Genome sequencing and analysis of the Tasmanian devil and its transmissible cancer.

The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations.

NxTrim: optimized trimming of Illumina mate pair reads.

MOTIVATION: Mate pair protocols add to the utility of paired-end sequencing by boosting the genomic distance spanned by each pair of reads, potentially allowing larger repeats to be bridged and resolved. The Illumina Nextera Mate Pair (NMP) protocol uses a circularization-based strategy that leaves behind 38-bp adapter sequences, which must be computationally removed from the data. While 'adapter trimming' is a well-studied area of bioinformatics, existing tools do not fully exploit the particular properties of NMP data and discard more data than is necessary. RESULTS: We present NxTrim, a tool that strives to discard as little sequence as possible from NMP reads. NxTrim makes full use of the sequence on both sides of the adapter site to build 'virtual libraries' of mate pairs, paired-end reads and single-ended reads. For bacterial data, we show that aggregating these datasets allows a single NMP library to yield an assembly whose quality compares favourably to that obtained from regular paired-end reads. AVAILABILITY AND IMPLEMENTATION: The source code is available at https://github.com/sequencing/NxTrim

A comprehensive catalogue of somatic mutations from a human cancer genome.

All cancers carry somatic mutations. A subset of these somatic alterations, termed driver mutations, confer selective growth advantage and are implicated in cancer development, whereas the remainder are passengers. Here we have sequenced the genomes of a malignant melanoma and a lymphoblastoid cell line from the same person, providing the first comprehensive catalogue of somatic mutations from an individual cancer. The catalogue provides remarkable insights into the forces that have shaped this cancer genome. The dominant mutational signature reflects DNA damage due to ultraviolet light exposure, a known risk factor for malignant melanoma, whereas the uneven distribution of mutations across the genome, with a lower prevalence in gene footprints, indicates that DNA repair has been preferentially deployed towards transcribed regions. The results illustrate the power of a cancer genome sequence to reveal traces of the DNA damage, repair, mutation and selection processes that were operative years before the cancer became symptomatic.

Specific correction of a splice defect in brain by nutritional supplementation.

Recent studies emphasize the importance of mRNA splicing in human genetic disease, as 20-30% of all disease-causing mutations are predicted to result in mRNA splicing defects. The plasticity of the mRNA splicing reaction has made these mutations attractive candidates for the development of therapeutics. Familial dysautonomia (FD) is a severe neurodegenerative disorder, and all patients have an intronic IVS20+6T>C splice site mutation in the IKBKAP gene, which results in tissue-specific skipping of exon 20 and a corresponding reduction in ikappaB kinase complex associated protein (IKAP) levels. We created transgenic mouse lines using a human IKBKAP bacterial artificial chromosome (BAC) into which we inserted the IKBKAP splice mutation (FD BAC) and have shown that the transgenic mice exhibit the same tissue-specific aberrant splicing patterns as seen in FD patients. We have previously demonstrated that the plant cytokinin kinetin can significantly improve the production of wild-type IKBKAP transcripts in FD lymphoblast cell lines by improving exon inclusion. In this study, we tested the ability of kinetin to alter IKBKAP splicing in the transgenic mice carrying the FD BAC and show that it corrects IKBKAP splicing in all major tissues assayed, including the brain. The amount of wild-type IKBKAP mRNA and IKAP protein was significantly higher in the kinetin-treated mice. These exciting results prove that treatment of FD, as well as other mechanistically related splicing disorders, with kinetin holds great promise as a potential therapeutic aimed at increasing normal protein levels, which may, in turn, slow disease progression.

Therapeutic potential and mechanism of kinetin as a treatment for the human splicing disease familial dysautonomia.

Mutations that affect the splicing of pre-mRNA are a major cause of human disease. Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a T to C transition at base pair 6 of IKBKAP intron 20. This mutation results in variable tissue-specific skipping of exon 20. Previously, we reported that the plant cytokinin kinetin dramatically increases exon 20 inclusion in RNA isolated from cultured FD cells. The goal of the current study was to investigate the nature of the FD splicing defect and the mechanism by which kinetin improves exon inclusion, as such knowledge will facilitate the development of future therapeutics aimed at regulating mRNA splicing. In this study, we demonstrate that treatment of FD lymphoblast cell lines with kinetin increases IKBKAP mRNA and IKAP protein to normal levels. Using a series of minigene constructs, we show that deletion of a region at the end of IKBKAP exon 20 disrupts the ability of kinetin to improve exon inclusion, pinpointing a kinetin responsive sequence element. We next performed a screen of endogenously expressed genes with multiple isoforms resulting from exon skipping events and show that kinetin's ability to improve exon inclusion is not limited to IKBKAP. Lastly, we highlight the potential of kinetin for the treatment of other human splicing disorders by showing correction of a splicing defect in neurofibromatosis.

Weak definition of IKBKAP exon 20 leads to aberrant splicing in familial dysautonomia.

Splicing mutations that lead to devastating genetic diseases are often located in nonconserved or weakly conserved sequences that normally do not affect splicing. Thus, the underlying reason for the splicing defect is not immediately obvious. An example of this phenomenon is observed in the neurodevelopmental disease familial dysautonomia (FD), which is caused by a single-base change in the 5' splice site (5'ss) of intron 20 in the IKBKAP gene (c.2204+6T>C). This mutation, which is in the sixth position of the intron and results in exon 20 skipping, has no phenotype in many other introns. To determine why the position 6 mutation causes aberrant splicing only in certain cases, we first used an in silico approach to identify potential sequences involved in exon 20 skipping. Computational analyses of the exon 20 5'ss itself predicted that this nine-nucleotide splicing signal, even when it contains the T>C mutation, is not sufficiently weak to explain the FD phenotype. However, the computational analysis predicted that both the upstream 3' splice site (3'ss) and exon 20 contain weak splicing signals, indicating that the FD 5'ss, together with the surrounding splicing signals, are not adequate for defining exon 20. These in silico predictions were corroborated using IKBKAP minigenes in a new rapid and simple in vitro coupled RNA polymerase (RNAP) II transcription/splicing assay. Finally, the weak splicing signals that flank the T>C mutation were validated as the underlying cause of familial dysautonomia in vivo using transient transfection assays. Together, our study demonstrates the general utility of combining in silico data with an in vitro RNAP II transcription/splicing system for rapidly identifying critical sequences that underlie the numerous splicing diseases caused by otherwise silent mutations.

Studying clonal dynamics in response to cancer therapy using high-complexity barcoding.

Resistance to cancer therapies presents a significant clinical challenge. Recent studies have revealed intratumoral heterogeneity as a source of therapeutic resistance. However, it is unclear whether resistance is driven predominantly by pre-existing or de novo alterations, in part because of the resolution limits of next-generation sequencing. To address this, we developed a high-complexity barcode library, ClonTracer, which enables the high-resolution tracking of more than 1 million cancer cells under drug treatment. In two clinically relevant models, ClonTracer studies showed that the majority of resistant clones were part of small, pre-existing subpopulations that selectively escaped under therapeutic challenge. Moreover, the ClonTracer approach enabled quantitative assessment of the ability of combination treatments to suppress resistant clones. These findings suggest that resistant clones are present before treatment, which would make up-front therapeutic combinations that target non-overlapping resistance a preferred approach. Thus, ClonTracer barcoding may be a valuable tool for optimizing therapeutic regimens with the goal of curative combination therapies for cancer.

Mutations in a protein target of the Pim-1 kinase associated with the RP9 form of autosomal dominant retinitis pigmentosa.

The RP9 form of autosomal dominant retinitis pigmentosa (adRP) maps to a locus on human chromosome 7p14. We now report two different disease associated mutations in a previously unidentified human gene, the mouse orthologue of which has been characterised by its interaction with the Pim-1 oncogene. In the original linked family we identified the missense mutation H137L. A second missense mutation, D170G, was found in a single RP patient. The putative RP9 gene appears to be expressed in a wide range of tissues, but its function is unknown and a pathogenic mechanism remains to be determined.

Loss of mouse Ikbkap, a subunit of elongator, leads to transcriptional deficits and embryonic lethality that can be rescued by human IKBKAP.

Familial dysautonomia (FD), a devastating hereditary sensory and autonomic neuropathy, results from an intronic mutation in the IKBKAP gene that disrupts normal mRNA splicing and leads to tissue-specific reduction of IKBKAP protein (IKAP) in the nervous system. To better understand the roles of IKAP in vivo, an Ikbkap knockout mouse model was created. Results from our study show that ablating Ikbkap leads to embryonic lethality, with no homozygous Ikbkap knockout (Ikbkap(-)(/)(-)) embryos surviving beyond 12.5 days postcoitum. Morphological analyses of the Ikbkap(-)(/)(-) conceptus at different stages revealed abnormalities in both the visceral yolk sac and the embryo, including stunted extraembryonic blood vessel formation, delayed entry into midgastrulation, disoriented dorsal primitive neural alignment, and failure to establish the embryonic vascular system. Further, we demonstrate downregulation of several genes that are important for neurulation and vascular development in the Ikbkap(-)(/)(-) embryos and show that this correlates with a defect in transcriptional elongation-coupled histone acetylation. Finally, we show that the embryonic lethality resulting from Ikbkap ablation can be rescued by a human IKBKAP transgene. For the first time, we demonstrate that IKAP is crucial for both vascular and neural development during embryogenesis and that protein function is conserved between mouse and human.

A humanized IKBKAP transgenic mouse models a tissue-specific human splicing defect.

Familial dysautonomia (FD) is a severe hereditary sensory and autonomic neuropathy, and all patients with FD have a splice mutation in the IKBKAP gene. The FD splice mutation results in variable, tissue-specific skipping of exon 20 in IKBKAP mRNA, which leads to reduced IKAP protein levels. The development of therapies for FD will require suitable mouse models for preclinical studies. In this study, we report the generation and characterization of a mouse model carrying the complete human IKBKAP locus with the FD IVS20+6T-->C splice mutation. We show that the mutant IKBKAP transgene is misspliced in this model in a tissue-specific manner that replicates the pattern seen in FD patient tissues. Creation of this humanized mouse is the first step toward development of a complex phenotypic model of FD. These transgenic mice are an ideal model system for testing the effectiveness of therapeutic agents that target the missplicing defect. Last, these mice will permit direct studies of tissue-specific splicing and the identification of regulatory factors that play a role in complex gene expression.

Rescue of a human mRNA splicing defect by the plant cytokinin kinetin.

The defective splicing of pre-mRNA is a major cause of human disease. Exon skipping is a common result of splice mutations and has been reported in a wide variety of genetic disorders, yet the underlying mechanism is poorly understood. Often, such mutations are incompletely penetrant, and low levels of normal transcript and protein are maintained. Familial dysautonomia (FD) is caused by mutations in IKBKAP, and all cases described to date involve an intron 20 mutation that results in a unique pattern of tissue-specific exon skipping. Accurate splicing of the mutant IKBKAP allele is particularly inefficient in the nervous system. Here we show that treatment with the plant cytokinin kinetin alters splicing of IKBKAP. Kinetin significantly increases inclusion of exon 20 from the endogenous gene, as well as from an IKBKAP minigene. By contrast the drug does not enhance inclusion of alternatively spliced exon 31 in MYO5A. Benzyladenine, the most closely related cytokinin, showed a similar but less dramatic effect. Our findings reveal a remarkable impact on splicing fidelity by these small molecules, which therefore provide new tools for the dissection of mechanisms controlling tissue-specific pre-mRNA splicing. Further, kinetin should be explored as a treatment for increasing the level of normal IKAP in FD, and for other splicing disorders that may share a similar mechanism.

Mutations in HPRP3, a third member of pre-mRNA splicing factor genes, implicated in autosomal dominant retinitis pigmentosa.

Retinitis pigmentosa (RP), the commonest form of inherited retinal dystrophies is a clinically and genetically heterogeneous disorder. It is characterized by progressive degeneration of the peripheral retina leading to night blindness and loss of peripheral visual field. RP is inherited either in an autosomal dominant, autosomal recessive or X-linked mode. A locus (RP18) for autosomal dominant RP was previously mapped by linkage analysis in two large pedigrees to chromosome 1p13-q21. The human HPRP3 gene, the orthologue of the yeast pre-mRNA splicing factor (PRP3), localizes within the RP18 disease interval. The recent identification of mutations in human splicing factors, PRPF31 and PRPC8, led us to screen HPRP3 as a candidate in three chromosome 1q-linked families. So far, two different missense mutations in two English, a Danish family and in three RP individuals have been identified. Both mutations are clustered within a two-codon stretch in the 11th exon of the HPRP3 gene. Interestingly, one of the mutations (T494M) is seen repeatedly in apparently unlinked families raising the possibility of a mutation hot spot. This has been confirmed by haplotype analysis using SNPs spanning the HPRP3 gene region supporting multiple origins of the mutation. The altered HPRP3 amino acids, which are highly conserved in all known HPRP3 orthologues, indicate a major function of that domain in the splicing process. The identification of mutations in a third pre-mRNA splicing factor gene further highlights a novel mechanism of photoreceptor degeneration due to defects in the splicing process.