Genetic and gene expression analyses of the polycystic ovary syndrome candidate gene fibrillin-3 and other fibrillin family members in human ovaries

Several studies have demonstrated an association between polycystic ovary syndrome (PCOS) and the dinucleotide repeat microsatellite marker D19S884, which is located in intron 55 of the fibrillin-3 (FBN3) gene. Fibrillins, including FBN1 and 2, interact with latent transforming growth factor (TGF)-β-binding proteins (LTBP) and thereby control the bioactivity of TGFβs. TGFβs stimulate fibroblast replication and collagen production. The PCOS ovarian phenotype includes increased stromal collagen and expansion of the ovarian cortex, features feasibly influenced by abnormal fibrillin expression. To examine a possible role of fibrillins in PCOS, particularly FBN3, we undertook tagging and functional single nucleotide polymorphism (SNP) analysis (32 SNPs including 10 that generate non-synonymous amino acid changes) using DNA from 173 PCOS patients and 194 controls. No SNP showed a significant association with PCOS and alleles of most SNPs showed almost identical population frequencies between PCOS and control subjects. No significant differences were observed for microsatellite D19S884. In human PCO stroma/cortex (n = 4) and non-PCO ovarian stroma (n = 9), follicles (n = 3) and corpora lutea (n = 3) and in human ovarian cancer cell lines (KGN, SKOV-3, OVCAR-3, OVCAR-5), FBN1 mRNA levels were approximately 100 times greater than FBN2 and 200–1000-fold greater than FBN3. Expression of LTBP-1 mRNA was 3-fold greater than LTBP-2. We conclude that FBN3 appears to have little involvement in PCOS but cannot rule out that other markers in the region of chromosome 19p13.2 are associated with PCOS or that FBN3 expression occurs in other organs and that this may be influencing the PCOS phenotype.

Association analysis of chromosome 1 migraine candidate genes

Migraine with aura (MA) is a subtype of typical migraine. Migraine with aura (MA) also encompasses a rare severe subtype Familial Hemiplegic Migraine (FHM) with several known genetic loci. The type 2 FHM (FHM-2) susceptibility locus maps to chromosome 1q23 and mutations in the ATP1A2 gene at this site have recently been implicated. We have previously provided evidence of linkage of typical migraine (predominantly MA) to microsatellite markers on chromosome 1, in the 1q31 and 1q23 regions. In this study, we have undertaken a large genomic investigation involving candidate genes that lie within the chromosome 1q23 and 1q31 regions using an association analysis approach. Methods We have genotyped a large population of case-controls (243 unrelated Caucasian migraineurs versus 243 controls) examining a set of 5 single nucleotide polymorphisms (SNPs) and the Fas Ligand dinucleotide repeat marker, located within the chromosome 1q23 and 1q31 regions. Results Several genes have been studied including membrane protein (ATP 1 subtype A4 and FasL), cytoplasmic glycoprotein (CASQ 1) genes and potassium (KCN J9 and KCN J10) and calcium (CACNA1E) channel genes in 243 migraineurs (including 85% MA and 15% of migraine without aura (MO)) and 243 matched controls. After correction for multiple testing, chi-square results showed non-significant P values (P > 0.008) across all SNPs (and a CA repeat) tested in these different genes, however results with the KCN J10 marker gave interesting results (P = 0.02) that may be worth exploring further in other populations. Conclusion These results do not show a significant role for the tested candidate gene variants and also do not support the hypothesis that a common chromosome 1 defective gene influences both FHM and the more common forms of migraine.

Association analysis of somatostatin receptor (SSTR1 and SSTR2) polymorphisms in breast cancer and solar keratosis

The presence of somatostatin receptors (SSTR1-5) in tumour cells indicates a potential for somatostatin to bind and suppress growth, as well as allowing for therapeutic treatment with somatostatin analogues. The genes for SSTR1 and SSTR2 have been shown to contain dinucleotide repeat polymorphisms. We have performed association studies on breast cancer and solar keratosis populations to determine whether these genes play a role in the development of these conditions. Results showed that there was no significant difference between SSTR1 and SSTR2 polymorphism frequencies in the tested breast cancer population (P = 0.59 and P = 0.54, respectively) nor the solar keratosis population (P = 0.10 and P = 0.883, respectively) as compared to unaffected populations. Hence, these studies do not support a role for these receptor genes in either breast cancer or solar keratosis lesions.

Association of estrogen receptor and glucocorticoid receptor gene polymorphisms with sporadic breast cancer

We have utilized a cross-sectional association approach to investigate sporadic breast cancer. Polymorphisms in 2 candidate genes, ESRalpha and GRL, were examined in an unrelated breast cancer-affected and age-matched control population. Several polymorphic regions within the ESRalpha gene have been identified, and some alleles of these polymorphisms have been found to occur at increased levels in breast-cancer patients. Additionally, variations in GRL have the potential to disrupt cell transcription and may be associated with cancer formation. We analyzed 3 polymorphisms, from codons 10 (TCT to TCC), 325 (CCC to CCG) and 594 (ACA to ACG) of ESRalpha, and a highly polymorphic dinucleotide repeat, D5S207, located within 200 kb of the GRL. When allelic frequencies of the codon 594 (exon 8) ESR polymorphism were compared between affected and unaffected populations, a significant difference was observed (p = 0.005). Results from the D5S207 dinucleotide repeat located near GRL also indicated a significant difference between the tested case and control populations (p = 0.001). Allelic frequencies of the codon 10 and codon 325 ESR polymorphisms were not significantly different between populations (p = 0.152 and 0.181, respectively). Our results indicate that specific alleles of the ESR gene (alpha subtype) and a marker for the GRL gene locus are associated with sporadic breast-cancer development in the tested Caucasian population and justify further investigation of the role of these and other nuclear steroid receptors in the etiology of breast cancer.

Cross-sectional study of a microsatellite marker in the low densitity lipoprotein receptor gene in obese normotensives

1. The low density lipoprotein receptor is an important regulator of serum cholesterol which may have implications for the development of both hypertension and obesity. In this study, genotypes for a low density lipoprotein receptor gene (LDLR) dinucleotide polymorphism were determined in both lean and obese normotensive populations. 2. In previous cross-sectional association studies an ApaLI and a HincII polymorphism for LDLR were shown to be associated with obesity in essential hypertensives. However, these polymorphisms did not show an association with obesity in normotensives. 3. In contrast, this study reports that preliminary results for an LDLR microsatellite marker, located more towards the 3' end of the gene, show a significant association with obesity in the normotensive population studied. These results indicate that LDLR could play an important role in the development of obesity, which might be independent of hypertension.

Evidence for allelic association of the dopamine β-hydroxylase gene (DBH) with susceptibility to typical migraine

Migraine is a debilitating neurological disorder characterized by recurrent attacks of severe headache. The disorder is highly prevalent, affecting approximately 12% of Caucasian populations. It is well known that migraine has a strong genetic component, although the type and number of genes involved is not yet clear. However, the calcium channel gene, CACNA1A, on chromosome 19 contains mutations responsible for familial hemiplegic migraine, a rare and severe subtype of migraine. There is also evidence to suggest that serotonin- and dopamine-related genes may be involved in the pathogenesis of migraine. This study employed a linkage and association approach to investigate neurotransmitter-related migraine candidate genes. Polymorphisms within the dopamine beta-hydroxylase (DBH) gene, serotonin transporter gene (SERT), and dopamine receptor gene (DRD2) were tested in 177 unrelated Caucasian migraineurs and 182 control individuals. In addition, an independent sample of 82 families affected with migraine was examined. Unrelated case-control association analysis of a DBH intragenic dinucleotide polymorphism indicated altered allelic distribution between migraine and control groups (L2=16.53, P=0.019). Furthermore, the transmission/disequilibrium test, which was implemented on the family data, also indicated distortion of allele transmission for the same DBH marker (L2=4.44, P=0.035). Together, these results provide evidence for allelic association of the DBH gene with typical migraine susceptibility (Fisher's combined P value=0.006) and indicate that further research into the role of the DBH gene in the etiology of migraine is warranted.

Genome stability pathways in head and neck cancers

Genomic instability underlies the transformation of host cells toward malignancy, promotes development of invasion and metastasis and shapes the response of established cancer to treatment. In this review, we discuss recent advances in our understanding of genomic stability in squamous cell carcinoma of the head and neck (HNSCC), with an emphasis on DNA repair pathways. HNSCC is characterized by distinct profiles in genome stability between similarly staged cancers that are reflected in risk, treatment response and outcomes. Defective DNA repair generates chromosomal derangement that can cause subsequent alterations in gene expression, and is a hallmark of progression toward carcinoma. Variable functionality of an increasing spectrum of repair gene polymorphisms is associated with increased cancer risk, while aetiological factors such as human papillomavirus, tobacco and alcohol induce significantly different behaviour in induced malignancy, underpinned by differences in genomic stability. Targeted inhibition of signalling receptors has proven to be a clinically-validated therapy, and protein expression of other DNA repair and signalling molecules associated with cancer behaviour could potentially provide a more refined clinical model for prognosis and treatment prediction. Development and expansion of current genomic stability models is furthering our understanding of HNSCC pathophysiology and uncovering new, promising treatment strategies. © 2013 Glenn Jenkins et al.

Differential expression of NADPH-diaphorase between electrophysiologically-defined classes of pyramidal neurons in rat ventral subiculum, in vitro

The subiculum is the major output region of the hippocampal formation. We have studied pyramidal neurons in slices of rat ventral subiculum to determine if there is a correlation between nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity and electrophysiological phenotype. The majority of NADPH-d-positive pyramidal neurons were found in the superficial cell layer (i.e. nearest to the hippocampal fissure) of the subiculum and appreciable NADPH-d activity was absent from pyramidal neurons in area CA1. This distribution of NADPH-d activity was mimicked by that of immunoreactivity for the neuronal isoform of nitric oxide synthase. Subicular pyramidal neurons were classified, electrophysiologically, as intrinsically burst-firing or regular spiking. After electrophysiological characterization, neurons were filled with Neurobiotin and revealed using fluorescence immunocytochemistry. The slices containing these neurons were also processed for NADPH-d. NADPH-d activity was found in six out of eight regular spiking neurons but was not found in any of 13 intrinsically burst-firing neurons (P=0.0008, Fisher's Exact Test). We conclude that in rat ventral subiculum, NADPH-d activity is present in a proportion of pyramidal neurons and indicates the presence of the neuronal isoform of nitric oxide synthase. Furthermore, amongst pyramidal neurons, NADPH-d activity is distributed preferentially to those with the regular spiking phenotype. The distribution of regular spiking neurons suggests that they may not be present to the same extent in all subicular output pathways. Thus, the actions of nitric oxide may be relatively specific to particular hippocampal connections.

A light and electron microscopic study of NADPH-diaphorase-,calretinin- and parvalbumin-containing neurons in the rat nucleus accumbens

The rat nucleus accumbens contains medium-sized, spiny projection neurons and intrinsic, local circuit neurons, or interneurons. Sub-classes of interneurons, revealed by calretinin (CR) or parvalbumin (PV) immunoreactivity or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, were compared in the nucleus accumbens core, shell and rostral pole. CR, PV and NADPH-diaphorase-containing neurons are shown to form three non-co-localising populations in these three areas. No significant differences in neuronal population densities were found between the subterritories. NADPH-diaphorase-containing neurons could be further separated morphologically into three sub-groups, but CR- and PV-immunoreactive neurons form homogeneous populations. Ultrastructurally, NADPH-diaphorase-, CR- and PV-containing neurons in the nucleus accumbens all possess nuclear indentations. These are deeper and fewer in neurons immunoreactive for PV than in CR- and NADPH-diaphorase-containing neurons. CR-immunoreactive boutons form asymmetrical and symmetrical synaptic specialisations on spines, dendrites and somata, while PV-immunoreactive boutons make only symmetrical synaptic specialisations. Both CR- and PV-immunoreactive boutons form symmetrical synaptic specialisations with medium-sized spiny neurons and contact other CR- and PV-immunoreactive somata, respectively. A novel non-carcinogenic substrate for the peroxidase reaction (Vector Slate Grey, SG) was found to be characteristically electron-dense and may be distinguishable from the diaminobenzidine reaction product. We conclude that the three markers used in this study are localised in distinct populations of nucleus accumbens interneurons. Our studies of their synaptic connections contribute to an increased understanding of the intrinsic circuitry of this area.

DNA methylation at the novel CpG sites in the promoter of MED15/PCQAP gene as a biomarker for head and neck cancers

Head and neck cancers (HNCs) represent a significant and ever-growing burden to the modern society, mainly due to the lack of early diagnostic methods. A significant number of HNCs is often associated with drinking, smoking, chewing beetle nut, and human papilloma virus (HPV) infections. We have analyzed DNA methylation patterns in tumor and normal tissue samples collected from head and neck squamous cell carcinoma (HNSCC) patients who were smokers. We have identified novel methylation sites in the promoter of the mediator complex subunit 15 (MED15/PCQAP) gene (encoing a co-factor important for regulation of transcription initiation for promoters of many genes), hypermethylated specifically in tumor cells. Two clusters of CpG dinucleotides methylated in tumors, but not in normal tissue from the same patients, were identified. These CpG methylation events in saliva samples were further validated in a separate cohort of HNSCC patients (who developed cancer due to smoking or HPV infections) and healthy controls using methylation-specific PCR (MSP). We used saliva as a biological medium because of its non-invasive nature, close proximity to the tumors, easiness and it is an economically viable option for large-scale screening studies. The methylation levels for the two identified CpG clusters were significantly different between the saliva samples collected from healthy controls and HNSCC individuals (Welch's t-test returning P, 0.05 and Mann-Whitney test P, 0.01 for both). The developed MSP assays also provided a good discriminative ability with AUC values of 0.70 (P, 0.01) and 0.63 (P, 0.05). The identified novel CpG methylation sites may serve as potential non-invasive biomarkers for detecting HNSCC. © the authors.

Computational analysis of epigenetic information in human DNA sequences

Over the last few years, investigations of human epigenetic profiles have identified key elements of change to be Histone Modifications, stable and heritable DNA methylation and Chromatin remodeling. These factors determine gene expression levels and characterise conditions leading to disease. In order to extract information embedded in long DNA sequences, data mining and pattern recognition tools are widely used, but efforts have been limited to date with respect to analyzing epigenetic changes, and their role as catalysts in disease onset. Useful insight, however, can be gained by investigation of associated dinucleotide distributions. The focus of this paper is to explore specific dinucleotides frequencies across defined regions within the human genome, and to identify new patterns between epigenetic mechanisms and DNA content. Signal processing methods, including Fourier and Wavelet Transformations, are employed and principal results are reported.

Cross-sectional study of a microsatellite marker in the low density lipoprotein receptor gene in obese normotensives

1. The low density lipoprotein receptor is an important regulator of serum cholesterol which may have implications for the development of both hypertension and obesity. In this study, genotypes for a low density lipoprotein receptor gene (LDLR) dinucleotide polymorphism were determined in both lean and obese normotensive populations. 2. In previous cross-sectional association studies an ApaLI and a HincII polymorphism for LDLR were shown to be associated with obesity in essential hypertensives. However, these polymorphisms did not show an association with obesity in normotensives. 3. In contrast, this study reports that preliminary results for an LDLR microsatellite marker, located more towards the 3' end of the gene, show a significant association with obesity in the normotensive population studied. These results indicate that LDLR could play an important role in the development of obesity, which might be independent of hypertension.