Showing papers by "Richard K. Wilson published in 2016"

TP53 and Decitabine in Acute Myeloid Leukemia and Myelodysplastic Syndromes

Abstract: BackgroundThe molecular determinants of clinical responses to decitabine therapy in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) are unclear. MethodsWe enrolled 84 adult patients with AML or MDS in a single-institution trial of decitabine to identify somatic mutations and their relationships to clinical responses. Decitabine was administered at a dose of 20 mg per square meter of body-surface area per day for 10 consecutive days in monthly cycles. We performed enhanced exome or gene-panel sequencing in 67 of these patients and serial sequencing at multiple time points to evaluate patterns of mutation clearance in 54 patients. An extension cohort included 32 additional patients who received decitabine in different protocols. ResultsOf the 116 patients, 53 (46%) had bone marrow blast clearance (<5% blasts). Response rates were higher among patients with an unfavorable-risk cytogenetic profile than among patients with an intermediate-risk or favorable-risk cytogenetic profile...

Long-read sequence assembly of the gorilla genome.

Abstract: Accurate sequence and assembly of genomes is a critical first step for studies of genetic variation. We generated a high-quality assembly of the gorilla genome using single-molecule, real-time sequence technology and a string graph de novo assembly algorithm. The new assembly improves contiguity by two to three orders of magnitude with respect to previously released assemblies, recovering 87% of missing reference exons and incomplete gene models. Although regions of large, high-identity segmental duplications remain largely unresolved, this comprehensive assembly provides new biological insight into genetic diversity, structural variation, gene loss, and representation of repeat structures within the gorilla genome. The approach provides a path forward for the routine assembly of mammalian genomes at a level approaching that of the current quality of the human genome.

Genetic alterations in uncommon low-grade neuroepithelial tumors: BRAF, FGFR1, and MYB mutations occur at high frequency and align with morphology

Abstract: Low-grade neuroepithelial tumors (LGNTs) are diverse CNS tumors presenting in children and young adults, often with a history of epilepsy. While the genetic profiles of common LGNTs, such as the pilocytic astrocytoma and ‘adult-type’ diffuse gliomas, are largely established, those of uncommon LGNTs remain to be defined. In this study, we have used massively parallel sequencing and various targeted molecular genetic approaches to study alterations in 91 LGNTs, mostly from children but including young adult patients. These tumors comprise dysembryoplastic neuroepithelial tumors (DNETs; n = 22), diffuse oligodendroglial tumors (d-OTs; n = 20), diffuse astrocytomas (DAs; n = 17), angiocentric gliomas (n = 15), and gangliogliomas (n = 17). Most LGNTs (84 %) analyzed by whole-genome sequencing (WGS) were characterized by a single driver genetic alteration. Alterations of FGFR1 occurred frequently in LGNTs composed of oligodendrocyte-like cells, being present in 82 % of DNETs and 40 % of d-OTs. In contrast, a MYB-QKI fusion characterized almost all angiocentric gliomas (87 %), and MYB fusion genes were the most common genetic alteration in DAs (41 %). A BRAF:p.V600E mutation was present in 35 % of gangliogliomas and 18 % of DAs. Pathogenic alterations in FGFR1/2/3, BRAF, or MYB/MYBL1 occurred in 78 % of the series. Adult-type d-OTs with an IDH1/2 mutation occurred in four adolescents, the youngest aged 15 years at biopsy. Despite a detailed analysis, novel genetic alterations were limited to two fusion genes, EWSR1-PATZ1 and SLMAP-NTRK2, both in gangliogliomas. Alterations in BRAF, FGFR1, or MYB account for most pathogenic alterations in LGNTs, including pilocytic astrocytomas, and alignment of these genetic alterations and cytologic features across LGNTs has diagnostic implications. Additionally, therapeutic options based upon targeting the effects of these alterations are already in clinical trials.

Deregulation of DUX4 and ERG in acute lymphoblastic leukemia

Abstract: Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL). Here we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG is a hallmark of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt uses a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivation domains of ERG, but it inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia in which DUX4 deregulation results in loss of function of ERG, either by deletion or induced expression of an isoform that is a dominant-negative inhibitor of wild-type ERG function.

The genomic landscape of core-binding factor acute myeloid leukemias

Abstract: Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving the core-binding factor (CBF) transcriptional complex To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n = 87) and adult (n = 78) samples, including cases with RUNX1-RUNX1T1 (n = 85) or CBFB-MYH11 (n = 80) rearrangements, by whole-genome or whole-exome sequencing In addition to known mutations in the Ras pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a previously unappreciated cooperating pathway in CBF-AML Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated remarkably different spectra of cooperating mutations, as RUNX1-RUNX1T1 cases harbored recurrent mutations in DHX15 and ZBTB7A, as well as an enrichment of mutations in epigenetic regulators, including ASXL2 and the cohesin complex This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscapes of cooperating mutations for these related AML subtypes

Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly

Abstract: The human reference genome assembly plays a central role in nearly all aspects of today's basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009 and reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that while the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health.

INTEGRATE: gene fusion discovery using whole genome and transcriptome data

Abstract: While next-generation sequencing (NGS) has become the primary technology for discovering gene fusions, we are still faced with the challenge of ensuring that causative mutations are not missed while minimizing false positives. Currently, there are many computational tools that predict structural variations (SV) and gene fusions using whole genome (WGS) and transcriptome sequencing (RNA-seq) data separately. However, as both WGS and RNA-seq have their limitations when used independently, we hypothesize that the orthogonal validation from integrating both data could generate a sensitive and specific approach for detecting high-confidence gene fusion predictions. Fortunately, decreasing NGS costs have resulted in a growing quantity of patients with both data available. Therefore, we developed a gene fusion discovery tool, INTEGRATE, that leverages both RNA-seq and WGS data to reconstruct gene fusion junctions and genomic breakpoints by split-read mapping. To evaluate INTEGRATE, we compared it with eight additional gene fusion discovery tools using the well-characterized breast cell line HCC1395 and peripheral blood lymphocytes derived from the same patient (HCC1395BL). The predictions subsequently underwent a targeted validation leading to the discovery of 131 novel fusions in addition to the seven previously reported fusions. Overall, INTEGRATE only missed six out of the 138 validated fusions and had the highest accuracy of the nine tools evaluated. Additionally, we applied INTEGRATE to 62 breast cancer patients from The Cancer Genome Atlas (TCGA) and found multiple recurrent gene fusions including a subset involving estrogen receptor. Taken together, INTEGRATE is a highly sensitive and accurate tool that is freely available for academic use.

Tumor Evolution in Two Patients with Basal-like Breast Cancer: A Retrospective Genomics Study of Multiple Metastases

Abstract: Background Metastasis is the main cause of cancer patient deaths and remains a poorly characterized process. It is still unclear when in tumor progression the ability to metastasize arises and whether this ability is inherent to the primary tumor or is acquired well after primary tumor formation. Next-generation sequencing and analytical methods to define clonal heterogeneity provide a means for identifying genetic events and the temporal relationships between these events in the primary and metastatic tumors within an individual. Methods and Findings We performed DNA whole genome and mRNA sequencing on two primary tumors, each with either four or five distinct tissue site-specific metastases, from two individuals with triple-negative/basal-like breast cancers. As evidenced by their case histories, each patient had an aggressive disease course with abbreviated survival. In each patient, the overall gene expression signatures, DNA copy number patterns, and somatic mutation patterns were highly similar across each primary tumor and its associated metastases. Almost every mutation found in the primary was found in a metastasis (for the two patients, 52/54 and 75/75). Many of these mutations were found in every tumor (11/54 and 65/75, respectively). In addition, each metastasis had fewer metastatic-specific events and shared at least 50% of its somatic mutation repertoire with the primary tumor, and all samples from each patient grouped together by gene expression clustering analysis. TP53 was the only mutated gene in common between both patients and was present in every tumor in this study. Strikingly, each metastasis resulted from multiclonal seeding instead of from a single cell of origin, and few of the new mutations, present only in the metastases, were expressed in mRNAs. Because of the clinical differences between these two patients and the small sample size of our study, the generalizability of these findings will need to be further examined in larger cohorts of patients. Conclusions Our findings suggest that multiclonal seeding may be common amongst basal-like breast cancers. In these two patients, mutations and DNA copy number changes in the primary tumors appear to have had a biologic impact on metastatic potential, whereas mutations arising in the metastases were much more likely to be passengers.

Pangolin genomes and the evolution of mammalian scales and immunity

Abstract: Pangolins, unique mammals with scales over most of their body, no teeth, poor vision, and an acute olfactory system, comprise the only placental order (Pholidota) without a whole-genome map. To investigate pangolin biology and evolution, we developed genome assemblies of the Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins. Strikingly, we found that interferon epsilon (IFNE), exclusively expressed in epithelial cells and important in skin and mucosal immunity, is pseudogenized in all African and Asian pangolin species that we examined, perhaps impacting resistance to infection. We propose that scale development was an innovation that provided protection against injuries or stress and reduced pangolin vulnerability to infection. Further evidence of specialized adaptations was evident from positively selected genes involving immunity-related pathways, inflammation, energy storage and metabolism, muscular and nervous systems, and scale/hair development. Olfactory receptor gene families are significantly expanded in pangolins, reflecting their well-developed olfaction system. This study provides insights into mammalian adaptation and functional diversification, new research tools and questions, and perhaps a new natural IFNE-deficient animal model for studying mammalian immunity.

The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling.

Abstract: Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases in Acanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer.

Thermostability of Well-Ordered HIV Spikes Correlates with the Elicitation of Autologous Tier 2 Neutralizing Antibodies.

Abstract: In the context of HIV vaccine design and development, HIV-1 spike mimetics displaying a range of stabilities were evaluated to determine whether more stable, well-ordered trimers would more efficiently elicit neutralizing antibodies. To begin, in vitro analysis of trimers derived from the cysteine-stabilized SOSIP platform or the uncleaved, covalently linked NFL platform were evaluated. These native-like trimers, derived from HIV subtypes A, B, and C, displayed a range of thermostabilities, and were "stress-tested" at varying temperatures as a prelude to in vivo immunogenicity. Analysis was performed both in the absence and in the presence of two different adjuvants. Since partial trimer degradation was detected at 37°C before or after formulation with adjuvant, we sought to remedy such an undesirable outcome. Cross-linking (fixing) of the well-ordered trimers with glutaraldehyde increased overall thermostability, maintenance of well-ordered trimer integrity without or with adjuvant, and increased resistance to solid phase-associated trimer unfolding. Immunization of unfixed and fixed well-ordered trimers into animals revealed that the elicited tier 2 autologous neutralizing activity correlated with overall trimer thermostability, or melting temperature (Tm). Glutaraldehyde fixation also led to higher tier 2 autologous neutralization titers. These results link retention of trimer quaternary packing with elicitation of tier 2 autologous neutralizing activity, providing important insights for HIV-1 vaccine design.

The Contribution of GWAS Loci in Familial Dyslipidemias.

Abstract: Familial combined hyperlipidemia (FCH) is a complex and common familial dyslipidemia characterized by elevated total cholesterol and/or triglyceride levels with over five-fold risk of coronary heart disease. The genetic architecture and contribution of rare Mendelian and common variants to FCH susceptibility is unknown. In 53 Finnish FCH families, we genotyped and imputed nine million variants in 715 family members with DNA available. We studied the enrichment of variants previously implicated with monogenic dyslipidemias and/or lipid levels in the general population by comparing allele frequencies between the FCH families and population samples. We also constructed weighted polygenic scores using 212 lipid-associated SNPs and estimated the relative contributions of Mendelian variants and polygenic scores to the risk of FCH in the families. We identified, across the whole allele frequency spectrum, an enrichment of variants known to elevate, and a deficiency of variants known to lower LDL-C and/or TG levels among both probands and affected FCH individuals. The score based on TG associated SNPs was particularly high among affected individuals compared to non-affected family members. Out of 234 affected FCH individuals across the families, seven (3%) carried Mendelian variants and 83 (35%) showed high accumulation of either known LDL-C or TG elevating variants by having either polygenic score over the 90th percentile in the population. The positive predictive value of high score was much higher for affected FCH individuals than for similar sporadic cases in the population. FCH is highly polygenic, supporting the hypothesis that variants across the whole allele frequency spectrum contribute to this complex familial trait. Polygenic SNP panels improve identification of individuals affected with FCH, but their clinical utility remains to be defined.

IRF6 mutation screening in non‐syndromic orofacial clefting: analysis of 1521 families

Abstract: Van der Woude syndrome (VWS) is an autosomal dominant malformation syndrome characterized by orofacial clefting (OFC) and lower lip pits. The clinical presentation of VWS is variable and can present as an isolated OFC, making it difficult to distinguish VWS cases from individuals with non-syndromic OFCs. About 70% of causal VWS mutations occur in IRF6, a gene that is also associated with non-syndromic OFCs. Screening for IRF6 mutations in apparently non-syndromic cases has been performed in several modestly sized cohorts with mixed results. In this study, we screened 1521 trios with presumed non-syndromic OFCs to determine the frequency of causal IRF6 mutations. We identified seven likely causal IRF6 mutations, although a posteriori review identified two misdiagnosed VWS families based on the presence of lip pits. We found no evidence for association between rare IRF6 polymorphisms and non-syndromic OFCs. We combined our results with other similar studies (totaling 2472 families) and conclude that causal IRF6 mutations are found in 0.24-0.44% of apparently non-syndromic OFC families. We suggest that clinical mutation screening for IRF6 be considered for certain family patterns such as families with mixed types of OFCs and/or autosomal dominant transmission.

Opsin Repertoire and Expression Patterns in Horseshoe Crabs: Evidence from the Genome of Limulus polyphemus (Arthropoda: Chelicerata)

Abstract: Horseshoe crabs are xiphosuran chelicerates, the sister group to arachnids. As such, they are important for understanding the most recent common ancestor of Euchelicerata and the evolution and diversification of Arthropoda. Limulus polyphemus is the most investigated of the four extant species of horseshoe crabs, and the structure and function of its visual system have long been a major focus of studies critical for understanding the evolution of visual systems in arthropods. Likewise, studies of genes encoding Limulus opsins, the protein component of the visual pigments, are critical for understanding opsin evolution and diversification among chelicerates, where knowledge of opsins is limited, and more broadly among arthropods. In the present study, we sequenced and assembled a high quality nuclear genomic sequence of L. polyphemus and used these data to annotate the full repertoire of Limulus opsins. We conducted a detailed phylogenetic analysis of Limulus opsins, including using gene structure and synteny information to identify relationships among different opsin classes. We used our phylogeny to identify significant genomic events that shaped opsin evolution and therefore the visual system of Limulus We also describe the tissue expression patterns of the 18 opsins identified and show that transcripts encoding a number, including a peropsin, are present throughout the central nervous system. In addition to significantly extending our understanding of photosensitivity in Limulus and providing critical insight into the genomic evolution of horseshoe crab opsins, this work provides a valuable genomic resource for addressing myriad questions related to xiphosuran physiology and arthropod evolution.

A High-Resolution SNP Array-Based Linkage Map Anchors a New Domestic Cat Draft Genome Assembly and Provides Detailed Patterns of Recombination.

Abstract: High-resolution genetic and physical maps are invaluable tools for building accurate genome assemblies, and interpreting results of genome-wide association studies (GWAS). Previous genetic and physical maps anchored good quality draft assemblies of the domestic cat genome, enabling the discovery of numerous genes underlying hereditary disease and phenotypes of interest to the biomedical science and breeding communities. However, these maps lacked sufficient marker density to order thousands of shorter scaffolds in earlier assemblies, which instead relied heavily on comparative mapping with related species. A high-resolution map would aid in validating and ordering chromosome scaffolds from existing and new genome assemblies. Here, we describe a high-resolution genetic linkage map of the domestic cat genome based on genotyping 453 domestic cats from several multi-generational pedigrees on the Illumina 63K SNP array. The final maps include 58,055 SNP markers placed relative to 6637 markers with unique positions, distributed across all autosomes and the X chromosome. Our final sex-averaged maps span a total autosomal length of 4464 cM, the longest described linkage map for any mammal, confirming length estimates from a previous microsatellite-based map. The linkage map was used to order and orient the scaffolds from a substantially more contiguous domestic cat genome assembly (Felis catus v8.0), which incorporated ∼20 × coverage of Illumina fragment reads. The new genome assembly shows substantial improvements in contiguity, with a nearly fourfold increase in N50 scaffold size to 18 Mb. We use this map to report probable structural errors in previous maps and assemblies, and to describe features of the recombination landscape, including a massive (∼50 Mb) recombination desert (of virtually zero recombination) on the X chromosome that parallels a similar desert on the porcine X chromosome in both size and physical location.

The impact of chromosomal translocation locus and fusion oncogene coding sequence in synovial sarcomagenesis.

Abstract: Synovial sarcomas are aggressive soft-tissue malignancies that express chromosomal translocation-generated fusion genes, SS18-SSX1 or SS18-SSX2 in most cases. Here, we report a mouse sarcoma model expressing SS18-SSX1, complementing our prior model expressing SS18-SSX2. Exome sequencing identified no recurrent secondary mutations in tumors of either genotype. Most of the few mutations identified in single tumors were present in genes that were minimally or not expressed in any of the tumors. Chromosome 6, either entirely or around the fusion gene expression locus, demonstrated a copy number gain in a majority of tumors of both genotypes. Thus, by fusion oncogene coding sequence alone, SS18-SSX1 and SS18-SSX2 can each drive comparable synovial sarcomagenesis, independent from other genetic drivers. SS18-SSX1 and SS18-SSX2 tumor transcriptomes demonstrated very few consistent differences overall. In direct tumorigenesis comparisons, SS18-SSX2 was slightly more sarcomagenic than SS18-SSX1, but equivalent in its generation of biphasic histologic features. Meta-analysis of human synovial sarcoma patient series identified two tumor-gentoype-phenotype correlations that were not modeled by the mice, namely a scarcity of male hosts and biphasic histologic features among SS18-SSX2 tumors. Re-analysis of human SS18-SSX1 and SS18-SSX2 tumor transcriptomes demonstrated very few consistent differences, but highlighted increased native SSX2 expression in SS18-SSX1 tumors. This suggests that the translocated locus may drive genotype-phenotype differences more than the coding sequence of the fusion gene created. Two possible roles for native SSX2 in synovial sarcomagenesis are explored. Thus, even specific partial failures of mouse genetic modeling can be instructive to human tumor biology.

North Carolina macular dystrophy (MCDR1) caused by a novel tandem duplication of the PRDM13 gene.

Abstract: PURPOSE To identify the underlying cause of disease in a large family with North Carolina macular dystrophy (NCMD) METHODS A large four-generation family (RFS355) with an autosomal dominant form of NCMD was ascertained Family members underwent comprehensive visual function evaluations Blood or saliva from six affected family members and three unaffected spouses was collected and DNA tested for linkage to the MCDR1 locus on chromosome 6q12 Three affected family members and two unaffected spouses underwent whole exome sequencing (WES) and subsequently, custom capture of the linkage region followed by next-generation sequencing (NGS) Standard PCR and dideoxy sequencing were used to further characterize the mutation RESULTS Of the 12 eyes examined in six affected individuals, all but two had Gass grade 3 macular degeneration features Large central excavation of the retinal and choroid layers, referred to as a macular caldera, was seen in an age-independent manner in the grade 3 eyes The calderas are unique to affected individuals with MCDR1 Genome-wide linkage mapping and haplotype analysis of markers from the chromosome 6q region were consistent with linkage to the MCDR1 locus Whole exome sequencing and custom-capture NGS failed to reveal any rare coding variants segregating with the phenotype Analysis of the custom-capture NGS sequencing data for copy number variants uncovered a tandem duplication of approximately 60 kb on chromosome 6q This region contains two genes, CCNC and PRDM13 The duplication creates a partial copy of CCNC and a complete copy of PRDM13 The duplication was found in all affected members of the family and is not present in any unaffected members The duplication was not seen in 200 ethnically matched normal chromosomes CONCLUSIONS The cause of disease in the original family with MCDR1 and several others has been recently reported to be dysregulation of the PRDM13 gene, caused by either single base substitutions in a DNase 1 hypersensitive site upstream of the CCNC and PRDM13 genes or a tandem duplication of the PRDM13 gene The duplication found in the RFS355 family is distinct from the previously reported duplication and provides additional support that dysregulation of PRDM13, not CCNC, is the cause of NCMD mapped to the MCDR1 locus

High-Resolution Longitudinal Study of HIV-1 Env Vaccine-Elicited B Cell Responses to the Virus Primary Receptor Binding Site Reveals Affinity Maturation and Clonal Persistence.

Abstract: Because of the genetic variability of the HIV-1 envelope glycoproteins (Env), the elicitation of neutralizing Abs to conserved neutralization determinants including the primary receptor binding site, CD4 binding site (CD4bs), is a major focus of vaccine development. To gain insight into the evolution of Env-elicited Ab responses, we used single B cell analysis to interrogate the memory B cell Ig repertoires from two rhesus macaques after five serial immunizations with Env/adjuvant. We observed that the CD4bs-specific repertoire displayed unique features in the third CDR of Ig H chains with minor alterations along the immunization course. Progressive affinity maturation occurred as evidenced by elevated levels of somatic hypermutation (SHM) in Ab sequences isolated at the late immunization time point compared with the early time point. Abs with higher SHM were associated with increased binding affinity and virus neutralization capacity. Moreover, a notable portion of the CD4bs-specific repertoire was maintained between early and late immunization time points, suggesting that persistent clonal lineages were induced by Env vaccination. Furthermore, we found that the predominant persistent CD4bs-specific clonal lineages had larger population sizes and higher affinities than that from the rest of the repertoires, underscoring the critical role of Ag affinity selection in Ab maturation and clonal expansion. Genetic and functional analyses revealed that the accumulation of SHM in both framework regions and CDRs contributed to the clonal affinity and antigenicity evolution. Our longitudinal study provides high-resolution understanding of the dynamically evolving CD4bs-specific B cell response after Env immunization in primates.

Dictyocaulus viviparus genome, variome and transcriptome elucidate lungworm biology and support future intervention.

Abstract: The bovine lungworm, Dictyocaulus viviparus (order Strongylida), is an important parasite of livestock that causes substantial economic and production losses worldwide. Here we report the draft genome, variome, and developmental transcriptome of D. viviparus. The genome (161 Mb) is smaller than those of related bursate nematodes and encodes fewer proteins (14,171 total). In the first genome-wide assessment of genomic variation in any parasitic nematode, we found a high degree of sequence variability in proteins predicted to be involved host-parasite interactions. Next, we used extensive RNA sequence data to track gene transcription across the life cycle of D. viviparus, and identified genes that might be important in nematode development and parasitism. Finally, we predicted genes that could be vital in host-parasite interactions, genes that could serve as drug targets, and putative RNAi effectors with a view to developing functional genomic tools. This extensive, well-curated dataset should provide a basis for developing new anthelmintics, vaccines, and improved diagnostic tests and serve as a platform for future investigations of drug resistance and epidemiology of the bovine lungworm and related nematodes.

Rare Variation in TET2 Is Associated with Clinically Relevant Prostate Carcinoma in African-Americans

Abstract: Background: Common variants have been associated with prostate cancer risk. Unfortunately, few are reproducibly linked to aggressive disease, the phenotype of greatest clinical relevance. One possible explanation is that rare genetic variants underlie a significant proportion of the risk for aggressive disease. Method: To identify such variants, we performed a two staged approach using whole exome sequencing followed by targeted sequencing of 800 genes in 652 aggressive prostate cancer patients and 752 disease-free controls in both African and European Americans. In each population, we tested rare variants for association using two gene-based aggregation tests. We established a study-wide significance threshold of 3.125 x 10-5 to correct for multiple testing. Results: TET2 in African-Americans was associated with aggressive disease with 24.4% of cases harboring a rare deleterious variant compared to 9.6% of controls (FET p = 1.84x10-5, OR=3.0; SKAT-O p= 2.74x10-5). We report 8 additional genes with suggestive evidence of association, including the DNA repair genes PARP2 and MSH6. Finally, we observed an excess of rare truncation variants in 5 genes including the DNA repair genes MSH6, BRCA1 and BRCA2. This adds to the growing body of evidence that DNA repair pathway defects may influence susceptibility to aggressive prostate cancer. Conclusion: Our findings suggest that rare variants influence risk of clinically relevant prostate cancer and if validated could serve to identify men for screening, prophylaxis and treatment. Impact: This study provides evidence that rare variants in TET2 may help identify African-American men at increased risk for clinically relevant prostate cancer.

Inactivation of RASA1 promotes melanoma tumorigenesis via R-Ras activation.

Abstract: // Hyeran Sung 1, 2 , Krishna L. Kanchi 3 , Xue Wang 1, 2 , Kristen S. Hill 1, 2 , Jane L. Messina 2, 4, 5 , Ji-Hyun Lee 6 , Youngchul Kim 7 , Nathan D. Dees 3 , Li Ding 3, 8, 9 , Jamie K. Teer 7 , Shengyu Yang 2, 10 , Amod A. Sarnaik 2, 4 , Vernon K. Sondak 2, 4 , James J. Mule 2, 4 , Richard K. Wilson 3, 9 , Jeffrey S. Weber 11 , Minjung Kim 1, 2, 4 1 Department of Molecular Oncology, Moffitt Cancer Center, Tampa, FL, USA 2 Comprehensive Melanoma Research Center, Moffitt Cancer Center, Tampa, FL, USA 3 The Genome Institute, Washington University, St. Louis, MO, USA 4 Department of Cutaneous Oncology, Moffitt Cancer Center, Tampa, FL, USA 5 Department of Pathology, Moffitt Cancer Center, Tampa, FL, USA 6 Department of Internal Medicine, University of New Mexico Comprehensive Cancer Center, Albuquerque, NM, USA 7 Department of Biostatistics and Bioinformatics, Moffitt Cancer Center, Tampa, FL, USA 8 Department of Medicine, Washington University, St. Louis, MO, USA 9 Department of Genetics, Washington University, St. Louis, MO, USA 10 Department of Tumor Biology, Moffitt Cancer Center, Tampa, FL, USA 11 Department of Medicine, NYU Langone Medical Center, New York, NY, USA Correspondence to: Minjung Kim, e-mail: Minjung.kim@moffitt.org Keywords: melanoma, RASA1, RasGAP, R-Ras, whole genome sequencing Received: January 15, 2016 Accepted: February 28, 2016 Published: March 16, 2016 ABSTRACT Inactivation of Ras GTPase activating proteins (RasGAPs) can activate Ras, increasing the risk for tumor development. Utilizing a melanoma whole genome sequencing (WGS) data from 13 patients, we identified two novel, clustered somatic missense mutations (Y472H and L481F) in RASA1 (RAS p21 protein activator 1, also called p120RasGAP). We have shown that wild type RASA1, but not identified mutants, suppresses soft agar colony formation and tumor growth of BRAF mutated melanoma cell lines via its RasGAP activity toward R-Ras (related RAS viral (r-ras) oncogene homolog) isoform. Moreover, R-Ras increased and RASA1 suppressed Ral-A activation among Ras downstream effectors. In addition to mutations, loss of RASA1 expression was frequently observed in metastatic melanoma samples on melanoma tissue microarray (TMA) and a low level of RASA1 mRNA expression was associated with decreased overall survival in melanoma patients with BRAF mutations. Thus, these data support that RASA1 is inactivated by mutation or by suppressed expression in melanoma and that RASA1 plays a tumor suppressive role by inhibiting R-Ras, a previously less appreciated member of the Ras small GTPases.

High-Quality Assembly of an Individual of Yoruban Descent

Abstract: De novo assembly of human genomes is now a tractable effort due in part to advances in sequencing and mapping technologies. We use PacBio single-molecule, real-time (SMRT) sequencing and BioNano genomic maps to construct the first de novo assembly of NA19240, a Yoruban individual from Africa. This chromosome-scaffolded assembly of 3.08 Gb with a contig N50 of 7.25 Mb and a scaffold N50 of 78.6 Mb represents one of the most contiguous high-quality human genomes. We utilize a BAC library derived from NA19240 DNA and novel haplotype-resolving sequencing technologies and algorithms to characterize regions of complex genomic architecture that are normally lost due to compression to a linear haploid assembly. Our results demonstrate that multiple technologies are still necessary for complete genomic representation, particularly in regions of highly identical segmental duplications. Additionally, we show that diploid assembly has utility in improving the quality of de novo human genome assemblies.

Ancient hybridization and strong adaptation to viruses across African vervet monkey populations

Abstract: Vervet monkeys (genus Chlorocebus , also known as African green monkeys), are highly abundant in savannahs and riverine forests throughout sub-Saharan Africa. They are amongst the most widely distributed nonhuman primates, show considerable phenotypic diversity, and have long been an important biomedical model for a variety of human diseases and in vaccine research. They are particularly interesting for HIV/AIDS research as they are the most abundant natural hosts of simian immunodeficiency virus (SIV), a close relative of HIV. Here we present the first genome-wide survey of polymorphism in vervets, using sequencing data from 163 individuals sampled from across Africa and the Caribbean islands where vervets were introduced during the colonial era. We find high diversity, within and between taxa, and clear evidence that taxonomic divergence was reticulate rather than following a simple branching pattern. A scan for diversifying selection across vervet taxa yields gene enrichments much stronger than in similar studies on humans. In particular, we report strong and highly polygenic selection signals affecting viral processes --- in line with recent evidence that proposes a driving role for viruses in protein evolution in mammals. Furthermore, selection scores are highly elevated in genes whose human orthologs interact with HIV, and in genes that show a response to experimental SIV infection in vervet monkeys but not in rhesus macaques, suggesting that part of the signal reflects taxon-specific adaptation to SIV. Intriguingly, rather than affecting genes with antiviral and inflammatory-related functions, selection in vervets appears to have primarily targeted genes involved in the transcriptional regulation of viruses, and in particular those that are harmful only under immunodeficiency, suggesting adaptation to living with SIV rather than defense against infection.

An HIV-1 Env–Antibody Complex Focuses Antibody Responses to Conserved Neutralizing Epitopes

Abstract: Elicitation of broadly neutralizing Ab (bNAb) responses to the conserved elements of the HIV-1 envelope glycoproteins (Env), including the primary receptor CD4 binding site (CD4bs), is a major focus of vaccine development yet to be accomplished. However, a large number of CD4bs-directed bNAbs have been isolated from HIV-1-infected individuals. Comparison of the routes of binding used by the CD4bs-directed bNAbs from patients and the vaccine-elicited CD4bs-directed mAbs indicates that the latter fail to neutralize primary virus isolates because they approach the Env spike with a vertical angle and contact the specific surface residues occluded in the native spike, including the bridging sheet on gp120. To preferentially expose the CD4bs and direct the immune response away from the bridging sheet, resulting in an altered angle of approach, we engineered an immunogen consisting of gp120 core in complex with the prototypic CD4-induced Ab, 17b. This mAb directly contacts the bridging sheet but not the CD4bs. The complex was further stabilized by chemical crosslinking to prevent dissociation. Rabbits immunized with the crosslinked complex displayed earlier affinity maturation, achieving tier 1 virus neutralization compared with animals immunized with gp120 core alone. Immunization with the crosslinked complex induced transient Ab responses with binding specificity similar to the CD4bs-directed bNAbs. mAbs derived from complex-immunized rabbits displayed footprints on gp120 more distal from the bridging sheet as compared with previous vaccine-elicited CD4bs Abs, indicating that Env-Ab complexes effectively dampen immune responses to undesired immunodominant bridging sheet determinants.

Cost-Effective Recruitment Strategies That Attract Underrepresented Minority Undergraduates Who Persist to STEM Doctorates:

Abstract: The paucity of underrepresented minorities (URMs) earning science, technology, engineering, and mathematics (STEM) degrees remains an issue in revitalizing the U.S. biomedical workforce. Due to reductions in federal funding, maintaining the integrity of programs that focus on URM retention and recruitment is crucial. We present data on the mechanisms used to recruit URM students to our program (e.g., email, events, referrals, website), which individually were equally effective in attracting applicants to the program. Recruitment mechanisms were grouped and further classified relative to their cost to implement as lower and higher cost. Our results indicate that lower cost mechanisms, statistically, were as effective as higher cost mechanisms in recruiting students who persisted to PhD programs. Using a binary logistic regression model to predict PhD matriculation, higher cost mechanisms were not significant predictors of PhD matriculation. Collectively, these data demonstrate for the first time that lower...

CIViC: A knowledgebase for expert-crowdsourcing the clinical interpretation of variants in cancer.

Abstract: CIViC is an expert crowdsourced knowledgebase for Clinical Interpretation of Variants in Cancer (www.civicdb.org) describing the therapeutic, prognostic, and diagnostic relevance of inherited and somatic variants of all types. CIViC is committed to open source code, open access content, public application programming interfaces (APIs), and provenance of supporting evidence to allow for the transparent creation of current and accurate variant interpretations for use in cancer precision medicine.

Identification of a Novel Gene on 10q22.1 Causing Autosomal Dominant Retinitis Pigmentosa (adRP).

Abstract: Whole-genome linkage mapping identified a region on chromosome 10q21.3-q22.1 with a maximum LOD score of 3.0 at 0 % recombination in a six-generation family with autosomal dominant retinitis pigmentosa (adRP). All known adRP genes and X-linked RP genes were excluded in the family by a combination of methods. Whole-exome next-generation sequencing revealed a missense mutation in hexokinase 1, HK1 c.2539G > A, p.Glu847Lys, tracking with disease in all affected family members. One severely-affected male is homozygous for this region by linkage analysis and has two copies of the mutation. No other potential mutations were detected in the linkage region nor were any candidates identified elsewhere in the genome. Subsequent testing detected the same mutation in four additional, unrelated adRP families, for a total of five mutations in 404 probands tested (1.2 %). Of the five families, three are from the Acadian population in Louisiana, one is French Canadian and one is Sicilian. Haplotype analysis of the affected chromosome in each family and the homozygous individual revealed a rare, shared haplotype of 450 kb, suggesting an ancient founder mutation. HK1 is a widely-expressed gene, with multiple, abundant retinal transcripts, coding for hexokinase 1. Hexokinase catalyzes phosphorylation of glucose to glusose-6-phospate, the first step in glycolysis. The Glu847Lys mutation is in a highly-conserved site, outside of the active site or known functional sites.