The benefits of selecting phenotype-specific variants for applications of mixed models in genomics.

Applications of linear mixed models (LMMs) to problems in genomics include phenotype prediction, correction for confounding in genome-wide association studies, estimation of narrow sense heritability, and testing sets of variants (e.g., rare variants) for association. In each of these applications, the LMM uses a genetic similarity matrix, which encodes the pairwise similarity between every two individuals in a cohort. Although ideally these similarities would be estimated using strictly variants relevant to the given phenotype, the identity of such variants is typically unknown. Consequently, relevant variants are excluded and irrelevant variants are included, both having deleterious effects. For each application of the LMM, we review known effects and describe new effects showing how variable selection can be used to mitigate them.

A powerful and efficient set test for genetic markers that handles confounders.

MOTIVATION: Approaches for testing sets of variants, such as a set of rare or common variants within a gene or pathway, for association with complex traits are important. In particular, set tests allow for aggregation of weak signal within a set, can capture interplay among variants and reduce the burden of multiple hypothesis testing. Until now, these approaches did not address confounding by family relatedness and population structure, a problem that is becoming more important as larger datasets are used to increase power. RESULTS: We introduce a new approach for set tests that handles confounders. Our model is based on the linear mixed model and uses two random effects-one to capture the set association signal and one to capture confounders. We also introduce a computational speedup for two random-effects models that makes this approach feasible even for extremely large cohorts. Using this model with both the likelihood ratio test and score test, we find that the former yields more power while controlling type I error. Application of our approach to richly structured Genetic Analysis Workshop 14 data demonstrates that our method successfully corrects for population structure and family relatedness, whereas application of our method to a 15 000 individual Crohn's disease case-control cohort demonstrates that it additionally recovers genes not recoverable by univariate analysis. AVAILABILITY: A Python-based library implementing our approach is available at http://mscompbio.codeplex.com.

An exhaustive epistatic SNP association analysis on expanded Wellcome Trust data.

We present an approach for genome-wide association analysis with improved power on the Wellcome Trust data consisting of seven common phenotypes and shared controls. We achieved improved power by expanding the control set to include other disease cohorts, multiple races, and closely related individuals. Within this setting, we conducted exhaustive univariate and epistatic interaction association analyses. Use of the expanded control set identified more known associations with Crohn's disease and potential new biology, including several plausible epistatic interactions in several diseases. Our work suggests that carefully combining data from large repositories could reveal many new biological insights through increased power. As a community resource, all results have been made available through an interactive web server.

Fine-mapping classical HLA variation associated with durable host control of HIV-1 infection in African Americans.

A small proportion of human immunodeficiency virus-1 (HIV-1) infected individuals, termed HIV-1 controllers, suppress viral replication to very low levels in the absence of therapy. Genetic investigations of this phenotype have strongly implicated variation in the class I major histocompatibility complex (MHC) region as key to HIV-1 control. We collected sequence-based classical class I HLA genotypes at 4-digit resolution in HIV-1-infected African American controllers and progressors (n = 1107), and tested them for association with host control using genome-wide single nucleotide polymorphism data to account for population structure. Several classical alleles at HLA-B were associated with host control, including B*57:03 [odds ratio (OR) = 5.1; P= 3.4 × 10(-18)] and B*81:01 (OR = 4.8; P= 1.3 × 10(-9)). Analysis of variable amino acid positions demonstrates that HLA-B position 97 is the most significant association with host control in African Americans (omnibus P = 1.2 × 10(-21)) and explains the signal of several HLA-B alleles, including B*57:03. Within HLA-B, we also identified independent effects at position 116 (omnibus P= 2.8 × 10(-15)) in the canonical F pocket, position 63 in the B pocket (P= 1.5 × 10(-3)) and the non-pocket position 245 (P= 8.8 × 10(-10)), which is thought to influence CD8-binding kinetics. Adjusting for these HLA-B effects, there is evidence for residual association in the MHC region. These results underscore the key role of HLA-B in affecting HIV-1 replication, likely through the molecular interaction between HLA-B and viral peptides presented by infected cells, and suggest that sites outside the peptide-binding pocket also influence HIV-1 control.

FaST linear mixed models for genome-wide association studies.

We describe factored spectrally transformed linear mixed models (FaST-LMM), an algorithm for genome-wide association studies (GWAS) that scales linearly with cohort size in both run time and memory use. On Wellcome Trust data for 15,000 individuals, FaST-LMM ran an order of magnitude faster than current efficient algorithms. Our algorithm can analyze data for 120,000 individuals in just a few hours, whereas current algorithms fail on data for even 20,000 individuals (http://mscompbio.codeplex.com/).

HIV-1 adaptation to NK-cell-mediated immune pressure.

Natural killer (NK) cells have an important role in the control of viral infections, recognizing virally infected cells through a variety of activating and inhibitory receptors. Epidemiological and functional studies have recently suggested that NK cells can also contribute to the control of HIV-1 infection through recognition of virally infected cells by both activating and inhibitory killer immunoglobulin-like receptors (KIRs). However, it remains unknown whether NK cells can directly mediate antiviral immune pressure in vivo in humans. Here we describe KIR-associated amino-acid polymorphisms in the HIV-1 sequence of chronically infected individuals, on a population level. We show that these KIR-associated HIV-1 sequence polymorphisms can enhance the binding of inhibitory KIRs to HIV-1-infected CD4(+) T cells, and reduce the antiviral activity of KIR-positive NK cells. These data demonstrate that KIR-positive NK cells can place immunological pressure on HIV-1, and that the virus can evade such NK-cell-mediated immune pressure by selecting for sequence polymorphisms, as was previously described for virus-specific T cells and neutralizing antibodies. NK cells might therefore have a previously underappreciated role in contributing to viral evolution.

Reduced replication capacity of NL4-3 recombinant viruses encoding reverse transcriptase-integrase sequences from HIV-1 elite controllers.

BACKGROUND: Identifying viral and host determinants of HIV-1 elite control may help inform novel therapeutic and/or vaccination strategies. Previously, we observed decreased replication capacity in controller-derived viruses suggesting that fitness consequences of human leukocyte antigen (HLA) class I-associated escape mutations in Gag may contribute to this phenotype. This study examines whether similar functional defects occur in Pol proteins of elite controllers. METHODS: Recombinant NL4-3 viruses encoding plasma RNA-derived reverse transcriptase-integrase sequences from 58 elite controllers and 50 untreated chronic progressors were constructed, and replication capacity measured in vitro using a green fluorescent protein (GFP) reporter T-cell assay. Sequences were analyzed for drug resistance and HLA-associated viral polymorphisms. RESULTS: Controller-derived viruses displayed significantly lower replication capacity compared with those from progressors (P < 0.0001). Among controllers, the most attenuated viruses were generated from individuals expressing HLA-B*57 or B*51. In viruses from B*57+ progressors (n = 8), a significant inverse correlation was observed between B*57-associated reverse transcriptase-integrase escape mutations and replication capacity (R = -0.89; P = 0.003); a similar trend was observed in B*57+ controller-derived viruses (n = 20, R = -0.36; P = 0.08). CONCLUSIONS: HIV-1 Pol function seemed to be compromised in elite controllers. As observed previously for Gag, HLA-associated immune pressure in Pol may contribute to viral attenuation and subsequent control of viremia.

The major genetic determinants of HIV-1 control affect HLA class I peptide presentation.

Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA-viral peptide interaction as the major factor modulating durable control of HIV infection.

Early selection in Gag by protective HLA alleles contributes to reduced HIV-1 replication capacity that may be largely compensated for in chronic infection.

Mutations that allow escape from CD8 T-cell responses are common in HIV-1 and may attenuate pathogenesis by reducing viral fitness. While this has been demonstrated for individual cases, a systematic investigation of the consequence of HLA class I-mediated selection on HIV-1 in vitro replication capacity (RC) has not been undertaken. We examined this question by generating recombinant viruses expressing plasma HIV-1 RNA-derived Gag-Protease sequences from 66 acute/early and 803 chronic untreated subtype B-infected individuals in an NL4-3 background and measuring their RCs using a green fluorescent protein (GFP) reporter CD4 T-cell assay. In acute/early infection, viruses derived from individuals expressing the protective alleles HLA-B*57, -B*5801, and/or -B*13 displayed significantly lower RCs than did viruses from individuals lacking these alleles (P < 0.05). Furthermore, acute/early RC inversely correlated with the presence of HLA-B-associated Gag polymorphisms (R = -0.27; P = 0.03), suggesting a cumulative effect of primary escape mutations on fitness during the first months of infection. At the chronic stage of infection, no strong correlations were observed between RC and protective HLA-B alleles or with the presence of HLA-B-associated polymorphisms restricted by protective alleles despite increased statistical power to detect these associations. However, RC correlated positively with the presence of known compensatory mutations in chronic viruses from B*57-expressing individuals harboring the Gag T242N mutation (n = 50; R = 0.36; P = 0.01), suggesting that the rescue of fitness defects occurred through mutations at secondary sites. Additional mutations in Gag that may modulate the impact of the T242N mutation on RC were identified. A modest inverse correlation was observed between RC and CD4 cell count in chronic infection (R = -0.17; P < 0.0001), suggesting that Gag-Protease RC could increase over the disease course. Notably, this association was stronger for individuals who expressed B*57, B*58, or B*13 (R = -0.27; P = 0.004). Taken together, these data indicate that certain protective HLA alleles contribute to early defects in HIV-1 fitness through the selection of detrimental mutations in Gag; however, these effects wane as compensatory mutations accumulate in chronic infection. The long-term control of HIV-1 in some persons who express protective alleles suggests that early fitness hits may provide lasting benefits.

Gag-protease-mediated replication capacity in HIV-1 subtype C chronic infection: associations with HLA type and clinical parameters.

The mechanisms underlying HIV-1 control by protective HLA class I alleles are not fully understood and could involve selection of escape mutations in functionally important Gag epitopes resulting in fitness costs. This study was undertaken to investigate, at the population level, the impact of HLA-mediated immune pressure in Gag on viral fitness and its influence on HIV-1 pathogenesis. Replication capacities of 406 recombinant viruses encoding plasma-derived Gag-protease from patients chronically infected with HIV-1 subtype C were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. Viral replication capacities varied significantly with respect to the specific HLA-B alleles expressed by the patient, and protective HLA-B alleles, most notably HLA-B81, were associated with lower replication capacities. HLA-associated mutations at low-entropy sites, especially the HLA-B81-associated 186S mutation in the TL9 epitope, were associated with lower replication capacities. Most mutations linked to alterations in replication capacity in the conserved p24 region decreased replication capacity, while most in the highly variable p17 region increased replication capacity. Replication capacity also correlated positively with baseline viral load and negatively with baseline CD4 count but did not correlate with the subsequent rate of CD4 decline. In conclusion, there is evidence that protective HLA alleles, in particular HLA-B81, significantly influence Gag-protease function by driving sequence changes in Gag and that conserved regions of Gag should be included in a vaccine aiming to drive HIV-1 toward a less fit state. However, the long-term clinical benefit of immune-driven fitness costs is uncertain given the lack of correlation with longitudinal markers of disease progression.

HLA-associated immune escape pathways in HIV-1 subtype B Gag, Pol and Nef proteins.

BACKGROUND: Despite the extensive genetic diversity of HIV-1, viral evolution in response to immune selective pressures follows broadly predictable mutational patterns. Sites and pathways of Human Leukocyte-Antigen (HLA)-associated polymorphisms in HIV-1 have been identified through the analysis of population-level data, but the full extent of immune escape pathways remains incompletely characterized. Here, in the largest analysis of HIV-1 subtype B sequences undertaken to date, we identify HLA-associated polymorphisms in the three HIV-1 proteins most commonly considered in cellular-based vaccine strategies. Results are organized into protein-wide escape maps illustrating the sites and pathways of HLA-driven viral evolution. METHODOLOGY/PRINCIPAL FINDINGS: HLA-associated polymorphisms were identified in HIV-1 Gag, Pol and Nef in a multicenter cohort of >1500 chronically subtype-B infected, treatment-naïve individuals from established cohorts in Canada, the USA and Western Australia. At q< or =0.05, 282 codons commonly mutating under HLA-associated immune pressures were identified in these three proteins. The greatest density of associations was observed in Nef (where close to 40% of codons exhibited a significant HLA association), followed by Gag then Pol (where approximately 15-20% of codons exhibited HLA associations), confirming the extensive impact of immune selection on HIV evolution and diversity. Analysis of HIV codon covariation patterns identified over 2000 codon-codon interactions at q< or =0.05, illustrating the dense and complex networks of linked escape and secondary/compensatory mutations. CONCLUSIONS/SIGNIFICANCE: The immune escape maps and associated data are intended to serve as a user-friendly guide to the locations of common escape mutations and covarying codons in HIV-1 subtype B, and as a resource facilitating the systematic identification and classification of immune escape mutations. These resources should facilitate research in HIV epitope discovery and host-pathogen co-evolution, and are relevant to the continued search for an effective CTL-based AIDS vaccine.