BMPER variants associated with a novel, attenuated subtype of diaphanospondylodysostosis.

Diaphanospondylodysostosis (DSD), caused by loss of bone morphogenetic protein-binding endothelial regulator (BMPER), has been considered a lethal skeletal dysplasia characterized by severe deficiency of vertebral body and sacral ossification, reduced rib number and cystic kidneys. In this study, however, we have demonstrated that variants in BMPER may cause a milder disorder, without renal anomalies, that is compatible with long-term survival. Four siblings, three males and one female, presented with severe congenital scoliosis associated with rib and vertebral malformations as well as strikingly delayed ossification of the pedicles. The female was stillborn from an unrelated cause. Stabilization of the scoliosis with expandable titanium rods was successful in the three boys, all of whom have short stature. An autosomal recessive mode of inheritance was hypothesized. Single nucleotide polymorphism microarray analysis was performed for three of the siblings to identify autosomal genes with shared allele patterns, suggesting possible linkage. Exome sequencing of one sibling was then performed. Rare variants were identified in 347 genes with shared alleles. Only one of these genes had bi-allelic variants in a gene strongly expressed in paraxial mesenchyme: BMPER, which is the cause of DSD, an autosomal recessive disorder. The disorder described herein could represent an attenuated form of DSD or could be designated a separate entity such as spondylopedicular dysplasia.

Epigenetic and transcriptional determinants of the human breast.

While significant effort has been dedicated to the characterization of epigenetic changes associated with prenatal differentiation, relatively little is known about the epigenetic changes that accompany post-natal differentiation where fully functional differentiated cell types with limited lifespans arise. Here we sought to address this gap by generating epigenomic and transcriptional profiles from primary human breast cell types isolated from disease-free human subjects. From these data we define a comprehensive human breast transcriptional network, including a set of myoepithelial- and luminal epithelial-specific intronic retention events. Intersection of epigenetic states with RNA expression from distinct breast epithelium lineages demonstrates that mCpG provides a stable record of exonic and intronic usage, whereas H3K36me3 is dynamic. We find a striking asymmetry in epigenomic reprogramming between luminal and myoepithelial cell types, with the genomes of luminal cells harbouring more than twice the number of hypomethylated enhancer elements compared with myoepithelial cells.

Single exon-resolution targeted chromosomal microarray analysis of known and candidate intellectual disability genes.

Intellectual disability affects about 3% of individuals globally, with∼50% idiopathic. We designed an exonic-resolution array targeting all known submicroscopic chromosomal intellectual disability syndrome loci, causative genes for intellectual disability, and potential candidate genes, all genes encoding glutamate receptors and epigenetic regulators. Using this platform, we performed chromosomal microarray analysis on 165 intellectual disability trios (affected child and both normal parents). We identified and independently validated 36 de novo copy-number changes in 32 trios. In all, 67% of the validated events were intragenic, involving only exon 1 (which includes the promoter sequence according to our design), exon 1 and adjacent exons, or one or more exons excluding exon 1. Seventeen of the 36 copy-number variants involve genes known to cause intellectual disability. Eleven of these, including seven intragenic variants, are clearly pathogenic (involving STXBP1, SHANK3 (3 patients), IL1RAPL1, UBE2A, NRXN1, MEF2C, CHD7, 15q24 and 9p24 microdeletion), two are likely pathogenic (PI4KA, DCX), two are unlikely to be pathogenic (GRIK2, FREM2), and two are unclear (ARID1B, 15q22 microdeletion). Twelve individuals with genomic imbalances identified by our array were tested with a clinical microarray, and six had a normal result. We identified de novo copy-number variants within genes not previously implicated in intellectual disability and uncovered pathogenic variation of known intellectual disability genes below the detection limit of standard clinical diagnostic chromosomal microarray analysis.

Mutation discovery in regions of segmental cancer genome amplifications with CoNAn-SNV: a mixture model for next generation sequencing of tumors.

Next generation sequencing has now enabled a cost-effective enumeration of the full mutational complement of a tumor genome-in particular single nucleotide variants (SNVs). Most current computational and statistical models for analyzing next generation sequencing data, however, do not account for cancer-specific biological properties, including somatic segmental copy number alterations (CNAs)-which require special treatment of the data. Here we present CoNAn-SNV (Copy Number Annotated SNV): a novel algorithm for the inference of single nucleotide variants (SNVs) that overlap copy number alterations. The method is based on modelling the notion that genomic regions of segmental duplication and amplification induce an extended genotype space where a subset of genotypes will exhibit heavily skewed allelic distributions in SNVs (and therefore render them undetectable by methods that assume diploidy). We introduce the concept of modelling allelic counts from sequencing data using a panel of Binomial mixture models where the number of mixtures for a given locus in the genome is informed by a discrete copy number state given as input. We applied CoNAn-SNV to a previously published whole genome shotgun data set obtained from a lobular breast cancer and show that it is able to discover 21 experimentally revalidated somatic non-synonymous mutations in a lobular breast cancer genome that were not detected using copy number insensitive SNV detection algorithms. Importantly, ROC analysis shows that the increased sensitivity of CoNAn-SNV does not result in disproportionate loss of specificity. This was also supported by analysis of a recently published lymphoma genome with a relatively quiescent karyotype, where CoNAn-SNV showed similar results to other callers except in regions of copy number gain where increased sensitivity was conferred. Our results indicate that in genomically unstable tumors, copy number annotation for SNV detection will be critical to fully characterize the mutational landscape of cancer genomes.

Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma.

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.

Retinoblastoma-binding proteins 4 and 9 are important for human pluripotent stem cell maintenance.

OBJECTIVE: The molecular mechanisms that maintain human pluripotent stem (PS) cells are not completely understood. Here we sought to identify new candidate PS cell regulators to facilitate future improvements in their generation, expansion, and differentiation. MATERIALS AND METHODS: We used bioinformatic analyses of multiple serial-analysis-of-gene-expression libraries (generated from human PS cells and their differentiated derivatives), together with small interfering RNA (siRNA) screening to identify candidate pluripotency regulators. Validation of candidate regulators involved promoter analyses, Affymetrix profiling, real-time PCR, and immunoprecipitation. RESULTS: Promoter analysis of genes differentially expressed across multiple serial-analysis-of-gene-expression libraries identified E2F motifs in the promoters of many PS cell-specific genes (e.g., POU5F1, NANOG, SOX2, FOXD3). siRNA analyses identified two retinoblastoma binding proteins (RBBP4, RBBP9) as required for maintenance of multiple human PS cell types. Both RBBPs were bound to RB in human PS cells, and E2F motifs were present in the promoters of genes whose expression was altered by decreasing RBBP4 and RBBP9 expression. Affymetrix and real-time PCR studies of siRNA-treated human PS cells showed that reduced RBBP4 or RBBP9 expression concomitantly decreased expression of POU5F1, NANOG, SOX2, and/or FOXD3 plus certain cell cycle genes (e.g., CCNA2, CCNB1), while increasing expression of genes involved in organogenesis (particularly neurogenesis). CONCLUSIONS: These results reveal new candidate positive regulators of human PS cells, providing evidence of their ability to regulate expression of pluripotency, cell cycle, and differentiation genes in human PS cells. These data provide valuable new leads for further elucidating mechanisms of human pluripotency.

Comparison of genome-wide array genomic hybridization platforms for the detection of copy number variants in idiopathic mental retardation.

BACKGROUND: Clinical laboratories are adopting array genomic hybridization as a standard clinical test. A number of whole genome array genomic hybridization platforms are available, but little is known about their comparative performance in a clinical context. METHODS: We studied 30 children with idiopathic MR and both unaffected parents of each child using Affymetrix 500 K GeneChip SNP arrays, Agilent Human Genome 244 K oligonucleotide arrays and NimbleGen 385 K Whole-Genome oligonucleotide arrays. We also determined whether CNVs called on these platforms were detected by Illumina Hap550 beadchips or SMRT 32 K BAC whole genome tiling arrays and tested 15 of the 30 trios on Affymetrix 6.0 SNP arrays. RESULTS: The Affymetrix 500 K, Agilent and NimbleGen platforms identified 3061 autosomal and 117 X chromosomal CNVs in the 30 trios. 147 of these CNVs appeared to be de novo, but only 34 (22%) were found on more than one platform. Performing genotype-phenotype correlations, we identified 7 most likely pathogenic and 2 possibly pathogenic CNVs for MR. All 9 of these putatively pathogenic CNVs were detected by the Affymetrix 500 K, Agilent, NimbleGen and the Illumina arrays, and 5 were found by the SMRT BAC array. Both putatively pathogenic CNVs identified in the 15 trios tested with the Affymetrix 6.0 were identified by this platform. CONCLUSIONS: Our findings demonstrate that different results are obtained with different platforms and illustrate the trade-off that exists between sensitivity and specificity. The large number of apparently false positive CNV calls on each of the platforms supports the need for validating clinically important findings with a different technology.

ARID1A mutations in endometriosis-associated ovarian carcinomas.

BACKGROUND: Ovarian clear-cell and endometrioid carcinomas may arise from endometriosis, but the molecular events involved in this transformation have not been described. METHODS: We sequenced the whole transcriptomes of 18 ovarian clear-cell carcinomas and 1 ovarian clear-cell carcinoma cell line and found somatic mutations in ARID1A (the AT-rich interactive domain 1A [SWI-like] gene) in 6 of the samples. ARID1A encodes BAF250a, a key component of the SWI­SNF chromatin remodeling complex. We sequenced ARID1A in an additional 210 ovarian carcinomas and a second ovarian clear-cell carcinoma cell line and measured BAF250a expression by means of immunohistochemical analysis in an additional 455 ovarian carcinomas. RESULTS: ARID1A mutations were seen in 55 of 119 ovarian clear-cell carcinomas (46%), 10 of 33 endometrioid carcinomas (30%), and none of the 76 high-grade serous ovarian carcinomas. Seventeen carcinomas had two somatic mutations each. Loss of the BAF250a protein correlated strongly with the ovarian clear-cell carcinoma and endometrioid carcinoma subtypes and the presence of ARID1A mutations. In two patients, ARID1A mutations and loss of BAF250a expression were evident in the tumor and contiguous atypical endometriosis but not in distant endometriotic lesions. CONCLUSIONS: These data implicate ARID1A as a tumor-suppressor gene frequently disrupted in ovarian clear-cell and endometrioid carcinomas. Since ARID1A mutation and loss of BAF250a can be seen in the preneoplastic lesions, we speculate that this is an early event in the transformation of endometriosis into cancer. (Funded by the British Columbia Cancer Foundation and the Vancouver General Hospital­University of British Columbia Hospital Foundation.).

LNCaP Atlas: gene expression associated with in vivo progression to castration-recurrent prostate cancer.

BACKGROUND: There is no cure for castration-recurrent prostate cancer (CRPC) and the mechanisms underlying this stage of the disease are unknown. METHODS: We analyzed the transcriptome of human LNCaP prostate cancer cells as they progress to CRPC in vivo using replicate LongSAGE libraries. We refer to these libraries as the LNCaP atlas and compared these gene expression profiles with current suggested models of CRPC. RESULTS: Three million tags were sequenced using in vivo samples at various stages of hormonal progression to reveal 96 novel genes differentially expressed in CRPC. Thirty-one genes encode proteins that are either secreted or are located at the plasma membrane, 21 genes changed levels of expression in response to androgen, and 8 genes have enriched expression in the prostate. Expression of 26, 6, 12, and 15 genes have previously been linked to prostate cancer, Gleason grade, progression, and metastasis, respectively. Expression profiles of genes in CRPC support a role for the transcriptional activity of the androgen receptor (CCNH, CUEDC2, FLNA, PSMA7), steroid synthesis and metabolism (DHCR24, DHRS7, ELOVL5, HSD17B4, OPRK1), neuroendocrine (ENO2, MAOA, OPRK1, S100A10, TRPM8), and proliferation (GAS5, GNB2L1, MT-ND3, NKX3-1, PCGEM1, PTGFR, STEAP1, TMEM30A), but neither supported nor discounted a role for cell survival genes. CONCLUSIONS: The in vivo gene expression atlas for LNCaP was sequenced and support a role for the androgen receptor in CRPC.

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/.

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications.

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.

Conserved role of intragenic DNA methylation in regulating alternative promoters.

Although it is known that the methylation of DNA in 5' promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear. In mammals, tissue- and cell type-specific methylation is present in a small percentage of 5' CpG island (CGI) promoters, whereas a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences. Tissue-specific intragenic methylation might reduce, or, paradoxically, enhance transcription elongation efficiency. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes. To investigate the role of intragenic methylation, we generated a map of DNA methylation from the human brain encompassing 24.7 million of the 28 million CpG sites. From the dense, high-resolution coverage of CpG islands, the majority of methylated CpG islands were shown to be in intragenic and intergenic regions, whereas less than 3% of CpG islands in 5' promoters were methylated. The CpG islands in all three locations overlapped with RNA markers of transcription initiation, and unmethylated CpG islands also overlapped significantly with trimethylation of H3K4, a histone modification enriched at promoters. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus and its mouse homologue demonstrated that this tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue- and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies.

High resolution analysis of follicular lymphoma genomes reveals somatic recurrent sites of copy-neutral loss of heterozygosity and copy number alterations that target single genes.

A multiplatform approach, including conventional cytogenetic techniques, BAC array comparative genomic hybridization, and Affymetrix 500K SNP arrays, was applied to the study of the tumor genomes of 25 follicular lymphoma biopsy samples with paired normal DNA samples to characterize balanced translocations, copy number imbalances, and copy-neutral loss of heterozygosity (cnLOH). In addition to the t(14;18), eight unique balanced translocations were found. Commonly reported FL-associated copy number regions were revealed including losses of 1p32-36, 6q, and 10q, and gains of 1q, 6p, 7, 12, 18, and X. The most frequent regions affected by copy-neutral loss of heterozygosity were 1p36.33 (28%), 6p21.3 (20%), 12q21.2-q24.33 (16%), and 16p13.3 (24%). We also identified by SNP analysis, 45 aberrant regions that each affected one gene, including CDKN2A, CDKN2B, FHIT, KIT, PEX14, and PTPRD, which were associated with canonical pathways involved in tumor development. This study illustrates the power of using complementary high-resolution platforms on paired tumor/normal specimens and computational analysis to provide potential insights into the significance of single-gene somatic aberrations in FL tumorigenesis.

A male with unilateral microphthalmia reveals a role for TMX3 in eye development.

Anophthalmia and microphthalmia are important birth defects, but their pathogenesis remains incompletely understood. We studied a patient with severe unilateral microphthalmia who had a 2.7 Mb deletion at chromosome 18q22.1 that was inherited from his mother. In-situ hybridization showed that one of the deleted genes, TMX3, was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TMX3 in 162 patients with anophthalmia or microphthalmia, and found two missense substitutions in unrelated patients: c.116G>A, predicting p.Arg39Gln, in a male with unilateral microphthalmia and retinal coloboma, and c.322G>A, predicting p.Asp108Asn, in a female with unilateral microphthalmia and severe micrognathia. We used two antisense morpholinos targeted against the zebrafish TMX3 orthologue, zgc:110025, to examine the effects of reduced gene expression in eye development. We noted that the morphant larvae resulting from both morpholinos had significantly smaller eye sizes and reduced labeling with islet-1 antibody directed against retinal ganglion cells at 2 days post fertilization. Co-injection of human wild type TMX3 mRNA rescued the small eye phenotype obtained with both morpholinos, whereas co-injection of human TMX3(p.Arg39Gln) mutant mRNA, analogous to the mutation in the patient with microphthalmia and coloboma, did not rescue the small eye phenotype. Our results show that haploinsufficiency for TMX3 results in a small eye phenotype and represents a novel genetic cause of microphthalmia and coloboma. Future experiments to determine if other thioredoxins are important in eye morphogenesis and to clarify the mechanism of function of TMX3 in eye development are warranted.

Digital gene expression by tag sequencing on the illumina genome analyzer.

This unit provides a protocol for performing digital gene expression profiling on the Illumina Genome Analyzer sequencing platform. Tag sequencing (Tag-seq) is an implementation of the LongSAGE protocol on the Illumina sequencing platform that increases utility while reducing both the cost and time required to generate gene expression profiles. The ultra-high-throughput sequencing capability of the Illumina platform allows the cost-effective generation of libraries containing an average of 20 million tags, a 200-fold improvement over classical LongSAGE. Tag-seq has less sequence composition bias, leading to a better representation of AT-rich tag sequences, and allows a more accurate profiling of a subset of the transcriptome characterized by AT-rich genes expressed at levels below the threshold of detection of LongSAGE (Morrissy et al., 2009).

A maternally inherited chromosome 18q22.1 deletion in a male with late-presenting diaphragmatic hernia and microphthalmia-evaluation of DSEL as a candidate gene for the diaphragmatic defect.

Using an Affymetrix GeneChip(R) Human Mapping 100K Set to study a patient with a late-presenting, right-sided diaphragmatic hernia and microphthalmia, we found a maternally inherited deletion that was 2.7 Mb in size at chromosome 18q22.1. Mapping of this deletion using fluorescence in situ hybridization revealed three deleted genes-CDH19, DSEL, and TXNDC10, and one gene that contained the deletion breakpoint, CCDC102B. We selected DSEL for further study in 125 patients with diaphragmatic hernias, as it is involved in the synthesis of decorin, a protein that is required for normal collagen formation and that is upregulated during myogenesis. We found p.Met14Ile in an unrelated patient with a late-presenting, anterior diaphragmatic hernia. In the murine diaphragm, Dsel was only weakly expressed at the time of diaphragm closure and its expression in C2C12 myoblast cells did not change significantly during myoblast differentiation, thus reducing the likelihood that the gene is involved in myogenesis of the diaphragm. Although it is possible that the 18q22.1 deletion and haploinsufficiency for DSEL contributed to the diaphragmatic defect in the patient, a definite role for DSEL and decorin in the formation of the collagen-containing, central tendon of the diaphragm has not yet been established.

Tumor-associated macrophages and survival in classic Hodgkin's lymphoma.

BACKGROUND: Despite advances in treatments for Hodgkin's lymphoma, about 20% of patients still die from progressive disease. Current prognostic models predict the outcome of treatment with imperfect accuracy, and clinically relevant biomarkers have not been established to improve on the International Prognostic Score. METHODS: Using gene-expression profiling, we analyzed 130 frozen samples obtained from patients with classic Hodgkin's lymphoma during diagnostic lymph-node biopsy to determine which cellular signatures were correlated with treatment outcome. We confirmed our findings in an independent cohort of 166 patients, using immunohistochemical analysis. RESULTS: Gene-expression profiling identified a gene signature of tumor-associated macrophages that was significantly associated with primary treatment failure (P=0.02). In an independent cohort of patients, we found that an increased number of CD68+ macrophages was correlated with a shortened progression-free survival (P=0.03) and with an increased likelihood of relapse after autologous hematopoietic stem-cell transplantation (P=0.008), resulting in shortened disease-specific survival (P=0.003). In multivariate analysis, this adverse prognostic factor outperformed the International Prognostic Score for disease-specific survival (P=0.003 vs. P=0.03). The absence of an elevated number of CD68+ cells in patients with limited-stage disease defined a subgroup of patients with a long-term disease-specific survival of 100% with the use of current treatment strategies. CONCLUSIONS: An increased number of tumor-associated macrophages was strongly associated with shortened survival in patients with classic Hodgkin's lymphoma and provides a new biomarker for risk stratification.

Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin.

Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B cells. Targeted resequencing studies have revealed mutations in various genes encoding proteins in the NF-kappaB pathway that contribute to the activated B-cell (ABC) DLBCL subtype, but thus far few GCB-specific mutations have been identified. Here we report recurrent somatic mutations affecting the polycomb-group oncogene EZH2, which encodes a histone methyltransferase responsible for trimethylating Lys27 of histone H3 (H3K27). After the recent discovery of mutations in KDM6A (UTX), which encodes the histone H3K27me3 demethylase UTX, in several cancer types, EZH2 is the second histone methyltransferase gene found to be mutated in cancer. These mutations, which result in the replacement of a single tyrosine in the SET domain of the EZH2 protein (Tyr641), occur in 21.7% of GCB DLBCLs and 7.2% of FLs and are absent from ABC DLBCLs. Our data are consistent with the notion that EZH2 proteins with mutant Tyr641 have reduced enzymatic activity in vitro.

Detection of pathogenic copy number variants in children with idiopathic intellectual disability using 500 K SNP array genomic hybridization.

BACKGROUND: Array genomic hybridization is being used clinically to detect pathogenic copy number variants in children with intellectual disability and other birth defects. However, there is no agreement regarding the kind of array, the distribution of probes across the genome, or the resolution that is most appropriate for clinical use. RESULTS: We performed 500 K Affymetrix GeneChip array genomic hybridization in 100 idiopathic intellectual disability trios, each comprised of a child with intellectual disability of unknown cause and both unaffected parents. We found pathogenic genomic imbalance in 16 of these 100 individuals with idiopathic intellectual disability. In comparison, we had found pathogenic genomic imbalance in 11 of 100 children with idiopathic intellectual disability in a previous cohort who had been studied by 100 K GeneChip array genomic hybridization. Among 54 intellectual disability trios selected from the previous cohort who were re-tested with 500 K GeneChip array genomic hybridization, we identified all 10 previously-detected pathogenic genomic alterations and at least one additional pathogenic copy number variant that had not been detected with 100 K GeneChip array genomic hybridization. Many benign copy number variants, including one that was de novo, were also detected with 500 K array genomic hybridization, but it was possible to distinguish the benign and pathogenic copy number variants with confidence in all but 3 (1.9%) of the 154 intellectual disability trios studied. CONCLUSION: Affymetrix GeneChip 500 K array genomic hybridization detected pathogenic genomic imbalance in 10 of 10 patients with idiopathic developmental disability in whom 100 K GeneChip array genomic hybridization had found genomic imbalance, 1 of 44 patients in whom 100 K GeneChip array genomic hybridization had found no abnormality, and 16 of 100 patients who had not previously been tested. Effective clinical interpretation of these studies requires considerable skill and experience.

Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution.

Recent advances in next generation sequencing have made it possible to precisely characterize all somatic coding mutations that occur during the development and progression of individual cancers. Here we used these approaches to sequence the genomes (>43-fold coverage) and transcriptomes of an oestrogen-receptor-alpha-positive metastatic lobular breast cancer at depth. We found 32 somatic non-synonymous coding mutations present in the metastasis, and measured the frequency of these somatic mutations in DNA from the primary tumour of the same patient, which arose 9 years earlier. Five of the 32 mutations (in ABCB11, HAUS3, SLC24A4, SNX4 and PALB2) were prevalent in the DNA of the primary tumour removed at diagnosis 9 years earlier, six (in KIF1C, USP28, MYH8, MORC1, KIAA1468 and RNASEH2A) were present at lower frequencies (1-13%), 19 were not detected in the primary tumour, and two were undetermined. The combined analysis of genome and transcriptome data revealed two new RNA-editing events that recode the amino acid sequence of SRP9 and COG3. Taken together, our data show that single nucleotide mutational heterogeneity can be a property of low or intermediate grade primary breast cancers and that significant evolution can occur with disease progression.