Human embryos commonly form abnormal nuclei during development: a mechanism of DNA damage, embryonic aneuploidy, and developmental arrest
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Dieter Egli
PROVIDER: E-GEOD-72150 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Methods of comprehensive microarray based analyses of single cell DNA are rapidly emerging. Whole genome amplification (WGA) remains a critical component for these methods to be successful. A number of commercially available WGA kits have been independently utilized in previous single cell microarray studies. However, direct comparison of their performance on single cells has not been conducted. The present study demonstrates that among previously published methods, a single cell GenomePlex WGA protocol provides the best combination of speed and accuracy for SNP microarray based copy number analysis when compared to a REPLI-g or GenomiPhi based protocol. Alternatively, for applications that do not have constraints on turn-around time and that are directed at accurate genotyping rather than copy number assignments, a REPLI-g based protocol may provide the best solution. Affymetrix SNP arrays were processed according to the manufacturer's directions on DNA extracted from human fibroblast cell lines and single fibroblast cells. Afflymetrix SNP array analysis was successfully completed on 46 lymphocyte single cell samples, 8 gDNA extracted from cell lines, 11 reference gDNA extracted from cell lines and 3 reference gDNA samples from the RMA of New Jersey DNA bank. GSM617116 to GSM617129: CEL files were processed using GTYPE version 4 (Affymetrix Inc., Genotyping Console 4.0 Manual) using the DM algorithm for genotype calls. Copy number and loss of heterozygosity were calculated from CHP files using CNAT version 4.1 (Affymetrix Inc., Genotyping Console 4.0 Manual) analysis against a reference set consisting of three normal females from in house gDNA bank, 11 normal females from Coriel cell lines and 16 normal females from the HapMap database (www.hapmap.org). The 16 normal females are NA10855, NA10863, NA11832, NA12057, NA12234, NA12717, NA12813, NA18505, NA18508, NA18517, NA19137, NA19152, NE00088, NE00091, NE00403, and NE01119.
2010-12-20 | E-GEOD-24690 | biostudies-arrayexpress
Project description:Embryo DNA fingerprinting represents an important tool for tracking embryo-specific outcomes after multiple embryo transfer during IVF. The situation in which 2 embryos are transferred and only one implants represents a unique opportunity for the most well-controlled comparison of competent and incompetent embryos. Specifically, this design eliminates all patient-related variables from the comparison of embryos with or without reproductive potential. However, in order to determine which embryo implanted, the investigator must wait until newborn DNA is available upon delivery. This study validates a non-invasive fetal DNA fingerprinting method that reduces the time to identify which embryo implanted by approximately 31 weeks. Thirty-four patients were studied to determine if fingerprinting of fetal DNA extracted from maternal plasma at 9 gestational weeks concurred with the buccal DNA results obtained from the newborn after delivery. This validation required single nucleotide polymorphism (SNP) profiles on each couples’ preimplantation embryos, enriched fetal DNA from maternal plasma at 9 weeks gestation, and newborn DNA obtained from buccal swabs after delivery. The predictions from fetal DNA-based embryo tracking and gender assignments made at 9 weeks gestation were 100% consistent with standardized methods of assessment performed after term delivery. This study demonstrates the first validated fetal DNA fingerprinting method which predicts both gender and which embryo implanted at 9 weeks gestation following multiple embryo transfer. Affymetrix SNP arrays were processed and successfully completed according to the manufacturer's directions on DNA extracted from 136 embryos, 33 parental blood samples, 17 enriched fetal DNA samples and 21 buccal DNA samples.
2011-04-14 | E-GEOD-26404 | biostudies-arrayexpress
Project description:Many studies estimate that chromosomal mosaicism within the cleavage stage human embryo is high. However, comparison of two unique methods of aneuploidy screening of blastomeres within the same embryo has not been conducted and may indicate whether mosaicism is overestimated due to technical inconsistency rather than biological phenomena. The present study investigates the prevalence of chromosomal abnormality and mosaicism found with two different single cell aneuploidy screening techniques.Thirteen arrested cleavage stage embryos were studied. Each was biopsied into individual cells (n=160). The cells from each embryo were randomized into two groups. Those destined for FISH based aneuploidy screening (n=75) were fixed, 1 cell per slide. Cells for SNP microarray based aneuploidy screening (n=85) were put into individual tubes. Microarray was significantly more reliable (96%) than FISH (83%) for providing an interpretable result (P=0.004). Markedly different results were obtained when comparing microarray and FISH results from individual embryos. Mosaicism was significantly less commonly observed by microarray (4 of 13 embryos; 31%) than by FISH (13 of 13 embryos; 100%)(P=0.0005). Although FISH evaluated fewer chromosomes per cell and fewer cells per embryo, FISH still displayed significantly more unique genetic diagnoses per embryo (3.2+0.2) than microarray (1.3+0.2)(P<0.0001). This is the first prospective, randomized, blinded, and paired comparison between microarray and FISH based aneuploidy screening. Aneuploidy and mosaicism were less common with microarray. While evaluating a smaller number of chromosomes with a proportionally smaller opportunity for finding mosaicism, FISH still had a dramatically higher level of inter-cell variation in diagnosis. SNP microarray based 24 chromosome aneuploidy screening provides more complete and consistent results than FISH. Affymetrix SNP arrays were processed according to the manufacturer's directions on DNA extracted from 13 arrested cleavage stage embryos and biopsied into individual blastomere cells. Individual blastomeres were randomized and blinded for SNP microarray analysis (n = 85). Afflymetrix SNP array analysis was successfully completed on 82 blastomeres.
2010-06-09 | E-GEOD-20975 | biostudies-arrayexpress
Project description:In the majority of colorectal cancers (CRC) under clinical suspicion for a hereditary cause, the disease-causing genetic factors are still to be discovered. In order to identify such genetic factors we stringently selected a discovery cohort of 41 CRC index patients with microsatellite-stable tumors. All patients were below 40 years of age at diagnosis and/or exhibited an overt family history. We employed genome-wide copy number profiling using high-resolution SNP-based array CGH on germline DNA, which resulted in the identification of novel copy number variants (CNVs) in 6 patients (15%) encompassing, among others, the cadherin gene CDH18, the bone morphogenetic protein antagonist family gene GREM1, and the breakpoint cluster region gene BCR. In addition, two genomic deletions were encountered encompassing two microRNA genes, hsa-mir-491/KIAA1797 and hsa-mir-646/AK309218. None of these CNVs has previously been reported in relation to CRC predisposition in humans, nor were they encountered in large control cohorts (>1,600 unaffected individuals). Since several of these newly identified candidate genes may be functionally linked to CRC development, our results illustrate the potential of this approach for the identification of novel candidate genes involved in CRC predisposition. Copy number detection was performed using CNAG2.0 software for 250k SNP arrays and using the Affymetrix Genotyping Console v2.1 software for SNP 6.0 arrays, Reference genomes are included in this data set. Germline genomic DNA from 41 patients with early-onset microsatellite stable colorectal cancer was hybridized on Affymetrix Nsp/6.0 SNP-based arrays according to manufacturer's procedures.
2010-11-01 | E-GEOD-13429 | biostudies-arrayexpress
Project description:Primitive neuro-ectodermal tumours (PNET) of the supratentorial region are rare, highly malignant embryonal brain tumours affecting young children. Although supratentorial PNET (sPNET) are histologically similar to infratentorial PNET/medulloblastoma, sPNET have more aggressive clinical phenotypes, which suggest sPNET represents distinct biological entities. In contrast to considerable progress in understanding the signalling pathways involved in medulloblastoma, little is known about sPNET pathogenesis. Prior low resolution CGH (comparative genomic hybridization) studies indicate sPNET have frequent genomic imbalances and copy number aberrations (CNAs). To define genes involved in sPNET pathogenesis, we utilized the Affymetrix 250K Nsp SNP (single nucleotide polymorphism) analysis to identify genes targeted by recurrent CNAs in primary human sPNET samples. Copy number analysis was conducted on 39 primary PNET samples. Select target genes were validated by genomic and/or RT-PCR. Our analysis revealed frequent CNA across the sPNET genome, encompassing large and focal chromosome segments, and corroborated previous reports that isochromosome 17q, an abnormality found in ~ 30% of medulloblastoma, is rare in sPNET. Keywords: single nucleotide polymorphism array, disease state analysis A total of 56 primary sPNET samples were collected for this study from The Hospital for Sick Children, John Hopkins University, St. Jude Research Hospital, University of Cambridge, Children’s Hospital Boston, Virginia Commonwealth University, Instituto nazionale per lo studio e la cura dei tumori, and Texas Children’s Hospital, with local Institutional Research Ethics Board approval. Pineoblastoma and rhaboid tumours were specifically excluded, leaving 52 samples, 39 of which had good quality DNA available. These samples, in addition to 28 diploid reference samples (a gift from Dr. S. Scherer, University of Toronto), were analyzed on the Affymetrix GeneChip Mapping 250K Nsp SNP array. Due to sample availability at the time the arrays were run, the diploid reference samples analyzed on the Sty arrays are different from reference samples analyzed on the Nsp arrays.
2009-12-21 | E-GEOD-14087 | biostudies-arrayexpress
Project description:Primitive neuro-ectodermal tumours (PNET) of the supratentorial region are rare, highly malignant embryonal brain tumours affecting young children. Although supratentorial PNET (sPNET) are histologically similar to infratentorial PNET/medulloblastoma, sPNET have more aggressive clinical phenotypes, which suggest sPNET represents distinct biological entities. In contrast to considerable progress in understanding the signalling pathways involved in medulloblastoma, little is known about sPNET pathogenesis. Here we identify a focal amplicon on chr19q13.41 that houses two polycistronic microRNA clusters. Functional analysese indicates that the microRNA clusters may promote oncogenesis in part by modulating cell survival. Copy number analysis was also performed on an overlapping set of PNET tumours, also available on GEO with accession code GSE14087. Keywords: single nucleotide polymorphism array, pediatric brain cancer A primitive neuroectodermal tumour sample with matched blood from the University of Cambridge was analyzed on the Affymetrix GeneChip Mapping 250K Nsp SNP array.
2010-04-03 | E-GEOD-14248 | biostudies-arrayexpress
Project description:Hypothesis: Non-small cell lung cancer (NSCLC) is characterized by a multitude of genetic aberrations with unknown clinical impact. In this study, we aimed to identify gene copy number changes that correlate with clinical outcome in NSCLC. To maximize the chance to identify clinically relevant events, we applied a strategy involving two prognostically extreme patient groups. Results: Genetic aberrations were strongly associated with tumor histology. In adenocarcinoma (n=50), gene copy number gains on chromosome 8q21-q24.3 (177 genes) were more frequent in long-term survivors. In squamous cell carcinoma (n=28), gains on chromosome 14q23.1-24.3 (133 genes) were associated with shorter survival, whereas losses in a neighboring region, 14q31.1-32.33 (110 genes), correlated with favorable outcome. In accordance with copy number gains and losses, mRNA expression levels of corresponding genes were increased or decreased, respectively. Conclusion: Comprehensive tumor profiling permits the integration of genomic, histologic and clinical data. We identified gene copy number gains and losses, with corresponding changes in mRNA levels, that were associated with prognosis in adenocarcinoma and squamous cell carcinoma of the lung. Short-term (<20 months; n=53) and long-term survivors (>58 months;n=47) were selected from a clinically well-characterized NSCLC patient cohort with available fresh-frozen tumor specimens. The samples were analyzed using high-resolution SNP-array technology. The molecular data was combined with information on clinical parameters.
2011-11-01 | E-GEOD-28572 | biostudies-arrayexpress
Project description:A method to infer genome-wide haplotypes from the analysis of one or two single (human) cells has tremendous applicative value. It would revolutionize not only preimplantation genetic diagnosis of in vitro fertilized human embryos in the clinic, but also animal breeding programs by enabling genome-wide quantitative trait loci selection at the embryonic level. In addition, it allows to further scrutinize drivers of haplotype diversity, mainly meiotic homologous recombination as well as somatic (homologous) recombination processes that occur often during (human) tumorigenesis. We developed a generic approach to type genome-wide single nucleotide polymorphisms in single human cells and to reconstruct genome-wide haplotypes of single or dual cell derived genotypes. Genome-wide sequences of syntenic alleles were determined for EBV-transformed lymphoblastoid cells as well as human blastomeres. To this end, multiple displacement amplified DNA samples of single cells were hybridized to Affymetrix 250K SNP-arrays. Different algorithmic designs were subsequently developed to assess from the single cell-derived SNP-probe intensities the sequence of syntenic alleles and to pinpoint accurately the majority of parental homologous recombination sites using a linkage-based approach. In total 12 amplified single human lymphoblastoid cell DNA samples, 3 amplified single human blastomere DNA samples and 19 non-amplified genomic DNA samples extracted from blood were analyzed by 250K Nsp I SNP arrays (GEO accession number GPL3718). The non-amplified genomic DNA samples extracted from blood were derived from 19 different persons belonging to 4 different families. The sample names of these 19 samples are respectively Family1_Individual1, Family1_mother, Family1_father, Family1_MaternalGrandmother, Family2_Individual2, Family2_mother, Family2_father, Family2_MaternalGrandmother, Family2_MaternalGrandfather, Family3_Individual3, Family3_Individual4, Family3_mother, Family3_father, Family3_MaternalGrandmother, Family3_MaternalGrandfather, Family4_mother, Family4_father, Family4_PaternalGrandmother, Family4_PaternalGrandfather. These 19 non-amplified genomic DNA samples extracted from blood were genotyped using the dynamic model algorithm embedded in the M-bM-^@M-^XGeneChip Genotyping Analysis Software (GTYPE) version 4.1 (Affymetrix)M-bM-^@M-^Y using a homozygous and heterozygous calling threshold of 0.12. The 12 amplified single lymphoblastoid cell DNA samples were derived from Epstein Barr virus transformed lymphoblastoid cell lines (EBV-line) of four different persons. Three amplified single lymphoblastoid cell DNA samples from individual M-bM-^@M-^\Family1_Individual1M-bM-^@M-^], three amplified single lymphoblastoid cell DNA samples from individual M-bM-^@M-^\Family2_Individual2M-bM-^@M-^], three amplified single lymphoblastoid cell DNA samples from individual M-bM-^@M-^\Family3_Individual3M-bM-^@M-^] and three amplified single lymphoblastoid cell DNA samples from individual M-bM-^@M-^\Family3_Individual4M-bM-^@M-^]. The samples names of these 12 are respectively: Family1_Individual1_SingleCell1, Family1_Individual1_SingleCell2, Family1_Individual1_SingleCell3, Family2_Individual2_SingleCell1, Family2_Individual2_SingleCell2, Family2_Individual2_SingleCell3, Family3_Individual3_SingleCell1, Family3_Individual3_SingleCell2, Family3_Individual3_SingleCell3, Family3_Individual4_SingleCell1, Family3_Individual4_SingleCell2, Family3_Individual4_SingleCell3. These 12 amplified single lymphoblastoid cell DNA samples were genotyped using (1) the dynamic model algorithm embedded in the M-bM-^@M-^XGeneChip Genotyping Analysis Software (GTYPE) version 4.1 (Affymetrix)M-bM-^@M-^Y using a homozygous and heterozygous calling threshold of 0.12, (2) the BRLMM algorithm within the M-bM-^@M-^XGenotyping console 3.0.1M-bM-^@M-^Y software (Affymetrix) using a scoring threshold of 0.1 and (3) the Birdseed algorithm of the APT-1.10.1 package (Affymetrix Power Tools) using the M-bM-^@M-^Xbirdseed-devM-bM-^@M-^Y command which is the most recently incorporated development version of birdseed from The Broad Institute (http://www.broadinstitute.org/mpg/birdsuite/birdseed.html). The 3 amplified single human blastomere DNA samples were derived from three different human blastomeres that belong to the same in vitro fertilized embryo. This embryo was conceived in vitro by using sperm of person M-bM-^@M-^\Family4_fatherM-bM-^@M-^] and an oocyte from person M-bM-^@M-^\Family4_motherM-bM-^@M-^]. The sample names of these 3 amplified single blastomere DNA samples are respectively: Family4_Blastomere1, Family4_Blastomere2, Family4_Blastomere3. These 3 amplified single human blastomere DNA samples were genotyped using the dynamic model algorithm embedded in the M-bM-^@M-^XGeneChip Genotyping Analysis Software (GTYPE) version 4.1 (Affymetrix)M-bM-^@M-^Y using a homozygous and heterozygous calling threshold of 0.12.
2012-10-01 | E-GEOD-17318 | biostudies-arrayexpress
Project description:Cancer development is associated with multiple genetic alterations and genomic instability either at the chromosomal or base pair level is generally thought to underlie these changes. However, it is still unknown whether genetic instability is absolutely required for tumorigenesis. Here we investigated the genomic instability status of four cytogenetically stable diploid cell lines CAL51, SK-UT-1B, A204 and CH1. We applied high resolution 500K single nucleotide polymorphism (SNP) array analysis and found that all the four cell lines have some sub-microscopic genomic copy number changes. Interestingly, there were many more sub-microscopic chromosome alterations in A204 and CH1 than in CAL51 and SK-UT-1B. Twenty-four-color fluorescence in situ hybridization (FISH) was used to analyse a large number of metaphases from CAL51, SK-UT-1B and CH1 cells. The rate of de novo chromosome rearrangements was significantly higher in CH1 than CAL51 and SK-UT-1B. Although this increased rate did not lead to many clonal cytogenetically apparent chromosome alterations in CH1 cells, it is consistent with a first step towards chromosomal instability. It is more striking that both cell lines CAL51 and SK-UT-1B which have a similar de novo chromosomal change rate to that of normal lymphocytes were microsatellite instability positive by BAT-26 microsatellite analysis. This study further strengths the current concept that genomic instability is associated with tumor development. Keywords: DNA copy number changes DNA from A204, CH1, CAL51 and SK-UT-1B were analyzed by Affymetrix Genechip Mapping 500K Set array. Data were compared with normal samples from HAPMAP database.
2008-03-14 | E-GEOD-8649 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE28571: Gene Copy Number Aberrations are Associated with Survival in Histological Subgroups of Non-Small Cell Lung Cancer (expression data) GSE28572: Gene Copy Number Aberrations are Associated with Survival in Histological Subgroups of Non-Small Cell Lung Cancer (copy number data) Refer to individual Series
2011-11-01 | E-GEOD-28582 | biostudies-arrayexpress