KMT2A-MAML2 rearrangement emerged and regressed during neuroblastoma therapy without leukemia after 12.8-year follow-up.

Twelvepatients without therapy-related leukemia were studied after completing TOP2 poison chemotherapy in a high-risk neuroblastoma regimen. One patient harbored an inv(11) that was a KMT2A rearrangement. The KMT2A-MAML2 transcript was expressed at low level. The patient was prospectively followed. The inv(11) was undetectable in ensuing samples. Leukemia never developed after a 12.8-year follow-up period. Enriched etoposide-induced TOP2A cleavage in the relevant MAML2 genomic region supports a TOP2A DNA damage mechanism. After completing TOP2 poison chemotherapies, covert KMT2A-R clones may occur in a small minority of patients; however, not all KMT2A rearrangements herald a therapy-related leukemia diagnosis.

Identification and characterization of a novel heparan sulfate-binding domain in Activin A longest variants and implications for function.

Activins regulate numerous processes including inflammation and are synthesized as precursors consisting of a long N-terminal pro-region and a mature protein. Genomic human databases currently list three activin A (Act A) variants termed X1, X2 and X3. The X3 variant is the shortest, lacks N-terminal segments present in X1 and X2, and has been the focus of most past literature. Here, we asked whether these variants are expressed by human cells and tissues and what structural features are contained within their pro-regions. Human monocytic-like cells THP1 and U937 expressed X1 and X2 variants after exposure to phorbol ester or granulocyte-macrophage colony-stimulating factor, while X2 transcripts were present in placenta. Expression vectors encoding full length X2 or X3 variants resulted in production and secretion of biologically active Act A from cultured cells. Previous studies reported a putative HS-binding domain (HBD) in the X3 pro-region. Here, we identified a novel HBD with consensus HS-binding motifs near the N-terminal end of X1 and X2 pro-regions. Peptides encompassing this new domain interacted with substrate-bound HS with nanomolar affinity, while peptides from putative X3 HBD did not. In good agreement, full length X2 pro-region interacted with heparin-agarose, while the X3 pro-region did not. In sum, our study reveals that Act A variants are expressed by inflammatory cells and placenta and yield biological activity. The high affinity HBD in X1 and X2 pro-region and its absence in X3 could greatly influence overall Act A distribution, availability and activity in physiological and pathological circumstances.

Correction to: HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression.

[This corrects the article DOI: 10.1038/celldisc.2016.45.].

Genome-wide TOP2A DNA cleavage is biased toward translocated and highly transcribed loci.

Type II topoisomerases orchestrate proper DNA topology, and they are the targets of anti-cancer drugs that cause treatment-related leukemias with balanced translocations. Here, we develop a high-throughput sequencing technology to define TOP2 cleavage sites at single-base precision, and use the technology to characterize TOP2A cleavage genome-wide in the human K562 leukemia cell line. We find that TOP2A cleavage has functionally conserved local sequence preferences, occurs in cleavage cluster regions (CCRs), and is enriched in introns and lincRNA loci. TOP2A CCRs are biased toward the distal regions of gene bodies, and TOP2 poisons cause a proximal shift in their distribution. We find high TOP2A cleavage levels in genes involved in translocations in TOP2 poison-related leukemia. In addition, we find that a large proportion of genes involved in oncogenic translocations overall contain TOP2A CCRs. The TOP2A cleavage of coding and lincRNA genes is independently associated with both length and transcript abundance. Comparisons to ENCODE data reveal distinct TOP2A CCR clusters that overlap with marks of transcription, open chromatin, and enhancers. Our findings implicate TOP2A cleavage as a broad DNA damage mechanism in oncogenic translocations as well as a functional role of TOP2A cleavage in regulating transcription elongation and gene activation.

HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression.

Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies.

The type 2 diabetes presumed causal variant within TCF7L2 resides in an element that controls the expression of ACSL5.

AIMS/HYPOTHESIS: One of the most strongly associated type 2 diabetes loci reported to date resides within the TCF7L2 gene. Previous studies point to the T allele of rs7903146 in intron 3 as the causal variant at this locus. We aimed to identify the actual gene(s) under the influence of this variant. METHODS: Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease, we generated a 1.4 kb deletion of the genomic region harbouring rs7903146 in the HCT116 cell line, followed by global gene expression analysis. We then carried out a combination of circularised chromosome conformation capture (4C) and Capture C in cell lines, HCT116 and NCM460 in order to ascertain which promoters of these perturbed genes made consistent physical contact with this genomic region. RESULTS: We observed 99 genes with significant differential expression (false discovery rate [FDR] cut-off:10%) and an effect size of at least twofold. The subsequent promoter contact analyses revealed just one gene, ACSL5, which resides in the same topologically associating domain as TCF7L2. The generation of additional, smaller deletions (66 bp and 104 bp) comprising rs7903146 showed consistently reduced ACSL5 mRNA levels across all three deletions of up to 30-fold, with commensurate loss of acyl-CoA synthetase long-chain family member 5 (ACSL5) protein. Notably, the deletion of this single-nucleotide polymorphism region abolished significantly detectable chromatin contacts with the ACSL5 promoter. We went on to confirm that contacts between rs7903146 and the ACSL5 promoter regions were conserved in human colon tissue. ACSL5 encodes ACSL5, an enzyme with known roles in fatty acid metabolism. CONCLUSIONS/INTERPRETATION: This 'variant to gene mapping' effort implicates the genomic location harbouring rs7903146 as a regulatory region for ACSL5.

Unique Familial MLL(KMT2A)-Rearranged Precursor B-Cell Infant Acute Lymphoblastic Leukemia in Non-twin Siblings.

BACKGROUND: Infant acute lymphoblastic leukemia (ALL) has never occurred in families except for the ∼100% concordant cases in monozygous twins attributed to twin-to-twin metastases. We report the first kindred with infant ALL in non-twin siblings. The siblings were diagnosed with MLL-rearranged (MLL-R) ALL 26 months apart. The second affected sibling had an unaffected dichorionic monozygous co-twin. Both had fatal outcomes. PROCEDURES: Translocations were characterized by karyotype, FISH, multiplex FISH, and MLL breakpoint cluster region (bcr) Southern blot analysis. Breakpoint junctions and fusion transcripts were cloned by PCR. TP53 mutation and NADPH quinone oxidorecuctase 1 (NQO1) C609T analyses were performed, and pedigree history and parental occupations were ascertained. The likelihood of chance occurrence of infant ALL in non-twin siblings was computed based on a binomial distribution. Zygosity was determined by single nucleotide polymorphism (SNP) array. RESULTS: The translocations were not related or vertically transmitted. The complex karyotype of the proband's ALL had chromosome 2, 3, 4, and 11 abnormalities causing a 5'-MLL-AFF1-3' fusion and a non-productive rearrangement of 3'MLL with a chromosome 3q intergenic region. The affected twin's ALL exhibited a simple t(4;11). The complex karyotype of the proband's ALL suggested a genotoxic insult, but no exposure was identified. There was no germline TP53 mutation. The NQO1 C609T risk allele was absent. The likelihood of infant ALL occurring in non-twin siblings by chance alone is one in 1.198 × 10(9) families. CONCLUSIONS: Whether because of a deleterious transplacental exposure, novel predisposition syndrome, or exceedingly rare chance occurrence, MLL-R infant ALL can occur in non-twin siblings. The discordant occurrence of infant ALL in the monozygous twins was likely because they were dichorionic.

ALK mutations confer differential oncogenic activation and sensitivity to ALK inhibition therapy in neuroblastoma.

Genetic studies have established anaplastic lymphoma kinase (ALK), a cell surface receptor tyrosine kinase, as a tractable molecular target in neuroblastoma. We describe comprehensive genomic, biochemical, and computational analyses of ALK mutations across 1,596 diagnostic neuroblastoma samples. ALK tyrosine kinase domain mutations occurred in 8% of samples--at three hot spots and 13 minor sites--and correlated significantly with poorer survival in high- and intermediate-risk neuroblastoma. Biochemical and computational studies distinguished oncogenic (constitutively activating) from nononcogenic mutations and allowed robust computational prediction of their effects. The mutated variants also showed differential in vitro crizotinib sensitivities. Our studies identify ALK genomic status as a clinically important therapeutic stratification tool in neuroblastoma and will allow tailoring of ALK-targeted therapy to specific mutations.

Progressive increase in mtDNA 3243A>G heteroplasmy causes abrupt transcriptional reprogramming.

Variation in the intracellular percentage of normal and mutant mitochondrial DNAs (mtDNA) (heteroplasmy) can be associated with phenotypic heterogeneity in mtDNA diseases. Individuals that inherit the common disease-causing mtDNA tRNA(Leu(UUR)) 3243A>G mutation and harbor ∼10-30% 3243G mutant mtDNAs manifest diabetes and occasionally autism; individuals with ∼50-90% mutant mtDNAs manifest encephalomyopathies; and individuals with ∼90-100% mutant mtDNAs face perinatal lethality. To determine the basis of these abrupt phenotypic changes, we generated somatic cell cybrids harboring increasing levels of the 3243G mutant and analyzed the associated cellular phenotypes and nuclear DNA (nDNA) and mtDNA transcriptional profiles by RNA sequencing. Small increases in mutant mtDNAs caused relatively modest defects in oxidative capacity but resulted in sharp transitions in cellular phenotype and gene expression. Cybrids harboring 20-30% 3243G mtDNAs had reduced mtDNA mRNA levels, rounded mitochondria, and small cell size. Cybrids with 50-90% 3243G mtDNAs manifest induction of glycolytic genes, mitochondrial elongation, increased mtDNA mRNA levels, and alterations in expression of signal transduction, epigenomic regulatory, and neurodegenerative disease-associated genes. Finally, cybrids with 100% 3243G experienced reduced mtDNA transcripts, rounded mitochondria, and concomitant changes in nuclear gene expression. Thus, striking phase changes occurred in nDNA and mtDNA gene expression in response to the modest changes of the mtDNA 3243G mutant levels. Hence, a major factor in the phenotypic variation in heteroplasmic mtDNA mutations is the limited number of states that the nucleus can acquire in response to progressive changes in mitochondrial retrograde signaling.

Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells.

Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here, we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 10(6) white blood cells (WBCs). Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control WBCs. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples of patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here, we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.

S(+)-ibuprofen destabilizes MYC/MYCN and AKT, increases p53 expression, and induces unfolded protein response and favorable phenotype in neuroblastoma cell lines.

Neuroblastoma is a common pediatric solid tumor that exhibits a striking clinical bipolarity: favorable and unfavorable. The survival rate of children with unfavorable neuroblastoma remains low among all childhood cancers. MYCN and MYC play a crucial role in determining the malignancy of unfavorable neuroblastomas, whereas high-level expression of the favorable neuroblastoma genes is associated with a good disease outcome and confers growth suppression of neuroblastoma cells. A small fraction of neuroblastomas harbors TP53 mutations at diagnosis, but a higher proportion of the relapse cases acquire TP53 mutations. In this study, we investigated the effect of S(+)-ibuprofen on neuroblastoma cell lines, focusing on the expression of the MYCN, MYC, AKT, p53 proteins and the favorable neuroblastoma genes in vitro as biomarkers of malignancy. Treatment of neuroblastoma cell lines with S(+)-ibuprofen resulted in a significant growth suppression. This growth effect was accompanied by a marked decrease in the expression of MYC, MYCN, AKT and an increase in p53 expression in neuroblastoma cell lines without TP53 mutation. In addition, S(+)-ibuprofen enhanced the expression of some favorable neuroblastoma genes (EPHB6, CD44) and genes involved in growth suppression and differentiation (EGR1, EPHA2, NRG1 and SEL1L). Gene expression profile and Ingenuity pathway analyses using TP53-mutated SKNAS cells further revealed that S(+)-ibuprofen suppressed molecular pathways associated with cell growth and conversely enhanced those of cell cycle arrest and the unfolded protein response. Collectively, these results suggest that S(+)-ibuprofen or its related compounds may have the potential for therapeutic and/or palliative use for unfavorable neuroblastoma.

Transient treatment with epigenetic modifiers yields stable neuroblastoma stem cells resembling aggressive large-cell neuroblastomas.

Cancer stem cells (CSCs) are plastic in nature, a characteristic that hampers cancer therapeutics. Neuroblastoma (NB) is a pediatric tumor of neural crest origin, and half of the cases are highly aggressive. By treating NB cell lines [SKNAS, SKNBE(2)C, CHP134, and SY5Y] with epigenetic modifiers for a short time, followed by sphere-forming culture conditions, we have established stem cell-like NB cells that are phenotypically stable for more than a year. These cells are characterized by their high expression of stemness factors, stem cell markers, and open chromatin structure. We referred to these cells as induced CSCs (iCSCs). SKNAS iCSC and SKNBE(2)C iCSC clones (as few as 100 cells) injected s.c. into SCID/Beige mice formed tumors, and in one case, SKNBE(2)C iCSCs metastasized to the adrenal gland, suggesting their increased metastatic potential. SKNAS iCSC xenografts showed the histologic appearance of totally undifferentiated large-cell NBs (LCNs), the most aggressive and deadly form of NB in humans. Immunohistochemical analyses showed that SKNAS iCSC xenografts expressed high levels of the stem cell marker CXCR4, whereas the SKNAS monolayer cell xenografts did not. The patterns of CXCR4 and MYC expression in SKNAS iCSC xenografts resembled those in the LCNs. The xenografts established from the NB iCSCs shared two common features: the LCN phenotype and high-level MYC/MYCN expression. These observations suggest both that NB cells with large and vesicular nuclei, representing their open chromatin structure, are indicative of stem cell-like tumor cells and that epigenetic changes may have contributed to the development of these most malignant NB cells.

Re-sequencing of ankyrin 3 exon 48 and case-control association analysis of rare variants in bipolar disorder type I.

OBJECTIVES: Genome-wide association studies (GWAS) recently identified ankyrin 3 (ANK3) as a candidate gene for bipolar disorder type I (BPD-I). Because the GWAS suggested multiple common haplotypes associated with BPD-I (with odds ratio ~1.3), we hypothesized that rare variants within these common haplotypes might increase risk for BPD-I. METHODS: We undertook a project in which the serine-rich domain-tail domain (SRD-TD)-encoding exon of ANK3 was amplified from genomic DNA (gDNA) of 384 BPD-I patients and re-sequenced by next generation sequencing (NGS; SOLiD™). RESULTS: We confirmed 18 novel mis-sense rare variants and one novel insertion/deletion variant within the SRD-TD exon, many of which change amino acid residues with extremely high evolutionary conservation. We genotyped most of these mis-sense variants in ≥ 1000 BPD-I and ≥ 1000 control individuals. We found no statistically significant association of any of the rare variants detected with BPD-I. CONCLUSIONS: Thus, we conclude that rare variants within the re-sequenced structural domains of ANK3 exon 48 do not contribute to BPD-I.

Common variation at BARD1 results in the expression of an oncogenic isoform that influences neuroblastoma susceptibility and oncogenicity.

The mechanisms underlying genetic susceptibility at loci discovered by genome-wide association study (GWAS) approaches in human cancer remain largely undefined. In this study, we characterized the high-risk neuroblastoma association at the BRCA1-related locus, BARD1, showing that disease-associated variations correlate with increased expression of the oncogenically activated isoform, BARD1ß. In neuroblastoma cells, silencing of BARD1ß showed genotype-specific cytotoxic effects, including decreased substrate-adherence, anchorage-independence, and foci growth. In established murine fibroblasts, overexpression of BARD1ß was sufficient for neoplastic transformation. BARD1ß stabilized the Aurora family of kinases in neuroblastoma cells, suggesting both a mechanism for the observed effect and a potential therapeutic strategy. Together, our findings identify BARD1ß as an oncogenic driver of high-risk neuroblastoma tumorigenesis, and more generally, they illustrate how robust GWAS signals offer genomic landmarks to identify molecular mechanisms involved in both tumor initiation and malignant progression. The interaction of BARD1ß with the Aurora family of kinases lends strong support to the ongoing work to develop Aurora kinase inhibitors for clinically aggressive neuroblastoma.

Mitochondrial disease genetic diagnostics: optimized whole-exome analysis for all MitoCarta nuclear genes and the mitochondrial genome.

Discovering causative genetic variants in individual cases of suspected mitochondrial disease requires interrogation of both the mitochondrial (mtDNA) and nuclear genomes. Whole-exome sequencing can support simultaneous dual-genome analysis, although currently available capture kits do not target the mtDNA genome and provide insufficient capture for some nuclear-encoded mitochondrial genes. To optimize interrogation of nuclear and mtDNA genes relevant to mitochondrial biology and disease, a custom SureSelect "Mito-Plus" whole-exome library was formulated by blending RNA "baits" from three separate designs: (A) Agilent Technologies SureSelectXT 50 Mb All Exon PLUS Targeted Enrichment Kit, (B) 16-gene nuclear panel targeting sequences for known MitoCarta proteins not included in the 50 Mb All Exon design, and (C) sequences targeting the entire mtDNA genome. The final custom formulations consisted of a 1:1 ratio of nuclear baits to which a 1 to 1,000-fold diluted ratio of mtDNA genome baits were blended. Patient sample capture libraries were paired-end sequenced on an Illumina HiSeq 2000 system using v3.0 SBS chemistry. mtDNA genome coverage varied depending on the mtDNA:nuclear blend ratio, where a 1:100 ratio provided optimal dual-genome coverage with 10X coverage for over 97.5% of all targeted nuclear regions and 1,000X coverage for 99.8% of the mtDNA genome. mtDNA mutations were reliably detected to at least an 8% heteroplasmy level, as discriminated both from sequencing errors and potential contamination from nuclear mtDNA transcripts (Numts). The "1:100 Mito-Plus Whole-Exome" Agilent capture kit offers an optimized tool for whole-exome analysis of nuclear and mtDNA genes relevant to the diagnostic evaluation of mitochondrial disease.

Preclinical evaluation of pemetrexed in pediatric solid tumors.

Renewed interest in antifols for the treatment of childhood cancers has resulted from identification of novel antifols with broad spectrums of anti-cancer activity. We evaluated the in vitro cytotoxicity of methotrexate and pemetrexed in a panel of 12 pediatric solid tumor cell lines using the sulforhodamine-B assay. The Ewing sarcoma (ES) cell lines demonstrated the greatest sensitivity to both methotrexate and pemetrexed. Expression of folate pathway genes (DHFR, TS, GARFT, GGH) did not correlate with in vitro drug sensitivity. Further evaluation of pemetrexed for the treatment of ES is warranted.

High-Resolution genomic arrays identify CNVs that phenocopy the chromosome 22q11.2 deletion syndrome.

The 22q11 Deletion Syndrome includes the overlapping phenotypes of DiGeorge/Velocardiofacial Syndromes, characterized by conotruncal heart defects, cleft palate, thymus, and parathyroid gland dysplasia. The majority (90%) of patients harbor detectable chr22q11.2 deletions, but a genetic etiology for the remainder of patients without a deletion can remain undefined despite major birth defects. We analyzed DNA from eight patients with normal 22q11 FISH studies by high-density single nucleotide polymorphism (SNP) arrays and identified potentially pathogenic copy number variants (CNVs) in four of eight patients. Two patients showed large CNVs in regions of known genomic disorders: one a deletion of distal chr22q11.2 and the other a duplication of chr5q35. A 3-Mb deletion of chr19p13.3 that includes a gene associated with conotruncal heart defects was found in a third patient. Two potentially pathogenic CNVs were found in a fourth patient: a large heterozygous deletion of chr6p24 and a smaller duplication of chr9p24. Our findings support a recent consensus statement advocating chromosomal microarray analysis as a first-line diagnostic approach for patients with multiple congenital anomalies. In patients with phenotypes suggestive of the 22q11.2 syndrome spectrum and normal FISH, microarray analysis can uncover the molecular basis of other genomic disorders whose features overlap those of 22q11.2 deletions.

Biological effects of induced MYCN hyper-expression in MYCN-amplified neuroblastomas.

Neuroblastoma is a childhood malignancy of the sympathetic nervous system. The tumor exhibits two different phenotypes: favorable and unfavorable. MYCN amplification is associated with rapid tumor progression and the worst neuroblastoma disease outcome. We have previously reported that inhibitors of histone deacetylase (HDAC) and proteasome enhance favorable neuroblastoma gene expression in neuroblastoma cell lines and inhibit growth of these cells. In this study, we investigated the effect of trichostatin A or TSA (an HDAC inhibitor), and epoxomycin (a proteasome inhibitor) on MYCN and p53 expression in MYCN-amplified neuroblastoma cells. It was found that TSA down-regulated MYCN expression, but Epoxomycin and the TSA/Epoxomycin combination led to MYCN hyper-expression in MYCN-amplified neuroblastoma cell lines. Despite their contrasting effects on MYCN expression, TSA and Epoxomycin caused growth suppression and cell death of the MYCN-amplified cell lines examined. Consistent with these data, forced hyper-expression of MYCN in MYCN-amplified IMR5 cells via transfection resulted in growth suppression and the increased expression of several genes known to suppress growth or induce cell death. Furthermore, Epoxomycin as a single agent and its combination with TSA enhance p53 expression in the MYCN-amplified neuroblastoma cell lines. Unexpectedly, co-transfection of TP53 and MYCN in IMR5 cells resulted in high p53 expression but a reduction of MYCN expression. Together our data suggest that either down regulation or hyper-expression of MYCN results in growth inhibition and/or apoptosis of MYCN-amplified neuroblastoma cells. In addition, elevated p53 expression has a suppressive effect on MYCN expression in these cells.

CNV Workshop: an integrated platform for high-throughput copy number variation discovery and clinical diagnostics.

BACKGROUND: Recent studies have shown that copy number variations (CNVs) are frequent in higher eukaryotes and associated with a substantial portion of inherited and acquired risk for various human diseases. The increasing availability of high-resolution genome surveillance platforms provides opportunity for rapidly assessing research and clinical samples for CNV content, as well as for determining the potential pathogenicity of identified variants. However, few informatics tools for accurate and efficient CNV detection and assessment currently exist. RESULTS: We developed a suite of software tools and resources (CNV Workshop) for automated, genome-wide CNV detection from a variety of SNP array platforms. CNV Workshop includes three major components: detection, annotation, and presentation of structural variants from genome array data. CNV detection utilizes a robust and genotype-specific extension of the Circular Binary Segmentation algorithm, and the use of additional detection algorithms is supported. Predicted CNVs are captured in a MySQL database that supports cohort-based projects and incorporates a secure user authentication layer and user/admin roles. To assist with determination of pathogenicity, detected CNVs are also annotated automatically for gene content, known disease loci, and gene-based literature references. Results are easily queried, sorted, filtered, and visualized via a web-based presentation layer that includes a GBrowse-based graphical representation of CNV content and relevant public data, integration with the UCSC Genome Browser, and tabular displays of genomic attributes for each CNV. CONCLUSIONS: To our knowledge, CNV Workshop represents the first cohesive and convenient platform for detection, annotation, and assessment of the biological and clinical significance of structural variants. CNV Workshop has been successfully utilized for assessment of genomic variation in healthy individuals and disease cohorts and is an ideal platform for coordinating multiple associated projects. AVAILABILITY AND IMPLEMENTATION: Available on the web at: http://sourceforge.net/projects/cnv.

Cross-platform expression microarray performance in a mouse model of mitochondrial disease therapy.

UNLABELLED: Microarray expression profiling has become a valuable tool in the evaluation of the genetic consequences of metabolic disease. Although 3'-biased gene expression microarray platforms were the first generation to have widespread availability, newer platforms are gradually emerging that have more up-to-date content and/or higher cost efficiency. Deciphering the relative strengths and weaknesses of these various platforms for metabolic pathway-level analyses can be daunting. We sought to determine the practical strengths and weaknesses of four leading commercially available expression array platforms relative to biologic investigations, as well as assess the feasibility of cross-platform data integration for purposes of biochemical pathway analyses. METHODS: Liver RNA from B6.Alb/cre,Pdss2(loxP/loxP) mice having primary coenzyme Q deficiency was extracted either at baseline or following treatment with an antioxidant/antihyperlipidemic agent, probucol. Target RNA samples were prepared and hybridized to Affymetrix 430 2.0, Affymetrix Gene 1.0 ST, Affymetrix Exon 1.0 ST, and Illumina Mouse WG-6 expression arrays. Probes on all platforms were re-mapped to coding sequences in the current version of the mouse genome. Data processing and statistical analysis were performed by R/Bioconductor functions, and pathway analyses were carried out by KEGG Atlas and GSEA. RESULTS: Expression measurements were generally consistent across platforms. However, intensive probe-level comparison suggested that differences in probe locations were a major source of inter-platform variance. In addition, genes expressed at low or intermediate levels had lower inter-platform reproducibility than highly expressed genes. All platforms showed similar patterns of differential expression between sample groups, with 'steroid biosynthesis' consistently identified as the most down-regulated metabolic pathway by probucol treatment. CONCLUSIONS: This work offers a timely guide for metabolic disease investigators to enable informed end-user decisions regarding choice of expression microarray platform best-suited to specific research project goals. Successful cross-platform integration of biochemical pathway expression data is also demonstrated, especially for well-annotated and highly expressed genes. However, integration of gene-level expression data is limited by individual platform probe design and the expression level of target genes. Cross-platform analyses of biochemical pathway data will require additional data processing and novel computational bioinformatics tools to address unique statistical challenges.