Whole-genome scan, in a complex disease, using 11,245 single-nucleotide polymorphisms: comparison with microsatellites

Despite the theoretical evidence of the utility of single-nucleotide polymorphisms (SNPs) for linkage analysis, no whole-genome scans of a complex disease have yet been published to directly compare SNPs with microsatellites. Here, we describe a whole-genome screen of 157 families with multiple cases of rheumatoid arthritis (RA), performed using 11,245 genomewide SNPs. The results were compared with those from a 10-cM microsatellite scan in the same cohort. The SNP analysis detected HLA*DRB1, the major RA susceptibility locus (P=.00004), with a linkage interval of 31 cM, compared with a 50-cM linkage interval detected by the microsatellite scan. In addition, four loci were detected at a nominal significance level (P<.05) in the SNP linkage analysis; these were not observed in the microsatellite scan. We demonstrate that variation in information content was the main factor contributing to observed differences in the two scans, with the SNPs providing significantly higher information content than the microsatellites. Reducing the number of SNPs in the marker set to 3,300 (1-cM spacing) caused several loci to drop below nominal significance levels, suggesting that decreases in information content can have significant effects on linkage results. In contrast, differences in maps employed in the analysis, the low detectable rate of genotyping error, and the presence of moderate linkage disequilibrium between markers did not significantly affect the results. We have demonstrated the utility of a dense SNP map for performing linkage analysis in a late-age-at-onset disease, where DNA from parents is not always available. The high SNP density allows loci to be defined more precisely and provides a partial scaffold for association studies, substantially reducing the resource requirement for gene-mapping studies.     

Transmembrane peptides as inhibitors of ErbB receptor signaling

Receptor tyrosine kinases have a single transmembrane (TM) segment that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, mutations within some of these receptors, and recent studies with the epidermal growth factor (EGF) and ErbB2 receptors have indicated that interactions between TM domains do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimerization and activation. One consequence of the importance of these interactions is that short hydrophobic peptides corresponding to these domains should act as specific inhibitors. To test this hypothesis, we constructed expression vectors encoding short fusion peptides encompassing native or mutated TM domains of the EGF, ErbB2, and insulin receptors. In human cell lines overexpressing the wild-type EGF receptor or ErbB2, we observed that the peptides are expressed at the cell surface and that they inhibit specifically the autophosphorylation and signaling pathway of their cognate receptor. Identical results were obtained with peptides chemically synthesized. Mechanism of action involves inhibition of dimerization of the receptors as shown by the lack of effects of mutant nondimerizing sequences, completed by density centrifugation and covalent cross-linking experiments. Our findings stress the role of TM domain interactions in ErbB receptor function, and possibly for other single-spanning membrane proteins.     

Increased Carbohydrate Induced Ghrelin Secretion in Obese vs. Normal‐weight Adolescent Girls

Orexigenic and anorexigenic pathways mediate food intake and may be affected by meal composition. Our objective was to determine whether changes in levels of active ghrelin and peptide YY (PYY) differ in obese vs. normal‐weight adolescent girls following specific macronutrient intake and predict hunger and subsequent food intake. We enrolled 26 subjects: 13 obese and 13 normal‐weight girls, 12–18 years old, matched for maturity (as assessed by bone age) and race. Subjects were assigned a high‐carbohydrate, high‐protein, and high‐fat breakfast in random order. Active ghrelin and PYY were assessed for 4 h after breakfast and 1 h after intake of a standardized lunch. Hunger was assessed using a standardized visual analog scale (VAS). No suppression in active ghrelin levels was noted following macronutrient intake in obese or normal‐weight girls. Contrary to expectations, active ghrelin increased in obese girls following the high‐carbohydrate breakfast, and the percent increase was higher than in controls (P = 0.046). Subsequent food intake at lunch was also higher (P = 0.03). Following the high‐fat breakfast, but not other breakfasts, percent increase in PYY was lower (P = 0.01) and subsequent lunch intake higher (P = 0.005) in obese compared with normal‐weight girls. In obese adolescents, specific intake of high‐carbohydrate and high‐fat breakfasts is associated with greater increases in ghrelin, lesser increases in PYY, and higher intake at a subsequent meal than in controls. Changes in anorexigenic and orexigenic hormones in obese vs. normal‐weight adolescents following high‐carbohydrate and high‐fat meals may influence hunger and satiety signals and subsequent food intake.     

Islet Endothelial Activation and Oxidative Stress Gene Expression Is Reduced by IL-1Ra Treatment in the Type 2 Diabetic GK Rat

Background

Inflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra).

Methodology/Principal Findings

Gene expression was analyzed by quantitative RT-PCR on islets isolated from 10-week-old diabetic GK and control Wistar rats. Furthermore, GK rats were treated s.c twice daily with IL-1Ra (Kineret, Amgen, 100 mg/kg/day) or saline, from 4 weeks of age onwards (onset of diabetes). Four weeks later, islet gene analysis and pancreas immunochemistry were performed. Thirty-two genes were selected encoding molecules involved in endothelial cell activation, particularly fibrinolysis, vascular tone, OS, angiogenesis and also inflammation. All genes except those encoding angiotensinogen and epoxide hydrolase (that were decreased), and 12-lipoxygenase and vascular endothelial growth factor (that showed no change), were significantly up-regulated in GK islets. After IL-1Ra treatment of GK rats in vivo, most selected genes implied in endothelium/OS/immune cells/fibrosis were significantly down-regulated. IL-1Ra also improved islet vascularization, reduced fibrosis and ameliorated glycemia.

Conclusions/Significance

GK rat islets have increased mRNA expression of markers of early islet endothelial cell activation, possibly triggered by several metabolic factors, and also some defense mechanisms. The beneficial effect of IL-1Ra on most islet endothelial/OS/immune cells/fibrosis parameters analyzed highlights a major endothelial-related role for IL-1 in GK islet alterations. Thus, metabolically-altered islet endothelium might affect the β-cell microenvironment and contribute to progressive type 2 diabetic β-cell dysfunction in GK rats. Counteracting islet endothelial cell inflammation might be one way to ameliorate/prevent β-cell dysfunction in type 2 diabetes.     

The Severity of Pandemic H1N1 Influenza in the United States,from April to July 2009: A Bayesian Analysis

Background

Accurate measures of the severity of pandemic (H1N1) 2009 influenza (pH1N1) are needed to assess the likely impact of an anticipated resurgence in the autumn in the Northern Hemisphere. Severity has been difficult to measure because jurisdictions with large numbers of deaths and other severe outcomes have had too many cases to assess the total number with confidence. Also, detection of severe cases may be more likely, resulting in overestimation of the severity of an average case. We sought to estimate the probabilities that symptomatic infection would lead to hospitalization, ICU admission, and death by combining data from multiple sources.

Methods and Findings

We used complementary data from two US cities: Milwaukee attempted to identify cases of medically attended infection whether or not they required hospitalization, while New York City focused on the identification of hospitalizations, intensive care admission or mechanical ventilation (hereafter, ICU), and deaths. New York data were used to estimate numerators for ICU and death, and two sources of data—medically attended cases in Milwaukee or self-reported influenza-like illness (ILI) in New York—were used to estimate ratios of symptomatic cases to hospitalizations. Combining these data with estimates of the fraction detected for each level of severity, we estimated the proportion of symptomatic patients who died (symptomatic case-fatality ratio, sCFR), required ICU (sCIR), and required hospitalization (sCHR), overall and by age category. Evidence, prior information, and associated uncertainty were analyzed in a Bayesian evidence synthesis framework. Using medically attended cases and estimates of the proportion of symptomatic cases medically attended, we estimated an sCFR of 0.048% (95% credible interval [CI] 0.026%–0.096%), sCIR of 0.239% (0.134%–0.458%), and sCHR of 1.44% (0.83%–2.64%). Using self-reported ILI, we obtained estimates approximately 7–9× lower. sCFR and sCIR appear to be highest in persons aged 18 y and older, and lowest in children aged 5–17 y. sCHR appears to be lowest in persons aged 5–17; our data were too sparse to allow us to determine the group in which it was the highest.

Conclusions

These estimates suggest that an autumn–winter pandemic wave of pH1N1 with comparable severity per case could lead to a number of deaths in the range from considerably below that associated with seasonal influenza to slightly higher, but with the greatest impact in children aged 0–4 and adults 18–64. These estimates of impact depend on assumptions about total incidence of infection and would be larger if incidence of symptomatic infection were higher or shifted toward adults, if viral virulence increased, or if suboptimal treatment resulted from stress on the health care system; numbers would decrease if the total proportion of the population symptomatically infected were lower than assumed. Please see later in the article for the Editors'' Summary     

Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro

We have established a detailed framework for the process of shoot regeneration from Arabidopsis root and hypocotyl explants grown in vitro . Using transgenic plant lines in which the GUS or GFP genes were fused to promoters of developmental genes ( WUS , CLV1 , CLV3 , STM , CUC1 , PLT1 , RCH1 , QC25 ), or to promoters of genes encoding indicators of the auxin response ( DR5 ) or transport ( PIN1 ), cytokinin (CK) response ( ARR5 ) or synthesis ( IPT5 ), or mitotic activity ( CYCB1 ), we showed that regenerated shoots originated directly or indirectly from the pericycle cells adjacent to xylem poles. In addition, shoot regeneration appeared to be partly similar to the formation of lateral root meristems (LRMs). During pre-culture on a 2, 4-dichlorophenoxyacetic acid (2, 4-D)-rich callus-inducing medium (CIM), xylem pericycle reactivation established outgrowths that were not true calli but had many characteristics of LRMs. Transfer to a CK-rich shoot-inducing medium (SIM) resulted in early LRM-like primordia changing to shoot meristems. Direct origin of shoots from the xylem pericycle occurred upon direct culture on CK-containing media without prior growth on CIM. Thus, it appeared that the xylem pericycle is more pluripotent than previously thought. This pluripotency was accompanied by the ability of pericycle derivatives to retain diploidy, even after several rounds of cell division. In contrast, the phloem pericycle did not display such developmental plasticity, and responded to CKs with only periclinal divisions. Such observations reinforce the view that the pericycle is an 'extended meristem' that comprises two types of cell populations. They also suggest that the founder cells for LRM initiation are not initially fully specified for this developmental pathway.     

Muscarinic receptor regulation of osmosensitive taurine transport in human SH-SY5Y neuroblastoma cells

The ability of G protein‐coupled receptors to regulate osmosensitive uptake of the organic osmolyte, taurine, into human SH‐SY5Y neuroblastoma cells has been examined. When monitored under isotonic conditions and in the presence of physiologically relevant taurine concentrations (1–100 μM), taurine influx was mediated exclusively by a Na⁺‐dependent, high‐affinity (K_m = 2.5 μM) saturable transport mechanism (V_max = 0.087 nmol/mg protein/min). Reductions in osmolarity of > 20% (attained under conditions of a constant NaCl concentration) resulted in an inhibition of taurine influx (> 30%) that could be attributed to a reduction in V_max, whereas the K_m for uptake remained unchanged. Inclusion of the muscarinic cholinergic agonist, oxotremorine‐M (Oxo‐M), also resulted in an attenuation of taurine influx (EC₅₀～0.7 μM). Although Oxo‐M‐mediated inhibition of taurine uptake could be observed under isotonic conditions (～25–30%), the magnitude of inhibition was significantly enhanced by hypotonicity (～55–60%), a result that also reflected a reduction in the V_max, but not the K_m, for taurine transport. Oxo‐M‐mediated inhibition of taurine uptake was dependent upon the availability of extracellular Ca²⁺ but was independent of protein kinase C activity. In addition to Oxo‐M, inclusion of either thrombin or sphingosine 1‐phosphate also attenuated volume‐dependent taurine uptake. The ability of Oxo‐M to inhibit the influx of taurine was attenuated by 4‐[(2‐butyl‐6,7‐dichloro‐2‐cyclopentyl‐2,3‐dihydro‐1‐oxo‐1H‐inden‐5‐yl)oxy]butanoic acid, an inhibitor of the volume‐sensitive organic osmolyte and anion channel. 4‐[(2‐Butyl‐6,7‐dichloro‐2‐cyclopentyl‐2,3‐dihydro‐1‐oxo‐1H‐inden‐5‐yl)oxy]butanoic acid also prevented receptor‐mediated changes in the efflux and influx of K⁺ under hypoosmotic conditions. The results suggest that muscarinic receptor activation can regulate both the volume‐dependent efflux and uptake of taurine and that these events may be functionally coupled.     

Multiple Loci within the Major Histocompatibility Complex Confer Risk of Psoriasis

Psoriasis is a common inflammatory skin disease characterized by thickened scaly red plaques. Previously we have performed a genome-wide association study (GWAS) on psoriasis with 1,359 cases and 1,400 controls, which were genotyped for 447,249 SNPs. The most significant finding was for SNP rs12191877, which is in tight linkage disequilibrium with HLA-Cw*0602, the consensus risk allele for psoriasis. However, it is not known whether there are other psoriasis loci within the MHC in addition to HLA-C. In the present study, we searched for additional susceptibility loci within the human leukocyte antigen (HLA) region through in-depth analyses of the GWAS data; then, we followed up our findings in an independent Han Chinese 1,139 psoriasis cases and 1,132 controls. Using the phased CEPH dataset as a reference, we imputed the HLA-Cw*0602 in all samples with high accuracy. The association of the imputed HLA-Cw*0602 dosage with disease was much stronger than that of the most significantly associated SNP, rs12191877. Adjusting for HLA-Cw*0602, there were two remaining association signals: one demonstrated by rs2073048 (p=2×10⁻⁶, OR=0.66), located within c6orf10, a potential downstream effecter of TNF-alpha, and one indicated by rs13437088 (p=9×10⁻⁶, OR=1.3), located 30 kb centromeric of HLA-B and 16 kb telomeric of MICA. When HLA-Cw*0602, rs2073048, and rs13437088 were all included in a logistic regression model, each of them was significantly associated with disease (p=3×10⁻⁴⁷, 6×10⁻⁸, and 3×10⁻⁷, respectively). Both putative loci were also significantly associated in the Han Chinese samples after controlling for the imputed HLA-Cw*0602. A detailed analysis of HLA-B in both populations demonstrated that HLA-B*57 was associated with an increased risk of psoriasis and HLA-B*40 a decreased risk, independently of HLA-Cw*0602 and the C6orf10 locus, suggesting the potential pathogenic involvement of HLA-B. These results demonstrate that there are at least two additional loci within the MHC conferring risk of psoriasis.     

Partitioning diversity for conservation analyses

Aim  Differentiation of sites or communities is often measured by partitioning regional or gamma diversity into additive or multiplicative alpha and beta components. The beta component and the ratio of within-group to total diversity (alpha/gamma) are then used to infer the compositional differentiation or similarity of the sites. There is debate about the appropriate measures and partitioning formulas for this purpose. We test the main partitioning methods, using empirical and simulated data, to see if some of these methods lead to false conclusions, and we show how to resolve the problems that we uncover.
Location  South America, Ecuador, Orellana province, Rio Shiripuno.
Methods  We construct sets of real and simulated tropical butterfly communities that can be unambiguously ranked according to their degree of differentiation. We then test whether beta and similarity measures from the different partitioning approaches rank these datasets correctly.
Results  The ratio of within-group diversity to total diversity does not reflect compositional similarity, when the Gini–Simpson index or Shannon entropy are used to measure diversity. Additive beta diversity based on the Gini–Simpson index does not reflect the degree of differentiation between N sites or communities.
Main conclusions  The ratio of within-group to total diversity (alpha/gamma) should not be used to measure the compositional similarity of groups, if diversity is equated with Shannon entropy or the Gini–Simpson index. Conversion of these measures to effective number of species solves these problems. Additive Gini–Simpson beta diversity does not directly reflect the differentiation of N samples or communities. However, when properly transformed onto the unit interval so as to remove the dependence on alpha and N , additive and multiplicative beta measures yield identical normalized measures of relative similarity and differentiation.