Novel, comprehensive approaches for biomarker discovery and validation are urgently needed. One particular area of methodologic need is for discovery of novel genetic biomarkers in complex diseases and traits. Here, we review recent successes in the use of genome wide association (GWA) approaches to identify genetic biomarkers in common human diseases and traits. Such studies are yielding initial insights into the allelic architecture of complex traits. In general, it appears that complex diseases are associated with many common polymorphisms, implying profound genetic heterogeneity between affected individuals.

Sepsis is caused by a heterogeneous group of infectious etiologies. Early diagnosis and the provision of appropriate antimicrobial therapy correlate with positive clinical outcomes. Current microbiological techniques are limited in their diagnostic capacities and timeliness. Multiplex PCR has the potential to rapidly identify bloodstream infections and fill this diagnostic gap. We identified patients from two large academic hospital emergency departments with suspected sepsis. The results of a multiplex PCR that could detect 25 bacterial and fungal pathogens were compared to those of blood culture. The results were analyzed with respect to the likelihood of infection, sepsis severity, the site of infection, and the effect of prior antibiotic therapy. We enrolled 306 subjects with suspected sepsis. Of these, 43 were later determined not to have infectious etiologies. Of the remaining 263 subjects, 70% had sepsis, 16% had severe sepsis, and 14% had septic shock. The majority had a definite infection (41.5%) or a probable infection (30.7%). Blood culture and PCR performed similarly with samples from patients with clinically defined infections (areas under the receiver operating characteristic curves, 0.64 and 0.60, respectively). However, blood culture identified more cases of septicemia than PCR among patients with an identified infectious etiology (66 and 46, respectively; P = 0.0004). The two tests performed similarly when the results were stratified by sepsis severity or infection site. Blood culture tended to detect infections more frequently among patients who had previously received antibiotics (P = 0.06). Conversely, PCR identified an additional 24 organisms that blood culture failed to detect. Real-time multiplex PCR has the potential to serve as an adjunct to conventional blood culture, adding diagnostic yield and shortening the time to pathogen identification.

Vesicular transport to and from the lysosome and late endosome is defective in patients with Chediak–Higashi syndrome (CHS) and in mutant beige (bg) mice1–4. CHS and bg cells have giant, perinuclear vesicles with characterises of late endosomes and lysosomes that arise from dysregulated homotypic fusion3–5. CHS and bg lysosomes also exhibit compartmental missorting of proteins, such as elastase, glucuronidase and cathepsin G2,3,6,7. Lyst, a candidate gene for bg, was identified by direct complementary DNA selection from a yeast artificial chromosome (YAC) clone containing a 650-kilobase segment of the bg-critical region on mouse chromosome 13. Lyst is disrupted by a 5-kilobase deletion in bg11J mice, and Lyst messenger RNA is markedly reduced in bg2J homozygotes. The homologous human gene, LYST, is highly conserved with mouse Lyst, and contains a frame-shift mutation at nucleotides 117–118 of the coding domain in a CHS patient. Thus bg mice and human CHS patients have homologous disorders associated with Lyst mutations. Lyst encodes a protein with a carboxy-terminal prenylation motif and multiple potential phosphorylation sites. Lyst protein is predicted to form extended helical domains, and has a region of sequence similar to stathmin, a coiled-coil phosphoprotein thought to act as a relay integrating cellular signal response coupling8–10.

Chediak–Higashi syndrome is an autosomal recessive, immune deficiency disorder of human (CHS) and mouse (beige, bg) that is characterized by abnormal intracellular protein transport to, and from, the lysosome. Recent reports have described the identification of homologous genes that are mutated in human CHS and bg mice. Here we report the sequences of two major mRNA isoforms of the CHS gene in human and mouse. These isoforms differ both in size and in sequence at the 3′ end of their coding domains, with the smaller isoform (~5.8 kb) arising from incomplete splicing and reading through an intron. These mRNAs also differ in tissue distribution of transcription and in predicted biological properties. Novel mutations were identified within the region of the coding domain common to both isoforms in three CHS patients: C→T transitions that generated stop codons (R50X and Q1029X) were found in two patients, and a novel frame-shift mutation (deletion of nucleotides 3073 and 3074 of the coding domain) was found in a third. Northern blots of lymphoblastoid mRNA from CHS patients revealed loss of the largest transcript (~13.5 kb) in two of seven CHS patients, while the small mRNA was undiminished in abundance. These results suggest that the small isoform alone cannot complement Chediak–Higashi syndrome.

Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1β, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-α treatment. Because microarrays can accommodat ~1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.

Although genetic studies have been critically important for the identification of therapeutic targets in Mendelian disorders, genetic approaches aiming to identify targets for common, complex diseases have traditionally had much more limited success. However, during the past year, a novel genetic approach — genome-wide association (GWA) — has demonstrated its potential to identify common genetic variants associated with complex diseases such as diabetes, inflammatory bowel disease and cancer. Here, we highlight some of these recent successes, and discuss the potential for GWA studies to identify novel therapeutic targets and genetic biomarkers that will be useful for drug discovery, patient selection and stratification in common diseases.

A number of mammalian antimicrobial proteins produced by neutrophils and cells of epithelial origin have chemotactic and activating effects on host cells, including cells of the immune system. Eosinophil granules contain an antimicrobial protein known as eosinophil-derived neurotoxin (EDN), which belongs to the RNase A superfamily. EDN has antiviral and chemotactic activities in vitro. In this study, we show that EDN, and to a lesser extent human pancreatic RNase (hPR), another RNase A superfamily member, activates human dendritic cells (DCs), leading to the production of a variety of inflammatory cytokines, chemokines, growth factors, and soluble receptors. Human angiogenin, a RNase evolutionarily more distant to EDN and hPR, did not display such activating effects. Additionally, EDN and hPR also induced phenotypic and functional maturation DCs. These RNases were as efficacious as TNF-α, but induced a different set of cytokine mediators. Furthermore, EDN production by human macrophages could be induced by proinflammatory stimuli. The results reveal the DC-activating activity of EDN and hPR and suggest that they are likely participants of inflammatory and immune responses. A number of endogenous mediators in addition to EDN have been reported to have both chemotactic and activating effects on APCs, and can thus amplify innate and Ag-specific immune responses to danger signals. We therefore propose these mediators be considered as endogenous multifunctional immune alarmins.

The muted (mu) mouse is a model for Hermansky–Pudlak Syndrome (HPS), an inherited disorder of humans causing hypopigmentation, hemorrhaging and early death due to lung abnormalities. The mu gene regulates the synthesis of specialized mammalian organelles such as melanosomes, platelet dense granules and lysosomes. Further, balance defects indicate that it controls the synthesis of otoliths of the inner ear. The mu gene has been identified by a positional/candidate approach involving large mouse interspecific backcrosses. It encodes a novel ubiquitously expressed transcript, specifying a predicted 185 amino acid protein, whose expression is abrogated in the mu allele which contains an insertion of an early transposon (ETn) retrotransposon. Expression is likewise expected to be lost in the mu J allele which contains a deletion of a single base pair within the coding region. The presence of structurally aberrant melanosomes within the eyes of mutant mice together with localization of the muted protein within vesicles in both the cell body and dendrites of transfected melan-a melanocytes emphasizes the role of the mu gene in vesicle trafficking. The mu gene is present only in mice and humans among analyzed genomes. As is true for several other recently identified mouse HPS genes, the mu gene is absent in lower eukaryotes. Therefore, the mu gene is a member of the novel gene set that has evolved in higher eukaryotes to regulate the synthesis/function of highly specialized subcellular organelles such as melanosomes and platelet dense granules.

High-throughput DNA sequencing has enabled systems biology to begin to address areas in health, agricultural and basic biological research. Concomitant with the opportunities is an absolute necessity to manage significant volumes of high-dimensional and inter-related data and analysis. Alpheus is an analysis pipeline, database and visualization software for use with massively parallel DNA sequencing technologies that feature multi-gigabase throughput characterized by relatively short reads, such as Illumina-Solexa (sequencing-by-synthesis), Roche-454 (pyrosequencing) and Applied Biosystem’s SOLiD (sequencing-by-ligation). Alpheus enables alignment to reference sequence(s), detection of variants and enumeration of sequence abundance, including expression levels in transcriptome sequence. Alpheus is able to detect several types of variants, including non-synonymous and synonymous single nucleotide polymorphisms (SNPs), insertions/deletions (indels), premature stop codons, and splice isoforms. Variant detection is aided by the ability to filter variant calls based on consistency, expected allele frequency, sequence quality, coverage, and variant type in order to minimize false positives while maximizing the identification of true positives. Alpheus also enables comparisons of genes with variants between cases and controls or bulk segregant pools. Sequence-based differential expression comparisons can be developed, with data export to SAS JMP Genomics for statistical analysis.

Through alternative processing of pre-mRNAs, individual mammalian genes often produce multiple mRNA and protein isoforms that may have related, distinct or even opposing functions. Here we report an in-depth analysis of 15 diverse human tissue and cell line transcriptomes based on deep sequencing of cDNA fragments, yielding a digital inventory of gene and mRNA isoform expression. Analysis of mappings of sequence reads to exon-exon junctions indicated that 92-94% of human genes undergo alternative splicing (AS), ∼86% with a minor isoform frequency of 15% or more. Differences in isoform-specific read densities indicated that a majority of AS and of alternative cleavage and polyadenylation (APA) events vary between tissues, while variation between individuals was ∼2- to 3-fold less common. Extreme or ‘switch-like’ regulation of splicing between tissues was associated with increased sequence conservation in regulatory regions and with generation of full-length open reading frames. Patterns of AS and APA were strongly correlated across tissues, suggesting coordinated regulation of these processes, and sequence conservation of a subset of known regulatory motifs in both alternative introns and 3′ UTRs suggested common involvement of specific factors in tissue-level regulation of both splicing and polyadenylation.

Vaccinia virus is reactogenic in a significant number of vaccinees, with the most common adverse events being fever, lymphadenopathy, and rash. Although the inoculation is given in the skin, these adverse events suggest a robust systemic inflammatory response. To elucidate the cytokine response signature of systemic adverse events, we used a protein microarray technique to precisely quantitate 108 serum cytokines and chemokines in vaccine recipients before and 1 week after primary immunization with Aventis Pasteur smallpox vaccine. We studied 74 individuals after vaccination, of whom 22 experienced a systemic adverse event and 52 did not. The soluble factors most associated with adverse events were selected on the basis of voting among a committee of machine-learning methods and statistical procedures, and the selected cytokines were used to build a final decision-tree model. On the basis of changes in protein expression, we identified 6 cytokines that accurately discriminate between individuals on the basis of adverse event status: granulocyte colony–stimulating factor, stem cell factor, monokine induced by interferon-γ (CXCL9), intercellular adhesion molecule–1, eotaxin, and tissue inhibitor of metalloproteinases–2. This cytokine signature is characteristic of particular inflammatory response pathways and suggests that the secretion of cytokines by fibroblasts plays a central role in systemic adverse events.

OBJECTIVES

Cytokines and growth factors play a major role in the dysregulated immune response in inflammatory bowel disease (IBD). We hypothesized that significant differences exist between the serum cytokine and growth factor profiles of pediatric IBD patients with active disease (AD) and those in remission, and that levels of some of these soluble mediators may be used to define regulators in IBD and determine disease activity.

METHODS

Eighty-eight, consecutive patients with confirmed Crohn’s disease (CD) and ulcerative colitis (UC) seen at the Duke Children’s Hospital were prospectively enrolled and a serum sample was obtained. Data were recorded at the time of serum collection to calculate disease activity indices. The relative expression of 78 cytokines, growth factors, and soluble receptors was determined using proprietary antibody-based protein microarrays amplified by rolling circle amplification. SPSS 8 (SPSS Inc., Chicago, IL) was used to compare protein profiles for CD and UC patients in clinical remission (CR) versus AD.

RESULTS

Sixty-five CD patients and 23 UC patients were enrolled. Forty-one CD patients had available samples and PCDAI results. Twenty-two patients were in remission PCDAI ≤ 12.5 (median 5), 19 patients had disease activity >15 (median 30). Univariate analysis revealed that PLGF, IL-7, IL-12p40, and TGF-β1 cytokine levels were significantly elevated for patients in CR versus AD (p < 0.01). Twelve UC serum samples had Seo/Truelove Witt AI for analysis. Five patients were in remission by TW AI and Seo AI ≤ 110 and 7 patients had active mild-to-severe disease by TW and Seo AI >110. Only one cytokine, IL12p40, showed significance between CR versus AD (p < 0.02).

CONCLUSIONS

Surprisingly, we found no differences in circulating levels of proinflammatory cytokines but found that pediatric IBD patients in remission compared to those with AD had higher levels of specific circulating cytokines, including the regulatory cytokines IL-12p40, and TGF-β1. It may be that these cytokines directly regulate intestinal inflammation in IBD or reflect the activity of T regulatory cells in negatively regulating the inflammatory response. Further studies will be needed to validate our results to define the molecular pathways involved in the intestinal immune response in man.

The ability to measure the abundance of many proteins precisely and simultaneously in experimental samples is an important, recent advance for static and dynamic, as well as descriptive and predictive, biological research. The value of multiplexed protein measurement is being established in applications such as comprehensive proteomic surveys, studies of protein networks and pathways, validation of genomic discoveries and clinical biomarker development. As standards do not yet exist that bridge all of these applications, the current recommended best practice for validation of results is to approach study design in an iterative process and to integrate data from several measurement technologies. This review describes current and emerging multiplexed protein measurement technologies and their applications, and discusses the remaining challenges in this field.

Background

Sepsis (bloodstream infection) is the leading cause of death in non-surgical intensive care units. It is diagnosed in 750,000 US patients per annum, and has high mortality. Current understanding of sepsis is predominately observational and correlational, with only a partial and incomplete understanding of the physiological dynamics underlying the syndrome. There exists a need for dynamical models of sepsis progression, based upon basic physiologic principles, which could eventually guide hourly treatment decisions.

Results

We present an initial mathematical model of sepsis, based on metabolic rate theory that links basic vascular and immunological dynamics. The model includes the rate of vascular circulation, a surrogate for the metabolic rate that is mechanistically associated with disease progression. We use the mass-specific rate of blood circulation (SRBC), a correlate of the body mass index, to build a differential equation model of circulation, infection, organ damage, and recovery. This introduces a vascular component into an infectious disease model that describes the interaction between a pathogen and the adaptive immune system.

Conclusion

The model predicts that deviations from normal SRBC correlate with disease progression and adverse outcome. We compare the predictions with population mortality data from cardiovascular disease and cancer and show that deviations from normal SRBC correlate with higher mortality rates.

BACKGROUND:Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis. MATERIALS AND METHODS: Fourteen cDNA fragments, covering the entire coding domain of LYST, were used as baits to screen five human cDNA libraries by a yeast two-hybrid method, modified to allow screening in the activation and the binding domain, three selectable markers, and more stringent confirmation procedures. Five of the interactions were confirmed by an in vitro binding assay. RESULTS: Twenty-one proteins that interact with LYST were identified in yeast two-hybrid screens. Four interactions, confirmed directly, were with proteins important in vesicular transport and signal transduction (the SNARE-complex protein HRS, 14-3-3, and casein kinase II). CONCLUSIONS:On the basis of protein interactions, LYST appears to function as an adapter protein that may juxtapose proteins that mediate intracellular membrane fusion reactions. The pathologic manifestations observed in CHS patients and in mice with the homologous mutation beige suggest that understanding the role of LYST may be relevant to the treatment of not only CHS but also of diseases such as asthma, urticaria, and lupus, as well as to the molecular dissection of the CHS-associated cancer predisposition.

The oomycete vegetable pathogen Phytophthora capsici has shown remarkable adaptation to fungicides and new hosts. Like other members of this destructive genus, P. capsici has an explosive epidemiology, rapidly producing massive numbers of asexual spores on infected hosts. In addition, P. capsici can remain dormant for years as sexually-recombined oospores, making it difficult to produce crops at infested sites, and allowing outcrossing populations to maintain significant genetic variation. Genome sequencing, development of a high-density genetic map, and integrative genomic/genetic characterization of P. capsici field isolates and intercross progeny revealed significant mitotic loss of heterozygosity (LOH) and higher levels of SNVs than those reported for humans, plants, and P. infestans. LOH was detected in clonally propagated field isolates and sexual progeny, cumulatively affecting >30% of the genome. LOH altered genotypes for more than 11,000 single nucleotide variant (SNV) sites and showed a strong association with changes in mating type and pathogenicity. Overall, it appears that LOH may provide a rapid mechanism for fixing alleles and may be an important component of adaptability for P. capsici.

Staphylococcus aureus causes a spectrum of human infection. Diagnostic delays and uncertainty lead to treatment delays and inappropriate antibiotic use. A growing literature suggests the host’s inflammatory response to the pathogen represents a potential tool to improve upon current diagnostics. The hypothesis of this study is that the host responds differently to S. aureus than to E. coli infection in a quantifiable way, providing a new diagnostic avenue. This study uses Bayesian sparse factor modeling and penalized binary regression to define peripheral blood gene-expression classifiers of murine and human S. aureus infection. The murine-derived classifier distinguished S. aureus infection from healthy controls and Escherichia coli-infected mice across a range of conditions (mouse and bacterial strain, time post infection) and was validated in outbred mice (AUC>0.97). A S. aureus classifier derived from a cohort of 94 human subjects distinguished S. aureus blood stream infection (BSI) from healthy subjects (AUC 0.99) and E. coli BSI (AUC 0.84). Murine and human responses to S. aureus infection share common biological pathways, allowing the murine model to classify S. aureus BSI in humans (AUC 0.84). Both murine and human S. aureus classifiers were validated in an independent human cohort (AUC 0.95 and 0.92, respectively). The approach described here lends insight into the conserved and disparate pathways utilized by mice and humans in response to these infections. Furthermore, this study advances our understanding of S. aureus infection; the host response to it; and identifies new diagnostic and therapeutic avenues.

There is great potential for host-based gene expression analysis to impact the early diagnosis of infectious diseases. In particular, the influenza pandemic of 2009 highlighted the challenges and limitations of traditional pathogen-based testing for suspected upper respiratory viral infection. We inoculated human volunteers with either influenza A (A/Brisbane/59/2007 (H1N1) or A/Wisconsin/67/2005 (H3N2)), and assayed the peripheral blood transcriptome every 8 hours for 7 days. Of 41 inoculated volunteers, 18 (44%) developed symptomatic infection. Using unbiased sparse latent factor regression analysis, we generated a gene signature (or factor) for symptomatic influenza capable of detecting 94% of infected cases. This gene signature is detectable as early as 29 hours post-exposure and achieves maximal accuracy on average 43 hours (p = 0.003, H1N1) and 38 hours (p-value = 0.005, H3N2) before peak clinical symptoms. In order to test the relevance of these findings in naturally acquired disease, a composite influenza A signature built from these challenge studies was applied to Emergency Department patients where it discriminates between swine-origin influenza A/H1N1 (2009) infected and non-infected individuals with 92% accuracy. The host genomic response to Influenza infection is robust and may provide the means for detection before typical clinical symptoms are apparent.

Monozygotic (MZ) or “identical” twins have been widely studied to dissect the relative contributions of genetics and environment in human diseases. In multiple sclerosis (MS), an autoimmune demyelinating disease and common cause of neurodegeneration and disability in young adults, disease discordance in MZ twins has been interpreted to indicate environmental importance in its pathogenesis1–8. However, genetic and epigenetic differences between MZ twins have been described, challenging the accepted experimental paradigm in disambiguating effects of nature and nurture.9–12 Here, we report the genome sequences of one MS-discordant MZ twin pair and messenger RNA (mRNA) transcriptome and epigenome sequences of CD4+ lymphocytes from three MS-discordant, MZ twin pairs. No reproducible differences were detected between co-twins among ~3.6 million single nucleotide polymorphisms (SNPs) or ~0.2 million insertion-deletion polymorphisms (indels). Nor were any reproducible differences observed between siblings of the three twin pairs in HLA haplotypes, confirmed MS-susceptibility SNPs, copy number variations, mRNA and genomic SNP and indel genotypes, or expression of ~19,000 genes in CD4+ T cells. Only two to 176 differences in methylation of ~2 million CpG dinucleotides were detected between siblings of the three twin pairs, in contrast to ~800 methylation differences between T cells of unrelated individuals and several thousand differences between tissues or normal and cancerous tissues. In the first systematic effort to estimate sequence variation among MZ co-twins, we did not find evidence for genetic, epigenetic or transcriptome differences that explained disease discordance. These are the first female, twin and autoimmune disease individual genome sequences reported.

Hermansky-Pudlak syndrome (HPS; MIM 203300) is a genetically heterogeneous disorder characterized by oculocutaneous albinism, prolonged bleeding and pulmonary fibrosis due to abnormal vesicle trafficking to lysosomes and related organelles, such as melanosomes and platelet dense granules1–3. In mice, at least 16 loci are associated with HPS4–6, including sandy (sdy; ref. 7). Here we show that the sdy mutant mouse expresses no dysbindin protein owing to a deletion in the gene Dtnbp1 (encoding dysbindin) and that mutation of the human ortholog DTNBP1 causes a novel form of HPS called HPS-7. Dysbindin is a ubiquitously expressed protein that binds to α- and β-dystrobrevins, components of the dystrophin-associated protein complex (DPC) in both muscle and nonmuscle cells8. We also show that dysbindin is a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1; refs. 9–11), which regulates trafficking to lysosome-related organelles and includes the proteins pallidin, muted and cappuccino, which are associated with HPS in mice. These findings show that BLOC-1 is important in producing the HPS phenotype in humans, indicate that dysbindin has a role in the biogenesis of lysosome-related organelles and identify unexpected interactions between components of DPC and BLOC-1.

Schizophrenia (SCZ) is a common, disabling mental illness with high heritability but complex, poorly understood genetic etiology. As the first phase of a genomic convergence analysis of SCZ, we generated 16.7 billion nucleotides of short read, shotgun sequences of cDNA from post-mortem cerebellar cortices of 14 patients and six, matched controls. A rigorous analysis pipeline was developed for analysis of digital gene expression studies. Sequences aligned to approximately 33,200 transcripts in each sample, with average coverage of 450 reads per gene. Following adjustments for confounding clinical, sample and experimental sources of variation, 215 genes differed significantly in expression between cases and controls. Golgi apparatus, vesicular transport, membrane association, Zinc binding and regulation of transcription were over-represented among differentially expressed genes. Twenty three genes with altered expression and involvement in presynaptic vesicular transport, Golgi function and GABAergic neurotransmission define a unifying molecular hypothesis for dysfunction in cerebellar cortex in SCZ.