The molecular mechanisms involved in the development of type 2 diabetes are poorly understood. Starting from genome-wide genotype data for 1,924 diabetic cases and 2,938 population controls generated by the Wellcome Trust Case Control Consortium, we set out to detect replicated diabetes association signals through analysis of 3,757 additional cases and 5,346 controls, and by integration of our findings with equivalent data from other international consortia. We detected diabetes susceptibility loci in and around the genes CDKAL1, CDKN2A/CDKN2B and IGF2BP2 and confirmed the recently described associations at HHEX/IDE and SLC30A8. Our findings provide insights into the genetic architecture of type 2 diabetes, emphasizing the contribution of multiple variants of modest effect. The regions identified underscore the importance of pathways influencing pancreatic beta cell development and function in the etiology of type 2 diabetes.
For most associations of common single nucleotide polymorphisms (SNPs) with common diseases, the genetic model of inheritance is unknown. The authors extended and applied a Bayesian meta-analysis approach to data from 19 studies on 17 replicated associations with type 2 diabetes. For 13 SNPs, the data fitted very well to an additive model of inheritance for the diabetes risk allele; for 4 SNPs, the data were consistent with either an additive model or a dominant model; and for 2 SNPs, the data were consistent with an additive or recessive model. Results were robust to the use of different priors and after exclusion of data for which index SNPs had been examined indirectly through proxy markers. The Bayesian meta-analysis model yielded point estimates for the genetic effects that were very similar to those previously reported based on fixed- or random-effects models, but uncertainty about several of the effects was substantially larger. The authors also examined the extent of between-study heterogeneity in the genetic model and found generally small between-study deviation values for the genetic model parameter. Heterosis could not be excluded for 4 SNPs. Information on the genetic model of robustly replicated association signals derived from genome-wide association studies may be useful for predictive modeling and for designing biologic and functional experiments.
Bayes theorem; diabetes mellitus, type 2; meta-analysis; models, genetic; polymorphism, genetic; population characteristics
For most associations of common polymorphisms with common diseases, the genetic model of inheritance is unknown. We extended and applied a Bayesian meta-analysis approach to data from 19 studies on 17 replicated associations for type 2 diabetes. For 13 polymorphisms, the data fit very well to an additive model, for 4 polymorphisms the data were consistent with either an additive or dominant model, and for 2 polymorphisms with an additive or recessive model of inheritance for the diabetes risk allele. Results were robust to using different priors and after excluding data where index polymorphisms had been examined indirectly through proxy markers. The Bayesian meta-analysis model yielded point estimates for the genetic effects that are very similar to those previously reported based on fixed or random effects models, but uncertainty about several of the effects was substantially larger. We also examined the extent of between-study heterogeneity in the genetic model and found generally small values of the between-study deviation for the genetic model parameter. Heterosis could not be excluded in 4 SNPs. Information on the genetic model of robustly replicated GWA-derived association signals may be useful for predictive modeling, and for designing biological and functional experiments.
To establish the incidence of nontraumatic lower-extremity amputation (LEA) in people with diabetes in Scotland.
RESEARCH DESIGN AND METHODS
This cohort study linked national morbidity records and diabetes datasets to establish the number of people with diabetes who underwent nontraumatic major and minor LEA in Scotland from 2004 to 2008.
Two thousand three hundred eighty-two individuals with diabetes underwent a nontraumatic LEA between 2004 and 2008; 57.1% (n = 1,359) underwent major LEAs. The incidence of any LEA among persons with diabetes fell over the 5-year study period by 29.8% (3.04 per 1,000 in 2004 to 2.13 per 1,000 in 2008, P < 0.001). Major LEA rates decreased by 40.7% from 1.87 per 1,000 in 2004 to 1.11 per 1,000 in 2008 (P < 0.001).
There has been a significant reduction in the incidence of LEA in persons with diabetes in Scotland between 2004 and 2008, principally explained by a reduction in major amputation.
Plasma sphingosine-1-phosphate (S1P) regulates vascular permeability, and plasma and lymph S1P guide lymphocyte egress from lymphoid organs. S1P is made intracellularly, and little is known about how S1P is delivered into circulatory fluids. Here we find that mice without the major facilitator superfamily transporter Spns2 have a profound reduction in lymph S1P, but only a minor decrease in plasma S1P. Spns2-deficient mice have a redistribution of lymphocytes from the spleen to lymph nodes and a loss of circulating lymphocytes, consistent with normal egress from the spleen directed by plasma S1P and blocked egress from lymph nodes directed by lymph S1P. Spns2 is needed in endothelial cells to supply lymph S1P and support lymphocyte circulation. As the first differential requirement for lymph and blood S1P to our knowledge, Spns2 may be an attractive target for immune suppressive drugs.
The purpose of this study was to determine effects of amlodipine, a dihydropyridine calcium channel blocker, on development of angiotensin II (AngII)-induced vascular pathologies.
Methods and Results
Male LDL receptor -/- mice were infused with vehicle, amlodipine (5 mg/kg/d), AngII (1,000 ng/kg/min), or AngII + amlodipine for 4 weeks through osmotic pumps (n=10/group). Mice were fed a saturated fat-enriched diet for 1 week prior to pump implantation and during 4 weeks of infusion. Infusion of amlodipine resulted in plasma concentrations of 32 ± 2 ng/ml and 27 ± 2 ng/ml for mice in saline + amlodipine and AngII + amlodipine groups, respectively. This infusion rate of amlodipine did not affect AngII-induced increases in systolic blood pressure. Three of 10 (30%) mice infused with AngII died of aortic rupture, while aortic rupture did not occur in mice co-infused with AngII + amlodipine. Suprarenal aortic width and intimal area of ascending aortas were measured to define aortic aneurysms. In the absence of AngII infusion, amlodipine did not change suprarenal aortic width and ascending aortic area. Infusion of AngII led to profound increases of suprarenal aortic width (saline + vehicle versus AngII + vehicle: 0.86 ± 0.02 versus 1.72 ± 0.26 mm; P=0.0006), whereas co-infusion of AngII and amlodipine diminished abdominal dilation (1.02 ± 0.14 mm; P=0.003). As expected, AngII infusion increased mean intimal area of ascending aortas (saline + vehicle versus AngII + vehicle: 8.5 ± 0.3 versus 12.5 ± 1.1 mm2; P=0.001), while co-infusion of AngII and amlodipine ablated dilation of the ascending aorta (8.6 ± 0.2 mm2; P=0.03). Co-administration of amlodipine also significantly attenuated AngII-induced atherosclerosis in the thoracic region as quantified by percent lesion area (AngII + vehicle versus AngII + amlodipine: 5.8 ± 2.1 % versus 0.3 ± 0.1%; P=0.05).
Amlodipine inhibited AngII-induced aortic aneurysms in both the abdominal and ascending regions, and atherosclerosis in hypercholesterolemic mice.
Without intervention, classic galactosemia is a potentially fatal disorder in infancy. With the benefit of early diagnosis and dietary restriction of galactose, the acute sequelae of classic galactosemia can be prevented or reversed. However, despite early and lifelong dietary treatment, many galactosemic patients go on to experience serious long-term complications including cognitive disability, speech problems, neurological and/or movement disorders and, in girls and women, ovarian dysfunction. Further, there remains uncertainty surrounding what constitutes a ‘best practice’ for treating this disorder. To explore the extent and implications of this uncertainty, we conducted a small but global survey of healthcare providers who follow patients with classic galactosemia, seeking to compare established protocols for diagnosis, intervention, and follow-up, as well as the outcomes and outcome frequencies seen in the patient populations cared for by these providers. We received 13 survey responses representing five continents and 11 countries. Respondents underscored disparities in approaches to diagnosis, management and follow-up care. Notably, we saw no clear relationship between differing approaches to care and long-term outcomes in the populations studied. Negative outcomes occurred in the majority of cases regardless of when treatment was initiated, how tightly galactose intake was restricted, or how closely patients were monitored. We document here what is, to our knowledge, the first global comparison of healthcare approaches to classic galactosemia. These data reinforce the idea that there is currently no one best practice for treating patients with classic galactosemia, and underscore the need for more extensive and statistically powerful comparative studies to reveal potential positive or negative impacts of differing approaches.
To further investigate susceptibility loci identified by genome-wide association studies, we genotyped 5,500 SNPs across 14 associated regions in 8,000 samples from a control group and 3 diseases: type 2 diabetes (T2D), coronary artery disease (CAD) and Graves’ disease. We defined, using Bayes theorem, credible sets of SNPs that were 95% likely, based on posterior probability, to contain the causal disease-associated SNPs. In 3 of the 14 regions, TCF7L2 (T2D), CTLA4 (Graves’ disease) and CDKN2A-CDKN2B (T2D), much of the posterior probability rested on a single SNP, and, in 4 other regions (CDKN2A-CDKN2B (CAD) and CDKAL1, FTO and HHEX (T2D)), the 95% sets were small, thereby excluding most SNPs as potentially causal. Very few SNPs in our credible sets had annotated functions, illustrating the limitations in understanding the mechanisms underlying susceptibility to common diseases. Our results also show the value of more detailed mapping to target sequences for functional studies.
We carried out a genome wide association study of type-2 diabetes (T2D) amongst 20,119 people of South Asian ancestry (5,561 with T2D); we identified 20 independent SNPs associated with T2D at P<10−4 for testing amongst a further 38,568 South Asians (13,170 with T2D). In combined analysis, common genetic variants at six novel loci (GRB14, ST6GAL1, VPS26A, HMG20A, AP3S2 and HNF4A) were associated with T2D (P=4.1×10−8 to P=1.9×10−11); SNPs at GRB14 were also associated with insulin sensitivity, and at ST6GAL1 and HNF4A with pancreatic beta-cell function respectively. Our findings provide additional insight into mechanisms underlying T2D, and demonstrate the potential for new discovery from genetic association studies in South Asians who have increased susceptibility to T2D.
Humans display structural and functional asymmetries in brain organization, strikingly with respect to language and handedness. The molecular basis of these asymmetries is unknown. We report a genome-wide association study meta-analysis for a quantitative measure of relative hand skill in individuals with dyslexia [reading disability (RD)] (n = 728). The most strongly associated variant, rs7182874 (P = 8.68×10−9), is located in PCSK6, further supporting an association we previously reported. We also confirmed the specificity of this association in individuals with RD; the same locus was not associated with relative hand skill in a general population cohort (n = 2,666). As PCSK6 is known to regulate NODAL in the development of left/right (LR) asymmetry in mice, we developed a novel approach to GWAS pathway analysis, using gene-set enrichment to test for an over-representation of highly associated variants within the orthologs of genes whose disruption in mice yields LR asymmetry phenotypes. Four out of 15 LR asymmetry phenotypes showed an over-representation (FDR≤5%). We replicated three of these phenotypes; situs inversus, heterotaxia, and double outlet right ventricle, in the general population cohort (FDR≤5%). Our findings lead us to propose that handedness is a polygenic trait controlled in part by the molecular mechanisms that establish LR body asymmetry early in development.
Humans have developed a population level bias towards right-handedness for tool-use. Understanding the genetic basis of handedness can help explain why this bias exists and may offer clues into the evolution of handedness and brain asymmetry. We have tested for correlation between relative hand skill and hundreds of thousands of genetic variants in a cohort of individuals with reading disability. The strongest associated variant is in the gene PCSK6, an enzyme that cleaves NODAL into an active form. NODAL plays a key role during the establishment of left/right (LR) asymmetry in diverse species, from snails to mammals. Pcsk6 knock-out mice display LR asymmetry defects like heterotaxia (abnormal organ positioning). We uncovered further variants associated with relative hand skill in the human versions of genes that also cause the LR asymmetry phenotypes heterotaxia, and situs inversus (reversal of organ asymmetry) when knocked out in mice. These results replicate in an independent general population cohort without reading disability. We propose that handedness is under the control of many variants, some of which are in genes that also contribute to the determination of body LR asymmetry.
Multiple rare variants either within or across genes have been hypothesised to collectively influence complex human traits. The increasing availability of high throughput sequencing technologies offers the opportunity to study the effect of rare variants on these traits. However, appropriate and computationally efficient analytical methods are required to account for collections of rare variants that display a combination of protective, deleterious and null effects on the trait. We have developed a novel method for the analysis of rare genetic variation in a gene, region or pathway that, by simply aggregating summary statistics at each variant, can: (i) test for the presence of a mixture of effects on a trait; (ii) be applied to both binary and quantitative traits in population-based and family-based data; (iii) adjust for covariates to allow for non-genetic risk factors and; (iv) incorporate imputed genetic variation. In addition, for preliminary identification of promising genes, the method can be applied to association summary statistics, available from meta-analysis of published data, for example, without the need for individual level genotype data. Through simulation, we show that our method is immune to the presence of bi-directional effects, with no apparent loss in power across a range of different mixtures, and can achieve greater power than existing approaches as long as summary statistics at each variant are robust. We apply our method to investigate association of type-1 diabetes with imputed rare variants within genes in the major histocompatibility complex using genotype data from the Wellcome Trust Case Control Consortium.
Rapid advances in sequencing technology mean that it is now possible to directly assay rare genetic variation. In addition, the availability of almost fully sequenced human genomes by the 1000 Genomes Project allows genotyping at rare variants that are not present on arrays commonly used in genome-wide association studies. Rare variants within a gene or region may act to collectively influence a complex trait. Methods for testing these rare variants should be able to account for a combination of those that serve to either increase, decrease or have no effect on the trait of interest. Here, we introduce a method for the analysis of a collection of rare genetic variants, within a gene or region, which assesses evidence for a mixture of effects. Our method simply aggregates summary statistics at each variant and, as such, can be applied to both population and family-based data, to binary or quantitative traits and to either directly genotyped or imputed data. In addition, it does not require individual level genotype or phenotype data, and can be adjusted for non-genetic risk factors. We illustrate our approach by examining imputed rare variants in the major histocompatibility complex for association with type-1 diabetes using genotype data from the Wellcome Trust case Control Consortium.
Lysophosphatidic acids are structurally simple lipid phosphate esters with a now widely appreciated role as extracellular signaling molecules. LPA binds to selective cell surface receptors to promote cell growth, survival, motility and differentiation. Studies using LPA receptor knockout mice and experimental therapeutics targeting these receptors identify roles for LPA signaling in processes that include cardiovascular disease and function, angiogenesis, reproduction, cancer progression and neuropathic pain. These studies identify considerable functional redundancy between these receptors and raise the possibility that additional lysophosphatidic acid receptors remain to be identified. Lysophosphatidic acid is present in the blood and other biological fluids at physiologically relevant concentrations and can likely be rapidly generated and degraded in different locations, for example at sites of inflammation, vascular injury and thrombosis or in the tumor micro environment. Recent work identifies a secreted enzyme, autotaxin, as the key component of an extracellular pathway for generation of lysophosphatidic acid by lysophospholipase D catalyzed hydrolysis of lysophospholipid substrates. In contrast to the apparently redundant functions of LPA receptors, studies using autotaxin knock out and transgenic mice indicate that this enzyme is uniquely required for LPA signaling during early development and serves as the primary determinant of circulating LPA levels in adult animals. Accordingly, pharmacological inhibition of autotaxin may be a viable and potentially effective way to interfere with LPA signaling in the cardiovascular system and possibly other settings such as tumor metastasis for therapeutic benefit. In this review we provide an update on recent advances in defining roles for LPA signaling in major disease processes and discuss recent progress in understanding the regulation and function of autotaxin focusing on strategies for the identification and initial evaluation of small molecule autotaxin inhibitors.
Lysophosphatidic acid; phospholipase; receptor; autotaxin; cancer; cardiovascular disease; molecular therapeutics
Left ventricular hypertrophy has multiple aetiologies including diabetes and genetic factors. We aimed to identify genetic variants predicting left ventricular hypertrophy in diabetic individuals.
Demographic, echocardiographic, prescribing, morbidity, mortality and genotyping databases connected with the Genetics of Diabetes Audit and Research in Tayside, Scotland project were accurately linked using a patient-specific identifier. Left ventricular hypertrophy cases were identified using echocardiographic data.
Genotyping data from 973 cases and 1443 non-left ventricular hypertrophy controls were analysed, investigating whether single nucleotide polymorphisms associated with left ventricular hypertrophy in previous Genome Wide Association Studies predicted left ventricular hypertrophy in our population of individuals with type 2 diabetes. Meta-analysis assessed overall significance of these single nucleotide polymorphisms, which were also used to create gene scores. Logistic regression assessed whether these scores predicted left ventricular hypertrophy.
Two single nucleotide polymorphisms previously associated with left ventricular hypertrophy were significant: rs17132261: OR 2.03, 95% CI 1.10-3.73, p-value 0.02 and rs2292462: OR 0.82, 95% CI 0.73-0.93 and p-value 2.26x10-3. Meta-analysis confirmed rs17132261 and rs2292462 were associated with left ventricular hypertrophy (p=1.03x10-8 and p=5.86x10-10 respectively) and one single nucleotide polymorphisms in IGF1R (rs4966014) became genome wide significant upon meta-analysis although was not significant in our study. Gene scoring based on published single nucleotide polymorphisms also predicted left ventricular hypertrophy in our study.
Rs17132261, within SLC25A46, encodes a mitochondrial phosphate transporter, implying abnormal myocardial energetics contribute to left ventricular hypertrophy development. Rs2292462 lies within the obesity-implicated neuromedin B gene. Rs4966014 lies within the IGF1R1 gene. IGF1 signalling is an established factor in cardiac hypertrophy.
We created a resource to study genetics of left ventricular hypertrophy in diabetes and validated our left ventricular hypertrophy phenotype in replicating single nucleotide polymorphisms identified by previous genome wide association studies investigating left ventricular hypertrophy.
Left ventricular hypertrophy; Genetics; Type 2 diabetes mellitus
SURF1 deficiency, a monogenic mitochondrial disorder, is the most frequent cause of cytochrome c oxidase (COX) deficient Leigh syndrome (LS). We report the first natural history study of SURF1 deficiency.
We conducted a multi-centre case notes review of 44 SURF1-deficient patients from ten different UK centres and two Australian centres. Survival data for LRPPRC-deficient LS and nuclear-encoded complex I-deficient LS patients were obtained from previous publications. The survival of SURF1-deficient patients was compared with these two groups using Kaplan-Meier survival analysis and logrank test.
The majority of patients (32/44, 73%) presented in infancy (median 9.5 months). Frequent symptoms were poor weight gain (95%, median age 10 months), hypotonia (93%, median age 14 months), poor feeding/vomiting (89%, median age 10 months), developmental delay (88%, median age 14 months), developmental regression (71%, median age 19 months), movement disorder (52%, median age 24 months), oculomotor involvement (52%, median age 29 months) and central respiratory failure (78%, median age 31 months). Hypertrichosis (41%), optic atrophy (23%), encephalopathy (20%), seizures (14%) and cardiomyopathy (2%) were observed less frequently.
Lactate was elevated in CSF (mean 4.3 mmol/L) in all patients (30/30) and in blood (mean 4.4 mmol/L) in 31/38 (81%). Fibroblast COX activity was universally decreased (25/25). Normal COX histochemistry was noted in 30% of biopsies, whereas muscle COX activity was reduced in 96% (25/26). Neuroimaging demonstrated lesions characteristic of LS in 28/33 (85%) and atypical findings in 3/33 (9%). Peripheral neuropathy was present in 13/16 (81%) (demyelinating 7/16, axonal 2/16). Kaplan-Meier analysis demonstrated that SURF1-deficient patients experience longer survival (median 5.4 years, p < 0.001) compared to LRPPRC deficiency (median 1.8 years) and nuclear-encoded complex I-deficient LS (median 1.6 years). Survival >10 years was observed in 7 patients, 6 of these patients did not experience neurological regression. The most frequent mutation was c.312_320del10insAT. Five novel mutations (c.468_469delTC, c.799_800delCT, c.575G>A (p.Arg192Gln), c.751+5G>A and c.752-2A>G) were identified.
SURF1-deficient patients have a homogeneous clinical and biochemical phenotype. Early recognition is essential to expedite diagnosis and enable prenatal diagnosis.
SURF1; Leigh syndrome; Cytochrome c oxidase; Complex IV; Mitochondrial disease
Lysophosphatidic acid (LPA) designates a family of bioactive phosphoglycerides that differ in the length and degree of saturation of their radyl chain. Additional diversity is provided by the linkage of the radyl chain to glycerol: acyl, alkyl, or alk-1-enyl. Acyl-LPAs are the predominate species in tissues and biological fluids. Alkyl-LPAs exhibit distinct pharmacodynamics at LPA receptors, potently drive platelet aggregation, and contribute to ovarian cancer aggressiveness. Multiple biosynthetic pathways exist for alkyl-LPA production. Herein we report that diacylglycerol kinases (DGKs) contribute to cell-associated alkyl-LPA production involving phosphorylation of 1-alkyl-2-acetyl glycerol and document the biosynthesis of alkyl-LPA by DGKs in SKOV-3 ovarian cancer cells, specifically identifying the contribution of DGKα. Concurrently, we discovered that treating SKOV-3 ovarian cancer cell with a sphingosine analog stimulates conversion of exogenous 1-alkyl-2-acetyl glycerol to alkyl-LPA, indicating that DGKα contributes significantly to the production of alkyl-LPA in SKOV-3 cells and identifying cross-talk between the sphingolipid and glycerol lipid pathways.
Alkylglycerol phosphate; alkyl lysophosphatidic acid; diacylglycerol kinase
Type 2 diabetes (T2D) is a complex disease characterized by beta cell dysfunctions. Islet amyloid polypeptide (IAPP) is highly conserved and co-secreted with insulin with over 40% of autopsy cases of T2D showing islet amyloid formation due to IAPP aggregation. Dysregulation in IAPP processing, stabilization and degradation can cause excessive oligomerization with beta cell toxicity. Previous studies examining genetic associations of pathways implicated in IAPP metabolism have yielded conflicting results due to small sample size, insufficient interrogation of gene structure and gene-gene interactions. In this multi-staged study, we screened 89 tag single nucleotide polymorphisms (SNPs) in 6 candidate genes implicated in IAPP metabolism and tested for independent and joint associations with T2D and beta cell dysfunctions. Positive signals in the stage-1 were confirmed by de novo and in silico analysis in a multi-centre unrelated case-control cohort. We examined the association of significant SNPs with quantitative traits in a subset of controls and performed bioinformatics and relevant functional analyses. Amongst the tag SNPs, rs1583645 in carboxypeptidase E (CPE) and rs6583813 in insulin degrading enzyme (IDE) were associated with 1.09 to 1.28 fold increased risk of T2D (PMeta = 9.4×10−3 and 0.02 respectively) in a meta-analysis of East Asians. Using genetic risk scores (GRS) with each risk variant scoring 1, subjects with GRS≥3 (8.2% of the cohort) had 56% higher risk of T2D than those with GRS = 0 (P = 0.01). In a subcohort of control subjects, plasma IAPP increased and beta cell function index declined with GRS (P = 0.008 and 0.03 respectively). Bioinformatics and functional analyses of CPE rs1583645 predicted regulatory elements for chromatin modification and transcription factors, suggesting differential DNA-protein interactions and gene expression. Taken together, these results support the importance of dysregulation of IAPP metabolism in T2D in East Asians.
We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.
Biofilm; wrinkled colony; rugose; Vibrio fischeri; Zeiss stemi; dissecting microscope
Lung cell migration is a crucial step for re-epithelialization that in turn is essential for remodeling and repair after lung injury. We hypothesize that secreted autotaxin (ATX), which exhibits lysophospholipase D (lysoPLD) activity, stimulates lung epithelial cell migration through lysophosphatidic acid (LPA) generation-dependent and -independent pathways. Release of endogenous ATX protein and activity was detected in lung epithelial cell culture medium. ATX with V5 tag (ATX-V5) overexpressed conditional medium had higher LPA levels compared to control medium and stimulated cell migration through Gαi-coupled LPA receptors, cytoskeleton rearrangement, phosphorylation of PKCδ and cortactin at the leading edge of migrating cells. Inhibition of PKCδ attenuated ATX-V5 overexpressed conditional medium-mediated phosphorylation of cortactin. In addition, a recombinant ATX mutant, lacking lysoPLD activity, or heat-inactived ATX also induced lung epithelial cell migration. Extracelluar ATX bound to LPA receptor and integrin β4 complex on A549 cell surface. Finally, intratracheal administration of lipopolysaccharide into mouse airway induced ATX release and LPA production in bronchoalveolar lavage fluid. These results suggested a significant role for ATX in lung epithelial cell migration and remodeling through its ability to induce LPA production-mediated phosphorylation of PKCδ and cortactin. In addition we also demonstrated assocation of ATX with epithelial cell surface LPA receptor and integrin β4.
ATX (autotoxin); lysoPLD; LPA; cell migration; signal transduction
Increased adiponectin levels have been shown to be associated with a lower risk of type 2 diabetes. To understand the relations between genetic variation at the adiponectin-encoding gene, ADIPOQ, and adiponectin levels, and subsequently its role in disease, we conducted a deep resequencing experiment of ADIPOQ in 14,002 subjects, including 12,514 Europeans, 594 African Americans, and 567 Indian Asians. We identified 296 single nucleotide polymorphisms (SNPs), including 30 amino acid changes, and carried out association analyses in a subset of 3,665 subjects from two independent studies. We confirmed multiple genome-wide association study findings and identified a novel association between a low-frequency SNP (rs17366653) and adiponectin levels (P = 2.2E–17). We show that seven SNPs exert independent effects on adiponectin levels. Together, they explained 6% of adiponectin variation in our samples. We subsequently assessed association between these SNPs and type 2 diabetes in the Genetics of Diabetes Audit and Research in Tayside Scotland (GO-DARTS) study, comprised of 5,145 case and 6,374 control subjects. No evidence of association with type 2 diabetes was found, but we were also unable to exclude the possibility of substantial effects (e.g., odds ratio 95% CI for rs7366653 [0.91–1.58]). Further investigation by large-scale and well-powered Mendelian randomization studies is warranted.
Bioactive lipids such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have been recently described as important regulators of pluripotency and differentiation of ES cells and neural progenitors. Here we show that LPP3, an enzyme that regulates the levels and biological activities of the aforementioned lipids, participates in neural differentiation and neuritogenesis. We find that Lpp3−/− ES cells differentiated in vitro into spinal neurons, show a considerable reduction in the amount of neural precursor and young neurons formed. In addition, differentiated Lpp3−/− neurons exhibit impaired neurite outgrowth. Surprisingly, when Lpp3−/− ES cells were differentiated, an unexpected appearance of smooth muscle actin positive cells was observed, an event that was dependent upon phosphorylated sphingosines. Our data suggest that LPP3 plays a fundamental role during early spinal neuroepithelium development and that it could also be instrumental in regulating neurite and axon outgrowth in vivo.
lipid phosphate phosphatase; ES cells; S1P; motor neurons; Ppap2b
It is thought that a proportion of the genetic susceptibility to complex diseases is due to low-frequency and rare variants. Next-generation sequencing in large populations facilitates the detection of rare variant associations to disease risk. In order to achieve adequate power to detect association at low-frequency and rare variants, locus-specific statistical methods are being developed that combine information across variants within a functional unit and test for association with this enriched signal through so-called burden tests.
We propose a hierarchical clustering approach and a similarity kernel-based association test for continuous phenotypes. This method clusters individuals into groups, within which samples are assumed to be genetically similar, and subsequently tests the group effects among the different clusters.
The power of this approach is comparable to that of collapsing methods when causal variants have the same direction of effect, but its power is significantly higher compared to burden tests when both protective and risk variants are present in the region of interest. Overall, we observe that the Sequence Kernel Association Test (SKAT) is the most powerful approach under the allelic architectures considered.
In our overall comparison, we find the analytical framework within which SKAT operates to yield higher power and to control type I error appropriately.
Allele match kernel; Genetic similarity; Next-generation sequencing; Single nucleotide polymorphism
We present an approximate conditional and joint association analysis that can use summary-level statistics from a meta-analysis of genome-wide association studies (GWAS) and estimated linkage disequilibrium (LD) from a reference sample with individual-level genotype data. Using this method, we analyzed meta-analysis summary data from the GIANT Consortium for height and body mass index (BMI), with the LD structure estimated from genotype data in two independent cohorts. We identified 36 loci with multiple associated variants for height (38 leading and 49 additional SNPs, 87 in total) via a genome-wide SNP selection procedure. The 49 new SNPs explain approximately 1.3% of variance, nearly doubling the heritability explained at the 36 loci. We did not find any locus showing multiple associated SNPs for BMI. The method we present is computationally fast and is also applicable to case-control data, which we demonstrate in an example from meta-analysis of type 2 diabetes by the DIAGRAM Consortium.
Vibrio fischeri produces a specific biofilm to promote colonization of its eukaryotic host, the squid Euprymna scolopes. Formation of this biofilm is induced by the sensor kinase RscS, which functions upstream of the response regulator SypG to regulate transcription of the symbiosis polysaccharide (syp) locus. Biofilm formation is also controlled by SypE, a multi-domain response regulator that consists of a central regulatory receiver (REC) domain flanked by an N-terminal serine kinase domain and a C-terminal serine phosphatase domain. SypE permits biofilm formation under rscS overexpression conditions, but inhibits biofilms induced by overexpression of sypG. We previously investigated the function of SypE in controlling biofilm formation induced by RscS. Here, we examined the molecular mechanism by which SypE naturally inhibits SypG-induced biofilms. We found that SypE’s N-terminal kinase domain was both required and sufficient to inhibit SypG-induced biofilms. This effect did not occur at the level of syp transcription. Instead, under sypG-overexpressing conditions, SypE inhibited biofilms by promoting the phosphorylation of another syp regulator, SypA, a putative anti-sigma factor antagonist. Inhibition by SypE of SypG-induced biofilm formation could be overcome by the expression of a non-phosphorylatable SypA mutant, indicating that SypE functions primarily if not exclusively to control SypA activity via phosphorylation. Finally, the presence of SypE was detrimental to colonization under sypG-overexpressing conditions, as cells deleted for sypE outcompeted wild-type cells for colonization when both strains overexpressed sypG. These results provide further evidence that biofilm formation is critical to symbiotic colonization, and support a model in which SypE naturally functions to restrict biofilm formation, and thus host colonization, to the appropriate environmental conditions.
Generation Scotland: Scottish Family Health Study (GS:SFHS) is a family-based biobank of 24,000 participants with rich phenotype and DNA available for genetic research. This paper describes the laboratory results from genotyping 32 single nucleotide polymorphisms (SNPs) on DNA from over 10,000 participants who attended GS:SFHS research clinics. The analysis described here was undertaken to test the quality of genetic information available to researchers. The success rate of each marker genotyped (call rate), minor allele frequency and adherence to Mendelian inheritance are presented. The few deviations in marker transmission in the 925 parent-child trios analysed were assessed as to whether they were likely to be miscalled genotypes, data or sample handling errors, or pedigree inaccuracies including non-paternity.
The first 10,450 GS:SFHS clinic participants who had spirometry and smoking data available and DNA extracted were selected. 32 SNPs were assayed, chosen as part of a replication experiment from a Genome-Wide Association Study meta-analysis of lung function.
In total 325,336 genotypes were returned. The overall project pass rate (32 SNPs on 10,450 samples) was 97.29%. A total of 925 parent-child trios were assessed for transmission of the SNP markers, with 16 trios indicating evidence of inconsistency in the recorded pedigrees.
The Generation Scotland: Scottish Family Health Study used well-validated study methods and can produce good quality genetic data, with a low error rate. The GS:SFHS DNA samples are of high quality and the family groups were recorded and processed with accuracy during collection of the cohort.
Genetics; SNP Genotyping; Parent-child trios; Error rate; Non paternity; Generation Scotland; Biobank
Rationale: Acute lung injury (ALI) is an important cause of morbidity and mortality, with no currently effective pharmacological therapies. Neutrophils have been specifically implicated in the pathogenesis of ALI, and there has been significant research into the mechanisms of early neutrophil recruitment, but those controlling the later phases of neutrophil emigration that characterize disease are poorly understood.
Objectives: To determine the influence of peripheral blood monocytes (PBMs) in established ALI.
Methods: In a murine model of LPS-induced ALI, three separate models of conditional monocyte ablation were used: systemic liposomal clodronate (sLC), inducible depletion using CD11b diphtheria toxin receptor (CD11b DTR) transgenic mice, and antibody-dependent ablation of CCR2hi monocytes.
Measurements and Main Results: PBMs play a critical role in regulating neutrophil emigration in established murine LPS-induced lung injury. Gr1hi and Gr1lo PBM subpopulations contribute to this process. PBM depletion is associated with a significant reduction in measures of lung injury. The specificity of PBM depletion was demonstrated by replenishment studies in which the effects were reversed by systemic PBM infusion but not by systemic or local pulmonary infusion of mature macrophages or lymphocytes.
Conclusions: These results suggest that PBMs, or the mechanisms by which they influence pulmonary neutrophil emigration, could represent therapeutic targets in established ALI.
acute lung injury; LPS; monocytes; neutrophils