Safety, tolerability, immunogenicity, and efficacy of UB-311 in participants with mild Alzheimer's disease: a randomised, double-blind, placebo-controlled, phase 2a study.

BACKGROUND: Anti-amyloid vaccines may offer a convenient, affordable, and accessible means of preventing and treating Alzheimer's disease. UB-311 is an anti-amyloid-ß active immunotherapeutic vaccine shown to be well-tolerated and to have a durable antibody response in a phase 1 trial. This phase 2a study assessed the safety, immunogenicity, and preliminary efficacy of UB-311 in participants with mild Alzheimer's disease. METHODS: A 78-week, randomised, double-blind, placebo-controlled, parallel-group, multicentre, phase 2a study was conducted in Taiwan. Participants were randomised in a 1:1:1 ratio to receive seven intramuscular injections of UB-311 (Q3M arm), or five doses of U311 with two doses of placebo (Q6M arm), or seven doses of placebo (placebo arm). The primary endpoints were safety, tolerability, and immunogenicity of UB-311. Safety was assessed in all participants who received at least one dose of investigational product. This study was registered at ClinicalTrials.gov (NCT02551809). FINDINGS: Between 7 December 2015 and 28 August 2018, 43 participants were randomised. UB-311 was safe, well-tolerated, and generated a robust immune response. The three treatment-emergent adverse events (TEAEs) with the highest incidence were injection-site pain (14 TEAEs in seven [16%] participants), amyloid-related imaging abnormality with microhaemorrhages and haemosiderin deposits (12 TEAEs in six [14%] participants), and diarrhoea (five TEAEs in five [12%] participants). A 97% antibody response rate was observed and maintained at 93% by the end of the study across both UB-311 arms. INTERPRETATION: These results support the continued development of UB-311. FUNDING: Vaxxinity, Inc. (Formerly United Neuroscience Ltd.).

Avoiding future controversies in the Alzheimer's disease space through understanding the aducanumab data and FDA review.

Key points of disagreement between the aducanumab FDA statistical review, which had primarily negative conclusions, and the clinical review, which had primarily positive conclusions, were investigated. Results from secondary endpoints in positive Study 302 were significant and these endpoints provided meaningful additional information. Findings indicate the statistical review of the aducanumab data was incorrect in a number of key areas. Greater placebo decline was not responsible for the significant results in Study 302. Correlations did exist between reduction in ß-amyloid and clinical outcomes. Missing data and functional unblinding did not likely bias results. In contrast, the clinical review went too far in saying the negative results in Study 301 did not detract from the positive results in Study 302, as all clinical data should be considered in the evaluation, and the clinical review accepted the company's explanation for divergence of the results between the studies although much of the divergence remained unexplained. Interestingly, both the statistical review and the clinical review considered the available efficacy evidence despite both studies being terminated early. Implications of these findings include that the divergence in results seen in the two phase 3 aducanumab studies can be expected in other studies with similar design and analysis. Therefore, further research is needed to determine if analysis methods other than MMRM and/or optimized outcomes will provide more consistent results across studies.

Meaningful Clinical Changes in Alzheimer Disease Measured With the iADRS and Illustrated Using the Donanemab TRAILBLAZER-ALZ Study Findings.

Purpose of Review: To provide relevant background of the Integrated Alzheimer's Disease Rating Scale (iADRS), with examples, to assist the reader with the interpretation of iADRS findings from the TRAILBLAZER-ALZ study. Recent Findings: The iADRS is an integrated measure of global Alzheimer disease (AD) severity for use in the clinical trial environment. It provides a single score that captures commonalities across cognitive and functional ability domains, reflecting disease-related impairment, while minimizing noise not related to disease progression that may exist within each domain. In AD, disease-modifying therapies (DMTs) are expected to slow the rate of clinical decline, changing the trajectory of disease progression. The overall percent slowing of disease progression with treatment is a more informative outcome of effect than absolute point differences between treatment and placebo groups at any given time point because the latter is influenced by treatment period and disease severity. The TRAILBLAZER-ALZ trial was a phase 2 study designed to evaluate the safety and efficacy of donanemab in participants with early symptomatic AD; the primary outcome measure was the change from baseline to 76 weeks on the iADRS. In the TRAILBLAZER-ALZ study, donanemab slowed disease progression by 32% at 18 months (p = 0.04 vs placebo), demonstrating clinical efficacy. At the patient level, one can assess whether the DMT effect is clinically meaningful by estimating the threshold of change consistent with clinically meaningful worsening; based on the TRAILBLAZER-ALZ findings, treatment with donanemab would delay reaching this threshold by approximately 6 months. Summary: The iADRS is capable of accurately describing clinical changes associated with disease progression and detecting treatment effects and is an effective assessment tool for use in clinical trials of individuals with early symptomatic AD.

Whole-Genome Sequencing to Predict Antimicrobial Susceptibility Profiles in Neisseria gonorrhoeae.

BACKGROUND: Neisseria gonorrhoeae is a major public health problem due to increasing incidence and antimicrobial resistance. Genetic markers of reduced susceptibility have been identified; the extent to which those are representative of global antimicrobial resistance is unknown. We evaluated the performance of whole-genome sequencing (WGS) used to predict susceptibility to ciprofloxacin and other antimicrobials using a global collection of N. gonorrhoeae isolates. METHODS: Susceptibility testing of common antimicrobials and the recently developed zolifodacin was performed using agar dilution to determine minimum inhibitory concentrations (MICs). We identified resistance alleles at loci known to contribute to antimicrobial resistance in N. gonorrhoeae from WGS data. We tested the ability of each locus to predict antimicrobial susceptibility. RESULTS: A total of 481 N. gonorrhoeae isolates, collected between 2004 and 2019 and making up 457 unique genomes, were sourced from 5 countries. All isolates with demonstrated susceptibility to ciprofloxacin (MIC ≤0.06 µg/mL) had a wild-type gyrA codon 91. Multilocus approaches were needed to predict susceptibility to other antimicrobials. All isolates were susceptible to zoliflodacin, defined by an MIC ≤0.25 µg/mL. CONCLUSIONS: Single marker prediction can be used to inform ciprofloxacin treatment of N. gonorrhoeae infection. A combination of molecular markers may be needed to determine susceptibility for other antimicrobials.

Survival analyses from the CENTAUR trial in amyotrophic lateral sclerosis: Evaluating the impact of treatment crossover on outcomes.

INTRODUCTION/AIMS: Trials incorporating placebo-to-active treatment crossover are encouraged in fatal conditions like amyotrophic lateral sclerosis (ALS) but may underestimate active treatment survival benefit. Here, we apply methods for modeling survival without crossover, including the rank-preserving structural failure time model (RPSFTM), to data from the CENTAUR trial of sodium phenylbutyrate and taurursodiol (PB and TURSO) in ALS incorporating both randomized placebo-controlled and open-label extension (OLE) phases. METHODS: Intent-to-treat (ITT) and RPSFTM survival analyses were performed with final data at a July 2020 cutoff date. Analyses of subgroups based on randomized treatment and OLE phase participation were also performed. RESULTS: Hazard ratios (95% confidence intervals) of death for PB and TURSO versus participants initially on placebo were 0.57 (0.35-0.92) on ITT analysis and 0.39 (0.17-0.88) in the primary on-treatment RPSFTM analysis (p = .023). Median ITT survival duration for PB and TURSO (25.8 mo) was 6.9 mo longer than placebo (18.9 mo) on ITT analysis and 10.6 mo longer than the median RPSFTM-adjusted survival duration for placebo (15.2 mo). Median survival duration was 18.8 mo longer in the PB and TURSO-randomized subgroup who continued into the OLE phase versus the placebo-randomized subgroup who did not continue into the OLE phase (p < .0001), although OLE phase selection bias may have potentially confounded these results. DISCUSSION: Similar to the prespecified ITT analysis, post hoc analyses adjusting for treatment crossover in CENTAUR showed a significant survival benefit for PB and TURSO. Such methods may provide clinical context for observed survival outcomes in future ALS crossover trials.

Effect of sodium phenylbutyrate/taurursodiol on tracheostomy/ventilation-free survival and hospitalisation in amyotrophic lateral sclerosis: long-term results from the CENTAUR trial.

BACKGROUND: Coformulated sodium phenylbutyrate/taurursodiol (PB/TURSO) was shown to prolong survival and slow functional decline in amyotrophic lateral sclerosis (ALS). OBJECTIVE: Determine whether PB/TURSO prolonged tracheostomy/ventilation-free survival and/or reduced first hospitalisation in participants with ALS in the CENTAUR trial. METHODS: Adults with El Escorial Definite ALS ≤18 months from symptom onset were randomised to PB/ TURSO or placebo for 6 months. Those completing randomised treatment could enrol in an open-label extension (OLE) phase and receive PB/TURSO for ≤30 months. Times to the following individual or combined key events were compared in the originally randomised treatment groups over a period spanning trial start through July 2020 (longest postrandomisation follow-up, 35 months): death, tracheostomy, permanent assisted ventilation (PAV) and first hospitalisation. RESULTS: Risk of any key event was 47% lower in those originally randomised to PB/TURSO (n=87) versus placebo (n=48, 71% of whom received delayed-start PB/TURSO in the OLE phase) (HR=0.53; 95% CI 0.35 to 0.81; p=0.003). Risks of death or tracheostomy/PAV (HR=0.51; 95% CI 0.32 to 0.84; p=0.007) and first hospitalisation (HR=0.56; 95% CI 0.34 to 0.95; p=0.03) were also decreased in those originally randomised to PB/TURSO. CONCLUSIONS: Early PB/TURSO prolonged tracheostomy/PAV-free survival and delayed first hospitalisation in ALS. TRIAL REGISTRATION NUMBER: NCT03127514; NCT03488524.

GenoRisk: A polygenic risk score for Alzheimer's disease.

INTRODUCTION: Recent clinical trials are considering inclusion of more than just apolipoprotein E (APOE) ε4 genotype as a way of reducing variability in analysis of outcomes. METHODS: Case-control data were used to compare the capacity of age, sex, and 58 Alzheimer's disease (AD)-associated single nucleotide polymorphisms (SNPs) to predict AD status using several statistical models. Model performance was assessed with Brier scores and tenfold cross-validation. Genotype and sex × age estimates from the best performing model were combined with age and intercept estimates from the general population to develop a personalized genetic risk score, termed age, and sex-adjusted GenoRisk. RESULTS: The elastic net model that included age, age x sex interaction, allelic APOE terms, and 29 additional SNPs performed the best. This model explained an additional 19% of the heritable risk compared to APOE genotype alone and achieved an area under the curve of 0.747. DISCUSSION: GenoRisk could improve the risk assessment of individuals identified for prevention studies.

Perspectives on statistical strategies for the regulatory biomarker qualification process.

Qualification of a biomarker for use in a medical product development program requires a statistical strategy that aligns available evidence with the proposed context of use (COU), identifies any data gaps to be filled and plans any additional research required to support the qualification. Accumulating, interpreting and analyzing available data is outlined, step-by-step, illustrated by a qualified enrichment biomarker example and a safety biomarker in the process of qualification. The detailed steps aid requestors seeking qualification of biomarkers, allowing them to organize the available evidence and identify potential gaps. This provides a statistical perspective for assessing evidence that parallels clinical considerations and is intended to guide the overall evaluation of evidentiary criteria to support a specific biomarker COU.

Value-Generating Exploratory Trials in Neurodegenerative Dementias.

Drug development for Alzheimer disease and other neurodegenerative dementias, including frontotemporal dementia, has experienced a long history of phase 2 and phase 3 clinical trials that failed to show efficacy of investigational drugs. Despite differences in clinical and behavioral characteristics, these disorders have shared pathologies and face common challenges in designing early-phase trials that are predictive of late-stage success. Here, we discuss exploratory clinical trials in neurodegenerative dementias. These are generally phase 1b or phase 2a trials that are designed to assess pharmacologic effects and rely on biomarker outcomes, with shorter treatment durations and fewer patients than traditional phase 2 studies. Exploratory trials can establish go/no-go decision points, support proof of concept and dose selection, and terminate drugs that fail to show target engagement with suitable exposure and acceptable safety profiles. Early failure saves valuable resources including opportunity costs. This is especially important for programs in academia and small biotechnology companies but may be applied to high-risk projects in large pharmaceutical companies to achieve proof of concept more rapidly at lower costs than traditional approaches. Exploratory studies in a staged clinical development program may provide promising data to warrant the substantial resources needed to advance compounds through late-stage development. To optimize the design and application of exploratory trials, the Alzheimer's Drug Discovery Foundation and the Association for Frontotemporal Degeneration convened an advisory panel to provide recommendations on outcome measures and statistical considerations for these types of studies and study designs that can improve efficiency in clinical development.

Long-term survival of participants in the CENTAUR trial of sodium phenylbutyrate-taurursodiol in amyotrophic lateral sclerosis.

An orally administered, fixed-dose coformulation of sodium phenylbutyrate-taurursodiol (PB-TURSO) significantly slowed functional decline in a randomized, placebo-controlled, phase 2 trial in ALS (CENTAUR). Herein we report results of a long-term survival analysis of participants in CENTAUR. In CENTAUR, adults with ALS were randomized 2:1 to PB-TURSO or placebo. Participants completing the 6-month (24-week) randomized phase were eligible to receive PB-TURSO in the open-label extension. An all-cause mortality analysis (35-month maximum follow-up post-randomization) incorporated all randomized participants. Participants and site investigators were blinded to treatment assignments through the duration of follow-up of this analysis. Vital status was obtained for 135 of 137 participants originally randomized in CENTAUR. Median overall survival was 25.0 months among participants originally randomized to PB-TURSO and 18.5 months among those originally randomized to placebo (hazard ratio, 0.56; 95% confidence interval, 0.34-0.92; P = .023). Initiation of PB-TURSO treatment at baseline resulted in a 6.5-month longer median survival as compared with placebo. Combined with results from CENTAUR, these results suggest that PB-TURSO has both functional and survival benefits in ALS.

Trial of Sodium Phenylbutyrate-Taurursodiol for Amyotrophic Lateral Sclerosis.

BACKGROUND: Sodium phenylbutyrate and taurursodiol have been found to reduce neuronal death in experimental models. The efficacy and safety of a combination of the two compounds in persons with amyotrophic lateral sclerosis (ALS) are not known. METHODS: In this multicenter, randomized, double-blind trial, we enrolled participants with definite ALS who had had an onset of symptoms within the previous 18 months. Participants were randomly assigned in a 2:1 ratio to receive sodium phenylbutyrate-taurursodiol (3 g of sodium phenylbutyrate and 1 g of taurursodiol, administered once a day for 3 weeks and then twice a day) or placebo. The primary outcome was the rate of decline in the total score on the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R; range, 0 to 48, with higher scores indicating better function) through 24 weeks. Secondary outcomes were the rates of decline in isometric muscle strength, plasma phosphorylated axonal neurofilament H subunit levels, and the slow vital capacity; the time to death, tracheostomy, or permanent ventilation; and the time to death, tracheostomy, permanent ventilation, or hospitalization. RESULTS: A total of 177 persons with ALS were screened for eligibility, and 137 were randomly assigned to receive sodium phenylbutyrate-taurursodiol (89 participants) or placebo (48 participants). In a modified intention-to-treat analysis, the mean rate of change in the ALSFRS-R score was -1.24 points per month with the active drug and -1.66 points per month with placebo (difference, 0.42 points per month; 95% confidence interval, 0.03 to 0.81; P = 0.03). Secondary outcomes did not differ significantly between the two groups. Adverse events with the active drug were mainly gastrointestinal. CONCLUSIONS: Sodium phenylbutyrate-taurursodiol resulted in slower functional decline than placebo as measured by the ALSFRS-R score over a period of 24 weeks. Secondary outcomes were not significantly different between the two groups. Longer and larger trials are necessary to evaluate the efficacy and safety of sodium phenylbutyrate-taurursodiol in persons with ALS. (Funded by Amylyx Pharmaceuticals and others; CENTAUR ClinicalTrials.gov number, NCT03127514.).

Individualized clinical management of patients at risk for Alzheimer's dementia.

INTRODUCTION: Multidomain intervention for Alzheimer's disease (AD) risk reduction is an emerging therapeutic paradigm. METHODS: Patients were prescribed individually tailored interventions (education/pharmacologic/nonpharmacologic) and rated on compliance. Normal cognition/subjective cognitive decline/preclinical AD was classified as Prevention. Mild cognitive impairment due to AD/mild-AD was classified as Early Treatment. Change from baseline to 18 months on the modified Alzheimer's Prevention Cognitive Composite (primary outcome) was compared against matched historical control cohorts. Cognitive aging composite (CogAging), AD/cardiovascular risk scales, and serum biomarkers were secondary outcomes. RESULTS: One hundred seventy-four were assigned interventions (age 25-86). Higher-compliance Prevention improved more than both historical cohorts (P = .0012, P < .0001). Lower-compliance Prevention also improved more than both historical cohorts (P = .0088, P < .0055). Higher-compliance Early Treatment improved more than lower compliance (P = .0007). Higher-compliance Early Treatment improved more than historical cohorts (P < .0001, P = .0428). Lower-compliance Early Treatment did not differ (P = .9820, P = .1115). Similar effects occurred for CogAging. AD/cardiovascular risk scales and serum biomarkers improved. DISCUSSION: Individualized multidomain interventions may improve cognition and reduce AD/cardiovascular risk scores in patients at-risk for AD dementia.

Cytochrome P450 2D-mediated metabolism is not necessary for tafenoquine and primaquine to eradicate the erythrocytic stages of Plasmodium berghei.

BACKGROUND: Due to the ability of the 8-aminoquinolines (8AQs) to kill different stages of the malaria parasite, primaquine (PQ) and tafenoquine (TQ) are vital for causal prophylaxis and the eradication of erythrocytic Plasmodium sp. parasites. Recognizing the potential role of cytochrome (CYP) 450 2D6 in the metabolism and subsequent hepatic efficacy of 8-aminoquinolines, studies were designed to explore whether CYP2D-mediated metabolism was related to the ability of single-dose PQ and TQ to eliminate the asexual and sexual erythrocytic stages of Plasmodium berghei. METHODS: An IV P. berghei sporozoite murine challenge model was utilized to directly compare causal prophylactic and erythrocytic activity (asexual and sexual parasite stages) dose-response relationships in C57BL/6 wild-type (WT) mice and subsequently compare the erythrocytic activity of PQ and TQ in WT and CYP2D knock-out (KO) mice. RESULTS: Single-dose administration of either 25 mg/kg TQ or 40 mg/kg PQ eradicated the erythrocytic stages (asexual and sexual) of P. berghei in C57BL WT and CYP2D KO mice. In WT animals, the apparent elimination of hepatic infections occurs at lower doses of PQ than are required to eliminate erythrocytic infections. In contrast, the minimally effective dose of TQ needed to achieve causal prophylaxis and to eradicate erythrocytic parasites was analogous. CONCLUSION: The genetic deletion of the CYP2D cluster does not affect the ability of PQ or TQ to eradicate the blood stages (asexual and sexual) of P. berghei after single-dose administration.

Characterization of the murine macrophage response to infection with virulent and avirulent Burkholderia species.

BACKGROUND: Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Gram-negative facultative intracellular pathogens, which are the causative agents of melioidosis and glanders, respectively. Depending on the route of exposure, aerosol or transcutaneous, infection by Bp or Bm can result in an extensive range of disease - from acute to chronic, relapsing illness to fatal septicemia. Both diseases are associated with difficult diagnosis and high fatality rates. About ninety five percent of patients succumb to untreated septicemic infections and the fatality rate is 50 % even when standard antibiotic treatments are administered. RESULTS: The goal of this study is to profile murine macrophage-mediated phenotypic and molecular responses that are characteristic to a collection of Bp, Bm, Burkholderia thailandensis (Bt) and Burkholderia oklahomensis (Bo) strains obtained from humans, animals, environment and geographically diverse locations. Burkholderia spp. (N = 21) were able to invade and replicate in macrophages, albeit to varying degrees. All Bp (N = 9) and four Bm strains were able to induce actin polymerization on the bacterial surface following infection. Several Bp and Bm strains showed reduced ability to induce multinucleated giant cell (MNGC) formation, while Bo and Bp 776 were unable to induce this phenotype. Measurement of host cytokine responses revealed a statistically significant Bm mediated IL-6 and IL-10 production compared to Bp strains. Hierarchical clustering of transcriptional data from 84 mouse cytokines, chemokines and their corresponding receptors identified 29 host genes as indicators of differential responses between the Burkholderia spp. Further validation confirmed Bm mediated Il-1b, Il-10, Tnfrsf1b and Il-36a mRNA expressions were significantly higher when compared to Bp and Bt. CONCLUSIONS: These results characterize the phenotypic and immunological differences in the host innate response to pathogenic and avirulent Burkholderia strains and provide insight into the phenotypic alterations and molecular targets underlying host-Burkholderia interactions.

Toll-like receptor agonist augments virus-like particle-mediated protection from Ebola virus with transient immune activation.

Identifying safe and effective adjuvants is critical for the advanced development of protein-based vaccines. Pattern recognition receptor (PRR) agonists are increasingly being explored as potential adjuvants, but there is concern that the efficacy of these molecules may be dependent on potentially dangerous levels of non-specific immune activation. The filovirus virus-like particle (VLP) vaccine protects mice, guinea pigs, and nonhuman primates from viral challenge. In this study, we explored the impact of a stabilized dsRNA mimic, polyICLC, on VLP vaccination of C57BL/6 mice and Hartley guinea pigs. We show that at dose levels as low as 100 ng, the adjuvant increased the efficacy of the vaccine in mice. Antigen-specific, polyfunctional CD4 and CD8 T cell responses and antibody responses increased significantly upon inclusion of adjuvant. To determine whether the efficacy of polyICLC correlated with systemic immune activation, we examined serum cytokine levels and cellular activation in the draining lymph node. PolyICLC administration was associated with increases in TNFα, IL6, MCP1, MIP1α, KC, and MIP1ß levels in the periphery and with the activation of dendritic cells (DCs), NK cells, and B cells. However, this activation resolved within 24 to 72 hours at efficacious adjuvant dose levels. These studies are the first to examine the polyICLC-induced enhancement of antigen-specific immune responses in the context of non-specific immune activation, and they provide a framework from which to consider adjuvant dose levels.

Leveraging prior information to detect causal variants via multi-variant regression.

Although many methods are available to test sequence variants for association with complex diseases and traits, methods that specifically seek to identify causal variants are less developed. Here we develop and evaluate a Bayesian hierarchical regression method that incorporates prior information on the likelihood of variant causality through weighting of variant effects. By simulation studies using both simulated and real sequence variants, we compared a standard single variant test for analyzing variant-disease association with the proposed method using different weighting schemes. We found that by leveraging linkage disequilibrium of variants with known GWAS signals and sequence conservation (phastCons), the proposed method provides a powerful approach for detecting causal variants while controlling false positives.

Prioritizing genetic variants for causality on the basis of preferential linkage disequilibrium.

To date, the widely used genome-wide association studies (GWASs) of the human genome have reported thousands of variants that are significantly associated with various human traits. However, in the vast majority of these cases, the causal variants responsible for the observed associations remain unknown. In order to facilitate the identification of causal variants, we designed a simple computational method called the "preferential linkage disequilibrium (LD)" approach, which follows the variants discovered by GWASs to pinpoint the causal variants, even if they are rare compared with the discovery variants. The approach is based on the hypothesis that the GWAS-discovered variant is better at tagging the causal variants than are most other variants evaluated in the original GWAS. Applying the preferential LD approach to the GWAS signals of five human traits for which the causal variants are already known, we successfully placed the known causal variants among the top ten candidates in the majority of these cases. Application of this method to additional GWASs, including those of hepatitis C virus treatment response, plasma levels of clotting factors, and late-onset Alzheimer disease, has led to the identification of a number of promising candidate causal variants. This method represents a useful tool for delineating causal variants by bringing together GWAS signals and the rapidly accumulating variant data from next-generation sequencing.

SVA: software for annotating and visualizing sequenced human genomes.

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.

A genome-wide comparison of the functional properties of rare and common genetic variants in humans.

One of the longest running debates in evolutionary biology concerns the kind of genetic variation that is primarily responsible for phenotypic variation in species. Here, we address this question for humans specifically from the perspective of population allele frequency of variants across the complete genome, including both coding and noncoding regions. We establish simple criteria to assess the likelihood that variants are functional based on their genomic locations and then use whole-genome sequence data from 29 subjects of European origin to assess the relationship between the functional properties of variants and their population allele frequencies. We find that for all criteria used to assess the likelihood that a variant is functional, the rarer variants are significantly more likely to be functional than the more common variants. Strikingly, these patterns disappear when we focus on only those variants in which the major alleles are derived. These analyses indicate that the majority of the genetic variation in terms of phenotypic consequence may result from a mutation-selection balance, as opposed to balancing selection, and have direct relevance to the study of human disease.

The characterization of twenty sequenced human genomes.

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.