Background & Aims
We studied the relationship between IL28B gene-related SNP rs12979860 and early viral kinetics (day 0–28) during peginterferon and ribavirin treatment, in 173 African Americans (AA) and 188 Caucasian Americans (CA) with HCV genotype 1.
We studied the relationship between IL28B 16 gene-related SNP rs12979860 and early viral kinetics (day 0–28) 17 during peginterferon and ribavirin treatment, in 171 African 18 Americans (AA) and 188 Caucasian Americans (CA) with HCV 19 genotype 1.
Compared to non-C/C genotypes, C/C was associated with greater declines in serum HCV RNA during phase 1 (day 0–2), phase 2 (day 7–28), and day 0–28 and higher response (undetected HCV RNA) rates at weeks 4 and 12 in AA and CA. A static phase and increases in HCV RNA from day 2 to 7 were more common in patients with non-C/C genotypes. C/C was also associated with higher week 24, 48, and 72 response rates in CA (p <0.01) but not in AA. At baseline, SNP genotype was the only independent predictor of phase 1; SNP genotype and phase 1 were independent predictors of phase 2 (p<0.001). There were no racial differences in HCV RNA declines during phase 1, day 2–7, phase 2, and day 0–28 with the same SNP genotype. AA with C/C and C/T genotypes had lower week 24, 48, and 72 (SVR) rates than did CA (p = 0.03). SNP C/C predicted higher SVR rates in AA and CA with high baseline HCV RNA (≥ 600,000 IU/ml), and in CA with ≥ 1 log10 IU/ml decrease in HCV RNA from day 0 to 28.
SNP rs12979860 is strongly associated with both phase 1 and phase 2 HCV RNA kinetics in AA and CA with HCV genotype 1.
IL28B gene; Hepatitis C virus; Viral kinetics; Single nucleotide polymorphism
The 12th International Meeting on Human Genome Variation and Complex Genome Analysis (HGV2011: Berkeley, California, USA, 8th–10th September 2011) was a stimulating workshop where researchers from academia and industry explored the latest progress, challenges, and opportunities in genome variation research. Key themes included progress beyond GWAS, variation in human populations, use of sequence data in medical settings, large-scale sequencing data analysis, and bioinformatics approaches to large datasets.
human variation; GWAS; SNP; medical genomics
A central challenge in interpreting personal genomes is determining which mutations most likely influence disease. Although progress has been made in scoring the functional impact of individual mutations, the characteristics of the genes in which those mutations are found remain largely unexplored. For example, genes known to carry few common functional variants in healthy individuals may be judged more likely to cause certain kinds of disease than genes known to carry many such variants. Until now, however, it has not been possible to develop a quantitative assessment of how well genes tolerate functional genetic variation on a genome-wide scale. Here we describe an effort that uses sequence data from 6503 whole exome sequences made available by the NHLBI Exome Sequencing Project (ESP). Specifically, we develop an intolerance scoring system that assesses whether genes have relatively more or less functional genetic variation than expected based on the apparently neutral variation found in the gene. To illustrate the utility of this intolerance score, we show that genes responsible for Mendelian diseases are significantly more intolerant to functional genetic variation than genes that do not cause any known disease, but with striking variation in intolerance among genes causing different classes of genetic disease. We conclude by showing that use of an intolerance ranking system can aid in interpreting personal genomes and identifying pathogenic mutations.
This work uses empirical single nucleotide variant data from the NHLBI Exome Sequencing Project to introduce a genome-wide scoring system that ranks human genes in terms of their intolerance to standing functional genetic variation in the human population. It is often inferred that genes carrying relatively fewer or relatively more common functional variants in healthy individuals may be judged respectively more or less likely to cause certain kinds of disease. We show that this intolerance score correlates remarkably well with genes already known to cause Mendelian diseases (P<10−26). Equally striking, however, are the differences in the relationship between standing genetic variation and disease causing genes for different disease types. Considering disorder classes defined by Goh et al (2007) human disease network, we show a nearly opposite pattern for genes linked to developmental disorders and those linked to immunological disorders, with the former being preferentially caused by genes that do not tolerate functional variation and the latter caused by genes with an excess of common functional variation. We conclude by showing that use of an intolerance ranking system can facilitate interpreting personal genomes and can facilitate identifying high impact mutations through the gene in which they occur.
Genetic variation in the IL28B region has been associated with sustained virological response (SVR) rates in chronic hepatitis C (CHC) patients treated with peginterferon-α and ribavirin. We hypothesized that IL28B polymorphism is associated with intrahepatic expression of interferon-stimulated genes (ISGs), known to influence treatment outcome. IL28B genotyping (rs12979860) and whole-genome RNA expression were performed using liver biopsies from 61 North American CHC patients. After correction for multiple testing (false discovery rate < 0.10), 164 transcripts were found to be differentially expressed by IL28B-type. The interferon signaling pathway was the most enriched canonical pathway differentially expressed by IL28B-type (p < 10−5), with most genes showing higher expression in livers of individuals carrying the poor-response IL28B-type. In 25 patients for which treatment response data were available, IL28B-type was associated with SVR (p = 0.0054). ISG expression was also associated with SVR; however, this was not independent of IL28B-type. Analysis of miR-122 expression in liver biopsies showed reduced miR-122 levels associated with poorer treatment outcome, independently of IL28B-type. No association was observed between IL28B-type and levels of liver IL28B or IL28A mRNA expression. IL28B protein sequence variants associated with rs12979860 were therefore investigated in vitro: no differences in ISG induction or inhibition of HCV replication were observed in Huh7.5 cells.
The good response IL28B variant was strongly associated with lower level ISG expression. The results suggest that IL28B genotype may explain the relationship between hepatic ISG expression and HCV treatment outcome, and this is independent of miR-122 expression. IL28B-type was not associated with intrahepatic IL28B mRNA expression in vivo. Further investigation of the precise molecular mechanism(s) by which IL28B genetic variation influences HCV outcomes is warranted.
interferon lambda; hepatitis C virus; gene expression
A single-nucleotide polymorphism (rs2395029) in the HCP5 gene associated with HLA-B*5701 is correlated with lower HIV-1 viral set point. The two allelic forms of coding region were ectopically expressed in TZM-bl cells for an effect on HIV-1 replication. No significant HIV-1 restriction was observed in the cells with infectivity assays throughout HIV-1 life cycle, suggesting that the association of HCP5 variant with viral control is likely due to HLA-B*5701-related effect or other functional variants in the haplotype or both.
A new study focuses attention on multigenic interactions influencing the risk of autism spectrum disorders.
Background. A recent genome-wide association study reported a strong association with a single-nucleotide polymorphism (SNP) in the inosine triphosphate (ITPA) gene and hemolytic anemia in patients infected with hepatitis C virus (HCV) receiving pegylated interferon and ribavirin. We investigate these polymorphisms in a cohort of human immunodeficiency virus (HIV)/HCV–coinfected patients.
Methods. DNA was available for 161 patients with validated outcomes. We analyzed the association between the variants and week 4 hemoglobin reduction. Anemia over the course of therapy, ribavirin (RBV) dose reduction, serum RBV level, and rapid virological response (RVR) and sustained virological response (SVR) were also investigated. Using a candidate gene approach, ITPA variants rs1127354 and rs7270101 were tested using the ABI TaqMan kit. Multivariable models were used to identify predictors of anemia.
Results. A significant minority (33%) of patients were predicted to have reduced ITPase activity. The minor allele of each variant was associated with protection against week 4 anemia. In multivariable models only the genetic variants, creatinine, and zidovudine exposure remained significant. ITPase deficiency was not associated with RBV-dose reduction, RVR, or SVR.
Conclusions. This study confirms that polymorphisms in the ITPA gene are associated with protection from RBV-induced anemia in HIV/HCV-coinfected patients but not improved clinical outcomes.
Topiramate is an antiepileptic drug that has marked, treatment-limiting side effects on specific aspects of cognitive performance in both patients and healthy volunteers. As these severe side-effects occur only in certain individuals, identifying genetic or environmental variables that influence cognitive response would be of great utility in determining whether to administer this drug to a patient. We gave an acute 100 mg oral dose of topiramate to 158 healthy volunteers and measured how the drug changed their performance on a diverse battery of cognitive tests. We found a wide range of responses to topiramate and demonstrated that not all tests in the battery were equally affected. There was no correlation between the effect of topiramate and either education level or baseline cognitive performance. Interestingly, there was up to 55-fold variation in the topiramate plasma levels of the participants. Our genome-wide association study (GWAS) of cognitive response did not reveal any genome-wide significant associations; the study was powered to find variants explaining at least 25% of the variation in cognitive response. Combining the results of this GWAS with a retrospective study of cognitive complaints in 290 epilepsy patients who received topiramate as part of their treatment also did not result in a significant association. Our results support the need for additional genetic studies of topiramate that utilize larger sample sizes.
topiramate; cognition; genome-wide association study; genetics; taste change
We sequenced the genomes of ten unrelated individuals and identified heterozygous stop gain variants in protein-coding genes: we then sequenced their transcriptomes and assessed the expression levels of the stop gain alleles. An ANOVA showed statistically significant differences between their expression levels (p=4×10-16). This difference was almost entirely accounted for by whether the stop gain variant had a second, non-protein-truncating function in or near an alternate transcript: stop gains without alternate functions were generally not found in the cDNA (p=3×10-5). Additionally, stop gain variants in two intronless genes were not expressed, an unexpected outcome given previous studies. In this study, stop gain variants were either well expressed in all individuals or were never expressed. Our finding that stop gain variants were generally expressed only when they had an alternate function suggests that most naturally occurring stop gain variants in protein-coding genes are either not transcribed or have their transcripts destroyed.
Nonsense-mediated decay; whole-genome sequencing; RNA-Seq; premature termination codons
With rapid advances in our knowledge of the human genome and increasing availability of high-throughput investigative technology, genome-wide association (GWA) studies have recently gained marked popularity. As an unbiased approach to identifying genomic regions of importance in complex human disease, the results of such studies have the potential to illuminate novel causal pathways, guide mechanistic research, and aid in prediction of disease risk. The use of a genome-wide approach presents considerable methodological and statistical challenges, and properly conducted studies are essential to avoid false-positive results. A total of 22 GWA studies have been published in pulmonary medicine thus far, implicating several intriguing genomic regions in the determination of pulmonary function measures, onset of asthma, and susceptibility to chronic obstructive pulmonary disease. Many questions remain, however, as most identified genetic variants contribute only nominally to overall disease risk, genetic disease mechanisms remain uncertain, and disease-associated variants are not consistent across studies. Perhaps most fundamentally, the association signals identified have not yet been traced to causal variants. This perspective will review the current state of GWA studies in pulmonary disease. We begin with an introduction to the hypothesis, principles, and limitations of this type of genome-wide approach, highlight key points from available studies, and conclude by addressing future approaches to better understand the genetics of complex pulmonary disease.
genetics; chronic obstructive pulmonary disease; asthma
Brain-derived neurotrophic factor (BDNF) is a neurotrophin which has been shown to regulate cell survival and proliferation, as well as synaptic growth and hippocampal long-term potentiation. A naturally occurring single nucleotide polymorphism in the human BDNF gene (val66met) has been associated with altered intercellular trafficking and regulated secretion of BDNF in met compared to val carriers. Additionally, previous studies have found a relationship between the BDNF val66met genotype and functional activity in the hippocampus during episodic and working memory tasks in healthy young adults. Specifically, studies have found that met carriers exhibit both poorer performance and reduced neural activity within the medial temporal lobe (MTL) when performing episodic memory tasks. However, these studies have not been well replicated and have not considered the role of behavioral differences in the interpretation of neural differences. The current study sought to control for cognitive performance in investigating the role of the BDNF val66met genotype on neural activity associated with episodic memory. Across item and relational memory tests, met carriers exhibited increased MTL activation during both encoding and retrieval stages, compared to non-carriers. The results suggest that met carriers are able to recruit MTL activity to support successful memory processes, and reductions in cognitive performance observed in prior studies are not a ubiquitous effect associated with variants of the BDNF val66met genotype.
There is considerable interest in the use of next-generation sequencing to help diagnose unidentified genetic conditions, but it is difficult to predict the success rate in a clinical setting that includes patients with a broad range of phenotypic presentations.
The authors present a pilot programme of whole-exome sequencing on 12 patients with unexplained and apparent genetic conditions, along with their unaffected parents. Unlike many previous studies, the authors did not seek patients with similar phenotypes, but rather enrolled any undiagnosed proband with an apparent genetic condition when predetermined criteria were met.
This undertaking resulted in a likely genetic diagnosis in 6 of the 12 probands, including the identification of apparently causal mutations in four genes known to cause Mendelian disease (TCF4, EFTUD2, SCN2A and SMAD4) and one gene related to known Mendelian disease genes (NGLY1). Of particular interest is that at the time of this study, EFTUD2 was not yet known as a Mendelian disease gene but was nominated as a likely cause based on the observation of de novo mutations in two unrelated probands. In a seventh case with multiple disparate clinical features, the authors were able to identify homozygous mutations in EFEMP1 as a likely cause for macular degeneration (though likely not for other features).
This study provides evidence that next-generation sequencing can have high success rates in a clinical setting, but also highlights key challenges. It further suggests that the presentation of known Mendelian conditions may be considerably broader than currently recognised.
Exome sequencing; unidentified genetic conditions; medical genetics; paediatrics; clinical genetics; complex traits; genetic screening/counselling; genetics; genome-wide; psychotic disorders (including schizophrenia); molecular genetics; gastroenterology; immunology (including allergy).
Large-scale association studies can identify the gene variants underlying common disease susceptibilities, but discovering how these variants produce the disease traits requires innovative biology, sadly lacking in most studies.
Understanding how human genetic variation impacts individual response to immunogens is fundamental for rational vaccine development. To explore host mechanisms involved in cellular immune responses to the MRKAd5 human immunodeficiency virus type 1 (HIV-1) gag/pol/nef vaccine tested in the Step trial, we performed a genome-wide association study of determinants of HIV-specific T cell responses, measured by interferon γ enzyme-linked immunospot assays. No human genetic variant reached genome-wide significance, but polymorphisms located in the major histocompatibility complex (MHC) region showed the strongest association with response to the HIV-1 Gag protein: HLA-B alleles known to be associated with differences in HIV-1 control were responsible for these associations. The implication of the same HLA alleles in vaccine-induced cellular immunity and in natural immune control is of relevance for vaccine design. Furthermore, our results demonstrate the importance of considering the host immunogenetic background in the analysis of immune responses to T cell vaccines.
Genomics is now a core element in the effort to develop a vaccine against HIV-1. Thanks to unprecedented progress in high-throughput genotyping and sequencing, in knowledge about genetic variation in humans, and in evolutionary genomics, it is finally possible to systematically search the genome for common genetic variants that influence the human response to HIV-1. The identification of such variants would help to determine which aspects of the response to the virus are the most promising targets for intervention. However, a key obstacle to progress remains the scarcity of appropriate human cohorts available for genomic research.
To date, CCR5 variants remain the only human genetic factors to be confirmed to impact HIV-1 acquisition. However, protective CCR5 variants are largely absent in African populations, in which sporadic resistance to HIV-1 infection is still unexplained. Here we perform a genome-wide association study (GWAS) in a population of 1,532 individuals from Malawi, a country with high prevalence of HIV-1 infection, to investigate whether common genetic variants associate with HIV-1 susceptibility in Africans. Using single nucleotide polymorphisms (SNPs) present on the genome-wide chip, we also investigated previously reported associations with HIV-1 susceptibility or acquisition. Recruitment was coordinated by the Center for HIV/AIDS Vaccine Immunology at two sexually transmitted infection clinics. HIV status was determined by HIV rapid tests and nucleic acid testing.
After quality control, the population consisted of 848 high-risk seronegative and 531 HIV-1 seropositive individuals. Logistic regression testing in an additive genetic model was performed for SNPs that passed quality control. No single SNP yielded a significant P-value after correction for multiple testing. The study was sufficiently powered to detect markers with genotype relative risk ≥ 2.0 and minor allele frequencies ≥12%. This is the first GWAS of host determinants of HIV-1 susceptibility, performed in an African population. The absence of any significant association can have many possible explanations: rarer genetic variants or common variants with weaker effect could be responsible for the resistance phenotype; alternatively, resistance to HIV-1 infection might be due to non-genetic parameters or to complex interactions between genes, immunity and environment.
Human immunodeficiency virus (HIV-1); acquisition; resistance; Genome Wide Association Study (GWAS); Africa
Polymorphisms of IL28B gene are highly associated with sustained virological response (SVR) in patients with chronic hepatitis C treated with peginterferon and ribavirin. Quantitation of Interferon-γ Inducible Protein-10 (IP-10) may also differentiate antiviral response. We evaluated IP-10 levels in pretreatment serum from 115 non-responders and 157 sustained responders in the VIRAHEP-C cohort, including African Americans (AA) and Caucasian Americans (CA). Mean IP-10 was lower in sustained responders compared to non-responders (460 ± 37 pg/ml vs 697 ± 49 pg/ml, p<0.001), both in AA and CA. The positive predictive value of low IP-10 levels (<600 pg/ml) for SVR was 69% while the negative predictive value of high IP-10 levels (>600 pg/ml) was 67%. We assessed the combination of pretreatment IP-10 levels with IL28B genotype as predictors of treatment response. The IL28B polymorphism rs12979860 was tested in 210 participants. CC, CT, or TT genotypes were found in 30%, 49%, and 21%, respectively, with corresponding SVR rates of 87%, 50%, and 39% (p<0.0001). Serum IP-10 levels within the IL28B genotype groups provided additional information regarding the likelihood of SVR (p< 0.0001). CT carriers with low IP-10 had 64% SVR versus 24% with high IP-10. Similarly, a higher SVR rate was identified for TT and CC carriers with low versus high IP-10 (TT: 48% versus 20%, CC: 89% versus 79%). IL28B genotype and baseline IP-10 levels were additive but independent when predicting SVR in both AA and CA.
When IL28B genotype is combined with pretreatment serum IP-10 measurement, the predictive value for discrimination between SVR and non-response is significantly improved, especially in non-CC genotypes. This relationship warrants further investigation to elucidate mechanisms of antiviral response and prospective validation.
Peginterferon; Ribavirin; Race; Biomarker; Genotype
Both clinical experience and a growing medical literature indicate that there are persons who have been exposed to HIV infection who have remained uninfected. While in some instances this may represent good fortune, cohorts of uninfected persons have been reported where risks for infection are thought to be high. In these cohorts a variety of characteristics have been proposed as mediating protection but to date only the 32 base pair deletion in the CCR5 gene that results in complete failure of cell surface expression of this co-receptor has been associated with high level protection from HIV infection. With this in mind, there are likely numerous other factors that may individually or in combination provide some level of protection from acquisition of HIV infection. As some of these factors are likely incompletely protective or inconsistently active, identifying them with confidence will be difficult. Nonetheless, clarifying the determinants of protection against HIV infection is a high priority that will require careful selection of high risk uninfected cohorts to which targeted studies of plausible mediators and broad screening for unexpected determinants of protection should be applied.
HIV infection; Exposed Seronegatives; High Risk Seronegatives; Interferon; Restriction factors; CCR5
Hepatitis C virus (HCV) infection is the most common blood borne infection in the U.S. with estimates of 4 million HCV-infected individuals in the U.S. and 170 million worldwide1. The majority (70%–80%) of HCV infections persist and about 30% of individuals with persistent infection develop chronic liver disease, including cirrhosis and hepatocellular carcinoma2. Epidemiological, viral, and host factors have been associated with the differences in HCV clearance or persistence and studies have demonstrated that a strong host immune response against HCV favors viral clearance3,4. Thus, variation in genes involved in the immune response may contribute to the ability to clear the virus. In a recent genome-wide association study, a single nucleotide polymorphism (rs12979860) 3kb upstream of the IL28B gene, which encodes the type III interferon IFN-λ3, was shown to associate strongly with more than a 2-fold difference in response to HCV drug treatment5. To determine the potential effect of rs12979860 variation on outcome to HCV infection in a natural history setting, we genotyped this variant in HCV cohorts comprised of individuals who spontaneously cleared the virus (N = 388) or had persistent infection (N = 620). We show that the C/C genotype strongly enhances resolution of HCV infection amongst individuals of both European and African ancestry (European: OR = 0.38, p = 10−7; African: OR = 0.32, p = 10−4; combined: OR = 0.33, p <10−12). To date, this is the strongest and most significant genetic effect associated with natural clearance of HCV, and these results implicate a primary role for IL28B in resolution of HCV infection.
One surprising feature of the recently completed waves of genome-wide association studies is the limited impact of common genetic variation in individually detectable polymorphisms on many human traits. This has been particularly pronounced for studies on psychiatric conditions, which have failed to produce clear, replicable associations for common variants. One popular explanation for these negative findings is that many of these traits may be genetically heterogeneous, leading to the idea that relevant endophenotypes may be more genetically tractable. Aspects of cognition may be the most important endophenotypes for psychiatric conditions such as schizophrenia, leading many researchers to pursue large-scale studies on the genetic contributors of cognitive performance in the normal population as a surrogate for aspects of liability to disease. Here, we perform a genome-wide association study with two tests of executive function, Digit Symbol and Stroop Color-Word, in 1086 healthy volunteers and with an expanded cognitive battery in 514 of these volunteers. We show that, consistent with published studies of the psychiatric conditions themselves, no single common variant has a large effect (explaining >4–8% of the population variation) on the performance of healthy individuals on standardized cognitive tests. Given that these are important endophenotypes, our work is consistent with the idea that identifying rare genetic causes of psychiatric conditions may be more important for future research than identifying genetically homogenous endophenotypes.
endophenotypes; genome-wide association; cognition; psychiatric conditions; common variants
Summary: Here we present Sequence Variant Analyzer (SVA), a software tool that assigns a predicted biological function to variants identified in next-generation sequencing studies and provides a browser to visualize the variants in their genomic contexts. SVA also provides for flexible interaction with software implementing variant association tests allowing users to consider both the bioinformatic annotation of identified variants and the strength of their associations with studied traits. We illustrate the annotation features of SVA using two simple examples of sequenced genomes that harbor Mendelian mutations.
Availability and implementation: Freely available on the web at http://www.svaproject.org.
Supplementary information: Supplementary data are available at Bioinformatics online.
BACKGROUND & AIMS
In a genome-wide association study of patients being treated for chronic hepatitis C, 2 functional variants in ITPA that cause inosine triphosphatase (ITPase) deficiency were shown to protect against ribavirin (RBV)-induced hemolytic anemia during early stages of treatment. We aimed to replicate this finding in an independent cohort from the Study of Viral Resistance to Antiviral Therapy of Chronic Hepatitis C and to investigate the effects of these variants beyond week 4.
Genetic material was available from 318 patients. The ITPA variants, rs1127354 (exon 2, P32T) and rs7270101 (intron 2, splice altering), were genotyped and tested for association with hemoglobin (Hb) reduction at week 4. An ITPase deficiency variable was defined that combined both ITPA variants according to documented effect on ITPase activity. We investigated the impact of ITPA variants on Hb levels over the course of therapy and on the need for RBV dose reduction.
The final analysis included 304 patients with genotype 1 hepatitis C virus (167 white patients and 137 black patients). The polymorphisms rs1127354 and rs7270101 were associated with Hb reduction at week 4 (P = 3.1 × 10−13 and 1.3 × 10−3, respectively). The minor alleles of each variant protected against Hb reduction. Combining the variants into the ITPase deficiency variable strengthened the association (P = 2.4 × 10−18). The ITPase deficiency variable was associated with lower rates of anemia over the entire treatment period (48 weeks), as well as a lower rate of anemia-related RBV dose reduction (hazard ratio, 0.52; P = .0037). No association with sustained virological response was observed.
Two polymorphisms that cause ITPase deficiency are strongly associated with protection from RBV-induced hemolytic anemia and decrease the need for RBV dose reduction.
Pharmacogenomics; Genome-Wide Association Study; Polymorphism; Single Nucleotide Polymorphism; HCV; Adverse Event
We performed a whole-genome association study on HIV-1 viral load setpoint in an African American cohort (n=515), and an intronic SNP in the HLA-B gene showed one of the strongest associations. Using a subset of patients, we show that this SNP reflects the effect of the HLA-B*5703 allele, which shows a genome-wide significant association with HIV-1 VL setpoint (p=5.6×10−10). These analyses therefore confirm a member of the HLA-B*57 group of alleles as the most important common variant influencing viral load variation in African Americans, consistent with what is observed in individuals of European ancestry in which the most important common variant is HLA-B*5701.
HIV; viral load setpoint; host genetics; association study; HLA