Abstract 3421: Serial whole exome sequencing showed the genetic aggravation of refractory osteosarcoma
Su Jin HeoJi Woong KimWoo Sun KwonHyo K. KimMin Hee HongWoo Ick YangSe-Kyu KimHyo Song KimHyun Cheol ChungTae Hyun HwangSun Young Rha
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Abstract Osteosarcoma is the most common malignant bone tumor in children and adolescents with frequent lung metastasis. As the survival has not been improved with chemotherapy for the last two decades, novel therapeutic approaches are required to efficiently treat osteosarcoma. Genomic analyses have revealed biologically useful information and genetically altered therapeutic targets. The subject of this study was a 25 year old patient with conventional high-grade osteosarcoma with multiple metastasis of lung, lymph nodes and chest wall, who had failed from several palliative systemic chemotherapies and radiotherapy. We performed whole exome sequencing of the gDNA from peripheral blood mononucleated cells (PBMC) and formalin fixed paraffin embedded (FFPE) tissues of primary tumor, secondary (recurrence 1) and tertiary (recurrence 2) metastatic tumors. Samples were sequenced using one lane of paired-end, 100 bp reads on Illumina Hiseq for each sample. Here, we present an analysis result using VarScan and custom-made programs for the detection of somatic mutations and loss of heterozygosity (LOHs) in exome data from changes of genetic variations throughout the disease progression compared to blood germ-line sequencing data. As a result, we observed the 36 common somatic mutated genes, including p73 and TSHR, in three tumor types. And we indentified the somatic mutations affecting the functions of known cancer genes, RBM15, SYNE1, GNAQ and XPA, which were only present in the secondary and/or tertiary metastatic tumors but not in the primary tumor. Especially, GNAQ mutations which is found in the metastatic tumors, is known to be mutated in 50% of melanoma and driven constitutive activity of the MAPK pathway. Therefore, GNAQ mutation in recurrent osteosarcoma patient might be a potential marker for new therapeutic strategies of inhibiting MAPK pathway. In conclusion, the ability to track clonal evolution in cancers may provide new strategies and opportunities for drug development, especially in refractory and rare diseases. Citation Format: Su Jin Heo, Ji Woong Kim, Woo Sun Kwon, Hyo Ki Kim, Min Hee Hong, Woo Ick Yang, Se-Kyu Kim, Hyo Song Kim, Hyun Cheol Chung, Tae Hyun Hwang, Sun Young Rha. Serial whole exome sequencing showed the genetic aggravation of refractory osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3421. doi:10.1158/1538-7445.AM2014-3421Keywords:
Exome
Primary tumor
Advances in technology are rapidly changing the field of medical genetics in both the research laboratory and the clinic. With the use of next-generation, or massively parallel, DNA sequencing, it is possible to determine the sequence of essentially all genes in an individual's genome — referred to as the exome — within a matter of days.This technology became widely available in 2005, and the first proof-of-principle experiment showing the power of exome sequencing for the discovery of genes associated with disease was published a few years later.1 Since then, exome analysis has been used in the research setting to . . .
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It is estimated that approximately 85% of human disease mutations are located in protein coding regions, therefore selectively sequencing all protein coding regions (exome) would be cost-effective and an alternative strategy to identify diseases' varaints. In 2009, scientists successfully identified one missense mutation in MYH3 among 4 individuals with Freeman Sheldon syndrome (one autosomal dominant disease) through exome sequencing. Since then, exome sequencing has been widely used to identify disease causative or susceptibility genes in Mendelian disorders and complex diseases. The application of exome sequencing in human diseases were summarized in this review.
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Mendelian inheritance
Human disease
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Illumina dye sequencing
Cancer genome sequencing
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We explored the feasibility of studying loss of heterozygosity (LOH) by using exome sequencing and compared the differences in genetic LOH between primary breast tumors and metastatic lesions. Exome sequencing was conducted to investigate the genetic LOH in the peripheral blood, a primary tumor and a metastatic lesion from the same patient. LOH was observed in 30 and 48 chromosomal loci of the primary tumor and metastatic lesion, respectively. The incidence of LOH was the highest on chromosome 19, followed by chromosomes 14, 3 and 11 in the metastatic lesion. Among these ‘hot’ regions, LOH was observed for multiple genes of the CECAM, MMP and ZNF families. Therefore, the use of exome sequencing for studying LOH is feasible. More members of gene families appeared with LOH in ‘hot’ regions, suggesting that these gene families had synergistic effects in tumorigenesis.
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Abstract Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques.
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Human genetics
Sequence (biology)
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Sequencing the coding regions, the exome, of the human genome is one of the major current strategies to identify low frequency and rare variants associated with human disease traits. So far, the most widely used commercial exome capture reagents have mainly targeted the consensus coding sequence (CCDS) database. We report the design of an extended set of targets for capturing the complete human exome, based on annotation from the GENCODE consortium. The extended set covers an additional 5594 genes and 10.3 Mb compared with the current CCDS-based sets. The additional regions include potential disease genes previously inaccessible to exome resequencing studies, such as 43 genes linked to ion channel activity and 70 genes linked to protein kinase activity. In total, the new GENCODE exome set developed here covers 47.9 Mb and performed well in sequence capture experiments. In the sample set used in this study, we identified over 5000 SNP variants more in the GENCODE exome target (24%) than in the CCDS-based exome sequencing.
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Reporting clinically actionable incidental genetic findings in the course of clinical exome testing is recommended by the American College of Medical Genetics and Genomics (ACMG). However, the performance of clinical exome methods for reporting small subsets of genes has not been previously reported.In this study, 57 exome data sets performed as clinical (n = 12) or research (n = 45) tests were retrospectively analyzed. Exome sequencing data was examined for adequacy in the detection of potentially pathogenic variant locations in the 56 genes described in the ACMG incidental findings recommendation. All exons of the 56 genes were examined for adequacy of sequencing coverage. In addition, nucleotide positions annotated in HGMD (Human Gene Mutation Database) were examined.The 56 ACMG genes have 18 336 nucleotide variants annotated in HGMD. None of the 57 exome data sets possessed a HGMD variant. The clinical exome test had inadequate coverage for >50% of HGMD variant locations in 7 genes. Six exons from 6 different genes had consistent failure across all 3 test methods; these exons had high GC content (76%-84%).The use of clinical exome sequencing for the interpretation and reporting of subsets of genes requires recognition of the substantial possibility of inadequate depth and breadth of sequencing coverage at clinically relevant locations. Inadequate depth of coverage may contribute to false-negative clinical exome results.
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Medical genetics
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Next-generation sequencing methods have revolutionized the possibilities for analyzing the human genome. Sequencing the exome, the protein-encoding portion of the genome, is used, in clinical medicine, especially in the diagnosis of rare hereditary diseases, congenital developmental disorders and cancer. Using exome sequencing as a diagnostic test is justified when the results could lead to an accurate diagnosis, significantly influence the treatment and genetic counseling. It is a reliable method for detecting single base mutations as minor deletions and insertions. However, with current methods the reliable analysis of larger changes in the number of copies, the length or repeats and areas present in multiple copies in the genome is challenging. Every human has many mutations in their exome, and distinguishing between insignificant and pathogenic mutations is thus a key challenge when interpreting the results of exome sequencing.
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Cancer genome sequencing
Human genetics
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Abstract Background Protein-coding regions in human genes harbor 85% of the mutations that are associated with disease-related traits. Compared with whole-genome sequencing of complex samples, exome sequencing serves as an alternative option because of its dramatically reduced cost. In fact, exome sequencing has been successfully applied to identify the cause of several Mendelian disorders, such as Miller and Schinzel-Giedio syndrome. However, there remain great challenges in handling the huge data generated by exome sequencing and in identifying potential disease-related genetic variations. Results In this study, Exome-assistant ( http://122.228.158.106/exomeassistant ), a convenient tool for submitting and annotating single nucleotide polymorphisms (SNPs) and insertion/deletion variations (InDels), was developed to rapidly detect candidate disease-related genetic variations from exome sequencing projects. Versatile filter criteria are provided by Exome-assistant to meet different users’ requirements. Exome-assistant consists of four modules: the single case module, the two cases module, the multiple cases module, and the reanalysis module. The two cases and multiple cases modules allow users to identify sample-specific and common variations. The multiple cases module also supports family-based studies and Mendelian filtering. The identified candidate disease-related genetic variations can be annotated according to their sample features. Conclusions In summary, by exploring exome sequencing data, Exome-assistant can provide researchers with detailed biological insights into genetic variation events and permits the identification of potential genetic causes of human diseases and related traits.
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