Lorentz Center - Karyotyping: from microscope to array - II from 28 Jan 2009 through 30 Jan 2009
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    Karyotyping: from microscope to array - II
    from 28 Jan 2009 through 30 Jan 2009

Title of Talk:

By Nigel Carter

CNVs, array-CGH and next generation sequencing”

In my talk I will touch on some of the latest developments in the identification and analysis of copy number variants both in normal populations and in disease.  As array-CGH resolution has developed we have been able to identify smaller copy number changes in individuals.  Using a set of 20 NimbleGen arrays each containing 2.1 million probes and tiling the human genome with long oligonucleotides we have been able to identify CNVs as small as 500 bp.  We have used this array set to screen 40 normal individuals and have made 51,981 CNV calls with  on average 1,300 CNVs per comparison (18-30 Mb) and corresponding to 9,299 discrete CNV loci which cover 4.8% of the human genome (135.6 Mb) thus greatly extending the number of known CNVs.  More importantly, the high resolution of this array set has allowed us to more accurately define CNV breakpoints and go on to design specific arrays for CNV genotyping which will allow the application of CNV in association studies.  We have been able to extend the number of CNV genotyping assays from a few hundred to approximately 5,000 and these are now being applied in large association studies such as the Wellcome Trust Case Control Consortium studies CCC1+ (19,000 samples) and CCC2 (~100,000 samples).  For patients with developmental disorders, these genotyping assays have the potential to allow us to determine the role of common, apparently benign CNVs in modifying disease severity and onset.

In the last part of my talk, I will describe how new generation sequencing is already being used to identify structural arrangements and mutations in normal individuals and patients and discuss the potential of this technology to replace DNA arrays in clinical diagnosis.



By Bert Eussen, Hannie Douben en Annelies de Klein


“Detection of copy number variation and allelic events”

To appreciate the significance of copy number variation (CNV) in our local patients, we have determined the overall pattern of CNV and copy neutral LOH in the GWA reference cohort. This local reference set of 471 normal, Caucasian females was generated by the Rotterdam “ERGO” Study. As platform we used the 550 dual Infinium array (Illumina), labeling and hybridization was fully automated, data was analyzed using Beadstudio v3 ( Illumina).  The Illumina Hapmap data set was used as reference and  copy-number segments (CBS algorithm) and  allelic event (LOH) were displayed as  frequency tables using  NexusCGH v4 (BioDiscovery) on a standard PC.  Nexus allows you to visualize your samples from frequency table to detailed analysis on gene level in an intuitive way.


Samples with waving, tailing patterns were excluded. In the remaining 417 samples and with the current setting 790 gains and losses were detected. Gains (n=371) ranged from 27 Kb-11Mb and losses (n=419) ranged from 14kb-5Mb, respectively. About 75% of the large (>0.5 MB) CNVs were single events. The B allele frequency was used to detect a shift in allele distribution, indicative of low mosaicism or LOH is also reported. Copy neutral LOH was observed at specific sites and two cases of allelic variants >90 MB


To show and explain the value of the combined analysis of copy-number and allelic variation a cases with a mos der(15)t(5;12)  will be shown.

The benefit of our platform choice (Illumina) and analysis software (Beadstudio / NexusCGH ) is dual: 1) large datasets can be handled and analyzed and 2) the information of copy number levels with the B-allele frequency  is importance in cases of chromosomal mosaicism, contamination, uniparental disomy (UPD).




By Victor Guryev and Ies Nijman

“Characterizing Structural genome variation in the RAT models of Human deseases with optical and paired-end maps”

Motivation. The previously unanticipated amount of structural variation (SV) in mammalian genomes poses new challenges in genome–wide discovery of SVs and the identification of their effect in natural and disease phenotypes. Due to inherent human heterozygosity, bioethical aspects and lack of invasive experiments, rat inbred strains may serve as a more convenient model for studying links between SVs and genetic disorders. We determined a detailed map of SVs in the genomes of thoroughly characterized hypertension model, HXB/BXH recombinant inbred rats. Since no single technology allows for exhaustive characterization we use several complimentary experimental approaches to obtain a comprehensive inventory of structural genome variants.


Optical mapping. The method is based on in situ digestion of DNA molecules with restriction enzyme, imaging and comparing restriction maps to in silico digest of genome assembly. It allows for high-resolution mapping of copy-number and copy-neutral structural variants. We have built optical maps (45x genome coverage) of Brown Norway rat genome to verify genome assembly and of two founder strains for HXB/BXH recombinant inbred strains, BN-Lx and SHR. The optical maps show that thousands of structural alterations can be found when comparing two rat strains. A comparable amount of SVs is seen when BN optical map is compared to the in silico digest of the reference genome assembly, indicating potential improvements for the current assembly.


Paired-end mapping. This method can provide a map of structural variants almost at base pair resolution. We used a combination of ultra-high throughput sequencing and paired-end mapping to characterize structural variation in BN and SHR rats. Next generation sequencers such as ABI SOLiD are capable of sequencing of about a hundred million paired tags per run, producing extensive clone coverage over the whole mammalian genome in a single run. Paired-end mapping reconfirms the variants observed previously by aCGH and optical mapping and extends the list of SVs by including small-sized polymorphisms.


Victor Guryev1, Sebastiaan A.A.C. van Heesch1, Veronika Boskova1, Isaac J. Nijman1, Michal Mokry1, Shiguo Zhou2, Steven Goldstein2, David Schwartz2 & Edwin Cuppen1


1Hubrecht Institute, University Medical Center Utrecht, Uppsalalaan 8, 3584CT Utrecht, The Netherlands. 2Laboratory of Genetics, University of Madison, WI 53706, USA.





By: Gunnar Houge, Trude Høysæther, Atle Brendehaug, Bjørn Ivar Haukanes and Randi Hovland

(Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Norway)


“The use of SNP-chip arrays in routine cytogenetic diagnostics”


The last year we have used Affymetrix SNP-chip arrays our routine diagnostic platform for genomic copy number analyses. After switching from HR-CGH to SNP-chip arrays, our diagnostic yield in patients with MR ±MCA/dysmorphism and normal G-banded karyotypes has increased from 7,2% (n=554) to 23,1% (n=160; 117 analysed on Affymetrix 250K arrays and 43 on Affymetrix Genome-Wide Human SNP Array 6.0). Compared to 250K arrays, the 1800K 6.0 array gives much less noise (at threshold 100 kb) and allows quite detailed examination of single candidate genes. In addition, uniparental isodisomies are automatically detected, the resolution in subtelomeric regions is much better, and the X-chromosome may be examined in detail for small deletions and duplications. Data from the same array can be used for haplotyping families, e.g. to look for regions that are identical-by-descent when recessive diseases are suspected. The main problems are no longer technical but clinical: What is the significance of some findings? Some copy number variations (CNVs) are inherited from a normal parent yet may still be significant, and other copy number changes (CNCs) are de novo but may still be normal variants. In other cases, a patient may have several CNVs/CNCs that all are likely contributors to the phenotype. Cytogenetics is now entering the exciting but challenging field of multifactorial genetics and susceptibility testing, and international collaboration on the clinical significance of findings is even more important than before.




By Jacqueline Schoumans


“CNV Detection in developmental delay; The Karolinska experience”


Microarray analysis for detection of chromosomal abnormalities in patients with developmental problems has brought a new era of cytogenetic testing, which in turn has significant impact on clinical practice. An important outcome of large scale testing of patients has been the identification of several ‘new’ microdeletion/duplication syndromes through so called ‘reverse dysmorphology’, that is, using a genotype to phenotype approach.


Although the implementation of array analysis in the diagnostic laboratories has been challenging at first, the availability of commercial array platforms has greatly facilitated to overcome technical challenges. Bioinformatics support and commercial developed software packages are also more widely available. However, the biggest challenge still remains; interpretation of the large number of copy number variants that are detected in each sample. At the Department of Clinical Genetics, Karolinska University Hospital, Stockholm we have offered microarray analysis as a clinical service since 2006.


Initially, we used arrays containing ~38 000 large insert clones. Later array analysis was facilitated by switching to the commercially available high density oligoarrays and aCGH analysis became ISO certified in our laboratory. Since the beginning of 2008, a new clinical investigation strategy has been implemented in our diagnostic setting. Patients with mental retardation and/ or malformation are first analysed with a dense CGH array instead of conventional karyotyping. Subsequently, the number of referrals per year has increased drastically. Today, a large number of genetic diagnostic centers across the globe offer whole genome analysis at high resolution and genotype as well as phenotype information is shared through online databases such as DECIPHER and ECARUCA. This approach facilitates the interpretation of detected CNVs and allows identification of novel microdeletion / microduplication syndromes. In collaboration with several other departments in Europe, USA and Australia, we have identified a, novel microdeletion syndrome in 17p13.3, distally located of Miller Dieker Syndrome (MDS) and Isolated Lissencepahly sequence (ILS). Copy number instability in chromosome region 17p13.3 and the range of specific pathological consequences which correlate to the underlying genomic abnormalities will be presented.




By Bert de Vries


New microdeletion / duplication phenotypes


Mental retardation occurs in 2-3% of the general population for which the cause remains largely unknown. Mental retardation may be seen in isolation or in combination with other malformations and/or dysmorphisms, resulting in specific syndromes.

Chromosome abnormalities, microscopically visible, are an important cause of mental retardation, detectable in ~10% of cases, depending on the clinical selection criteria and techniques used. In selected groups of mentally retarded patients subtelomeric rearrangements, below the level that can be detected by the light microscope (< 5Mb), have been found in ~ 5% of the patients, whereas Ravnan et al. (2006) detected ~ 2.5% in a large series of 11,688 patients using FISH.

Recently new techniques such as MAPH and MLPA have become available to test the subtelomeric integrity. Targeting the telomeric regions using the various new techniques has led to the identification of novel microdeletion syndromes related to the telomeres, such as 1qter, 3qter, 9qter and 22qter. In addition to microdeletions also novel microduplications of certain telomeric regions are found. So far, the latter are predominantly single cases and therefore not allowing for recognizing overlapping clinical features leading to the characterisation of a specific syndrome. 

Tiling resolution arrays covering the whole genome are used leading to identification of micro-aberrations in 10-15% of mentally retarded patients with additional dysmorphisms. 

To date, novel syndromes are being characterised such as the 15q13.3, 15q24, 16p11.2-p12.2 and 17q21.3 microdeletion syndromes. In these novel syndromes specific genomic structures, namely homologous flanking segmental duplications, are regarded as the underlying cause through nonallelic homologous recombination (NAHR). 

These new full coverage array CGH techniques prove the importance of chromosomal microdeletions and - duplications as a common cause of mental retardation and congenital abnormalities, and have led to the identification of novel microdeletion/duplication syndromes.




By Bauke Ylstra


“Array CGH in oncology for diagnosis and patient treatment”


Patient tailored medicine requires matching of the most effective therapy with the molecular characteristics of a cancer. Therefore, we need to recognize the molecular heterogeneity of the individual patient's tumor. Chromosomal copy number aberrations (CNAs) underlie almost all human cancers and offer considerable opportunities to stratify patient samples for therapy (Costa et al., 2008). Array Comparative Genomic Hybridization (array CGH) is the high-resolution laboratory technique of choice to detect such CNAs with high resolution and on genome-wide scale (Ylstra et al., 2006). The array CGH technique can be applied to archival tissue specimens (formalin fixed paraffin embedded, FFPE). This has allowed us to identify; therapeutic targets in lung cancer (Gallegos Ruiz et al., 2008), pivotal genes in pathogenesis of colorectal and DLBCL (Oudejans et al., 2009; Carvalho et al., 2009) chromosomal regions responsible for resistancy to therapies in colorectal cancers (van de Wiel et al., 2005), and different (sub-) types of medduloblastomas, gastric, colorectal, cervical and head&neck tumors (for a review see (Costa et al., 2008)). 

Unsupervised clustering of chromosomal copy number profiles has also allowed us to distinguish subclasses within mammary tumors which are tightly linked to survival in independent datasets (Fridlyand et al., 2006; Chin et al., 2006; Chin et al., 2007). Recently, we have shown that chromosomal copy number profiles could function as markers to predictors response to chemotherapy in advanced colorectal cancer.  With these examples I hope to be able to convince you during my seminar that copy number aberrations can serve as a marker for better cancer classification, prognosis, and outcome prediction.




Carvalho B, Postma C, Mongera S, Hopmans E, Diskin S, van de Wiel MA, van CW, Thas O, Matthai A, Cuesta MA, Terhaar Sive Droste JS, Craanen M, Schrock E, Ylstra B, Meijer GA. 2009. Multiple putative oncogenes at the chromosome 20q amplicon contribute to colorectal adenoma to carcinoma progression. GUT 58:79-89.

Chin K, Devries S, Fridlyand J, Spellman PT, Roydasgupta R, Kuo WL, Lapuk A, Neve RM, Qian Z, Ryder T, Chen F, Feiler H, Tokuyasu T, Kingsley C, Dairkee S, Meng Z, Chew K, Pinkel D, Jain A, Ljung BM, Esserman L, Albertson DG, Waldman FM, Gray JW. 2006. Genomic and transcriptional aberrations linked to breast cancer pathophysiologies. Cancer Cell 10:529-541.

Chin SF, Teschendorff AE, Marioni JC, Wang Y, Barbosa-Morais NL, Thorne NP, Costa JL, Pinder SE, van de Wiel MA, Green AR, Ellis IO, Porter PL, Tavare S, Brenton JD, Ylstra B, Caldas C. 2007. High-resolution array-CGH and expression profiling identifies a novel genomic subtype of ER negative breast cancer. Genome Biol. 8:R215-

Costa JL, Meijer G, Ylstra B, Caldas C. 2008. Array comparative genomic hybridization copy number profiling: a new tool for translational research in solid malignancies. Semin.Radiat.Oncol. 18:98-104.

Fridlyand J, Snijders AM, Ylstra B, Li H, Olshen A, Segraves R, Dairkee S, Tokuyasu T, Ljung BM, Jain AN, McLennan J, Ziegler J, Chin K, Devries S, Feiler H, Gray JW, Waldman F, Pinkel D, Albertson DG. 2006. Breast tumor copy number aberration phenotypes and genomic instability. BMC Cancer 6:96-

Gallegos Ruiz MI, Floor K, Roepman P, Rodriguez JA, Meijer GA, Mooi WJ, Jassem E, Niklinski J, Muley T, van ZN, Smit EF, Beebe K, Neckers L, Ylstra B, Giaccone G. 2008. Integration of Gene Dosage and Gene Expression in Non-Small Cell Lung Cancer, Identification of HSP90 as Potential Target. PLoS.ONE. 3:e0001722-

Oudejans JJ, van Wieringen WN, Smeets SJ, Tijssen M, Vosse SJ, Meijer CJ, Meijer GA, van de Wiel MA, Ylstra B. 2009. Identification of genes putatively involved in the pathogenesis of diffuse large B-cell lymphomas by integrative genomics. Genes Chromosomes.Cancer 48:250-260.

van de Wiel MA, Costa JL, Smid K, Oudejans CB, Bergman AM, Meijer GA, Peters GJ, Ylstra B. 2005. Expression microarray analysis and oligo array comparative genomic hybridization of acquired gemcitabine resistance in mouse colon reveals selection for chromosomal aberrations. Cancer Res. 65:10208-10213.

Ylstra B, van den IJssel P, Carvalho B, Brakenhoff RH, Meijer GA. 2006. BAC to the future! or oligonucleotides: a perspective for micro array comparative genomic hybridization (array CGH). Nucleic Acids Res. 34:445-450.