Heike Fiegler

Publications of Heike Fiegler

• Sites of Differential DNA Methylation between Placenta and Peripheral Blood. Molecular Markers for Noninvasive Prenatal Diagnosis of Aneuploidies.

  Authors: Elisavet A Papageorgiou, Heike Fiegler, Vardhman Rakyan, Stephan Beck, Maj Hulten, Klea Lamnissou, Nigel P Carter, Philippos C Patsalis

  The American journal of pathology. 05/2009;

  The use of epigenetic differences between maternal whole blood and fetal (placental) DNA is one of the main areas of interest for the development of noninvasive prenatal diagnosis of aneuploidies.The use of epigenetic differences between maternal whole blood and fetal (placental) DNA is one of the main areas of interest for the development of noninvasive prenatal diagnosis of aneuploidies. However, the lack of detailed chromosome-wide identification of differentially methylated sites has limited the application of this approach. In this study, we describe an analysis of chromosome-wide methylation status using methylation DNA immunoprecipitation coupled with high-resolution tiling oligonucleotide array analysis specific for chromosomes 21, 18, 13, X, and Y using female whole blood and placental DNA. We identified more than 2000 regions of differential methylation between female whole blood and placental DNA on each of the chromosomes tested. A subset of the differentially methylated regions identified was validated by real-time quantitative polymerase chain reaction. Additionally, correlation of these regions with CpG islands, genes, and promoter regions was investigated. Between 56 to 83% of the regions were located within nongenic regions whereas only 1 to 11% of the regions overlapped with CpG islands; of these, up to 65% were found in promoter regions. In summary, we identified a large number of previously unreported fetal epigenetic molecular markers that have the potential to be developed into targets for noninvasive prenatal diagnosis of trisomy 21 and other common aneuploidies. In addition, we demonstrated the effectiveness of the methylation DNA immunoprecipitation approach in the enrichment of hypermethylated fetal DNA.

• Clinical implication of recurrent copy number alterations in hepatocellular carcinoma and putative oncogenes in recurrent gains on 1q.

  Authors: Tae Min Kim, Seon-Hee Yim, Seung-Hun Shin, Hai-Dong Xu, Yu-Chae Jung, Cheol Keun Park, Jong Young Choi, Won Sang Park, Mi Seon Kwon, Heike Fiegler, Nigel P Carter, Mun-Gan Rhyu, Yeun-Jun Chung

  International journal of cancer. Journal international du cancer. 10/2008;

  To elucidate the pathogenesis of hepatocellular carcinoma (HCC) and develop useful prognosis predictors, it is necessary to identify biologically relevant genomic alterations in HCC. In our study, weTo elucidate the pathogenesis of hepatocellular carcinoma (HCC) and develop useful prognosis predictors, it is necessary to identify biologically relevant genomic alterations in HCC. In our study, we defined recurrently altered regions (RARs) common to many cases of HCCs, which may contain tumor-related genes, using whole-genome array-CGH and explored their associations with the clinicopathologic features. Gene set enrichment analysis was performed to investigate functional implication of RARs. On an average, 23.1% of the total probes were altered per case. Mean numbers of altered probes are significantly higher in high-grade, bigger and microvascular invasion (MVI) positive tumors. In total, 32 RARs (14 gains and 18 losses) were defined and 4 most frequent RARs are gains in 1q21.1-q32.1 (64.5%), 1q32.1-q44 (59.2%), 8q11.21-q24.3 (48.7%) and a loss in 17p13.3-p12 (51.3%). Through focusing on RARs, we identified genes and functional pathways likely to be involved in hepatocarcinogenesis. Among genes in the recurrently gained regions on 1q, expression of KIF14 and TPM3 was significantly increased, suggesting their oncogenic potential in HCC. Some RARs showed the significant associations with the clinical features. Especially, the recurrent loss in 9p24.2-p21.1 and gain in 8q11.21-q24.3 are associated with the high tumor grade and MVI, respectively. Functional analysis showed that cytokine receptor binding and defense response to virus pathways are significantly enriched in high grade-related RARs. Taken together, our results and the strategy of analysis will help to elucidate pathogenesis of HCC and to develop biomarkers for predicting behaviors of HCC. (c) 2008 Wiley-Liss, Inc.

• An integrated resource for genome-wide identification and analysis of human tissue-specific differentially methylated regions (tDMRs).

  Authors: Vardhman Rakyan, Thomas Down, Natalie Thorne, Paul Flicek, Eugene Kulesha, Stefan Graf, Eleni Tomazou, Liselotte Backdahl, Nathan Johnson, Marils Herberth, Kevin Howe, David Jackson, Marcos Miretti, Heike Fiegler, John Marioni, Ewan Birney, Tim Hubbard, Nigel Carter, Simon Tavare, Stephan Beck

  Genome research. 07/2008;

  We report a novel resource (methylation profiles of DNA, mPod) for human genome-wide tissue-specific DNA methylation profiles. mPod consists of three fully integrated parts, genome-wide DNAWe report a novel resource (methylation profiles of DNA, mPod) for human genome-wide tissue-specific DNA methylation profiles. mPod consists of three fully integrated parts, genome-wide DNA methylation reference profiles of 13 normal somatic tissues, placenta, sperm and an immortalized cell line, a visualization tool that has been integrated with the Ensembl genome browser and a new algorithm for the analysis of immunoprecipitation-based DNA methylation profiles. We demonstrate the utility of our resource by identifying the first comprehensive genome-wide set of tissue-specific differentially methylated regions (tDMRs) that may play a role in cellular identity and the regulation of tissue-specific genome function. We also discuss the implications of our findings with respect to the regulatory potential of regions with varied CpG density, gene expression, transcription factor motifs, gene ontology and correlation with other epigenetic marks such as histone modifications.

• Replication-timing-correlated spatial chromatin arrangements in cancer and in primate interphase nuclei.

  Authors: Florian Grasser, Michaela Neusser, Heike Fiegler, Tobias Thormeyer, Marion Cremer, Nigel P Carter, Thomas Cremer, Stefan Müller

  Journal of cell science. 07/2008; 121(Pt 11):1876-86.

  Using published high-resolution data on S-phase replication timing, we determined the three-dimensional (3D) nuclear arrangement of 33 very-early-replicating and 31 very-late-replicating loci. WeUsing published high-resolution data on S-phase replication timing, we determined the three-dimensional (3D) nuclear arrangement of 33 very-early-replicating and 31 very-late-replicating loci. We analyzed diploid human, non-human primate and rearranged tumor cells by 3D fluorescence in situ hybridization with the aim of investigating the impact of chromosomal structural changes on the nuclear organization of these loci. Overall, their topology was found to be largely conserved between cell types, species and in tumor cells. Early-replicating loci were localized in the nuclear interior, whereas late-replicating loci showed a broader distribution with a higher preference for the periphery than for late-BrdU-incorporation foci. However, differences in the spatial arrangement of early and late loci of chromosome 2, as compared with those from chromosome 5, 7 and 17, argue against replication timing as a major driving force for the 3D radial genome organization in human lymphoblastoid cell nuclei. Instead, genomic properties, and local gene density in particular, were identified as the decisive parameters. Further detailed comparisons of chromosome 7 loci in primate and tumor cells suggest that the inversions analyzed influence nuclear topology to a greater extent than the translocations, thus pointing to geometrical constraints in the 3D conformation of a chromosome territory.

• Autosomal-dominant microtia linked to five tandem copies of a copy-number-variable region at chromosome 4p16.

  Authors: Irina Balikova, Kevin Martens, Cindy Melotte, Mustapha Amyere, Steven Van Vooren, Yves Moreau, David Vetrie, Heike Fiegler, Nigel P Carter, Thomas Liehr, Miikka Vikkula, Gert Matthijs, Jean-Pierre Fryns, Ingele Casteels, Koen Devriendt, Joris Robert Vermeesch

  American journal of human genetics. 02/2008; 82(1):181-7.

  Recently, large-scale benign copy-number variations (CNVs)--encompassing over 12% of the genome and containing genes considered to be dosage tolerant for human development--were uncovered in theRecently, large-scale benign copy-number variations (CNVs)--encompassing over 12% of the genome and containing genes considered to be dosage tolerant for human development--were uncovered in the human population. Here we present a family with a novel autosomal-dominantly inherited syndrome characterized by microtia, eye coloboma, and imperforation of the nasolacrimal duct. This phenotype is linked to a cytogenetically visible alteration at 4pter consisting of five copies of a copy-number-variable region, encompassing a low-copy repeat (LCR)-rich sequence. We demonstrate that the approximately 750 kb amplicon occurs in exact tandem copies. This is the first example of an amplified CNV associated with a Mendelian disorder, a discovery that implies that genome screens for genetic disorders should include the analysis of so-called benign CNVs and LCRs.

• Germline rates of de novo meiotic deletions and duplications causing several genomic disorders.

  Authors: Daniel J Turner, Marcos Miretti, Diana Rajan, Heike Fiegler, Nigel P Carter, Martyn L Blayney, Stephan Beck, Matthew E Hurles

  Nature genetics. 02/2008; 40(1):90-5.

  Meiotic recombination between highly similar duplicated sequences (nonallelic homologous recombination, NAHR) generates deletions, duplications, inversions and translocations, and it is responsibleMeiotic recombination between highly similar duplicated sequences (nonallelic homologous recombination, NAHR) generates deletions, duplications, inversions and translocations, and it is responsible for genetic diseases known as 'genomic disorders', most of which are caused by altered copy number of dosage-sensitive genes. NAHR hot spots have been identified within some duplicated sequences. We have developed sperm-based assays to measure the de novo rate of reciprocal deletions and duplications at four NAHR hot spots. We used these assays to dissect the relative rates of NAHR between different pairs of duplicated sequences. We show that (i) these NAHR hot spots are specific to meiosis, (ii) deletions are generated at a higher rate than their reciprocal duplications in the male germline and (iii) some of these genomic disorders are likely to have been underascertained clinically, most notably that resulting from the duplication of 7q11, the reciprocal of the deletion causing Williams-Beuren syndrome.

• Guidelines for molecular karyotyping in constitutional genetic diagnosis.

  Authors: Joris Robert Vermeesch, Heike Fiegler, Nicole de Leeuw, Karoly Szuhai, Jacqueline Schoumans, Roberto Ciccone, Frank Speleman, Anita Rauch, Jill Clayton-Smith, Conny Van Ravenswaaij, Damien Sanlaville, Philippos C Patsalis, Helen Firth, Koen Devriendt, Orsetta Zuffardi

  European journal of human genetics : EJHG. 12/2007; 15(11):1105-14.

  Array-based whole genome investigation or molecular karyotyping enables the genome-wide detection of submicroscopic imbalances. Proof-of-principle experiments have demonstrated that molecularArray-based whole genome investigation or molecular karyotyping enables the genome-wide detection of submicroscopic imbalances. Proof-of-principle experiments have demonstrated that molecular karyotyping outperforms conventional karyotyping with regard to detection of chromosomal imbalances. This article identifies areas for which the technology seems matured and areas that require more investigations. Molecular karyotyping should be part of the genetic diagnostic work-up of patients with developmental disorders. For the implementation of the technique for other constitutional indications and in prenatal diagnosis, more research is appropriate. Also, the article aims to provide best practice guidelines for the application of array comparative genomic hybridisation to ensure both technical and clinical quality criteria that will optimise and standardise results and reports in diagnostic laboratories. In short, both the specificity and the sensitivity of the arrays should be evaluated in every laboratory offering the diagnostic test. Internal and external quality control programmes are urgently needed to evaluate and standardise the test results between laboratories.

• Diet and the evolution of human amylase gene copy number variation.

  Authors: George H Perry, Nathaniel J Dominy, Katrina G Claw, Arthur S Lee, Heike Fiegler, Richard Redon, John Werner, Fernando A Villanea, Joanna L Mountain, Rajeev Misra, Nigel P Carter, Charles Lee, Anne C Stone

  Nature genetics. 11/2007; 39(10):1256-60.

  Starch consumption is a prominent characteristic of agricultural societies and hunter-gatherers in arid environments. In contrast, rainforest and circum-arctic hunter-gatherers and some pastoralistsStarch consumption is a prominent characteristic of agricultural societies and hunter-gatherers in arid environments. In contrast, rainforest and circum-arctic hunter-gatherers and some pastoralists consume much less starch. This behavioral variation raises the possibility that different selective pressures have acted on amylase, the enzyme responsible for starch hydrolysis. We found that copy number of the salivary amylase gene (AMY1) is correlated positively with salivary amylase protein level and that individuals from populations with high-starch diets have, on average, more AMY1 copies than those with traditionally low-starch diets. Comparisons with other loci in a subset of these populations suggest that the extent of AMY1 copy number differentiation is highly unusual. This example of positive selection on a copy number-variable gene is, to our knowledge, one of the first discovered in the human genome. Higher AMY1 copy numbers and protein levels probably improve the digestion of starchy foods and may buffer against the fitness-reducing effects of intestinal disease.

• Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.

  Authors: Ewan Birney, John A Stamatoyannopoulos, Anindya Dutta, Roderic Guigó, Thomas R Gingeras, Elliott H Margulies, Zhiping Weng, Michael Snyder, Emmanouil T Dermitzakis, Robert E Thurman [......] David B Jaffe, Jean L Chang, Kerstin Lindblad-Toh, Eric S Lander, Maxim Koriabine, Mikhail Nefedov, Kazutoyo Osoegawa, Yuko Yoshinaga, Baoli Zhu, Pieter J de Jong

  Nature. 07/2007; 447(7146):799-816.

  We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These dataWe report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

• Spreading of mammalian DNA-damage response factors studied by ChIP-chip at damaged telomeres.

  Authors: Andreas Meier, Heike Fiegler, Purificacion Muñoz, Peter Ellis, Diane Rigler, Cordelia Langford, Maria A Blasco, Nigel Carter, Stephen P Jackson

  The EMBO journal. 07/2007; 26(11):2707-18.

  Phosphorylated histone H2AX (gammaH2AX) is generated in nucleosomes flanking sites of DNA double-strand breaks, triggering the recruitment of DNA-damage response proteins such as MDC1 and 53BP1.Phosphorylated histone H2AX (gammaH2AX) is generated in nucleosomes flanking sites of DNA double-strand breaks, triggering the recruitment of DNA-damage response proteins such as MDC1 and 53BP1. Here, we study shortened telomeres in senescent human cells. We show that most telomeres trigger gammaH2AX formation, which spreads up to 570 kb into the subtelomeric regions. Furthermore, we reveal that the spreading patterns of 53BP1 and MDC1 are very similar to that of gammaH2AX, consistent with a structural link between these factors. Moreover, different subsets of telomeres signal in different cell lines, with those that signal tending to equate to the shortest telomeres of the corresponding cell line, thus linking telomere attrition with DNA-damage signalling. Notably, we find that, in some cases, gammaH2AX spreading is modulated in a manner suggesting that H2AX distribution or its ability to be phosphorylated is not uniform along the chromosome. Finally, we observe weak gammaH2AX signals at telomeres of proliferating cells, but not in hTERT immortalised cells, suggesting that low telomerase activity leads to telomere uncapping and senescence in proliferating primary cells.

• Radial chromatin positioning is shaped by local gene density, not by gene expression.

  Authors: Katrin Küpper, Alexandra Kölbl, Dorothee Biener, Sandra Dittrich, Johann von Hase, Tobias Thormeyer, Heike Fiegler, Nigel P Carter, Michael R Speicher, Thomas Cremer, Marion Cremer

  Chromosoma. 07/2007; 116(3):285-306.

  G- and R-bands of metaphase chromosomes are characterized by profound differences in gene density, CG content, replication timing, and chromatin compaction. The preferential localization ofG- and R-bands of metaphase chromosomes are characterized by profound differences in gene density, CG content, replication timing, and chromatin compaction. The preferential localization of gene-dense, transcriptionally active, and early replicating chromatin in the nuclear interior and of gene-poor, later replicating chromatin at the nuclear envelope has been demonstrated to be evolutionary-conserved in various cell types. Yet, the impact of different local chromatin features on the radial nuclear arrangement of chromatin is still not well understood. In particular, it is not known whether radial chromatin positioning is preferentially shaped by local gene density per se or by other related parameters such as replication timing or transcriptional activity. The interdependence of these distinct chromatin features on the linear deoxyribonucleic acid (DNA) sequence precludes a simple dissection of these parameters with respect to their importance for the reorganization of the linear DNA organization into the distinct radial chromatin arrangements observed in the nuclear space. To analyze this problem, we generated probe sets of pooled bacterial artificial chromosome (BAC) clones from HSA 11, 12, 18, and 19 representing R/G-band-assigned chromatin, segments with different gene density and gene loci with different expression levels. Using multicolor 3D flourescent in situ hybridization (FISH) and 3D image analysis, we determined their localization in the nucleus and their positions within or outside the corresponding chromosome territory (CT). For each BAC data on local gene density within 2- and 10-Mb windows, as well as GC (guanine and cytosine) content, replication timing and expression levels were determined. A correlation analysis of these parameters with nuclear positioning revealed regional gene density as the decisive parameter determining the radial positioning of chromatin in the nucleus in contrast to band assignment, replication timing, and transcriptional activity. We demonstrate a polarized distribution of gene-dense vs gene-poor chromatin within CTs with respect to the nuclear border. Whereas we confirm previous reports that a particular gene-dense and transcriptionally highly active region of about 2 Mb on 11p15.5 often loops out from the territory surface, gene-dense and highly expressed sequences were not generally found preferentially at the CT surface as previously suggested.

• High resolution array-CGH analysis of single cells.

  Authors: Heike Fiegler, Jochen B Geigl, Sabine Langer, Diane Rigler, Keith Porter, Kristian Unger, Nigel P Carter, Michael R Speicher

  Nucleic acids research. 02/2007; 35(3):e15.

  Heterogeneity in the genome copy number of tissues is of particular importance in solid tumor biology. Furthermore, many clinical applications such as pre-implantation and non-invasive prenatalHeterogeneity in the genome copy number of tissues is of particular importance in solid tumor biology. Furthermore, many clinical applications such as pre-implantation and non-invasive prenatal diagnosis would benefit from the ability to characterize individual single cells. As the amount of DNA from single cells is so small, several PCR protocols have been developed in an attempt to achieve unbiased amplification. Many of these approaches are suitable for subsequent cytogenetic analyses using conventional methodologies such as comparative genomic hybridization (CGH) to metaphase spreads. However, attempts to harness array-CGH for single-cell analysis to provide improved resolution have been disappointing. Here we describe a strategy that combines single-cell amplification using GenomePlex library technology (GenomePlex) Single Cell Whole Genome Amplification Kit, Sigma-Aldrich, UK) and detailed analysis of genomic copy number changes by high-resolution array-CGH. We show that single copy changes as small as 8.3 Mb in single cells are detected reliably with single cells derived from various tumor cell lines as well as patients presenting with trisomy 21 and Prader-Willi syndrome. Our results demonstrate the potential of this technology for studies of tumor biology and for clinical diagnostics.

• Breaking the waves: improved detection of copy number variation from microarray-based comparative genomic hybridization.

  Authors: John C Marioni, Natalie P Thorne, Armand Valsesia, Tomas Fitzgerald, Richard Redon, Heike Fiegler, T Daniel Andrews, Barbara E Stranger, Andrew G Lynch, Emmanouil T Dermitzakis, Nigel P Carter, Simon Tavaré, Matthew E Hurles

  Genome biology. 02/2007; 8(10):R228.

  BACKGROUND: Large-scale high throughput studies using microarray technology have established that copy number variation (CNV) throughout the genome is more frequent than previously thought. SuchBACKGROUND: Large-scale high throughput studies using microarray technology have established that copy number variation (CNV) throughout the genome is more frequent than previously thought. Such variation is known to play an important role in the presence and development of phenotypes such as HIV-1 infection and Alzheimer's disease. However, methods for analyzing the complex data produced and identifying regions of CNV are still being refined. RESULTS: We describe the presence of a genome-wide technical artifact, spatial autocorrelation or 'wave', which occurs in a large dataset used to determine the location of CNV across the genome. By removing this artifact we are able to obtain both a more biologically meaningful clustering of the data and an increase in the number of CNVs identified by current calling methods without a major increase in the number of false positives detected. Moreover, removing this artifact is critical for the development of a novel model-based CNV calling algorithm - CNVmix - that uses cross-sample information to identify regions of the genome where CNVs occur. For regions of CNV that are identified by both CNVmix and current methods, we demonstrate that CNVmix is better able to categorize samples into groups that represent copy number gains or losses. CONCLUSION: Removing artifactual 'waves' (which appear to be a general feature of array comparative genomic hybridization (aCGH) datasets) and using cross-sample information when identifying CNVs enables more biological information to be extracted from aCGH experiments designed to investigate copy number variation in normal individuals.

• Construction and use of spotted large-insert clone DNA microarrays for the detection of genomic copy number changes.

  Authors: Heike Fiegler, Richard Redon, Nigel P Carter

  Nature protocols. 02/2007; 2(3):577-87.

  Microarray-based comparative genomic hybridization has become a widespread method for the analysis of DNA copy number changes across the human genome. Initial methods for microarray constructionMicroarray-based comparative genomic hybridization has become a widespread method for the analysis of DNA copy number changes across the human genome. Initial methods for microarray construction using large-insert clones required the preparation of DNA from large-scale cultures. This rapidly became an expensive and time-consuming process when expanded to the number of clones needed for higher resolution arrays. To overcome this problem, several PCR-based strategies have been developed to enable array construction from small amounts of cloned DNA. Here, we describe the construction of microarrays composed of human-specific large-insert clones (40-200 kb) using a specific degenerate oligonucleotide PCR strategy. In addition, we also describe array hybridization using manual and automated procedures and methods for array analysis. The technology and protocols described in this article can easily be adapted for other species dependent on the availability of clone libraries. According to our protocols, the procedure will take approximately 3 days from labeling the DNA to scanning the hybridized slides.

• Accurate and reliable high-throughput detection of copy number variation in the human genome.

  Authors: Heike Fiegler, Richard Redon, Dan Andrews, Carol Scott, Robert Andrews, Carol Carder, Richard Clark, Oliver Dovey, Peter Ellis, Lars Feuk [......] Keith Porter, Rachel E Rigby, Diane Rigler, Armand Valsesia, Cordelia Langford, Sean J Humphray, Stephen W Scherer, Charles Lee, Matthew E Hurles, Nigel P Carter

  Genome research. 01/2007; 16(12):1566-74.

  This study describes a new tool for accurate and reliable high-throughput detection of copy number variation in the human genome. We have constructed a large-insert clone DNA microarray covering theThis study describes a new tool for accurate and reliable high-throughput detection of copy number variation in the human genome. We have constructed a large-insert clone DNA microarray covering the entire human genome in tiling path resolution that we have used to identify copy number variation in human populations. Crucial to this study has been the development of a robust array platform and analytic process for the automated identification of copy number variants (CNVs). The array consists of 26,574 clones covering 93.7% of euchromatic regions. Clones were selected primarily from the published "Golden Path," and mapping was confirmed by fingerprinting and BAC-end sequencing. Array performance was extensively tested by a series of validation assays. These included determining the hybridization characteristics of each individual clone on the array by chromosome-specific add-in experiments. Estimation of data reproducibility and false-positive/negative rates was carried out using self-self hybridizations, replicate experiments, and independent validations of CNVs. Based on these studies, we developed a variance-based automatic copy number detection analysis process (CNVfinder) and have demonstrated its robustness by comparison with the SW-ARRAY method.

• Global variation in copy number in the human genome.

  Authors: Richard Redon, Shumpei Ishikawa, Karen R Fitch, Lars Feuk, George H Perry, T Daniel Andrews, Heike Fiegler, Michael H Shapero, Andrew R Carson, Wenwei Chen [......] Lluis Armengol, Donald F Conrad, Xavier Estivill, Chris Tyler-Smith, Nigel P Carter, Hiroyuki Aburatani, Charles Lee, Keith W Jones, Stephen W Scherer, Matthew E Hurles

  Nature. 12/2006; 444(7118):444-54.

  Copy number variation (CNV) of DNA sequences is functionally significant but has yet to be fully ascertained. We have constructed a first-generation CNV map of the human genome through the study ofCopy number variation (CNV) of DNA sequences is functionally significant but has yet to be fully ascertained. We have constructed a first-generation CNV map of the human genome through the study of 270 individuals from four populations with ancestry in Europe, Africa or Asia (the HapMap collection). DNA from these individuals was screened for CNV using two complementary technologies: single-nucleotide polymorphism (SNP) genotyping arrays, and clone-based comparative genomic hybridization. A total of 1,447 copy number variable regions (CNVRs), which can encompass overlapping or adjacent gains or losses, covering 360 megabases (12% of the genome) were identified in these populations. These CNVRs contained hundreds of genes, disease loci, functional elements and segmental duplications. Notably, the CNVRs encompassed more nucleotide content per genome than SNPs, underscoring the importance of CNV in genetic diversity and evolution. The data obtained delineate linkage disequilibrium patterns for many CNVs, and reveal marked variation in copy number among populations. We also demonstrate the utility of this resource for genetic disease studies.

• A complex rearrangement on chromosome 22 affecting both homologues; haplo-insufficiency of the Cat eye syndrome region may have no clinical relevance.

  Authors: Marjolein Kriek, Karoly Szuhai, Sarina G Kant, Stefan J White, Hans Dauwerse, Heike Fiegler, Nigel P Carter, Jeroen Knijnenburg, Johan T den Dunnen, Hans J Tanke, Martijn H Breuning, Carla Rosenberg

  Human genetics. 09/2006; 120(1):77-84.

  The presence of highly homologous sequences, known as low copy repeats, predisposes for unequal recombination within the 22q11 region. This can lead to genomic imbalances associated with severalThe presence of highly homologous sequences, known as low copy repeats, predisposes for unequal recombination within the 22q11 region. This can lead to genomic imbalances associated with several known genetic disorders. We report here a developmentally delayed patient carrying different rearrangements on both chromosome 22 homologues, including a previously unreported rearrangement within the 22q11 region. One homologue carries a deletion of the proximal part of chromosome band 22q11. To our knowledge, a 'pure' deletion of this region has not been described previously. Four copies of this 22q11 region, however, are associated with Cat eye syndrome (CES). While the phenotypic impact of this deletion is unclear, familial investigation revealed five normal relatives carrying this deletion, suggesting that haplo-insufficiency of the CES region has little clinical relevance. The other chromosome 22 homologue carries a duplication of the Velocardiofacial/DiGeorge syndrome (VCFS/DGS) region. In addition, a previously undescribed deletion of 22q12.1, located in a relatively gene-poor region, was identified. As the clinical features of patients suffering from a duplication of the VCFS/DGS region have proven to be extremely variable, it is impossible to postulate as to the contribution of the 22q12.1 deletion to the phenotype of the patient. Additional patients with a deletion within this region are needed to establish the consequences of this copy number alteration. This study highlights the value of using different genomic approaches to unravel chromosomal alterations in order to study their phenotypic impact.

• Combined array-comparative genomic hybridization and single-nucleotide polymorphism-loss of heterozygosity analysis reveals complex changes and multiple forms of chromosomal instability in colorectal cancers.

  Authors: Michelle Gaasenbeek, Kimberley Howarth, Andrew J Rowan, Patricia A Gorman, Angela Jones, Tracy Chaplin, Ying Liu, David Bicknell, Eleanor J Davison, Heike Fiegler, Nigel P Carter, Rebecca R Roylance, Ian P M Tomlinson

  Cancer research. 05/2006; 66(7):3471-9.

  Cancers with chromosomal instability (CIN) are held to be aneuploid/polyploid with multiple large-scale gains/deletions, but the processes underlying CIN are unclear and different types of CIN mightCancers with chromosomal instability (CIN) are held to be aneuploid/polyploid with multiple large-scale gains/deletions, but the processes underlying CIN are unclear and different types of CIN might exist. We investigated colorectal cancer cell lines using array-comparative genomic hybridization (CGH) for copy number changes and single-copy number polymorphism (SNP) microarrays for allelic loss (LOH). Many array-based CGH changes were not found by LOH because they did not cause true reduction-to-homozygosity. Conversely, many regions of SNP-LOH occurred in the absence of copy number change, comprising an average per cell line of 2 chromosomes with complete LOH; 1-2 terminal regions of LOH (mitotic recombination); and 1 interstitial region of LOH. SNP-LOH detected many novel changes, representing possible locations of uncharacterized tumor suppressor loci. Microsatellite unstable (MSI+) lines infrequently showed gains/deletions or whole-chromosome LOH, but their near-diploid karyotypes concealed mitotic recombination frequencies similar to those of MSI- lines. We analyzed p53 and chromosome 18q (SMAD4) in detail, including mutation screening. Almost all MSI- lines showed LOH and/or deletion of p53 and 18q; some near-triploid lines had acquired three independent changes at these loci. We found consistent results in primary colorectal cancers. Overall, the distributions of mitotic recombination and whole-chromosome LOH in the MSI- cell lines differed significantly from random, with some lines having much higher than expected levels of these changes. Moreover, lines with more LOH changes had significantly fewer copy number changes. These data suggest that CIN is not synonymous with copy number change and some cancers have a specific tendency to whole-chromosome deletion and regain or to mitotic recombination.

• Genomic profiling identifies discrete deletions associated with translocations in glioblastoma multiforme.

  Authors: Paul J Mulholland, Heike Fiegler, Chiara Mazzanti, Patricia Gorman, Peter Sasieni, Joanna Adams, Tania A Jones, Jane W Babbage, Radost Vatcheva, Koichi Ichimura, Philip East, Chrysanthos Poullikas, V Peter Collins, Nigel P Carter, Ian P M Tomlinson, Denise Sheer

  Cell cycle (Georgetown, Tex.). 05/2006; 5(7):783-91.

  Glioblastoma multiforme is the most common tumor arising in the central nervous system. Patients with these tumors have limited treatment options and their disease is invariably fatal. MolecularlyGlioblastoma multiforme is the most common tumor arising in the central nervous system. Patients with these tumors have limited treatment options and their disease is invariably fatal. Molecularly targeted agents offer the potential to improve patient treatment, however the use of these will require a fuller understanding of the genetic changes in these complex tumors. In this study, we identify copy number changes in a series of glioblastoma multiforme tumors and cell lines by applying high-resolution microarray comparative genomic hybridization. Molecular cytogenetic characterization of the cell lines revealed that copy number changes define translocation breakpoints. We focused on chromosome 6 and further characterized three regions of copy number change associated with translocations including a discrete deletion involving IGF2R, PARK2, PACRG and QKI and an unbalanced translocation involving POLH, GTPBP2 and PTPRZ1.

• Micro-array analyses decipher exceptional complex familial chromosomal rearrangement.

  Authors: Christine Fauth, Susan M Gribble, Keith M Porter, Montserrat Codina-Pascual, Bee Ling Ng, Jürgen Kraus, Sabine Uhrig, Jürgen Leifheit, Thomas Haaf, Heike Fiegler, Nigel P Carter, Michael R Speicher

  Human genetics. 04/2006; 119(1-2):145-53.

  Recently there has been an increased interest in large-scale genomic variation and clinically in the consequences of haploinsufficiency of genomic segments or disruption of normal gene function byRecently there has been an increased interest in large-scale genomic variation and clinically in the consequences of haploinsufficiency of genomic segments or disruption of normal gene function by chromosome rearrangements. Here, we present an extraordinary case in which both mother and daughter presented with unexpected chromosomal rearrangement complexity, which we characterized with array-CGH, array painting and multicolor large insert clone hybridizations. We found the same 12 breakpoints involving four chromosomes in both mother and daughter. In addition, the daughter inherited a microdeletion from her father. We mapped all breakpoints to the resolution level of breakpoint spanning clones. Genes were found within 7 of the 12 breakpoint regions, some of which were disrupted by the chromosome rearrangement. One of the rearrangements disrupted a locus, which has been discussed as a quantitative trait locus for fetal hemoglobin expression in adults. Interestingly, both mother and daughter show persistent fetal hemoglobin levels. We detail the most complicated familial complex chromosomal rearrangement reported to date and thus an extreme example of inheritance of chromosomal rearrangements without error in meiotic segregation.