Examination of the role of nitric oxide synthase and renal kallikrein as candidate genes for essential hypertension

Nitric oxide synthase and renal kallikrein are both involved in blood pressure regulation. Genes for these enzymes may, therefore, be considered candidates for hypertension pathogenesis. 2. In the present study, genotypes for nitric oxide synthase and renal kallikrein microsatellite markers were determined in a cross-sectional association analysis of hypertensive patients and normotensive control subjects. 3. Results from this study did not indicate an association of either of the candidate gene polymorphisms with essential hypertension. Hence, findings for this study do not support a role for these genes in human hypertension.

Independent, marked associations of alleles of the insulin receptor and dipeptidyl carboxypeptidase-I genes with essential hypertension

1. There is evidence to suggest that essential hypertension is a polygenic disorder and that it arises from yet-to-be-identified predisposing variants of certain genes that influence blood pressure. The cloning of various hormone, enzyme, adrenoceptor and hormone receptor genes whose products are involved in blood pressure control and the identification of polymorphisms of these has permitted us to test their genetic association with hypertension. 2. Cross-sectional analyses of a number of candidate gene markers were performed in hypertensive and normotensive subjects who were selected on the basis of both parents being either hypertensive or normotensive, respectively, and the difference in total alleles on all chromosomes for each polymorphism between the hypertensive and normotensive groups was test by χ analysis with one degree of freedom. 3. A marked association was observed between hypertension and insertion alleles of polymorphisms of the insulin receptor gene (INSR) (P<0.0040) and the dipeptidyl carboxypeptidase-1 (angiotensin I-converting enzyme; kininase II) gene (DCP1) (P<0.0018). No association with hypertension was evident, however, for polymorphisms of the growth hormone, low-density lipoprotein receptor, renal kallikrein, α2- and β1-adrenoreceptor, atrial natriuretic factor and insulin genes. 4. All but one of the hypertensive subjects had at least one of the hypertension-associated alleles, and although subjects homozygous for both were three times more frequent in the hypertensive group, examination of the nine possible genotypes suggested that the INSR and DCP1 alleles are independent markers for hypertension. 5. The present results suggest that genetic variant(s) in close linkage disequilibrium with polymorphisms at INSR and DCP1 may be involved in part in the aetiology of essential hypertension.

Expression of glucocorticoid and progesterone nuclear receptor genes in archival breast cancer tissue.

BACKGROUND: Previous studies in our laboratory have shown associations of specific nuclear receptor gene variants with sporadic breast cancer. In order to investigate these findings further, we conducted the present study to determine whether expression levels of the progesterone and glucocorticoid nuclear receptor genes vary in different breast cancer grades. METHODS: RNA was extracted from paraffin-embedded archival breast tumour tissue and converted into cDNA. Sample cDNA underwent PCR using labelled primers to enable quantitation of mRNA expression. Expression data were normalized against the 18S ribosomal gene multiplex and analyzed using analysis of variance. RESULTS: Analysis of variance indicated a variable level of expression of both genes with regard to breast cancer grade (P = 0.00033 for glucocorticoid receptor and P = 0.023 for progesterone receptor). CONCLUSION: Statistical analysis indicated that expression of the progesterone nuclear receptor is elevated in late grade breast cancer tissue.

Genetic variation in cytokine-related genes and migraine susceptibility

Migraine is classified by the World Health Organization (WHO) as being one of the top 20 most debilitating diseases. According to the neurovascular hypothesis, neuroinflammation may promote the activation and sensitisation of meningeal nociceptors, inducing the persistent throbbing headache characterized in migraine. The tumor necrosis factor (TNF) gene cluster, made up of TNFα, lymphotoxin α (LTA), and lymphotoxin β (LTB), has been implicated to influence the intensity and duration of local inflammation. It is thought that sterile inflammation mediated by LTA, LTB, and TNFα contributes to threshold brain excitability, propagation of neuronal hyperexcitability and thus initiation and maintenance of a migraine attack. Previous studies have investigated variants within the TNF gene cluster region in relation to migraine susceptibility, with largely conflicting results. The aim of this study was to expand on previous research and utilize a large case-control cohort and range of variants within the TNF gene cluster to investigate the role of the TNF gene cluster in migraine. Nine single nucleotide polymorphisms (SNPs) were selected for investigation as follows: rs1800683, rs2229094, rs2009658, rs2071590, rs2239704, rs909253, rs1800630, rs1800629, and rs3093664. No significant association with migraine susceptibility was found for any of the SNPs tested, with further testing according to migraine subtype and gender also showing no association for disease risk. Haplotype analysis showed that none of the tested haplotypes were significantly associated with migraine.

Cyclin A/CDK2 regulates multiple G2 phase pathways

The cell cycle is a carefully choreographed series of phases that when executed successfully will allow the complete replication of the genome and the equal division of the genome and other cellular content into two independent daughter cells. The inability of the cell to execute cell division successfully can result in either checkpoint activation to allow repair and/or apoptosis and/or mutations/errors that may or may not lead to tumourgenesis. Cyclin A/CDK2 is the primary cyclin/CDK regulating G2 phase progression of the cell cycle. Cyclin A/CDK2 activity peaks in G2 phase and its inhibition causes a G2 phase delay that we have termed 'the cyclin A/CDK2 dependent G2 delay'. Understanding the key pathways that are involved in the cyclin A/CDK2 dependent G2 delay has been the primary focus of this study. Characterising the cyclin A/CDK2 dependent G2 delay revealed accumulated levels of the inactive form of the mitotic regulator, cyclin B/CDK1. Surprisingly, there was also increased microtubule nucleation at the centrosomes, and the centrosomes stained for markers of cyclin B/CDK1 activity. Both microtubule nucleation at the centrosomes and phosphoprotein markers were lost with short-term treatment of CDK1/2 inhibition. Cyclin A/CDK2 localised at the centrosomes in late G2 phase after separation of the centrosomes but before the start of prophase. Thus G2 phase cyclin A/CDK2 controls the timing of entry into mitosis by controlling the subsequent activation of cyclin B/CDK1, but also has an unexpected role in coordinating the activation of cyclin B/CDK1 at the centrosome and in the nucleus. In addition to regulating the timing of cyclin B/CDK1 activation and entry into mitosis in the unperturbed cell cycle, cyclin A/CDK2 also was shown to have a role in G2 phase checkpoint recovery. Known G2 phase regulators were investigated to determine whether they had a role in imposing the cyclin A/ CDK2 dependent G2 delay. Examination of the critical G2 checkpoint arrest protein, Chk1, which also has a role during unperturbed G2/M phases revealed the presence of activated Chk1 in G2 phase, in a range of cell lines. Activated Chk1 levels were shown to accumulate in cyclin A/CDK2 depleted/inhibited cells. Further investigations revealed that Chk1, but not Chk2, depletion could reverse the cyclin A/CDK2 dependent G2 delay. It was confirmed that the accumulative activation of Chk1 was not a consequence of DNA damage induced by cyclin A depletion. The potential of cyclin A/CDK2 to regulate Chk1 revealed that the inhibitory phosphorylations, Ser286 and Ser301, were not directly catalysed by cyclin A/CDK2 in G2 phase to regulate mitotic entry. It appeared that the ability of cyclin A/CDK2 to regulate cyclin B/CDK1 activation impacted cyclin B/CDK1s phosphorylation of Chk1 on Ser286 and Ser301, thereby contributing to the delay in G2/M phase progression. Chk1 inhibition/depletion partially abrogated the cyclin A/CDK2 dependent G2 delay, and was less effective in abrogating G2 phase checkpoint suggesting that other cyclin A/CDK2 dependent mechanisms contributed to these roles of cyclin A/CDK2. In an attempt to identify these other contributing factors another G2/M phase regulator known to be regulated by cyclin A/CDK2, Cdh1 and its substrates Plk1 and Claspin were examined. Cdh1 levels were reduced in cyclin A/CDK2 depleted/inhibited cells although this had little effect on Plk1, a known Cdh1 substrate. However, the level of another substrate, Claspin, was increased. Cdh1 depletion mimicked the effect of cyclin A depletion but to a weaker extent and was sufficient at increasing Claspin levels similar to the increase caused by cyclin A depletion. Co-depletion of cyclin A and Claspin blocked the accumulation of activated Chk1 normally seen with cyclin A depletion alone. However Claspin depletion alone did not reduce the cyclin A/CDK2 dependent G2 delay but this is likely to be a result of inhibition of S phase roles of Claspin. Together, these data suggest that cyclin A/CDK2 regulates a number of different mechanisms that contribute to G2/M phase progression. Here it has been demonstrated that in normal G2/M progression and possibly to a lesser extent in G2 phase checkpoint recovery, cyclin A/CDK2 regulates the level of Cdh1 which in turn affects at least one of its substrates, Claspin, and consequently results in the increased level of activated Chk1 observed. However, the involvement of Cdh1 and Claspin alone does not explain the G2 phase delay observed with cyclin A/CDK2 depletion/inhibition. It is likely that other mechanisms, possibly including cyclin A/CDK2 regulation of Wee1 and FoxM1, as reported by others, combine with the mechanism described here to regulate normal G2/M phase progression and G2 phase checkpoint recovery. These findings support the critical role for cyclin A/CDK2 in regulating progression into mitosis and suggest that upstream regulators of cyclin A/CDK2 activation will also be critical controllers of this cell cycle transition. The pathways that work to co-ordinate cell cycle progression are very intricate and deciphering these pathways, required for normal cell cycle progression, is key to understanding tumour development. By understanding cell cycle regulatory pathways it will allow the identification of the pathway/s and their mechanism/s that become affected in tumourgenesis. This will lead to the development of better targeted therapies, inferring better efficacy with fewer side effects than commonly seen with the use of traditional therapies, such as chemotherapy. Furthermore, this has the potential to positively impact the development of personalised medicines and the customisation of healthcare.

Gene expression profiling in human breast cancer – toward personalised therapeutics?

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.

Closing the case of APOE in multiple sclerosis : no association with disease risk in over 29,000 subjects

Background Single nucleotide polymorphisms (SNPs) rs429358 (ε4) and rs7412 (ε2), both invoking changes in the amino-acid sequence of the apolipoprotein E (APOE) gene, have previously been tested for association with multiple sclerosis (MS) risk. However, none of these studies was sufficiently powered to detect modest effect sizes at acceptable type-I error rates. As both SNPs are only imperfectly captured on commonly used microarray genotyping platforms, their evaluation in the context of genome-wide association studies has been hindered until recently. Methods We genotyped 12 740 subjects hitherto not studied for their APOE status, imputed raw genotype data from 8739 subjects from five independent genome wide association studies datasets using the most recent high-resolution reference panels, and extracted genotype data for 8265 subjects from previous candidate gene assessments. Results Despite sufficient power to detect associations at genome-wide significance thresholds across a range of ORs, our analyses did not support a role of rs429358 or rs7412 on MS susceptibility. This included meta-analyses of the combined data across 13 913 MS cases and 15 831 controls (OR=0.95, p=0.259, and OR 1.07, p=0.0569, for rs429358 and rs7412, respectively). Conclusion Given the large sample size of our analyses, it is unlikely that the two APOE missense SNPs studied here exert any relevant effects on MS susceptibility.

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.

HLA-DRB1 associations with disease susceptibility and clinical course in Australians with multiple sclerosis

Human leucocyte antigen (HLA)-DRB1*1501 and other class II alleles influence susceptibility to multiple sclerosis (MS), but their contribution if any to the clinical course of MS remains uncertain. Here, we have investigated DRB1 alleles in a large sample of 1230 Australian MS cases, with some enrichment for subjects with primary progressive (PPMS) disease (n = 246) and 1210 healthy controls. Using logistic regression, we found that DRB1*1501 was strongly associated with risk (P = 7 x 10-45), as expected, and after adjusting for DRB1*1501, a predisposing effect was also observed for DRB1*03 (P = 5 x 10-7). Individuals homozygous for either DRB1*15 or DRB1*03 were considerably more at risk of MS than heterozygotes and non-carriers. Both the DRB1*04 and the DRB1*01/DRB1*15 genotype combination, respectively, protected against PPMS in comparison to subjects with relapsing disease. Together, these data provide further evidence of heterogeneity at the DRB1 locus and confirm the importance of HLA variants in the phenotypic expression of MS.

Identification of TFG (TRK-fused gene) as a putative metastatic melanoma tumor suppressor gene

High density SNP arrays can be used to identify DNA copy number changes in tumors such as homozygous deletions of tumor suppressor genes and focal amplifications of oncogenes. Illumina Human CNV370 Bead chip arrays were used to assess the genome for unbalanced chromosomal events occurring in 39 cell lines derived from stage III metastatic melanomas. A number of genes previously recognized to have an important role in the development and progression of melanoma were identified including homozygous deletions of CDKN2A (13 of 39 samples), CDKN2B (10 of 39), PTEN (3 of 39), PTPRD (3 of 39), TP53 (1 of 39), and amplifications of CCND1 (2 of 39), MITF (2 of 39), MDM2 (1 of 39), and NRAS (1 of 39). In addition, a number of focal homozygous deletions potentially targeting novel melanoma tumor suppressor genes were identified. Because of their likely functional significance for melanoma progression, FAS, CH25H, BMPR1A, ACTA2, and TFG were investigated in a larger cohort of melanomas through sequencing. Nonsynonymous mutations were identified in BMPR1A (1 of 43), ACTA2 (3 of 43), and TFG (5 of 103). A number of potentially important mutation events occurred in TFG including the identification of a mini mutation ‘‘hotspot’’ at amino acid residue 380 (P380S and P380L) and the presence of multiple mutations in two melanomas. Mutations in TFG may have important clinical relevance for current therapeutic strategies to treat metastatic melanoma.

Multiple sclerosis and continence issues : an exploratory study

The study described in this article aimed to identify issues relating to incontinence and assess the impact of referral to a continence adviser on the lives of people with multiple sclerosis (MS). The study design used an in-depth, two-phase anonymous mail survey within a general community as nominated by the participants. Fifty-six people participated in phase 1 and eleven people completed phase 2. The results indicated that incontinence is a problem for the vast majority of participants — people with MS. One-third of the eligible participants took up the option of a consultation, assessment and treatment from a continence nurse. Reasons for not taking up the visit from the continence nurse included ‘managing OK’, ‘didn’t think it would help’, ‘embarrassed’ and ‘too busy’. Increasing awareness of urinary incontinence in the community is important and education needs to focus on at-risk groups in presenting the range of options available to assist people experiencing incontinence.

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 pattern of the cannabinoid receptor genes in the frontal cortex of mood disorder patients and mice selectively bred for high and low fear

Although the endocannabinoid system (ECS) has been implicated in brain development and various psychiatric disorders, precise mechanisms of the ECS on mood and anxiety disorders remain unclear. Here, we have investigated developmental and disease-related expression pattern of the cannabinoid receptor 1 (CB1) and the cannabinoid receptor 2 (CB2) genes in the dorsolateral prefrontal cortex (PFC) of humans. Using mice selectively bred for high and low fear, we further investigated potential association between fear memory and the cannabinoid receptor expression in the brain. The CB1, not the CB2, mRNA levels in the PFC gradually decrease during postnatal development ranging in age from birth to 50 years (r 2 > 0.6 & adj. p < 0.05). The CB1 levels in the PFC of major depression patients were higher when compared to the age-matched controls (adj. p < 0.05). In mice, the CB1, not the CB2, levels in the PFC were positively correlated with freezing behavior in classical fear conditioning (p < 0.05). These results suggest that the CB1 in the PFC may play a significant role in regulating mood and anxiety symptoms. Our study demonstrates the advantage of utilizing data from postmortem brain tissue and a mouse model of fear to enhance our understanding of the role of the cannabinoid receptors in mood and anxiety disorders