Combination Of Whole-genome Sequencing Research Articles

Letter by Finsterer and Zarrouk-Mahjoub Regarding Article, “Combination of Whole Genome Sequencing, Linkage, and Functional Studies Implicates a Missense Mutation in Titin as a Cause of Autosomal Dominant Cardiomyopathy With Features of Left Ventricular Noncompaction”

With interest, we read the article by Hastings et al1 about a 3-generation family in whom 7 members carried the titin mutation p.A178D , and 5 (3 on echocardiography and 2 on cardiac magnetic resonance imaging [MRI]) had left ventricular hypertrabeculation/noncompaction (LVHT). We have the following comments and concerns. To date, a causal relation between mutations in any of the >40 genes and many chromosomal defects associated with LVHT and LVHT has never been proven. Arguments against a causal relation are that only a small number of patients with a certain mutation in any of these genes regarded as causative truly develop LVHT; …

Combination of Whole Genome Sequencing, Linkage, and Functional Studies Implicates a Missense Mutation in Titin as a Cause of Autosomal Dominant Cardiomyopathy With Features of Left Ventricular Noncompaction.

High throughput next-generation sequencing techniques have made whole genome sequencing accessible in clinical practice; however, the abundance of variation in the human genomes makes the identification of a disease-causing mutation on a background of benign rare variants challenging. Here we combine whole genome sequencing with linkage analysis in a 3-generation family affected by cardiomyopathy with features of autosomal dominant left ventricular noncompaction cardiomyopathy. A missense mutation in the giant protein titin is the only plausible disease-causing variant that segregates with disease among the 7 surviving affected individuals, with interrogation of the entire genome excluding other potential causes. This A178D missense mutation, affecting a conserved residue in the second immunoglobulin-like domain of titin, was introduced in a bacterially expressed recombinant protein fragment and biophysically characterized in comparison to its wild-type counterpart. Multiple experiments, including size exclusion chromatography, small-angle x ray scattering, and circular dichroism spectroscopy suggest partial unfolding and domain destabilization in the presence of the mutation. Moreover, binding experiments in mammalian cells show that the mutation markedly impairs binding to the titin ligand telethonin. Here we present genetic and functional evidence implicating the novel A178D missense mutation in titin as the cause of a highly penetrant familial cardiomyopathy with features of left ventricular noncompaction. This expands the spectrum of titin's roles in cardiomyopathies. It furthermore highlights that rare titin missense variants, currently often ignored or left uninterpreted, should be considered to be relevant for cardiomyopathies and can be identified by the approach presented here.

Abstract 3901: Identification of novel tumor suppressor candidates and characterizing their potential driver role in familial cholangiocarcinoma

Abstract Cholangiocarcinoma (bile duct cancer) is an epithelial malignancy originating from the bile duct that connects the liver to the small intestine. This carcinoma, while rare, has an extremely poor prognosis with a 5-year survival of less than 10%. Initial clinical presentation and diagnosis is frequently at an advanced stage and while amenable to specific combination chemotherapy regimens, inevitably progresses towards distal metastasis. Only recently has the molecular genetic underpinnings of this cancer been explored with a number of genome sequencing studies of a limited number of primary tumors. For this study, we used a multi-faceted approach that integrates the genetic analysis of an extended familial cholangiocarcinoma pedigree with identification of somatic genetic aberrations discovered via primary tumor sequencing of affected family members. Our goal was to identify potential novel tumor suppressor(s) that have not yet been described and are associated with an increased familial risk of cholangiocarcinoma. The extended pedigree was characterized by multiple members with relatively young onset of cholangiocarcinoma and displayed a segregation pattern consistent with an autosomal dominant inheritance pattern. Several members of the family had undergone biopsy or resection and as a result, had primary tumor tissue available for analysis. Relying on a combination of whole genome sequencing, experimentally-derived haplotype phasing, exome sequencing and tumor subclonal identification, we performed a combined germline and somatic analysis on this pedigree. Our analysis included family members both affected and unaffected by biliary cancers. Somatic variant analysis of the tumor samples was used to identify driver mutations that may work in concert with candidate germline mutation to initiate and maintain tumorigenesis. Remarkably, the cancer genome sequencing analysis identified a number of common somatic features among primary tumors found in different family members. Analysis of tumor clonality was derived from copy number and mutation data and further informed our analysis of possible contributing drivers. To further inform the analysis of suspected candidate germline loci, we conducted experimentally-based haplotype phasing covering Megabase segments to better delineate blocks that segregated with affected members. We identified several novel candidate genes with germline mutations with a predicted deleterious effect that segregated to affected individuals and had not been previously noted as polymorphisms or rare variants. Thus, we identified several candidate genes that may be novel tumor suppressor candidates for cholangiocarcinoma and increase the susceptibility to this lethal tumor. Overall, our study has significant implications by shedding light on the genetic basis of cholangiocarcinoma. Citation Format: Stephanie Greer, Lincoln D. Nadauld, Billy Lau, Laura Miotke, Erik Hopmans, Christina M. Wood, John M. Bell, Hanlee P. Ji. Identification of novel tumor suppressor candidates and characterizing their potential driver role in familial cholangiocarcinoma. [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 3901. doi:10.1158/1538-7445.AM2015-3901

Tracing Origins of the Salmonella Bareilly Strain Causing a Food-borne Outbreak in the United States.

Using a novel combination of whole-genome sequencing (WGS) analysis and geographic metadata, we traced the origins of Salmonella Bareilly isolates collected in 2012 during a widespread food-borne outbreak in the United States associated with scraped tuna imported from India. Using next-generation sequencing, we sequenced the complete genome of 100 Salmonella Bareilly isolates obtained from patients who consumed contaminated product, from natural sources, and from unrelated historically and geographically disparate foods. Pathogen genomes were linked to geography by projecting the phylogeny on a virtual globe and produced a transmission network. Phylogenetic analysis of WGS data revealed a common origin for outbreak strains, indicating that patients in Maryland and New York were infected from sources originating at a facility in India. These data represent the first report fully integrating WGS analysis with geographic mapping and a novel use of transmission networks. Results showed that WGS vastly improves our ability to delimit the scope and source of bacterial food-borne contamination events. Furthermore, these findings reinforce the extraordinary utility that WGS brings to global outbreak investigation as a greatly enhanced approach to protecting the human food supply chain as well as public health in general.

The genome sequence of the popular hexose-transport-deficient Saccharomyces cerevisiae strain EBY.VW4000 reveals LoxP/Cre-induced translocations and gene loss.

Saccharomyces cerevisiae harbours a large group of tightly controlled hexose transporters with different characteristics. Construction and characterization of S. cerevisiae EBY.VW4000, a strain devoid of glucose import, was a milestone in hexose-transporter research. This strain has become a widely used platform for discovery and characterization of transporters from a wide range of organisms. To abolish glucose uptake, 21 genes were knocked out, involving 16 successive deletion rounds with the LoxP/Cre system. Although such intensive modifications are known to increase the risk of genome alterations, the genome of EBY.VW4000 has hitherto not been characterized. Based on a combination of whole genome sequencing, karyotyping and molecular confirmation, the present study reveals that construction of EBY.VW4000 resulted in gene losses and chromosomal rearrangements. Recombinations between the LoxP scars have led to the assembly of four neo-chromosomes, truncation of two chromosomes and loss of two subtelomeric regions. Furthermore, sporulation and spore germination are severely impaired in EBY.VW4000. Karyotyping of the EBY.VW4000 lineage retraced its current chromosomal architecture to four translocations events occurred between the 6th and the 12th rounds of deletion. The presented data facilitate further studies on EBY.VW4000 and highlight the risks of genome alterations associated with repeated use of the LoxP/Cre system.

Genomic catastrophes frequently arise in esophageal adenocarcinoma and drive tumorigenesis.

Oesophageal adenocarcinoma (EAC) incidence is rapidly increasing in Western countries. A better understanding of EAC underpins efforts to improve early detection and treatment outcomes. While large EAC exome sequencing efforts to date have found recurrent loss-of-function mutations, oncogenic driving events have been underrepresented. Here we use a combination of whole-genome sequencing (WGS) and single-nucleotide polymorphism-array profiling to show that genomic catastrophes are frequent in EAC, with almost a third (32%, n = 40/123) undergoing chromothriptic events. WGS of 22 EAC cases show that catastrophes may lead to oncogene amplification through chromothripsis-derived double-minute chromosome formation (MYC and MDM2) or breakage-fusion-bridge (KRAS, MDM2 and RFC3). Telomere shortening is more prominent in EACs bearing localized complex rearrangements. Mutational signature analysis also confirms that extreme genomic instability in EAC can be driven by somatic BRCA2 mutations. These findings suggest that genomic catastrophes have a significant role in the malignant transformation of EAC.

The genomic landscape of the Ewing Sarcoma family of tumors reveals recurrent STAG2 mutation.

The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.

Selected insights from application of whole-genome sequencing for outbreak investigations

The advent of high-throughput whole-genome sequencing has the potential to revolutionize the conduct of outbreak investigation. Because of its ultimate resolution power for differentiating between closely related pathogen strains, whole-genome sequencing could augment the traditional epidemiologic investigations of infectious disease outbreaks. The combination of whole-genome sequencing and intensive epidemiologic analysis provided new insights on the sources and transmission dynamics of large-scale epidemics caused by Escherichia coli and Vibrio cholerae, nosocomial outbreaks caused by methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae, Mycobacterium abscessus, community-centered outbreaks caused by Mycobacterium tuberculosis, and natural disaster-associated outbreaks caused by environmentally acquired molds. When combined with traditional epidemiologic investigation, whole-genome sequencing has proven useful for elucidating the sources and transmission dynamics of disease outbreaks. Development of a fully automated bioinformatics pipeline for the analysis of whole-genome sequence data is much needed to make this powerful tool more widely accessible.

Abstract 1781: Whole-genome and RNA sequencing identify a novel recurrent translocation in adrenocortical carcinoma.

Abstract Adrenocortical carcinoma (ACC) is a lethal tumor of the adrenal cortex with limited treatment options. The only possibility for cure is complete surgical resection, but 50-70% of patients present with metastatic disease, precluding curative surgery. The outcome for patients with ACC has remained unchanged in the past 25 years, with the overall 5-year survival of patients undergoing surgical resection being ∼40%. Patients that present with advanced disease fare considerably worse with 5-year survival rates of 10-20%. The standard chemotherapy regimen of mitotane (the only approved agent), etoposide, doxorubicin, and cisplatin, has a response rate of 23.2%. A better understanding of pathobiology is needed to improve the treatment results for this disease. Previous genomic studies combined with expression microarray studies have implicated lack of H19 expression coupled with IGF2 over-expression, beta-catenin activation, and loss of p53 pathway function as important events in ACC, but no single alteration has been seen in all ACC. We used a combination of whole genome sequencing, exome sequencing, and RNA-sequencing on three tumor/normal pairs from patients. Using data from spectral karyotyping of the two ACC cell lines, SW-13 and NCI-H295R, we mined the sequencing data for structural rearrangements in regions of common breakpoints. We identified a novel recurrent translocation, t(4;8)(p16.2;p23.1) in all three tumors that was not present in the normal sequence. Using fluorescent in situ hybridization (FISH) we validated the translocation in the three tumors and both SW-13 and H295R. An additional five of five ACC scored positive for the translocation, as did three malignant non-ACC tumors (malignant pheochromocytoma, pancreatic neuroendocrine tumor, and carcinoid) suggesting that this finding has implications beyond ACC. The recurrent translocation t(4;8) (p16.2; p23.1) involves two genes of unknown function, BC042823 (uc003gho.2, IMAGE:5275587) on chromosome 4 and BC030294 (ucoo3wrb.1, IMAGE:5396854) on chromosome 8. RNA- sequencing data and RT-PCR with primers designed to both derivative chromosomes revealed transcription from both derivative chromosomes. The cDNA products were sequenced by Sanger sequencing and show partial homology to a non-coding RNA, LOC100506990, at chr8:12,338,893-12,452,929. RNA-sequencing data revealed this locus is highly transcribed in normal adrenal gland but reduced in expression in the three ACCs. In contrast to other recurrent translocations previously found in other cancers, this translocation is only present in 18-30% of the tumor nuclei scored by FISH, suggesting that it is a necessary alteration in malignant progression, but underscoring the question of how it might cooperate with events in other sub-clones to initiate tumor formation and progression. Citation Format: Michael J. Demeure, Kaiti Schwartz, Aditi Bapat, Claire Linnehan, Hollie Benson, Rebecca Reiman, Lori Phillips, Christophe Legendre, Raghu Metpally, Ahmet Kurdoglu, David Craig, John Carpten, Winnie S. Liang, Waibhav Tembe, Kimberly J. Bussey. Whole-genome and RNA sequencing identify a novel recurrent translocation in adrenocortical carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1781. doi:10.1158/1538-7445.AM2013-1781