Splice Site Single Nucleotide Variants Research Articles

Whole-Exome Sequencing Identifies Two Discrete Druggable Signaling Pathways in Follicular Thyroid Cancer

Thyroid cancer is the most common endocrine malignancy, with continuously increasing incidence. Follicular thyroid cancer (FTC) accounts for approximately 10% to 15% of these cases and is known to be associated with several gene mutations. The purpose of this study was to identify novel therapeutic targets in FTC using whole-exome sequencing (WES) and bioinformatics analysis. Whole-exome sequencing was performed on 6 established FTC cell lines. Stringent false-proof filtering and exclusion of synonymous and known polymorphisms yielded novel missense, nonsense, and splice-site single nucleotide variants (SNV). Gene variants were analyzed for structural, functional, and evolutionary properties using GO (Gene Ontology), Pfam (Protein Families), and KEGG (Kyoto Encyclopedia of Genes and Genomes) searches by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) and GORILLA (Gene Ontology enRIchment anaLysis and visuaLizAtion tool) analyses. A false discovery rate of <0.5 was used to denote significantly enriched signaling pathways. An average of 657 (range 366 to 1,158) SNVs including 31 (range 12 to 53) known cancer driver genes were identified in FTC cell line exomes. The SNV burden, distribution, frequency, and signature followed the known thyroid mutation profiles, without chromosomal bias. Recurrently mutated cancer driver genes included FRG1 (6/6), CDC27, NCOR1, PRSS1 (5/6), AHCTF1, MUC20, PABPC1, and PABPC3 (4/6). Pathway analysis using bioinformatics tools STRING and GORILLA segregated FTC cell lines into 2 druggable signaling groups showing dominant RAS/ERK1-2/AKT and CDK1/CyclinB signaling pathway targets. Next-generation sequencing tools can be used to identify druggable signaling targets for precision treatment of FTCs.

Using Whole Exome Sequencing to Identify Genetic Variation and Polymorphisms Associated with Graft Versus Host Disease in Allogeneic Stem Cell Transplant Recipients

Objective: Allogeneic stem cell transplant (ASCT) is a potentially curative treatment for many hematologic malignancies. HLA match is the principal factor in donor selection but the total antigenic landscape as seen by the donor immune cells extends beyond the HLA and minor HLA antigens. Thus, despite "perfect" HLA match graft versus host disease (GVHD) caused by alloreactive donor immune cells reacting to disparate antigens in the recipient, remains a significant cause of morbidity and mortality. Whole exome sequencing (WES) which assays all the protein coding nucleotide sequences provides a powerful means to assess the entire antigenic makeup of the individual. We examined the WES of sibling donor-recipient pairs undergoing BMT to assess the influence of the extent of genetic disparity and specific gene polymorphisms on GVHD.Methods: Sixteen matched sibling donor-recipient pairs receiving allogeneic stem cell transplants between June 2008 and April 2013 had samples available for retrospective WES. Transplant data including conditioning regimen, age of donor and recipient at the time of transplant, development and type of GVHD and cause of death were recorded. The sequencing was performed using Illumina HiSeq2000 and the single nucleotide variants (SNVs) and small insertions and deletions (INDELs) were identified using the haplotype caller within the Genome Analysis Toolkit GATK v2.7. Functional variants including non-synonymous, frame-shift, truncating, and splice site SNVs and INDELs identified were compared between donor and recipient and among those with GVHD and those without GVHD (non-GVHD). One-sided Fisher's exact test was used to determine the statistical significance of the SNPs in GVHD and non-GVHD. Candidate genes with a p value of <0.05 were further evaluated for their biochemical pathway and possible role in inflammatory or immune responses.Results: Nine of 16 patients had a diagnosis of AML or high grade myelodysplastic syndrome, 3 had a diagnosis of non- Hodgkin lymphoma, 1 had Hodgkin lymphoma, 1 had multiple myeloma, and 2 had CML. Six patients were conditioned with a myeloablative regimen, others with a reduced intensity conditioning regimen. Eight of 16 patients were female and 7 of 16 donors were female. Nine of 16 patients developed stage II-IV acute and/or chronic GVHD with 7 of them developing GVHD of the skin. A total of 12,623 genes with 43,670 functional variants were identified using WES. 134 genes had a p value < 0.05 and enrichment analysis was performed on these genes. 12 out of 134 genes were associated with gene ontology (GO) term "immune system development" with an unadjusted enrichment p-value 0.0015. Three (IL31RA, OSMR, SIRPA) out of 134 genes were associated with "inflammation_Jak-STAT pathway" with an adjusted enrichment p-value 0.07. Variants of IL31RA, OSMR, and PTX3 were identified as genes with polymorphisms associated with GVHD (p= 0.003, 0.01, and 0.01, respectively) with 5 variants identified in IL31RA, 7 variants in OSMR, and 2 variants in PTX3. Further investigation of these three genes revealed their role in inflammatory or immune responses.Discussion: GVHD is a cytokine mediated immune-inflammatory process that can occur despite "perfect" HLA matching. WES identified the extensive genetic variability between donors and recipients implying large allo-reactivity potential that can lead to GVHD. Quantification of genetic variation may allow for more precise determination of GVHD risk and better selection of donors. In our cohort, WES identified polymorphisms in IL31RA, OSMR, and PTX3 as statistically significantly associated with development of GVHD. The protein products of these three genes have each been shown to be involved in inflammatory processes and are influenced by other cytokines. IL31RA and OSMR are found in many tissues including the skin and lungs. They function both independently and as a heterodimer receptor capable of binding multiple cytokines including IL-31, IL-6, and TNF-α. PTX3 is a reactive protein that increases in response to cytokine release related to systemic inflammation. Levels of this protein have been used as a prognostic marker in many disease processes with high levels correlating with poor outcomes. The small sample size of our cohort precludes any firm conclusions but provides promising hypotheses for further evaluation in larger studies. DisclosuresNo relevant conflicts of interest to declare.

Abstract 482: Integrated genetic and epigenetic analysis defines novel molecular clusters in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood. Recent sequencing studies have revealed a number of recurrent mutational targets of RMS including multiple components of the FGFR4/RAS/AKT pathway. However, due to the limited number of the mutational targets, our knowledge about molecular mechanisms of RMS is still incomplete. Moreover, relatively low mutation numbers in RMS suggests involvement of other mechanisms, such as epigenetic alterations, which have not been addressed in the previous studies. To address these issues, we conducted an integrated genetic/epigenetic study using whole exome sequencing (WES) and transcriptome sequencing (WTS), SNP array and methylation array. We first performed WES of 16 paired tumors, of which 8 tumors were analyzed by WTS. We first sequenced the exome of 16 paired tumors/normal samples, of which 3 were also analyzed for 2 relapsed and one metastatic samples. Among 690 candidate somatic changes detected by our pipeline, 604 in 531 genes (88%) were validated by deep sequencing and included 512 missense, 31 nonsense, and 21 splice site single nucleotide variants (SNVs) as well as 32 frameshift and 8 in-frame indels. Although novel recurrent fusion genes were not detected by WTS, we identified 18 recurrent mutated genes by whole exome sequencing. Among these, 14 putative driver genes were selected for subsequently targeted deep sequencing in the entire cohort of 60 RMS cases. Most frequently mutated genes were TP53, followed by NRAS, FGFR4, BCOR, and ARID1A. FGFR4/RAS/AKT pathway genes were mutated as many as 40% of RMS cases. In addition, CN changes involving FGFR4/RAS/AKT pathway genes as well as cell cycle regulators, such as CN gains in 2p24.3 (MYCN), 12q15 (FRS2), loss of 9p21 (CDKN2A/B) and 17p13.2 (TP53) were also recurrently detected. Most of these FGFR4/RAS/AKT pathway alterations, except for GAB1, were predominantly found in ERMS, suggesting that deregulated FGFR4/RAS/AKT signaling plays an important role in ERMS pathogenesis. We also performed methylation array in 50 cases. Based on the DNA methylation patterns, we identified 2 sample clusters, that exhibit remarkable correlation with histological phenotypes (named as cluster E and A). Cluster E was composed almost exclusively of ERMS, whereas all ARMS cases were grouped into cluster A. Accordingly, all tumors positive for PAX3 or 7-FOXO1 fusions were grouped into cluster A. Cluster E has been divided into 2 distinct signatures (named as E1 and E2), characterized by high frequencies of multiple chromosomal copy number changes, compared to cluster A, including gains of chromosomes 2, 8, and 12, and a higher frequency of FGFR4/RAS/AKT pathway mutations, the latter being especially prominent in the E2 cluster (46% in E1 and 73% in E2 vs. 18% in A). Our integrated analyses provide a framework for the identification of genetic/epigenetic alterations underlying pathogenesis of RMS. Citation Format: Masafumi Seki, Riki Nishimura, Kenichi Yoshida, Teppei Shimamura, Yuichi Shiraishi, Yusuke Sato, Motohiro Kato, Kenichi Chiba, HIroko Tanaka, Genta Nagae, Yusuke Okuno, Hajime Hosoi, Yukichi Tanaka, Hajime Ohkita, Mitsuru Miyashi, Tomoaki Taguchi, Katsuyoshi Koh, Ryoji Hanada, Akira Oka, Satoru Miyano, Hiroyuki Aburatani, Yasuhide Hayashi, Seishi Ogawa, Junko Takita. Integrated genetic and epigenetic analysis defines novel molecular clusters in rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 482. doi:10.1158/1538-7445.AM2015-482

Whole-genome sequencing of individuals from a founder population identifies candidate genes for asthma.

Asthma is a complex genetic disease caused by a combination of genetic and environmental risk factors. We sought to test classes of genetic variants largely missed by genome-wide association studies (GWAS), including copy number variants (CNVs) and low-frequency variants, by performing whole-genome sequencing (WGS) on 16 individuals from asthma-enriched and asthma-depleted families. The samples were obtained from an extended 13-generation Hutterite pedigree with reduced genetic heterogeneity due to a small founding gene pool and reduced environmental heterogeneity as a result of a communal lifestyle. We sequenced each individual to an average depth of 13-fold, generated a comprehensive catalog of genetic variants, and tested the most severe mutations for association with asthma. We identified and validated 1960 CNVs, 19 nonsense or splice-site single nucleotide variants (SNVs), and 18 insertions or deletions that were out of frame. As follow-up, we performed targeted sequencing of 16 genes in 837 cases and 540 controls of Puerto Rican ancestry and found that controls carry a significantly higher burden of mutations in IL27RA (2.0% of controls; 0.23% of cases; nominal p = 0.004; Bonferroni p = 0.21). We also genotyped 593 CNVs in 1199 Hutterite individuals. We identified a nominally significant association (p = 0.03; Odds ratio (OR) = 3.13) between a 6 kbp deletion in an intron of NEDD4L and increased risk of asthma. We genotyped this deletion in an additional 4787 non-Hutterite individuals (nominal p = 0.056; OR = 1.69). NEDD4L is expressed in bronchial epithelial cells, and conditional knockout of this gene in the lung in mice leads to severe inflammation and mucus accumulation. Our study represents one of the early instances of applying WGS to complex disease with a large environmental component and demonstrates how WGS can identify risk variants, including CNVs and low-frequency variants, largely untested in GWAS.

Identification of Novel Variants in Colorectal Cancer Families by High-Throughput Exome Sequencing

Colorectal cancer (CRC) in densely affected families without Lynch Syndrome may be due to mutations in undiscovered genetic loci. Familial linkage analyses have yielded disparate results; the use of exome sequencing in coding regions may identify novel segregating variants. We completed exome sequencing on 40 affected cases from 16 multicase pedigrees to identify novel loci. Variants shared among all sequenced cases within each family were identified and filtered to exclude common variants and single-nucleotide variants (SNV) predicted to be benign. We identified 32 nonsense or splice-site SNVs, 375 missense SNVs, 1,394 synonymous or noncoding SNVs, and 50 indels in the 16 families. Of particular interest are two validated and replicated missense variants in CENPE and KIF23, which are both located within previously reported CRC linkage regions, on chromosomes 1 and 15, respectively. Whole-exome sequencing identified DNA variants in multiple genes. Additional sequencing of these genes in additional samples will further elucidate the role of variants in these regions in CRC susceptibility. Exome sequencing of familial CRC cases can identify novel rare variants that may influence disease risk.