To complement our special issue on exome sequencing, Genome Biology asked several leaders in the field for their views on this new approach. Leslie G Biesecker (LGB), Jim C Mullikin (JM) and Kevin V Shianna (KVS) discuss the reasons for the popularity of exome sequencing and its contribution to genomics.

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.

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.

Aims

An association between carbamazepine-induced hypersensitivity and HLA-A*3101 has been reported in populations of both European and Asian descent. We aimed to investigate HLA-A*3101 and other common variants across the genome as markers for cutaneous adverse drug reactions (cADRs) attributed to lamotrigine and phenytoin.

Materials & methods

We recruited patients with lamotrigine-induced cADRs (n = 46) and patients with phenytoin-cADRs (n = 44) and the 1958 British birth cohort was used as a control (n = 1296). HLA-A*3101 was imputed from genome-wide association study data. We applied genome-wide association to study lamotrigine- and phenytoin-induced cADR, and total cADR cases combined.

Results

Neither HLA-A*3101 nor any other genetic marker significantly predicted lamotrigine- or phenytoin-induced cADRs.

Conclusion

HLA-A*3101 does not appear to be a predictor for lamotrigine- and phenytoin-induced cADRs in Europeans. Our genome-wide association study results do not support the existence of a clinically relevant common variant for the development of lamotrigine- or phenytoin-induced cADRs. As a predictive marker, HLA-A*3101 appears to be specific for carbamazepine-induced cADRs.

Background

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.

Methods

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.

Results

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).

Conclusions

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.

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.

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.

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.

Conclusions

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.

The authors that the number of activating and inhibitory KIR genes varies between individuals and plays a role in the regulation of immune mechanisms that determine HIV-1 control.

A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection.

Author Summary

There is marked intrinsic variation in the extent to which individuals are able to control HIV-1. We have identified a genetic copy number variable region (CNV) in humans that plays a significant role in the control of HIV-1. This CNV is located in the genomic region that encodes the killer cell immunoglobulin-like receptors (KIRs) and specifically affects the KIR3DS1 and KIR3DL1 genes, encoding two KIRs that interact with human leukocyte antigen B (HLA-B) ligands. KIRs are expressed on the surface of natural killer (NK) cells, which serve as important players in the innate immune response, and are involved in the recognition of infected and malignant cells through a loss or alteration in “self” ligands. We use both genetic association and functional evidence to show a strong interaction between KIR3DL1 and KIR3DS1, indicating that increasing gene counts for KIR3DL1 confer increasing levels of protection against HIV-1, but only in the presence of at least one copy of KIR3DS1. This effect was associated with a dramatic increase in the abundance of KIR3DS1+ NK cells in the peripheral blood, and strongly associated with a more robust capacity of peripheral NK cells to suppress HIV-1 replication in vitro. This work provides one of the few examples of an association between a relatively common CNV and a human complex trait.

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.

Contact: d.ge@duke.edu

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.

METHODS

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.

RESULTS

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.

CONCLUSIONS

Two polymorphisms that cause ITPase deficiency are strongly associated with protection from RBV-induced hemolytic anemia and decrease the need for RBV dose reduction.

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.

Psychiatric disorders such as schizophrenia are commonly accompanied by cognitive impairments that are treatment resistant and crucial to functional outcome. There has been great interest in studying cognitive measures as endophenotypes for psychiatric disorders, with the hope that their genetic basis will be clearer. To investigate this, we performed a genome-wide association study involving 11 cognitive phenotypes from the Cambridge Neuropsychological Test Automated Battery. We showed these measures to be heritable by comparing the correlation in 100 monozygotic and 100 dizygotic twin pairs. The full battery was tested in ∼750 subjects, and for spatial and verbal recognition memory, we investigated a further 500 individuals to search for smaller genetic effects. We were unable to find any genome-wide significant associations with either SNPs or common copy number variants. Nor could we formally replicate any polymorphism that has been previously associated with cognition, although we found a weak signal of lower than expected P-values for variants in a set of 10 candidate genes. We additionally investigated SNPs in genomic loci that have been shown to harbor rare variants that associate with neuropsychiatric disorders, to see if they showed any suggestion of association when considered as a separate set. Only NRXN1 showed evidence of significant association with cognition. These results suggest that common genetic variation does not strongly influence cognition in healthy subjects and that cognitive measures do not represent a more tractable genetic trait than clinical endpoints such as schizophrenia. We discuss a possible role for rare variation in cognitive genomics.

This is a crucial transition time for human genetics in general, and for HIV host genetics in particular. After years of equivocal results from candidate gene analyses, several genome-wide association studies have been published that looked at plasma viral load or disease progression. Results from other studies that used various large-scale approaches (siRNA screens, transcriptome or proteome analysis, comparative genomics) have also shed new light on retroviral pathogenesis. However, most of the inter-individual variability in response to HIV-1 infection remains to be explained: genome resequencing and systems biology approaches are now required to progress toward a better understanding of the complex interactions between HIV-1 and its human host.

We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten “case” genomes from individuals with severe hemophilia A and ten “control” genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs) discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.

Author Summary

We report here the nearly complete genomic sequence of 20 different individuals, determined using “next-generation” sequencing technologies. We use these data to characterize the type of genetic variation carried by humans in a sample of this size, which is to our knowledge the largest set of unrelated genomic sequences that have been reported. We summarize different categories of variation in each genome, and in total across all 20 of the genomes, finding a surprising number of variants predicted to reduce or remove the proteins encoded by many different genes. This work provides important fundamental information about the scope of human genetic variation, and suggests ways to further explore the relationship between these genetic variants and human disease.

Studies of the genetics of gene expression reveal expression SNPs that explain variation in transcript abundance. Here we address the robustness of eSNP associations to environmental geography and population structure in a comparison of 194 Arab and Amazigh individuals from a city and two villages in southern Morocco. Gene expression differed between pairs of locations for up to a third of all transcripts, with notable enrichment for ribosomal biosynthesis and oxidative phosphorylation. Robust associations were observed in the leukocyte samples with cis-eSNPs (P < 10−08) for 346 genes, and trans-eSNPs (P < 10−11) with 10 genes. All of these were consistent across the three sample locations and after controlling for ethnicity and relatedness. No evidence for large-effect trans-acting mediators of the pervasive environmental influence was found and instead genetic and environmental factors acted in a largely additive manner.

Although more than 2,400 genes have been shown to contain variants that cause Mendelian disease, there are still several thousand such diseases yet to be molecularly defined. The ability of new whole-genome sequencing technologies to rapidly indentify most of the genetic variants in any given genome opens an exciting opportunity to identify these disease genes. Here we sequenced the whole genome of a single patient with the dominant Mendelian disease, metachondromatosis (OMIM 156250), and used partial linkage data from her small family to focus our search for the responsible variant. In the proband, we identified an 11 bp deletion in exon four of PTPN11, which alters frame, results in premature translation termination, and co-segregates with the phenotype. In a second metachondromatosis family, we confirmed our result by identifying a nonsense mutation in exon 4 of PTPN11 that also co-segregates with the phenotype. Sequencing PTPN11 exon 4 in 469 controls showed no such protein truncating variants, supporting the pathogenicity of these two mutations. This combination of a new technology and a classical genetic approach provides a powerful strategy to discover the genes responsible for unexplained Mendelian disorders.

Author Summary

Metachondromatosis (MC) is an autosomal dominant condition characterized by exostoses (osteochondromas), commonly of the hands and feet, and enchondromas of long bone metaphyses and iliac crests. MC exostoses may regress or even resolve over time, and short stature is not characteristic of MC. Here, we sequenced the whole genome of a single patient with MC and used partial linkage data from her small family to focus our search for the responsible variant. In the proband, we identified an 11 bp deletion in exon four of PTPN11, which results in premature translation termination and co-segregates with the phenotype. In a second metachondromatosis family, we identified a nonsense mutation in exon 4 of PTPN11 that also co-segregates with the phenotype. Germline gain-of-function missense mutations in PTPN11 cause an overlapping but distinct group of dominant disorders with involvement of the face, heart, skeleton, skin, and brain, including Noonan syndrome (OMIM 163950), Noonan-like disorder with multiple giant cell lesion syndrome (OMIM 163955), and LEOPARD syndrome (OMIM 151100). Nonsense mutations in PTPN11 have not been described in humans and the loss-of-function PTPN11 mutations we report here are the first to be described in human disease.

Partial epilepsies have a substantial heritability. However, the actual genetic causes are largely unknown. In contrast to many other common diseases for which genetic association-studies have successfully revealed common variants associated with disease risk, the role of common variation in partial epilepsies has not yet been explored in a well-powered study. We undertook a genome-wide association-study to identify common variants which influence risk for epilepsy shared amongst partial epilepsy syndromes, in 3445 patients and 6935 controls of European ancestry. We did not identify any genome-wide significant association. A few single nucleotide polymorphisms may warrant further investigation. We exclude common genetic variants with effect sizes above a modest 1.3 odds ratio for a single variant as contributors to genetic susceptibility shared across the partial epilepsies. We show that, at best, common genetic variation can only have a modest role in predisposition to the partial epilepsies when considered across syndromes in Europeans. The genetic architecture of the partial epilepsies is likely to be very complex, reflecting genotypic and phenotypic heterogeneity. Larger meta-analyses are required to identify variants of smaller effect sizes (odds ratio <1.3) or syndrome-specific variants. Further, our results suggest research efforts should also be directed towards identifying the multiple rare variants likely to account for at least part of the heritability of the partial epilepsies. Data emerging from genome-wide association-studies will be valuable during the next serious challenge of interpreting all the genetic variation emerging from whole-genome sequencing studies.

Background

There is considerable interest in the development of methods to efficiently identify all coding variants present in large sample sets of humans. There are three approaches possible: whole-genome sequencing, whole-exome sequencing using exon capture methods, and RNA-Seq. While whole-genome sequencing is the most complete, it remains sufficiently expensive that cost effective alternatives are important.

Results

Here we provide a systematic exploration of how well RNA-Seq can identify human coding variants by comparing variants identified through high coverage whole-genome sequencing to those identified by high coverage RNA-Seq in the same individual. This comparison allowed us to directly evaluate the sensitivity and specificity of RNA-Seq in identifying coding variants, and to evaluate how key parameters such as the degree of coverage and the expression levels of genes interact to influence performance. We find that although only 40% of exonic variants identified by whole genome sequencing were captured using RNA-Seq; this number rose to 81% when concentrating on genes known to be well-expressed in the source tissue. We also find that a high false positive rate can be problematic when working with RNA-Seq data, especially at higher levels of coverage.

Conclusions

We conclude that as long as a tissue relevant to the trait under study is available and suitable quality control screens are implemented, RNA-Seq is a fast and inexpensive alternative approach for finding coding variants in genes with sufficiently high expression levels.

Ma’am – We read with great interest the article by He et al. [2008] describing the effects on HIV acquisition and disease progression of a single-nucleotide polymorphism (SNP, rs2814778, -46T→C) that disrupts the promoter region of the Duffy antigen receptor for chemokines (DARC) gene and abolishes gene expression in red blood cells. He et al. reported that HIV-infected African Americans have a frequency of the null homozygous genotype (-46C/C) of 70% while non-HIV infected individuals have a null genotype frequency of 60%. Based on this frequency difference they argued that the null allele confers susceptibility to infection with HIV-1. They also reported that the null genotype is associated with better outcomes amongst those who do become infected, including longer survival, slower loss of CD4+ T-lymphocytes, and delayed progression to HIV-associated dementia.

There is great interindividual variability in HIV-1 viral setpoint after seroconversion, some of which is known to be due to genetic differences among infected individuals. Here, our focus is on determining, genome-wide, the contribution of variable gene expression to viral control, and to relate it to genomic DNA polymorphism. RNA was extracted from purified CD4+ T-cells from 137 HIV-1 seroconverters, 16 elite controllers, and 3 healthy blood donors. Expression levels of more than 48,000 mRNA transcripts were assessed by the Human-6 v3 Expression BeadChips (Illumina). Genome-wide SNP data was generated from genomic DNA using the HumanHap550 Genotyping BeadChip (Illumina). We observed two distinct profiles with 260 genes differentially expressed depending on HIV-1 viral load. There was significant upregulation of expression of interferon stimulated genes with increasing viral load, including genes of the intrinsic antiretroviral defense. Upon successful antiretroviral treatment, the transcriptome profile of previously viremic individuals reverted to a pattern comparable to that of elite controllers and of uninfected individuals. Genome-wide evaluation of cis-acting SNPs identified genetic variants modulating expression of 190 genes. Those were compared to the genes whose expression was found associated with viral load: expression of one interferon stimulated gene, OAS1, was found to be regulated by a SNP (rs3177979, p = 4.9E-12); however, we could not detect an independent association of the SNP with viral setpoint. Thus, this study represents an attempt to integrate genome-wide SNP signals with genome-wide expression profiles in the search for biological correlates of HIV-1 control. It underscores the paradox of the association between increasing levels of viral load and greater expression of antiviral defense pathways. It also shows that elite controllers do not have a fully distinctive mRNA expression pattern in CD4+ T cells. Overall, changes in global RNA expression reflect responses to viral replication rather than a mechanism that might explain viral control.

Author Summary

There has been recent progress in understanding the genetic factors that modulate susceptibility to HIV-1 infection. Genetic variation explains to a certain extent differences in disease progression among individuals. Less is known regarding the contribution of differences in gene expression to viral control. The present study evaluated, genome-wide, gene expression levels in CD4+ T cell, the main target of HIV-1. Thereafter, it searched for genetic variants that would modify gene expression. Specific expression profiles associated with high levels of viremia—in particular, the upregulation of genes of the antiviral defense. In contrast, no expression profile associated with effective viral control. Multiple genetic variants modulated gene expression in CD4+ T cells; however, none had a strong influence on viral control. This integrated genome-wide assessment suggests that viral replication drives gene expression rather than expression pointing to mechanisms of viral control.

To extend the understanding of host genetic determinants of HIV-1 control, we performed a genome-wide association study in a cohort of 2,554 infected Caucasian subjects. The study was powered to detect common genetic variants explaining down to 1.3% of the variability in viral load at set point. We provide overwhelming confirmation of three associations previously reported in a genome-wide study and show further independent effects of both common and rare variants in the Major Histocompatibility Complex region (MHC). We also examined the polymorphisms reported in previous candidate gene studies and fail to support a role for any variant outside of the MHC or the chemokine receptor cluster on chromosome 3. In addition, we evaluated functional variants, copy-number polymorphisms, epistatic interactions, and biological pathways. This study thus represents a comprehensive assessment of common human genetic variation in HIV-1 control in Caucasians.

Author Summary

The ability to spontaneously control HIV-1 upon infection is highly variable between individuals. To evaluate the contribution of variation in human genes to differences in plasma viral load and in disease progression rates, we performed a genome-wide association study in >2,500 HIV–infected individuals. This study achieved two goals: it completed the analysis of common variation influencing viral control, and it re-assessed the majority of previously reported genetic associations. We show that genetic variants located near the HLA-B and HLA-C genes are the strongest determinants of viral control, and that other independent associations exist in the same region of chromosome 6, the Major Histocompatibility Complex, known to contain a large number of genes involved in immune defense. We could not replicate most of the previously published associations with HIV candidate genes in this large, well-characterized cohort. Overall, common human genetic variation, together with demographic variables, explains up to 22% of the variability in viral load in the Caucasian population.

The tumor suppressor p53 has been implicated in multiple functions that play key roles in health and disease, including ribosome biogenesis, control of aging, and cell cycle regulation. A genetic screen for negative regulators of innate immunity in Caenorhabditis elegans led to the identification of a mutation in NOL-6, a nucleolar RNA-associated protein (NRAP), which is involved in ribosome biogenesis and conserved across eukaryotic organisms. Mutation or silencing of NOL-6 and other nucleolar proteins results in an enhanced resistance to bacterial infections. A full-genome microarray analysis on animals with altered immune function due to mutation in nol-6 shows increased transcriptional levels of genes regulated by a p53 homologue, CEP-1. Further studies indicate that the activation of innate immunity by inhibition of nucleolar proteins requires p53/CEP-1 and its transcriptional target SYM-1. Since nucleoli and p53/CEP-1 are conserved, our results reveal an ancient immune mechanism by which the nucleolus may regulate immune responses against bacterial pathogens.

Author Summary

Innate immunity comprises a variety of defense mechanisms used by metazoans to prevent microbial infections. These nonspecific defense responses used by the innate immune system are governed by interacting and intersecting pathways that control not only immune responses but also longevity and responses to different stresses. Increasing evidence highlights the plurifunctional nature of the nucleolus, which appears to control various cellular processes involved in health and disease, from ribosome biogenesis to regulation of the cell cycle and the cellular stress response. We provide evidence indicating that the nucleolus suppresses innate immunity against bacteria by preventing the transcriptional activity of the tumor suppressor p53. We found that animals lacking nucleolar proteins are highly resistant to infections by bacterial pathogens. We also found that the activation of innate immunity by inhibition of nucleolar proteins requires potential immune effectors whose expression in response to stress is regulated by p53. Our study links the nucleolus, p53, and innate immunity against bacterial infections for the first time, and highlights a new mechanism that can potentially be exploited to alleviate bacterial infections.