Non-synonymous Single Nucleotide Variants Research Articles

Association of Variants in BAG3 With Cardiomyopathy Outcomes in African American Individuals

The prevalence of nonischemic dilated cardiomyopathy (DCM) is greater in individuals of African ancestry than in individuals of European ancestry. However, little is known about whether the difference in prevalence or outcomes is associated with functional genetic variants. We hypothesized that Bcl2-associated anthanogene 3 (BAG3) genetic variants were associated with outcomes in individuals of African ancestry with DCM. This multicohort study of the BAG3 genotype in patients of African ancestry with dilated cardiomyopathy uses DNA obtained from African American individuals enrolled in 3 clinical studies: the Genetic Risk Assessment of African Americans With Heart Failure (GRAHF) study; the Intervention in Myocarditis and Acute Cardiomyopathy Trial-2 (IMAC-2) study; and the Genetic Risk Assessment of Cardiac Events (GRACE) study. Samples of DNA were also acquired from the left ventricular myocardium of patients of African ancestry who underwent heart transplant at the University of Colorado and University of Pittsburgh. The primary end points were the prevalence of BAG3 mutations in African American individuals and event-free survival in participants harboring functional BAG3 mutations. Four BAG3 genetic variants were identified; these were expressed in 42 of 402 African American individuals (10.4%) with nonischemic heart failure and 9 of 107 African American individuals (8.4%) with ischemic heart failure but were not present in a reference population of European ancestry (P < .001). The variants included 2 nonsynonymous single-nucleotide variants; 1 three-nucleotide in-frame insertion; and 2 single-nucleotide variants that were linked in cis. The presence of BAG3 variants was associated with a nearly 2-fold (hazard ratio, 1.97 [95% CI, 1.19-3.24]; P = .01) increase in cardiac events in carriers compared with noncarriers. Transfection of transformed adult human ventricular myocytes with plasmids expressing the 4 variants demonstrated that each variant caused an increase in apoptosis and a decrease in autophagy when samples were subjected to the stress of hypoxia-reoxygenation. This study demonstrates that genetic variants in BAG3 found almost exclusively in individuals of African ancestry were not causative of disease but were associated with a negative outcome in patients with a dilated cardiomyopathy through modulation of the function of BAG3. The results emphasize the importance of biological differences in causing phenotypic variance across diverse patient populations, the need to include diverse populations in genetic cohorts, and the importance of determining the pathogenicity of genetic variants.

Targeted next-generation sequencing as a comprehensive test for Mendelian diseases: a cohort diagnostic study

With the development of next generation sequencing, more and more common inherited diseases have been reported. However, accurate and convenient molecular diagnosis cannot be achieved easily because of the enormous size of disease causing mutations. In this study, we introduced a new single-step method for the genetic analysis of patients and carriers in real clinical settings. All kinds of disease causing mutations can be detected at the same time in patients with Mendelian diseases or carriers. First, we evaluated this technology using YH cell line DNA and 9 samples with known mutations. Accuracy and stability of 99.80% and 99.58% were achieved respectively. Then, a total of 303 patients were tested using our targeted NGS approaches, 50.17% of which were found to have deleterious mutations and molecular confirmation of the clinical diagnosis. We identified 219 disease causing mutations, 43.84% (96/219) of which has never been reported before. Additionally, we developed a new deleteriousness prediction method for nonsynonymous SNVs, and an automating annotation and diagnosis system for Mendelian diseases, thus greatly assisting and enhancing Mendelian diseases diagnosis and helping to make a precise diagnosis for patients with Mendelian diseases.

Pan-Cancer Analysis Reveals the Functional Importance of Protein Lysine Modification in Cancer Development.

Large-scale tumor genome sequencing projects have revealed a complex landscape of genomic mutations in multiple cancer types. A major goal of these projects is to characterize somatic mutations and discover cancer drivers, thereby providing important clues to uncover diagnostic or therapeutic targets for clinical treatment. However, distinguishing only a few somatic mutations from the majority of passenger mutations is still a major challenge facing the biological community. Fortunately, combining other functional features with mutations to predict cancer driver genes is an effective approach to solve the above problem. Protein lysine modifications are an important functional feature that regulates the development of cancer. Therefore, in this work, we have systematically analyzed somatic mutations on seven protein lysine modifications and identified several important drivers that are responsible for tumorigenesis. From published literature, we first collected more than 100,000 lysine modification sites for analysis. Another 1 million non-synonymous single nucleotide variants (SNVs) were then downloaded from TCGA and mapped to our collected lysine modification sites. To identify driver proteins that significantly altered lysine modifications, we further developed a hierarchical Bayesian model and applied the Markov Chain Monte Carlo (MCMC) method for testing. Strikingly, the coding sequences of 473 proteins were found to carry a higher mutation rate in lysine modification sites compared to other background regions. Hypergeometric tests also revealed that these gene products were enriched in known cancer drivers. Functional analysis suggested that mutations within the lysine modification regions possessed higher evolutionary conservation and deleteriousness. Furthermore, pathway enrichment showed that mutations on lysine modification sites mainly affected cancer related processes, such as cell cycle and RNA transport. Moreover, clinical studies also suggested that the driver proteins were significantly associated with patient survival, implying an opportunity to use lysine modifications as molecular markers in cancer diagnosis or treatment. By searching within protein-protein interaction networks using a random walk with restart (RWR) algorithm, we further identified a series of potential treatment agents and therapeutic targets for cancer related to lysine modifications. Collectively, this study reveals the functional importance of lysine modifications in cancer development and may benefit the discovery of novel mechanisms for cancer treatment.

Combination of Cytokine-Induced Killer Cells and Programmed Cell Death-1 Blockade Works Synergistically to Enhance Therapeutic Efficacy in Metastatic Renal Cell Carcinoma and Non-Small Cell Lung Cancer.

Programmed cell death-1 (PD-1) inhibition therapy has changed the treatment paradigm of metastatic renal cell carcinoma (MRCC) and non-small cell lung cancer (NSCLC). However, attempts to use the drug as a single agent have achieved only limited clinical success. To further enhance the clinical benefits of monotherapy, combination therapies will likely be necessary. Cytokine-induced killer (CIK) cells are a heterogeneous subset of ex vivo expanded T lymphocytes that have been shown to prolong the survival of cancer patients. We are conducting a study to evaluate the efficacy of PD-1 inhibitor in combination with CIK cells in relapsed/refractory MRCC and NSCLC and to analyze potential biomarkers to predict which patients will benefit most from the combined therapy. The results of two patients treated in an ongoing clinical trial for MRCC and NSCLC are described here. The tumor biopsy from Patient 1 exhibited moderate CD3+ T cell infiltration, but no PD-1 or PD-L1 expression. The tumor cells from Patient 2 strongly expressed PD-L1, and there was extensive tumor infiltration by CD3+ T cells; however, no PD-1 staining was seen. Non-synonymous single nucleotide variant (nsSNVs), along with higher indel mutations, in Patient 1 and nsSNVs along with higher tumor mutation burden in Patient 2 correlate with tumor-infiltrating CD3+ lymphocyte density. Patient 1 achieved a complete response, and Patient 2 achieved a near-complete response. A PD-1 inhibitor in combination with CIK cells led to potent antitumor activity in MRCC and NSCLC; CD3+ T cell infiltration in baseline tumor biopsies is a potential predictive biomarker. This approach is being further investigated in an ongoing phase I trial.

CHCHD10 variants in amyotrophic lateral sclerosis: Where is the evidence?

After the initial report of a CHCHD10 mutation in mitochondrial disease with features resembling amyotrophic lateral sclerosis (ALS), CHCHD10 mutations have been considered to be a frequent cause for ALS. However, the exact pathogenicity and clinical significance of these mutations remain unclear. Here, we aimed to determine the role of CHCHD10 mutations in ALS. We analyzed 4,365 whole genome sequenced ALS patients and 1,832 controls from 7 different countries and examined all nonsynonymous single nucleotide variants in CHCHD10. These were tested for association with ALS, independently and in aggregate using several genetic burden tests (including sequence kernel association test [SKAT], optimal unified test [SKAT-O], and Firth logistic regression). We identified 3 new variants in cases, but only 1 was ALS-specific. Also, 1 control-specific mutation was identified. There was no increased burden of rare coding mutations among ALS patients compared to controls (p = 0.86, p = 0.86, and p = 0.88 for SKAT, SKAT-O, and Firth, respectively). The few carriers with potential pathogenic CHCHD10 mutations exhibited a slowly progressive ALS-like phenotype with atypical features such as myopathy and deafness. CHCHD10 mutations seem to be a far less prevalent cause of pure ALS than previously suggested, and instead appear related to more complex phenotypes. There appears to be insufficient evidence for the pathogenicity of most previously reported variants in pure ALS. This study shows that routine testing for CHCHD10 mutations in pure ALS is not recommended and illustrates the importance of sufficient genetic and functional evidence in establishing pathogenicity of genetic variants. Ann Neurol 2018;83:110-116.

Abstract 3631: Passenger fusion genes are novel predictive biomarker of anti-PD-1 antibody nivolumab for ovarian cancer

Abstract Background: Immune checkpoint PD-1 signal blockade provides significant clinical efficacy and durable response for several cancer patients. However it is crucial to identify a biomarker to predict the response to PD-1 signal blockade. Herein we analyzed the somatic mutanomes and transcriptomes of tumor samples from ovarian cancer patients treated with nivolumab to identify potential biomarkers of antitumor response. Methods: We previously reported a phase II clinical trial for resistant ovarian cancer with nivolumab (UMIN000005714) and showed disease control rate was 45% including 2 complete responders. To estimate the somatic mutation burden (TMB) of tumor samples, the number of non-synonymous single nucleotide variants (nsSNVs), insertions and deletions of genes was analyzed by whole exome sequencing of formalin fixed paraffin embedded tumor tissues. Next, to evaluate whole transcriptome of these samples, we performed an RNAseq analysis and detected several fusion genes in some patients. We evaluated the correlation between clinical anti-tumor response for nivolumab treatment with the numbers of somatic mutations or transcriptomic alignments containing gene fusions. Results: 16 tumor samples (7 responders, 9 non-responders and 1 loss) for whole exome sequencing and 17 samples (7 responders and 10 non-responders) for RNA sequencing were available. Contrary to expectations generated by previously reported papers in melanoma or lung cancers, the levels of TMB including nsSNVs, insertions and deletions of genes were not significantly correlated to antitumor response. However, more than 2 fusion genes were detected (mean=6.3, range 0-15) in 6 of 7 responders, while 8 of 10 non-responders did not have any fusion genes and the remaining non-responders had only 1 fusion gene each (mean=0.2, range, 0-1). Therefore an antitumor response to nivolumab was strongly correlated to the number of fusion genes in ovarian cancer (p=0.0003, sensitivity: 86% [6/7] specificity: 100% [10/10]). Conclusions: This study, to our knowledge, is the first to describe passenger fusion genes of tumors, and might provide a clinically useful and novel predictive biomarker of antitumor response to anti-PD-1/PD-L1 antibody therapy for ovarian cancer. Citation Format: Junzo Hamanishi, Ryusuke Murakami, John B. Brown, Tsukasa Baba, Kaoru Abiko, Eiji Suzuki, Masaki Mandai. Passenger fusion genes are novel predictive biomarker of anti-PD-1 antibody nivolumab for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3631.

Abstract LB-196: EGFR-mutant lung adenocarcinomas mutation profiles reveal ARID1A might be a novel tyrosine kinase resistance pathway

Abstract Lung cancer has the highest mortality across all cancers in the world. Epidermal growth factor receptor (EGFR) is commonly mutated in lung adenocarcinomas from Asian non-smoking females. These tumors usually respond to EGFR tyrosine kinase inhibitors (EGFR-TKI), but the outcomes vary in different patients. Though EGFR T790M, amplification of MET and other mechanisms have been shown to confer EGFR-TKI resistance, the molecular mechanisms in around 30% of patients are still unknown. To uncover novel candidate pathways of EGFR-TKI resistance, we analyzed the whole exome mutation profiles of pre-treatment, EGFR T790M-free lung adenocarcinomas with typical activating mutations (L858R, exon 19 deletions). Amongst 19 non-responding (NR) tumors without significant shrinkage after gefitinib or erlotinib treatment, 67 cancer-related genes showed coding region non-synonymous single nucleotide variations (SNV) or insertion-deletions (indels) but they were not detected in 22 responding tumors. Gene set enrichment analysis (GSEA) showed 14 of the 67 genes were involved in the EGFR and WNT signaling network (false discovery rate q=1.14 e-8 for EGFR and q=4.3 e-9 for WNT), including well-known TKI resistance-related genes PIK3CA, PTEN and NF1. Notably, ARID1A, which encodes a subunit of the chromatin remodeling complex, showed nonsense mutations in 2 NR but not responding tumors, which were validated by Sanger sequencing. More than 40% of ARID1A mutations in lung adenocarcinomas recorded in COSMIC were nonsense mutations or frameshift indels. Loss of function mutations of ARID1A have also been reported in various cancers, together suggesting ARID1A might be a tumor suppressor but its involvement in EGFR mutant lung cancers is unknown. To investigate the potential role of ARID1A, its expression was first studied using Kaplan-Meier plotter. Low expression of ARID1A is associated with significantly better overall survival than high expression level in lung adenocarcinoma patients (p=8.5 e-7). To test whether loss of ARID1A could cause TKI resistance, ARID1A was knocked down by shRNA in HCC827 lung cancer cell line harboring EGFR exon19 deletion. MTT cell viability assay using gefitinib showed increase of IC50. Western blot assay further showed ARID1A knockdown increased phosphorylation of AKT at Ser473. Together, the findings suggest loss of ARID1A could lead to EGFR bypass and promote activation of the downstream PI3K/AKT pathway, leading to EGFR TKI resistance. Overall, our findings suggest EGFR-TKI resistance mutations could be detected before TKI drug selection. Resistant gene mutations present in pre-treatment tumors could help to predict the treatment response. More specifically, our study identified a promising gene list involved in EGFR-TKI resistance, and suggested ARID1A loss could be a novel EGFR-TKI resistance mechanism. Citation Format: Xuyuan Gao, Hang Xu, James CM Ho, Oscar SH Chan, Feng Xu, Junwen Wang, Victor HF Lee, Vicky PC Tin, Zhijie Xiao, Siqi Wang, Judy WP Yam, Maria P Wong. EGFR-mutant lung adenocarcinomas mutation profiles reveal ARID1A might be a novel tyrosine kinase resistance pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-196.

Toll-like receptor 4: a target for chemoprevention of hepatocellular carcinoma in obesity and steatohepatitis.

The incidence of hepatocellular carcinoma (HCC) associated with non-alcoholic fatty liver disease (NAFLD) is rapidly increasing. We aimed to elucidate the genetic basis of NAFLD-associated HCC and identify candidate targets for chemoprevention. Twenty HCC tumors, distant liver and matched tails from mice with hepatocyte-deletion of Pten (HepPten-) were subjected to whole-exome sequencing. A total of 162 genes with somatic non-synonymous single nucleotide variants or exonic small insertions and deletions in tumors were identified. Ingenuity Pathway Analysis of these 162 genes, further identified Toll-like receptor (TLR) 4, a key mediator of proinflammatory responses, and resatorvid, a TLR4 inhibitor, as the main causal networks of this dataset. Resatorvid treatment strongly prevented HCC development in these mice (p < 0.001). Remarkably, HCC patients with high tumoral TLR4 mRNA expression were more likely to be diagnosed with NAFLD and obese. TLR4 mRNA expression positively correlated with IL-6 and IL-10 mRNA expression in HCC tumors and the correlation was stronger in obese HCC patients. We have identified tumor mutation signatures and associated causal networks in NAFLD-associated HCC in HepPten- mice and further demonstrated the important role of TLR4 in promoting HCC development. This study also identified IL-6 and IL-10 as markers of TLR4 activation in HCC and subjects with NAFLD and obesity as the target population who would benefit from TLR4 inhibition treatment for HCC chemoprevention.

Evaluation of computational techniques for predicting non-synonymous single nucleotide variants pathogenicity

The human genetic diseases associated with many factors, one of these factors is the non-synonymous Single Nucleotide Variants (nsSNVs) cause single amino acid change with another resulting in protein function change leading to disease. Many computational techniques have been released to expect the impacts of amino acid alteration on protein function and classify mutations as pathogenic or neutral. Here in this article, we assessed the performance of eight techniques; FATHMM, SIFT, Provean, iFish, Mutation Assessor, PANTHER, SNAP2, and PON- P2 using a VaribenchSelectedPure dataset of 2144 pathogenic variants and 3777 neutral variants extracted from the free standard database “Varibench.” The first five techniques achieve (45.60–83.75) % specificity, (52.64–94.13) % sensitivity, (51.00–88.90) % AUC, and (49.76–88.24) % ACC on whole dataset, while all eight techniques achieve (36.54–77.88) % specificity, (50.00–75.00) % sensitivity, (51.00–76.40) % AUC, and (25.00–77.78) % ACC on random sample dataset. We also created a Meta classifier (CSTJ48) that combines FATHMM, iFish, and Mutation Assessor. It registers 96.33% specificity, 86.07% sensitivity, 91.20% AUC, and 91.89 ACC. By comparing the results, it's clear that FATHMM gives the highest performance over the seven individual techniques, where it achieves 83.75% and 77.88% specificity, 94.13%, and 75.00% sensitivity, 88.90% and 76.40% AUC, and 88.24% and 77.78% ACC on whole and random sample dataset, respectively. Also, the launched Meta classifier (CSTJ48) is outperforming over all the eight individual tools that compared here.

Stochastic processes constrain the within and between host evolution of influenza virus.

The evolutionary dynamics of influenza virus ultimately derive from processes that take place within and between infected individuals. Here we define influenza virus dynamics in human hosts through sequencing of 249 specimens from 200 individuals collected over 6290 person-seasons of observation. Because these viruses were collected from individuals in a prospective community-based cohort, they are broadly representative of natural infections with seasonal viruses. Consistent with a neutral model of evolution, sequence data from 49 serially sampled individuals illustrated the dynamic turnover of synonymous and nonsynonymous single nucleotide variants and provided little evidence for positive selection of antigenic variants. We also identified 43 genetically-validated transmission pairs in this cohort. Maximum likelihood optimization of multiple transmission models estimated an effective transmission bottleneck of 1-2 genomes. Our data suggest that positive selection is inefficient at the level of the individual host and that stochastic processes dominate the host-level evolution of influenza viruses.

Aneuploid CTC and CEC.

Conventional circulating tumor cell (CTC) detection technologies are restricted to large tumor cells (> white blood cells (WBCs)), or those unique carcinoma cells with double positive expression of surface epithelial cell adhesion molecule (EpCAM) for isolation, and intracellular structural protein cytokeratins (CKs) for identification. With respect to detecting the full spectrum of highly heterogeneous circulating rare cells (CRCs), including CTCs and circulating endothelial cells (CECs), it is imperative to develop a strategy systematically coordinating all tri-elements of nucleic acids, biomarker proteins, and cellular morphology, to effectively enrich and comprehensively identify CRCs. Accordingly, a novel strategy integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH), independent of cell size variation and free of hypotonic damage as well as anti-EpCAM perturbing, has been demonstrated to enable in situ phenotyping multi-protein expression, karyotyping chromosome aneuploidy, and detecting cytogenetic rearrangements of the ALK gene in non-hematologic CRCs. Symbolic non-synonymous single nucleotide variants (SNVs) of both the TP53 gene (P33R) in each single aneuploid CTCs, and the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor suppressor gene in each examined aneuploid CECs, were identified for the first time across patients with diverse carcinomas. Comprehensive co-detecting observable aneuploid CTCs and CECs by SE-iFISH, along with applicable genomic and/or proteomic single cell molecular profiling, are anticipated to facilitate elucidating how those disparate categories of aneuploid CTCs and CECs cross-talk and functionally interplay with tumor angiogenesis, therapeutic drug resistance, tumor progression, and cancer metastasis.

Screening for susceptibility genes in hereditary non-polyposis colorectal cancer.

In the present study, hereditary non-polyposis colorectal cancer (HNPCC) susceptibility genes were screened for using whole exome sequencing in 3 HNPCC patients from 1 family and using single nucleotide polymorphism (SNP) genotyping assays in 96 other colorectal cancer and control samples. Peripheral blood was obtained from 3 HNPCC patients from 1 family; the proband and the proband's brother and cousin. High-throughput sequencing was performed using whole exome capture technology. Sequences were aligned against the HAPMAP, dbSNP130 and 1,000 Genome Project databases. Reported common variations and synonymous mutations were filtered out. Non-synonymous single nucleotide variants in the 3 HNPCC patients were integrated and the candidate genes were identified. Finally, SNP genotyping was performed for the genes in 96 peripheral blood samples. In total, 60.4 Gb of data was retrieved from the 3 HNPCC patients using whole exome capture technology. Subsequently, according to certain screening criteria, 15 candidate genes were identified. Among the 96 samples that had been SNP genotyped, 92 were successfully genotyped for 15 gene loci, while genotyping for HTRA1 failed in 4 sporadic colorectal cancer patient samples. In 12 control subjects and 81 sporadic colorectal cancer patients, genotypes at 13 loci were wild-type, namely DDX20, ZFYVE26, PIK3R3, SLC26A8, ZEB2, TP53INP1, SLC11A1, LRBA, CEBPZ, ETAA1, SEMA3G, IFRD2 and FAT1. The CEP290 genotype was mutant in 1 sporadic colorectal cancer patient and was wild-type in all other subjects. A total of 5 of the 12 control subjects and 30 of the 81 sporadic colorectal cancer patients had a mutant HTRA1 genotype. In all 3 HNPCC patients, the same mutant genotypes were identified at all 15 gene loci. Overall, 13 potential susceptibility genes for HNPCC were identified, namely DDX20, ZFYVE26, PIK3R3, SLC26A8, ZEB2, TP53INP1, SLC11A1, LRBA, CEBPZ, ETAA1, SEMA3G, IFRD2 and FAT1.

MP54-16 URINARY EXOSOMAL AND CELL-FREE DNA DETECTS SOMATIC MUTATION AND COPY NUMBER ALTERATION IN UROTHELIAL CARCINOMA OF BLADDER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I1 Apr 2018MP54-16 URINARY EXOSOMAL AND CELL-FREE DNA DETECTS SOMATIC MUTATION AND COPY NUMBER ALTERATION IN UROTHELIAL CARCINOMA OF BLADDER Kwang Hyun Kim, Bong Suk Sim, and Dong Hyeon Lee Kwang Hyun KimKwang Hyun Kim More articles by this author , Bong Suk SimBong Suk Sim More articles by this author , and Dong Hyeon LeeDong Hyeon Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1707AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urothelial bladder canrcinoma (UBC) is characterized by a large number of genetic alteration. Urinary DNA is promising resources for liquid biopsy in urological malignancies. In this study, we performed genomic profiling of UBC and matched urinary cell free DNA (cfDNA) and exosomal DNA (exoDNA). METHODS We included 9 patients who underwent surgery for UBC. Fresh frozen tumor sample and normal blood sample was used for genomic profiling of UBC. We also performed genomic profiling of matched urinary DNA to investigate whether genomic alteration in tumor samples are echoed in urinary DNA. Urinary exoDNA was extracted from urinary exosome which was isolated by ExoQuick and urinary cfDNA was extracted by commercial kit using magnetic bead. We performed 9 gene target sequencing for somatic mutation analysis and low depth whole genome sequencing (ldWGS) for copy number analysis. RESULTS We found 17 somatic mutations in 6 patients, and 17 included 6 nonsynonymous SNVs, 3 stopgain SNVs, 2 frameshift deletion and 6 synonymous SNVs. Of 17 somatic mutations, 12 were identified in cfDNA and exoDNA with the mean allele frequency of 54.5% and 65.6%, respectively. Mean depth of cfDNA and exoDNA was 1721X and 1627X, respectively (Figure 1). In copy number analysis, mean 20.4% of whole genome region was covered by >1X. Copy number plots of cfDNA and exoDNA showed similar pattern with those of tumor samples. When we compare the log2 ratio of 100k bin size in whole genome regions, Pearson correlation coefficients of tumor vs cfDNA (0.481) and tumor vs exoDNA (0.455) were higher than that of tumor vs normal (0.086) (Figure 2). CONCLUSIONS Both urinary cfDNA and exoDNA were representative of the entire human genome and allowed genomic profiling of UBC. Specifically, copy number analysis using ldWGS has potential to be used as tools developing biomarker with low cost and whole genome coverage. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e718 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Kwang Hyun Kim More articles by this author Bong Suk Sim More articles by this author Dong Hyeon Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Generalising better: Applying deep learning to integrate deleteriousness prediction scores for whole-exome SNV studies.

Many automatic classifiers were introduced to aid inference of phenotypical effects of uncategorised nsSNVs (nonsynonymous Single Nucleotide Variations) in theoretical and medical applications. Lately, several meta-estimators have been proposed that combine different predictors, such as PolyPhen and SIFT, to integrate more information in a single score. Although many advances have been made in feature design and machine learning algorithms used, the shortage of high-quality reference data along with the bias towards intensively studied in vitro models call for improved generalisation ability in order to further increase classification accuracy and handle records with insufficient data. Since a meta-estimator basically combines different scoring systems with highly complicated nonlinear relationships, we investigated how deep learning (supervised and unsupervised), which is particularly efficient at discovering hierarchies of features, can improve classification performance. While it is believed that one should only use deep learning for high-dimensional input spaces and other models (logistic regression, support vector machines, Bayesian classifiers, etc) for simpler inputs, we still believe that the ability of neural networks to discover intricate structure in highly heterogenous datasets can aid a meta-estimator. We compare the performance with various popular predictors, many of which are recommended by the American College of Medical Genetics and Genomics (ACMG), as well as available deep learning-based predictors. Thanks to hardware acceleration we were able to use a computationally expensive genetic algorithm to stochastically optimise hyper-parameters over many generations. Overfitting was hindered by noise injection and dropout, limiting coadaptation of hidden units. Although we stress that this work was not conceived as a tool comparison, but rather an exploration of the possibilities of deep learning application in ensemble scores, our results show that even relatively simple modern neural networks can significantly improve both prediction accuracy and coverage. We provide open-access to our finest model via the web-site: http://score.generesearch.ru/services/badmut/.

Mutation, methylation, and gene expression profiles in dup(1q)-positive pediatric B-cell precursor acute lymphoblastic leukemia

High-throughput sequencing was applied to investigate the mutation/methylation patterns on 1q and gene expression profiles in pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL) with/without (w/wo) dup(1q). Sequencing of the breakpoint regions and all exons on 1q in seven dup(1q)-positive cases revealed non-synonymous somatic single nucleotide variants (SNVs) in BLZF1, FMN2, KCNT2, LCE1C, NES, and PARP1. Deep sequencing of these in a validation cohort w (n = 17)/wo (n = 94) dup(1q) revealed similar SNV frequencies in the two groups (47% vs. 35%; P = 0.42). Only 0.6% of the 36,259 CpGs on 1q were differentially methylated between cases w (n = 14)/wo (n = 13) dup(1q). RNA sequencing of high hyperdiploid (HeH) and t(1;19)(q23;p13)-positive cases w (n = 14)/wo (n = 52) dup(1q) identified 252 and 424 differentially expressed genes, respectively; only seven overlapped. Of the overexpressed genes in the HeH and t(1;19) groups, 23 and 31%, respectively, mapped to 1q; 60-80% of these encode nucleic acid/protein binding factors or proteins with catalytic activity. We conclude that the pathogenetically important consequence of dup(1q) in BCP ALL is a gene-dosage effect, with the deregulated genes differing between genetic subtypes, but involving similar molecular functions, biological processes, and protein classes.

Loss and gain of N-linked glycosylation sequons due to single-nucleotide variation in cancer

Despite availability of sequence site-specific information resulting from years of sequencing and sequence feature curation, there have been few efforts to integrate and annotate this information. In this study, we update the number of human N-linked glycosylation sequons (NLGs), and we investigate cancer-relatedness of glycosylation-impacting somatic nonsynonymous single-nucleotide variation (nsSNV) by mapping human NLGs to cancer variation data and reporting the expected loss or gain of glycosylation sequon. We find 75.8% of all human proteins have at least one NLG for a total of 59,341 unique NLGs (includes predicted and experimentally validated). Only 27.4% of all NLGs are experimentally validated sites on 4,412 glycoproteins. With respect to cancer, 8,895 somatic-only nsSNVs abolish NLGs in 5,204 proteins and 12,939 somatic-only nsSNVs create NLGs in 7,356 proteins in cancer samples. nsSNVs causing loss of 24 NLGs on 23 glycoproteins and nsSNVs creating 41 NLGs on 40 glycoproteins are identified in three or more cancers. Of all identified cancer somatic variants causing potential loss or gain of glycosylation, only 36 have previously known disease associations. Although this work is computational, it builds on existing genomics and glycobiology research to promote identification and rank potential cancer nsSNV biomarkers for experimental validation.

Effect of SMARCB1 deficiency in renal medullary carcinoma (RMC) on genes associated with nucleosome assembly and telomere organization.

614 Background: RMC is a highly aggressive tumor with close to universal fatality despite therapy. It is almost exclusively found in young African-Americans with sickle cell trait, and is characterized by complete loss of expression of SMARCB1, a major chromatin remodeler involved in regulation of gene expression. We investigated the effects of SMARCB1 loss on mutation frequency, gene expression, and cell growth in RMC. Methods: Whole exome sequencing (WES) and RNA sequencing (RNA-seq) were performed in RMC tissues from 15 and 11 patients respectively, each with matched adjacent normal kidney tissue controls. In vitro experiments were performed in a cell line (RMC2C) we established from a patient with RMC. SMARCB1 was conditionally re-expressed using a tetracycline-inducible lentivector. Gene ontology (GO) analysis was performed using DAVID. Results: WES showed that RMC harbors a low number (median of &lt; 25/tumor sample) of non-synonymous exomic single nucleotide variants (SNVs) or small indels. GO analysis revealed that the most significant pathways upregulated in RMC compared with normal tissue were those associated with nucleosome assembly and telomere organization (p values &lt; 0.0001). Re-expression of SMARCB1 at near-endogenous levels suppressed the growth rate of RMC2C cells. Subsequent silencing of SMARCB1 expression restored the growth rate of these cells. RNA-seq of RMC2C cells expressing SMARCB1 demonstrated that the most significant downregulated pathways compared with SMARCB1-negative RMC2C cells were those associated with nucleosome assembly and telomere organization (p values &lt; 0.0001). Conclusions: RMC harbors a remarkably simple genome, as evidenced by our WES analysis. Therefore, consistently detected alterations, such as SMARCB1 loss, are likely to serve as drivers for this disease. Indeed, in vitro restoration of SMARCB1 expression suppressed the growth of RMC cells and repressed genes associated with nucleosome assembly and telomere organization, identifying for the first time a causal link between loss of SMARCB1 and dysregulation of these genes. These results provide the basis for future therapeutic strategies targeting SMARCB1 loss in RMC.

Hot-spot KIF5A mutations cause familial ALS.

Heterozygous missense mutations in the N-terminal motor or coiled-coil domains of the kinesin family member 5A (KIF5A) gene cause monogenic spastic paraplegia (HSP10) and Charcot-Marie-Tooth disease type 2 (CMT2). Moreover, heterozygous de novo frame-shift mutations in the C-terminal domain of KIF5A are associated with neonatal intractable myoclonus, a neurodevelopmental syndrome. These findings, together with the observation that many of the disease genes associated with amyotrophic lateral sclerosis disrupt cytoskeletal function and intracellular transport, led us to hypothesize that mutations in KIF5A are also a cause of amyotrophic lateral sclerosis. Using whole exome sequencing followed by rare variant analysis of 426 patients with familial amyotrophic lateral sclerosis and 6137 control subjects, we detected an enrichment of KIF5A splice-site mutations in amyotrophic lateral sclerosis (2/426 compared to 0/6137 in controls; P = 4.2 × 10-3), both located in a hot-spot in the C-terminus of the protein and predicted to affect splicing exon 27. We additionally show co-segregation with amyotrophic lateral sclerosis of two canonical splice-site mutations in two families. Investigation of lymphoblast cell lines from patients with KIF5A splice-site mutations revealed the loss of mutant RNA expression and suggested haploinsufficiency as the most probable underlying molecular mechanism. Furthermore, mRNA sequencing of a rare non-synonymous missense mutation (predicting p.Arg1007Gly) located in the C-terminus of the protein shortly upstream of the splice donor of exon 27 revealed defective KIF5A pre-mRNA splicing in respective patient-derived cell lines owing to abrogation of the donor site. Finally, the non-synonymous single nucleotide variant rs113247976 (minor allele frequency = 1.00% in controls, n = 6137), also located in the C-terminal region [p.(Pro986Leu) in exon 26], was significantly enriched in familial amyotrophic lateral sclerosis patients (minor allele frequency = 3.40%; P = 1.28 × 10-7). Our study demonstrates that mutations located specifically in a C-terminal hotspot of KIF5A can cause a classical amyotrophic lateral sclerosis phenotype, and underline the involvement of intracellular transport processes in amyotrophic lateral sclerosis pathogenesis.

WITHDRAWN: Comprehensive analysis of gene mutation and expression profiles in fibrous dysplasia of bone via next-generation sequencing

// Hongbo He 1 , Qing Liu 1 , Can Zhang 1 , Zhan Liao 1 , Yupeng Liu 1 , Jun Wan 1 , Jian Tian 1 , Xiaopeng Tong 1 , Lichun Sun 1,2,3 and Wei Luo 1 1 Department of Orthopedics, Xiangya Hospital, Central South University, Changsha, Hunan, China 2 Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, Institute of Pharmacy & Pharmacology, University of South China, Hengyang, China 3 Department of Medicine, School of Medicine, Tulane Health Sciences Center, New Orleans, LA, USA Correspondence to: Wei Luo, email: luowei0928@126.com Keywords : fibrous dysplasia; whole-exome sequencing (WES); transcriptome sequencing (RNAseq); single gene sequencing; SNV/InDel Received: May 07, 2017 Accepted: November 13, 2017 Epub: January 03, 2018 Abstract Fibrous dysplasia (FD) of bone is a rare and non-inheritable bone disease. Its precise molecular mechanisms remain unclear. We collected FD patient samples and applied the innovative technology of whole-exome sequencing (WES) and transcriptome sequencing (RNAseq) to analyze the gene mutations and expression profiles. By comparing gene mutations with both WES and RNAseq, and further filtering these mutations with gene expressional significance (average > 1TPM), we eventually identified 20 genes with non-synonymous single nucleotide variations (SNVs) occurring in the exonic regions of all FD samples. About 40 genes with SNVs were mutated in 50% of the FD samples, including GNAS (Guanine Nucleotide Binding Protein Alpha) and RTN4 (Reticulon 4). Particularly, in three of these patients (Patient 1, 3 and 4), GNAS were identified as having SNVs, with the nucleotide changed in exon 8 and the amino acid residue changed in position 201 (GNAS: NM_000516: exon8: c.G602A: p.R201H; or c.C601T: p.R201C). We only observed 2 genes, FAM21B (mutation type: deletion) and GAGE2A (mutation type: amplification), with copy number variations (CNVs) occurred in FD patients. When analyzing signaling pathways and biological functions, we predicted some key genes, such as GPCRs, G proteins, chemokine and ANK-associated genes, and their associated signaling networks might play critical roles in FD development. We further confirmed these observations via single gene sequencing and Q-PCR. Thus, the mutations of these genes may be connected with fibroblast and bone development disorders. These findings may provide a clue for our next investigation of FD pathogenesis and its molecular mechanisms.

TP53, ETV6 and RUNX1 Germline Variants in Patients Developing Secondary Neoplasms after Treatment for Childhood Acute Lymphoblastic Leukemia

Introduction: Today most of the children treated for acute lymphoblastic leukemia (ALL) can be cured by the application of intensive combination chemotherapy regimens. However, up to 10% develop a secondary malignancy (SMN) after undergoing ALL treatment with cure rates being often dismal. Thus, strategies are needed for an early identification of patients at risk for SMN development, an improved understanding of the underlying pathobiology and, ideally, the development of preventive actions. Analyzing three candidates associated with predisposition to neoplastic diseases - TP53, ETV6 and RUNX1 - we aimed to determine the frequency of single nucleotide variants (SNV) in these genes in a cohort of SMN patients.Methods: Using targeted sequencing, we analyzed a cohort of childhood ALL patients with SMN after treatment on one of seven consecutive ALL-BFM protocols - ALL-BFM 79 to AIEOP-BFM ALL 2000.In the observation period from 1984 to 2008, 168 SMN patients were identified. The median follow-up for the entire patient cohort was 10.6 years as of April, 2013. With the exception of ALL-BFM 79, treatment was stratified into 3 branches, mainly according to the initial leukemic cell load, adverse genetic aberrations and treatment response. DNA isolated from remission bone marrow smears was analyzed employing two different multiplex PCR-based Ion AmpliSeq™ Panels (Life Technologies, Darmstadt, Germany) according to the manufacturer's instructions. The first panel, covering all coding exons of TP53, was employed in 49 patients with adequate material available for analysis. The second panel, interrogating the complete coding regions of ETV6 and RUNX1, was applied in 38 patients with sufficient material available after TP53 analysis. SPSS (IBM Deutschland GmbH, Ehningen, Germany) was used for computerized calculations.Results: In our TP53 analyses, only 1/49 (2%) genotyped SMN patients carried a heterozygous non-synonymous SNV within the coding region. This rare missense variant, p.Asn235Ser (rs144340710), was detected in a patient developing a small round cell sarcoma after treatment for ALL. The in silico scores from Polyphen and Sift algorithms indicated a benign effect for this change. Regarding ETV6, 3/38 (8%) patients carried a heterozygous non-synonymous SNV; two of them developed a hematologic and one patient a solid SMN. One frameshift insertion (p.Ile48Glyfs*2) and a missense variant (p.Leu79Pro) were observed in exon 2, part of the pointed domain. A further missense variant (p.Arg399Gly) was detected in exon 7 within the C-terminal DNA-binding ETS domain. Although ETV6 p.Arg399 is a hotspot mutation site for recurrent somatic mutations in malignancies, none of these variants was recorded in the databases dbSNP, the 1000Genomes Project, the NHLBI GO Exome Sequencing Project, Exome Aggregation Consortium or the Catalogue of Somatic Mutations in Cancer. For both missense variants in silico scores from Sift and Polyphen algorithms predicted a probably damaging effect. Molecular modeling of p.Arg399Gly suggests that the change might directly modulate the DNA binding qualities of the ETS domain. Furthermore, 2/38 patients (5%) had coding variants in RUNX1, both developed a hematologic SMN. One patient carried a nonsense insertion (Leu253Argfs*3). A second patient had a missense SNV (p.Leu56Ser, rs111527738) within the N-terminal RUNT domain, with a probably damaging effect (Polyphen). The described ETV6 and RUNX1 SNV were mutually exclusive. Compared to published data on childhood ALL patients (T. Moriyama et al. Lancet Oncol. 2015 and J. Zhang et al. N Engl J Med. 2015), we found approximately 10-fold and 4-fold higher frequencies of ETV6 and RUNX1 SNV, respectively, in patients developing a SMN after ALL treatment on ALL-BFM protocols.Conclusions: The low frequency of TP53 SNV in our case series suggests that alterations in this frequently mutated tumor suppressor gene may not play a major role in the pathobiology of SMN associated with the treatment of ALL. However, our findings in ETV6 and RUNX1 analyses suggest that genetic variation in these genes may be involved in the development of SMN after undergoing ALL treatment and warrant additional studies employing appropriate control groups for further clarification. DisclosuresSchrappe:Novartis: Consultancy, Research Funding; Medac: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding.