Suggestive linkage of the parathyroid receptor type 1 to osteoporosis

We have investigated the role of 23 candidate genes in the control of bone mineral density (BMD) by linkage studies in families of probands with osteoporosis (lumbar spine [LS] or femoral neck [FN] BMD T score < -2.5) and low BMD relative to an age- and gender-matched cohort (Z score < -2.0). One hundred and fifteen probands (35 male, 80 female) and 499 of their first- or second-degree relatives (223 males and 276 females) were recruited for the study. BMD was measured at the LS and FN using dual-energy X-ray absorptiometry and expressed as age- and gender-matched Z scores corrected for body mass index. The candidate genes studied were the androgen receptor, type I collagen A1 (COLIA1), COLIA2, COLIIA1, vitamin D receptor (VDR), colony-stimulating factor 1, calcium-sensing receptor, epidermal growth factor (EGF), estrogen receptor 1 (ESR1), fibrillin type 1, insulin-like growth factor 1, interleukin-1 alpha (IL-1α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-11 (IL-11), osteopontin, parathyroid hormone (PTH), PTH-related peptide, PTH receptor type 1 (PTHR1), transforming growth factor-beta 1, and tumor necrosis factors alpha and beta. Sixty-four microsatellites lying close to or within these genes were investigated for linkage with BMD. Using the program MapMaker/Sibs there was suggestive evidence of linkage between BMD and PTHR1 (maximum LOD score obtained [MLS] 2.7-3.5). Moderate evidence of linkage was also observed with EGF (MLS 1.8), COLIA1 (MLS 1.7), COLIIA1/VDR (MLS 1.7), ESR1 (MLS 1.4), IL-1α (MLS 1.4), IL-4 (MLS 1.2), and IL-6 (MLS 1.2). Variance components analysis using the program ACT, correcting for proband-wise ascertainment, also showed evidence of linkage (p ≤0.05) at markers close to or within the candidate genes IL- 1α, PTHR1, IL-6, and COLIIA1/VDR. Further studies will be required to confirm these findings, to refine the location of gene responsible for the observed linkage, and to screen the candidate genes targeted at these loci for mutations.

Candidate gene analysis for quantitative traits using the transmission disequilibrium test : the example of the melanocortin 4-receptor in pigs

Population-wide associations between loci due to linkage disequilibrium can be used to map quantitative trait loci (QTL) with high resolution. However, spurious associations between markers and QTL can also arise as a consequence of population stratification. Statistical methods that cannot differentiate between loci associations due to linkage disequilibria from those caused in other ways can render false-positive results. The transmission-disequilibrium test (TDT) is a robust test for detecting QTL. The TDT exploits within-family associations that are not affected by population stratification. However, some TDTs are formulated in a rigid-form, with reduced potential applications. In this study we generalize TDT using mixed linear models to allow greater statistical flexibility. Allelic effects are estimated with two independent parameters: one exploiting the robust within-family information and the other the potentially biased between-family information. A significant difference between these two parameters can be used as evidence for spurious association. This methodology was then used to test the effects of the fourth melanocortin receptor (MC4R) on production traits in the pig. The new analyses supported the previously reported results; i.e., the studied polymorphism is either causal of in very strong linkage disequilibrium with the causal mutation, and provided no evidence for spurious association.

Association study of the calcitonin gene-related polypeptide-alpha (CALCA) and the receptor activity modifying 1 (RAMP1) genes with migraine

Migraine is a common neurovascular brain disorder characterised by recurrent attacks of severe headache that may be accompanied by various neurological symptoms. Migraine is thought to result from activation of the trigeminovascular system followed by vasodilation of pain-producing intracranial blood vessels and activation of second-order sensory neurons in the trigeminal nucleus caudalis. Calcitonin gene-related peptide (CGRP) is a mediator of neurogenic inflammation and the most powerful vasodilating neuropeptide, and has been implicated in migraine pathophysiology. Consequently, genes involved in CGRP synthesis or CGRP receptor genes may play a role in migraine and/or increase susceptibility. This study investigates whether variants in the gene that encodes CGRP, calcitonin-related polypeptide alpha (CALCA) or in the gene that encodes a component of its receptor, receptor activity modifying protein 1 (RAMP1), are associated with migraine pathogenesis and susceptibility. The single nucleotide polymorphisms (SNPs) rs3781719 and rs145837941 in the CALCA gene, and rs3754701 and rs7590387 at the RAMP1 locus, were analysed in an Australian Caucasian population of migraineurs and matched controls. Although we find no significant association of any of the SNPs tested with migraine overall, we detected a nominally significant association (p = 0.031) of the RAMP1 rs3754701 variant in male migraine subjects, although this is non-significant after Bonferroni correction for multiple testing.

Investigation of the [−/A]8 and C1236T genetic variations within the human toll-like receptor 3 gene for association with multiple sclerosis

Multiple sclerosis (MS) is a serious cause of neurological disability among young adults. The clinical course remains difficult to predict, and the pathogenesis of the disease is still modestly understood. Autoimmunity is thought to be a key aspect of the disease, with autoreactive T cells thought to mediate central nervous system (CNS) inflammation to some extent. Toll-like receptors are known to mediate cellular recognition of pathogens by way of patterns of molecular presentation. Toll-like receptor 3 is coded by the gene TLR3 and is recognized as an important factor in virus recognition and is known to be involved in the expression of neuroprotective mediators. We set out to investigate two variations within the TLR3 gene, an 8 bp insertion-deletion \[-/A](8) and a single base-pair variation C1236T, in subjects with MS and matched healthy controls to determine whether significant differences exist in these markers in an Australian population. We used capillary gel electrophoresis and TaqMan genotyping assay techniques to resolve genotypes for each marker, respectively. Our work found no significant difference between frequencies for TLR3 \[-/A](8) by genotype (chi(2)=1.03, p=0.60) or allele (chi(2)=1.09, p=0.30). Similarly, we found no evidence for the association of TLR3 C1236T by genotype (chi(2)=0.35, p=0.84) or allele frequency (chi(2)=0.31, p=0.58). This work reveals no evidence to suggest that these markers are associated with MS in the tested population. Although the role of TLR3 and the wider toll-like receptor family remain significant in neurological and CNS inflammatory disorders, our current work does not support a role for the two tested variants in this gene with regard to MS susceptibility.

An investigation of the C77G and C772T variations within the human protein tyrosine phosphatase receptor type C gene for association with multiple sclerosis in an Australian population

Multiple sclerosis (MS) is a common cause of neurological disability in young adults. The disease generally manifests in early to middle adulthood and causes various neurological deficits. Autoreactive T lymphocytes and their associated antigens have long been presumed important features of MS pathogenesis. The Protein tyrosine phosphatase receptor type C gene (PTPRC) encodes the T-cell receptor CD45. Variations within PTPRC have been previously associated with diseases of autoimmune origin such as type 1 diabetes mellitus and Graves' disease. We set out to investigate two variants within the PTPRC gene, C77G and C772T in subjects with MS and matched healthy controls to determine whether significant differences exist in these markers in an Australian population. We employed high resolution melt analysis (HRM) and restriction length polymorphism (RFLP) techniques to determine genotypic and allelic frequencies. Our study found no significant difference between frequencies for PTPRC C77G by either genotype (Χ2 = 0.65, P = 0.72) or allele (Χ2 = 0.48, P = 0.49). Similarly, we did not find evidence to suggest an association between PTPRC C772T by genotype (Χ2 = 1.06, P = 0.59) or allele (Χ2 = 0.20, P = 0.66). Linkage disequilibrium (LD) analysis showed strong linkage disequilibrium between the two tested markers (D' = 0.9970, SD = 0.0385). This study reveals no evidence to suggest that these markers are associated with MS in the tested Australian Caucasian population. Although the PTPRC gene has a significant role in regulating CD4+ and CD8+ autoreactive T-cells, interferon-beta responsiveness, and potentially other important processes, our study does not support a role for the two tested variants of this gene in MS susceptibility in the Australian population.

No role for estrogen receptor 1 gene intron 1 Pvu II and exon 4 C325G polymorphisms in migraine susceptibility

Background We have previously reported an association between the estrogen receptor 1 (ESR1) gene exon 8 G594A polymorphism and migraine susceptibility in two independent Australian cohorts. In this paper we report results of analysis of two further single nucleotide polymorphisms (SNPs) in the ESR1 gene in the same study group, the T/C Pvu II SNP in intron 1 and the C325G SNP in exon 4, as well as results of linkage disequilibrium (LD) analysis on these markers. Methods We investigated these variants by case-control association analysis in a cohort of 240 migraineurs and 240 matched controls. The SNPs were genotyped using specific restriction enzyme assays. Results were analysed using contingency table methods incorporating the chi-squared statistic. LD results are presented as D' statistics with associated P values. Results We found no evidence for association of the Pvu II T/C polymorphism and the C325G polymorphism and migraine susceptibility and no evidence for LD between these two SNPs and the previously implicated exon 8 G594A marker. Conclusion We have found no role for the polymorphisms in intron 1 and exon 4 with migraine susceptibility. To further investigate our previously implicated exon 8 marker, we suggest the need for studies with a high density of polymorphisms be undertaken, with particular focus on markers in LD with the exon 8 marker.

Cyclooxygenase-2 protein levels are independent of epidermal growth factor receptor expression or activation in operable non-small cell lung cancer

Both cyclooxygenase (COX)-2 and epidermal growth factor receptor (EGFR) are thought to play important roles in the pathogenesis of non-small cell lung cancer (NSCLC). A number of in vitro studies have postulated a link between EGFR activation and subsequent COX-2 upregulation. The relationship between these factors has not been established in patients with NSCLC. COX-2 and EGFR expression were studied in 172 NSCLC specimens using standard immunohistochemical techniques. Western blotting was used to determine COX-2 and EGFR levels in five NSCLC cell lines. The effect of treatment with EGF on COX-2 expression in A549 cells was assessed. Results: Both EGFR and COX-2 are overexpressed in NSCLC. The predominant pattern of COX-2 and EGFR staining was cytoplasmic. Membranous EGFR staining was seen in 23.3% of cases. There was no relationship between COX-2 and EGFR expression and survival or any clinicopathological features. No correlation was seen between EGFR expression and COX-2 expression in the immunohistochemical series or in the cell lines. Treatment with EGF did not upregulate COX-2 levels in A549 cells, either in serum free or serum-supplemented conditions. Conclusions: Although COX-2 and EGFR are over-expressed in NSCLC neither was of prognostic significance in this series of cases. There is no correlation between these two factors in either tumour samples or cell lines. Although these factors show no correlation in NSCLC, they remain potential, though independent targets for treatment. © 2004 Elsevier Ireland Ltd. All rights reserved.

Estrogen receptor β activation impairs mitochondrial oxidative metabolism and affects malignant mesothelioma cell growth in vitro and in vivo

Estrogen receptor (ER)-β has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERβ coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERβ, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERβ agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERβ-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma.

Interleukin-6 is a potent inducer of S100P, which is up-regulated in androgen-refractory and metastatic prostate cancer

Elevated circulating interleukin-6 (IL6) and up-regulated S100P in prostate cancer (PCa) specimens correlate independently with progression to androgen-independent and metastatic PCa. The cause of up-regulated S100P levels in advanced PCa remains to be determined. We investigated the possibility that IL6 is an inducer of S100P. Determination of mRNA and protein levels by real-time PCR and Western blotting revealed that IL6 is a more potent inducer of S100P than the synthetic androgen, R1881, in the LNCaP/C4-2B model of PCa progression. IL6 did not require androgen to induce S100P in these cells, which express a functional androgen receptor (AR). Like R1881, IL6 was unable to induce S100P in PC3 cells that lack a functional AR. IL6 did not strongly induce the AR-dependent genes PSA and KLK2 and, contrary to R1881, down-regulated Cyr61/CCN1, a potential marker that is down-regulated in PCa. Epidermal growth factor (EGF), which like IL6 is a non-androgen activator of the AR, did not induce S100P. The data identifies a unique gene-induction profile for IL6 and suggests that IL6 may require a functional AR for S100P induction. A link between elevated IL6 and up-regulated S100P in androgen-refractory and metastatic PCa is postulated.

A conjugative plasmid carrying the carbapenem resistance gene blaOXA-23 in AbaR4 in an extensively resistant GC1 Acinetobacter baumannii isolate

OBJECTIVES: To locate the acquired bla(OXA-23) carbapenem resistance gene in an Australian A. baumannii global clone 1 (GC1) isolate. METHODS: The genome of the extensively antibiotic-resistant GC1 isolate A85 harbouring bla(OXA-23) in Tn2006 was sequenced using Illumina HiSeq, and the reads were used to generate a de novo assembly. PCR was used to assemble relevant contigs. Sequences were compared with ones in GenBank. Conjugation experiments were conducted. RESULTS: The sporadic GC1 isolate A85, recovered in 2003, was extensively resistant, exhibiting resistance to imipenem, meropenem and ticarcillin/clavulanate, to cephalosporins and fluoroquinolones and to the older antibiotics gentamicin, kanamycin and neomycin, sulfamethoxazole, trimethoprim and tetracycline. Genes for resistance to older antibiotics are in the chromosome, in an AbaR3 resistance island. A second copy of the ampC gene in Tn6168 confers cephalosporin resistance and the gyrA and parC genes have mutations leading to fluoroquinolone resistance. An 86 335 bp repAci6 plasmid, pA85-3, carrying bla(OXA-23) in Tn2006 in AbaR4, was shown to transfer imipenem, meropenem and ticarcillin/clavulanate resistance into a susceptible recipient. A85 also contains two small cryptic plasmids of 2.7 and 8.7 kb. A85 is sequence type ST126 (Oxford scheme) and carries a novel KL15 capsule locus and the OCL3 outer core locus. CONCLUSIONS: A85 represents a new GC1 lineage identified by the novel capsule locus but retains AbaR3 carrying genes for resistance to older antibiotics. Resistance to imipenem, meropenem and ticarcillin/clavulanate has been introduced into A85 by pA85-3, a repAci6 conjugative plasmid carrying Tn2006 in AbaR4.

Relationship of a variant in the NTRK1 gene to white matter microstructure in young adults

The NTRK1 gene (also known as TRKA) encodes a high-affinity receptor for NGF, a neurotrophin involved in nervous system development and myelination. NTRK1 has been implicated in neurological function via links between the T allele at rs6336 (NTRK1-T) and schizophrenia risk. A variant in the neurotrophin gene, BDNF, was previously associated with white matter integrity in young adults, highlighting the importantce of neurotrophins to white matter development. We hypothesized that NTRK1-T would relate to lower fractional anisotropy in healthy adults. We scanned 391 healthy adult human twins and their siblings (mean age: 23.6 ± 2.2 years; 31 NTRK1-T carriers, 360 non-carriers) using 105-gradient diffusion tensor imaging at 4 tesla. We evaluated in brain white matter how NTRK1-T and NTRK1 rs4661063 allele A (rs4661063-A, which is in moderate linkage disequilibrium with rs6336) related to voxelwise fractional anisotropy-acommondiffusion tensor imaging measure of white matter microstructure. We used mixed-model regression to control for family relatedness, age, and sex. The sample was split in half to test reproducibility of results. The false discovery rate method corrected for voxelwise multiple comparisons. NTRK1-T and rs4661063-A correlated with lower white matter fractional anisotropy, independent of age and sex (multiple-comparisons corrected: false discovery rate critical p=0.038 forNTRK1-Tand0.013 for rs4661063-A). In each half-sample, theNTRK1-T effectwasreplicated in the cingulum, corpus callosum, superior and inferior longitudinal fasciculi, inferior fronto-occipital fasciculus, superior corona radiata, and uncinate fasciculus. Our results suggest that NTRK1-T is important for developing white matter microstructure.