High-resolution Comparative Genomic Hybridization Array Research Articles

Characterization of copy-number variants in a large cohort of patients with von Willebrand disease reveals a relationship between disrupted regions and disease type

BackgroundGenetic analysis for von Willebrand disease (VWD) commonly utilizes DNA sequencing to identify variants in the von Willebrand factor (VWF) gene; however, this technique cannot always detect copy-number variants (CNVs). Additional mapping of CNVs in patients with VWD is needed. ObjectivesThis study aimed to characterize CNVs in a large sample of VWF mutation-negative VWD patients. MethodsTo determine the role of CNVs in VWD, a VWF high-resolution comparative genomic hybridization array was custom-designed to avoid multiple sequence variations, repeated sequences, and the VWF pseudogene. This was performed on 204 mutation-negative subjects for whom clinical variables were also available. ResultsAmong the 204 patients, 7 unique CNVs were found, with a total of 24 CNVs (12%). Of the 7 unique CNVs, 1 was novel, 1 was found in a VWF database, and 5 were previously reported. All patients with type 1C VWD and a CNV had the same exon 33 and 34 in-frame deletion. Certain clinical variables were also significantly different between those with and without CNVs. ConclusionThe in-frame deletion in patients with type 1C VWD exactly matches the D4N module of the D4 domain, a region where mutations and deletions are known to affect clearance. We observed significantly higher VWF–to–ristocetin cofactor levels in patients with type 1C VWD and a CNV than in patients without a CNV, suggesting a relationship between CNVs and the increased clearance observed in patients with type 1C VWD. Glycoprotein IbM activity was significantly lower in patients with type 1 VWD and a CNV than in patients without a CNV, suggesting that platelet binding is more affected by CNVs than single base pair mutations. This work elucidates some of the underlying genetic mechanisms of CNVs in these patients.

Rare Copy Number Variants in Array-Based Comparative Genomic Hybridization in Early-Onset Skeletal Fragility.

Early-onset osteoporosis is characterized by low bone mineral density (BMD) and fractures since childhood or young adulthood. Several monogenic forms have been identified but the contributing genes remain inadequately characterized. In search for novel variants and novel candidate loci, we screened a cohort of 70 young subjects with mild to severe skeletal fragility for rare copy-number variants (CNVs). Our study cohort included 15 subjects with primary osteoporosis before age 30 years and 55 subjects with a pathological fracture history and low or normal BMD before age 16 years. A custom-made high-resolution comparative genomic hybridization array with enriched probe density in >1,150 genes important for bone metabolism and ciliary function was used to search for CNVs. We identified altogether 14 rare CNVs. Seven intronic aberrations were classified as likely benign. Five CNVs of unknown clinical significance affected coding regions of genes not previously associated with skeletal fragility (ETV1-DGKB, AGBL2, ATM, RPS6KL1-PGF, and SCN4A). Finally, two CNVs were pathogenic and likely pathogenic, respectively: a 4 kb deletion involving exons 1–4 of COL1A2 (NM_000089.3) and a 12.5 kb duplication of exon 3 in PLS3 (NM_005032.6). Although both genes have been linked to monogenic forms of osteoporosis, COL1A2 deletions are rare and PLS3 duplications have not been described previously. Both CNVs were identified in subjects with significant osteoporosis and segregated with osteoporosis within the families. Our study expands the number of pathogenic CNVs in monogenic skeletal fragility and shows the validity of targeted CNV screening to potentially pinpoint novel candidate loci in early-onset osteoporosis.

Distinct Patterns of Genomic Alterations in Adult T-Cell Leukemia-Lymphoma Endemic in the Western World

Introduction: Acute T-cell leukemia/lymphoma (ATLL) is a highly aggressive malignancy caused by HTLV-I, which is endemic in Japan, the Caribbean, and South America. ATLL carries a dismal prognosis and is generally incurable with conventional chemotherapy. ATLL is challenging to study at the molecular level, in part due to its complex genetic alterations likely resulting from years of HTLV-I driven T-cell proliferation and accumulation of genetic damage prior to malignant transformation. While no specific chromosome or genetic abnormalities have been proven to contribute to the pathogenesis of ATLL, older comparative genomic hybridization (CGH) studies performed in Japanese patients have demonstrated frequent genetic lesions (gains and losses) involving specific chromosomal regions, thus limited information exists about the chromosomal abnormalities occurring in the African ATLL variant commonly seen in the Western World.Methods: In this study, we used a high-density oligo array 244K platform CGH platform (Agilent Technologies) with an average resolution of 8.9 Kb, to perform a comprehensive genomic analysis of 47 ATLL patient tumor specimens obtained from African-descendants in the United States, Caribbean and Brazil. Patients were sub-classified as acute-type (A) ATLL (n=31), lymphomatous (L) (n=8), chronic (n=7, six whom had unfavorable features), and one with smouldering type according to Shimoyama criteria. DNA samples were extracted from peripheral blood mononuclear cells or tumor samples from these patients and checked for quality.Results: ATLL tumors exhibited complex genomic abnormalities and high copy number changes (CNCs). The average of copy number (CN) aberrations per sample was 238 in the L-group vs. 114 in acute/unfavorable chronic (A/UC) group. However, many chromosomal alterations were observed in this cohort, which had not been previously reported by other studies. The common CNCs were gains at 1q21-q44, 3, 3p, 7q22-q36, 8, 18, 19p13.1-p13.3, 21q21.1-q22.3, 22q12-q13 and losses at 5q13.2-q32, 6q11-q15, 9q13-q21. Gains in the 14 q32 (IGH) regions and losses in the 7p14.1 (TCRG), 7q34 (TCRB) and 14q11.2 (TCRA) regions involving small DNA segments were frequently observed. Genomic losses involving at least one or more known or candidate tumor suppressor genes were found in nearly all tumors, including some genes not previously implicated in ATLL. Some of the most significant gene or locus specific losses occurring in at least 20 % of the tumors in aggressive ATLL subtypes (A/UC and L) are summarized in Table 1. Losses of CDKN2A and CDKN2B tumor suppressor genes have been previously implicated in ATLL and other cancers. Several other genes found by this analysis have been implicated in apoptosis or cancer (i.e. CBLB, ANKRD11, IKZF1, and EPC1). IMMP2L deletion was associated with shorter survival time (2.3 weeks) compared with those cases without this gene deletion (29 weeks) in the A/UC group (p=0.005). ANKRD11 homo- or heterozygous deletions were seen in 37% of L-type and 19% of acute-type cases, and were associated with a shorter survival (13 vs. 43 weeks, p=<0.05) in the A group. CPN2/LRRC15 locus gains in 32% of A-type were linked to poorer survival (16 vs. 42 weeks, p=0.05).Table 1GENEFUNCTIONLOSSES (n) GAINS (n) NRXN3Membrane receptor, cell adhesion23 A/UC 3 LNSIMMP2LMitochondrial inner membrane peptidase16 A/UC* 5 L4 A/UCCDKN2A/ CDKN2B (P16INK4/p15INK4b)Cyclin-dependent kinase inhibitors, tumor suppressors16 A/UC 2 L1 A/UC 1 LCBLBE3 ubiquitin protein ligase12 A/UC 1 LNSANKRD11Transcriptional inhibitor, co-activator of p536 A/UC* 3 LNSCPN2CarboxypeptidaseNS10 A/UC* 2 LIKZF1Zinc-finger DNA binding proteins , lymphocyte differentiationNS6 A/UC 3 LINSIG1Endoplasmic reticulum membrane protein, intracellular lipid metabolismNS10 A/UC 1 LEPC1Member of the polycomb group family, transcriptional activator and repressor10 A/UC 2 LNS* are genomic imbalances associated with a statistically significant reduction in survival. NS: non-significant.Conclusion: In sum, using a high resolution CGH array we observed distinct patterns ofgenetic aberrations in ATLL endemic in the Western World. We have successfully narrowed the genomic regions containing potential candidate genes that could be relevant to the pathogenesis of this fatal disease. Functional studies are required to determine the role of some of these genes in the pathogenesis of ATLL. DisclosuresO’Hara:BioDiscovery, Inc.: Employment.

TIARA genome database: update 2013

The Total Integrated Archive of short-Read and Array (TIARA; http://tiara.gmi.ac.kr) database stores and integrates human genome data generated from multiple technologies including next-generation sequencing and high-resolution comparative genomic hybridization array. The TIARA genome browser is a powerful tool for the analysis of personal genomic information by exploring genomic variants such as SNPs, indels and structural variants simultaneously. As of September 2012, the TIARA database provides raw data and variant information for 13 sequenced whole genomes, 16 sequenced transcriptomes and 33 high resolution array assays. Sequencing reads are available at a depth of ∼30× for whole genomes and 50× for transcriptomes. Information on genomic variants includes a total of ∼9.56 million SNPs, 23 025 of which are non-synonymous SNPs, and ∼1.19 million indels. In this update, by adding high coverage sequencing of additional human individuals, the TIARA genome database now provides an extensive record of rare variants in humans. Following TIARA’s fundamentally integrative approach, new transcriptome sequencing data are matched with whole-genome sequencing data in the genome browser. Users can here observe, for example, the expression levels of human genes with allele-specific quantification. Improvements to the TIARA genome browser include the intuitive display of new complex and large-scale data sets.

Archival Fine-Needle Aspiration Cytopathology (FNAC) Samples: Untapped Resource for Clinical Molecular Profiling

Microarray technologies provide high-resolution maps of chromosome imbalances and epigenomic aberrations in the cancer cell genome. Such assays are often sensitive to sample DNA integrity, voiding the utility of many archival pathology specimens and necessitating the special handling of prospective clinical specimens. We have identified the remarkable preservation of higher-molecular weight DNA in archival fine-needle aspiration cytopathology specimens from patients greater than 10 years of age. We further demonstrate the outstanding technical performance of 57 fine-needle aspiration cytopathology samples for aberration detection on high-resolution comparative genomic hybridization array, DNA methylation, and single nucleotide polymorphism genotyping platforms. Forty-four of 46 malignant aspirates in this study manifested unequivocal genomic aberrations. Importantly, matched Papanicolaou and Diff-Quik fine-needle aspiration cytopathology samples showed critical differences in DNA preservation and DNA integrity. Overall, this study identifies a largely untapped reserve of human pathology specimens for molecular profiling studies, with ramifications for the prospective collection of clinical biospecimens.

Genomic Profiling of Breast Cancer (BC) in Mali by Oligonucleotide Comparative Genomic Hybridization (CGH) Array.

Abstract Background: Epidemiological studies suggest that BC in Africa exhibit specific biological characteristics as compared to BC from US/Europe. In order to identify therapeutic targets that would be specific from Africa, we analyzed 28 samples from Mali by high resolution CGH array, and compared gene gains/losses to the ones observed in USA [André F et al, Clin Cancer Res, 2009]Patients and methods: 71 paraffin-embedded samples of invasive BC from patients treated by surgery at Bamako were retrieved, 28 of them (from 2 pts stage 1; 13 pts stage 2; 11 pts stage 3 and 2 pts stage 4) were eligible for CGH array (1) (Agilent, 4*44K) (cancer cells&amp;gt;50%; undamaged DNA &amp;gt;1µg).Results were compared to 106 samples from MD Anderson, profiled in the sample genomic platform using the same array (1)Results: The median age was 49 year old. Twenty-one samples (75%) were ER-negative. No difference was observed regarding the frequency of gene gains in 17q12-21 (ERBB2) nor 8p11-12 (FGFR1). Six regions were more frequently gained in BC from Mali, as compared to those from US (FDR&amp;lt;5%). Two regions included targetable molecular abnormalities. 9p34 region was gained in 43% of samples and included Abl1, a tyrosine kinase involved in CML, and VAV2, a guanine exchange factor. 22q11-13.1 region was gained in 34% of samples and included bcr, ewsr1, pdgfb and MAF-E. These two regions were gained in less than 5% of BC from US. Finally, a region located in 6p21 was gained in 56% of BC from Mali. This region included a cluster of genes involved in immune response (lymphotoxin A, MICA/MICB).Conclusion: BC from Mali present some candidate specific targetable alterations as gains of Abl1, bcr and pdgf-B. FISH and IHC are being done in order to validate targets.Additional high throughput analyses will be performed to characterize such tumors, including virus arrays, exon arrays and hot spot mutations. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3059.

Targeted comparative genomic hybridization array for the detection of single- and multiexon gene deletions and duplications

PurposeTo develop a high resolution microarray based method to detect single- and multiexons gene deletions and duplications. MethodsWe have developed a high-resolution comparative genomic hybridization array to detect single- and multiexon deletions and duplications in a large set of genes on a single microarray, using the NimbleGen 385K array with an exon-centric design. ResultsWe have successfully developed, validated, and implemented a targeted gene comparative genomic hybridization arrays for detecting single- and multiexon deletions and duplication in autosomal and X-linked disease-associated genes. ConclusionThe comparative genomic hybridization arrays can be adopted readily by clinical molecular diagnostic laboratories as a rapid, cost-effective, highly sensitive, and accurate approach for the detection of single- and multiexon deletions or duplications, particularly in cases where direct sequencing fails to identify a mutation.

DNA Copy Number Variation and Gene Expression Analyses Reveal the Implication of Specific Oncogenes and Genes in GBM

To understand the pathogenesis of glioblastoma multiforme (GBM) we used high-resolution comparative genomic hybridization arrays and gene expression microarrays to identify DNA copy number alterations and gene expression changes in comparable sets of GBM samples. Gains were detected at chromosomes 1, 2, 7, 9, 12, 19, and 20 and losses at 6, 9, and 10. Gene expression analyses identified specific genes overexpressed in GBM mapping at amplified chromosomal regions. Among these genes we found genes involved in angiogenesis, extracellular matrix remodeling and several oncogenes. DNA copy number analysis along with gene expression profiles provides a powerful strategy to understand tumor progression and identification of genes involved in GBM pathogenesis.

Genomic analysis of the 8p11‐12 amplicon in familial breast cancer

Amplification of 8p11-12 has been recurrently reported in sporadic breast cancer. These studies define a complex molecular structure with a set of minimal amplified regions, and different putative oncogenes that show a strong correlation between amplification and over-expression such as ZNF703/FLJ14299, SPFH2/C8orf2, BRF2 and RAB11FIP. However, none of these studies were carried out on familial breast malignancies. We have studied the incidence, molecular features and clinical value of this amplification in familial breast tumors associated with BRCA1, BRCA2 and non-BRCA1/2 gene mutations. We detected 9 out of 80 familial tumors with this amplicon by chromosomal comparative genomic hybridization. Next, we used a high-resolution comparative genomic hybridization array covering the 8p11-12 region to characterize this chromosomal region. This approach allowed us to define 2 cores of common amplification that largely overlap with those reported in sporadic tumors. Our findings confirm the molecular complexity of this chromosomal region and indicate that this genomic event is a common alteration in breast cancer, present not only in sporadic but also in familial tumors. Finally, we found correlation between the 8p11-12 amplification and proliferation (Ki-67) and cyclin E expression, which further proves in familial tumors the poor prognosis association previously reported in sporadic breast cancer.

Array-Based Comparative Genomic Hybridization Analysis Identified Cyclin D1 as a Target Oncogene at 11q13.3 in Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma is highly prevalent in Southern China and Southeast Asia. To unveil the molecular basis of this endemic disease, high-resolution comparative genomic hybridization arrays were used for systematic investigation of genomic abnormalities in 26 nasopharyngeal carcinoma samples. A comprehensive picture of genetic lesions associated with tumorigenesis of nasopharyngeal carcinoma was generated. Consistent chromosomal gains were frequently found on 1q, 3q, 8q, 11q, 12p, and 12q. High incidences of nonrandom losses were identified on chromosomes 3p, 9p, 11q, 14q, and 16q. In addition to previously characterized regions, we have identified several novel minimal regions of gains, including 3q27.3-28, 8q21-24, 11q13.1-13.3, and 12q13, which may harbor candidate nasopharyngeal carcinoma-associated oncogenes. In this study, gain of 11q13.1-13.3 was the most frequently detected chromosomal aberration and a 5.3-Mb amplicon was delineated at this region. Within this 11q13 amplicon, concordant amplification and overexpression of cyclin D1 (CCND1) oncogene was found in nasopharyngeal carcinoma cell lines, xenografts, and primary tumors. Knockdown of cyclin D1 by small interfering RNA in nasopharyngeal carcinoma cell lines led to significant decrease of cell proliferation. The findings suggest that cyclin D1 is a target oncogene at 11q13 in nasopharyngeal carcinoma and its activation plays a significant role in nasopharyngeal carcinoma tumorigenesis.

A 1 Mb minimal amplicon at 8p11–12 in breast cancer identifies new candidate oncogenes

Amplification of 8p11-12 is a well-known alteration in human breast cancers but the driving oncogene has not been identified. We have developed a high-resolution comparative genomic hybridization array covering 8p11-12 and analysed 33 primary breast tumors, 20 primary ovarian tumors and 27 breast cancer cell lines. Expression analysis of the genes in the region was carried out by using real-time quantitative PCR and/or oligo-microarray profiling. In all, 24% (8/33) of the breast tumors, 5% (1/20) of the ovary tumors and 15% (4/27) of the cell lines showed 8p11-12 amplification. We identified a 1 Mb segment of common amplification that excludes previously proposed candidate genes. Some of the amplified genes did not show overexpression, whereas for others, overexpression was not specifically attributable to amplification. The genes FLJ14299, C8orf2, BRF2 and RAB11FIP, map within the 8p11-12 minimal amplicon, two have a putative function consistent with an oncogenic role, these four genes showed a strong correlation between amplification and overexpression and are therefore the best candidate driver oncogenes at 8p12.