Beyond missing heritability: prediction of complex traits

Despite rapid advances in genomic technology, our ability to account for phenotypic variation using genetic information remains limited for many traits. This has unfortunately resulted in limited application of genetic data towards preventive and personalized medicine, one of the primary impetuses of genome-wide association studies. Recently, a large proportion of the "missing heritability" for human height was statistically explained by modeling thousands of single nucleotide polymorphisms concurrently. However, it is currently unclear how gains in explained genetic variance will translate to the prediction of yet-to-be observed phenotypes. Using data from the Framingham Heart Study, we explore the genomic prediction of human height in training and validation samples while varying the statistical approach used, the number of SNPs included in the model, the validation scheme, and the number of subjects used to train the model. In our training datasets, we are able to explain a large proportion of the variation in height (h(2) up to 0.83, R(2) up to 0.96). However, the proportion of variance accounted for in validation samples is much smaller (ranging from 0.15 to 0.36 depending on the degree of familial information used in the training dataset). While such R(2) values vastly exceed what has been previously reported using a reduced number of pre-selected markers (<0.10), given the heritability of the trait (～ 0.80), substantial room for improvement remains.     

Critical roles of arginine in growth and biofilm development by Streptococcus gordonii

Streptococcus gordonii is an oral commensal and an early coloniser of dental plaque. In vitro, S. gordonii is conditionally auxotrophic for arginine in monoculture but biosynthesises arginine when coaggregated with Actinomyces oris. Here, we investigated the arginine‐responsive regulatory network of S. gordonii and the basis for conditional arginine auxotrophy. ArcB, the catabolic ornithine carbamoyltransferase involved in arginine degradation, was also essential for arginine biosynthesis. However, arcB was poorly expressed following arginine depletion, indicating that arcB levels may limit S. gordonii arginine biosynthesis. Arginine metabolism gene expression was tightly co‐ordinated by three ArgR/AhrC family regulators, encoded by argR, ahrC and arcR genes. Microarray analysis revealed that > 450 genes were regulated in response to rapid shifts in arginine concentration, including many genes involved in adhesion and biofilm formation. In a microfluidic salivary biofilm model, low concentrations of arginine promoted S. gordonii growth, whereas high concentrations (> 5 mM arginine) resulted in dramatic reductions in biofilm biomass and changes to biofilm architecture. Collectively, these data indicate that arginine metabolism is tightly regulated in S. gordonii and that arginine is critical for gene regulation, cellular growth and biofilm formation. Manipulating exogenous arginine concentrations may be an attractive approach for oral biofilm control.     

Conditional Survival in de novo Metastatic Urothelial Carcinoma

Background

Second-line therapy is frequently utilized for metastatic urothelial carcinoma, but there are limited data to guide this approach. While an assessment of overall survival based on registry data may not capture the impact of second- and third-line therapies on clinical outcome, this may be reflected in relative conditional survival (RCS).

Methods

Patients with stage IV urothelial carcinoma diagnosed from 1990–2010 were identified from the Surveillance, Epidemiology and End Results (SEER) dataset. The association of clinicopathologic variables with disease specific survival (DSS) was explored through univariate and multivariate analyses. DSS in subgroups divided by time period (1990–2000 v 2001–2010) was compared using the Kaplan-Meier method and log-rank test. One-year RCS at annual landmarks up to 5 years was compared in subgroups divided by time period.

Results

Of 261,987 patients diagnosed with urothelial carcinoma from 1990–2010, 3,110 patients met criteria for the current analysis. Characteristics of patients diagnosed between 1990 and 2000 (n = 810) and 2001 to 2010 (n = 2,300) were similar and there was no significant difference in DSS between the two groups. On multivariate analysis, older age (age ≥ 80) was associated with shorter DSS (HR 1.79, 95%CI 1.48–2.15), but no association was found between time period of diagnosis and outcome. One-year RCS improved substantially through successive annual landmarks up to 5 years, but no differences were seen in subgroups divided by time of diagnosis.

Conclusions

No difference in RCS was observed amongst patients with stage IV urothelial carcinoma diagnosed from 1990–2000 and 2001–2010. A lack of difference in RCS (more so than cumulative DSS) may reflect a lack of progress in salvage therapies for the disease.     

Histamine release factor from Dermanyssus gallinae (De Geer): characterization and in vitro assessment as a protective antigen

A cDNA encoding a 174-amino-acid orthologue of a tick histamine release factor (HRF) was identified from the haematophagous poultry red mite Dermanyssus gallinae. The predicted D. gallinae HRF protein (Dg-HRF-1) sequence is highly conserved with the tick HRFs (identity 52-54%) and to a lesser degree with translationally controlled tumour proteins (TCTP) from mammals and other invertebrates (range 38-47%). Phylogenetically, Dg-HRF-1 partitions with the tick HRF clade suggesting a shared linage and potentially similar function(s). A recombinant Dg-HRF-1 protein (rDg-HRF-1) was produced and shown to induce degranulation of rat peritoneal mast cells in vitro, confirming conservation of the histamine-releasing function in D. gallinae. Polyclonal antibodies were generated in rabbits and hens to rDg-HRF-1. Western blotting demonstrated that native Dg-HRF is a soluble protein and immunohistochemical staining of mite sections revealed that the distribution of Dg-HRF, although ubiquitous, is more common in mite reproductive, digestive and synganglion tissues. A survey of hens housed continuously in a mite-infested commercial poultry unit failed to identify IgY specific for recombinant or native Dg-HRF, indicating that Dg-HRF is not exposed to the host during infestation/feeding and may therefore have potential as a vaccine using the concealed antigen approach. To test the protective capability of rDg-HRF-1, fresh heparinised chicken blood was enriched with yolk-derived anti-Dg-HRF IgY antibodies and fed to semi-starved mites using an in vitro feeding system. A statistically significant increase in mortality was shown (P = 0.004) in mites fed with anti-Dg-HRF IgY after just one blood meal. The work presented here demonstrates, to our knowledge for the first time, the feasibility of vaccinating hens with recombinant D. gallinae antigens to control mite infestation and the potential of rDg-HRF-1 as a vaccine antigen.     

Crystal structure of Leishmania major oligopeptidase B gives insight into the enzymatic properties of a trypanosomatid virulence factor

Oligopeptidase B (OPB) is a serine peptidase with dibasic substrate specificity. It is found in bacteria, plants, and trypanosomatid pathogens, where it has been identified as a virulence factor and potential drug target. In this study we expressed active recombinant Leishmania major OPB and provide the first structure of an oligopeptidase B at high resolution. The crystallographic study reveals that OPB comprises two domains, a catalytic and a propeller domain, linked together by a hinge region. The structure has been determined in complex with the oligopeptide, protease-inhibitor antipain, giving detailed information on the enzyme active site and extended substrate binding pockets. It shows that Glu-621 plays a critical role in the S1 binding pocket and, along with Phe-603, is largely responsible for the enzyme substrate specificity in P1. In the S2 binding pocket, Tyr-499 was shown to be important for substrate stability. The structure also allowed an investigation into the function of residues highlighted in other studies including Glu-623, which was predicted to be involved in the S1 binding pocket but is found forming an inter-domain hydrogen bond. Additional important salt bridges/hydrogen bonds between the two domains were observed, highlighting the significance of the domain interface in OPB. This work provides a foundation for the study of the role of OPBs as virulence factors in trypanosomatids. It could facilitate the development of specific OPB inhibitors with therapeutic potential by exploiting its unique substrate recognition properties as well as providing a model for OPBs in general.     

Disruption of the murine protein kinase Cbeta gene promotes gallstone formation and alters biliary lipid and hepatic cholesterol metabolism

The protein kinase C (PKC) family of Ca(2+) and/or lipid-activated serine-threonine protein kinases is implicated in the pathogenesis of obesity and insulin resistance. We recently reported that protein kinase Cβ (PKCβ), a calcium-, diacylglycerol-, and phospholipid-dependent kinase, is critical for maintaining whole body triglyceride homeostasis. We now report that PKCβ deficiency has profound effects on murine hepatic cholesterol metabolism, including hypersensitivity to diet-induced gallstone formation. The incidence of gallstones increased from 9% in control mice to 95% in PKCβ(-/-) mice. Gallstone formation in the mutant mice was accompanied by hyposecretion of bile acids with no alteration in fecal bile acid excretion, increased biliary cholesterol saturation and hydrophobicity indices, as well as hepatic p42/44(MAPK) activation, all of which enhance susceptibility to gallstone formation. Lithogenic diet-fed PKCβ(-/-) mice also displayed decreased expression of hepatic cholesterol-7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8b1). Finally, feeding a modified lithogenic diet supplemented with milk fat, instead of cocoa butter, both increased the severity of and shortened the interval for gallstone formation in PKCβ(-/-) mice and was associated with dramatic increases in cholesterol saturation and hydrophobicity indices. Taken together, the findings reveal a hitherto unrecognized role of PKCβ in fine tuning diet-induced cholesterol and bile acid homeostasis, thus identifying PKCβ as a major physiological regulator of both triglyceride and cholesterol homeostasis.     

Predicting community structure of ground-foraging ant assemblages with Markov models of behavioral dominance

Although interference competition is a conspicuous component of many animal communities, it is still uncertain whether the competitive ability of a species determines its relative abundance and patterns of association with other species. We used replicated arena tests to quantify behavioral dominance of eight common species of co-occurring ground-foraging ants in the Siskiyou Mountains of southern Oregon. We found that behavior recorded in laboratory assays was an accurate representation of a colony's ability to monopolize resources in the field. We used interaction frequencies from the behavioral tests to estimate transition probabilities in a simple Markov chain model to predict patterns of relative abundance in a metacommunity that is dominated by behavioral interactions. We also tested whether behavioral interactions between each pair of species could be used to predict patterns of species co-occurrence. We found that the Markov model did not accurately predict patterns of observed relative abundance on either the local or the regional scale. However, we did detect a significant negative correlation at the local scale in which behaviorally dominant species occupied relatively few baits. Pairwise behavioral data also did not predict species co-occurrence in any site. Although interference competition is a conspicuous process in ant communities, our results suggest that it may not contribute much to patterns of relative abundance and species co-occurrence in the system studied here. However, the negative correlation between behavioral dominance and bait occupancy at the local scale suggests that competition-colonization trade-offs may be important in resource acquisition and persistence of behaviorally subordinate species.     

Cell type-specific proteasomal processing of HIV-1 Gag-p24 results in an altered epitope repertoire

Proteasomes are critical for the processing of antigens for presentation through the major histocompatibility complex (MHC) class I pathway. HIV-1 Gag protein is a component of several experimental HIV-1 vaccines. Therefore, understanding the processing of HIV-1 Gag protein and the resulting epitope repertoire is essential. Purified proteasomes from mature dendritic cells (DC) and activated CD4(+) T cells from the same volunteer were used to cleave full-length Gag-p24 protein, and the resulting peptide fragments were identified by mass spectrometry. Distinct proteasomal degradation patterns and peptide fragments were unique to either mature DC or activated CD4(+) T cells. Almost half of the peptides generated were cell type specific. Two additional differences were observed in the peptides identified from the two cell types. These were in the HLA-B35-Px epitope and the HLA-B27-KK10 epitope. These epitopes have been linked to HIV-1 disease progression. Our results suggest that the source of generation of precursor MHC class I epitopes may be a critical factor for the induction of relevant epitope-specific cytotoxic T cells.     

Why Is There a Lack of Consensus on Molecular Subgroups of Glioblastoma? Understanding the Nature of Biological and Statistical Variability in Glioblastoma Expression Data

Introduction

Gene expression patterns characterizing clinically-relevant molecular subgroups of glioblastoma are difficult to reproduce. We suspect a combination of biological and analytic factors confounds interpretation of glioblastoma expression data. We seek to clarify the nature and relative contributions of these factors, to focus additional investigations, and to improve the accuracy and consistency of translational glioblastoma analyses.

Methods

We analyzed gene expression and clinical data for 340 glioblastomas in The Cancer Genome Atlas (TCGA). We developed a logic model to analyze potential sources of biological, technical, and analytic variability and used standard linear classifiers and linear dimensional reduction algorithms to investigate the nature and relative contributions of each factor.

Results

Commonly-described sources of classification error, including individual sample characteristics, batch effects, and analytic and technical noise make measurable but proportionally minor contributions to inconsistent molecular classification. Our analysis suggests that three, previously underappreciated factors may account for a larger fraction of classification errors: inherent non-linear/non-orthogonal relationships among the genes used in conjunction with classification algorithms that assume linearity; skewed data distributions assumed to be Gaussian; and biologic variability (noise) among tumors, of which we propose three types.

Conclusions

Our analysis of the TCGA data demonstrates a contributory role for technical factors in molecular classification inconsistencies in glioblastoma but also suggests that biological variability, abnormal data distribution, and non-linear relationships among genes may be responsible for a proportionally larger component of classification error. These findings may have important implications for both glioblastoma research and for translational application of other large-volume biological databases.