, may not benefit from vaccination in terms of enhanced immune responses against re-exposure. The final section of this study aimed to determine if the vaccination regimes mentioned above caused in vivo persistence of C. muridarum in the upper reproductive tracts of mice. As there has been no characterization of C. muridarum persistence in vitro, either ultrastructurally or via transcriptome analysis, this was the first aim of this section. Once it had been shown that C. muridarum could be induced into a persistent state, the gene transcriptional profiles of the selected persistent marker genes were used to determine if persistent infections were indeed present in the upper reproductive tracts of the mice. We found that intranasal immunization during an active infection induced persistent infections in the oviducts, but not the uterine horns, and that intranasal immunization after clearance of infection, caused persistent infections in both the uterine horns and the oviducts of the mice. This is a significant finding, not only because it is the first time that C. muridarum persistence has been characterized in vitro, but also due to the fact that there is minimal characterization of in vivo persistence of any chlamydial species. It is possible that the induction of persistent infections in the reproductive tract might enhance the development of pathology and thereby enhance the risk of infertility, factors that need to be prevented by vaccination, not enhanced. Overall, this study has shown that the inoculating dose does not affect pathology development in the female reproductive tract of infected mice, but does alter the degree and rate of ascending infection. It has also been shown that intranasal immunization during an active genital infection, or after clearance of one, induces persistent infections in the uterine horns and oviducts of mice. This suggests that potential vaccine candidates will need to have these factors closely examined before progressing to clinical trials. This is significant, because if the same situation occurs in humans, a vaccine administered to an asymptomatic, or previously exposed individual may not afford any extra protection and may in fact enhance the risk of development of infection-associated sequelae. This suggests that a vaccine may serve the community better if administered before the commencement of sexual activity.

The role of insulin and IGF2 signalling on metabolic pathways in prostate cancer progression

Prostate cancer (CaP) is the most commonly diagnosed cancer in males in Australia, North America, and Europe. If found early and locally confined, CaP can be treated with radical prostatectomy or radiation therapy; however, 25-40% patients will relapse and go on to advanced disease. The most common therapy in these cases is androgen deprivation therapy (ADT), which suppresses androgen production from the testis. Lack of the testicular androgen supply causes cells of the prostate to undergo apoptosis. However, in some cases the regression initially seen with ADT eventually gives way to a growth of a population of cancerous cells that no longer require testicular androgens. This phenotype is essentially fatal and is termed castrate resistant prostate cancer (CRPC). In addition to eventual regression, there are many undesirable side effects which accompany ADT, including development of a metabolic syndrome, which is defined by the U.S. National Library of Medicine as “a combination of medical disorders that increase the risk of developing cardiovascular disease and diabetes.” This project will focus on the effect of ADT induced hyperinsulinemia, as mimicked by treating androgen receptor positive CaP cells with insulin in a serum (hormone) deprived environment. While this side effect is not widely explored, in this thesis it is demonstrated for the first time that insulin upregulates pathways important to CaP progression. Our group has previously shown that during CaP progression, the enzymes necessary for de novo steroidogenesis are upregulated in the LNCaP xenograft model, total steroid levels are increased in tumours compared to pre castrate levels, and de novo steroidogenesis from radio-labelled acetate has been demonstrated. Because of the CaP dependence on AR for survival, we and other groups believe that CaP cells carry out de novo steroidogenesis to survive in androgen deprived conditions. Because (a) men on ADT often develop metabolic syndrome, and (b) men with lifestyle-induced obesity and hyperinsulinemia have worse prognosis and faster disease progression, and because (c) insulin causes steroidogenesis in other cell lines, the hypothesis that insulin may contribute to CaP progression through upregulation of steroidogenesis was explored. Insulin upregulates steroidogenesis enzymes at the mRNA level in three AR positive cell lines, as well as upregulating these enzymes at the protein level in two cell lines. It has also been demonstrated that insulin increases mitochondrial (functional) levels of steroid acute regulatory protein (StAR). Furthermore, insulin causes increased levels of total steroids in and induction of de novo steroid synthesis by insulin has been demonstrated at levels induced sufficient to activate AR. The effect of insulin analogs on CaP steroidogenesis in LNCaP and VCaP cells has also been investigated because epidemiological studies suggest that some of the analogs developed may have more cancer stimulatory effects than normal insulin. In this project, despite the signalling differences between glargine, X10, and insulin, these analogs did not appear to induce steroidogenesis any more potently that normal insulin. The effect of insulin of MCF7breast cancer cells was also investigated with results suggesting that breast cancer cells may be capable of de novo steroidogenesis, and that increase in estradiol production may be exacerbated by insulin. Insulin has also been long known to stimulate lipogenesis in the liver and adipocytes, and has been demonstrated to increase lipogenesis in breast cancer cells; therefore, investigation of the effect of insulin on lipogenesis, which is a hallmark of aggressive cancers, was investigated. In CaP progression sterol regulatory element binding protein (SREBP) is dysregulated and upregulates fatty acid synthase (FASN), acetyl CoA-carboxylase, and other lipogenesis genes. SREBP is important for steroidogenesis and in this project has been shown to be upregulated by insulin in CaP cells. Fatty acid synthesis provides building blocks of membrane growth, provides substrates for acid oxidation, the main energy source for CaP cells, provides building blocks for anti-apoptotic and proinflammatory molecules, and provides molecules that stimulate steroidogenesis. In this project it has been shown that insulin upregulates FASN and ACC, which synthesize fatty acids, as well as upregulating hormone sensitive lipase (HSL), diazepam-binding inhibitor (DBI), and long-chain acyl-CoA synthetase 3 (ACSL3), which contribute to lipid activation of steroidogenesis. Insulin also upregulates total lipid levels and de novo lipogenesis, which can be suppressed by inhibition of the insulin receptor (INSR). The fatty acids synthesized after insulin treatment are those that have been associated with CaP; furthermore, microarray data suggests insulin may upregulate fatty acid biosynthesis, metabolism and arachidonic acid metabolism pathways, which have been implicated in CaP growth and survival. Pharmacological agents used to treat patients with hyperinsulinemia/ hyperlipidemia have gained much interest in regards to CaP risk and treatment; however, the scientific rationale behind these clinical applications has not been examined. This thesis explores whether the use of metformin or simvastatin would decrease either lipogenesis or steroidogenesis or both in CaP cells. Simvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitor, which blocks synthesis of cholesterol, the building block of steroids/ androgens. It has also been postulated to down regulate SREBP in other metabolic disorders. It has been shown in this thesis, in LNCaP cells, that simvastatin inhibited and decreased insulin induced steroidogenesis and lipogenesis, respectively, but increased these pathways in the absence of insulin. Conversely, metformin, which activates AMP-activated protein kinase (AMPK) to shut down lipogenesis, cholesterol synthesis, and protein synthesis, highly suppresses both steroidogenesis and lipogenesis in the presence and absence of insulin. Lastly, because it has been demonstrated to increase steroidogenesis in other cell lines, and because the elucidation of any factors affecting steroidogenesis is important to understanding CaP, the effect of IGF2 on steroidogenesis in CaP cells was investigated. In patient samples, as men progress to CRPC, IGF2 mRNA and the protein levels of the receptors it may signal through are upregulated. It has also been demonstrated that IGF2 upregulates steroidogenic enzymes at both the mRNA and protein levels in LNCaP cells, increases intracellular and secreted steroid/androgen levels in LNCaPs to levels sufficient to stimulate the AR, and upregulated de novo steroidogenesis in LNCaPs and VCaPs. As well, inhibition of INSR and insulin-like growth factor 1 receptor (IGF1R), which IGF2 signals through, suggests that induction of steroidogenesis may be occurring predominantly through IGF1R. In summary, this project has illuminated for the first time that insulin is likely to play a large role in cancer progression, through upregulation of the steroidogenesis and lipogenesis pathways at the mRNA and protein levels, and production levels, and demonstrates a novel role for IGF-II in CaP progression through stimulation of steroidogenesis. It has also been demonstrated that metformin and simvastatin drugs may be useful in suppressing the insulin induction of these pathways. This project affirms the pathways by which ADT- induced metabolic syndrome may exacerbate CaP progression and strongly suggests that the monitoring and modulation of the metabolic state of CaP patients could have a strong impact on their therapeutic outcomes.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines : targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines: targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

Genotypes of the MTHFR C677T and MTRR A66G genes act independently to reduce migraine disability in response to vitamin supplementation

BACKGROUND: Migraine is a chronic disabling neurovascular condition that may in part be caused by endothelial and cerebrovascular disruption induced by hyperhomocysteinaemia. We have previously provided evidence indicating that reduction of homocysteine by vitamin supplementation can reduce the occurrence of migraine in women. The current study examined the genotypic effects of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) gene variants on the occurrence of migraine in response to vitamin supplementation. METHODS: This was a 6-month randomized, double-blinded placebo-controlled trial of daily vitamin B supplementation (B(6), B(9) and B(12)) on reduction of homocysteine and of the occurrence of migraine in 206 female patients diagnosed with migraine with aura. RESULTS: Vitamin supplementation significantly reduced homocysteine levels (P<0.001), severity of headache in migraine (P=0.017) and high migraine disability (P=0.022) in migraineurs compared with the placebo effect (P>0.1). When the vitamin-treated group was stratified by genotype, the C allele carriers of the MTHFR C677T variant showed a higher reduction in homocysteine levels (P<0.001), severity of pain in migraine (P=0.01) and percentage of high migraine disability (P=0.009) compared with those with the TT genotypes. Similarly, the A allele carriers of the MTRR A66G variants showed a higher level of reduction in homocysteine levels (P<0.001), severity of pain in migraine (P=0.002) and percentage of high migraine disability (P=0.006) compared with those with the GG genotypes. Genotypic analysis for both genes combined indicated that the treatment effect modification of the MTRR variant was independent of the MTHFR variant. CONCLUSION: This provided further evidence that vitamin supplementation is effective in reducing migraine and also that both MTHFR and MTRR gene variants are acting independently to influence treatment response in female migraineurs.

Investigation of homocysteine-pathway-related variants in essential hypertension

Hyperhomocysteinemia (hHcy) has been associated with an increased risk of cardiovascular disease and stroke. Essential hypertension (EH), a polygenic condition, has also been associated with increased risk of cardiovascular related disorders. To investigate the role of the homocysteine (Hcy) metabolism pathway in hypertension we conducted a case-control association study of Hcy pathway gene variants in a cohort of Caucasian hypertensives and age- and sex-matched normotensives. We genotyped two polymorphisms in the methylenetetrahydrofolate reductase gene (MTHFR C677T and MTHFR A1298C), one polymorphism in the methionine synthase reductase gene (MTRR A66G), and one polymorphism in the methylenetetrahydrofolate dehydrogenase 1 gene (MTHFD1 G1958A) and assessed their association with hypertension using chi-square analysis. We also performed a multifactor dimensionality reduction (MDR) analysis to investigate any potential epistatic interactions among the four polymorphisms and EH. None of the four polymorphisms was significantly associated with EH and although we found a moderate synergistic interaction between MTHFR A1298C and MTRR A66G, the association of the interaction model with EH was not statistically significant (

The role of the MTHFR gene in migraine

Migraine is a common neurological disorder and is characterized by debilitating head pain and an assortment of additional symptoms which can include nausea, emesis, photophobia, phonophobia, and occasionally, visual sensory disturbances. A number of genes have been implicated in the pathogenesis of this disease, including genes involved in regulating the vascular system. Of particular importance are the methylenetetrahydrofolate reductase (MTHFR) gene and the role it plays in migraine with aura. Migraine with aura has previously been shown to have a significant comorbidity with stroke, making the vascular class of genes a priority for migraine studies. In this report, we outline the importance of the MTHFR gene in migraine and also discuss the use of a genetic isolate to investigate MTHFR genetic variants. From this study, 3 MTHFR single nucleotide polymorphisms showing association with migraine in the Norfolk Island population have been identified, thus reinforcing the potential role of MTHFR in migraine susceptibility. Further studies will continue to build a gene profile of variants involved in the complex disease migraine and improve understanding of the underlying genetic causes of this disorder.

Analysis of the MTHFR C677T variant with migraine phenotypes

Background The methylenetetrahydrofolate reductase (MTHFR) gene variant C677T has been implicated as a genetic risk factor in migraine susceptibility, particularly in Migraine with Aura. Migraine, with and without aura (MA and MO) have many diagnostic characteristics in common. It is postulated that migraine symptomatic characteristics might themselves be influenced by MTHFR. Here we analysed the clinical profile, migraine symptoms, triggers and treatments of 267 migraineurs previously genotyped for the MTHFR C677T variant. The chi-square test was used to analyse all potential relationships between genotype and migraine clinical variables. Regression analyses were performed to assess the association of C677T with all migraine clinical variables after adjusting for gender. Findings The homozygous TT genotype was significantly associated with MA (P < 0.0001) and unilateral head pain (P = 0.002). While the CT genotype was significantly associated with physical activity discomfort (P < 0.001) and stress as a migraine trigger (P = 0.002). Females with the TT genotype were significantly associated with unilateral head pain (P < 0.001) and females with the CT genotype were significantly associated with nausea (P < 0.001), osmophobia (P = 0.002), and the use of natural remedy for migraine treatment (P = 0.003). Conversely, male migraineurs with the TT genotype experienced higher incidences of bilateral head pain (63% vs 34%) and were less likely to use a natural remedy as a migraine treatment compared to female migraineurs (5% vs 20%). Conclusions MTHFR genotype is associated with specific clinical variables of migraine including unilateral head pain, physical activity discomfort and stress.

No association between MTHFR A1298C and MTRR A66G polymorphisms, and MS in an Australian cohort

Multiple sclerosis (MS) is a complex neurological disease that affects the central nervous system (CNS) resulting in debilitating neuropathology. Pathogenesis is primarily defined by CNS inflammation and demyelination of nerve axons. Methionine synthase reductase (MTRR) is an enzyme that catalyzes the remethylation of homocysteine (Hcy) to methionine via cobalamin and folate dependant reactions. Cobalamin acts as an intermediate methyl carrier between methylenetetrahydrofolate reductase (MTHFR) and Hcy. MTRR plays a critical role in maintaining cobalamin in an active form and is consequently an important determinant of total plasma Hcy (pHcy) concentrations. Elevated intracellular pHcy levels have been suggested to play a role in CNS dysfunction, neurodegenerative, and cerebrovascular diseases. Our investigation entailed the genotyping of a cohort of 140 cases and matched controls for MTRR and MTHFR, by restriction length polymorphism (RFLP) techniques. Two polymorphisms: MTRR A66G and MTHFR A1298C were investigated in an Australian age and gender matched case-control study. No significant allelic frequency difference was observed between cases and controls at the α = 0.05 level (MTRR χ2 = 0.005, P = 0.95, MTHFR χ2 = 1.15, P = 0.28). Our preliminary findings suggest no association between the MTRR A66G and MTHFR A1298C polymorphisms and MS

Endophenotype profiles of MTHFR and ACE gene polymorphisms in migraine susceptibility

Background Migraine is a debilitating neurological disorder affecting approximately 12% of the Caucasian population. There are two main sub-types of migraine, migraine without aura (MO) and migraine with aura (MA). Migraine exhibits varied phenotypic expression with sufferers experiencing a range of neurological and other symptoms. It is likely that multiple susceptibility genes play a role in this varied phenotypic expression, thus investigation of genotype-phenotype relationships may provide valuable insights into the role of susceptibility genes in this disorder. Methods This study investigated the links between migraine susceptibility genes, methylenetetrahydrofolate reductase (MTHFR) and angiotensin converting enzyme (ACE), and clinical manifestation through statistical analyses. Results The result showed that for the MTHFR genotypes, there was a statistically significant correlation with the TT homozygous genotype and visual disturbances, unilateral head pain and physical activity discomforts. It was also found that bilateral head pain was associated with the male gender. Conclusion From these study results, it is plausible to state that MTHFR genotypes affect the phenotypic expression of migraine disease manifestation.

Expression Profiles of Mitochondrial Genes in the Frontal Cortex and the Caudate Nucleus of Developing Humans and Mice Selectively Bred for High and Low Fear

A growing body of evidence suggests that mitochondrial function may be important in brain development and psychiatric disorders. However, detailed expression profiles of those genes in human brain development and fear-related behavior remain unclear. Using microarray data available from the public domain and the Gene Ontology analysis, we identified the genes and the functional categories associated with chronological age in the prefrontal cortex (PFC) and the caudate nucleus (CN) of psychiatrically normal humans ranging in age from birth to 50 years. Among those, we found that a substantial number of genes in the PFC (115) and the CN (117) are associated with the GO term: mitochondrion (FDR qv <0.05). A greater number of the genes in the PFC (91%) than the genes in the CN (62%) showed a linear increase in expression during postnatal development. Using quantitative PCR, we validated the developmental expression pattern of four genes including monoamine oxidase B (MAOB), NADH dehydrogenase flavoprotein (NDUFV1), mitochondrial uncoupling protein 5 (SLC25A14) and tubulin beta-3 chain (TUBB3). In mice, overall developmental expression pattern of MAOB, SLC25A14 and TUBB3 in the PFC were comparable to the pattern observed in humans (p<0.05). However, mice selectively bred for high fear did not exhibit normal developmental changes of MAOB and TUBB3. These findings suggest that the genes associated with mitochondrial function in the PFC play a significant role in brain development and fear-related behavior.

Expression and prognostic significance of a panel of tissue hypoxia markers in head-and-neck squamous cell carcinomas

Purpose: To investigate the expression pattern of hypoxia-induced proteins identified as being involved in malignant progression of head-and-neck squamous cell carcinoma (HNSCC) and to determine their relationship to tumor pO 2 and prognosis. Methods and Materials: We performed immunohistochemical staining of hypoxia-induced proteins (carbonic anhydrase IX [CA IX], BNIP3L, connective tissue growth factor, osteopontin, ephrin A1, hypoxia inducible gene-2, dihydrofolate reductase, galectin-1, IκB kinase β, and lysyl oxidase) on tumor tissue arrays of 101 HNSCC patients with pretreatment pO 2 measurements. Analysis of variance and Fisher's exact tests were used to evaluate the relationship between marker expression, tumor pO 2, and CA IX staining. Cox proportional hazard model and log-rank tests were used to determine the relationship between markers and prognosis. Results: Osteopontin expression correlated with tumor pO 2 (Eppendorf measurements) (p = 0.04). However, there was a strong correlation between lysyl oxidase, ephrin A1, and galectin-1 and CA IX staining. These markers also predicted for cancer-specific survival and overall survival on univariate analysis. A hypoxia score of 0-5 was assigned to each patient, on the basis of the presence of strong staining for these markers, whereby a higher score signifies increased marker expression. On multivariate analysis, increasing hypoxia score was an independent prognostic factor for cancer-specific survival (p = 0.015) and was borderline significant for overall survival (p = 0.057) when adjusted for other independent predictors of outcomes (hemoglobin and age). Conclusions: We identified a panel of hypoxia-related tissue markers that correlates with treatment outcomes in HNSCC. Validation of these markers will be needed to determine their utility in identifying patients for hypoxia-targeted therapy. © 2007 Elsevier Inc. All rights reserved.

A phase II study of the modulation of 5-fluorouracil and folinic acid with high-dose infusional hydroxyurea in metastatic colorectal carcinoma

Background: Hydroxyurea (HU), an inhibitor of ribonucleotide reductase, may potentiate the activity of 5-fluorouracil (5-FU) and folinic acid (FA) by reducing the deoxyribonucleotide pool available for DNA synthesis and repair. However as HU may inhibit the formation of 5-fluoro-2-deoxyuridine-5- monophosphate (FdUMP), one of the principal active metabolites of 5-FU, the scheduling of HU may be critical. In vitro experiments suggest that administration of HU following 5-FU, maintaining the concentration in the region of I mM for six or more hours, significantly enhances the efficacy of 5-FU. Patients and methods: 5-FU/FA was given as follows: days 1 and 2 - FA 250 mg/m 2 (max. 350 mg) over two hours followed by 5-FU 400 mg/m 2 by intravenous bolus (ivb) over 15 minutes and subsequently 5-FU 400 mg/m 2 infusion (ivi) over 22 hours. HU was administered on day 3 immediately after the 5-FU with 3 g ivb over 15 minutes followed by 12 g ivi over 12 hours. Results: Thirty patients were entered into the study. Median survival was nine months (range 1-51 + months). There were eight partial responses (28%, 95% CI: 13%-47%). The median duration of response was 6.5 (range 4-9 months). Grade 3-4 toxicities included neutropenia (grade 3 in eight patients and grade 4 in five), anaemia (grade 3 in one patient) and diarrhoea (grade 3 in two patients). Neutropenia was associated with pyrexia in two patients. Phlebitis at the infusion site occurred in five patients. The treatment was complicated by pulmonary embolism in one patient and deep venous thrombosis in another. Conclusion: HU administered in this schedule is well tolerated. Based on these results and those of other phase II studies, a randomised phase III study of 5-FU, FA and HU versus 5-FU and FA using the standard de Gramont schedule is recommended.