Investigating the genetic association between ERAP1 and ankylosing spondylitis

A strong association between ERAP1 and ankylosing spondylitis (AS) was recently identified by the Wellcome Trust Case Control Consortium and the Australo-Anglo-American Spondylitis Consortium (WTCCC-TASC) study. ERAP1 is highly polymorphic with strong linkage disequilibrium evident across the gene. We therefore conducted a series of experiments to try to identify the primary genetic association(s) with ERAP1. We replicated the original associations in an independent set of 730 patients and 1021 controls, resequenced ERAP1 to define the full extent of coding polymorphisms and tested all variants in additional association studies. The genetic association with ERAP1 was independently confirmed; the strongest association was with rs30187 in the replication set (P = 3.4 × 103). When the data were combined with the original WTCCC-TASC study the strongest association was with rs27044 (P = 1.1 × 10-9). We identified 33 sequence polymorphisms in ERAP1, including three novel and eight known non-synonymous polymorphisms. We report several new associations between AS and polymorphisms distributed across ERAP1 from the extended case-control study, the most significant of which was with rs27434 (P = 4.7 × 10-7). Regression analysis failed to identify a primary association clearly; we therefore used data from HapMap to impute genotypes for an additional 205 non-coding SNPs located within and adjacent to ERAP1. A number of highly significant associations (P < 5 × 10-9) were identified in regulatory sequences which are good candidates for causing susceptibility to AS, possibly by regulating ERAP1 expression. © 2009 The Author(s).

Genetic analyses in a sample of individuals with high or low BMD shows association with multiple Wnt pathway genes

INTRODUCTION Although the high heritability of BMD variation has long been established, few genes have been conclusively shown to affect the variation of BMD in the general population. Extreme truncate selection has been proposed as a more powerful alternative to unselected cohort designs in quantitative trait association studies. We sought to test these theoretical predictions in studies of the bone densitometry measures BMD, BMC, and femoral neck area, by investigating their association with members of the Wnt pathway, some of which have previously been shown to be associated with BMD in much larger cohorts, in a moderate-sized extreme truncate selected cohort (absolute value BMD Z-scores = 1.5-4.0; n = 344). MATERIALS AND METHODS Ninety-six tag-single nucleotide polymorphism (SNPs) lying in 13 Wnt signaling pathway genes were selected to tag common genetic variation (minor allele frequency [MAF] > 5% with an r(2) > 0.8) within 5 kb of all exons of 13 Wnt signaling pathway genes. The genes studied included LRP1, LRP5, LRP6, Wnt3a, Wnt7b, Wnt10b, SFRP1, SFRP2, DKK1, DKK2, FZD7, WISP3, and SOST. Three hundred forty-four cases with either high or low BMD were genotyped by Illumina Goldengate microarray SNP genotyping methods. Association was tested either by Cochrane-Armitage test for dichotomous variables or by linear regression for quantitative traits. RESULTS Strong association was shown with LRP5, polymorphisms of which have previously been shown to influence total hip BMD (minimum p = 0.0006). In addition, polymorphisms of the Wnt antagonist, SFRP1, were significantly associated with BMD and BMC (minimum p = 0.00042). Previously reported associations of LRP1, LRP6, and SOST with BMD were confirmed. Two other Wnt pathway genes, Wnt3a and DKK2, also showed nominal association with BMD. CONCLUSIONS This study shows that polymorphisms of multiple members of the Wnt pathway are associated with BMD variation. Furthermore, this study shows in a practical trial that study designs involving extreme truncate selection and moderate sample sizes can robustly identify genes of relevant effect sizes involved in BMD variation in the general population. This has implications for the design of future genome-wide studies of quantitative bone phenotypes relevant to osteoporosis.

Breakthroughs in genetic studies of ankylosing spondylitis

Ankylosing spondylitis (AS), the prototypic seronegative arthropathy, is known to be highly heritable, with >90% of the risk of developing the disease determined genetically. As with most common heritable diseases, progress in identifying the genes involved using family-based or candidate gene approaches has been slow. The recent development of the genome-wide association study approach has revolutionized genetic studies of such diseases. Early studies in ankylosing spondylitis have produced two major breakthroughs in the identification of genes contributing roughly one third of the population attributable risk of the disease, and pointing directly to a potential therapy. These exciting findings highlight the potential of future more comprehensive genetic studies of determinants of disease risk and clinical manifestations, and are the biggest advance in our understanding of the causation of the disease since the discovery of the association with HLA-B27.

Genetic, functional, and histopathological evaluation of two C-terminal BRCA1 missense variants

Background: The vast majority of BRCA1 missense sequence variants remain uncharacterised for their possible effect on protein expression and function, and therefore are unclassified in terms of their pathogenicity. BRCA1 plays diverse cellular roles and it is unlikely that any single functional assay will accurately reflect the total cellular implications of missense mutations in this gene. Objective: To elucidate the effect of two BRCA1 variants, 5236G>C (G1706A) and 5242C>A (A1708E) on BRCA1 function, and to survey the relative usefulness of several assays to direct the characterisation of other unclassified variants in BRCA genes. Methods and Results: Data from a range of bioinformatic, genetic, and histopathological analyses, and in vitro functional assays indicated that the 1708E variant was associated with the disruption of different cellular functions of BRCA1. In transient transfection experiments in T47D and 293T cells, the 1708E product was mislocalised to the cytoplasm and induced centrosome amplification in 293T cells. The 1708E variant also failed to transactivate transcription of reporter constructs in mammalian transcriptional transactivation assays. In contrast, the 1706A variant displayed a phenotype comparable to wildtype BRCA1 in these assays. Consistent with functional data, tumours from 1708E carriers showed typical BRCA1 pathology, while tumour material from 1706A carriers displayed few histopathological features associated with BRCA1 related tumours. Conclusions: A comprehensive range of genetic, bioinformatic, and functional analyses have been combined for the characterisation of BRCA1 unclassified sequence variants. Consistent with the functional analyses, the combined odds of causality calculated for the 1706A variant after multifactorial likelihood analysis (1:142) indicates a definitive classification of this variant as "benign". In contrast, functional assays of the 1708E variant indicate that it is pathogenic, possibly through subcellular mislocalisation. However, the combined odds of 262:1 in favour of causality of this variant does not meet the minimal ratio of 1000:1 for classification as pathogenic, and A1708E remains formally designated as unclassified. Our findings highlight the importance of comprehensive genetic information, together with detailed functional analysis for the definitive categorisation of unclassified sequence variants. This combination of analyses may have direct application to the characterisation of other unclassified variants in BRCA1 and BRCA2.

The v-MFG test: Investigating maternal, offspring and maternal-fetal genetic incompatibility effects on disease and viability

The MFG test is a family-based association test that detects genetic effects contributing to disease in offspring, including offspring allelic effects, maternal allelic effects and MFG incompatibility effects. Like many other family-based association tests, it assumes that the offspring survival and the offspring-parent genotypes are conditionally independent provided the offspring is affected. However, when the putative disease-increasing locus can affect another competing phenotype, for example, offspring viability, the conditional independence assumption fails and these tests could lead to incorrect conclusions regarding the role of the gene in disease. We propose the v-MFG test to adjust for the genetic effects on one phenotype, e.g., viability, when testing the effects of that locus on another phenotype, e.g., disease. Using genotype data from nuclear families containing parents and at least one affected offspring, the v-MFG test models the distribution of family genotypes conditional on offspring phenotypes. It simultaneously estimates genetic effects on two phenotypes, viability and disease. Simulations show that the v-MFG test produces accurate genetic effect estimates on disease as well as on viability under several different scenarios. It generates accurate type-I error rates and provides adequate power with moderate sample sizes to detect genetic effects on disease risk when viability is reduced. We demonstrate the v-MFG test with HLA-DRB1 data from study participants with rheumatoid arthritis (RA) and their parents, we show that the v-MFG test successfully detects an MFG incompatibility effect on RA while simultaneously adjusting for a possible viability loss.

Genetic disorders of the LRP5-Wnt signalling pathway affecting the skeleton

Osteoporosis is a common, increasingly prevalent and potentially debilitating condition of men and women. Genetic factors are major determinants of bone mass and the risk of fracture, but few genes have been definitively demonstrated to be involved. The identification of these factors will provide novel insights into the processes of bone formation and loss and thus the pathogenesis of osteoporosis, enabling the rational development of novel therapies. In this article, we present the extensive genetic and functional data indicating that the LRP5 gene and the Wnt signalling pathway are key players in bone formation and the risk of osteoporosis, and that LRP5 signalling is essential for normal morphology, developmental processes and bone health.

Genetic and genomic studies of PADI4 in rheumatoid arthritis

Objectives. Strong genetic association of rheumatoid arthritis (RA) with PADI4 (peptidyl arginine deiminase) has previously been described in Japanese, although this was not confirmed in a subsequent study in the UK. We therefore undertook a further study of genetic association between PADI4 and RA in UK Caucasians and also studied expression of PADI4 in the peripheral blood of patients with RA. Methods. Seven single-nucleotide polymorphisms (SNP) were genotyped using polymerase chain reaction (PCR)-restriction fragment length polymorphism in 111 RA cases and controls. A marker significantly associated with RA (PADI4_100, rs#2240339) in this first data set (P = 0.03) was then tested for association in a larger group of 439 RA patients and 428 controls. PADI4 transcription was also assessed by real-time quantitative PCR using RNA extracted from peripheral blood mononuclear cells from 13 RA patients and 11 healthy controls. Results. A single SNP was weakly associated with RA (P = 0.03) in the initial case-control study, a single SNP (PADI4_100) and a two marker haplotype of that SNP and the neighbouring SNP (PADI4_04) were significantly associated with RA (P = 0.02 and P = 0.03 respectively). PADI4_100 was not associated with RA in a second sample set. PADI4 expression was four times greater in cases than controls (P = 0.004), but expression levels did not correlate with the levels of markers of inflammation. Conclusion. PADI4 is significantly overexpressed in the blood of RA patients but genetic variation within PADI4 is not a major risk factor for RA in Caucasians.

Genetic studies of disorders of calcium crystal deposition

In summary, although many factors are likely to be involved in regulating calcification and ossification processes, studies of the causation of articular chondrocalcinosis and disorders of spinal ossification, such as DISH and OPLL, implicate control over inorganic pyrophosphate levels as being one of the most important factors in their aetiopathogenesis. The findings of these studies may prove relevant to other rheumatic diseases in which ectopic ossification occurs, such as AS.

Genetic control of bone density and turnover: Role of the collagen 1α1, estrogen receptor, and vitamin D receptor genes

Genetic factors are known to influence both the peak bone mass and probably the rate of change in bone density. A range of regulatory and structural genes has been proposed to be involved including collagen 1α1 (COL1A1), the estrogen receptor (ER), and the vitamin D receptor (VDR), but the actual genes involved are uncertain. We therefore studied the role of the COL1A1 and VDR loci in control of bone density by linkage in 45 dizygotic twin pairs and 29 nuclear families comprising 120 individuals. The influences on bone density of polymorphisms of COL1A1, VDR, and ER were studied by association both cross-sectionally and longitudinally in 193 elderly postmenopausal women (average age, 69 years) over a mean follow-up time of 6.3 years. Weak linkage of the COL1A1 locus with bone density was observed in both twins and families (p = 0.02 in both data sets), confirming previous observations of linkage of this locus with bone density. Association between the MscI polymorphism of COL1A1 and rate of lumbar spine bone loss was observed with significant gene-environment interaction related to dietary calcium intake (p = 0.0006). In the lowest tertile of dietary calcium intake, carriers of "s" alleles lost more bone than "SS" homozygotes (p = 0.01), whereas the opposite was observed in the highest dietary calcium intake (p = 0.003). Association also was observed between rate of bone loss at both the femoral neck and the lumbar spine and the TaqI VDR polymorphism (p = 0.03). This association was strongest in those in the lowest tertile of calcium intake, also suggesting the presence of gene-environment interaction involving dietary calcium and VDR, influencing bone turnover. No significant association was observed between the PvuII ER polymorphism alone or in combination with VDR or COL1A1 genotypes, with either bone density or its rate of change. These data support the involvement of COL1A1 in determination of bone density and the interaction of both COL1A1 and VDR with calcium intake in regulation of change of bone density over time.

The emerging role of extracellular vesicle-mediated drug resistance in cancers: Implications in advanced prostate cancer

Emerging evidence has shown that the extracellular vesicles (EVs) regulate various biological processes and can control cell proliferation and survival, as well as being involved in normal cell development and diseases such as cancers. In cancer treatment, development of acquired drug resistance phenotype is a serious issue. Recently it has been shown that the presence of multidrug resistance proteins such as Pgp-1 and enrichment of the lipid ceramide in EVs could have a role in mediating drug resistance. EVs could also mediate multidrug resistance through uptake of drugs in vesicles and thus limit the bioavailability of drugs to treat cancer cells. In this review, we discussed the emerging evidence of the role EVs play in mediating drug resistance in cancers and in particular the role of EVs mediating drug resistance in advanced prostate cancer. The role of EV-associated multidrug resistance proteins, miRNA, mRNA, and lipid as well as the potential interaction(s) among these factors was probed. Lastly, we provide an overview of the current available treatments for advanced prostate cancer, considering where EVs may mediate the development of resistance against these drugs.

Growth hormone resistance and somatomedins in children with end-stage liver disease awaiting transplantation

Background: The success of orthotopic liver transplantation as treatment for end-stage liver disease has prompted investigation of strategies to maintain or improve nutrition and growth in children awaiting transplantation, because malnutrition is an adverse prognostic factor. The purpose of this study was to evaluate the effect of recombinant human growth hormone therapy on body composition and indices of liver function in patients awaiting transplant. Methods: The study was designed as a placebo- controlled, double-blind, crossover trial. Patients received 0.2 U/kg growth hormone, subcutaneously, or placebo daily for 28 days during two treatment periods, separated by a 2-week washout period. Ten patients (mean age, 3.06 ± 1.15 years; range, 0.51-11.65 years, five men), with extrahepatic biliary atresia (n = 8) or two with Alagille's syndrome (n = 2), with end-stage liver disease, completed the trial while awaiting orthotopic liver transplantation. Height, weight, total body potassium, total body fat, resting energy expenditure, respiratory quotient, hematologic and multiple biochemical profile, number of albumin infusions, insulin-like growth factor-1 and 1, growth hormone binding protein (GHBP), and insulin-like growth factor binding protein-1 (IGFBP-1) and insulin-like growth factor binding protein (IGFBP-3) were measured at the beginning and end of each treatment period. Results: Growth hormone treatment was associated with a significant decline in serum bilirubin (-34.6 ± 16.5 μmol/l vs. 18.2 ± 11.59 μmol/l; p < 0.02) but there was no significant effect on any anthropometric or body composition measurements, or on any biochemical or hematologic parameters. Conclusions: These children with end-stage liver disease displayed growth hormone resistance, particularly in relation to the somatomedin axis. Exogenous growth hormone administration may be of limited value in these patients

Electrical Conductivity and Magneto-Resistance Studies on Polyaniline-Pmma Blends at Very Low Temperature

D.C. electrical conductivity of polyaniline (33%,40%) blended with PMMA was measured from 5K to 300mK. The conductivity behaviour is consistent with fluctuation induced tunneling. Magneto-resistance (MR) was measured between 300K and 2K. From 20K to 2K, a large positive MR was observed. At 2K, for low magnetic fields (<1 Tesla), a deviation from the normal H-2 behaviour was observed.

Engineering cellulose nanofibre suspensions to control filtration resistance and sheet permeability

This study examines and quantifies the effect of adding polyelectrolytes to cellulose nanofibre suspensions on the gel point of cellulose nanofibre suspensions, which is the lowest solids concentration at which the suspension forms a continuous network. The lower the gel point, the faster the drainage time to produce a sheet and the higher the porosity of the final sheet formed. Two new techniques were designed to measure the dynamic compressibility and the drainability of nanocellulose–polyelectrolyte suspensions. We developed a master curve which showed that the independent variable controlling the behaviour of nanocellulose suspensions and its composite is the structure of the flocculated suspension which is best quantified as the gel point. This was independent of the type of polyelectrolyte used. At an addition level of 2 mg/g of nanofibre, a reduction in gel point over 50 % was achieved using either a high molecular weight (13 MDa) linear cationic polyacrylamide (CPAM, 40 % charge), a dendrimer polyethylenimine of high molecular weight of 750,000 Da (HPEI) or even a low molecular weight of 2000 Da (LPEI). There was no significant difference in the minimum gel point achieved, despite the difference in polyelectrolyte morphology and molecular weight. In this paper, we show that the gel point controls the flow through the fibre suspension, even when comparing fibre suspensions with solids content above the gel point. A lower gel point makes it easier for water to drain through the fibre network,reducing the pressure required to achieve a given dewatering rate and reducing the filtering time required to form a wet laid sheet. We further show that the lower gel point partially controls the structure of the wet laid sheet after it is dried. Halving the gel point increased the air permeability of the dry sheet by 37, 46 and 25 %, when using CPAM, HPEI and LPEI, respectively. The resistance to liquid flow was reduced by 74 and 90 %, when using CPAM and LPEI. Analysing the paper formed shows that sheet forming process and final sheet properties can be engineered and controlled by adding polyelectrolytes to the nanofibre suspension.

Resistance Noise in Electrically Biased Bilayer Graphene

We demonstrate that the low-frequency resistance uctuations, or noise, in bilayer graphene is strongly connected to its band structure, and displays a minimum when the gap between the conduction and valence band is zero. Using double-gated bilayer graphene devices we have tuned the zero gap and charge neutrality points independently, which oers a versatile mechanism to investigate the low-energy band structure, charge localization and screening properties of bilayer graphene.

Nature and extent of genetic diversity of dengue viruses determined by 454 pyrosequencing

Dengue virus (DENV) populations are characteristically highly diverse. Regular lineage extinction and replacement is an important dynamic DENV feature, and most DENV lineage turnover events are associated with increased incidence of disease. The role of genetic diversity in DENV lineage extinctions is not understood. We investigated the nature and extent of genetic diversity in the envelope (E) gene of DENV serotype 1 representing different lineages histories. A region of the DENV genome spanning the E gene was amplified and sequenced by Roche/454 pyrosequencing. The pyrosequencing results identified distinct sub-populations (haplotypes) for each DENV-1 E gene. A phylogenetic tree was constructed with the consensus DENV-1 E gene nucleotide sequences, and the sequences of each constructed haplotype showed that the haplotypes segregated with the Sanger consensus sequence of the population from which they were drawn. Haplotypes determined through pyrosequencing identified a recombinant DENV genome that could not be identified through Sanger sequencing. Nucleotide level sequence diversities of DENV-1 populations determined from SNP analysis were very low, estimated from 0.009-0.01. There were also no stop codon, frameshift or non-frameshift mutations observed in the E genes of any lineage. No significant correlations between the accumulation of deleterious mutations or increasing genetic diversity and lineage extinction were observed (p>0.5). Although our hypothesis that accumulation of deleterious mutations over time led to the extinction and replacement of DENV lineages was ultimately not supported by the data, our data does highlight the significant technical issues that must be resolved in the way in which population diversity is measured for DENV and other viruses. The results provide an insight into the within-population genetic structure and diversity of DENV-1 populations.