Improving genetic risk prediction by leveraging pleiotropy

An important task of human genetics studies is to predict accurately disease risks in individuals based on genetic markers, which allows for identifying individuals at high disease risks, and facilitating their disease treatment and prevention. Although hundreds of genome-wide association studies (GWAS) have been conducted on many complex human traits in recent years, there has been only limited success in translating these GWAS data into clinically useful risk prediction models. The predictive capability of GWAS data is largely bottlenecked by the available training sample size due to the presence of numerous variants carrying only small to modest effects. Recent studies have shown that different human traits may share common genetic bases. Therefore, an attractive strategy to increase the training sample size and hence improve the prediction accuracy is to integrate data from genetically correlated phenotypes. Yet, the utility of genetic correlation in risk prediction has not been explored in the literature. In this paper, we analyzed GWAS data for bipolar and related disorders and schizophrenia with a bivariate ridge regression method, and found that jointly predicting the two phenotypes could substantially increase prediction accuracy as measured by the area under the receiver operating characteristic curve. We also found similar prediction accuracy improvements when we jointly analyzed GWAS data for Crohn’s disease and ulcerative colitis. The empirical observations were substantiated through our comprehensive simulation studies, suggesting that a gain in prediction accuracy can be obtained by combining phenotypes with relatively high genetic correlations. Through both real data and simulation studies, we demonstrated pleiotropy can be leveraged as a valuable asset that opens up a new opportunity to improve genetic risk prediction in the future.     

Interleukin-2

Between October 1987 and October 1989 we have treated 110 patients with advanced solid tumors with recombinant interleukin-2 (rIL2) based immunotherapy. In renal cell cancer we have studied rIL2 alone, rIL2 combined with rIL2 activated lymphocytes (LAK), and in an ongoing study rIL2 and LAK and alpha-interferon (alpha IFN). There is suggestive evidence of increasingly good results in these consecutive studies. In melanoma the combination of rIL2 and chemotherapy was investigated, followed by an ongoing study of rIL2 and alpha IFN. In these studies rIL2 has been administered as a continuous intravenous infusion of 18 x 10(6) International Units/m2/day for 5 days (18 x 10(6) IU = 3 x 10(6) Cetus Units = 6.9 Biological Response Modifiers Program (BRMP) Units). Patients with non-small cell lung cancer are entered in a phase I-II study of rIL2 and alpha IFN. The rIL2 administration differs from the above mentioned schedule in that rIL2 is given at a maximum dose of 6 x 10(6) IU/m2/day for 28 days on an outpatient basis. In a phase I study we have searched for the maximum tolerated dose of a daily time 4 schedule of rIL2. In the second part of this study a daily time 4 schedule, every week for 4 weeks is being investigated. Finally, we are investigating the safety and efficacy of local regional administration of rIL2 in patients with head and neck cancer, mesothelioma, and liver metastasis of colorectal cancer.     

Viral security proteins: counteracting host defences

Interactions with host defences are key aspects of viral infection. Various viral proteins perform counter-defensive functions, but a distinct class, called security proteins, is dedicated specifically to counteracting host defences. Here, the properties of the picornavirus security proteins L and 2A are discussed. These proteins have well-defined positions in the viral polyprotein, flanking the capsid precursor, but they are structurally and biochemically unrelated. Here, we consider the impact of these two proteins, as well as that of a third security protein, L(*), on viral reproduction, pathogenicity and evolution. The concept of security proteins could serve as a paradigm for the dedicated counter-defensive proteins of other viruses.     

One hundred years of pleiotropy: a retrospective

Pleiotropy is defined as the phenomenon in which a single locus affects two or more distinct phenotypic traits. The term was formally introduced into the literature by the German geneticist Ludwig Plate in 1910, 100 years ago. Pleiotropy has had an important influence on the fields of physiological and medical genetics as well as on evolutionary biology. Different approaches to the study of pleiotropy have led to incongruence in the way that it is perceived and discussed among researchers in these fields. Furthermore, our understanding of the term has changed quite a bit since 1910, particularly in light of modern molecular data. This review traces the history of the term "pleiotropy" and reevaluates its current place in the field of genetics.     

Subcellular compartmentalization of adeno-associated virus type 2 assembly.

Using immunofluorescence and in situ hybridization techniques, we studied the intracellular localization of adeno-associated virus type 2 (AAV-2) Rep proteins, VP proteins, and DNA during the course of an AAV-2/adenovirus type 2 coinfection. In an early stage, the Rep proteins showed a punctate distribution pattern over the nuclei of infected cells, reminiscent of replication foci. At this stage, no capsid proteins were detectable. At later stages, the Rep proteins were distributed more homogeneously over the nuclear interior and finally became redistributed into clusters slightly enriched at the nuclear periphery. During an intermediate stage, they also appeared at an interior part of the nucleolus for a short period, whereas most of the time the nucleoli were Rep negative. AAV-2 DNA colocalized with the Rep proteins. All three capsid proteins were strongly enriched in the nucleolus in a transient stage of infection, when the Rep proteins homogeneously filled the nucleoplasm. Thereafter, they became distributed over the whole nucleus and colocalized in nucleoplasmic clusters with the Rep proteins and AAV-2 DNA. While VP1 and VP2 strongly accumulated in the nucleus, VP3 was almost equally distributed between the nucleus and cytoplasm. Capsids, visualized by a conformation-specific antibody, were first detectable in the nucleoli and then spread over the whole nucleoplasm. This suggests that nucleolar components are involved in initiation of capsid assembly whereas DNA packaging occurs in the nucleoplasm. Expression of a transfected full-length AAV-2 genome followed by adenovirus infection showed all stages of an AAV-2/adenovirus coinfection, whereas after expression of the cap gene alone, capsids were restricted to the nucleoli and did not follow the nuclear redistribution observed in the presence of the whole AAV-2 genome. Coexpression of Rep proteins released the restriction of capsids to the nucleolus, suggesting that the Rep proteins are involved in nuclear redistribution of AAV capsids during viral infection. Capsid formation was dependent on the concentration of expressed capsid protein.     

Subcellular compartmentalization of activation and desensitization of responses mediated by NK2 neurokinin receptors

A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells. On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer. This can be quantified for nM to microM agonist concentrations and monitored in parallel with intracellular calcium responses. On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence. Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area. Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application. Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains.     

Ghrelin: a striking example of neuroendocrine peptide pleiotropy

Ghrelin, a peptide predominantly produced by the stomach, has been discovered as a natural ligand of the growth hormone secretagogue receptor (GHS-R) type 1a. Shortly there after, it attracted enormous interest since it appeared as the first peripheral orexigenic factor. Besides, ghrelin exerts other neuroendocrine metabolic and non-endocrine actions (e.g. cardiovascular activities) that may rely on the widespread distribution of ghrelin and its receptor (GHS-R). The existence of several GHS-R subtypes and evidences that neuroendocrine and metabolic but not all other ghrelin actions are dependent on acylation on serine 3 add further complexity to the system whose major physiological role remains to be definitely elucidated. Ghrelin knockout(-/-) mice are neither anorectic nor dwarf though GHS-R-/- are slightly underweight and do not respond to ghrelin with increased GH secretion or appetite. Thus, the continuation of the fascinating ghrelin story as well as its potential pathophysiological implications in endocrinology and internal medicine remain open avenues for future investigations.     

Differential membrane compartmentalization of Ret by PTB-adaptor engagement

Glial cell line-derived neurotrophic factor family ligands act through the receptor tyrosine kinase Ret, which plays important roles during embryonic development for cell differentiation, survival, and migration. Ret signaling is markedly affected by compartmentalization of receptor complexes into membrane subdomains. Ret can propagate biochemical signaling from within concentrates in cholesterol-rich membrane microdomains or lipid rafts, or outside such regions, but the mechanisms for, and consequences of, Ret translocation between these membrane compartments remain largely unclear. Here we investigate the interaction of Shc and Frs2 phosphotyrosine-binding domain-containing adaptor molecules with Ret and their function in redistributing Ret to specialized membrane compartments. We found that engagement of Ret with the Frs2 adaptor results in an enrichment of Ret in lipid rafts and that signal transduction pathways and chemotaxis responses depend on the integrity of such rafts. The competing Shc adaptor did not promote Ret translocation to equivalent domains, and Shc-mediated effects were less affected by disruption of lipid rafts. However, by expressing a chimeric Shc protein that localizes to lipid rafts, we showed that biochemical signaling downstream of Ret resembled that of Ret signaling via Frs2. We have identified a previously unknown mechanism in which phosphotyrosine-binding domain-containing adaptors, by means of relocating Ret receptor complexes to lipid rafts, segregate diverse signaling and cellular functions mediated by Ret. These results reveal the existence of a novel mechanism that could, by subcellular relocation of Ret, work to amplify ligand gradients during chemotaxis.     

Subcellular compartmentalization by local differentiation of cytoplasmic structure

The compartmentalization of eukaryotic cells by internal membranes and the subcellular localization of endogenous macromolecules by specific binding mechanisms are familiar concepts. In this report we present evidence that the cytoplasmic ground substance, which surrounds and contains the membrane-bound compartments, may also be compartmentalized by local differentiations of its submicroscopic structure that sort subcellular particles on the basis of size. The subcellular distribution of size-fractionated, fluorescent tracer particles was studied in living cells by ratio imaging and fluorescence recovery after photobleaching (FRAP). Large and small particles showed different distributions within the cytoplasmic volume, suggesting that the large particles were relatively excluded from some domains. While the structural basis for this phenomenon is not yet understood in detail, ratio imaging of large and small particles can be used as an empirical tool to identify cytoplasmic compartments for further study. The cytoplasmic diffusion coefficient (Dcyto) and % mobile fraction of the large particles showed considerable spatial variation over the projected area of the cell, while Dcyto and % mobile fraction of the small particles did not. A model is presented to account for this difference. Based on this model, a method is proposed by which FRAP can be used to detect sol-gel transitions in the cytoplasmic ground substance of living cells.     

Interleukin-2 production during murine infection by Leishmania mexicana amazonensis

Highly susceptible BALB/c mice, resistant C57B1/6 and their F1 progeny (BDF1) were infected subcutaneously in the foot pad with Leishmania mexicana amazonensis. At various times after infection, spleen or draining popliteal lymph node cells were assayed for their capacity to generate Interleukin-2 (I1-2) by Concanavalin A (ConA) stimulation. In both BALB/c and C57B1/6 strains there was a transient increase in their capacity to produce I1-2, from the 3rd to the 10th week post-infection. Return to pre-infection levels occurred between 13th to 16th week post-infection in all three strains. BALB/c mice always produced higher titers of I1-2 than C57B1/6, but such differences were statistically significant only at 3 and 10 weeks post-infection. BDF1 mice had titers similar to those observed in BALB/c mice. I1-2 production by ConA-stimulated lymph node cells was lower as compared to the spleen, but with a similar pattern among the three mice strains. Our data show that susceptibility to infection by L. mexicana amazonensis is not associated with deficient ConA-stimulated I1-2 production.     

CSN facilitates Cullin-RING ubiquitin ligase function by counteracting autocatalytic adapter instability

The COP9 signalosome (CSN) is known to bind cullin-RING ubiquitin ligases (CRLs) and to promote their activity in vivo. The mechanism of this stimulation has remained enigmatic because CSN's intrinsic and associated enzymatic activities paradoxically inhibit CRL activity in vitro. Reconciling this paradox, we show here that Csn5-catalysed cullin (Cul) deneddylation and Ubp12-mediated deubiquitination cooperate in maintaining the stability of labile substrate adapters, thus facilitating CRL function. Various fission-yeast csn and ubp12 deletion mutants have lower levels of the Cul3p adapter Btb3p. This decrease is due to increased autocatalytic, Cul3p-dependent, ubiquitination and the subsequent degradation of Btb3p. The CSN-Ubp12p pathway also maintains the stability of the Cul1p adapter Pop1p, a mechanism required for the efficient destruction of its cognate substrate Rum1p. Emphasizing the physiological importance of this mechanism, we found that the dispensable csn5 and ubp12 genes become essential for viability when adapter recruitment to Cul1p is compromised. Our data suggest that maintenance of adapter stability is a general mechanism of CRL control by the CSN.     

Interleukin-6 regulation of transforming growth factor (TGF)-beta receptor compartmentalization and turnover enhances TGF-beta1 signaling

Transforming growth factor (TGF)-beta1 is a key cytokine involved in the pathogenesis of fibrosis in many organs, whereas interleukin (IL)-6 plays an important role in the regulation of inflammation. Recent reports demonstrate interaction between the two cytokines in disease states. We have assessed the effect of IL-6 on TGF-beta1 signaling and defined the mechanism by which this occurred. Stimulation of Smad-responsive promoter (SBE)4-Lux activity by TGF-beta1 was significantly greater in the presence of IL-6 than that induced by TGF-beta1 alone. Augmented TGF-beta1 signaling following the addition of IL-6 appeared to be mediated through binding to the cognate IL-6 receptor, the presence of which was confirmed by fluorescence-activated cell sorting and Stat-specific signaling. TGF-beta1 receptors internalize by both caveolin-1 (Cav-1) lipid raft and early endosome antigen 1 (EEA-1) non-lipid raft pathways, with non-lipid raft-associated internalization increasing TGF-beta1 signaling. Affinity labeling of TGF-beta1 receptors demonstrated that IL-6 stimulation resulted in increased partitioning of TGF-beta receptors to the non-lipid raft fraction. There was no change in expression of Cav-1; however, following IL-6 stimulation, co-immunoprecipitation demonstrated decreased association of IL-6 receptor with Cav-1. Increased TGF-beta1-dependent Smad signaling by IL-6 was significantly attenuated by inhibition of clathrin-mediated endocytosis and augmented by depletion of membrane cholesterol. These results indicate that IL-6 increased trafficking of TGF-beta1 receptors to non-lipid raft-associated pools results in augmented TGF-beta1 Smad signaling.