Broad-Spectrum Detection of HPV in Male Genital Samples Using Target-Enriched Whole-Genome Sequencing.

Most human papillomavirus (HPV) surveillance studies target 30-50 of the more than 200 known types. We applied our recently described enriched whole-genome sequencing (eWGS) assay to demonstrate the impact of detecting all known and novel HPV types in male genital samples (n = 50). HPV was detected in nearly all (82%) samples, (mean number of types/samples 13.6; range 1-85), and nearly all HPV-positive samples included types in multiple genera (88%). A total of 560 HPV detections (237 unique HPV types: 46 alpha, 55 beta, 135 gamma, and 1 mu types) were made. The most frequently detected HPV types were alpha (HPV90, 43, and 74), beta (HPV115, 195, and 120), and gamma (HPV134, mSD2, and HPV50). High-risk alpha types (HPV16, 18, 31, 39, 52, and 58) were not common. A novel gamma type was identified (now officially HPV229) along with 90 unclassified types. This pilot study demonstrates the utility of the eWGS assay for broad-spectrum type detection and suggests a significantly higher type diversity in males compared to females that warrants further study.

Enhanced Immunogenicity of Adjuvanted Microparticulate HPV16 Vaccines Administered via the Transdermal Route.

Human papillomavirus (HPV) causes cervical cancer among women and is associated with other anogenital cancers in men and women. Prophylactic particulate vaccines that are affordable, self-administered and efficacious could improve uptake of HPV vaccines world-wide. The goal of this research is to develop a microparticulate HPV16 vaccine for transdermal administration using AdminPatch® and assess its immunogenicity in a pre-clinical mouse model. HPV16 microparticles were prepared using a biocompatible polymer and characterized in terms of size, zeta potential, encapsulation efficiency and microparticle yield. Scanning and transmission electron microscopy were conducted to confirm particle image and to visualize the conformation of HPV16 vaccine particles released from microparticle formulation. In vivo studies performed to evaluate the potential of the microparticulate vaccine initiated a robust and sustained immune response. HPV16 IgG antibodies were significantly elevated in the microparticle group compared to antigen solutions administered by the transdermal route. Results show significant expansion of CD4+, CD45R, CD27 and CD62L cell populations in the vaccinated mice group, indicating the high efficacy of the microparticulate vaccine when administered via transdermal route. The findings of this study call attention to the use of minimally invasive, pain-free routes to deliver vaccine.

NanoString Technology for Human Papillomavirus Typing.

High-throughput HPV typing assays with increased automation, faster turnaround and type-specific digital readout would facilitate studies monitoring the impact of HPV vaccination. We evaluated the NanoString nCounter® platform for detection and digital readout of 48 HPV types in a single reaction. NanoString (NS) used proprietary software to design CodeSets: type-specific probe pairs targeting 48 HPV types and the globin gene. We tested residual DNA extracts from epidemiologic specimens and defined samples (HPV plasmids at 10 to 104 copies/reaction) directly (No-PCR) as well as after L1 consensus PCR of 45 (PCR-45) or 15 cycles (PCR-15). Assay and interpretation followed NS recommendations. We evaluated analytic performance by comparing NanoString results for types included in prior assays: Roche Linear Array (LA) or HPV TypeSeq assay. No-PCR results on 40 samples showed good type-specific agreement with LA (k = 0.621) but sensitivity was 65% with lower limit of detection (LOD) at 104 plasmid copies. PCR-45 results showed almost perfect type-specific agreement with LA (k = 0.862), 82% sensitivity and LOD at 10 copies. PCR-15 results on 75 samples showed substantial type-specific agreement with LA (k = 0.796, 92% sensitivity) and TypeSeq (k = 0.777, 87% sensitivity), and LOD at 10 copies of plasmids. This proof-of-principle study demonstrates the efficacy of the NS platform with HPV CodeSet for type-specific detection using a low number of PCR cycles (PCR-15). Studies are in progress to evaluate assay reproducibility and analytic validation with a larger number of samples.

Bioinformatics Pipeline for Human Papillomavirus Short Read Genomic Sequences Classification Using Support Vector Machine.

We recently developed a test based on the Agilent SureSelect target enrichment system capturing genomic fragments from 191 human papillomaviruses (HPV) types for Illumina sequencing. This enriched whole genome sequencing (eWGS) assay provides an approach to identify all HPV types in a sample. Here we present a machine learning algorithm that calls HPV types based on the eWGS output. The algorithm based on the support vector machine (SVM) technique was trained on eWGS data from 122 control samples with known HPV types. The new algorithm demonstrated good performance in HPV type detection for designed samples with 25 or greater HPV plasmid copies per sample. We compared the results of HPV typing made by the new algorithm for 261 residual epidemiologic samples with the results of the typing delivered by the standard HPV Linear Array (LA). The agreement between methods (97.4%) was substantial (kappa= 0.783). However, the new algorithm identified additionally 428 instances of HPV types not detectable by the LA assay by design. Overall, we have demonstrated that the bioinformatics pipeline is an accurate tool for calling HPV types by analyzing data generated by eWGS processing of DNA fragments extracted from control and epidemiological samples.

Pilot Study of Markers for High-grade Anal Dysplasia in a Southern Cohort From the Women's Interagency Human Immunodeficiency Virus Study.

BACKGROUND: Anal cancer rates have increased, particularly in human immunodeficiency virus (HIV)-infected (HIV+) women. We assessed factors associated with anal precancer in HIV+ and at-risk HIV-negative women from the Atlanta Women's Interagency HIV Study cohort. METHODS: All participants underwent high-resolution anoscopy and anal cytology and had anal and cervical samples collected. Specimens were tested for 37 human papillomavirus (HPV) types and for FAM19A4 and microRNA124-2 promoter methylation. Binary logistic regression and multivariate analysis were conducted with histologic anal high-grade squamous intraepithelial lesion (A-HSIL) as the dependent variable. RESULTS: Seventy-five women were enrolled: 52 (69%) were HIV+ with three-fourths having undetectable viral load; 64 (86%) were black; mean age was 49 ± 8 years. Forty-nine (65%) anal cytology samples were abnormal, and 38 (51%) of anal samples were positive for at least 1 of 13 high-risk HPV (hrHPV) types. Thirteen (18%) anal biopsies identified A-HSIL. Hypermethylation of FAM19A4 and/or microRNA124-2 was found in 69 (95%) anal samples and 19 (26%) cervical samples. In multivariate analyses, the odds of having A-HSIL were >6 times higher in women with anal hrHPV (adjusted odds ratio [aOR], 6.08 [95% confidence interval {CI}, 1.27-29.18], P = .02) and with positive cervical methylation (aOR, 6.49 [95% CI, 1.66-25.35], P = .007), but not significantly higher in women with positive anal methylation. CONCLUSIONS: Anal hrHPV and promoter hypermethylation in the cervix show promise as biomarkers for anal cancer screening in HIV+ and at-risk HIV-negative women. Greater understanding of gene silencing by promoter hypermethylation in anal carcinogenesis is needed.

Universal human papillomavirus typing by whole genome sequencing following target enrichment: evaluation of assay reproducibility and limit of detection.

BACKGROUND: We recently described a method for unbiased detection of all known human papillomaviruses (HPV) types with the potential for the determination of their variant and integration from the resulting whole genome sequence data. Considering the complex workflow for target-enriched next generation sequencing (NGS), we focused on the reproducibility and limit of detection (LOD) of this new universal HPV typing assay in this study. RESULTS: We evaluated the reproducibility and LOD for HPV genotyping based on our recently published method that used RNA-baits targeting whole genomes of 191 HPV types, Agilent SureSelect protocol for target enrichment and Illumina HiSeq 2500 for sequencing (eWGS, enriched whole genome sequencing). Two libraries, prepared from pooled plasmids representing 9 vaccine HPV types at varying input (1-625 copies/reaction), were sequenced twice giving four replicates for evaluating reproducibility and LOD. eWGS showed high correlation in the number of reads mapped to HPV reference genomes between the two flow-cell lanes within (R2 = 1) and between experiments (R2 = 0.99). The number of mapped reads was positively correlated to copy number (ß = 13.9, p < 0.0001). The limit of blank (LOB) could be calculated based on mapped reads to HPV types not included in each sample. HPV genotyping was reproducible for all 9 types at 625 copies using multiple cut-off criteria but LOD was 25 copies based on number of reads above LOB even when multiple types were present. eWGS showed no bias for HPV genotyping under single or multiple infection (p = 0.16-0.99). CONCLUSIONS: The universal eWGS method for HPV genotyping has sensitivity, competitive with widely used consensus PCR methods with reduced type competition, and with the potential for determination of variant and integration status. The protocol used in this study, using defined samples varying in complexity and copy number, analyzed in replicate and duplicate assays, is applicable to most WGS methods.

Association of chronic fatigue syndrome with premature telomere attrition.

BACKGROUND: Chronic fatigue syndrome (CFS), also known as myalgic encephalomyelitis (ME), is a severely debilitating condition of unknown etiology. The symptoms and risk factors of ME/CFS share features of accelerated aging implicated in several diseases. Using telomere length as a marker, this study was performed to test the hypothesis that ME/CFS is associated with accelerated aging. METHODS: Participant (n = 639) data came from the follow-up time point of the Georgia CFS surveillance study. Using the 1994 CFS Research Case Definition with questionnaire-based subscale thresholds for fatigue, function, and symptoms, participants were classified into four illness groups: CFS if all criteria were met (n = 64), CFS-X if CFS with exclusionary conditions (n = 77), ISF (insufficient symptoms/fatigue) if only some criteria were met regardless of exclusionary conditions (n = 302), and NF (non-fatigued) if no criteria and no exclusionary conditions (n = 196). Relative telomere length (T/S ratio) was measured using DNA from whole blood and real-time PCR. General linear models were used to estimate the association of illness groups or T/S ratio with demographics, biological measures and covariates with significance set at p < 0.05. RESULTS: The mean T/S ratio differed significantly by illness group (p = 0.0017); the T/S ratios in CFS (0.90 ± 0.03) and ISF (0.94 ± 0.02) were each significantly lower than in NF (1.06 ± 0.04). Differences in T/S ratio by illness groups remained significant after adjustment for covariates of age, sex, body mass index, waist-hip ratio, post-exertional malaise and education attainment. Telomere length was shorter by 635, 254 and 424 base pairs in CFS, CFS-X and ISF, respectively, compared to NF. This shorter telomere length translates to roughly 10.1-20.5, 4.0-8.2 and 6.6-13.7 years of additional aging in CFS, CFS-X and ISF compared to NF respectively. Further, stratified analyses based on age and sex demonstrated that the association of ME/CFS with short telomeres is largely moderated by female subjects < 45 years old. CONCLUSIONS: This study found a significant association of ME/CFS with premature telomere attrition that is largely moderated by female subjects < 45 years old. Our results indicate that ME/CFS could be included in the list of conditions associated with accelerated aging. Further work is needed to evaluate the functional significance of accelerated aging in ME/CFS.

Multi-Site Clinical Assessment of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (MCAM): Design and Implementation of a Prospective/Retrospective Rolling Cohort Study.

In the Multi-Site Clinical Assessment of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (MCAM), we relied on expert clinician diagnoses to enroll patients from 7 specialty clinics in the United States in order to perform a systematic collection of data on measures of myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS). Healthy persons and those with other illnesses that share some features with ME/CFS were enrolled in comparison groups. The major objectives were to: 1) use standardized questionnaires to measure illness domains of ME/CFS and to evaluate patient heterogeneity overall and between clinics; 2) describe the course of illness, identify the measures that best correlate with meaningful clinical differences, and assess the performances of questionnaires as patient/person-reported outcome measures; 3) describe prescribed medications, orders for laboratory and other tests, and management tools used by expert clinicians to care for persons with ME/CFS; 4) collect biospecimens for future hypothesis testing and for evaluation of morning cortisol profiles; and 5) identify measures that best distinguish persons with ME/CFS from those in the comparison groups and detect subgroups of persons with ME/CFS who may have different underlying causes. Enrollment began in 2012 and is planned to continue in multiple stages through 2017. We present the MCAM methods in detail, along with an initial description of the 471 patients with ME/CFS who were enrolled in stage 1.

Precision Medicine Requires Precision Laboratories.

This commentary highlights the validation study by Lih et al that supports the use of precision medicine for improved clinical trials.

Universal Human Papillomavirus Typing Assay: Whole-Genome Sequencing following Target Enrichment.

We designed a universal human papillomavirus (HPV) typing assay based on target enrichment and whole-genome sequencing (eWGS). The RNA bait included 23,941 probes targeting 191 HPV types and 12 probes targeting beta-globin as a control. We used the Agilent SureSelect XT2 protocol for library preparation, Illumina HiSeq 2500 for sequencing, and CLC Genomics Workbench for sequence analysis. Mapping stringency for type assignment was determined based on 8 (6 HPV-positive and 2 HPV-negative) control samples. Using the optimal mapping conditions, types were assigned to 24 blinded samples. eWGS results were 100% concordant with Linear Array (LA) genotyping results for 9 plasmid samples and fully or partially concordant for 9 of the 15 cervical-vaginal samples, with 95.83% overall type-specific concordance for LA genotyping. eWGS identified 7 HPV types not included in the LA genotyping. Since this method does not involve degenerate primers targeting HPV genomic regions, PCR bias in genotype detection is minimized. With further refinements aimed at reducing cost and increasing throughput, this first application of eWGS for universal HPV typing could be a useful method to elucidate HPV epidemiology.

Pathway-focused genetic evaluation of immune and inflammation related genes with chronic fatigue syndrome.

Recent evidence suggests immune and inflammatory alterations are important in chronic fatigue syndrome (CFS). This study was done to explore the association of functionally important genetic variants in inflammation and immune pathways with CFS. Peripheral blood DNA was isolated from 50 CFS and 121 non-fatigued (NF) control participants in a population-based study. Genotyping was performed with the Affymetrix Immune and Inflammation Chip that covers 11K single nucleotide polymorphisms (SNPs) following the manufacturer's protocol. Genotyping accuracy for specific genes was validated by pyrosequencing. Golden Helix SVS software was used for genetic analysis. SNP functional annotation was done using SPOT and GenomePipe programs. CFS was associated with 32 functionally important SNPs: 11 missense variants, 4 synonymous variants, 11 untranslated regulatory region (UTR) variants and 6 intronic variants. Some of these SNPs were in genes within pathways related to complement cascade (SERPINA5, CFB, CFH, MASP1 and C6), chemokines (CXCL16, CCR4, CCL27), cytokine signaling (IL18, IL17B, IL2RB), and toll-like receptor signaling (TIRAP, IRAK4). Of particular interest is association of CFS with two missense variants in genes of complement activation, rs4151667 (L9H) in CFB and rs1061170 (Y402H) in CFH. A 5' UTR polymorphism (rs11214105) in IL18 also associated with physical fatigue, body pain and score for CFS case defining symptoms. This study identified new associations of CFS with genetic variants in pathways including complement activation providing additional support for altered innate immune response in CFS. Additional studies are needed to validate the findings of this exploratory study.

Prediction of complex human diseases from pathway-focused candidate markers by joint estimation of marker effects: case of chronic fatigue syndrome.

BACKGROUND: The current practice of using only a few strongly associated genetic markers in regression models results in generally low power in prediction or accounting for heritability of complex human traits. PURPOSE: We illustrate here a Bayesian joint estimation of single nucleotide polymorphism (SNP) effects principle to improve prediction of phenotype status from pathway-focused sets of SNPs. Chronic fatigue syndrome (CFS), a complex disease of unknown etiology with no laboratory methods for diagnosis, was chosen to demonstrate the power of this Bayesian method. For CFS, such a genetic predictive model in combination with clinical evidence might lead to an earlier diagnosis than one based solely on clinical findings. METHODS: One of our goals is to model disease status using Bayesian statistics which perform variable selection and parameter estimation simultaneously and which can induce the sparseness and smoothness of the SNP effects. Smoothness of the SNP effects is obtained by explicit modeling of the covariance structure of the SNP effects. RESULTS: The Bayesian model achieved perfect goodness of fit when tested within the sampled data. Tenfold cross-validation resulted in 80% accuracy, one of the best so far for CFS in comparison to previous prediction models. Model reduction aspects were investigated in a computationally feasible manner. Additionally, genetic variation estimates provided by the model identified specific genetic markers for their biological role in the disease pathophysiology. CONCLUSIONS: This proof-of-principle study provides a powerful approach combining Bayesian methods, SNPs representing multiple pathways and rigorous case ascertainment for accurate genetic risk prediction modeling of complex diseases like CFS and other chronic diseases.

Human papillomavirus prevalence in invasive anal cancers in the United States before vaccine introduction.

OBJECTIVE: This study aimed to conduct a representative survey of human papillomavirus (HPV) prevalence and its genotype distribution in invasive anal cancer specimens in the United States. MATERIALS AND METHODS: Population-based archival anal cancer specimens were identified from Florida, Kentucky, Louisiana, and Michigan cancer registries and Surveillance, Epidemiology, and End Results (SEER) tissue repositories in Hawaii, Iowa, and Los Angeles. Sections from 1 representative block per case were used for DNA extraction. All extracts were assayed first by linear array and retested with INNO-LiPA if inadequate or HPV negative. RESULTS: Among 146 unique invasive anal cancer cases, 93 (63.7%) were from women, and 53 (36.3%) were from men. Human papillomavirus (any type) was detected in 133 cases (91.1%) and 129 (88.4%) contained at least 1 high risk-type, most (80.1%) as a single genotype. Human papillomavirus type 16 had the highest prevalence (113 cases, 77.4%); HPV types 6, 11, 18, and 33 were also found multiple times. Among HPV-16-positive cases, 37% were identified as prototype variant Ep, and 63% were nonprototypes: 33% Em, 12% E-G131G, 5% Af1, 4% AA/NA-1, 3% E-C109G, 3% E-G131T, 2% As, and 1% Af2. No significant differences in the distributions of HPV (any), high-risk types, or HPV-16/18 were seen between sex, race, or age group. CONCLUSIONS: The establishment of prevaccine HPV prevalence in the United States is critical to the surveillance of vaccine efficacy. Almost 80% of anal cancers were positive for the vaccine types HPV-16 or HPV-18, and in 70%, these were the only types detected, suggesting that a high proportion might be preventable by current vaccines.

Convergent genomic studies identify association of GRIK2 and NPAS2 with chronic fatigue syndrome.

BACKGROUND: There is no consistent evidence of specific gene(s) or molecular pathways that contribute to the pathogenesis, therapeutic intervention or diagnosis of chronic fatigue syndrome (CFS). While multiple studies support a role for genetic variation in CFS, genome-wide efforts to identify associated loci remain unexplored. We employed a novel convergent functional genomics approach that incorporates the findings from single-nucleotide polymorphism (SNP) and mRNA expression studies to identify associations between CFS and novel candidate genes for further investigation. METHODS: We evaluated 116,204 SNPs in 40 CFS and 40 nonfatigued control subjects along with mRNA expression of 20,160 genes in a subset of these subjects (35 CFS subjects and 27 controls) derived from a population-based study. RESULTS: Sixty-five SNPs were nominally associated with CFS (p<0.001), and 165 genes were differentially expressed (≥4-fold; p≤0.05) in peripheral blood mononuclear cells of CFS subjects. Two genes, glutamate receptor, ionotropic, kinase 2 (GRIK2) and neuronal PAS domain protein 2 (NPAS2), were identified by both SNP and gene expression analyses. Subjects with the G allele of rs2247215 (GRIK2) were more likely to have CFS (p=0.0005), and CFS subjects showed decreased GRIK2 expression (10-fold; p=0.015). Subjects with the T allele of rs356653 (NPAS2) were more likely to have CFS (p=0.0007), and NPAS2 expression was increased (10-fold; p=0.027) in those with CFS. CONCLUSION: Using an integrated genomic strategy, this study suggests a possible role for genes involved in glutamatergic neurotransmission and circadian rhythm in CFS and supports further study of novel candidate genes in independent populations of CFS subjects.

Functional genomics of serotonin receptor 2A (HTR2A): interaction of polymorphism, methylation, expression and disease association.

Serotonergic neurotransmission plays a key role in the pathophysiology of neuropsychiatric illnesses. The functional significance of a promoter polymorphism, -1438G/A (rs6311), in one of the major genes of this system (serotonin receptor 2A, HTR2A) remains poorly understood in the context of epigenetic factors, transcription factors and endocrine influences. We used functional and structural equation modeling (SEM) approaches to assess the contributions of the polymorphism (rs6311), DNA methylation and clinical variables to HTR2A expression in chronic fatigue syndrome (CFS) subjects from a population-based study. HTR2A was up-regulated in CFS through allele-specific expression modulated by transcription factors at critical sites in its promoter: an E47 binding site at position -1,438, (created by the A-allele of rs6311 polymorphism), a glucocorticoid receptor (GR) binding site encompassing a CpG at position -1,420, and Sp1 binding at CpG methylation site -1,224. Methylation at -1,420 was strongly correlated with methylation at -1,439, a CpG site that is dependent upon the G-allele of rs6311 at position -1,438. SEM revealed a strong negative interaction between E47 and GR binding (in conjunction with cortisol level) on HTR2A expression. This study suggests that the promoter polymorphism (rs6311) can affect both transcription factor binding and promoter methylation, and this along with an individual's stress response can impact the rate of HTR2A transcription in a genotype and methylation-dependent manner. This study can serve as an example for deciphering the molecular determinants of transcriptional regulation of major genes of medical importance by integrating functional genomics and SEM approaches. Confirmation in an independent study population is required.

Transcriptional control of complement activation in an exercise model of chronic fatigue syndrome.

Complement activation resulting in significant increases of C4a split product may be a marker of postexertional malaise in individuals with chronic fatigue syndrome (CFS). This study focused on identification of the transcriptional control that may contribute to the increased C4a in CFS subjects after exercise. We used quantitative reverse-transcription polymerase chain reaction to evaluate differential expression of genes in the classical and lectin pathways in peripheral blood mononuclear cells (PBMCs). Calibrated expression values were normalized to the internal reference gene peptidylpropyl isomerase B (PPIB), the external reference gene ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL), or the geometric mean (GM) of the genes ribosomal protein, large, P0 (RPLP0) and phosphoglycerate kinase 1 (PGK1). All nine genes tested, except mannose-binding lectin 2 (MBL2), were expressed in PBMCs. At 1 hour postexercise, C4, mannan-binding lectin serine protease 2 (MASP2) and ficolin 1 (FCN1) transcripts were detected at higher levels (> or = 2-fold) in at least 50% (4 of 8) of CFS subjects and were detected in 88% (7 of 8) CFS subjects when subjects with overexpression of either C4 or MASP2 were combined. Only an increase in the MASP2 transcript was statistically significant (PPIB, P = 0.001; GM, P = 0.047; rbcL, P = 0.045). This result may be due to the significant but transient downregulation of MASP2 in control subjects (PPIB, P = 0.023; rbcL, P = 0.027). By 6 hours postexercise, MASP2 expression was similar in both groups. In conclusion, lectin pathway responded to exercise differentially in CFS than in control subjects. MASP2 down-regulation may act as an antiinflammatory acute-phase response in healthy subjects, whereas its elevated level may account for increased C4a and inflammation-mediated postexertional malaise in CFS subjects.

An angiotensin-1 converting enzyme polymorphism is associated with allostatic load mediated by C-reactive protein, interleukin-6 and cortisol.

Allostatic load (AL) is a theoretical framework that describes the cumulative physiologic effects of adaptation to change or stress throughout the lifespan. AL is operationalized by a composite index of multiple biomarkers. Accordingly, genes, behavior and environment contribute to AL. To determine if individual differences in AL may be influenced by inherent genetic variation, we calculated an allostatic load index (ALI) for 182 Caucasian subjects derived from a population-based study of chronic fatigue syndrome. Nearly 65% of the subjects in this study sample reported fatiguing illness. ALI was calculated based on 11 measures representing metabolic, cardiovascular, inflammatory, hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system (SNS) activities. Subjects were dichotomized into high (ALI > or = 3) or low (ALI < 3) AL groups, and the association between high AL and 129 polymorphisms in 32 genes related to the HPA axis, neurotransmission, inflammation, cardiovascular and metabolic functions were evaluated. Polymorphisms in angiotensin-1 converting enzyme (ACE), corticotropin-releasing hormone receptor 1 (CRHR1), and serotonin receptors (HTR3A and HTR4) were associated with AL (p=0.0007-0.0486), but only one polymorphism, rs4968591, in ACE remained significant after correction for multiple comparisons. The T allele of ACE rs4968591 was more common in subjects with high AL (67.5%) than in subjects with low AL (49.3%) (p=0.0007), and this effect appeared independent of age, sex, body mass index and fatigue status. Additionally, high interleukin-6 (IL-6; p(trend)=0.04), and C-reactive protein (CRP; p(trend)=0.01) levels, as well as low urinary cortisol levels in females (p=0.03) were associated with the T allele, which may result in allele-specific binding of the transcription factor, E2F1. Our results suggest a role for ACE in the bidirectional communication between the central nervous and immune systems in response to stress. Further studies will be needed (a) to replicate the association between AL and ACE polymorphisms in population studies designed to differentiate the effects of sex, age and racial/ethnic background, (b) to evaluate the effect of allele-specific binding of E2F1 at rs4968591, and (c) to examine the role of ACE in the co-regulation of CRP, IL-6 and cortisol.

Integrated weighted gene co-expression network analysis with an application to chronic fatigue syndrome.

BACKGROUND: Systems biologic approaches such as Weighted Gene Co-expression Network Analysis (WGCNA) can effectively integrate gene expression and trait data to identify pathways and candidate biomarkers. Here we show that the additional inclusion of genetic marker data allows one to characterize network relationships as causal or reactive in a chronic fatigue syndrome (CFS) data set. RESULTS: We combine WGCNA with genetic marker data to identify a disease-related pathway and its causal drivers, an analysis which we refer to as "Integrated WGCNA" or IWGCNA. Specifically, we present the following IWGCNA approach: 1) construct a co-expression network, 2) identify trait-related modules within the network, 3) use a trait-related genetic marker to prioritize genes within the module, 4) apply an integrated gene screening strategy to identify candidate genes and 5) carry out causality testing to verify and/or prioritize results. By applying this strategy to a CFS data set consisting of microarray, SNP and clinical trait data, we identify a module of 299 highly correlated genes that is associated with CFS severity. Our integrated gene screening strategy results in 20 candidate genes. We show that our approach yields biologically interesting genes that function in the same pathway and are causal drivers for their parent module. We use a separate data set to replicate findings and use Ingenuity Pathways Analysis software to functionally annotate the candidate gene pathways. CONCLUSION: We show how WGCNA can be combined with genetic marker data to identify disease-related pathways and the causal drivers within them. The systems genetics approach described here can easily be used to generate testable genetic hypotheses in other complex disease studies.

Genetic evaluation of the serotonergic system in chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a debilitating disorder of unknown etiology with no known lesions, diagnostic markers or therapeutic intervention. The pathophysiology of CFS remains elusive, although abnormalities in the central nervous system (CNS) have been implicated, particularly hyperactivity of the serotonergic (5-hydroxytryptamine; 5-HT) system and hypoactivity of the hypothalamic-pituitary-adrenal (HPA) axis. Since alterations in 5-HT signaling can lead to physiologic and behavioral changes, a genetic evaluation of the 5-HT system was undertaken to identify serotonergic markers associated with CFS and potential mechanisms for CNS abnormality. A total of 77 polymorphisms in genes related to serotonin synthesis (TPH2), signaling (HTR1A, HTR1E, HTR2A, HTR2B, HTR2C, HTR3A, HTR3B, HTR4, HTR5A, HTR6, and HTR7), transport (SLC6A4), and catabolism (MAOA) were examined in 137 clinically evaluated subjects (40 CFS, 55 with insufficient fatigue, and 42 non-fatigued, NF, controls) derived from a population-based CFS surveillance study in Wichita, Kansas. Of the polymorphisms examined, three markers (-1438G/A, C102T, and rs1923884) all located in the 5-HT receptor subtype HTR2A were associated with CFS when compared to NF controls. Additionally, consistent associations were observed between HTR2A variants and quantitative measures of disability and fatigue in all subjects. The most compelling of these associations was with the A allele of -1438G/A (rs6311) which is suggested to have increased promoter activity in functional studies. Further, in silico analysis revealed that the -1438 A allele creates a consensus binding site for Th1/E47, a transcription factor implicated in the development of the nervous system. Electrophoretic mobility shift assay supports allele-specific binding of E47 to the A allele but not the G allele at this locus. These data indicate that sequence variation in HTR2A, potentially resulting in its enhanced activity, may be involved in the pathophysiology of CFS.

Evaluation of RNA markers for early detection of cervical neoplasia in exfoliated cervical cells.

Numerous molecular biomarkers have been suggested for early detection of cervical cancer, but their usefulness in routinely collected exfoliated cells remains uncertain. We used quantitative reverse transcription-PCR to evaluate expression of 40 candidate genes as markers for high-grade cervical intraepithelial neoplasia (CIN) in exfoliated cervical cells collected at the time of colposcopy. Samples from the 93 women with CIN3 or cancer were compared with those from 186 women without disease matched (1:2) for age, race, and high-risk human papillomavirus status. Normalized threshold cycles (C(t)) for each gene were analyzed by receiver operating characteristics to determine their diagnostic performance in a split sample validation approach. Six markers were confirmed by an area under the curve >0.6 in both sample sets: claudin 1 (0.75), minichromosome maintenance deficient 5 (0.71) and 7 (0.64), cell division cycle 6 homologue (0.71), antigen identified by monoclonal antibody Ki-67 (0.66), and SHC SH2-domain binding protein 1 (0.61). The sensitivity for individual markers was relatively low and a combination of five genes to a panel resulted in 60% sensitivity with 76% specificity, not positively increasing this performance. Although the results did not indicate superiority of RNA markers for cervical cancer screening, their performance in detecting disease in women referred for colposcopy suggests that the genes and pathways they highlight could be useful in alternative detection formats or in combination with other screening indicators.