Correction: The genetic consequences of dog breed formation-Accumulation of deleterious genetic variation and fixation of mutations associated with myxomatous mitral valve disease in cavalier King Charles spaniels.

[This corrects the article DOI: 10.1371/journal.pgen.1009726.].

The genetic consequences of dog breed formation-Accumulation of deleterious genetic variation and fixation of mutations associated with myxomatous mitral valve disease in cavalier King Charles spaniels.

Selective breeding for desirable traits in strictly controlled populations has generated an extraordinary diversity in canine morphology and behaviour, but has also led to loss of genetic variation and random entrapment of disease alleles. As a consequence, specific diseases are now prevalent in certain breeds, but whether the recent breeding practice led to an overall increase in genetic load remains unclear. Here we generate whole genome sequencing (WGS) data from 20 dogs per breed from eight breeds and document a ~10% rise in the number of derived alleles per genome at evolutionarily conserved sites in the heavily bottlenecked cavalier King Charles spaniel breed (cKCs) relative to in most breeds studied here. Our finding represents the first clear indication of a relative increase in levels of deleterious genetic variation in a specific breed, arguing that recent breeding practices probably were associated with an accumulation of genetic load in dogs. We then use the WGS data to identify candidate risk alleles for the most common cause for veterinary care in cKCs-the heart disease myxomatous mitral valve disease (MMVD). We verify a potential link to MMVD for candidate variants near the heart specific NEBL gene in a dachshund population and show that two of the NEBL candidate variants have regulatory potential in heart-derived cell lines and are associated with reduced NEBL isoform nebulette expression in papillary muscle (but not in mitral valve, nor in left ventricular wall). Alleles linked to reduced nebulette expression may hence predispose cKCs and other breeds to MMVD via loss of papillary muscle integrity.

Transcriptomic analysis of the woodchuck model of chronic hepatitis B.

UNLABELLED: The Eastern woodchuck (Marmota monax) is naturally infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to the human hepatitis B virus (HBV). The woodchuck is used as an animal model for studying chronic hepatitis B (CHB) and HBV-associated hepatocellular carcinoma (HCC) in humans, but the lack of sequence information has hitherto precluded functional genomics analysis. To address this major limitation of the model, we report here the sequencing, assembly, and annotation of the woodchuck transcriptome, together with the generation of custom woodchuck microarrays. Using this new platform, we characterized the transcriptional response to persistent WHV infection and WHV-induced HCC. This revealed that chronic WHV infection, like HBV, is associated with (1) a limited intrahepatic type I interferon response; (2) intrahepatic induction of markers associated with T cell exhaustion; (3) elevated levels of suppressor of cytokine signaling 3 (SOCS3) in the liver; and (4) intrahepatic accumulation of neutrophils. Underscoring the translational value of the woodchuck model, this study also determined that WHV-induced HCC shares molecular characteristics with a subtype of human HCC with poor prognosis. CONCLUSION: Our data establish the translational value of the woodchuck model and provide new insight into immune pathways which may play a role either in the persistence of HBV infection or the sequelae of CHB.

Analysis of TSC cortical tubers by deep sequencing of TSC1, TSC2 and KRAS demonstrates that small second-hit mutations in these genes are rare events.

Tuberous sclerosis complex (TSC) is an often severe neurocutaneous syndrome. Cortical tubers are the predominant neuropathological finding in TSC, and their number and location has been shown to correlate roughly with the severity of neurologic features in TSC. Past studies have shown that genomic deletion events in TSC1 or TSC2 are very rare in tubers, and suggested the potential involvement of the MAPK pathway in their pathogenesis. We used deep sequencing to assess all coding exons of TSC1 and TSC2, and the activating mutation hot spots within KRAS in 46 tubers from TSC patients. Germline heterozygous mutations were identified in 81% of tubers. The same secondary mutation in TSC2 was identified in six tuber samples from one individual. Further study showed that this second hit mutation was widely distributed in the cortex from one cerebral hemisphere of this individual at frequencies up to 10%. No other secondary mutations were found in the other 40 tubers analyzed. These data indicate that small second hit mutations in any of these three genes are very rare in TSC tubers. However, in one TSC individual, a second hit TSC2 point mutation occurred early during brain development, and likely contributed to tuber formation.

Second generation sequencing of the mesothelioma tumor genome.

The current paradigm for elucidating the molecular etiology of cancers relies on the interrogation of small numbers of genes, which limits the scope of investigation. Emerging second-generation massively parallel DNA sequencing technologies have enabled more precise definition of the cancer genome on a global scale. We examined the genome of a human primary malignant pleural mesothelioma (MPM) tumor and matched normal tissue by using a combination of sequencing-by-synthesis and pyrosequencing methodologies to a 9.6X depth of coverage. Read density analysis uncovered significant aneuploidy and numerous rearrangements. Method-dependent informatics rules, which combined the results of different sequencing platforms, were developed to identify and validate candidate mutations of multiple types. Many more tumor-specific rearrangements than point mutations were uncovered at this depth of sequencing, resulting in novel, large-scale, inter- and intra-chromosomal deletions, inversions, and translocations. Nearly all candidate point mutations appeared to be previously unknown SNPs. Thirty tumor-specific fusions/translocations were independently validated with PCR and Sanger sequencing. Of these, 15 represented disrupted gene-encoding regions, including kinases, transcription factors, and growth factors. One large deletion in DPP10 resulted in altered transcription and expression of DPP10 transcripts in a set of 53 additional MPM tumors correlated with survival. Additionally, three point mutations were observed in the coding regions of NKX6-2, a transcription regulator, and NFRKB, a DNA-binding protein involved in modulating NFKB1. Several regions containing genes such as PCBD2 and DHFR, which are involved in growth factor signaling and nucleotide synthesis, respectively, were selectively amplified in the tumor. Second-generation sequencing uncovered all types of mutations in this MPM tumor, with DNA rearrangements representing the dominant type.

Ultra deep sequencing detects a low rate of mosaic mutations in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous syndrome caused by mutations in TSC1 and TSC2. However, 10-15% TSC patients have no mutation identified with conventional molecular diagnostic studies. We used the ultra-deep pyrosequencing technique of 454 Sequencing to search for mosaicism in 38 TSC patients who had no TSC1 or TSC2 mutation identified by conventional methods. Two TSC2 mutations were identified, each at 5.3% read frequency in different patients, consistent with mosaicism. Both mosaic mutations were confirmed by several methods. Five of 38 samples were found to have heterozygous non-mosaic mutations, which had been missed in earlier analyses. Several other possible low-frequency mosaic mutations were identified by deep sequencing, but were discarded as artifacts by secondary studies. The low frequency of detection of mosaic mutations, two (6%) of 33, suggests that the majority of TSC patients who have no mutation identified are not due to mosaicism, but rather other causes, which remain to be determined. These findings indicate the ability of deep sequencing, coupled with secondary confirmatory analyses, to detect low-frequency mosaic mutations.

Transcriptome sequencing of malignant pleural mesothelioma tumors.

Cancers arise by the gradual accumulation of mutations in multiple genes. We now use shotgun pyrosequencing to characterize RNA mutations and expression levels unique to malignant pleural mesotheliomas (MPMs) and not present in control tissues. On average, 266 Mb of cDNA were sequenced from each of four MPMs, from a control pulmonary adenocarcinoma (ADCA), and from normal lung tissue. Previously observed differences in MPM RNA expression levels were confirmed. Point mutations were identified by using criteria that require the presence of the mutation in at least four reads and in both cDNA strands and the absence of the mutation from sequence databases, normal adjacent tissues, and other controls. In the four MPMs, 15 nonsynonymous mutations were discovered: 7 were point mutations, 3 were deletions, 4 were exclusively expressed as a consequence of imputed epigenetic silencing, and 1 was putatively expressed as a consequence of RNA editing. Notably, each MPM had a different mutation profile, and no mutated gene was previously implicated in MPM. Of the seven point mutations, three were observed in at least one tumor from 49 other MPM patients. The mutations were in genes that could be causally related to cancer and included XRCC6, PDZK1IP1, ACTR1A, and AVEN.

The Melanoma Vascular Mimicry Phenotype Defined in Gene Expression and Microsome Sequencing Analysis.

The phenomenon of vasculogenic mimicry in melanoma has been recently described to be an important factor relating to melanoma progression. Large scale gene expression profiling by real-time quantitative RT-QPCR of a panel of 40 normal tissues and 54 cancer cell lines revealed that two genetically related melanoma cell lines, one derived from a primary lesion Hs.688(A) and one derived from a lymph node metastasis Hs.688(B), displayed a unique expression pattern when compared to other cancer cell lines and tissue samples in the panel. Quantitative-RT-PCR data indicated that these melanoma cells expressed a number of activated endothelial cell-associated genes such as tissue inhibitors of matrix metalloproteinases TIMP-2, matrix metalloproteinase (MMP-1, MMP-2), thrombospondin 1 (TSP1), proto-oncogene c-MET and vascular endothelial growth factor (VEGF). To examine the gene expression profile of these unique melanoma cells in greater depth, cDNA libraries were made from isolated microsome complexes to enrich those transcripts that were destined to be translated into cell surface or secreted proteins. High throughput sequencing analysis revealed that this library contained over 7000 cDNAs and was enriched by over 80% of secreted or membrane-bound proteins. The presence in the cDNA library of genes such as acetyl LDL receptor, tumor endothelial markers-1, 5 and 8 (TEMs), flow-induced endothelial G protein coupled receptor-1 and VEGF-related protein (VRP), all of which are known to be expressed uniquely by endothelial cells, supported the hypothesis that Hs.688(A) and Hs.688(B) cells were mimicking an activated vascular phenotype. Ultimately the goal is to investigate the biological roles of endothelial cell-associated genes in the behavior of Hs.688(A) and Hs.688 (B) melanoma cells.

Simultaneous gene expression analysis of steady-state and actively translated mRNA populations from osteosarcoma MG-63 cells in response to IL-1alpha via an open expression analysis platform.

Pro-inflammatory cytokines play a key role in various forms of metabolic bone diseases, including osteopenia and osteoporosis. Human MG-63 cells treated with IL-1alpha were used as a model system to identify potential marker genes that are differentially expressed. This study is designed to quantitate gene expression of actively translated mRNAs as compared to the steady-state mRNA population. Both steady-state mRNAs and actively translated mRNAs from control MG-63 cells and MG-63 cells treated with IL-1alpha were isolated and converted to cDNA. The gene expression analysis from these samples was then quantitated with an open expression analysis platform with no requirement for a priori knowledge of sequence information. As a result, many differentially regulated genes were discovered via IL-1alpha treatment. Some of the genes have been described previously as playing important roles in the regulation of inflammation and cell adhesion. These comparisons provided a panoramic overview of gene expression at both the total transcript and post-transcriptional levels. In addition, the quantitation of actively translated mRNAs associated with polysomes also provided a better estimation of protein expression levels. This methodology allows for the identification of genes acutely regulated during translation. Furthermore, the process may aid in the identification of new drug targets or biomarkers.