ABSTRACTObjectiveTo develop a novel prenatal assay based on selective analysis of cell‐free DNA in maternal blood for evaluation of fetal Trisomy 21 (T21) and Trisomy 18 (T18).MethodsTwo hundred ninety‐eight pregnancies, including 39 T21 and seven T18 confirmed fetal aneuploidies, were analyzed using a novel, highly multiplexed assay, termed digital analysis of selected regions (DANSR™). Cell‐free DNA from maternal blood samples was analyzed using DANSR assays for loci on chromosomes 21 and 18. Products from 96 separate patients were pooled and sequenced together. A standard Z‐test of chromosomal proportions was used to distinguish aneuploid samples from average‐risk pregnancy samples. DANSR aneuploidy discrimination was evaluated at various sequence depths.ResultsAt the lowest sequencing depth, corresponding to 204 000 sequencing counts per sample, average‐risk cases where distinguished from T21 and T18 cases, with Z statistics for all cases exceeding 3.6. Increasing the sequencing depth to 410 000 counts per sample substantially improved separation of aneuploid and average‐risk cases. A further increase to 620 000 counts per sample resulted in only marginal improvement. This depth of sequencing represents less than 5% of that required by massively parallel shotgun sequencing approaches.ConclusionDigital analysis of selected regions enables highly accurate, cost efficient, and scalable noninvasive fetal aneuploidy assessment. © 2012 John Wiley & Sons, Ltd.

ObjectiveUse of massively parallel shotgun sequencing (MPSS) to precisely quantify the chromosomal composition of cell free DNA (cfDNA) from maternal blood has shown promise for non-invasive detection of fetal aneuploidy. Because MPSS involves analysis of DNA from the entire genome, the number of sequencing reads required for aneuploidy detection constrains the efficiency, scalability, and thus, clinical utility of this approach. To address these limitations, we developed and evaluated a novel method, Digital ANalysis of Selected Regions (DANSR(TM)), which uses ligation of locus-specific oligos to produce sequencing template only from selected genomic loci.Study DesignProspective analysis of 405 pregnant women involving a training and validation cohort.ResultsWe developed DANSR assays for 576 loci on each of chromosomes 18 and 21, and used these assays to evaluate cfDNA from 405 pregnant women, including 72 trisomy 21 (T21) and 16 trisomy 18 (T18) pregnancies. Sequencing template from up to 96 patient samples was analyzed in a single flow cell lane on an Illumina HiSeq 2000; 97% of the resulting sequencing reads mapped to expected loci. We trained our aneuploidy detection algorithm on an un-blinded set of 190 normal, 36 T21 and 8 T18 pregnancies. Of the 222 (95%) samples that passed quality control (QC), 35/35 (100%) T21 and 7/7 (100%) T18 samples had chromosome proportion Z-scores >3, and 179/180 (99.4%) normal samples had Z-scores <3. We next performed a blinded validation study on an independent set of cfDNA samples from 171 pregnant women, including 36 T21 and 8 T18 pregnancies. Only one sample (0.6%) failed to pass QC. All 36 T21 samples (100%) and 8 T18 samples (100%) had Z-scores greater than 3 and all of the 126 normal samples had Z-scores less than 3 (100%). These results were generated from an average of 789K raw sequencing reads per sample, i.e., <5% of the raw reads required for aneuploidy detection using MPSS.ConclusionDANSR represents a high accuracy, cost efficient, scalable approach to non-invasive detection of fetal aneuploidy. ObjectiveUse of massively parallel shotgun sequencing (MPSS) to precisely quantify the chromosomal composition of cell free DNA (cfDNA) from maternal blood has shown promise for non-invasive detection of fetal aneuploidy. Because MPSS involves analysis of DNA from the entire genome, the number of sequencing reads required for aneuploidy detection constrains the efficiency, scalability, and thus, clinical utility of this approach. To address these limitations, we developed and evaluated a novel method, Digital ANalysis of Selected Regions (DANSR(TM)), which uses ligation of locus-specific oligos to produce sequencing template only from selected genomic loci. Use of massively parallel shotgun sequencing (MPSS) to precisely quantify the chromosomal composition of cell free DNA (cfDNA) from maternal blood has shown promise for non-invasive detection of fetal aneuploidy. Because MPSS involves analysis of DNA from the entire genome, the number of sequencing reads required for aneuploidy detection constrains the efficiency, scalability, and thus, clinical utility of this approach. To address these limitations, we developed and evaluated a novel method, Digital ANalysis of Selected Regions (DANSR(TM)), which uses ligation of locus-specific oligos to produce sequencing template only from selected genomic loci. Study DesignProspective analysis of 405 pregnant women involving a training and validation cohort. Prospective analysis of 405 pregnant women involving a training and validation cohort. ResultsWe developed DANSR assays for 576 loci on each of chromosomes 18 and 21, and used these assays to evaluate cfDNA from 405 pregnant women, including 72 trisomy 21 (T21) and 16 trisomy 18 (T18) pregnancies. Sequencing template from up to 96 patient samples was analyzed in a single flow cell lane on an Illumina HiSeq 2000; 97% of the resulting sequencing reads mapped to expected loci. We trained our aneuploidy detection algorithm on an un-blinded set of 190 normal, 36 T21 and 8 T18 pregnancies. Of the 222 (95%) samples that passed quality control (QC), 35/35 (100%) T21 and 7/7 (100%) T18 samples had chromosome proportion Z-scores >3, and 179/180 (99.4%) normal samples had Z-scores <3. We next performed a blinded validation study on an independent set of cfDNA samples from 171 pregnant women, including 36 T21 and 8 T18 pregnancies. Only one sample (0.6%) failed to pass QC. All 36 T21 samples (100%) and 8 T18 samples (100%) had Z-scores greater than 3 and all of the 126 normal samples had Z-scores less than 3 (100%). These results were generated from an average of 789K raw sequencing reads per sample, i.e., <5% of the raw reads required for aneuploidy detection using MPSS. We developed DANSR assays for 576 loci on each of chromosomes 18 and 21, and used these assays to evaluate cfDNA from 405 pregnant women, including 72 trisomy 21 (T21) and 16 trisomy 18 (T18) pregnancies. Sequencing template from up to 96 patient samples was analyzed in a single flow cell lane on an Illumina HiSeq 2000; 97% of the resulting sequencing reads mapped to expected loci. We trained our aneuploidy detection algorithm on an un-blinded set of 190 normal, 36 T21 and 8 T18 pregnancies. Of the 222 (95%) samples that passed quality control (QC), 35/35 (100%) T21 and 7/7 (100%) T18 samples had chromosome proportion Z-scores >3, and 179/180 (99.4%) normal samples had Z-scores <3. We next performed a blinded validation study on an independent set of cfDNA samples from 171 pregnant women, including 36 T21 and 8 T18 pregnancies. Only one sample (0.6%) failed to pass QC. All 36 T21 samples (100%) and 8 T18 samples (100%) had Z-scores greater than 3 and all of the 126 normal samples had Z-scores less than 3 (100%). These results were generated from an average of 789K raw sequencing reads per sample, i.e., <5% of the raw reads required for aneuploidy detection using MPSS. ConclusionDANSR represents a high accuracy, cost efficient, scalable approach to non-invasive detection of fetal aneuploidy. DANSR represents a high accuracy, cost efficient, scalable approach to non-invasive detection of fetal aneuploidy.

BackgroundExposure of the brain to environmental agents during critical periods of neuronal development is considered a key factor underlying many neurologic disorders.ObjectivesIn this study we examined the influence of genotoxicants on cerebellar function during early development by measuring global gene expression changes.MethodsWe measured global gene expression in immature cerebellar neurons (i.e., granule cells) after treatment with two distinct alkylating agents, methylazoxymethanol (MAM) and nitrogen mustard (HN2). Granule cell cultures were treated for 24 hr with MAM (10–1,000 μM) or HN2 (0.1–20 μM) and examined for cell viability, DNA damage, and markers of apoptosis.ResultsNeuronal viability was significantly reduced (p < 0.01) at concentrations > 500 μM for MAM and > 1.0 μM for HN2; this correlated with an increase in both DNA damage and markers of apoptosis. Neuronal cultures treated with sublethal concentrations of MAM (100 μM) or HN2 (1.0 μM) were then examined for gene expression using large-scale mouse cDNA microarrays (27,648). Gene expression results revealed that a) global gene expression was predominantly up-regulated by both genotoxicants; b) the number of down-regulated genes was approximately 3-fold greater for HN2 than for MAM; and c) distinct classes of molecules were influenced by MAM (i.e, neuronal differentiation, the stress and immune response, and signal transduction) and HN2 (i.e, protein synthesis and apoptosis).ConclusionsThese studies demonstrate that individual genotoxicants induce distinct gene expression signatures. Further study of these molecular networks may explain the variable response of the developing brain to different types of environmental genotoxicants.

Desmoplastic small round cell tumor (DSRCT) is a primitive sarcoma characterized by a recurrent chromosomal translocation, t(11;22)(p13;q12), which fuses the 5' exons of the EWS gene to the 3' exons of the WT1 gene. EWS-WT1 chimeras are heterogeneous as a result of fusions of different regions of the EWS gene to the WT1 gene. We report here a rare and novel EWS-WT1 variant, EWS-WT1 5/10, in a 6-year-old boy diagnosed with DSRCT and analyze the potential transactivation effect of the fusion oncoprotein. The predicted product is comprised of the N-terminal transactivation domain of EWS and lacks any sequence derived from the WT1 gene product. Nonetheless, the truncated protein was able to stimulate expression of the insulin-like growth factor-I receptor gene, a potent antiapoptotic receptor tyrosine kinase with potentially important roles in DSRCT etiology. These findings raise the possibility that the oncogenic potential of EWS-WT1 fusions is not necessarily a consequence of the fusion protein product per se.

Rafael A Irizarry1, Daniel Warren2, Forrest Spencer3, Irene F Kim4, Shyam Biswal5, Bryan C Frank6, Edward Gabrielson7, Joe G N Garcia8, Joel Geoghegan9, Gregory Germino4, Constance Griffin10, Sara C Hilmer11, Eric Hoffman11, Anne E Jedlicka12, Ernest Kawasaki9, Francisco Martı́nez-Murillo13, Laura Morsberger10, Hannah Lee5, David Petersen9, John Quackenbush6,14, Alan Scott12, Michael Wilson15,17, Yanqin Yang2, Shui Qing Ye8 & Wayne Yu16

To facilitate collaborative research efforts between multi-investigator teams using DNA microarrays, we identified sources of error and data variability between laboratories and across microarray platforms, and methods to accommodate this variability. RNA expression data were generated in seven laboratories, which compared two standard RNA samples using 12 microarray platforms. At least two standard microarray types (one spotted, one commercial) were used by all laboratories. Reproducibility for most platforms within any laboratory was typically good, but reproducibility between platforms and across laboratories was generally poor. Reproducibility between laboratories increased markedly when standardized protocols were implemented for RNA labeling, hybridization, microarray processing, data acquisition and data normalization. Reproducibility was highest when analysis was based on biological themes defined by enriched Gene Ontology (GO) categories. These findings indicate that microarray results can be comparable across multiple laboratories, especially when a common platform and set of procedures are used.

The gonadal steroid estrogen is a pleiotropic hormone that has multiple effects on numerous cellular functions. One of estrogen's major targets is the brain, where the steroid not only affects growth, differentiation, and survival of neurons, but also regulates cell excitability. Because estrogen modulates multiple, overlapping signaling pathways, it has been difficult to scrutinize the transcriptional activity of the steroid. Therefore, we still lack a global picture of how different genes interact and are regulated by estrogen. Herein we report the use of suppression subtractive hybridization followed by custom microarray analysis of thousands of genes that are differentially expressed during the negative feedback phase of the female reproductive cycle. We have found a number of key transcripts that are regulated by estrogen and contribute to the alteration in synaptic transmission and hence excitability of hypothalamic neurons (e.g., GABA neurons). These include gec-1, GABABR2, PI3 kinase subunit p55γ, and a number of proteins containing pleckstrin homology domains that are critical for plasma membrane targeting. Studies are underway to refine our analysis to individual nuclei and individual cells. However, what has emerged from this highly sensitive microarray analysis is that estrogen affects neuronal plasticity in hypothalamic neurons not only by transcription of new membrane proteins (e.g., receptors and channels), but also by altering expression of downstream signaling molecules and proteins involved in neurosecretory pathways.

Purpose Neuroblastoma (NB) is a common childhood malignancy characterized by heterogeneous clinical behaviour. The purpose of this study was to identify potential NB biomarkers that may improve outcome prediction.Patients and Methods The suppression subtractive hybridization (SSH) technique was used to identify the genes differentially expressed between NB and control tissue. RNA isolated from 235 primary NB tumor samples obtained from the Children's Cancer Group was evaluated for expression of the candidate markers using quantitative reverse transcriptase polymerase chain reaction (Taqman assays). The association between the mRNA expression levels in the identified candidate genes and clinical outcome was evaluated.Results SSH analysis identified differential expression of members of the GABAergic gene family in NB. Lower levels of gamma-aminobutyric acid (GABA) receptor-associated protein (GABARAP) gene expression predict decreased survival among all patients. GABA(A) delta receptor subunit gene expression was predictive of a poor outcome among Evans stage IV-S patients. An index of five coexpressed GABA(A) receptor subunits was identified (GABA(A) profile [GAP score]). Patients with a higher GAP score > -1) had a survival advantage. Multivariate analysis showed that GABARAP and GABA(A) alpha2 receptor subunit gene expression levels and GAP score remained predictors of clinical outcome after accounting for current prognostic indicators.Conclusion Dysregulation of the GABAergic system may constitute a fundamental event in the development of NB, and assessment of GABAergic system gene expression could provide improved patient stratification and potential new therapies. (C) 2004 by American Society of Clinical Oncology.

Asian Indians with type 2 diabetes mellitus (T2D) have higher susceptibility to diabetic nephropathy (T2DN), the leading cause of end-stage renal disease and morbidity in diabetes. Peripheral blood cells (PBCs) play an important role in diabetes, yet very little is known about the molecular mechanisms of PBCs regulated in insulin homeostasis. In this study we explored the global gene expression changes in PBCs in diabetes and diabetic nephropathy to identify the potential candidate genes and molecular networks regulated in diabetes and nephropathy.