Genomic Array CGH Research Articles

Identification of ectodysplasin-A receptor gene deletion at 2q12.2 and a potential autosomal MR locus

Mental retardation (MR) is not a common feature observed in patients with classical ectodermal dysplasias (EDs). Several genes responsible for EDs and MR have been identified. However, the causation has yet to be identified in a significant number of patients with either ED or MR. Here, we have molecularly characterized a de novo balanced translocation t(1;6)(p22.1;p22.1) in a female patient who had mild features of ED with hypodontia, microcephaly, and cognitive impairment. Mapping of the translocation breakpoints in the patient revealed no obvious causative gene for either ED or MR. Whole genome array CGH analysis unveiled two novel submicroscopic deletions at 2q12.2 and 6q22.3, unrelated to the translocation in the patient. The 2q12.2 deletion contains a known ED gene, ectodysplasin-A receptor (EDAR), and the loss of one copy of this gene is considered to be responsible for the ectodermal phenotype in the patient. It is plausible that a potential autosomal MR gene deleted at 2q12.2 or 6q22.3 is likely the cause of the neurodevelopmental defects in the patient.

Philadelphia Negative BCR/ABL1 Positive CML: FISH Mapping and aCGH Reveal Breakpoint Hotspots.

Chronic myeloid leukaemia (CML) is a pluripotent haematopoietic stem cell disorder defined by expression of the BCR/ABL1 fusion gene, a constitutively activated tyrosine kinase. The fusion gene is a result of t(9;22)(q34;q11) or related variant rearrangements leading to formation of the Philadelphia chromosome (Ph). In less than 2% of the CML patients the bone marrow cells appear to have a normal karyogram although the fusion gene can be identified by molecular means and located by FISH on 22q11 and less frequently on 9q34 or on a third chromosome. The biology and clinical significance of the genetic rearrangements in Ph negative BCR/ABL1 positive disease are controversial and widely discussed. Two alternative mechanisms have been postulated but not confirmed: simple insertion or multiple step model. Although sequencing analysis as early as 1990 (Morris and Heisterkamp) showed that a large part of the 9q34 region is present in the masked Ph, little has been done to clarify the underlying rearrangements. Aided by genome array CGH and FISH, we constructed a comprehensive map of the regions flanking ABL1 and BCR genes in 9 patients and one cell line with this form of CML. The main results show:1.the formation of the BCR/ABL1 fusion is associated with relocation of sequences well in excess of the ABL1 and BCR gene in all 10 samples,2.the involvement of downstream 3′ ABL1 is diversely associated with distal breakpoint clusters in the regions of the RAP-GEF1 and RXRA genes,3.the disease progression can be accompanied by sequential breakages/reunions within the regions flanking the BCR/ABL1 fusion and4.the presence of multiple cryptic rearrangements of both 9q34 and 22q11 segments identifies a sub-group of patients with shorter survival thus demonstrating the clinical significance of genome instability in Ph negative, BCR/ABL1 positive CML.

Complex balanced translocation t(1;5;7)(p32.1;q14.3;p21.3) and two microdeletions del(1)(p31.1p31.1) and del(7)(p14.1p14.1) in a patient with features of Greig cephalopolysyndactyly and mental retardation

Complex chromosome rearrangements (CCRs) are rare structural abnormalities that involve at least two chromosomes and more than two breakpoints and are often associated with developmental delay, mental retardation, and congenital anomalies. We report on a de novo, apparently balanced translocation t(1;5;7)(p32.1;q14.3;p21.3) involving three chromosomes in a 7-year-old boy with severe psychomotor retardation, neonatal muscular hypertonia, congenital heart defect, polysyndactyly of hands and feet, and dysmorphic features resembling Greig cephalopolysyndactyly syndrome. Analysis of the chromosome breakpoints using fluorescence in situ hybridization (FISH) with locus-specific BAC clones and long-range PCR products did not identify chromosome imbalance at any of the interrogated regions. High-resolution comparative genomic hybridization (HR-CGH) and array CGH (aCGH) revealed two additional cryptic de novo deletions, del(1)(p31.1p31.1) and del(7)(p14.1p14.1), respectively, that are not associated with the translocation breakpoints. FISH and polymorphic marker analyses showed that the deletion on derivative chromosome 1 is between 4.2 and 6.1 Mb, and the deletion on derivative chromosome 7 is approximately 5.1 Mb, and that both are paternal in origin. The deletion on chromosome 7p encompasses the GLI3 gene that is causative for the Greig cephalopolysyndactyly, Pallister-Hall and some cases of Acrocallosal syndromes. We discuss the potential mechanisms of formation of the described CCR.

A6-05: Clinicopathologic Implications of minimal genomic alteration regions (MAR) identified in non-small cell lung cancer by using whole genome array-CGH

A6-05: Clinicopathologic Implications of minimal genomic alteration regions (MAR) identified in non-small cell lung cancer by using whole genome array-CGH

Identification and characterization of DNA imbalances in neuroblastoma by high-resolution oligonucleotide array comparative genomic hybridization

Neuroblastoma (NB) is a pediatric tumor characterized by high genetic heterogeneity. Although the prognostic significance of some genomic abnormalities (i.e., MYCN amplification, 1p loss, and 17q gain) is recognized, genes that are involved in chromosome rearrangements remain largely unknown. Considerable progress has been made over the last years in characterizing DNA abnormalities by metaphase comparative genomic hybridization (mCGH) and array CGH (aCGH). Here we report a pilot study of 5 localized and 12 disseminated NB by 44,000 and of 4 out of 17 cases by 244,000 oligonucleotide aCGH. Localized tumors were predominantly characterized by losses of whole chromosomes 3, 4, 10, and 16, and gains of 6, 7, 8, 13, 17, 18, and 20, whereas disseminated tumors showed several structural aberrations including 17q gain and 3p and 11q losses. Characterization of chromosome 2p in MYCN-amplified NB revealed several structural rearrangements with regions of gain interspersed among sites of amplification, indicating that the MYCN amplicon may encompass several genes. The high-resolution zooming in chromosomal aberrant regions detected several micro-deletions and micro-amplifications in the NB genome. Our results indicate that the increased sensitivity of the aCGH also allows the identification of DNA aberrations in challenging samples (i.e., NB showing tissue and genetic heterogeneity).

A girl with deletion 9q22.1–q22.32 including thePTCHandROR2genes identified by genome‐wide array‐CGH

The underlying genetic cause of mental retardation (MR) remains unknown in about half of the cases. Recently, using whole genome array comparative genomic hybridization (array-CGH), submicroscopic genetic imbalances have been detected in up to 20% of patients with an unexplained MR, dysmorphic features, and apparently normal karyotype. Here, we present a 12-year-old girl with features of basal cell nevus syndrome (BCNS), pulmonary valve stenosis, and MR, in whom array-CGH identified a 7.7 Mb deletion on 9q22.1-q22.32. The deleted region includes, among others, the ROR2 and PTCH genes. Haploinsufficiency of PTCH causes the BCNS syndrome and mutations in ROR2 have been found in an autosomal recessive Robinow syndrome and a dominantly inherited brachydactyly type 1B. We speculate that haploinsufficiency of ROR2 may contribute to pulmonary valve stenosis. Because of an age-dependent penetrance, BCNS may be challenging for diagnosis particularly when the features are not part of a typical clinical spectrum of BCNS. Early diagnosis of BCNS is important for preventing the development of associated tumors and better care of the patient. Our data confirm the previous observations that application of the whole genome array-CGH should be considered in selected patients with undiagnosed MR and dysmorphic features.

Molecular Characterization of Genomic Structural Alterations through CGH Arrays in Acute Myeloid Leukemia with Normal Cytogenetic Findings.

Acute myeloid leukemia (AML) is an heterogeneous disease. Cytogenetic findings are one of the main prognosis criteria beside age, leucocytosis, and antecedent of previous hemopathy. More than 50% of AML patients, mainly with normal cytogenetic, belong to the intermediate cytogenetic risk group. Even in this group, cryptic molecular abnormalities as mutation are frequent with prognosis value. In this work we have studied 31 AML patients (Pts) with normal caryotype by high resolution comparative genomic hybridization array CGH using agilent 185k chips to detect gene copy number alterations (CNA). Tumor DNA was obtained from bone marrow diagnosis samples (>60% of blasts) and control DNA from pooled blood samples obtained from 30 healthy donors or from AML patients in CR during the follow up. The design of the study was first to hybridize tumor DNA from the 31 patients against pooled control DNA to detect both copy number polymorphisms (CNP) and acquired CNA in AML and in a second time to hybridize tumor DNA from the 10 pts with the greater number of abnormalities against autologous DNA obtained at CR time to distinguish acquired CNA from CNP. Briefly DNA labelling was performed using Cy3dUTP and Cy5 dUTP respectively for control and tumor samples. Fluorescence ratios were normalized and spots with value that deviated significantly from background were retained as significant evidence of DNA copy number alterations after statistical analysis using Feature Extraction and CGH Analytics AGILENT software. BAC from the Human Genome Center (Wellcome Trust Sanger) were used to obtain FISH probes to confirm abnormalities observed by CGH arrays on metaphase spreads obtained from bone marrow diagnosis sample when available. In each case, two colour FISH was performed by the simultaneous hybridization on a green BAC probe located in the CNA regions and a red control BAC probe located in the vicinity of the CNA regions obtained from the UCSC genome browser data.Results98 DNA copy number alterations were observed with 56 losses and 42 gains cases. Those CNA were variable in size from 8,6 Mb to <100 Kb. 8 CNA regions were recurrent (5 losses in 7q31 (12pts), 4q13 (3pts), 1q31 (4pts), 2q37 (2pts) et 22q13 (3pts) and 3 gains in 1q23 (3pts), 2p11(2pts) et 17q21(2pts). Non recurrent CNA were located in 1p36.2, 1p34.3, 1p21.1, 1p22, 1q31.2, 3q12.2, 4p13, 5p15.3, 5q14.1, 6p22.1, 7p22.1, 7q11.23, 10q26.3, 11q13.2, 15q11.2, 15q15.3, 15q25.3, 15q26.1, 16q24.2, 17p13.2, 17p11.2, 22q13,33 for gains and in 1p33, 1q32.1, 1q32.2, 3p14.1, 3p13-p12.3, 4q26, 7p11.2, 7p14.3, 7q35, 8p23.2, 8q23.1, 9p24.1, 9p13.1-p12, 9q34.11-q34.12, 12p13.31, 13q14.2, 14q21.2, 14q22.1, 14q23.2, 15q22.31, 16q22.1, 21q21.2, 21q22.12 for losses. No one of the patients had a genome without CNA and 6 patients (20%) had more than 5 CNAs. The CGH data showed clearly that 80% of the CNA observed with pool control DNA were not present when tumor DNA was hybridized against the autologous CR DNA confirming the high frequency of CNP as reported on http://projects.tcag.ca/variation website. All of the acquired CNA regions observed were concordant with the FISH results involving genes known to be crucial in leukemia as RUNX1, Abl, CEBPa but also many other genes. In conclusion cryptic abnormalities are frequent in normal caryotype AML and 20% of the patients studied had more than 5 CNA traducing a great instability of the genome of leukemic cells.

Chromosomal Aberration Patterns of Aggressive B-Cell Lymphomas Closely Correlate with Molecularly Defined Disease Subtypes and Regional mRNA Expression. An Analysis of 213 Tumor Samples within the German MMML Network.

Recently, it has been demonstrated that aggressive B-cell non-Hodgkin lymphomas (B-NHL) split up into distinct subgroups such as molecular Burkitt (mBL) and molecular non-Burkitt lymphomas (non-mBL). The non-mBL group can further be devided in activated B-cell (ABC) and germinal center B-cell (GCB) like lymphomas. In the present investigation we aimed at unravelling correlations of single or multiple chromosomal imbalances, their respective regional mRNA gene expression levels and the above mentioned molecular subgroups. Aggressive B-NHL samples (n=255) were investigated within the German network project Molecular Mechanisms in Malignant Lymphomas (MMML). Data from genomic arrayCGH analysis were evaluable for 213 lymphoma cases. Consensus review diagnoses according to WHO criteria were: DLBCL (n=154), atypical BL (n=20), typical BL (n=15) and other aggressive B-NHL (n=24). For all cases gene expression analysis was done using the Affymetrix U133A GeneChip. FISH data were available for translocations involving the MYC locus and for t(14;18). Using arrayCGH, chromosomal gains in more than 10% of all cases have been identified on 18q(24.4%), 3q(19.8%), 1q(19.1%), 12q(16.7%) 2p(16.3 %), 7p and 7q(16.3%), 6p(13.2%) and 11q(12.5%). Deletions occurred most frequently on chromosome arm 6q(20.3%), 17p(15.4%), 9p(14.8%) and 8p(10%). Molecular BLs had significantly less chromosomal aberrations than non-mBLs but frequently show gains of chromosome arm 1q. Non-mBLs show higher genomic complexity and strikingly differing aberration patterns for GCB (more than 20% gains on 1q, 2p, 7q, 11q, 12q) and ABC (more than 20% gains on 3q, 9p, 18q, loss on 6q) type lymphomas. These findings were also supported by results of genomic clustering algorithms such as non negative matrix factorization. For a large number of recurrent chromosomal aberrations we were able to delineate minimal aberrant chromosomal regions and to correlate respective regional mRNA gene expression levels. Strong proportional correlations were found for gains of 18q21.1-q21.33 and losses of 6q21-q24.3. In conclusion, genomic aberration patterns closely correlate with distinct molecular subtypes of aggressive B-NHL. These aberration patterns might serve as surrogate markers providing diagnostic and prognostic information as well as a basis for targeted gene investigations and risk adapted therapy studies.

Recent advances in array comparative genomic hybridization technologies and their applications in human genetics

Array comparative genomic hybridization (array CGH) is a method used to detect segmental DNA copy number alterations. Recently, advances in this technology have enabled high-resolution examination for identifying genetic alterations and copy number variations on a genome-wide scale. This review describes the current genomic array platforms and CGH methodologies, highlights their applications for studying cancer genetics, constitutional disease and human variation, and discusses visualization and analytical software programs for computational interpretation of array CGH data.

A variant Cri du Chat phenotype and autism spectrum disorder in a subject withde novocryptic microdeletions involving 5p15.2 and 3p24.3‐25 detected using whole genomic array CGH

Cri du Chat syndrome (CdCs) is a well-defined clinical entity, with an incidence of 1/15,000 to 1/50,000. The critical region for CdCs has been mapped to 5p15, with the hallmark cat-like cry sublocalized to 5p15.3 and the remaining clinical features to 5p15.2. We report findings in a subject with a de novo t(5;7)(p15.2;p12.2) and an inv(3)(p24q24), who was found to have a cryptic microdeletion in the critical region for CdCs detected using a 1-Mb genomic microarray. In addition to 5p deletion, the proband had a de novo single clone loss at the 3p breakpoint of inv(3)(p24q24) and a familial single clone deletion at 18q12. Deletions were confirmed using microsatellite analysis and fluorescence in situ hybridization. The 5p deletion encompasses approximately 3 Mb, mapping to the border between bands 5p15.2 and 5p15.31. The single clone deletion on chromosome 3 maps to 3p24.3-3p25, for which there is no known phenotype. The clinical features of our proband differ from the characteristic CdC phenotype, which may reflect the combined effect of the two de novo microdeletions and/or may further refine the critical region for CdCs. Typical features of CdCs that are present in the proband include moderate intellectual disability, speech, and motor delay as well as dysmorphic features (e.g. broad and high nasal root, hypertelorism, and coarse facies). Expected CdCs features that are not present are growth delay, microcephaly, round facies, micrognathia, epicanthal folds, and the signature high-pitched cry. Behavioral traits in this subject included autism spectrum disorder, attention-deficit hyperactivity disorder, and unmanageable behavior including aggression, tantrums, irritability, and self-destructive behavior. Several of these behaviors have been previously reported in patients with 5p deletion syndrome. Although most agree on the cat-cry critical region (5p15.3), there is discrepancy in the precise location and size of the region associated with the more severe manifestations of CdCs. The clinical description of this proband and the characterization of his 5p deletion may help to further refine the phenotype-genotype associations in CdCs and autism spectrum disorder.

Combined Array Comparative Genomic Hybridization and Tissue Microarray Analysis Suggest PAK1 at 11q13.5-q14 as a Critical Oncogene Target in Ovarian Carcinoma

Amplification of chromosomal regions leads to an increase of DNA copy numbers and expression of oncogenes in many human tumors. The identification of tumor-specific oncogene targets has potential diagnostic and therapeutic implications. To identify distinct spectra of oncogenic alterations in ovarian carcinoma, metaphase comparative genomic hybridization (mCGH), array CGH (aCGH), and ovarian tumor tissue microarrays were used in this study. Twenty-six primary ovarian carcinomas and three ovarian carcinoma cell lines were analyzed by mCGH. Frequent chromosomal overrepresentation was observed on 2q (31%), 3q (38%), 5p (38%), 8q (52%), 11q (21%), 12p (21%), 17q (21%), and 20q (52%). The role of oncogenes residing in gained chromosomal loci was determined by aCGH with 59 genetic loci commonly amplified in human tumors. DNA copy number gains were most frequently observed for PIK3CA on 3q (66%), PAK1 on 11q (59%), KRAS2 on 12p (55%), and STK15 on 20q (55%). The 11q13-q14 amplicon, represented by six oncogenes (CCND1, FGF4, FGF3, EMS1, GARP, and PAK1) revealed preferential gene copy number gains of PAK1, which is located at 11q13.5-q14. Amplification and protein expression status of both PAK1 and CCND1 were further examined by fluorescence in situ hybridization and immunohistochemistry using a tissue microarray consisting of 268 primary ovarian tumors. PAK1 copy number gains were observed in 30% of the ovarian carcinomas and PAK1 protein was expressed in 85% of the tumors. PAK1 gains were associated with high grade (P < 0.05). In contrast, CCND1 gene alterations and protein expression were less frequent (10.6% and 25%, respectively), suggesting that the critical oncogene target of amplicon 11q13-14 lies distal to CCND1. This study demonstrates that aCGH facilitates further characterization of oncogene candidates residing in amplicons defined by mCGH.