ExpansionHunter Denovo: a computational method for locating known and novel repeat expansions in short-read sequencing data.

Repeat expansions are responsible for over 40 monogenic disorders, and undoubtedly more pathogenic repeat expansions remain to be discovered. Existing methods for detecting repeat expansions in short-read sequencing data require predefined repeat catalogs. Recent discoveries emphasize the need for methods that do not require pre-specified candidate repeats. To address this need, we introduce ExpansionHunter Denovo, an efficient catalog-free method for genome-wide repeat expansion detection. Analysis of real and simulated data shows that our method can identify large expansions of 41 out of 44 pathogenic repeats, including nine recently reported non-reference repeat expansions not discoverable via existing methods.

Genome sequencing in persistently unsolved white matter disorders.

Genetic white matter disorders have heterogeneous etiologies and overlapping clinical presentations. We performed a study of the diagnostic efficacy of genome sequencing in 41 unsolved cases with prior exome sequencing, resolving an additional 14 from an historical cohort (n = 191). Reanalysis in the context of novel disease-associated genes and improved variant curation and annotation resolved 64% of cases. The remaining diagnoses were directly attributable to genome sequencing, including cases with small and large copy number variants (CNVs) and variants in deep intronic and technically difficult regions. Genome sequencing, in combination with other methodologies, achieved a diagnostic yield of 85% in this retrospective cohort.

Copy-number variants in clinical genome sequencing: deployment and interpretation for rare and undiagnosed disease.

PURPOSE: Current diagnostic testing for genetic disorders involves serial use of specialized assays spanning multiple technologies. In principle, genome sequencing (GS) can detect all genomic pathogenic variant types on a single platform. Here we evaluate copy-number variant (CNV) calling as part of a clinically accredited GS test. METHODS: We performed analytical validation of CNV calling on 17 reference samples, compared the sensitivity of GS-based variants with those from a clinical microarray, and set a bound on precision using orthogonal technologies. We developed a protocol for family-based analysis of GS-based CNV calls, and deployed this across a clinical cohort of 79 rare and undiagnosed cases. RESULTS: We found that CNV calls from GS are at least as sensitive as those from microarrays, while only creating a modest increase in the number of variants interpreted (~10 CNVs per case). We identified clinically significant CNVs in 15% of the first 79 cases analyzed, all of which were confirmed by an orthogonal approach. The pipeline also enabled discovery of a uniparental disomy (UPD) and a 50% mosaic trisomy 14. Directed analysis of select CNVs enabled breakpoint level resolution of genomic rearrangements and phasing of de novo CNVs. CONCLUSION: Robust identification of CNVs by GS is possible within a clinical testing environment.

Multiple Routes to Oncogenesis Are Promoted by the Human Papillomavirus-Host Protein Network.

We have mapped a global network of virus-host protein interactions by purification of the complete set of human papillomavirus (HPV) proteins in multiple cell lines followed by mass spectrometry analysis. Integration of this map with tumor genome atlases shows that the virus targets human proteins frequently mutated in HPV- but not HPV+ cancers, providing a unique opportunity to identify novel oncogenic events phenocopied by HPV infection. For example, we find that the NRF2 transcriptional pathway, which protects against oxidative stress, is activated by interaction of the NRF2 regulator KEAP1 with the viral protein E1. We also demonstrate that the L2 HPV protein physically interacts with the RNF20/40 histone ubiquitination complex and promotes tumor cell invasion in an RNF20/40-dependent manner. This combined proteomic and genetic approach provides a systematic means to study the cellular mechanisms hijacked by virally induced cancers.Significance: In this study, we created a protein-protein interaction network between HPV and human proteins. An integrative analysis of this network and 800 tumor mutation profiles identifies multiple oncogenesis pathways promoted by HPV interactions that phenocopy recurrent mutations in cancer, yielding an expanded definition of HPV oncogenic roles. Cancer Discov; 8(11); 1474-89. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333.

Epigenetic aging signatures in mice livers are slowed by dwarfism, calorie restriction and rapamycin treatment.

BACKGROUND: Global but predictable changes impact the DNA methylome as we age, acting as a type of molecular clock. This clock can be hastened by conditions that decrease lifespan, raising the question of whether it can also be slowed, for example, by conditions that increase lifespan. Mice are particularly appealing organisms for studies of mammalian aging; however, epigenetic clocks have thus far been formulated only in humans. RESULTS: We first examined whether mice and humans experience similar patterns of change in the methylome with age. We found moderate conservation of CpG sites for which methylation is altered with age, with both species showing an increase in methylome disorder during aging. Based on this analysis, we formulated an epigenetic-aging model in mice using the liver methylomes of 107 mice from 0.2 to 26.0 months old. To examine whether epigenetic aging signatures are slowed by longevity-promoting interventions, we analyzed 28 additional methylomes from mice subjected to lifespan-extending conditions, including Prop1df/df dwarfism, calorie restriction or dietary rapamycin. We found that mice treated with these lifespan-extending interventions were significantly younger in epigenetic age than their untreated, wild-type age-matched controls. CONCLUSIONS: This study shows that lifespan-extending conditions can slow molecular changes associated with an epigenetic clock in mice livers.

Interaction Landscape of Inherited Polymorphisms with Somatic Events in Cancer.

Recent studies have characterized the extensive somatic alterations that arise during cancer. However, the somatic evolution of a tumor may be significantly affected by inherited polymorphisms carried in the germline. Here, we analyze genomic data for 5,954 tumors to reveal and systematically validate 412 genetic interactions between germline polymorphisms and major somatic events, including tumor formation in specific tissues and alteration of specific cancer genes. Among germline-somatic interactions, we found germline variants in RBFOX1 that increased incidence of SF3B1 somatic mutation by 8-fold via functional alterations in RNA splicing. Similarly, 19p13.3 variants were associated with a 4-fold increased likelihood of somatic mutations in PTEN. In support of this association, we found that PTEN knockdown sensitizes the MTOR pathway to high expression of the 19p13.3 gene GNA11 Finally, we observed that stratifying patients by germline polymorphisms exposed distinct somatic mutation landscapes, implicating new cancer genes. This study creates a validated resource of inherited variants that govern where and how cancer develops, opening avenues for prevention research.Significance: This study systematically identifies germline variants that directly affect tumor evolution, either by dramatically increasing alteration frequency of specific cancer genes or by influencing the site where a tumor develops. Cancer Discovery; 7(4); 410-23. ©2017 AACR.See related commentary by Geeleher and Huang, p. 354This article is highlighted in the In This Issue feature, p. 339.

A Network of Conserved Synthetic Lethal Interactions for Exploration of Precision Cancer Therapy.

An emerging therapeutic strategy for cancer is to induce selective lethality in a tumor by exploiting interactions between its driving mutations and specific drug targets. Here we use a multi-species approach to develop a resource of synthetic lethal interactions relevant to cancer therapy. First, we screen in yeast ∼169,000 potential interactions among orthologs of human tumor suppressor genes (TSG) and genes encoding drug targets across multiple genotoxic environments. Guided by the strongest signal, we evaluate thousands of TSG-drug combinations in HeLa cells, resulting in networks of conserved synthetic lethal interactions. Analysis of these networks reveals that interaction stability across environments and shared gene function increase the likelihood of observing an interaction in human cancer cells. Using these rules, we prioritize ∼10(5) human TSG-drug combinations for future follow-up. We validate interactions based on cell and/or patient survival, including topoisomerases with RAD17 and checkpoint kinases with BLM.

Methylome-wide Analysis of Chronic HIV Infection Reveals Five-Year Increase in Biological Age and Epigenetic Targeting of HLA.

HIV-infected individuals are living longer on antiretroviral therapy, but many patients display signs that in some ways resemble premature aging. To investigate and quantify the impact of chronic HIV infection on aging, we report a global analysis of the whole-blood DNA methylomes of 137 HIV+ individuals under sustained therapy along with 44 matched HIV- individuals. First, we develop and validate epigenetic models of aging that are independent of blood cell composition. Using these models, we find that both chronic and recent HIV infection lead to an average aging advancement of 4.9 years, increasing expected mortality risk by 19%. In addition, sustained infection results in global deregulation of the methylome across >80,000 CpGs and specific hypomethylation of the region encoding the human leukocyte antigen locus (HLA). We find that decreased HLA methylation is predictive of lower CD4 / CD8 T cell ratio, linking molecular aging, epigenetic regulation, and disease progression.

Analysis of Matched Tumor and Normal Profiles Reveals Common Transcriptional and Epigenetic Signals Shared across Cancer Types.

To identify the transcriptional regulatory changes that are most widespread in solid tumors, we performed a pan-cancer analysis using over 600 pairs of tumors and adjacent normal tissues profiled in The Cancer Genome Atlas (TCGA). Frequency of upregulation was calculated across mRNA expression levels, microRNA expression levels and CpG methylation sites and is provided here as a resource. Frequent tumor-associated alterations were identified using a simple statistical approach. Many of the identified changes were consistent with the increased rate of cell division in cancer, such as the overexpression of cell cycle genes and hypermethylation of PRC2 binding sites. However, we also identified proliferation-independent alterations, which highlight novel pathways essential to tumor formation. Nearly all of the GABA receptors are frequently downregulated, with the gene encoding the delta subunit (GABRD) strongly upregulated as the notable exception. Metabolic genes are also frequently downregulated, particularly alcohol dehydrogenases and others consistent with the decreased role of oxidative phosphorylation in cancerous cells. Alterations in the composition of GABA receptors and metabolism may play a key role in the differentiation of cancer cells, independent of proliferation.

ERCC1 and TS Expression as Prognostic and Predictive Biomarkers in Metastatic Colon Cancer.

In patients with metastatic colon cancer, response to first line chemotherapy is a strong predictor of overall survival (OS). Currently, oncologists lack diagnostic tests to determine which chemotherapy regimen offers the greatest chance for response in an individual patient. Here we present the results of gene expression analysis for two genes, ERCC1 and TS, measured with the commercially available ResponseDX: Colon assay (Response Genetics, Los Angeles, CA) in 41 patients with de novo metastatic colon cancer diagnosed between July 2008 and August 2013 at the University of California, San Diego. In addition ERCC1 and TS expression levels as determined by RNAseq and survival data for patients in TCGA were downloaded from the TCGA data portal. We found that patients with low expression of ERCC1 (n = 33) had significantly longer median OS (36.0 vs. 10.1 mo, HR 0.29, 95% CI .095 to .84, log-rank p = 9.0x10-6) and median time to treatment to failure (TTF) following first line chemotherapy (14.1 vs. 2.4 mo, HR 0.17, 95% CI 0.048 to 0.58, log-rank p = 5.3x10-4) relative to those with high expression (n = 4). After accounting for the covariates age, sex, tumor grade and ECOG performance status in a Cox proportional hazard model the association of low ERCC1 with longer OS (HR 0.18, 95% CI 0.14 to 0.26, p = 0.0448) and TTF (HR 0.16, 95% CI 0.14 to 0.21, p = 0.0053) remained significant. Patients with low TS expression (n = 29) had significantly longer median OS (36.0 vs. 14.8 mo, HR 0.25, 95% CI 0.074 to 0.82, log-rank p = 0.022) relative to those with high expression (n = 12). The combined low expression of ERCC1/TS was predictive of response in patients treated with FOLFOX (40% vs. 91%, RR 2.3, Fisher's exact test p = 0.03, n = 27), but not with FOLFIRI (71% vs. 71%, RR 1.0, Fisher's exact test p = 1, n = 14). Overall, these findings suggest that measurement of ERCC1 and TS expression has potential clinical utility in managing patients with metastatic colorectal cancer.

Combined TP53 mutation/3p loss correlates with decreased radiosensitivity and increased matrix-metalloproteinase activity in head and neck carcinoma.

OBJECTIVE: Patients with head and neck squamous cell carcinoma (HNSCC) containing TP53 mutation and 3p deletion ("double-hit") have poorer prognosis compared to patients with either event alone ("single-hit"). The etiology for worse clinical outcomes in patients with "double-hit" cancers is unclear. We compared radiosensitivity of cell lines containing both TP53 mutations and deletion of Fragile Histidine Triad (FHIT, the gene most commonly associated with 3p deletion) to "single-hit" lines with only TP53 mutation. We compared radiosensitivity in a "single-hit" cell line with TP53 mutation converted to "double-hit" using RNA interference targeting FHIT. Finally, we compared matrixmetalloproteinase-2/9 (MMP-2/9) activity, a previously-established biomarker for tumor aggressiveness, in xenograft tumors derived from these cell lines. MATERIALS/METHODS: TP53 mutation and FHIT deletion profiles of HNSCC lines were established using Cancer Cell Line Encyclopedia (CCLE). We used RNA-interference to convert a "single-hit" cell line (SCC4) to "double-hit". Cultured cells were examined for radiosensitivity and cisplatin sensitivity. MMP-2/9 activity was evaluated in "double-hit" versus "single-hit" tumors using ratiometric activatable cell-penetrating peptide (RACPP) in tongue (n=17) and flank xenografts (n=4). RESULTS: Radiotherapy caused greater double-stranded DNA breaks in "single-hit" vs naturally occurring and engineered "double-hit" cells. In-vivo, "double-hit" xenografts demonstrated higher MMP-2/9 activity compared to "single-hit" xenografts (p<0.01). There was no difference in cisplatin sensitivity between the cell lines. CONCLUSIONS: TP53 mutation combined with FHIT deletion correlates with decreased radiosensitivity in HNC cell lines. Xenograft from "double-hit" cells exhibit increased MMP-2/9 activity. These findings may in part account for the worse clinical outcome seen in patients with HNSCC "double-hit" tumors.

Multi-tiered genomic analysis of head and neck cancer ties TP53 mutation to 3p loss.

Head and neck squamous cell carcinoma (HNSCC) is characterized by aggressive behavior with a propensity for metastasis and recurrence. Here we report a comprehensive analysis of the molecular and clinical features of HNSCC that govern patient survival. We find that TP53 mutation is frequently accompanied by loss of chromosome 3p and that the combination of these events is associated with a surprising decrease in survival time (1.9 years versus >5 years for TP53 mutation alone). The TP53-3p interaction is specific to chromosome 3p and validates in HNSCC and pan-cancer cohorts. In human papillomavirus (HPV)-positive tumors, in which HPV inactivates TP53, 3p deletion is also common and is associated with poor outcomes. The TP53-3p event is modified by mir-548k expression, which decreases survival further, and is mutually exclusive with mutations affecting RAS signaling. Together, the identified markers underscore the molecular heterogeneity of HNSCC and enable a new multi-tiered classification of this disease.

Matrix-metalloproteinases in head and neck carcinoma-cancer genome atlas analysis and fluorescence imaging in mice.

OBJECTIVE: (1) Obtain matrix-metalloproteinase (MMP) expression profiles for head and neck squamous cell carcinoma (HNSCC) specimens from the Cancer Genomic Atlas (TCGA). (2) Demonstrate HNSCC imaging using MMP-cleavable, fluorescently labeled ratiometric activatable cell-penetrating peptide (RACPP). STUDY DESIGN: Retrospective human cohort study; prospective animal study. SETTING: Translational research laboratory. SUBJECTS AND METHODS: Patient clinical data and mRNA expression levels of MMP genes were downloaded from TCGA data portal. RACPP provides complementary ratiometric fluorescent contrast (increased Cy5 and decreased Cy7 intensities) when cleaved by MMP2/9. HNSCC-tumor bearing mice were imaged in vivo after RACPP injection. Histology was evaluated by a pathologist blinded to experimental conditions. Zymography confirmed MMP-2/9 activity in xenografts. RACPP was applied to homogenized human HNSCC specimens, and ratiometric fluorescent signal was measured on a microplate reader for ex vivo analysis. RESULTS: Expression of multiple MMPs including MMP2/9 is greater in patient HNSCC tumors than matched control tissue. In patients with human papilloma virus positive (HPV+) tumors, higher MMP2 and MMP14 expression correlates with worse 5-year survival. Orthotopic tongue HNSCC xenografts showed excellent ratiometric fluorescent labeling with MMP2/9-cleavable RACPP (sensitivity = 95.4%, specificity = 95.0%). Fluorescence ratios were greater in areas of higher tumor burden (P < .03), which is useful for intraoperative margin assessment. Ex vivo, human HNSCC specimens showed greater cleavage of RACPP when compared to control tissue (P = .009). CONCLUSIONS: Human HNSCC tumors show increased mRNA expression of multiple MMPs including MMP2/9. We used RACPP, a ratiometric fluorescence assay of MMP2/9 activity, to show improved occult tumor identification and margin clearance. Ex vivo assays using RACPP in biopsy specimens may identify patients who will benefit from intraoperative RACPP use.

Brevity of haptic force perturbations induces heightened adaptive sensitivity.

We have exposed human participants to both full-movement and pulsatile viscous force perturbations to study the effect of force duration on the incremental transformation of sensation into adaptation. Traditional views of movement biomechanics could suggest that pulsatile forces would largely be attenuated as stiffness and viscosity act as a natural low-pass filter. Sensory transduction, however, tends to react to changes in stimuli and therefore could underlie heightened sensitivity to briefer, pulsatile forces. Here, participants adapted within perturbation duration conditions in a manner proportionate to sensed force and positional errors. Across perturbation conditions, we found participants had greater adaptive sensitivity when experiencing pulsatile forces rather than full-movement forces. In a follow-up experiment, we employed error-clamped, force channel trials to determine changes in predictive force generation. We found that while participants learned to closely compensate for the amplitude and breadth of full-movement forces, they exhibited a persistent mismatch in amplitude and breadth between adapted motor output and experienced pulsatile forces. This mismatch could generate higher salience of error signals that contribute to heightened sensitivity to pulsatile forces.

Arabidopsis circadian clock protein, TOC1, is a DNA-binding transcription factor.

The first described feedback loop of the Arabidopsis circadian clock is based on reciprocal regulation between Timing of CAB Expression 1 (TOC1) and Circadian Clock-associated 1 (CCA1)/late elongated hypocotyl (LHY). CCA1 and LHY are Myb transcription factors that bind directly to the TOC1 promoter to negatively regulate its expression. Conversely, the activity of TOC1 has remained less well characterized. Genetic data support that TOC1 is necessary for the reactivation of CCA1/LHY, but there is little description of its biochemical function. Here we show that TOC1 occupies specific genomic regions in the CCA1 and LHY promoters. Purified TOC1 binds directly to DNA through its CCT domain, which is similar to known DNA-binding domains. Chemical induction and transient overexpression of TOC1 in Arabidopsis seedlings cause repression of CCA1/LHY expression, demonstrating that TOC1 can repress direct targets, and mutation or deletion of the CCT domain prevents this repression showing that DNA-binding is necessary for TOC1 action. Furthermore, we use the Gal4/UAS system in Arabidopsis to show that TOC1 acts as a general transcriptional repressor, and that repression activity is in the pseudoreceiver domain of the protein. To identify the genes regulated by TOC1 on a genomic scale, we couple TOC1 chemical induction with microarray analysis and identify previously unexplored potential TOC1 targets and output pathways. Taken together, these results define a biochemical action for the core clock protein TOC1 and refine our perspective on how plant clocks function.

Tensor decomposition reveals concurrent evolutionary convergences and divergences and correlations with structural motifs in ribosomal RNA.

Evolutionary relationships among organisms are commonly described by using a hierarchy derived from comparisons of ribosomal RNA (rRNA) sequences. We propose that even on the level of a single rRNA molecule, an organism's evolution is composed of multiple pathways due to concurrent forces that act independently upon different rRNA degrees of freedom. Relationships among organisms are then compositions of coexisting pathway-dependent similarities and dissimilarities, which cannot be described by a single hierarchy. We computationally test this hypothesis in comparative analyses of 16S and 23S rRNA sequence alignments by using a tensor decomposition, i.e., a framework for modeling composite data. Each alignment is encoded in a cuboid, i.e., a third-order tensor, where nucleotides, positions and organisms, each represent a degree of freedom. A tensor mode-1 higher-order singular value decomposition (HOSVD) is formulated such that it separates each cuboid into combinations of patterns of nucleotide frequency variation across organisms and positions, i.e., "eigenpositions" and corresponding nucleotide-specific segments of "eigenorganisms," respectively, independent of a-priori knowledge of the taxonomic groups or rRNA structures. We find, in support of our hypothesis that, first, the significant eigenpositions reveal multiple similarities and dissimilarities among the taxonomic groups. Second, the corresponding eigenorganisms identify insertions or deletions of nucleotides exclusively conserved within the corresponding groups, that map out entire substructures and are enriched in adenosines, unpaired in the rRNA secondary structure, that participate in tertiary structure interactions. This demonstrates that structural motifs involved in rRNA folding and function are evolutionary degrees of freedom. Third, two previously unknown coexisting subgenic relationships between Microsporidia and Archaea are revealed in both the 16S and 23S rRNA alignments, a convergence and a divergence, conferred by insertions and deletions of these motifs, which cannot be described by a single hierarchy. This shows that mode-1 HOSVD modeling of rRNA alignments might be used to computationally predict evolutionary mechanisms.