Clinical Genomics: Challenges and Opportunities.

Next-generation sequencing (NGS) approaches are highly applicable to clinical studies. We review recent advances in sequencing technologies, as well as their benefits and tradeoffs, to provide an overview of clinical genomics from study design to computational analysis. Sequencing technologies enable genomic, transcriptomic, and epigenomic evaluations. Studies that use a combination of whole genome, exome, mRNA, and bisulfite sequencing are now feasible due to decreasing sequencing costs. Single-molecule sequencing increases read length, with the MinIONTM nanopore sequencer, which offers a uniquely portable option at a lower cost. Many of the published comparisons we review here address the challenges associated with different sequencing methods. Overall, NGS techniques, coupled with continually improving analysis algorithms, are useful for clinical studies in many realms, including cancer, chronic illness, and neurobiology. We, and others in the field, anticipate the clinical use of NGS approaches will continue to grow, especially as we shift into an era of precision medicine.

A Sonomyography-Based Muscle Computer Interface for Individuals With Spinal Cord Injury.

Impairment of hand functions in individuals with spinal cord injury (SCI) severely disrupts activities of daily living. Recent advances have enabled rehabilitation assisted by robotic devices to augment the residual function of the muscles. Traditionally, electromyography-based muscle activity sensing interfaces have been utilized to sense volitional motor intent to drive robotic assistive devices. However, the dexterity and fidelity of control that can be achieved with electromyography-based control have been limited due to inherent limitations in signal quality. We have developed and tested a muscle-computer interface (MCI) utilizing sonomyography to provide control of a virtual cursor for individuals with motor-incomplete spinal cord injury. We demonstrate that individuals with SCI successfully gained control of a virtual cursor by utilizing contractions of muscles of the wrist joint. The sonomyography-based interface enabled control of the cursor at multiple graded levels demonstrating the ability to achieve accurate and stable endpoint control. Our sonomyography-based muscle-computer interface can enable dexterous control of upper-extremity assistive devices for individuals with motor-incomplete SCI.

Development, Content Validity and Reliability of Upper Extremity Functional Skill Measure in C5-C7 Spinal Cord Injury.

STUDY DESIGN: A methodological research design. OBJECTIVE: To create an objective measure for assessing hand functions in C5-C7 spinal cord injury (SCI) and estimation of its content validity and internal consistency reliability. METHOD: This study was executed in three phases. Phase 1 included a thorough review of the literature, semi-structured in-depth interviews of participants with tetraplegia and interviews of caregivers of SCI individuals and healthcare workers dealing with SCI to understand the hand functions of individuals with C5-C7 SCI. Phase 2 consisted of the development of the tool. The content validity ratio (CVR) method and the opinion of the expert validated the content of the upper extremity functional skill measure (UEFSM). Phase 3 included a quantitative evaluation of the tool which was done on a targeted group of 30 subjects with C5-C7 SCI. RESULTS: Through the review of the literature and in-depth interview of the participants, 11 items were developed under four content areas: grasp, grip, pinch and gross movement. Items with a minimum CVR of 0.56 were retained at a significance level of p = 0.05 resulting in a 10-item tool for assessing the hand function of individuals with C5-C7 SCI categorized under four subscales. Pilot testing on 10 subjects reveals an average time of 2 minutes and 25 seconds to complete the task. The Cronbach's alpha was found to be 0.878. CONCLUSION: UEFSM is a 10-item tool with good content validity and internal consistency reliability for the assessment of hand functions in individuals with C5-C7 SCI.

Activation of RAS/MAPK pathway confers MCL-1 mediated acquired resistance to BCL-2 inhibitor venetoclax in acute myeloid leukemia.

Despite high initial response rates, acute myeloid leukemia (AML) treated with the BCL-2-selective inhibitor venetoclax (VEN) alone or in combinations commonly acquires resistance. We performed gene/protein expression, metabolomic and methylation analyses of isogenic AML cell lines sensitive or resistant to VEN, and identified the activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein, as a major acquired mechanism of VEN resistance. MCL-1 sustained survival and maintained mitochondrial respiration in VEN-RE cells, which had impaired electron transport chain (ETC) complex II activity, and MCL-1 silencing or pharmacologic inhibition restored VEN sensitivity. In support of the importance of RAS/MAPK activation, we found by single-cell DNA sequencing rapid clonal selection of RAS-mutated clones in AML patients treated with VEN-containing regimens. In summary, these findings establish RAS/MAPK/MCL-1 and mitochondrial fitness as key survival mechanisms of VEN-RE AML and provide the rationale for combinatorial strategies effectively targeting these pathways.

Epigenetic Forensics for Suspect Identification and Age Prediction.

Background: Genetic testing at crime scenes is an instrumental molecular technique to identify or eliminate suspects, as well as to overturn wrongful convictions. Yet, genotyping alone cannot reveal the age of a sample, which could help advance the utility of crime scene samples for suspect identification. The distribution of cytosine methylation within a DNA sample can be leveraged to determine the epigenetic age of someone's blood. Methodology: We sought to demonstrate the ability of DNA methylation markers to accurately discern the age of blood spots from an actual crime scene, a "mock" crime scene, and also from a tube of blood stored in ethylenediaminetetraacetic acid for >20 years. This was achieved by quantifying methylation within known age-associated genetic loci across each DNA sample. We observed a strong linear coefficient (0.91) and high overall correlation (R 2 = 0.963) between the known age of a sample and the predicted age. Conclusion: We show that novel methods for targeted methylation and low-input whole-genome bisulfite sequencing can enable a novel and improved forensic profile of a crime scene that discerns not only who was present at the crime, but also their age. Finally, we use this model to discern the age and provenance of a blood sample that was used in a criminal investigation.

Acquired Resistance to BET-PROTACs (Proteolysis-Targeting Chimeras) Caused by Genomic Alterations in Core Components of E3 Ligase Complexes.

Proteolysis-targeting chimeras (PROTAC) are bifunctional molecules that hijack endogenous E3 ubiquitin ligases to induce ubiquitination and subsequent degradation of protein of interest. Recently, it has been shown that PROTACs with robust in vitro and in vivo activities and, in some cases, drug-like pharmaceutical properties can be generated using small-molecule ligands for the E3 ligases VHL and CRBN. These findings stoked tremendous enthusiasm on using PROTACs for therapeutics development. Innate and acquired drug resistance often underlies therapeutic failures, particularly for cancer therapy. With the PROTAC technology progressing rapidly toward therapeutic applications, it would be important to understand whether and how resistance to these novel agents may emerge. Using BET-PROTACs as a model system, we demonstrate that resistance to both VHL- and CRBN-based PROTACs can occur in cancer cells following chronic treatment. However, unlike what was often observed for many targeted therapeutics, resistance to BET-PROTACs did not result from secondary mutations that affect compound binding to the target. In contrast, acquired resistance to both VHL- and CRBN-based BET-PROTACs was primarily caused by genomic alterations that compromise core components of the relevant E3 ligase complexes.

Distinct evolution and dynamics of epigenetic and genetic heterogeneity in acute myeloid leukemia.

Genetic heterogeneity contributes to clinical outcome and progression of most tumors, but little is known about allelic diversity for epigenetic compartments, and almost no data exist for acute myeloid leukemia (AML). We examined epigenetic heterogeneity as assessed by cytosine methylation within defined genomic loci with four CpGs (epialleles), somatic mutations, and transcriptomes of AML patient samples at serial time points. We observed that epigenetic allele burden is linked to inferior outcome and varies considerably during disease progression. Epigenetic and genetic allelic burden and patterning followed different patterns and kinetics during disease progression. We observed a subset of AMLs with high epiallele and low somatic mutation burden at diagnosis, a subset with high somatic mutation and lower epiallele burdens at diagnosis, and a subset with a mixed profile, suggesting distinct modes of tumor heterogeneity. Genes linked to promoter-associated epiallele shifts during tumor progression showed increased single-cell transcriptional variance and differential expression, suggesting functional impact on gene regulation. Thus, genetic and epigenetic heterogeneity can occur with distinct kinetics likely to affect the biological and clinical features of tumors.

Geospatial Resolution of Human and Bacterial Diversity with City-Scale Metagenomics.

The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station's history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of cities.

Cell-cycle reprogramming for PI3K inhibition overrides a relapse-specific C481S BTK mutation revealed by longitudinal functional genomics in mantle cell lymphoma.

UNLABELLED: Despite the unprecedented clinical activity of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in mantle cell lymphoma (MCL), acquired resistance is common. By longitudinal integrative whole-exome and whole-transcriptome sequencing and targeted sequencing, we identified the first relapse-specific C481S mutation at the ibrutinib binding site of BTK in MCL cells at progression following a durable response. This mutation enhanced BTK and AKT activation and tissue-specific proliferation of resistant MCL cells driven by CDK4 activation. It was absent, however, in patients with primary resistance or progression following transient response to ibrutinib, suggesting alternative mechanisms of resistance. Through synergistic induction of PIK3IP1 and inhibition of PI3K-AKT activation, prolonged early G1 arrest induced by PD 0332991 (palbociclib) inhibition of CDK4 sensitized resistant lymphoma cells to ibrutinib killing when BTK was unmutated, and to PI3K inhibitors independent of C481S mutation. These data identify a genomic basis for acquired ibrutinib resistance in MCL and suggest a strategy to override both primary and acquired ibrutinib resistance. SIGNIFICANCE: We have discovered the first relapse-specific BTK mutation in patients with MCL with acquired resistance, but not primary resistance, to ibrutinib, and demonstrated a rationale for targeting the proliferative resistant MCL cells by inhibiting CDK4 and the cell cycle in combination with ibrutinib in the presence of BTK(WT) or a PI3K inhibitor independent of BTK mutation. As drug resistance remains a major challenge and CDK4 and PI3K are dysregulated at a high frequency in human cancers, targeting CDK4 in genome-based combination therapy represents a novel approach to lymphoma and cancer therapy. Cancer Discov; 4(9); 1022-35. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973.

Dramatic fertility decline in aging C. elegans males is associated with mating execution deficits rather than diminished sperm quality.

Although much is known about female reproductive aging, fairly little is known about the causes of male reproductive senescence. We developed a method that facilitates culture maintenance of Caenorhabditis elegans adult males, which enabled us to measure male fertility as populations age, without profound loss of males from the growth plate. We find that the ability of males to sire progeny declines rapidly in the first half of adult lifespan and we examined potential factors that contribute towards reproductive success, including physical vigor, sperm quality, mating apparatus morphology, and mating ability. Of these, we find little evidence of general physical decline in males or changes in sperm number, morphology, or capacity for activation, at time points when reproductive senescence is markedly evident. Rather, it is the loss of efficient mating ability that correlates most strongly with reproductive senescence. Low insulin signaling can extend male ability to sire progeny later in life, although insulin impact on individual facets of mating behavior is complex. Overall, we suggest that combined modest deficits, predominantly affecting the complex mating behavior rather than sperm quality, sum up to block effective C. elegans male reproduction in middle adult life.

An Antioxidant potential of Hingot and Hadjod.

The antioxidant activity of two plants - Hadjod i.e. Cissus quadrangularis (CQ) and Hingot i.e. Balanites aegyptiaca (BA) was determined by the thiocyanate method. The antioxidant activity of both the plants increased with increasing amount of extract (200 g-1000 g) added to the linoleic emulsion. The ethanolic extract of CQ was more effective than the other. Like antioxidant activity, the reducing power was also dependent upon the concentration. The ethanolic extract of BA shows more reducing power than the other. The result obtained in the present study indicates that the both the plants are potential source of natural antioxidants. In addition, we could suggest that although the reducing power of a substance may be an indicator of its potential antioxidant activity, there is not necessarily a linear correlation between these two activities.