Neoantigen Landscape Supports Feasibility of Personalized Cancer Vaccine for Follicular Lymphoma.

Personalized cancer vaccines designed to target neoantigens represent a promising new treatment paradigm in oncology. In contrast to classical idiotype vaccines, we hypothesized that 'polyvalent' vaccines could be engineered for the personalized treatment of follicular lymphoma (FL) using neoantigen discovery by combined whole exome sequencing (WES) and RNA sequencing (RNA-Seq). Fifty-eight tumor samples from 57 patients with FL underwent WES and RNA-Seq. Somatic and B-cell clonotype neoantigens were predicted and filtered to identify high-quality neoantigens. B-cell clonality was determined by alignment of B-cell receptor (BCR) CDR3 regions from RNA-Seq data, grouping at the protein level, and comparison to the BCR repertoire from healthy individuals using RNA-Seq data. An average of 52 somatic mutations per patient (range: 2-172) were identified, and two or more (median: 15) high-quality neoantigens were predicted for 56 of 58 FL samples. The predicted neoantigen peptides were composed of missense mutations (77%), indels (9%), gene fusions (3%), and BCR sequences (11%). Building off of these preclinical analyses, we initiated a pilot clinical trial using personalized neoantigen vaccination combined with PD-1 blockade in patients with relapsed or refractory FL (#NCT03121677). Synthetic long peptide (SLP) vaccines targeting predicted high-quality neoantigens were successfully synthesized for and administered to all four patients enrolled. Initial results demonstrate feasibility, safety, and potential immunologic and clinical responses. Our study suggests that a genomics-driven personalized cancer vaccine strategy is feasible for patients with FL, and this may overcome prior challenges in the field.

Ultra-Deep Sequencing Reveals the Mutational Landscape of Classical Hodgkin Lymphoma.

The malignant Hodgkin and Reed Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) are scarce in affected lymph nodes, creating a challenge to detect driver somatic mutations. As an alternative to cell purification techniques, we hypothesized that ultra-deep exome sequencing would allow genomic study of HRS cells, thereby streamlining analysis and avoiding technical pitfalls. To test this, 31 cHL tumor/normal pairs were exome sequenced to approximately 1,000× median depth of coverage. An orthogonal error-corrected sequencing approach verified >95% of the discovered mutations. We identified mutations in genes novel to cHL including: CDH5 and PCDH7, novel stop gain mutations in IL4R, and a novel pattern of recurrent mutations in pathways regulating Hippo signaling. As a further application of our exome sequencing, we attempted to identify expressed somatic single-nucleotide variants (SNV) in single-nuclei RNA sequencing (snRNA-seq) data generated from a patient in our cohort. Our snRNA analysis identified a clear cluster of cells containing a somatic SNV identified in our deep exome data. This cluster has differentially expressed genes that are consistent with genes known to be dysregulated in HRS cells (e.g., PIM1 and PIM3). The cluster also contains cells with an expanded B-cell clonotype further supporting a malignant phenotype. This study provides proof-of-principle that ultra-deep exome sequencing can be utilized to identify recurrent mutations in HRS cells and demonstrates the feasibility of snRNA-seq in the context of cHL. These studies provide the foundation for the further analysis of genomic variants in large cohorts of patients with cHL. SIGNIFICANCE: Our data demonstrate the utility of ultra-deep exome sequencing in uncovering somatic variants in Hodgkin lymphoma, creating new opportunities to define the genes that are recurrently mutated in this disease. We also show for the first time the successful application of snRNA-seq in Hodgkin lymphoma and describe the expression profile of a putative cluster of HRS cells in a single patient.

Mutations associated with progression in follicular lymphoma predict inferior outcomes at diagnosis: Alliance A151303.

Follicular lymphoma (FL) is clinically heterogeneous, with select patients tolerating extended watch-and-wait, whereas others require prompt treatment, suffer progression of disease within 24 months of treatment (POD24), and/or experience aggressive histologic transformation (t-FL). Because our understanding of the relationship between genetic alterations in FL and patient outcomes remains limited, we conducted a clinicogenomic analysis of 370 patients with FL or t-FL (from Cancer and Leukemia Group B/Alliance trials 50402/50701/50803, or real-world cohorts from Washington University School of Medicine, Cleveland Clinic, or University of Miami). FL subsets by grade, stage, watch-and-wait, or POD24 status did not differ by mutation burden, whereas mutation burden was significantly higher in relapsed/refractory (rel/ref) FL and t-FL than in newly diagnosed (dx) FL. Nonetheless, mutation burden in dx FL was not associated with frontline progression-free survival (PFS). CREBBP was the only gene more commonly mutated in FL than in t-FL yet mutated CREBBP was associated with shorter frontline PFS in FL. Mutations in 20 genes were more common in rel/ref FL or t-FL than in dx FL, including 6 significantly mutated genes (SMGs): STAT6, TP53, IGLL5, B2M, SOCS1, and MYD88. We defined a mutations associated with progression (MAP) signature as ≥2 mutations in these 7 genes (6 rel/ref FL or t-FL SMGs plus CREBBP). Patients with dx FL possessing a MAP signature had shorter frontline PFS, revealing a 7-gene set offering insight into FL progression risk potentially more generalizable than the m7-Follicular Lymphoma International Prognostic Index (m7-FLIPI), which had modest prognostic value in our cohort. Future studies are warranted to validate the poor prognosis associated with a MAP signature in dx FL, potentially facilitating novel trials specifically in this high-risk subset of patients.

Phase 1/dose expansion trial of brentuximab vedotin and lenalidomide in relapsed or refractory diffuse large B-cell lymphoma.

New therapies are needed for patients with relapsed/refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL) who do not benefit from or are ineligible for stem cell transplant and chimeric antigen receptor therapy. The CD30-targeted, antibody-drug conjugate brentuximab vedotin (BV) and the immunomodulator lenalidomide (Len) have demonstrated promising activity as single agents in this population. We report the results of a phase 1/dose expansion trial evaluating the combination of BV/Len in rel/ref DLBCL. Thirty-seven patients received BV every 21 days, with Len administered continuously for a maximum of 16 cycles. The maximum tolerated dose of the combination was 1.2 mg/kg BV with 20 mg/d Len. BV/Len was well tolerated with a toxicity profile consistent with their use as single agents. Most patients required granulocyte colony-stimulating factor support because of neutropenia. The overall response rate was 57% (95% CI, 39.6-72.5), complete response rate, 35% (95% CI, 20.7-52.6); median duration of response, 13.1 months; median progression-free survival, 10.2 months (95% CI, 5.5-13.7); and median overall survival, 14.3 months (95% CI, 10.2-35.6). Response rates were highest in patients with CD30+ DLBCL (73%), but they did not differ according to cell of origin (P = .96). NK cell expansion and phenotypic changes in CD8+ T-cell subsets in nonresponders were identified by mass cytometry. BV/Len represents a potential treatment option for patients with rel/ref DLBCL. This combination is being further explored in a phase 3 study (registered on https://clinicaltrials.org as NCT04404283). This trial was registered on https://clinicaltrials.gov as NCT02086604.

Genetic Ancestry Correlations with Driver Mutations Suggest Complex Interactions between Somatic and Germline Variation in Cancer.

Carrot-Zhang and colleagues describe associations between Native American ancestry and the somatic mutational landscape in lung cancer, including tumor mutation burden and specific driver mutations in EGFR, KRAS, and STK11. Local ancestry analysis suggests that specific germline loci, and not environment, underlie these associations.See related article by Carrot-Zhang et al., p. 591.

Standard operating procedure for somatic variant refinement of sequencing data with paired tumor and normal samples.

PURPOSE: Following automated variant calling, manual review of aligned read sequences is required to identify a high-quality list of somatic variants. Despite widespread use in analyzing sequence data, methods to standardize manual review have not been described, resulting in high inter- and intralab variability. METHODS: This manual review standard operating procedure (SOP) consists of methods to annotate variants with four different calls and 19 tags. The calls indicate a reviewer's confidence in each variant and the tags indicate commonly observed sequencing patterns and artifacts that inform the manual review call. Four individuals were asked to classify variants prior to, and after, reading the SOP and accuracy was assessed by comparing reviewer calls with orthogonal validation sequencing. RESULTS: After reading the SOP, average accuracy in somatic variant identification increased by 16.7% (p value = 0.0298) and average interreviewer agreement increased by 12.7% (p value < 0.001). Manual review conducted after reading the SOP did not significantly increase reviewer time. CONCLUSION: This SOP supports and enhances manual somatic variant detection by improving reviewer accuracy while reducing the interreviewer variability for variant calling and annotation.

Single-agent ibrutinib in relapsed or refractory follicular lymphoma: a phase 2 consortium trial.

Most patients with follicular lymphoma (FL) experience multiple relapses necessitating subsequent lines of therapy. Ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor approved for the treatment of several B-cell malignancies, showed promising activity in FL in a phase 1 study. We report the results of a phase 2 trial evaluating ibrutinib in recurrent FL. Forty patients with recurrent FL were treated with ibrutinib 560 mg/d until progression or intolerance. The primary end point was overall response rate (ORR). Exploratory analyses included correlations of outcome with recurrent mutations identified in a cancer gene panel that used next-generation sequencing in pretreatment biopsies from 31 patients and results of early interim positron emission tomography/computed tomography scans in 20 patients. ORR was 37.5% with a complete response rate of 12.5%, median progression-free survival (PFS) of 14 months, and 2-year PFS of 20.4%. Response rates were significantly higher among patients whose disease was sensitive to rituximab (52.6%) compared with those who were rituximab refractory (16.7%) (P = .04). CARD11 mutations were present in 16% of patients (5 of 31) and predicted resistance to ibrutinib with only wild-type patients responding (P = .002). Maximum standardized uptake value at cycle 1 day 8 correlated with response and PFS. Ibrutinib was well-tolerated with a toxicity profile similar to labeled indications. Ibrutinib is a well-tolerated treatment with modest activity in relapsed FL. Evaluation of BTK inhibitors in earlier lines of therapy may be warranted on the basis of improved response rates in rituximab-sensitive disease. Somatic mutations such as CARD11 may have an impact on response to ibrutinib, may inform clinical decisions, and should be evaluated in larger data sets. This trial was registered at www.clinicaltrials.gov as #NCT01849263.

Corrigendum: 1000 Genomes-based meta-analysis identifies 10 novel loci for kidney function.

This corrects the article DOI: 10.1038/srep45040.

1000 Genomes-based meta-analysis identifies 10 novel loci for kidney function.

HapMap imputed genome-wide association studies (GWAS) have revealed >50 loci at which common variants with minor allele frequency >5% are associated with kidney function. GWAS using more complete reference sets for imputation, such as those from The 1000 Genomes project, promise to identify novel loci that have been missed by previous efforts. To investigate the value of such a more complete variant catalog, we conducted a GWAS meta-analysis of kidney function based on the estimated glomerular filtration rate (eGFR) in 110,517 European ancestry participants using 1000 Genomes imputed data. We identified 10 novel loci with p-value < 5 × 10-8 previously missed by HapMap-based GWAS. Six of these loci (HOXD8, ARL15, PIK3R1, EYA4, ASTN2, and EPB41L3) are tagged by common SNPs unique to the 1000 Genomes reference panel. Using pathway analysis, we identified 39 significant (FDR < 0.05) genes and 127 significantly (FDR < 0.05) enriched gene sets, which were missed by our previous analyses. Among those, the 10 identified novel genes are part of pathways of kidney development, carbohydrate metabolism, cardiac septum development and glucose metabolism. These results highlight the utility of re-imputing from denser reference panels, until whole-genome sequencing becomes feasible in large samples.

Relación entre la presión de pulso y la enfermedad cerebrovascular aterotrombótica / Relation between pulse pressure and atherothrombotic cerebrovascular disease

Introducción: la presión de pulso es un importante marcador y/o predictor de riesgo de complicaciones ateroscleróticas agudas, donde está incluida la enfermedad cerebrovascular. Objetivos: determinar la relación entre la presión de pulso y la enfermedad cerebrovascular aterotrombótica en pacientes hipertensos y no hipertensos. Métodos: estudio explicativo, analítico, caso/control. El grupo de casos fue de 226 pacientes con diagnóstico de enfermedad cerebrovascular aterotrombótica, ingresados en la Sala de Ictus del Hospital General Docente Enrique Cabrera entre 2014-2016 y el grupo control de 226 sin enfermedad cerebrovascular. Se formaron dos grupos, uno con presión de pulso 60 mmhg y otro con presión de pulso < 60 mmHg en pacientes hipertensos y no hipertensos. Resultados: la media de la edad fue de 69,65 años y el 61,45 por ciento eran masculinos; la frecuencia de hipertensión arterial fue de 70,4 por ciento y la de fumadores, 35,4 por ciento y con PP≥ 60 mmHg, 62,0 por ciento. La asociación de la presión de pulso 60 mmHg con enfermedad cerebrovascular aterotrombótica en pacientes hipertensos resultó significativa con odds-ratio 4,72, Intervalo de Confianza 95 por ciento(2,79-7,98) y en pacientes no hipertensos también resultó significativa con odds-ratio 6,86 Intervalo de Confianza 95 por ciento (3,33-14,07). El riesgo atribuible en expuestos fue de 88,6 por ciento y el riesgo atribuible en la población de 50,4 por ciento. Conclusiones: la presión de pulso se asoció de forma significativa a la enfermedad cerebrovascular aterotrombótica en pacientes hipertensos y no hipertensos(AU)

Introduction: Pulse pressure is an important marker or predictor of risk for acute atherosclerotic complications, including cerebrovascular disease. Objectives: To determine the relationship between pulse pressure and atherothrombotic cerebrovascular disease in hypertensive and nonhypertensive patients. Methods: Explanatory, analytical, case control study. The case group consisted of 226 patients diagnosed with atherothrombotic cerebrovascular disease admitted to the Ictus Room at Enrique Cabrera General Teaching Hospital between 2014 and 2016, and the control group consisted of 226 patients without cerebrovascular disease. Two groups were formed, one with a pulse pressure higher than or equal to 60 mmHg and one with a pulse pressure under 60 mmHg in hypertensive and nonhypertensive patients. Results: Mean age was 69.65 years and 61.45 percent were male. The frequency of hypertension was 70.4 percent and that of smokers was 35.4 percent, and with PP≥60 mmHg, which represented 62.0 percent. The association of pulse pressure higher than or equal to 60 mmHg with atherothrombotic cerebrovascular disease in hypertensive patients was significant with odds ratio of 4.72, and confidence interval of 95 percent (2.79-7.98), while in nonhypertensive patients it was also significant with odd ratios of 6.86, and confidence interval of 95 percent CI (3.33-14.07). The attributable risk in exposed people was 88.6 percent and the attributable risk in the population was 50.4 percent. Conclusions: Pulse pressure was significantly associated with atherothrombotic cerebrovascular disease in both hypertensive and nonhypertensive patients(AU)

Relación entre la presión de pulso y la enfermedad cerebrovascular aterotrombótica / Relation between pulse pressure and atherothrombotic cerebrovascular disease

Introducción: la presión de pulso es un importante marcador y/o predictor de riesgo de complicaciones ateroscleróticas agudas, donde está incluida la enfermedad cerebrovascular. Objetivos: determinar la relación entre la presión de pulso y la enfermedad cerebrovascular aterotrombótica en pacientes hipertensos y no hipertensos. Métodos: estudio explicativo, analítico, caso/control. El grupo de casos fue de 226 pacientes con diagnóstico de enfermedad cerebrovascular aterotrombótica, ingresados en la Sala de Ictus del Hospital General Docente Enrique Cabrera entre 2014-2016 y el grupo control de 226 sin enfermedad cerebrovascular. Se formaron dos grupos, uno con presión de pulso 60 mmhg y otro con presión de pulso < 60 mmHg en pacientes hipertensos y no hipertensos. Resultados: la media de la edad fue de 69,65 años y el 61,45 por ciento eran masculinos; la frecuencia de hipertensión arterial fue de 70,4 por ciento y la de fumadores, 35,4 por ciento y con PP≥ 60 mmHg, 62,0 por ciento. La asociación de la presión de pulso 60 mmHg con enfermedad cerebrovascular aterotrombótica en pacientes hipertensos resultó significativa con odds-ratio 4,72, Intervalo de Confianza 95 por ciento(2,79-7,98) y en pacientes no hipertensos también resultó significativa con odds-ratio 6,86 Intervalo de Confianza 95 por ciento (3,33-14,07). El riesgo atribuible en expuestos fue de 88,6 por ciento y el riesgo atribuible en la población de 50,4 por ciento. Conclusiones: la presión de pulso se asoció de forma significativa a la enfermedad cerebrovascular aterotrombótica en pacientes hipertensos y no hipertensos(AU)

Introduction: Pulse pressure is an important marker or predictor of risk for acute atherosclerotic complications, including cerebrovascular disease. Objectives: To determine the relationship between pulse pressure and atherothrombotic cerebrovascular disease in hypertensive and nonhypertensive patients. Methods: Explanatory, analytical, case control study. The case group consisted of 226 patients diagnosed with atherothrombotic cerebrovascular disease admitted to the Ictus Room at Enrique Cabrera General Teaching Hospital between 2014 and 2016, and the control group consisted of 226 patients without cerebrovascular disease. Two groups were formed, one with a pulse pressure higher than or equal to 60 mmHg and one with a pulse pressure under 60 mmHg in hypertensive and nonhypertensive patients. Results: Mean age was 69.65 years and 61.45 percent were male. The frequency of hypertension was 70.4 percent and that of smokers was 35.4 percent, and with PP≥60 mmHg, which represented 62.0 percent. The association of pulse pressure higher than or equal to 60 mmHg with atherothrombotic cerebrovascular disease in hypertensive patients was significant with odds ratio of 4.72, and confidence interval of 95 percent (2.79-7.98), while in nonhypertensive patients it was also significant with odd ratios of 6.86, and confidence interval of 95 percent CI (3.33-14.07). The attributable risk in exposed people was 88.6 percent and the attributable risk in the population was 50.4 percent. Conclusions: Pulse pressure was significantly associated with atherothrombotic cerebrovascular disease in both hypertensive and nonhypertensive patients(AU)

Recurrent somatic mutations affecting B-cell receptor signaling pathway genes in follicular lymphoma.

Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma, yet it remains only partially characterized at the genomic level. To improve our understanding of the genetic underpinnings of this incurable and clinically heterogeneous disease, whole-exome sequencing was performed on tumor/normal pairs from a discovery cohort of 24 patients with FL. Using these data and mutations identified in other B-cell malignancies, 1716 genes were sequenced in 113 FL tumor samples from 105 primarily treatment-naive individuals. We identified 39 genes that were mutated significantly above background mutation rates. CREBBP mutations were associated with inferior PFS. In contrast, mutations in previously unreported HVCN1, a voltage-gated proton channel-encoding gene and B-cell receptor signaling modulator, were associated with improved PFS. In total, 47 (44.8%) patients harbor mutations in the interconnected B-cell receptor (BCR) and CXCR4 signaling pathways. Histone gene mutations were more frequent than previously reported (identified in 43.8% of patients) and often co-occurred (17.1% of patients). A novel, recurrent hotspot was identified at a posttranslationally modified residue in the histone H2B family. This study expands the number of mutated genes described in several known signaling pathways and complexes involved in lymphoma pathogenesis (BCR, Notch, SWitch/sucrose nonfermentable (SWI/SNF), vacuolar ATPases) and identified novel recurrent mutations (EGR1/2, POU2AF1, BTK, ZNF608, HVCN1) that require further investigation in the context of FL biology, prognosis, and treatment.

Parent-of-Origin Effects of the APOB Gene on Adiposity in Young Adults.

Loci identified in genome-wide association studies (GWAS) of cardio-metabolic traits account for a small proportion of the traits' heritability. To date, most association studies have not considered parent-of-origin effects (POEs). Here we report investigation of POEs on adiposity and glycemic traits in young adults. The Jerusalem Perinatal Family Follow-Up Study (JPS), comprising 1250 young adults and their mothers was used for discovery. Focusing on 18 genes identified by previous GWAS as associated with cardio-metabolic traits, we used linear regression to examine the associations of maternally- and paternally-derived offspring minor alleles with body mass index (BMI), waist circumference (WC), fasting glucose and insulin. We replicated and meta-analyzed JPS findings in individuals of European ancestry aged ≤50 belonging to pedigrees from the Framingham Heart Study, Family Heart Study and Erasmus Rucphen Family study (total N≅4800). We considered p<2.7x10-4 statistically significant to account for multiple testing. We identified a common coding variant in the 4th exon of APOB (rs1367117) with a significant maternally-derived effect on BMI (ß = 0.8; 95%CI:0.4,1.1; p = 3.1x10-5) and WC (ß = 2.7; 95%CI:1.7,3.7; p = 2.1x10-7). The corresponding paternally-derived effects were non-significant (p>0.6). Suggestive maternally-derived associations of rs1367117 were observed with fasting glucose (ß = 0.9; 95%CI:0.3,1.5; p = 4.0x10-3) and insulin (ln-transformed, ß = 0.06; 95%CI:0.03,0.1; p = 7.4x10-4). Bioinformatic annotation for rs1367117 revealed a variety of regulatory functions in this region in liver and adipose tissues and a 50% methylation pattern in liver only, consistent with allelic-specific methylation, which may indicate tissue-specific POE. Our findings demonstrate a maternal-specific association between a common APOB variant and adiposity, an association that was not previously detected in GWAS. These results provide evidence for the role of regulatory mechanisms, POEs specifically, in adiposity. In addition this study highlights the benefit of utilizing family studies for deciphering the genetic architecture of complex traits.

Admixture mapping of coronary artery calcification in African Americans from the NHLBI family heart study.

BACKGROUND: Coronary artery calcification (CAC) is an imaging biomarker of coronary atherosclerosis. In European Americans, genome-wide association studies (GWAS) have identified several regions associated with coronary artery disease. However, few large studies have been conducted in African Americans. The largest meta-analysis of CAC in African Americans failed to identify genome-wide significant variants despite being powered to detect effects comparable to effects identified in European Americans. Because CAC is different in prevalence and severity in African Americans and European Americans, admixture mapping is a useful approach to identify loci missed by GWAS. RESULTS: We applied admixture mapping to the African American cohort of the Family Heart Study and identified one genome-wide significant region on chromosome 12 and three potential regions on chromosomes 6, 15, and 19 that are associated with CAC. Follow-up studies using previously reported GWAS meta-analysis data suggest that the regions identified on chromosome 6 and 15 contain variants that are possibly associated with CAC. The associated region on chromosome 6 contains the gene for BMP-6, which is expressed in vascular calcific lesions. CONCLUSIONS: Our results suggest that admixture mapping can be a useful hypothesis-generating tool to identify genomic regions that contribute to complex diseases in genetically admixed populations.

Selection of models for the analysis of risk-factor trees: leveraging biological knowledge to mine large sets of risk factors with application to microbiome data.

MOTIVATION: Establishment of a statistical association between microbiome features and clinical outcomes is of growing interest because of the potential for yielding insights into biological mechanisms and pathogenesis. Extracting microbiome features that are relevant for a disease is challenging and existing variable selection methods are limited due to large number of risk factor variables from microbiome sequence data and their complex biological structure. RESULTS: We propose a tree-based scanning method, Selection of Models for the Analysis of Risk factor Trees (referred to as SMART-scan), for identifying taxonomic groups that are associated with a disease or trait. SMART-scan is a model selection technique that uses a predefined taxonomy to organize the large pool of possible predictors into optimized groups, and hierarchically searches and determines variable groups for association test. We investigate the statistical properties of SMART-scan through simulations, in comparison to a regular single-variable analysis and three commonly-used variable selection methods, stepwise regression, least absolute shrinkage and selection operator (LASSO) and classification and regression tree (CART). When there are taxonomic group effects in the data, SMART-scan can significantly increase power by using bacterial taxonomic information to split large numbers of variables into groups. Through an application to microbiome data from a vervet monkey diet experiment, we demonstrate that SMART-scan can identify important phenotype-associated taxonomic features missed by single-variable analysis, stepwise regression, LASSO and CART.

Novel approach identifies SNPs in SLC2A10 and KCNK9 with evidence for parent-of-origin effect on body mass index.

The phenotypic effect of some single nucleotide polymorphisms (SNPs) depends on their parental origin. We present a novel approach to detect parent-of-origin effects (POEs) in genome-wide genotype data of unrelated individuals. The method exploits increased phenotypic variance in the heterozygous genotype group relative to the homozygous groups. We applied the method to >56,000 unrelated individuals to search for POEs influencing body mass index (BMI). Six lead SNPs were carried forward for replication in five family-based studies (of ∼4,000 trios). Two SNPs replicated: the paternal rs2471083-C allele (located near the imprinted KCNK9 gene) and the paternal rs3091869-T allele (located near the SLC2A10 gene) increased BMI equally (beta = 0.11 (SD), P<0.0027) compared to the respective maternal alleles. Real-time PCR experiments of lymphoblastoid cell lines from the CEPH families showed that expression of both genes was dependent on parental origin of the SNPs alleles (P<0.01). Our scheme opens new opportunities to exploit GWAS data of unrelated individuals to identify POEs and demonstrates that they play an important role in adult obesity.

Genetic variation and adaptation in Africa: implications for human evolution and disease.

Because modern humans originated in Africa and have adapted to diverse environments, African populations have high levels of genetic and phenotypic diversity. Thus, genomic studies of diverse African ethnic groups are essential for understanding human evolutionary history and how this leads to differential disease risk in all humans. Comparative studies of genetic diversity within and between African ethnic groups creates an opportunity to reconstruct some of the earliest events in human population history and are useful for identifying patterns of genetic variation that have been influenced by recent natural selection. Here we describe what is currently known about genetic variation and evolutionary history of diverse African ethnic groups. We also describe examples of recent natural selection in African genomes and how these data are informative for understanding the frequency of many genetic traits, including those that cause disease susceptibility in African populations and populations of recent African descent.

Identifying Darwinian selection acting on different human APOL1 variants among diverse African populations.

Disease susceptibility can arise as a consequence of adaptation to infectious disease. Recent findings have suggested that higher rates of chronic kidney disease (CKD) in individuals with recent African ancestry might be attributed to two risk alleles (G1 and G2) at the serum-resistance-associated (SRA)-interacting-domain-encoding region of APOL1. These two alleles appear to have arisen adaptively, possibly as a result of their protective effects against human African trypanosomiasis (HAT), or African sleeping sickness. In order to explore the distribution of potential functional variation at APOL1, we studied nucleotide variation in 187 individuals across ten geographically and genetically diverse African ethnic groups with exposure to two Trypanosoma brucei subspecies that cause HAT. We observed unusually high levels of nonsynonymous polymorphism in the regions encoding the functional domains that are required for lysing parasites. Whereas allele frequencies of G2 were similar across all populations (3%-8%), the G1 allele was only common in the Yoruba (39%). Additionally, we identified a haplotype (termed G3) that contains a nonsynonymous change at the membrane-addressing-domain-encoding region of APOL1 and is present in all populations except for the Yoruba. Analyses of long-range patterns of linkage disequilibrium indicate evidence of recent selection acting on the G3 haplotype in Fulani from Cameroon. Our results indicate that the G1 and G2 variants in APOL1 are geographically restricted and that there might be other functional variants that could play a role in HAT resistance and CKD risk in African populations.

Patterns of nucleotide and haplotype diversity at ICAM-1 across global human populations with varying levels of malaria exposure.

Malaria is one of the strongest selective pressures in recent human evolution. African populations have been and continue to be at risk for malarial infections. However, few studies have re-sequenced malaria susceptibility loci across geographically and genetically diverse groups in Africa. We examined nucleotide diversity at Intercellular adhesion molecule-1 (ICAM-1), a malaria susceptibility candidate locus, in a number of human populations with a specific focus on diverse African ethnic groups. We used tests of neutrality to assess whether natural selection has impacted this locus and tested whether SNP variation at ICAM-1 is correlated with malaria endemicity. We observe differing patterns of nucleotide and haplotype variation in global populations and higher levels of diversity in Africa. Although we do not observe a deviation from neutrality based on the allele frequency distribution, we do observe several alleles at ICAM-1, including the ICAM-1 (Kilifi) allele, that are correlated with malaria endemicity. We show that the ICAM-1 (Kilifi) allele, which is common in Africa and Asia, exists on distinct haplotype backgrounds and is likely to have arisen more recently in Asia. Our results suggest that correlation analyses of allele frequencies and malaria endemicity may be useful for identifying candidate functional variants that play a role in malaria resistance and susceptibility.

Gene-centric meta-analysis of lipid traits in African, East Asian and Hispanic populations.

Meta-analyses of European populations has successfully identified genetic variants in over 100 loci associated with lipid levels, but our knowledge in other ethnicities remains limited. To address this, we performed dense genotyping of ∼2,000 candidate genes in 7,657 African Americans, 1,315 Hispanics and 841 East Asians, using the IBC array, a custom ∼50,000 SNP genotyping array. Meta-analyses confirmed 16 lipid loci previously established in European populations at genome-wide significance level, and found multiple independent association signals within these lipid loci. Initial discovery and in silico follow-up in 7,000 additional African American samples, confirmed two novel loci: rs5030359 within ICAM1 is associated with total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) (p = 8.8×10(-7) and p = 1.5×10(-6) respectively) and a nonsense mutation rs3211938 within CD36 is associated with high-density lipoprotein cholesterol (HDL-C) levels (p = 13.5×10(-12)). The rs3211938-G allele, which is nearly absent in European and Asian populations, has been previously found to be associated with CD36 deficiency and shows a signature of selection in Africans and African Americans. Finally, we have evaluated the effect of SNPs established in European populations on lipid levels in multi-ethnic populations and show that most known lipid association signals span across ethnicities. However, differences between populations, especially differences in allele frequency, can be leveraged to identify novel signals, as shown by the discovery of ICAM1 and CD36 in the current report.