Joint host-pathogen genomic analysis identifies hepatitis B virus mutations associated with human NTCP and HLA class I variation.

Evolutionary changes in the hepatitis B virus (HBV) genome could reflect its adaptation to host-induced selective pressure. Leveraging paired human exome and ultra-deep HBV genome-sequencing data from 567 affected individuals with chronic hepatitis B, we comprehensively searched for the signatures of this evolutionary process by conducting "genome-to-genome" association tests between all human genetic variants and viral mutations. We identified significant associations between an East Asian-specific missense variant in the gene encoding the HBV entry receptor NTCP (rs2296651, NTCP S267F) and mutations within the receptor-binding region of HBV preS1. Through in silico modeling and in vitro preS1-NTCP binding assays, we observed that the associated HBV mutations are in proximity to the NTCP variant when bound and together partially increase binding affinity to NTCP S267F. Furthermore, we identified significant associations between HLA-A variation and viral mutations in HLA-A-restricted T cell epitopes. We used in silico binding prediction tools to evaluate the impact of the associated HBV mutations on HLA presentation and observed that mutations that result in weaker binding affinities to their cognate HLA alleles were enriched. Overall, our results suggest the emergence of HBV escape mutations that might alter the interaction between HBV PreS1 and its cellular receptor NTCP during viral entry into hepatocytes and confirm the role of HLA class I restriction in inducing HBV epitope variations.

The best of both worlds: Blending cutting-edge research with clinical processes for a productive exome clinic.

Genomic medicine has been transformed by next-generation sequencing (NGS), inclusive of exome sequencing (ES) and genome sequencing (GS). Currently, ES is offered widely in clinical settings, with a less prevalent alternative model consisting of hybrid programs that incorporate research ES along with clinical patient workflows. We were among the earliest to implement a hybrid ES clinic, have provided diagnoses to 45% of probands, and have identified several novel candidate genes. Our program is enabled by a cost-effective investment by the health system and is unique in encompassing all the processes that have been variably included in other hybrid/clinical programs. These include careful patient selection, utilization of a phenotype-agnostic bioinformatics pipeline followed by manual curation of variants and phenotype integration by clinicians, close collaborations between the clinicians and the bioinformatician, pursuit of interesting variants, communication of results to patients in categories that are predicated upon the certainty of a diagnosis, and tracking changes in results over time and the underlying mechanisms for such changes. Due to its effectiveness, scalability to GS and its resource efficiency, specific elements of our paradigm can be incorporated into existing clinical settings, or the entire hybrid model can be implemented within health systems that have genomic medicine programs, to provide NGS in a scientifically rigorous, yet pragmatic setting.

Genome-wide association study (GWAS) of human host factors influencing viral severity of herpes simplex virus type 2 (HSV-2).

Herpes simplex virus type 2 (HSV-2) is an incurable viral infection with severity ranging from asymptomatic to frequent recurrences. The viral shedding rate has been shown as a reproducible HSV-2 severity end point that correlates with lesion rates. We used a genome-wide association study (GWAS) to investigate the role of common human genetic variation in HSV-2 severity. We performed a GWAS on 223 HSV-2-positive participants of European ancestry. Severity was measured by viral shedding rate, as defined by the percent of days PCR+ for HSV-2 DNA over at least 30 days. Analyses were performed under linear regression models, adjusted for age, sex, and ancestry. There were no genome-wide significant (p < 5E-08) associations with HSV-2 viral shedding rate. The top nonsignificant SNP (rs75932292, p = 6.77E-08) associated with HSV-2 viral shedding was intergenic, with the nearest known biologically interesting gene (ABCA1) ~130 kbp downstream. Several other SNPs approaching significance were in or near genes with viral or neurological associations, including four SNPs in KIF1B. The current study is the first comprehensive genome-wide investigation of human genetic variation in virologic severity of established HSV-2 infection. However, no significant associations were observed with HSV-2 virologic severity, leaving the exact role of human variation in HSV-2 severity unclear.

Association of CYP2B6 Single-Nucleotide Polymorphisms Altering Efavirenz Metabolism With Hepatitis C Virus (HCV) Treatment Relapse Among Human Immunodeficiency Virus/HCV-Coinfected African Americans Receiving Ledipasvir/Sofosbuvir in the ION-4 Trial.

In the ION-4 trial, hepatitis C virus relapse was rare, occurring only in African American patients, 80% receiving efavirenz for human immunodeficiency virus infection. We observed no indication that CYP2B6 polymorphisms associated with increased plasma efavirenz exposure explained the relapses.

Polymorphisms of large effect explain the majority of the host genetic contribution to variation of HIV-1 virus load.

Previous genome-wide association studies (GWAS) of HIV-1-infected populations have been underpowered to detect common variants with moderate impact on disease outcome and have not assessed the phenotypic variance explained by genome-wide additive effects. By combining the majority of available genome-wide genotyping data in HIV-infected populations, we tested for association between ∼8 million variants and viral load (HIV RNA copies per milliliter of plasma) in 6,315 individuals of European ancestry. The strongest signal of association was observed in the HLA class I region that was fully explained by independent effects mapping to five variable amino acid positions in the peptide binding grooves of the HLA-B and HLA-A proteins. We observed a second genome-wide significant association signal in the chemokine (C-C motif) receptor (CCR) gene cluster on chromosome 3. Conditional analysis showed that this signal could not be fully attributed to the known protective CCR5Δ32 allele and the risk P1 haplotype, suggesting further causal variants in this region. Heritability analysis demonstrated that common human genetic variation-mostly in the HLA and CCR5 regions-explains 25% of the variability in viral load. This study suggests that analyses in non-European populations and of variant classes not assessed by GWAS should be priorities for the field going forward.

Evaluating the Impact of Functional Genetic Variation on HIV-1 Control.

Background: Previous genetic association studies of human immunodeficiency virus-1 (HIV-1) progression have focused on common human genetic variation ascertained through genome-wide genotyping. Methods: We sought to systematically assess the full spectrum of functional variation in protein coding gene regions on HIV-1 progression through exome sequencing of 1327 individuals. Genetic variants were tested individually and in aggregate across genes and gene sets for an influence on HIV-1 viral load. Results: Multiple single variants within the major histocompatibility complex (MHC) region were observed to be strongly associated with HIV-1 outcome, consistent with the known impact of classical HLA alleles. However, no single variant or gene located outside of the MHC region was significantly associated with HIV progression. Set-based association testing focusing on genes identified as being essential for HIV replication in genome-wide small interfering RNA (siRNA) and clustered regularly interspaced short palindromic repeats (CRISPR) studies did not reveal any novel associations. Conclusions: These results suggest that exonic variants with large effect sizes are unlikely to have a major contribution to host control of HIV infection.

No Evidence for Association of ß-Defensin Genomic Copy Number with HIV Susceptibility, HIV Load during Clinical Latency, or Progression to AIDS.

Common single-nucleotide variation in the host accounts for 25% of the variability in the plasma levels of HIV during the clinical latency stage (viral load set point). However, the role of rare variants and copy number variants remains relatively unexplored. Previous work has suggested copy number variation of a cluster of ß-defensin genes affects HIV load in treatment-naïve sub-Saharan Africans and rate of response to antiretroviral treatment. Here we analyse a total of 1827 individuals from two cohorts of HIV-infected individuals from Europe and sub-Saharan Africa to investigate the role of ß-defensin copy number variation on HIV load at set point. We find no evidence for association of copy number with viral load. We also compare distribution of ß-defensin copy number between European cases and controls and find no differences, arguing against a role of ß-defensin copy number in HIV acquisition. Taken together, our data argue against an effect of copy number variation of the ß-defensin region in the spontaneous control of HIV infection.

Testing for risk and protective trends in genetic analyses of HIV acquisition.

Host genetics studies of HIV-1 acquisition are critically important for the identification of new targets for drug and vaccine development. Analyses of such studies typically focus on pairwise comparisons of three different groups: HIV-1 positive individuals, HIV-1 high-risk seronegative individuals, and population controls. Because there is a clear expectation of how gene frequencies of risk or protective alleles would be ordered in the three groups, we are able to construct a statistical framework that offers a consistent increase in power over a wide-range of the magnitude of risk/protective effects. In this paper, we develop tests that constrain the alternative hypothesis to appropriately reflect risk or protective trends jointly across the three groups and show that they lead to a substantial increase in power over the naive pairwise approach. We develop both likelihood-ratio and score statistics that test for genetic effects across the three groups while constraining the alternative hypothesis to reflect biologically motivated trends of risk or protection. The asymptotic distribution of both statistics (likelihood ratio and score) is derived. We investigate the performance of our approach via extensive simulation studies using a biologically motivated model of HIV-1 acquisition, and find that our proposed approach leads to an increase in power of roughly 10-28%. We illustrate our approach with an analysis of the effect of the CCR5Δ32 mutation on HIV acquisition.

Host genetics of HIV acquisition and viral control.

Since the discovery of HIV as the cause of AIDS, numerous insights have been gained from studies of its natural history and epidemiology. It has become clear that there are substantial interindividual differences in the risk of HIV acquisition and course of disease. Meanwhile, the field of human genetics has undergone a series of rapid transitions that have fundamentally altered the approach to studying HIV host genetics. We aim to describe the field as it has transitioned from the era of candidate-gene studies and the era of genome-wide association studies (GWAS) to its current state in the infancy of comprehensive sequencing. In some ways the field has come full circle, having evolved from being driven almost exclusively by our knowledge of immunology, to a bias-free GWAS approach, to a point where our ability to catalogue human variation far outstrips our ability to biologically interpret it.

Association study of common genetic variants and HIV-1 acquisition in 6,300 infected cases and 7,200 controls.

Multiple genome-wide association studies (GWAS) have been performed in HIV-1 infected individuals, identifying common genetic influences on viral control and disease course. Similarly, common genetic correlates of acquisition of HIV-1 after exposure have been interrogated using GWAS, although in generally small samples. Under the auspices of the International Collaboration for the Genomics of HIV, we have combined the genome-wide single nucleotide polymorphism (SNP) data collected by 25 cohorts, studies, or institutions on HIV-1 infected individuals and compared them to carefully matched population-level data sets (a list of all collaborators appears in Note S1 in Text S1). After imputation using the 1,000 Genomes Project reference panel, we tested approximately 8 million common DNA variants (SNPs and indels) for association with HIV-1 acquisition in 6,334 infected patients and 7,247 population samples of European ancestry. Initial association testing identified the SNP rs4418214, the C allele of which is known to tag the HLA-B*57:01 and B*27:05 alleles, as genome-wide significant (p = 3.6 × 10⁻¹¹). However, restricting analysis to individuals with a known date of seroconversion suggested that this association was due to the frailty bias in studies of lethal diseases. Further analyses including testing recessive genetic models, testing for bulk effects of non-genome-wide significant variants, stratifying by sexual or parenteral transmission risk and testing previously reported associations showed no evidence for genetic influence on HIV-1 acquisition (with the exception of CCR5Δ32 homozygosity). Thus, these data suggest that genetic influences on HIV acquisition are either rare or have smaller effects than can be detected by this sample size.

Distinct signatures of diversifying selection revealed by genome analysis of respiratory tract and invasive bacterial populations.

Many pathogens colonize different anatomical sites, but the selective pressures contributing to survival in the diverse niches are poorly understood. Group A Streptococcus (GAS) is a human-adapted bacterium that causes a range of infections. Much effort has been expended to dissect the molecular basis of invasive (sterile-site) infections, but little is known about the genomes of strains causing pharyngitis (streptococcal "sore throat"). Additionally, there is essentially nothing known about the genetic relationships between populations of invasive and pharyngitis strains. In particular, it is unclear if invasive strains represent a distinct genetic subpopulation of strains that cause pharyngitis. We compared the genomes of 86 serotype M3 GAS pharyngitis strains with those of 215 invasive M3 strains from the same geographical location. The pharyngitis and invasive groups were highly related to each other and had virtually identical phylogenetic structures, indicating they belong to the same genetic pool. Despite the overall high degree of genetic similarity, we discovered that strains from different host environments (i.e., throat, normally sterile sites) have distinct patterns of diversifying selection at the nucleotide level. In particular, the pattern of polymorphisms in the hyaluronic acid capsule synthesis operon was especially different between the two strain populations. This finding was mirrored by data obtained from full-genome analysis of strains sequentially cultured from nonhuman primates. Our results answer the long-standing question of the genetic relationship between GAS pharyngitis and invasive strains. The data provide previously undescribed information about the evolutionary history of pathogenic microbes that cause disease in different anatomical sites.

Investigating the Relationship Between Rare Genetic Variants and Fibrosis in Pediatric Nonalcoholic Fatty Liver Disease.

Background and Aims: Nonalcoholic Fatty Liver Disease (NAFLD) is a complex human disease. Common genetic variation in the patatin-like phospholipase domain containing 3 (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2) genes have been associated with an increased risk of developing NAFLD, nonalcoholic steatohepatitis (NASH), and fibrosis in adults. The role of rare genetic variants in the development and progression of NAFLD in children is not well known. We aimed to explore the role of rare genetic variants in pediatric patients with advanced fibrosis. Methods: Whole exome sequencing data was generated for 229 pediatric patients diagnosed with NAFLD recruited from the NASH Clinical Research Network (NASH CRN). Case-control single variant and gene-based collapsing analyses were used to test for rare variants that were enriched or depleted within the pediatric NAFLD cohort specifically for advanced fibrosis (cases) versus those without fibrosis (controls) or six other histologic characteristics. Exome data from non-NAFLD population controls were also used for additional analyses. All results were adjusted for multiple testing using a Bonferroni correction. Results: No genome-wide significant associations were found between rare variation and presence of advanced fibrosis or NASH, nor the severity of steatosis, inflammation, or hepatocellular ballooning. Significantly, no enrichment of rare variants in PNPLA3 or TM6SF2 was observed across phenotypes. Conclusion: In a cohort of children with histologically proven NAFLD, no genome-wide significant associations were found between rare genetic variation and advanced fibrosis or six other histologic features. Of particular interest was the lack of association with genes of interest in adults: PNPLA3 and TM6SF2, though limitations in sample size may reduce the ability to detect associations, particularly with rare variation.

Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.

Group A Streptococcus (GAS) causes an exceptionally broad range of infections in humans, from relatively mild pharyngitis and skin infections to life-threatening necrotizing fasciitis and toxic shock syndrome. An epidemic of severe invasive human infections caused by type emm59 GAS, heretofore an exceedingly rare cause of disease, spread west to east across Canada over a 3-year period (2006 to 2008). By sequencing the genomes of 601 epidemic, historic, and other emm59 organisms, we discovered that a recently emerged, genetically distinct emm59 clone is responsible for the Canadian epidemic. Using near-real-time genome sequencing, we were able to show spread of the Canadian epidemic clone into the United States. The extensive genome data permitted us to identify patterns of geographic dissemination as well as links between emm59 subclonal lineages that cause infections. Mouse and nonhuman primate models of infection demonstrated that the emerged clone is unusually virulent. Transmission of epidemic emm59 strains may have occurred primarily by skin contact, as suggested by an experimental model of skin transmission. In addition, the emm59 strains had a significantly impaired ability to persist in human saliva and to colonize the oropharynx of mice, and seldom caused human pharyngitis. Our study contributes new information to the rapidly emerging field of molecular pathogenomics of bacterial epidemics and illustrates how full-genome data can be used to precisely illuminate the landscape of strain dissemination during a bacterial epidemic.

Molecular complexity of successive bacterial epidemics deconvoluted by comparative pathogenomics.

Understanding the fine-structure molecular architecture of bacterial epidemics has been a long-sought goal of infectious disease research. We used short-read-length DNA sequencing coupled with mass spectroscopy analysis of SNPs to study the molecular pathogenomics of three successive epidemics of invasive infections involving 344 serotype M3 group A Streptococcus in Ontario, Canada. Sequencing the genome of 95 strains from the three epidemics, coupled with analysis of 280 biallelic SNPs in all 344 strains, revealed an unexpectedly complex population structure composed of a dynamic mixture of distinct clonally related complexes. We discovered that each epidemic is dominated by micro- and macrobursts of multiple emergent clones, some with distinct strain genotype-patient phenotype relationships. On average, strains were differentiated from one another by only 49 SNPs and 11 insertion-deletion events (indels) in the core genome. Ten percent of SNPs are strain specific; that is, each strain has a unique genome sequence. We identified nonrandom temporal-spatial patterns of strain distribution within and between the epidemic peaks. The extensive full-genome data permitted us to identify genes with significantly increased rates of nonsynonymous (amino acid-altering) nucleotide polymorphisms, thereby providing clues about selective forces operative in the host. Comparative expression microarray analysis revealed that closely related strains differentiated by seemingly modest genetic changes can have significantly divergent transcriptomes. We conclude that enhanced understanding of bacterial epidemics requires a deep-sequencing, geographically centric, comparative pathogenomics strategy.

Interactome analysis of longitudinal pharyngeal infection of cynomolgus macaques by group A Streptococcus.

Relatively little is understood about the dynamics of global host-pathogen transcriptome changes that occur during bacterial infection of mucosal surfaces. To test the hypothesis that group A Streptococcus (GAS) infection of the oropharynx provokes a distinct host transcriptome response, we performed genome-wide transcriptome analysis using a nonhuman primate model of experimental pharyngitis. We also identified host and pathogen biological processes and individual host and pathogen gene pairs with correlated patterns of expression, suggesting interaction. For this study, 509 host genes and seven biological pathways were differentially expressed throughout the entire 32-day infection cycle. GAS infection produced an initial widespread significant decrease in expression of many host genes, including those involved in cytokine production, vesicle formation, metabolism, and signal transduction. This repression lasted until day 4, at which time a large increase in expression of host genes was observed, including those involved in protein translation, antigen presentation, and GTP-mediated signaling. The interactome analysis identified 73 host and pathogen gene pairs with correlated expression levels. We discovered significant correlations between transcripts of GAS genes involved in hyaluronic capsule production and host endocytic vesicle formation, GAS GTPases and host fibrinolytic genes, and GAS response to interaction with neutrophils. We also identified a strong signal, suggesting interaction between host gammadelta T cells and genes in the GAS mevalonic acid synthesis pathway responsible for production of isopentenyl-pyrophosphate, a short-chain phospholipid that stimulates these T cells. Taken together, our results are unique in providing a comprehensive understanding of the host-pathogen interactome during mucosal infection by a bacterial pathogen.

Polymorphisms in regulator of protease B (RopB) alter disease phenotype and strain virulence of serotype M3 group A Streptococcus.

Whole-genome sequencing of serotype M3 group A streptococci (GAS) from oropharyngeal and invasive infections in Ontario recently showed that the gene encoding regulator of protease B (RopB) is highly polymorphic in this population. To test the hypothesis that ropB is under diversifying selective pressure among all serotype M3 GAS strains, we sequenced this gene in 1178 strains collected from different infection types, geographic regions, and time periods. The results confirmed our hypothesis and discovered a significant association between mutant ropB alleles, decreased activity of its major regulatory target SpeB, and pharyngitis. Additionally, isoallelic strains with ropB polymorphisms were significantly less virulent in a mouse model of necrotizing fasciitis. These studies provide a model strategy for applying whole-genome sequencing followed by deep single-gene sequencing to generate new insight to the rapid evolution and virulence regulation of human pathogens.

The diagnostic yield of exome sequencing in liver diseases from a curated gene panel.

Exome sequencing (ES) has been used in a variety of clinical settings but there are limited data on its utility for diagnosis and/or prediction of monogenic liver diseases. We developed a curated list of 502 genes for monogenic disorders associated with liver phenotypes and analyzed ES data for these genes in 758 patients with chronic liver diseases (CLD). For comparison, we examined ES data in 7856 self-declared healthy controls (HC), and 2187 patients with chronic kidney disease (CKD). Candidate pathogenic (P) or likely pathogenic (LP) variants were initially identified in 19.9% of participants, most of which were attributable to previously reported pathogenic variants with implausibly high allele frequencies. After variant annotation and filtering based on population minor allele frequency (MAF ≤ 10-4 for dominant disorders and MAF ≤ 10-3 for recessive disorders), we detected a significant enrichment of P/LP variants in the CLD cohort compared to the HC cohort (X2 test OR 5.00, 95% CI 3.06-8.18, p value = 4.5e-12). A second-level manual annotation was necessary to capture true pathogenic variants that were removed by stringent allele frequency and quality filters. After these sequential steps, the diagnostic rate of monogenic disorders was 5.7% in the CLD cohort, attributable to P/LP variants in 25 genes. We also identified concordant liver disease phenotypes for 15/22 kidney disease patients with P/LP variants in liver genes, mostly associated with cystic liver disease phenotypes. Sequencing results had many implications for clinical management, including familial testing for early diagnosis and management, preventative screening for associated comorbidities, and in some cases for therapy. Exome sequencing provided a 5.7% diagnostic rate in CLD patients and required multiple rounds of review to reduce both false positive and false negative findings. The identification of concordant phenotypes in many patients with P/LP variants and no known liver disease also indicates a potential for predictive testing for selected monogenic liver disorders.

A genome-wide screen for variants influencing certolizumab pegol response in a moderate to severe rheumatoid arthritis population.

Certolizumab pegol (CZP) is a PEGylated Fc-free tumor necrosis factor (TNF) inhibitor antibody approved for use in the treatment of rheumatoid arthritis (RA), Crohn's disease, psoriatic arthritis, axial spondyloarthritis and psoriasis. In a clinical trial of patients with severe RA, CZP improved disease symptoms in approximately half of patients. However, variability in CZP efficacy remains a problem for clinicians, thus, the aim of this study was to identify genetic variants predictive of CZP response. We performed a genome-wide association study (GWAS) of 302 RA patients treated with CZP in the REALISTIC trial to identify common single nucleotide polymorphisms (SNPs) associated with treatment response. Whole-exome sequencing was also performed for 74 CZP extreme responders and non-responders within the same population, as well as 1546 population controls. No common SNPs or rare functional variants were significantly associated with CZP response, though a non-significant enrichment in the RA-implicated KCNK5 gene was observed. Two SNPs near spondin-1 and semaphorin-4G approached genome-wide significance. The results of the current study did not provide an unambiguous predictor of CZP response.

Group A Streptococcus emm gene types in pharyngeal isolates, Ontario, Canada, 2002-2010.

Group A Streptococcus (GAS) is a human-adapted pathogen that causes a variety of diseases, including pharyngitis and invasive infections. GAS strains are categorized by variation in the nucleotide sequence of the gene (emm) that encodes the M protein. To identify the emm types of GAS strains causing pharyngitis in Ontario, Canada, we sequenced the hypervariable region of the emm gene in 4,635 pharyngeal GAS isolates collected during 2002-2010. The most prevalent emm types varied little from year to year. In contrast, fine-scale geographic analysis identified inter-site variability in the most common emm types. Additionally, we observed fluctuations in yearly frequency of emm3 strains from pharyngitis patients that coincided with peaks of emm3 invasive infections. We also discovered a striking increase in frequency of emm89 strains among isolates from patients with pharyngitis and invasive disease. These findings about the epidemiology of GAS are potentially useful for vaccine research.

Lower respiratory tract infection in cynomolgus macaques (Macaca fascicularis) infected with group A Streptococcus.

Group A Streptococcus (GAS), a human-specific pathogen, is best known for causing pharyngitis ("strep-throat") and necrotizing fasciitis ("flesh-eating disease"). However, the organism is also an uncommon but important cause of community-acquired bronchopneumonia, an infection with an exceptionally high mortality rate. Inasmuch as little is known about the molecular pathogenesis of GAS lower respiratory tract infection, we sought to develop a relevant human infection model. Nine cynomolgus macaques were infected by intra-bronchial instillation of either sterile saline or GAS (10(5) or 10(7) CFU). Animals were continuously monitored and sacrificed at five days post-inoculation. Serial bronchial alveolar lavage specimens and tissues collected at necropsy were used for histologic and immunohistochemical examination, quantitative microbial culture, lung and blood biomarker analysis, and in vivo GAS gene expression studies. The lower respiratory tract disease observed in cynomolgus macaques mimicked the clinical and pathological features of severe GAS bronchopneumonia in humans. This new monkey model will be useful for testing hypotheses bearing on the molecular pathogenesis of GAS in the lower respiratory tract.