Sophia Derdak

A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing

As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy.

Development and validation of a 20K single nucleotide polymorphism (SNP) whole genome genotyping array for apple (Malus × domestica Borkh).

High-density SNP arrays for genome-wide assessment of allelic variation have made high resolution genetic characterization of crop germplasm feasible. A medium density array for apple, the IRSC 8K SNP array, has been successfully developed and used for screens of bi-parental populations. However, the number of robust and well-distributed markers contained on this array was not sufficient to perform genome-wide association analyses in wider germplasm sets, or Pedigree-Based Analysis at high precision, because of rapid decay of linkage disequilibrium. We describe the development of an Illumina Infinium array targeting 20K SNPs. The SNPs were predicted from re-sequencing data derived from the genomes of 13 Malus × domestica apple cultivars and one accession belonging to a crab apple species (M. micromalus). A pipeline for SNP selection was devised that avoided the pitfalls associated with the inclusion of paralogous sequence variants, supported the construction of robust multi-allelic SNP haploblocks and selected up to 11 entries within narrow genomic regions of ±5 kb, termed focal points (FPs). Broad genome coverage was attained by placing FPs at 1 cM intervals on a consensus genetic map, complementing them with FPs to enrich the ends of each of the chromosomes, and by bridging physical intervals greater than 400 Kbps. The selection also included ∼3.7K validated SNPs from the IRSC 8K array. The array has already been used in other studies where ∼15.8K SNP markers were mapped with an average of ∼6.8K SNPs per full-sib family. The newly developed array with its high density of polymorphic validated SNPs is expected to be of great utility for Pedigree-Based Analysis and Genomic Selection. It will also be a valuable tool to help dissect the genetic mechanisms controlling important fruit quality traits, and to aid the identification of marker-trait associations suitable for the application of Marker Assisted Selection in apple breeding programs.

Whole-exome sequencing in splenic marginal zone lymphoma reveals mutations in genes involved in marginal zone differentiation

Splenic marginal zone lymphoma (SMZL) is a B-cell neoplasm whose molecular pathogenesis remains fundamentally unexplained, requiring more precise diagnostic markers. Previous molecular studies have revealed 7q loss and mutations of nuclear factor κB (NF-κB), B-cell receptor (BCR) and Notch signalling genes. We performed whole-exome sequencing in a series of SMZL cases. Results confirmed that SMZL is an entity distinct from other low-grade B-cell lymphomas, and identified mutations in multiple genes involved in marginal zone development, and others involved in NF-κB, BCR, chromatin remodelling and the cytoskeleton.

Extreme genomic erosion after recurrent demographic bottlenecks in the highly endangered Iberian lynx

BackgroundGenomic studies of endangered species provide insights into their evolution and demographic history, reveal patterns of genomic erosion that might limit their viability, and offer tools for their effective conservation. The Iberian lynx (Lynx pardinus) is the most endangered felid and a unique example of a species on the brink of extinction.ResultsWe generate the first annotated draft of the Iberian lynx genome and carry out genome-based analyses of lynx demography, evolution, and population genetics. We identify a series of severe population bottlenecks in the history of the Iberian lynx that predate its known demographic decline during the 20th century and have greatly impacted its genome evolution. We observe drastically reduced rates of weak-to-strong substitutions associated with GC-biased gene conversion and increased rates of fixation of transposable elements. We also find multiple signatures of genetic erosion in the two remnant Iberian lynx populations, including a high frequency of potentially deleterious variants and substitutions, as well as the lowest genome-wide genetic diversity reported so far in any species.ConclusionsThe genomic features observed in the Iberian lynx genome may hamper short- and long-term viability through reduced fitness and adaptive potential. The knowledge and resources developed in this study will boost the research on felid evolution and conservation genomics and will benefit the ongoing conservation and management of this emblematic species.

Shared Oncogenic Pathways Implicated in Both Virus-Positive and UV-Induced Merkel Cell Carcinomas

Merkel cell carcinoma (MCC) is a highly malignant neuroendocrine tumor of the skin whose molecular pathogenesis is not completely understood, despite the role that Merkel cell polyomavirus can play in 55-90% of cases. To study potential mechanisms driving this disease in clinically characterized cases, we searched for somatic mutations using whole-exome sequencing, and extrapolated our findings to study functional biomarkers reporting on the activity of the mutated pathways. Confirming previous results, Merkel cell polyomavirus-negative tumors had higher mutational loads with UV signatures and more frequent mutations in TP53 and RB compared with their Merkel cell polyomavirus-positive counterparts. Despite important genetic differences, the two Merkel cell carcinoma etiologies both exhibited nuclear accumulation of oncogenic transcription factors such as NFAT or nuclear factor of activated T cells (NFAT), P-CREB, and P-STAT3, indicating commonly deregulated pathogenic mechanisms with the potential to serve as targets for therapy. A multivariable analysis identified phosphorylated CRE-binding protein as an independent survival factor with respect to clinical variables and Merkel cell polyomavirus status in our cohort of Merkel cell carcinoma patients.

Genetic Diversity and Population Structure of Rice Varieties Cultivated in Temperate Regions

BackgroundAfter its domestication, rice cultivation expanded from tropical regions towards northern latitudes with temperate climate in a progressive process to overcome limiting photoperiod and temperature conditions. This process has originated a wide range of diversity that can be regarded as a valuable resource for crop improvement. In general, current rice breeding programs have to deal with a lack of both germplasm accessions specifically adapted to local agro-environmental conditions and adapted donors carrying desired agronomical traits. Comprehensive maps of genome variability and population structure would facilitate genome-wide association studies of complex traits, functional gene investigations and the selection of appropriate donors for breeding purposes.ResultsA collection of 217 rice varieties mainly cultivated in temperate regions was generated. The collection encompasses modern elite and old cultivars, as well as traditional landraces covering a wide genetic diversity available for rice breeders. Whole Genome Sequencing was performed on 14 cultivars representative of the collection and the genomic profiles of all cultivars were constructed using a panel of 2697 SNPs with wide coverage throughout the rice genome, obtained from the sequencing data. The population structure and genetic relationship analyses showed a strong substructure in the temperate rice population, predominantly based on grain type and the origin of the cultivars. Dendrogram also agrees population structure results.ConclusionsBased on SNP markers, we have elucidated the genetic relationship and the degree of genetic diversity among a collection of 217 temperate rice varieties possessing an enormous variety of agromorphological and physiological characters. Taken together, the data indicated the occurrence of relatively high gene flow and elevated rates of admixture between cultivars grown in remote regions, probably favoured by local breeding activities. The results of this study significantly expand the current genetic resources available for temperate varieties of rice, providing a valuable tool for future association mapping studies.

Genomic and Molecular Screenings Identify Different Mechanisms for Acquired Resistance to MET Inhibitors in Lung Cancer Cells

The development of resistance to tyrosine kinase inhibitors (TKI) limits the long-term efficacy of cancer treatments involving them. We aimed to understand the mechanisms that underlie acquired resistance (AR) to MET inhibitors in lung cancer. EBC1 cells, which have MET amplification and are sensitive to TKIs against MET, were used to generate multiple clones with AR to a MET-TKI. Whole-exome sequencing, RNA sequencing, and global DNA methylation analysis were used to scrutinize the genetic and molecular characteristics of the resistant cells. AR to the MET-TKI involved changes common to all resistant cells, that is, phenotypic modifications, specific changes in gene expression, and reactivation of AKT, ERK, and mTOR. The gene expression, global DNA methylation, and mutational profiles distinguished at least two groups of resistant cells. In one of these, the cells have acquired sensitivity to erlotinib, concomitantly with mutations of the KIRREL, HDAC11, HIATL1, and MAPK1IP1L genes, among others. In the other group, some cells have acquired inactivation of neurofibromatosis type 2 (NF2) concomitantly with strong overexpression of NRG1 and a mutational profile that includes changes in LMLN and TOMM34. Multiple independent and simultaneous strategies lead to AR to the MET-TKIs in lung cancer cells. The acquired sensitivity to erlotinib supports the known crosstalk between MET and the HER family of receptors. For the first time, we show inactivation of NF2 during acquisition of resistance to MET-TKI that may explain the refractoriness to erlotinib in these cells. Mol Cancer Ther; 16(7); 1366–76. ©2017 AACR.

Colorectal adenomas contain multiple somatic mutations that do not coincide with synchronous adenocarcinoma specimens

We have performed a comparative ultrasequencing study of multiple colorectal lesions obtained simultaneously from four patients. Our data show that benign lesions (adenomatous or hyperplastic polyps) contain a high mutational load. Additionally multiple synchronous colorectal lesions show non overlapping mutational signatures highlighting the degree of heterogeneity between multiple specimens in the same patient. Observations in these cases imply that considering not only the number of mutations but an effective oncogenic combination of mutations can determine the malignant progression of colorectal lesions.

Whole exome sequencing analysis reveals TRPV3 as a risk factor for cardioembolic stroke.

Genetic studies suggest that hundreds of genes associated with stroke remain unidentified. Exome sequencing proves useful for finding new genes associated with stroke. We aimed to find new genetic risk factors for cardioembolic stroke by analysing exome sequence data using new strategies. For discovery, we analysed 42 cardioembolic stroke cases and controls with extreme phenotypes (cohort 1), and for replication, 32 cardioembolic stroke cases and controls (cohort 2) using the SeqCapExome capture kit. We then analysed the replicated genes in two new cohorts that comprised 834 cardioembolic strokes and controls (cohort 3) and 64,373 cardioembolic strokes and controls (cohort 4). Transcriptomic in-silico functional analyses were also performed. We found 26 coding regions with a higher frequency of mutations in cardioembolic strokes after correcting for the number of mutations found in the whole exome of every patient. The TRPV3 gene was associated with cardioembolic stroke after replication of exome sequencing analysis (p-value-discovery: 0.018, p-value-replication: 0.014). The analysis of the TRPV3 gene using polymorphisms in cohort 3 and 4 revealed two polymorphisms associated with cardioembolic stroke in both cohorts, the most significant polymorphism being rs151091899 (p-value: 3.1 × 10-05; odds ratio: 5.4) in cohort 3. The genotype of one polymorphism of TRPV3 was associated with a differential expression of genes linked to cardiac malformations. In conclusion, new strategies using exome sequence data have revealed TRPV3 as a new gene associated with cardioembolic stroke. This strategy among others might be useful in finding new genes associated with complex genetic diseases.

Identification of genetic variation in the swine toll-like receptors and development of a porcine TLR genotyping array

BackgroundToll-like receptors (TLR) are crucial in innate immunity for the recognition of a broad range of microbial pathogens and are expressed in multiple cell types. There are 10 TLR genes described in the pig genome.ResultsWith a twofold objective i.e. to catalogue genetic variants in porcine TLR genes and develop a genotyping array for genetic association studies on immune-related traits, we combined targeted sub-genome enrichment and high-throughput sequencing to sequence the 10 porcine TLR genes in 266 pigs from 10 breeds and wild boars using a DNA-pooling strategy. We identified 306 single nucleotide variants across the 10 TLR and 11 populations, 87 of which were novel. One hundred and forty-seven positions i.e. six stop-gains and 141 non-synonymous substitutions were predicted to alter the protein sequence. Three positions were unique to a single breed with alternative allele frequencies equal to or higher than 0.5. We designed a genotyping array for future applications in genetic association studies, with a selection of 126 variants based on their predicted impact on protein sequence. Since TLR4, TLR7 and TLR9 were underrepresented in this selection, we also included three variants that were located in the 3′UTR of these genes. We tested the array by genotyping 214 of the 266 sequenced pigs. We found that 93 variants that involved the 10 TLR genes were polymorphic in these animals. Twelve of these variants were novel. Furthermore, seven known variants that are associated with immune-related phenotypes are present on the array and can thus be used to test such associations in additional populations.ConclusionsWe identified genetic variations that potentially have an impact on the protein sequence of porcine TLR. A genotyping array with 80 non-synonymous, 10 synonymous and three 3′UTR polymorphisms in the 10 TLR genes is now available for association studies in swine populations with measures on immune-related traits.