Array-based Comparative Genomic Hybridization Analysis Research Articles

Genetic Alteration of Keap1 Confers Constitutive Nrf2 Activation and Resistance to Chemotherapy in Gallbladder Cancer

Biliary tract cancer (BTC) is a highly malignant tumor, and identification of effective therapeutic targets to improve prognosis is urgently required. Oncogenic activation of survival genes is important for cancer cells to overcome oxidative stresses induced by their microenvironments that include chronic inflammation or exposure to anticancer drugs. We attempted to examine whether deregulation of Nrf2, a master transcriptional factor of various cytoprotective genes against oxidative stress, plays a role in the carcinogenesis of BTC. We screened genetic alteration of Keap1, a negative regulator of Nrf2, in BTC including tumors originated from gallbladder and extra- and intrahepatic bile ducts. Functional analysis of cancer-related mutant Keap1 in Nrf2 repression and the association between Nrf2 activation and resistance to 5-fluorouracil (5-FU) were investigated. Recurrent (in 1/11 cell lines and 6/53 primary tumors) Keap1 gene alterations were observed in BTC and were especially frequent (4/13, 30.7%) in gallbladder cancer (GBC). These alterations led to a considerable loss of Nrf2 repression activity, caused constitutive activation of Nrf2, and promoted cell proliferation. Down-regulation of Nrf2 activity by either Keap1 complementation or Nrf2 short interference RNA increased sensitivity to 5-FU in Keap1-altered BTC cells. Keap1 mutation occurs frequently in GBC. Aberrant Nrf2 activation provoked by Keap1 alteration is one of the molecular mechanisms for chemotherapeutic resistance in GBC and will be a novel therapeutic target as an enhancer of sensitivity to 5-FU-based regimens.

Total deletion and a missense mutation of ITPR1 in Japanese SCA15 families

Spinocerebellar ataxia type 15 (SCA15) is a progressive neurodegenerative disorder characterized by pure cerebellar ataxia, very slow progression, and distinct cerebellar atrophy. The locus for SCA15 was first mapped to 3p24.2-3pter in an Australian family. We have subsequently mapped two Japanese families presenting with ataxia and postural tremor of the head, arm, or trunk to the SCA15 locus. Recently, partial deletions involving both the type 1 inositol 1,4,5-triphosphate receptor (ITPR1) and sulfatase modifying factor 1 (SUMF1) genes have been identified in Australian and British families with SCA15. We conducted fine haplotype analysis on the region including ITPR1. To identify the deletion, we conducted gene dosage analysis and array-based comparative genomic hybridization (aCGH) analysis. Gene expression analysis was performed using quantitative real-time reverse transcription PCR. Mutational analyses of ITPR1 and SUMF1 were also performed. We have identified a 414-kb deletion including the entire ITPR1 and exon 1 of SUMF1 in patients in family A. The expression levels of ITPR1 and SUMF1 mRNAs of the patient were half those of the normal control. Furthermore, in family B, we have identified a C-to-T substitution at position 8581 of ITPR1, resulting in the amino acid substitution of leucine for proline at codon 1059, which is highly conserved among species. Our results strongly confirm that ITPR1 is the causative gene for SCA15 and suggest that we need to investigate the point mutation in ITPR1 in the patients with autosomal dominant cerebellar ataxia and tremor.

Array-based Comparative Genomic Hybridization (aCGH) Analysis is a Useful Tool for Distinguishing Primary Pulmonary From Metastatic Neuroendocrine Carcinoma to the Lung

Distinguishing primary from metastatic disease is vital for patient prognosis and care. This becomes a difficult task for both clinicians and pathologists when the clinical data, morphology, and immunohistochemistry (IHC) are not congruent. Array-based comparative genomic hybridization (aCGH) is a fairly recent technique that enables the analysis of whole tumor genome for discreet genetic abnormalities. As it is applicable to paraffin-embedded tissue, it could be suited for surgical pathology settings. We report a case of a 71-year-old woman diagnosed with uterine carcinosarcoma associated with high-grade neuroendocrine carcinoma (NEC). Six months later, the patient presented with pulmonary nodules. A wedge resection of one the nodules was performed and the histology and IHC were consistent with NEC. However, the diagnostic dilemma was whether the pulmonary lesions were primary of the lung or metastatic from the NEC component of the endometrium. Clinical, morphologic, and IHC analysis were not helpful to answer the above question. However, by revealing different genetic profiles of each of the 2 tumors, aCGH was suggestive of the existence of 2 independent primary tumors one endometrial and second pulmonary. In conclusion, aCGH is a powerful tool that is capable of revealing tumor gene fingerprinting and it can be used in routine surgical pathology especially in those cases where the other conventional techniques are not helpful.

Low‐level mosaicism of trisomy 14: Phenotypic and molecular characterization

Trisomy 14 mosaicism is a rare cytogenetic abnormality with a defined and recognizable clinical phenotype. We present a detailed clinical history and physical findings of five patients with low-level mosaicism of trisomy 14 detected by array-based comparative genomic hybridization (array-CGH) analysis or by routine chromosome analysis. These patients exhibited growth and developmental delays with variable severity, congenital anomalies, pigmentary skin lesions, and dysmorphic features. The phenotype of our patients was compared with previously described cases. This report suggests that trisomy 14 mosaicism may be more common than has been previously appreciated and also illustrates the important application of array-CGH to detect low-level mosaic chromosome abnormalities. We predict that a wider application of the array-CGH technology will significantly increase the detection rate of low-level mosaicism and will subsequently improve our ability to provide a diagnosis for patients with dysmorphic features, congenital anomalies, and developmental delay.

Establishment, characterization and drug sensitivity testing in primary cultures of human thymoma and thymic carcinoma

Thymomas and thymic carcinomas are peculiar epithelial tumors of the anterior mediastinum. They may show aggressive clinical behavior and are a paradigm for the interaction between the tumor and the immune system. So far, adequate functional studies enabling a better understanding of this malignancy have not been performed, since human thymoma/thymic carcinoma cell lines have not been available. Here, the authors describe the establishment, characterization and functional analyses of epithelial cell lines from a Type B1-thymoma and a poorly differentiated thymic carcinoma. By Fluorescence-activated cell sorting (FACS) analyses, both cell lines were aneuploid. The aneuploid cell fraction of the thymic carcinoma cell line was characterized by a high proliferation index of 55.9%, in contrast to a lower proliferation rate of the aneuploid cell fraction of the thymoma (19.7%). Array-based comparative genomic hybridization (aCGH) and conventional cytogenetic analysis of the thymoma revealed only minor imbalances whereas the thymic carcinoma was characterized by a complex karyotype in the hyperdiploid range that was readily defined with multicolor FISH (mFISH). Application of a selective COX-2 inhibitor reduced cell viability in both cell lines in a dose-dependent manner. In conclusion, these first cell lines of a thymoma and a CD5-positive thymic carcinoma are useful tools for further in vitro studies of cellular, molecular and genetic aspects of the disease and for functional tests to evaluate new therapeutic targets.

Identification of SMURF1 as a possible target for 7q21.3‐22.1 amplification detected in a pancreatic cancer cell line by in‐house array‐based comparative genomic hybridization

Pancreatic cancer (PC) cell lines provide a useful starting point for the discovery and functional analysis of genes driving the genesis and progression of this lethal cancer. To increase our understanding of the gene copy number changes in pancreatic carcinomas and to identify key amplification and deletion targets, we applied genome-wide array-based comparative genomic hybridization using in-house array (MCG Cancer Array-800) to 24 PC cell lines. Overall, the analyses revealed high genomic complexity, with several copy number changes detected in each line. Homozygous deletions (log(2)ratio < -2) of eight genes (clones) were seen in 14 of the 24 cell lines, whereas high-level amplifications (log(2)ratio > 2) of 10 genes (clones) were detected in seven lines. Among them, we focused on high-level amplification at 7q22.1, because target genes for this alteration remain unknown. Through precise mapping of the altered region by fluorescence in situ hybridization, determination of the expression status of genes located within those regions, and functional analysis using knockdown of the gene expression or the ectopic overexpression approach in PC cell lines, as well as immunohistochemical analyses of candidates in primary tumors of PC, we successfully identified SMURF1 as having the greatest potential as a 7q21.3-22.1 amplification target. SMURF1 may work as a growth-promoting gene in PC through overexpression and might be a good candidate as a therapeutic target. Our results suggest that array-based comparative genomic hybridization analysis combined with further genetic and functional examinations is a useful approach for identifying novel tumor-associated genes involved in the pathogenesis of this lethal disease.

Array-based comparative genomic hybridization analysis of 1176 consecutive clinical genetics investigations

PurposeCytogenetic investigations are useful for etiologic determinations of mental retardation, developmental delay, multiple congenital anomalies, and pregnancy complications; however, the causes remain elusive in a majority of cases despite high-resolution cytogenetic studies and multiple fluorescence in situ hybridization examinations. Array-based comparative genomic hybridization has the ability to examine the genome at a higher resolution and may yield an increased detection of genetic abnormalities. The purpose of this study was to assess the use of array-based comparative genomic hybridization in a clinical genetics setting. MethodsDNA from 1176 patients was analyzed using a bacterial artificial chromosome array-based comparative genomic hybridization platform. All abnormal cases were confirmed by fluorescence in situ hybridization and parental studies were completed when possible. ResultsOf the 1176 patients included in this survey, 163 showed a genomic imbalance identified by array-based comparative genomic hybridization. Of these 163 cases, 116 had a clinically relevant genetic abnormality. A total of 9.8% (116 of 1176 cases) were determined to exhibit a causative genomic imbalance. Twenty-five of the 116 abnormal cases had a previously identified cytogenetic abnormality yielding an increased detection rate of 7.9% (91 of 1146) in cases with normal or no cytogenetics. ConclusionArray-based comparative genomic hybridization increases the overall abnormality detection rate, thus improving the diagnostic potential of clinical cytogenetics investigations.

22q11.2 Distal Deletion: A Recurrent Genomic Disorder Distinct from DiGeorge Syndrome and Velocardiofacial Syndrome

Microdeletions within chromosome 22q11.2 cause a variable phenotype, including DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS). About 97% of patients with DGS/VCFS have either a common recurrent approximately 3 Mb deletion or a smaller, less common, approximately 1.5 Mb nested deletion. Both deletions apparently occur as a result of homologous recombination between nonallelic flanking low-copy repeat (LCR) sequences located in 22q11.2. Interestingly, although eight different LCRs are located in proximal 22q, only a few cases of atypical deletions utilizing alternative LCRs have been described. Using array-based comparative genomic hybridization (CGH) analysis, we have detected six unrelated cases of deletions that are within 22q11.2 and are located distal to the approximately 3 Mb common deletion region. Further analyses revealed that the rearrangements had clustered breakpoints and either a approximately 1.4 Mb or approximately 2.1 Mb recurrent deletion flanked proximally by LCR22-4 and distally by either LCR22-5 or LCR22-6, respectively. Parental fluorescence in situ hybridization (FISH) analyses revealed that none of the available parents (11 out of 12 were available) had the deletion, indicating de novo events. All patients presented with characteristic facial dysmorphic features. A history of prematurity, prenatal and postnatal growth delay, developmental delay, and mild skeletal abnormalities was prevalent among the patients. Two patients were found to have a cardiovascular malformation, one had truncus arteriosus, and another had a bicuspid aortic valve. A single patient had a cleft palate. We conclude that distal deletions of chromosome 22q11.2 between LCR22-4 and LCR22-6, although they share some characteristic features with DGS/VCFS, represent a novel genomic disorder distinct genomically and clinically from the well-known DGS/VCF deletion syndromes.

Periventricular heterotopia in a boy with interstitial deletion of chromosome 4p

We report on a 4-year-old boy with a proximal interstitial deletion in the short arm of chromosome 4p with the karyotype 46,XY,del(4)(p14p15.32),inv(9)(p13q13). For a precise delineation of the deleted region, an array-based comparative genomic hybridization (a-CGH) analysis was performed. The proband's phenotype and cytogenetic findings are compared with previously reported cases with proximal 4p deletion syndrome. The syndrome is associated with normal growth, varying degrees of mental retardation, characteristic facial appearance and minor dysmorphic features. Additionally, our patient developed a seizure disorder due to abnormal neuronal migration, i.e., periventricular heterotopia.

Chronic Oxidative Stress Causes Amplification and Overexpression of ptprz1 Protein Tyrosine Phosphatase to Activate β-Catenin Pathway

Ferric nitrilotriacetate induces oxidative renal tubular damage via Fenton-reaction, which subsequently leads to renal cell carcinoma (RCC) in rodents. Here, we used gene expression microarray and array-based comparative genomic hybridization analyses to find target oncogenes in this model. At the common chromosomal region of amplification (4q22) in rat RCCs, we found ptprz1, a tyrosine phosphatase (also known as protein tyrosine phosphatase zeta or receptor tyrosine phosphatase beta) highly expressed in the RCCs. Analyses revealed genomic amplification up to eightfold. Despite scarcity in the control kidney, the amounts of PTPRZ1 were increased in the kidney after 3 weeks of oxidative stress, and mRNA levels were increased 16 approximately 552-fold in the RCCs. Network analysis of the expression revealed the involvement of the beta-catenin pathway in the RCCs. In the RCCs, dephosphorylated beta-catenin was translocated to nuclei, resulting in the expression of its target genes cyclin D1, c-myc, c-jun, fra-1, and CD44. Furthermore, knockdown of ptprz1 with small interfering RNA (siRNA), in FRCC-001 and FRCC-562 cell lines established from the induced RCCs, decreased the amounts of nuclear beta-catenin and suppressed cellular proliferation concomitant with a decrease in the expression of target genes. These results demonstrate that chronic oxidative stress can induce genomic amplification of ptprz1, activating beta-catenin pathways without the involvement of Wnt signaling for carcinogenesis. Thus, iron-mediated persistent oxidative stress confers an environment for gene amplification.

Genetically Distinct and Clinically Relevant Classification of Hepatocellular Carcinoma: Putative Therapeutic Targets

The biological aggressiveness of hepatocellular carcinoma (HCC) and the lack of optimal therapeutic strategies have rendered the disease a major challenge. Highly heterogeneous genetic alteration profiles of HCC have made it difficult to identify effective tailor-made molecular therapeutic targets. Therefore, classification of HCC into genetically homogeneous subclasses would be of great worth to develop novel therapeutic strategies. We clarified genome-scale chromosomal copy number alteration profiles and mutational statuses of p53 and beta-catenin in 87 HCC tumors. We investigated the possibility that HCC might be classifiable into a number of homogeneous subclasses based solely on their genetic alteration profiles. We also explored putative molecular therapeutic targets specific for each HCC subgroup. Unsupervised hierarchical cluster analysis based on chromosomal alteration profiles suggested that HCCs with heterogeneous genetic backgrounds are divisible into homogeneous subclasses that are highly associated with a range of clinicopathologic features of the tumors and moreover with clinical outcomes of the patients (P < .05). These genetically homogeneous subclasses could be characterized distinctively by pathognomonic chromosomal amplifications (eg, c-Myc-induced HCC, 6p/1q-amplified HCC, and 17q-amplified HCC). An in vitro experiment raised a possibility that Rapamycin would significantly inhibit the proliferative activities of HCCs with 17q amplification. HCC is composed of several genetically homogeneous subclasses, each of which harbors characteristic genetic alterations that can be putative tailor-made molecular therapeutic targets for HCCs with specific genetic backgrounds. Our results offer an opportunity for developing novel individualized therapeutic modalities for distinctive genome types of HCC.

Amplicon Mapping and Expression Profiling Identify the Fas-Associated Death Domain Gene as a New Driver in the 11q13.3 Amplicon in Laryngeal/Pharyngeal Cancer

Amplification of the 11q13 region is a frequent event in human cancer. The highest incidence (36%) is found in head and neck squamous cell carcinomas. Recently, we reported that the amplicon size in 30 laryngeal and pharyngeal carcinomas with 11q13 amplification is determined by unique genomic structures, resulting in the amplification of a set of genes rather than a single gene. To investigate which gene(s) drive the 11q13 amplicon, we determined the smallest region of overlap with amplification and the expression levels of all genes within this amplicon. Using array-based comparative genomic hybridization analysis, we detected a region of approximately 1.7 Mb containing 13 amplified genes in more than 25 of the 29 carcinomas. Quantitative reverse transcription-PCR revealed that overexpression of 8 potential driver genes including, cyclin D1, cortactin, and Fas-associated death domain (FADD), correlated significantly with DNA amplification. FADD protein levels correlated well with DNA amplification, implicating that FADD is also a candidate driver gene in the 11q13 amplicon. Analysis of 167 laryngeal carcinomas showed that increased expression of FADD (P = 0.007) and Ser(194) phosphorylated FADD (P = 0.011) were associated with a worse disease-specific survival. FADD was recently reported to be involved in cell cycle regulation, and cancer cells expressing high levels of the Ser(194) phosphorylated isoform of FADD proved to be more sensitive to Taxol-induced cell cycle arrest. Because of the frequent amplification of the 11q13 region and concomitant overexpression of FADD in head and neck squamous cell carcinomas, we hypothesize that FADD is a marker to select patients that might benefit from Taxol-based chemoradiotherapy.

Exclusion of APC and VHL gene deletions by array-based comparative hybridization in two patients with microscopically visible chromosomal aberrations

Karyotyping is a major component of the genetic work-up of patients with dysmorphism. Cytogenetic aberrations close to a known tumor suppressor gene raise important clinical issues because deletion of that tumor suppressor gene can cause genetic predisposition to cancer. We present two cancer-free dysmorphic patients with karyotypes of 46,XX,del(5)(q15q22.3) and 46,XX,del(3)(p25.2∼pter). These deletions are close to the APC and VHL genes that confer susceptibility to familial Adenomatous polyposis (OMIM #17510) and von-Hippel-Lindau syndrome (OMIM #193300), respectively. The array-based comparative genomic hybridization (array-CGH) analysis using a custom Agilent 44K oligonucleotide array demonstrated an interstitial 20.7-megabase (Mb) deletion on 5q (chr5: 89,725,638-110,491,345) and a terminal 9.45-Mb deletion on 3p (chr3:pter-9,450,984). According to the March 2006 human reference sequence, the APC gene is located at chr5: 112,101,483-112,209,835 and the VHL gene is located at chr3: 10,158,319-10,168,746. These results indicate that the APC gene is 2,300 kilobases (kb) and the VHL gene is 700 kb away from deleted regions. Southern blot analysis for APC and VHL genes were negative, consistent with array-CGH findings. These results demonstrate the power of array-CCH to assess potential tumor suppressor gene involvement and cancer risk in patients with microscopically visible deletions in areas near tumor suppressors.

A de novo 1.1–1.6 Mb subtelomeric deletion of chromosome 20q13.33 in a patient with learning difficulties but without obvious dysmorphic features

We report on a de novo submicroscopic deletion of 20q13.33 identified by subtelomeric fluorescence in situ hybridization (FISH) in a 4-year-old girl with learning difficulties, hyperlaxity and strabismus, but without obvious dysmorphic features. Further investigations by array-based comparative genomic hybridization (array-CGH) and FISH analysis allowed us to delineate the smallest reported subterminal deletion of chromosome 20q, spanning a 1.1-1.6 Mb with a breakpoint localized between BAC RP5-887L7 and RP11-261N11. The genes CHRNA4 and KCNQ2 implicated in autosomal dominant epilepsy are included in the deletion interval. Subterminal 20q deletions as found in the present patient have, to our knowledge, only been reported in three patients. We review the clinical and behavioral phenotype of such "pure" subterminal 20q deletions.

Genetic analysis of multifocal superficial urothelial cancers by array-based comparative genomic hybridisation

The purpose of this study was to investigate the accumulation of genetic alterations during metachronous and/or synchronous development of multifocal low-grade superficial urothelial tumours in the same patient, by using array-based comparative genomic hybridisation (array-CGH) and FGFR mutation analysis. We analysed 24 tumours (pTa-1 G1-2) from five patients. We had previously identified a clonal relationship among the tumours of each patient by microsatellite analysis. This time, unsupervised hierarchical cluster analysis revealed that the tumours from each patient were clustered together independently of the tumours from the other patients. All of the tumours from a single patient showed a set of 2–7 identical regional or whole-arm chromosomal changes. In addition, several individual alterations were also found. Cladistic diagrams revealed that the accumulation of genetic alterations could not be explained by a linear model, and the existence of a hypothetical precursor cell was assumed in four patients. In some cases, FGFR mutation seemed to occur later during multifocal tumour development. Taken together, these findings suggest that low-grade superficial urothelial tumours accumulate minor genetic alterations during multifocal development, although these tumours are genetically stable.

Partial tandem duplication of GRIA3 in a male with mental retardation

The genetic factors underlying mental retardation (MR) are very heterogeneous. Recent studies have identified a number of genes involved in MR, several of which lie on the X-chromosome, but the current understanding of the monogenic causes of MR is far from complete. Investigation of chromosomal rearrangements in patients with MR has proven particularly informative in the search for novel genes. Using array-based comparative genomic hybridization analysis, we identified a small copy number gain at Xq25, which was undetectable by conventional G-band analysis, in a boy with unexplained MR. Further characterization revealed a partial tandem duplication of GRIA3, an alteration also present on one allele in his mother. RT-PCR analysis of lymphoblastoid cell RNA revealed remarkably reduced GRIA3 transcript levels in the patient. The mother, whose cognitive level is normal, also demonstrated remarkably reduced GRIA3 transcript levels in lymphoblastoid cells, and X-chromosome inactivation (XCI) was completely skewed in her peripheral lymphocytes. It is possible that XCI in the brain is not completely skewed and that GRIA3 expression from the normal allele may account for the mother's normal cognitive function. Taken together with previous findings of GRIA3 disruptions in the patients with MR, our study strengthens the idea that GRIA3 is a candidate gene for X-linked MR and that severely reduced GRIA3 expression results in MR.

Epigenetic identification of ADAMTS18 as a novel 16q23.1 tumor suppressor frequently silenced in esophageal, nasopharyngeal and multiple other carcinomas

Tumor suppressor genes (TSGs) often locate at chromosomal regions with frequent deletions in tumors. Loss of 16q23 occurs frequently in multiple tumors, indicating the presence of critical TSGs at this locus, such as the well-studied WWOX. Herein, we found that ADAMTS18, located next to WWOX, was significantly downregulated in multiple carcinoma cell lines. No deletion of ADAMTS18 was detected with multiplex differential DNA-PCR or high-resolution 1-Mb array-based comparative genomic hybridization (CGH) analysis. Instead, methylation of the ADAMTS18 promoter CpG Island was frequently detected with methylation-specific PCR and bisulfite genome sequencing in multiple carcinoma cell lines and primary carcinomas, but not in any nontumor cell line and normal epithelial tissue. Both pharmacological and genetic demethylation dramatically induced the ADAMTS18 expression, indicating that CpG methylation directly contributes to the tumor-specific silencing of ADAMTS18. Ectopic ADAMTS18 expression led to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells lacking the expression. Thus, through functional epigenetics, we identified ADAMTS18 as a novel functional tumor suppressor, being frequently inactivated epigenetically in multiple carcinomas.

Gene expression profiles in squamous cell cervical carcinoma using array-based comparative genomic hybridization analysis

Our aim was to identify novel genomic regions of interest and provide highly dynamic range information on correlation between squamous cell cervical carcinoma and its related gene expression patterns by a genome-wide array-based comparative genomic hybridization (array-CGH). We analyzed 15 cases of cervical cancer from KangNam St Mary's Hospital of the Catholic University of Korea. Microdissection assay was performed to obtain DNA samples from paraffin-embedded cervical tissues of cancer as well as of the adjacent normal tissues. The bacterial artificial chromosome (BAC) array used in this study consisted of 1440 human BACs and the space among the clones was 2.08 Mb. All the 15 cases of cervical cancer showed the differential changes of the cervical cancer–associated genetic alterations. The analysis limit of average gains and losses was 53%. A significant positive correlation was found in 8q24.3, 1p36.32, 3q27.1, 7p21.1, 11q13.1, and 3p14.2 changes through the cervical carcinogenesis. The regions of high level of gain were 1p36.33–1p36.32, 8q24.3, 16p13.3, 1p36.33, 3q27.1, and 7p21.1. And the regions of homozygous loss were 2q12.1, 22q11.21, 3p14.2, 6q24.3, 7p15.2, and 11q25. In the high level of gain regions, GSDMDC1, RECQL4, TP73, ABCF3, ALG3, HDAC9, ESRRA, and RPS6KA4 were significantly correlated with cervical cancer. The genes encoded by frequently lost clones were PTPRG, GRM7, ZDHHC3, EXOSC7, LRP1B, and NR3C2. Therefore, array-CGH analyses showed that specific genomic alterations were maintained in cervical cancer that were critical to the malignant phenotype and may give a chance to find out possible target genes present in the gained or lost clones.

The Role of EXT1 in Nonhereditary Osteochondroma: Identification of Homozygous Deletions

Multiple osteochondromas is a hereditary syndrome that is characterized by the formation of cartilage-capped bony neoplasms (osteochondromas), for which exostosis (multiple)-1 (EXT1) has been identified as a causative gene. However, 85% of all osteochondromas present as solitary (nonhereditary) lesions in which somatic mutations in EXT1 are extremely rare, but loss of heterozygosity and clonal rearrangement of 8q24 (the chromosomal locus of EXT1) are common. We examined whether EXT1 might act as a classical tumor suppressor gene for nonhereditary osteochondromas. Eight nonhereditary osteochondromas were subjected to high-resolution array-based comparative genomic hybridization (array-CGH) analysis for chromosome 8q. The array-CGH results were validated by subjecting tumor DNA to multiple ligation-dependent probe amplification (MLPA) analysis for EXT1. EXT1 locus-specific fluorescent in situ hybridization (FISH) was performed on nuclei isolated from the three tissue components of osteochondroma (cartilage cap, perichondrium, bony stalk) to examine which parts of the tumor are of clonal origin. Array-CGH analysis of tumor DNA revealed that all eight osteochondromas had a large deletion of 8q; five tumors had an additional small deletion of the other allele of 8q that contained the EXT1 gene. MLPA analysis of tumor DNA confirmed these findings and identified two additional deletions that were smaller than the limit of resolution of array-CGH. FISH analysis of the cartilage cap, perichondrium, and bony stalk showed that these homozygous EXT1 deletions were present only in the cartilage cap of osteochondroma. EXT1 functions as a classical tumor suppressor gene in the cartilage cap of nonhereditary osteochondromas.

Duplication of 17(p11.2p11.2) in a male child with autism and severe language delay

Duplications of 17(p11.2p11.2) have been associated with various behavioral manifestations including attention deficits, obsessive-compulsive symptoms, autistic traits, and language delay. We are conducting a genetic study of autism and are screening all cases for submicroscopic chromosomal abnormalities, in addition to standard karyotyping, and fragile X testing. Using array-based comparative genomic hybridization analysis of data from the Affymetrix GeneChip(R) Human Mapping Array set, we detected a duplication of approximately 3.3 Mb on chromosome 17p11.2 in a male child with autism and severe expressive language delay. The duplication was confirmed by measuring the copy number of genomic DNA using quantitative polymerase chain reaction. Gene expression analyses revealed increased expression of three candidate genes for the Smith-Magenis neurobehavioral phenotype, RAI1, DRG2, and RASD1, in transformed lymphocytes from Case 81A, suggesting gene dosage effects. Our results add to a growing body of evidence suggesting that duplications of 17(p11.2p11.2) result in language delay as well as autism and related phenotypes. As Smith-Magenis syndrome is also associated with language delay, a gene involved in acquisition of language may lie within this interval. Whether a parent of origin effect, gender of the case, the presence of allelic variation, or changes in expression of genes outside the breakpoints influence the resultant phenotype remains to be determined.