Adrian Ally

The genetic landscape of high-risk neuroblastoma

Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents with widespread metastatic disease, resulting in survival rates of less than 50%. To determine the spectrum of somatic mutation in high-risk neuroblastoma, we studied 240 affected individuals (cases) using a combination of whole-exome, genome and transcriptome sequencing as part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Here we report a low median exonic mutation frequency of 0.60 per Mb (0.48 nonsilent) and notably few recurrently mutated genes in these tumors. Genes with significant somatic mutation frequencies included ALK (9.2% of cases), PTPN11 (2.9%), ATRX (2.5%, and an additional 7.1% had focal deletions), MYCN (1.7%, causing a recurrent p.Pro44Leu alteration) and NRAS (0.83%). Rare, potentially pathogenic germline variants were significantly enriched in ALK, CHEK2, PINK1 and BARD1. The relative paucity of recurrent somatic mutations in neuroblastoma challenges current therapeutic strategies that rely on frequently altered oncogenic drivers.

Oligonucleotide Microarray Analysis of Genomic Imbalance in Children with Mental Retardation

The cause of mental retardation in one-third to one-half of all affected individuals is unknown. Microscopically detectable chromosomal abnormalities are the most frequently recognized cause, but gain or loss of chromosomal segments that are too small to be seen by conventional cytogenetic analysis has been found to be another important cause. Array-based methods offer a practical means of performing a high-resolution survey of the entire genome for submicroscopic copy-number variants. We studied 100 children with idiopathic mental retardation and normal results of standard chromosomal analysis, by use of whole-genome sampling analysis with Affymetrix GeneChip Human Mapping 100K arrays. We found de novo deletions as small as 178 kb in eight cases, de novo duplications as small as 1.1 Mb in two cases, and unsuspected mosaic trisomy 9 in another case. This technology can detect at least twice as many potentially pathogenic de novo copy-number variants as conventional cytogenetic analysis can in people with mental retardation.

Alternative expression analysis by RNA sequencing

In alternative expression analysis by sequencing (ALEXA-seq), we developed a method to analyze massively parallel RNA sequence data to catalog transcripts and assess differential and alternative expression of known and predicted mRNA isoforms in cells and tissues. As proof of principle, we used the approach to compare fluorouracil-resistant and -nonresistant human colorectal cancer cell lines. We assessed the sensitivity and specificity of the approach by comparison to exon tiling and splicing microarrays and validated the results with reverse transcription–PCR, quantitative PCR and Sanger sequencing. We observed global disruption of splicing in fluorouracil-resistant cells characterized by expression of new mRNA isoforms resulting from exon skipping, alternative splice site usage and intron retention. Alternative expression annotation databases, source code, a data viewer and other resources to facilitate analysis are available at http://www.alexaplatform.org/alexa_seq/.

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

BackgroundGenomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.ResultsWe evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.ConclusionWe observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.

LongSAGE profiling of nine human embryonic stem cell lines

To facilitate discovery of novel human embryonic stem cell (ESC) transcripts, we generated 2.5 million LongSAGE tags from 9 human ESC lines. Analysis of this data revealed that ESCs express proportionately more RNA binding proteins compared with terminally differentiated cells, and identified novel ESC transcripts, at least one of which may represent a marker of the pluripotent state.

Spatial heterogeneity in medulloblastoma

Spatial heterogeneity of transcriptional and genetic markers between physically isolated biopsies of a single tumor poses major barriers to the identification of biomarkers and the development of targeted therapies that will be effective against the entire tumor. We analyzed the spatial heterogeneity of multiregional biopsies from 35 patients, using a combination of transcriptomic and genomic profiles. Medulloblastomas (MBs), but not high-grade gliomas (HGGs), demonstrated spatially homogeneous transcriptomes, which allowed for accurate subgrouping of tumors from a single biopsy. Conversely, somatic mutations that affect genes suitable for targeted therapeutics demonstrated high levels of spatial heterogeneity in MB, malignant glioma, and renal cell carcinoma (RCC). Actionable targets found in a single MB biopsy were seldom clonal across the entire tumor, which brings the efficacy of monotherapies against a single target into question. Clinical trials of targeted therapies for MB should first ensure the spatially ubiquitous nature of the target mutation.

ALEXA: a microarray design platform for alternative expression analysis

To the editor: Eukaryotic genomes are predicted to contain about 7,000–29,000 genes1. Each of these genes may be alternatively processed to produce multiple distinct mRNAs by alternative transcript initiation, splicing and polyadenylation (collectively referred to as alternative expression). Although analysis of available transcript resources indicates that up to ~75% of genes are alternatively processed, most microarray expression platforms cannot detect alternative transcripts2. Proof-of-principle experiments have described the use of oligonucleotide microarrays to profile transcript isoforms generated by alternative expression, but resources to create such arrays are lacking3,4. To address this limitation we created a microarray design platform for alternative expression analysis (ALEXA), which is capable of designing arrays that can detect all of the major categories of alternative expression. The ALEXA platform facilitates selection and annotation of oligonucleotide probes representing alternative expression events for any species in the EnsEMBL database1. For each target gene, probes are selected within every exon, intron, exon junction and exon boundary. This approach allows for the detection of constitutive and alternative exons, canonical exon junctions, junctions of known or new exon-skipping events, alternative exon boundaries and retained introns (Supplementary Fig. 1 online). We designed the platform to be flexible to the user’s experimental interests and preferred array manufacturer. The user may limit probe selection to known alternative expression events or include all possible exon junctions and boundaries to drive the discovery of transcripts. Probes may be designed for an arbitrary subset of genes or for all genes. Most technical parameters of the design can be modified by the user, including: the amount and types of control probes; the use of varying or fixed probe length; and the thresholds for filtering of probe sequences. The probe design process begins with retrieval of genomic sequences from EnsEMBL, removal of pseudogenes, masking of repeat elements and extraction of probe sequences. Random probe sequences are generated to uniformly represent the melting temperature and length of all experimental probes. Extracted and randomly generated probes are scored according to their melting temperature, folding potential, complexity and specificity (Supplementary Methods online). Although several publications have described using microarrays to study alternative expression in model organisms and specific tissues2, to our knowledge ours is the first report of a resource that makes alternative expression microarray designs readily available. Using the ALEXA approach, we precomputed microarray designs representing ~100 million probe sequences for ten EnsEMBL genomes (Supplementary Table 1 online). We assessed the ALEXA approach by using a prototype human array to profile the expression of alternative mRNA isoforms in 5-fluorouracil (5-FU)sensitive and resistant colorectal cancer cell lines5 and compared the results to those from the Affymetrix ‘GeneChip Human Exon 1.0 ST’ array (see Supplementary Results, Supplementary Fig. 2 and Supplementary Table 2 online). Genes and exons differentially expressed between 5-FU–sensitive and resistant cells were identified by both platforms (with significant overlap), but ALEXA arrays provided additional information on the connectivity and boundaries of exons (Table 1). Furthermore, alternative expression events identified by ALEXA were significantly enriched for known alternative expression events represented in publicly available mRNA and expressed sequence tag (EST) databases (Supplementary Results and Supplementary Data 1 online). Finally, we demonstrated the advantage of the ALEXA approach by identifying several differentially expressed known and predicted isoforms with potential relevance to 5-FU resistance (Supplementary Fig. 3 and Supplementary Tables 3 and 4 online). The approach and resources described in this work have considerable potential to advance studies of gene regulation, transcript processing, human disease and evolutionary biology (Supplementary Discussion online). The source code, precomputed array designs and related materials to assist in the creation of custom alternative expression microarrays are available on the ALEXA website (http://www.alexaplatform.org).

Novel mRNA isoforms and mutations of uridine monophosphate synthetase and 5-fluorouracil resistance in colorectal cancer

The drug fluorouracil (5-FU) is a widely used antimetabolite chemotherapy in the treatment of colorectal cancer. The gene uridine monophosphate synthetase (UMPS) is thought to be primarily responsible for conversion of 5-FU to active anticancer metabolites in tumor cells. Mutation or aberrant expression of UMPS may contribute to 5-FU resistance during treatment. We undertook a characterization of UMPS mRNA isoform expression and sequence variation in 5-FU-resistant cell lines and drug-naive or -exposed primary and metastatic tumors. We observed reciprocal differential expression of two UMPS isoforms in a colorectal cancer cell line with acquired 5-FU resistance relative to the 5-FU-sensitive cell line from which it was derived. A novel isoform arising as a consequence of exon skipping was increased in abundance in resistant cells. The underlying mechanism responsible for this shift in isoform expression was determined to be a heterozygous splice site mutation acquired in the resistant cell line. We developed sequencing and expression assays to specifically detect alternative UMPS isoforms and used these to determine that UMPS was recurrently disrupted by mutations and aberrant splicing in additional 5-FU-resistant colorectal cancer cell lines and colorectal tumors. The observed mutations, aberrant splicing and downregulation of UMPS represent novel mechanisms for acquired 5-FU resistance in colorectal cancer.

The Genome of the Northern Sea Otter (Enhydra lutris kenyoni)

The northern sea otter inhabits coastal waters of the northern Pacific Ocean and is the largest member of the Mustelidae family. DNA sequencing methods that utilize microfluidic partitioned and non-partitioned library construction were used to establish the sea otter genome. The final assembly provided 2.426 Gbp of highly contiguous assembled genomic sequences with a scaffold N50 length of over 38 Mbp. We generated transcriptome data derived from a lymphoma to aid in the determination of functional elements. The assembled genome sequence and underlying sequence data are available at the National Center for Biotechnology Information (NCBI) under the BioProject accession number PRJNA388419.

The Genome of the Beluga Whale (Delphinapterus leucas)

The beluga whale is a cetacean that inhabits arctic and subarctic regions, and is the only living member of the genus Delphinapterus. The genome of the beluga whale was determined using DNA sequencing approaches that employed both microfluidic partitioning library and non-partitioned library construction. The former allowed for the construction of a highly contiguous assembly with a scaffold N50 length of over 19 Mbp and total reconstruction of 2.32 Gbp. To aid our understanding of the functional elements, transcriptome data was also derived from brain, duodenum, heart, lung, spleen, and liver tissue. Assembled sequence and all of the underlying sequence data are available at the National Center for Biotechnology Information (NCBI) under the Bioproject accession number PRJNA360851A.