Comprehensive molecular profiling of multiple myeloma identifies refined copy number and expression subtypes.

Multiple myeloma is a treatable, but currently incurable, hematological malignancy of plasma cells characterized by diverse and complex tumor genetics for which precision medicine approaches to treatment are lacking. The Multiple Myeloma Research Foundation's Relating Clinical Outcomes in Multiple Myeloma to Personal Assessment of Genetic Profile study ( NCT01454297 ) is a longitudinal, observational clinical study of newly diagnosed patients with multiple myeloma (n = 1,143) where tumor samples are characterized using whole-genome sequencing, whole-exome sequencing and RNA sequencing at diagnosis and progression, and clinical data are collected every 3 months. Analyses of the baseline cohort identified genes that are the target of recurrent gain-of-function and loss-of-function events. Consensus clustering identified 8 and 12 unique copy number and expression subtypes of myeloma, respectively, identifying high-risk genetic subtypes and elucidating many of the molecular underpinnings of these unique biological groups. Analysis of serial samples showed that 25.5% of patients transition to a high-risk expression subtype at progression. We observed robust expression of immunotherapy targets in this subtype, suggesting a potential therapeutic option.

Assessing the Effects of Pulsed Electromagnetic Therapy on Painful Diabetic Distal Symmetric Peripheral Neuropathy: A Double-Blind Randomized Controlled Trial.

BACKGROUND: Significant complications of diabetes include pain and the loss of sensation in peripheral limbs. Pain management of diabetic symmetric peripheral neuropathy (DSPN) remains challenging. This study reports on utilizing pulsed electromagnetic field therapy (PEMF) to reduce pain and improve skin perfusion pressure (SPP) in subjects with DSPN. METHODS: A randomized, sham-controlled, double-blind, clinical trial was conducted on subjects afflicted with foot pain associated with DSPN. Following informed consent, 182 subjects with diabetes and confirmed DSPN were entered into the trial for a period of 18 weeks. Subjects were randomized into active PEMF treatment or nonactive sham and instructed to treat to their feet for 30 minutes, twice daily and report daily pain scores. Some patients in the active arm experienced a transient low field strength notification (LFSN) due to improper pad placement during treatment. Skin perfusion pressure measurements were also collected at two and seven weeks to assess peripheral arterial disease effects via measurement of local microcirculatory flow and blood pressure. RESULTS: Patients in the active arm who did not receive an LFSN experienced a clinically significant 30% reduction in pain from baseline compared to sham (P < .05). Though not statistically significant, SPP in the active group trended toward improvement compared to sham. CONCLUSIONS: Pulsed electromagnetic field therapy appears effective as a nonpharmacological means for reduction of pain associated with diabetic peripheral neuropathy and holds promise for improvement of vascular physiology in microcirculatory dysfunction associated with diabetic peripheral arterial disease.

Identifying treatment options for BRAFV600 wild-type metastatic melanoma: A SU2C/MRA genomics-enabled clinical trial.

Although combination BRAF and MEK inhibitors are highly effective for the 40-50% of cutaneous metastatic melanomas harboring BRAFV600 mutations, targeted agents have been ineffective for BRAFV600wild-type (wt) metastatic melanomas. The SU2C Genomics-Enabled Medicine for Melanoma Trial utilized a Simon two-stage optimal design to assess whether comprehensive genomic profiling improves selection of molecular-based therapies for BRAFV600wt metastatic melanoma patients who had progressed on standard-of-care therapy, which may include immunotherapy. Of the response-evaluable patients, binimetinib was selected for 20 patients randomized to the genomics-enabled arm, and nine were treated on the alternate treatment arm. Response rates for 27 patients treated with targeted recommendations included one (4%) partial response, 18 (67%) with stable disease, and eight (30%) with progressive disease. Post-trial genomic and protein pathway activation mapping identified additional drug classes that may be considered for future studies. Our results highlight the complexity and heterogeneity of metastatic melanomas, as well as how the lack of response in this trial may be associated with limitations including monotherapy drug selection and the dearth of available single and combination molecularly-driven therapies to treat BRAFV600wt metastatic melanomas.

Evolutionary stability, landscape heterogeneity, and human land-usage shape population genetic connectivity in the Cape Floristic Region biodiversity hotspot.

As human-induced change eliminates natural habitats, it impacts genetic diversity and population connectivity for local biodiversity. The South African Cape Floristic Region (CFR) is the most diverse extratropical area for plant biodiversity, and much of its habitat is protected as a UNESCO World Heritage site. There has long been great interest in explaining the underlying factors driving this unique diversity, especially as much of the CFR is endangered by urbanization and other anthropogenic activity. Here, we use a population and landscape genetic analysis of SNP data from the CFR endemic plant Leucadendron salignum or "common sunshine conebush" as a model to address the evolutionary and environmental factors shaping the vast CFR diversity. We found that high population structure, along with relatively deeper and older genealogies, is characteristic of the southwestern CFR, whereas low population structure and more recent lineage coalescence depict the eastern CFR. Population network analyses show genetic connectivity is facilitated in areas of lower elevation and higher seasonal precipitation. These population genetic signatures corroborate CFR species-level patterns consistent with high Pleistocene biome stability and landscape heterogeneity in the southwest, but with coincident instability in the east. Finally, we also find evidence of human land-usage as a significant gene flow barrier, especially in severely threatened lowlands where genetic connectivity has been historically the highest. These results help identify areas where conservation plans can prioritize protecting high genetic diversity threatened by contemporary human activities within this unique cultural UNESCO site.

ACValidator: A novel assembly-based approach for in silico verification of circular RNAs.

Circular RNAs (circRNAs) are evolutionarily conserved RNA species that are formed when exons "back-splice" to each other. Current computational algorithms to detect these back-splicing junctions produce divergent results, and hence there is a need for a method to distinguish true-positive circRNAs. To this end, we developed Assembly based CircRNA Validator (ACValidator) for in silico verification of circRNAs. ACValidator extracts reads from a user-defined window on either side of a circRNA junction and assembles them to generate contigs. These contigs are aligned against the circRNA sequence to find contigs spanning the back-spliced junction. When evaluated on simulated datasets, ACValidator achieved over ∼80% sensitivity on datasets with an average of 10 circRNA-supporting reads and with read lengths of at least 100 bp. In experimental datasets, ACValidator produced higher verification percentages for samples treated with ribonuclease R compared to nontreated samples. Our workflow is applicable to non-polyA-selected RNAseq datasets and can also be used as a candidate selection strategy for prioritizing experimental validations. All workflow scripts are freely accessible on our GitHub page https://github.com/tgen/ACValidator along with detailed instructions to set up and run ACValidator.

Leveraging Spatial Variation in Tumor Purity for Improved Somatic Variant Calling of Archival Tumor Only Samples.

Archival tumor samples represent a rich resource of annotated specimens for translational genomics research. However, standard variant calling approaches require a matched normal sample from the same individual, which is often not available in the retrospective setting, making it difficult to distinguish between true somatic variants and individual-specific germline variants. Archival sections often contain adjacent normal tissue, but this tissue can include infiltrating tumor cells. As existing comparative somatic variant callers are designed to exclude variants present in the normal sample, a novel approach is required to leverage adjacent normal tissue with infiltrating tumor cells for somatic variant calling. Here we present lumosVar 2.0, a software package designed to jointly analyze multiple samples from the same patient, built upon our previous single sample tumor only variant caller lumosVar 1.0. The approach assumes that the allelic fraction of somatic variants and germline variants follow different patterns as tumor content and copy number state change. lumosVar 2.0 estimates allele specific copy number and tumor sample fractions from the data, and uses a to model to determine expected allelic fractions for somatic and germline variants and to classify variants accordingly. To evaluate the utility of lumosVar 2.0 to jointly call somatic variants with tumor and adjacent normal samples, we used a glioblastoma dataset with matched high and low tumor content and germline whole exome sequencing data (for true somatic variants) available for each patient. Both sensitivity and positive predictive value were improved when analyzing the high tumor and low tumor samples jointly compared to analyzing the samples individually or in-silico pooling of the two samples. Finally, we applied this approach to a set of breast and prostate archival tumor samples for which tumor blocks containing adjacent normal tissue were available for sequencing. Joint analysis using lumosVar 2.0 detected several variants, including known cancer hotspot mutations that were not detected by standard somatic variant calling tools using the adjacent tissue as presumed normal reference. Together, these results demonstrate the utility of leveraging paired tissue samples to improve somatic variant calling when a constitutional sample is not available.

De novo transcriptome assemblies of four xylem sap-feeding insects.

Background: Spittle bugs and sharpshooters are well-known xylem sap-feeding insects and vectors of the phytopathogenic bacterium Xylella fastidiosa (Wells), a causal agent of Pierce's disease of grapevines and other crop diseases. Specialized feeding on nutrient-deficient xylem sap is relatively rare among insect herbivores, and only limited genomic and transcriptomic information has been generated for xylem-sap feeders. To develop a more comprehensive understanding of biochemical adaptations and symbiotic relationships that support survival on a nutritionally austere dietary source, transcriptome assemblies for three sharpshooter species and one spittlebug species were produced. Findings: Trinity-based de novo transcriptome assemblies were generated for all four xylem-sap feeders using raw sequencing data originating from whole-insect preps. Total transcripts for each species ranged from 91 384 for Cuerna arida to 106 998 for Homalodisca liturata with transcript totals for Graphocephala atropunctata and the spittlebug Clastoptera arizonana falling in between. The percentage of transcripts comprising complete open reading frames ranged from 60% for H. liturata to 82% for C. arizonana. Bench-marking universal single-copy orthologs analyses for each dataset indicated quality assemblies and a high degree of completeness for all four species. Conclusions: These four transcriptomes represent a significant expansion of data for insect herbivores that feed exclusively on xylem sap, a nutritionally deficient dietary source relative to other plant tissues and fluids. Comparison of transcriptome data with insect herbivores that utilize other dietary sources may illuminate fundamental differences in the biochemistry of dietary specialization.

Prediction of a peptidome for the western tarnished plant bug Lygus hesperus.

Many strategies for controlling insect pests require an understanding of their hormonal signaling agents, peptides being the largest and most diverse single class of these molecules. Lygus hesperus is a pest species of particular concern, as it is responsible for significant damage to a wide variety of commercially important plant crops. At present, little is known about the peptide hormones of L. hesperus. Here, transcriptomic data were used to predict a peptidome for L. hesperus. Fifty-three L. hesperus transcripts encoding peptide precursors were identified, with a subset amplified by PCR for sequence verification. The proteins deduced from these transcripts allowed for the prediction of a 119-sequence peptidome for L. hesperus. The predicted peptides include isoforms of allatostatin A, allatostatin B (AST-B), allatostatin C, allatotropin, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/ion transport peptide, diuretic hormone 31, GSEFLamide, insulin-like peptide, myosuppressin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, pyrokinin, short neuropeptide F, SIFamide, sulfakinin and tachykinin-related peptide. Of note were several isoforms of AST-B that possess -WX7Wamide carboxyl-termini rather than the stereotypical -WX6Wamide (e.g., KWQDMQNPGWamide), an allatotropin ending in -SARGFamide rather than -TARGFamide (GLKNGPLNSARGFamide), a GSEFLamide ending in -GTEFLamide (TVGTEFLamide), several orcokinins with PMDEIDR- rather than NFDEIDR- amino-termini (e.g., PMDEIDRAGFTHFV), and an eight rather than 12 amino acid long isoform of SIFamide (PPFNGSIFamide). Collectively, the L. hesperus peptidome predicted here provides a resource for initiating physiological investigations of peptidergic signaling in this species, including studies directed at the biological control of this agricultural pest.

Sequencing, de novo assembly and annotation of a pink bollworm larval midgut transcriptome.

BACKGROUND: The pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) is one of the world's most important pests of cotton. Insecticide sprays and transgenic cotton producing toxins of the bacterium Bacillus thuringiensis (Bt) are currently used to manage this pest. Bt toxins kill susceptible insects by specifically binding to and destroying midgut cells, but they are not toxic to most other organisms. Pink bollworm is useful as a model for understanding insect responses to Bt toxins, yet advances in understanding at the molecular level have been limited because basic genomic information is lacking for this cosmopolitan pest. Here, we have sequenced, de novo assembled and annotated a comprehensive larval midgut transcriptome from a susceptible strain of pink bollworm. FINDINGS: A de novo transcriptome assembly for the midgut of P. gossypiella was generated containing 46,458 transcripts (average length of 770 bp) derived from 39,874 unigenes. The size of the transcriptome is similar to published midgut transcriptomes of other Lepidoptera and includes up to 91 % annotated contigs. The dataset is publicly available in NCBI and GigaDB as a resource for researchers. CONCLUSIONS: Foundational knowledge of protein-coding genes from the pink bollworm midgut is critical for understanding how this important insect pest functions. The transcriptome data presented here represent the first large-scale molecular resource for this species, and may be used for deciphering relevant midgut proteins critical for xenobiotic detoxification, nutrient digestion and allocation, as well as for the discovery of protein receptors important for Bt intoxication.

Population Genomic Analysis Reveals Differential Evolutionary Histories and Patterns of Diversity across Subgenomes and Subpopulations of Brassica napus L.

The allotetraploid species Brassica napus L. is a global crop of major economic importance, providing canola oil (seed) and vegetables for human consumption and fodder and meal for livestock feed. Characterizing the genetic diversity present in the extant germplasm pool of B. napus is fundamental to better conserve, manage and utilize the genetic resources of this species. We used sequence-based genotyping to identify and genotype 30,881 SNPs in a diversity panel of 782 B. napus accessions, representing samples of winter and spring growth habits originating from 33 countries across Europe, Asia, and America. We detected strong population structure broadly concordant with growth habit and geography, and identified three major genetic groups: spring (SP), winter Europe (WE), and winter Asia (WA). Subpopulation-specific polymorphism patterns suggest enriched genetic diversity within the WA group and a smaller effective breeding population for the SP group compared to WE. Interestingly, the two subgenomes of B. napus appear to have different geographic origins, with phylogenetic analysis placing WE and WA as basal clades for the other subpopulations in the C and A subgenomes, respectively. Finally, we identified 16 genomic regions where the patterns of diversity differed markedly from the genome-wide average, several of which are suggestive of genomic inversions. The results obtained in this study constitute a valuable resource for worldwide breeding efforts and the genetic dissection and prediction of complex B. napus traits.

De novo construction of an expanded transcriptome assembly for the western tarnished plant bug, Lygus hesperus.

BACKGROUND: The plant bug Lygus hesperus Knight is a polyphagous pest of many economically important crops. Despite its pest status, little is known about the molecular mechanisms responsible for much of the biology of this species. Earlier Lygus transcriptome assemblies were limited by low read depth, or because they focused on specific conditions. To generate a more comprehensive transcriptome, we supplemented previous datasets with new reads corresponding to specific tissues (heads, antennae, and male reproductive tissues). This transcriptome augments current Lygus molecular resources and provides the foundational knowledge critical for future comparative studies. FINDINGS: An expanded, Trinity-based de novo transcriptome assembly for L. hesperus was generated using previously published whole body Illumina data, supplemented with 293 million bp of new raw sequencing data corresponding to five tissue-specific cDNA libraries and 11 Illumina sequencing runs. The updated transcriptome consists of 22,022 transcripts (average length of 2075 nt), 62 % of which contain complete open reading frames. Significant coverage of the BUSCO (benchmarking universal single-copy orthologs) dataset and robust metrics indicate that the transcriptome is a quality assembly with a high degree of completeness. Initial assessment of the new assembly's utility revealed that the length and abundance of transcripts predicted to regulate insect physiology and chemosensation have improved, compared with previous L. hesperus assemblies. CONCLUSIONS: This transcriptome represents a significant expansion of Lygus transcriptome data, and improves foundational knowledge about the molecular mechanisms underlying L. hesperus biology. The dataset is publically available in NCBI and GigaDB as a resource for researchers.

Mutations in the Notch pathway alter the patterning of multifidus.

Clinical studies have suggested that defects in the epaxial muscles, particularly multifidus, may contribute to the etiology of idiopathic scoliosis. While the epaxial muscles and the vertebrae derive from the same embryonic segmentation process, the mechanisms that pattern the multisegmental back muscles are still unclear. The process of segmentation is regulated by the Notch signaling pathway, and mutations in the modulators delta-like 3 (Dll3) and lunatic fringe (Lfng) are genetic models for spinal disorders such as scoliosis. Osteological defects have been characterized in these genetic models, but myological phenotypes have not previously been studied. We analyzed the multifidus muscle in the mouse (Mus musculus) and observed intriguing changes in the cranio-caudal borders of multifidus in Dll3 and Lfng models. Statistical analysis did not find a significant association between the majority of the multifidus anomalies and the vertebral defects, suggesting a previously unappreciated role for Notch signaling in patterning epaxial muscle groups. These findings indicate an additional mechanism by which DLL3 and LFNG may play a role in the etiology of human idiopathic scoliosis.