De novo mutations in schizophrenia implicate synaptic networks.

Inherited alleles account for most of the genetic risk for schizophrenia. However, new (de novo) mutations, in the form of large chromosomal copy number changes, occur in a small fraction of cases and disproportionally disrupt genes encoding postsynaptic proteins. Here we show that small de novo mutations, affecting one or a few nucleotides, are overrepresented among glutamatergic postsynaptic proteins comprising activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-d-aspartate receptor (NMDAR) complexes. Mutations are additionally enriched in proteins that interact with these complexes to modulate synaptic strength, namely proteins regulating actin filament dynamics and those whose messenger RNAs are targets of fragile X mental retardation protein (FMRP). Genes affected by mutations in schizophrenia overlap those mutated in autism and intellectual disability, as do mutation-enriched synaptic pathways. Aligning our findings with a parallel case-control study, we demonstrate reproducible insights into aetiological mechanisms for schizophrenia and reveal pathophysiology shared with other neurodevelopmental disorders.

Autotaxin-lysophosphatidic acid receptor signalling regulates hepatitis C virus replication.

BACKGROUND & AIMS: Chronic hepatitis C is a global health problem with an estimated 170 million hepatitis C virus (HCV) infected individuals at risk of progressive liver disease and hepatocellular carcinoma (HCC). Autotaxin (ATX, gene name: ENPP2) is a phospholipase with diverse roles in the physiological and pathological processes including inflammation and oncogenesis. Clinical studies have reported increased ATX expression in chronic hepatitis C, however, the pathways regulating ATX and its role in the viral life cycle are not well understood. METHODS: In vitro hepatocyte and ex vivo liver culture systems along with chimeric humanized liver mice and HCC tissue enabled us to assess the interplay between ATX and the HCV life cycle. RESULTS: HCV infection increased hepatocellular ATX RNA and protein expression. HCV infection stabilizes hypoxia inducible factors (HIFs) and we investigated a role for these transcription factors to regulate ATX. In vitro studies show that low oxygen increases hepatocellular ATX expression and transcriptome analysis showed a positive correlation between ATX mRNA levels and hypoxia gene score in HCC tumour tissue associated with HCV and other aetiologies. Importantly, inhibiting ATX-lysophosphatidic acid (LPA) signalling reduced HCV replication, demonstrating a positive role for this phospholipase in the viral life cycle. LPA activates phosphoinositide-3-kinase that stabilizes HIF-1α and inhibiting the HIF signalling pathway abrogates the pro-viral activity of LPA. CONCLUSIONS: Our data support a model where HCV infection increases ATX expression which supports viral replication and HCC progression. LAY SUMMARY: Chronic hepatitis C is a global health problem with infected individuals at risk of developing liver disease that can progress to hepatocellular carcinoma. Autotaxin generates the biologically active lipid lysophosphatidic acid that has been reported to play a tumorigenic role in a wide number of cancers. In this study we show that hepatitis C virus infection increases autotaxin expression via hypoxia inducible transcription factor and provides an environment in the liver that promotes fibrosis and liver injury. Importantly, we show a new role for lysophosphatidic acid in positively regulating hepatitis C virus replication.

The broad assessment of HCV genotypes 1 and 3 antigenic targets reveals limited cross-reactivity with implications for vaccine design.

OBJECTIVE: Developing a vaccine that is cross-reactive between HCV genotypes requires data on T cell antigenic targets that extends beyond genotype-1. We characterised T cell immune responses against HCV genotype-3, the most common infecting genotype in the UK and Asia, and assessed within genotype and between genotype cross-reactivity. DESIGN: T cell targets were identified in 140 subjects with either acute, chronic or spontaneously resolved HCV genotype-3 infection using (1) overlapping peptides and (2) putative human leucocyte antigens (HLA)-class-I wild type and variant epitopes through the prior assessment of polymorphic HCV genomic sites associated with host HLA, in IFNγ-ELISpot assays. CD4+/CD8+ T cell subsets were defined and viral variability at T cell targets was determined through population analysis and viral sequencing. T cell cross-reactivity between genotype-1 and genotype-3 variants was assessed. RESULTS: In resolved genotype-3 infection, T cells preferentially targeted non-structural proteins at a high magnitude, whereas in chronic disease T cells were absent or skewed to target structural proteins. Additional responses to wild type but not variant HLA predicted peptides were defined. Major sequence viral variability was observed within genotype-3 and between genotypes 1 and 3 HCV at T cell targets in resolved infection and at dominant epitopes, with limited T cell cross-reactivity between viral variants. Overall 41 CD4/CD8+ genotype-3 T cell targets were identified with minimal overlap with those described for HCV genotype-1. CONCLUSIONS: HCV T cell specificity is distinct between genotypes with limited T cell cross-reactivity in resolved and chronic disease. Therefore, viral regions targeted in natural HCV infection may not serve as attractive targets for a vaccine that aims to protect against multiple HCV genotypes.

Global distribution and prevalence of hepatitis C virus genotypes.

UNLABELLED: Hepatitis C virus (HCV) exhibits high genetic diversity, characterized by regional variations in genotype prevalence. This poses a challenge to the improved development of vaccines and pan-genotypic treatments, which require the consideration of global trends in HCV genotype prevalence. Here we provide the first comprehensive survey of these trends. To approximate national HCV genotype prevalence, studies published between 1989 and 2013 reporting HCV genotypes are reviewed and combined with overall HCV prevalence estimates from the Global Burden of Disease (GBD) project. We also generate regional and global genotype prevalence estimates, inferring data for countries lacking genotype information. We include 1,217 studies in our analysis, representing 117 countries and 90% of the global population. We calculate that HCV genotype 1 is the most prevalent worldwide, comprising 83.4 million cases (46.2% of all HCV cases), approximately one-third of which are in East Asia. Genotype 3 is the next most prevalent globally (54.3 million, 30.1%); genotypes 2, 4, and 6 are responsible for a total 22.8% of all cases; genotype 5 comprises the remaining <1%. While genotypes 1 and 3 dominate in most countries irrespective of economic status, the largest proportions of genotypes 4 and 5 are in lower-income countries. CONCLUSION: Although genotype 1 is most common worldwide, nongenotype 1 HCV cases­which are less well served by advances in vaccine and drug development­still comprise over half of all HCV cases. Relative genotype proportions are needed to inform healthcare models, which must be geographically tailored to specific countries or regions in order to improve access to new treatments. Genotype surveillance data are needed from many countries to improve estimates of unmet need.

CompARE: study protocol for a phase III randomised controlled platform trial comparing alternative regimens for escalating treatment of intermediate and high-risk oropharyngeal cancer.

BACKGROUND: Patients with intermediate and high-risk oropharyngeal cancer (OPC) have poorer response to standard treatment and poorer overall survival compared to low-risk OPC. CompARE is designed to test alternative approaches to intensified treatment for these patients to improve survival. METHODS: CompARE is a pragmatic phase III, open-label, multicenter randomised controlled trial with an adaptive multi-arm, multi-stage design and an integrated QuinteT Recruitment Intervention. Eligible OPC patients include those with human papillomavirus (HPV) negative, T1-T4, N1-N3 or T3-4, N0, or HPV positive N3, T4, or current smokers (or ≥ 10 pack years previous smoking history) with T1-T4, N2b-N3. CompARE was originally designed with four arms (one control [arm 1] and three experimental: arm 2-induction chemotherapy followed by arm 1; arm 3-dose-escalated radiotherapy plus concomitant cisplatin; and arm 4-resection of primary followed by arm 1). The three original experimental arms have been closed to recruitment and a further experimental arm opened (arm 5-induction durvalumab followed by arm 1 and then adjuvant durvalumab). Currently recruiting are arm 1 (control): standard treatment of 3-weekly cisplatin 100 mg/m2 or weekly 40 mg/m2 with intensity-modulated radiotherapy using 70 Gy in 35 fractions ± neck dissection determined by clinical and radiological assessment 3 months post-treatment, and arm 5 (intervention): one cycle of induction durvalumab 1500 mg followed by standard treatment then durvalumab 1500 mg every 4 weeks for a total of 6 months. The definitive and interim primary outcome measures are overall survival time and event-free survival (EFS) time, respectively. Secondary outcome measures include quality of life, toxicity, swallowing outcomes, feeding tube incidence, surgical complication rates, and cost-effectiveness. The design anticipates that after approximately 7 years, 84 required events will have occurred to enable analysis of the definitive primary outcome measure for this comparison. Planned interim futility analyses using EFS will also be performed. DISCUSSION: CompARE is designed to be efficient and cost-effective in response to new data, emerging new treatments or difficulties, with the aim of bringing new treatment options for these patients. TRIAL REGISTRATION: ISRCTN ISRCTN41478539 . Registered on 29 April 2015.

HCV genotype-3a T cell immunity: specificity, function and impact of therapy.

BACKGROUND: Hepatitis C virus (HCV) genotype-3a infection is now the dominant strain in South Asia and the UK. Characteristic features include a favourable response to therapy; the reasons for this are unknown but may include distinct genotype-3a-specific T cell immunity. In contrast to genotype-1 infection, T cell immunity to this subtype is poorly defined. OBJECTIVES: The aims of the study were to (1) define the frequency, specificity and cross-reactivity of T cell immunity across the whole viral genome in genotype-3a infection and (2) assess the impact of interferon (IFN)-α/ribavirin on T cell immunity. DESIGN: T cell responses in chronic and resolved HCV genotype-3a were analysed in comparison with genotype-1 infection (total n=85) using specific peptide panels in IFN-γ ELISpot assays. T cell responses were followed longitudinally in a subset of genotype-3a infected patients receiving therapy. Responses were further defined by CD4 and CD8 subset analysis, sequencing of autologous virus and cross-reactivity of genotype-3a with genotype-1a/-1b antigens. RESULTS: CD8 T cell responses commonly targeted the non-structural (NS) proteins in chronic genotype-3a infection whereas in genotype-1 infection CD4 responses targeting HCV core predominated (p=0.0183). Resolved infection was associated with CD4 T cells targeting NS proteins. Paradoxically, a sustained response to therapy was associated with a brisk decline in virus-specific and total lymphocyte counts that recovered after treatment. CONCLUSION: HCV genotype-3a exhibits a distinct T cell specificity with implications for vaccine design. However, our data do not support the theory that genotype-3a viral clearance with therapy is associated with an enhanced antiviral T cell response. Paradoxically, a reduction in these responses may serve as a biomarker of IFN responsiveness.

Protective effect of human leukocyte antigen B27 in hepatitis C virus infection requires the presence of a genotype-specific immunodominant CD8+ T-cell epitope.

UNLABELLED: Human leukocyte antigen B27 (HLA-B27) is associated with protection in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27-restricted CD8+ T-cell epitopes in both infections. In order to define the relative contribution of a specific HLA-B27-restricted epitope to the natural course of HCV infection, we compared the biological impact of the highly conserved HCV genotype 1 epitope, for which the protective role has been described, with the corresponding region in genotype 3 that differs in its sequence by three amino acid residues. The genotype 3a peptide was not recognized by CD8+ T cells specific for the genotype 1 peptide. Furthermore, patients with acute or chronic infection with HCV genotype 3a did not mount T-cell responses to this epitope region, and their autologous viral sequences showed no evidence of T-cell pressure. Finally, we found a significantly higher frequency of HLA-B27 positivity in patients with chronic HCV genotype 3a infection compared to genotype 1 infection, indicating that there is no protection by HLA-B27 in HCV genotype 3 infection. CONCLUSION: Our data indicate that the protective effect of HLA-B27 is limited to HCV genotype 1 infection and does not expand to other genotypes such as genotype 3a. This can most likely be explained by intergenotype sequence diversity leading to the loss of the immunodominant HLA-B27 epitope in viral strains other than genotype 1. Our results underline the central role of a single HLA-B27-restricted epitope-specific CD8+ T-cell response in mediating protection in HCV genotype 1 infection.

Genetic history of hepatitis C virus in East Asia.

The hepatitis C virus (HCV), which currently infects an estimated 3% of people worldwide, has been present in some human populations for several centuries, notably HCV genotypes 1 and 2 in West Africa and genotype 6 in Southeast Asia. Here we use newly developed methods of sequence analysis to conduct the first comprehensive investigation of the epidemic and evolutionary history of HCV in Asia. Our analysis includes new HCV core (n = 16) and NS5B (n = 14) gene sequences, obtained from serum samples of jaundiced patients from Laos. These exceptionally diverse isolates were analyzed in conjunction with all available reference strains using phylogenetic and Bayesian coalescent methods. We performed statistical tests of phylogeographic structure and applied a recently developed "relaxed molecular clock" approach to HCV for the first time, which indicated an unexpectedly high degree of rate variation. Our results reveal a >1,000-year-long development of genotype 6 in Asia, characterized by substantial phylogeographic structure and two distinct phases of epidemic history, before and during the 20th century. We conclude that HCV lineages representing preexisting and spatially restricted strains were involved in multiple, independent local epidemics during the 20th century. Our analysis explains the generation and maintenance of HCV diversity in Asia and could provide a template for further investigations of HCV spread in other regions.

Full-length characterization of hepatitis C virus subtype 3a reveals novel hypervariable regions under positive selection during acute infection.

Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies.

Hepatitis C virus drug resistance and immune-driven adaptations: relevance to new antiviral therapy.

UNLABELLED: The efficacy of specifically targeted anti-viral therapy for hepatitis C virus (HCV) (STAT-C), including HCV protease and polymerase inhibitors, is limited by the presence of drug-specific viral resistance mutations within the targeted proteins. Genetic diversity within these viral proteins also evolves under selective pressures provided by host human leukocyte antigen (HLA)-restricted immune responses, which may therefore influence STAT-C treatment response. Here, the prevalence of drug resistance mutations relevant to 27 developmental STAT-C drugs, and the potential for drug and immune selective pressures to intersect at sites along the HCV genome, is explored. HCV nonstructural (NS) 3 protease or NS5B polymerase sequences and HLA assignment were obtained from study populations from Australia, Switzerland, and the United Kingdom. Four hundred five treatment-naïve individuals with chronic HCV infection were considered (259 genotype 1, 146 genotype 3), of which 38.5% were coinfected with human immunodeficiency virus (HIV). We identified preexisting STAT-C drug resistance mutations in sequences from this large cohort. The frequency of the variations varied according to individual STAT-C drug and HCV genotype/subtype. Of individuals infected with subtype 1a, 21.5% exhibited genetic variation at a known drug resistance site. Furthermore, we identified areas in HCV protease and polymerase that are under both potential HLA-driven pressure and therapy selection and identified six HLA-associated polymorphisms (P

Divergent adaptation of hepatitis C virus genotypes 1 and 3 to human leukocyte antigen-restricted immune pressure.

UNLABELLED: Many hepatitis C virus (HCV) infections worldwide are with the genotype 1 and 3 strains of the virus. Cellular immune responses are known to be important in the containment of HCV genotype 1 infection, and many genotype 1 T cell targets (epitopes) that are presented by host human leukocyte antigens (HLAs) have been identified. In contrast, there is almost no information known about the equivalent responses to genotype 3. Immune escape mechanisms used by HCV include the evolution of viral polymorphisms (adaptations) that abrogate this host-viral interaction. Evidence of HCV adaptation to HLA-restricted immune pressure on HCV can be observed at the population level as viral polymorphisms associated with specific HLA types. To evaluate the escape patterns of HCV genotypes 1 and 3, we assessed the associations between viral polymorphisms and specific HLA types from 187 individuals with genotype 1a and 136 individuals with genotype 3a infection. We identified 51 HLA-associated viral polymorphisms (32 for genotype 1a and 19 for genotype 3a). Of these putative viral adaptation sites, six fell within previously published epitopes. Only two HLA-associated viral polymorphisms were common to both genotypes. In the remaining sites with HLA-associated polymorphisms, there was either complete conservation or no significant HLA association with viral polymorphism in the alternative genotype. This study also highlights the diverse mechanisms by which viral evasion of immune responses may be achieved and the role of genotype variation in these processes. CONCLUSION: There is little overlap in HLA-associated polymorphisms in the nonstructural proteins of HCV for the two genotypes, implying differences in the cellular immune pressures acting on these viruses and different escape profiles. These findings have implications for future therapeutic strategies to combat HCV infection, including vaccine design.

Identifying gene-environment interactions in schizophrenia: contemporary challenges for integrated, large-scale investigations.

Recent years have seen considerable progress in epidemiological and molecular genetic research into environmental and genetic factors in schizophrenia, but methodological uncertainties remain with regard to validating environmental exposures, and the population risk conferred by individual molecular genetic variants is small. There are now also a limited number of studies that have investigated molecular genetic candidate gene-environment interactions (G × E), however, so far, thorough replication of findings is rare and G × E research still faces several conceptual and methodological challenges. In this article, we aim to review these recent developments and illustrate how integrated, large-scale investigations may overcome contemporary challenges in G × E research, drawing on the example of a large, international, multi-center study into the identification and translational application of G × E in schizophrenia. While such investigations are now well underway, new challenges emerge for G × E research from late-breaking evidence that genetic variation and environmental exposures are, to a significant degree, shared across a range of psychiatric disorders, with potential overlap in phenotype.

The infective causes of hepatitis and jaundice amongst hospitalised patients in Vientiane, Laos.

There is little information on the diverse infectious causes of jaundice and hepatitis in the Asiatic tropics. Serology (hepatitis A, B, C and E, leptospirosis, dengue, rickettsia), antigen tests (dengue), PCR assays (hepatitis A, C and E) and blood cultures (septicaemia) were performed on samples from 392 patients admitted with jaundice or raised transaminases (> or =x3) to Mahosot Hospital, Vientiane, Laos over 3 years. Conservative definitions suggested diagnoses of dengue (8.4%), rickettsioses (7.3%), leptospirosis (6.8%), hepatitis B (4.9%), hepatitis C (4.9%), community-acquired septicaemia (3.3%) and hepatitis E (1.6%). Although anti-hepatitis A virus (HAV) IgM antibody results suggested that 35.8% of patients had acute HAV infections, anti-HAV IgG antibody avidity and HAV PCR suggested that 82% had polyclonal activation and not acute HAV infections. Scrub typhus, murine typhus or leptospirosis were present in 12.8% of patients and were associated with meningism and relatively low AST and ALT elevation. These patients would be expected to respond to empirical doxycycline therapy which, in the absence of virological diagnosis and treatment, may be an appropriate cost-effective intervention in Lao patients with jaundice/hepatitis.

Cellular immune responses during high-dose interferon-alpha induction therapy for hepatitis C virus infection.

BACKGROUND: The effect that high-dose interferon (IFN)-alpha induction therapy for hepatitis C virus (HCV) infection has on cellular immune responses is currently unknown. METHODS: Thirty-one treatment-naive patients with chronic HCV infection received amantadine and ribavirin, combined with 6 weeks of high-dose IFN-alpha-2b induction therapy followed by weekly pegylated IFN-alpha-2b, for 24 or 48 weeks. Using IFN-gamma and interleukin (IL)-2 enzyme-linked immunospot (ELISpot) assays, we analyzed the pattern of cytokine secretion by structural and nonstructural HCV- and cytomegalovirus (CMV)-specific T cells before, during, and after therapy. RESULTS: HCV-specific T cell responses, which were predominantly IFN-gamma secreting and which correlated with alanine transaminase levels (r2 = 0.45; P = .001), were found before treatment in 10 of 15 patients with a sustained virological response (SVR) and in 11 of 16 in the non-SVR group. There was a striking loss of IFN-gamma and IL-2 HCV-specific T cells during therapy, predominantly in the SVR group. This response recovered after cessation of therapy, regardless of outcome. Suppression of CMV-specific T cell responses, in addition to total lymphocyte counts, was also observed. CONCLUSIONS: High-dose IFN-alpha induction therapy leads to a profound decline in IL-2- and IFN-gamma-secreting HCV- and CMV-specific T cells. These data indicate that restoration of T cell responses is unlikely to be causally linked to an early response or SVR to therapy.