Comparative genomic hybridization analysis of genetic aberrations associated with development of esophageal squamous cell carcinoma in Henan, China

AIM: To characterize cytogenetic alterations in esophageal squamous cell carcinoma (ESCC) and its metastasis. METHODS: A total of 37 cases of primary ESCC and 15 pairs of primary ESCC tumors and their matched metastatic lymph nodes cases were enrolled from Linzhou, the high incidence area for ESCC in Henan, northern China. The comparative genomic hybridization (CGH) was applied to determine the chromosomal aberrations on the DNA extracted from the frozen ESCC and metastatic lymph node samples from these patients. RESULTS: CGH showed chromosomal aberrations in all the cases. In 37 cases of primary ESCC, chromosomal profile of DNA copy number was characterized by frequently detected gains at 8q (29/37, 78%), 3q (24/37, 65%), 5p (19/37, 51%); and frequently detected losses at 3p (21/37, 57%), 8p and 9q (14/37, 38%). In 15 pairs of primary ESCC tumors and their matched metastatic lymph node cases, the majority of the chromosomal aberrations in both primary tumor and metastatic lymph node lesions were consistent with the primary ESCC cases, but new candidate regions of interest were also detected. The most significant finding is the gains of chromosome 6p with a minimum high-level amplification region at 6p12-6q12 in 7 metastatic lymph nodes butonly in 2 corresponding primary tumors (P = 0.05) and 20p with a minimum high-level amplification region at 20p12 in 11 metastatic lymph nodes but only in 5 corresponding primary tumors (P < 0.05). Another interesting finding is the loss of chromosome 10p and 10q in 8 and 7 metastatic lymph nodes but only in 2 corresponding primary tumors (P < 0.05). CONCLUSION: Using the CGH technique to detect chromosomal aberrations in both the primary tumor and its metastatic lymph nodes of ESCC, gains of 8q, 3q and 5p and loss of 3p, 8p, 9q and 13q were specifically implicated in ESCC in Linzhou population. Gains of 6p and 20p and loss of 10pq may contribute to the lymph node metastasis of ESCC. These findings suggest that the gains and losses of chromosomal regions may contain ESCC-related oncogenes and tumor suppressor genes and provide important theoretic information for identifying and cloning novel ESCC-related oncogenes and tumor suppressor genes.

Association between chromosomal aberration of COX8C and tethered spinal cord syndrome:array-based comparative genomic hybridization analysis

Copy number variations have been found in patients with neural tube abnormalities.In this study,we performed genome-wide screening using high-resolution array-based comparative genomic hybridization in three children with tethered spinal cord syndrome and two healthy parents.Of eight copy number variations,four were non-polymorphic.These non-polymorphic copy number variations were associated with Angelman and Prader-Willi syndromes,and microcephaly.Gene function enrichment analysis revealed that COX8 C,a gene associated with metabolic disorders of the nervous system,was located in the copy number variation region of Patient 1.Our results indicate that array-based comparative genomic hybridization can be used to diagnose tethered spinal cord syndrome.Our results may help determine the pathogenesis of tethered spinal cord syndrome and prevent occurrence of this disease.

Detection of chromosomal imbalance in oligodendroglial tumors by comparative genomic hybridization

Objective To investigate the relationship between genomic DNA imbalance in oligodendroglial tumors and its different classification. Methods 16 oligodendrogliomas and 17 anaplastic oligodendrogliomas were investigated by comparative genomic hybridization on Paraffin-Embedded tissue samples,and the chromosomal genomic DNA imbalances were analyzed. Results Chromosome DNA imbalance rates in oligodendrogliomas

Methyl-CpG-Binding protein 2 duplication syndrome in a Chinese patient:A case report and review of the literature

BACKGROUND Chromosomal Xq28 region duplication encompassing methyl-CpG-binding protein 2(MECP2)results in an identifiable phenotype and global developmental delay known as MECP2 duplication syndrome(MDS).This syndrome has a wide range of clinical manifestations,including abnormalities in appearance,neurodevelopment,and gastrointestinal motility;recurrent infections;and spasticity.Here,we report a case of confirmed MDS at our institution.CASE SUMMARY A 12-year-old Chinese boy presented with intellectual disability(poor intellectual[reasoning,judgment,abstract thinking,and learning]and adaptive[lack of communication and absent social skills,apraxia,and ataxia]functioning)and dysmorphism.He had no history of recurrent infections,seizures,or bowel dysfunction,which is different from that in reported cases.Microarray comparative genomic hybridization confirmed MECP2 duplication in the patient and his mother who is a carrier.The duplication size was the same in the patient and his mother.No prophylactic antibiotic or anti-seizure therapy was offered to the patient or his mother before or after the consultation.CONCLUSION MDS is rare and has various clinical presentations.Clinical suspicion is critical in patients presenting with developmental delays.

Analysis of comparative genomic hybridization and loss of heterozygosity in 43 primary gastric carcinomas

To investigate common chromosomal changes and the LOH frequency of microsatellite loci in primary gastric cancer samples in order to locate the deleted regions in which human gastric cancer related genes might exist Methods Comparative genomic hybridization (CGH) was used to define global chromosomal aberrations in 43 primary gastric tumors Based on the results of CGH, analysis of loss of heterozygosity (LOH) was performed in chromosome 19 in which the loss was first discovered in the gastric cancers The PCR-based approach was used to investigate 22 loci, which are spaced at 1 1-10 9 cM intervals throughout chromosome 19 The amplified PCR fragments were subjected to electrophoresis in PAGE gel and analyzed with Genescan TM and Genotyper TM Results CGH analysis revealed gains in chromosome 3p(8/43), 8q(8/43), 20 [20 (9/43), 20p(7/43), 20q(4/43)], 12q(16/43), 13q(12/43) and losses in 19 [19 (15/43)], 7 [17 (8/43), 17p (10/43)], 16 (10/43) and 1p (11/43) Among the 43 evaluated samples, the most frequent LOH was detected at locus D19S571 (27 81%) Conclusions The tumorigenesis of gastric cancer includes several chromosomal changes The aberration of chromosome 19 was the first common change founded in gastric cancer The region near the D19S571 might harbor potential genes related to the tumorigenesis of gastric cancer

Novel chromosomal alterations detected in primary nasopharyngeal carcinoma by comparative genomic hybridization

Objective To gain a better understanding of genetic changes in Cantonese nasopharyngeal carcinoma (NPC) Methods Comparative genomic hybridization (CGH) was performed on 17 primary nasopharyngeal carcinomas Results A novel copy number gain an chromosome 4q and loss of chromosome 1p were found at a high frequency (>50%) Conclusions Current analysis revealed a comprehensive profile of the chromosomal regions showing gain of chromosomes 4q, 12q, and 1q as well as loss of chromosomes 1p, 3p, 11q, 14q, 15q, 13q, Xq, 9q, 10p, 10q, and 16q Frequently altered loci may encode oncogenes or tumor suppressor genes involved in the development of primary NPC

Copy number changes of target genes in chromosome 3q25.3-qter of esophageal squamous cell carcinoma: TP63is amplified in early carcinogenesis but down-regulated as disease progressed

AIM: By using comparative genomic hybridization, gain of 3q was found in 45-86% cases of esophageal squamous cell carcinoma (EC-SCC). Chromosome 3q25.3-qter is the minimal common region with several oncogenes found within this region. However, amplification patterns of these genes in EC-SCC have never been reported. The possible association of copy number changes of these genes with pathologic characteristics is still not clear. METHODS: Real-time quantitative PCR (Q-PCR) was performed to analyze the copy number changes of 13 candidate genes within this region in 60 primary tumors of EC-SCC, and possible association of copy number changes with pathologic characteristics was analyzed by statistics. Immunohistochemistry (IHC) study was also performed on another set of 111 primary tumors of EC-SCC to verify the association between TP63 expression change and lymph node metastasis status. RESULTS: The average copy numbers (±SE) per haploid genome of individual genes in 60 samples were (from centromere to telomere): SSR3: 4.19 (±0.69); CCNL1: 5.24 (±0.67); SMC4L1: 2.01 (±0.16); EVI1: 2.02 (±0.12); hTERC. 5.28 (±0.54); SKIL 2.71 (±0.14); EIF5A2. 1.95 (±0.12); ECT2: 9.18 (±1.68); PIK3CA: 8.13 (±1.17); EIF4G1: 1.07 (±0.05); 557: 3.07 (±0.25); TP63: 2.51 (±0.22); TFRC. 2.42 (±0.19). Four clusters of amplification were found: SSR3 and CCLN1 at 3q25.31; hTERC and SKIL at 3q26.2; ECT2 and PIK3CA at 3q26.31-q26.32; and 55T, TP63 and TFRC at 3q27.3-q29. Patients with lymph node metastasis had significantly lower copy number of TP63 in the primary tumor than those without lymph node metastasis. IHC study on tissue arrays also showed that patients with lymph node metastasis have significantly lower TP63 staining score in the primary tumor than those without lymph node metastasis. CONCLUSION: This study showed that different amplification patterns were seen among different genes within 3q25.3-qter in EC-SCC, and several novel candidate oncogenes (SSR3, SMC4L1, ECT2, and SST) were identified. TP63 is amplified in early stage of EC-SCC carcinogenesis but down-regulated in advanced stage of disease.

Analysis of Chinese women with primary ovarian insufficiency by high resolution array-comparative genomic hybridization

Background Primary ovarian insufficiency （POI） is defined as a primary ovarian defect characterized by absent menarche （primary amenorrhea） or premature depletion of ovarian follicles before the age of 40 years. The etiology of primary ovarian insufficiency in human female patients is still unclear. The purpose of this study is to investigate the potential genetic causes in primary amenorrhea patients by high resolution array based comparative genomic hybridization （array-CGH） analysis. Methods Following the standard karyotyping analysis, genomic DNA from whole blood of 15 primary amenorrhea patients and 15 normal control women was hybridized with Affymetrix cytogenetic 2.7M arrays following the standard protocol. Copy number variations identified by array-CGH were confirmed by real time polymerase chain reaction.

Spectrum of Cytogenomic Abnormalities Revealed by Array Comparative Genomic Hybridization on Products of Conception Culture Failure and Normal Karyotype Samples

Approximately 30% of pregnancies after implantation end up in spontaneous abortions, and 50% of them are caused by chromosomal abnormalities. However, the spectrum of genomic copy number variants （CNVs） in products of conception （POC） and the underlying gene- dosage-sensitive mechanisms causing spontaneous abortions remain largely unknown. In this study, array comparative genornic hybridiza- tion （aCGH） analysis was performed as a salvage procedure for 128 POC culture failure （POC-CF） samples and as a supplemental procedure for 106 POC normal karyotype （POC-NK） samples. Chromosomal abnormalities were detected in 10% of POC-CF and pathogenic CNVs were detected in 3.9% of POC-CF and 5.7% of POC-NK samples. Compiled results from this study and relevant case series through a literature review demonstrated an abnormality detection rate （ADR） of 35% for chromosomal abnormalities in POC-CF samples, 3.7% for pathogenic CNVs in POC-CF samples, and 4.6% for pathogenic CNVs in POC-NK samples. Ingenuity Pathway Analysis （IPA） was performed on the genes from pathogenic CNVs found in POC samples. The denoted primary gene networks suggested that apoptosis and cell proliferation pathways are involved in miscarriage. In summary, a similar spectrum of cytogenomic abnormalities was observed in POC culture success and POC-CF samples. A threshold effect correlating the number of dosage-sensitive genes in a chromosome with the observed frequency of autosomai trisomy is proposed. A rationalized approach using firstly fluorescence in situ hybridization （FISH） testing with probes of chromosomes X/Y/ 18, 13/21, and 15/16/22 for common aneuploidies and polyploidies and secondly aCGH for other cytogenomic abnormalities is recommended for POC-CF samples.

Chromosomal imbalances revealed in primary rhabdomyosarcomas by comparative genomic hybridization

Background Previous cytogenetic studies revealed rhabdomyosarcoma. We profiled chromosomal imbalances aberrations varied among the three subtypes of n the different subtypes and investigated the relationships between clinical parameters and genomic aberrations. Methods Comparative genomic hybridization was used to investigate genomic imbalances in 25 cases of primary rhabdomyosarcomas and two rhabdomyosarcoma cell lines. Specimens were reviewed to determine histological type, pathological grading and clinical staging. Results Changes involving one or more regions of the genome were seen in all rhabdomyosarcomal patients. For rhabdomyosarcoma, DNA sequence gains were most frequently （〉30%） seen in chromosomes 2p, 12q, 6p, 9q, 10q, lp, 2q, 6q, 8q, 15q and 18q; losses from 3p, 11p and 6p. In aggressive alveolar rhabdomyosarcoma, frequent gains were seen on chromosomes 12q, 2p, 6p, 2q, 4q, 10q and 15q; losses from 3p, 6p, lq and 5q. For embryonic rhabdomyosarcoma, frequent gains were on 7p, 9q, 2p, 18q, lp and 8q; losses only from 11p. Frequently gained chromosome arms of translocation associated with rhabdomyosarcoma were 12q, 2, 6, 10q, 4q and 15q; losses from 3p, 6p and 5q. The frequently gained chromosome arms of nontranslocation associated with rhabdomyosarcoma were 2p, 9q and 18q, while 11p and 14q were the frequently lost chromosome arms. Gains on chromosome 12q were significantly correlated with translocation type. Gains on chromosome 9q were significantly correlated with clinical staging. Conclusions Gains on chromosomes 2p, 12q, 6p, 9q, 10q, lp, 2q, 6q, 8q, 15q and 18q and losses on chromosomes 3p, 11p and 6p may be related to rhabdomyosarcomal carcinogenesis. Furthermore, gains on chromosome 12q may be correlated with translocation and gains on chromosome 9q with the early stages of rhabdomyosarcoma.

Analyses of Genotypes and Phenotypes of Ten Chinese Patients with Wolf-Hirschhorn Syndrome by Multiplex Ligation-dependent Probe Amplification and Array Comparative Genomic Hybridization

Background： Wolf-Hirschhorn syndrome （WHS） is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the features of Chinese WHS patients. This study aimed to characterize the clinical and molecular cytogenetic features of Chinese WHS patients using the combination of multiplex ligation-dependent probe amplification （MLPA） and array comparative genomic hybridization （array CGH）. Methods： Clinical information was collected from ten patients with WHS. Genomic DNA was extracted from the peripheral blood of the patients. The deletions were analyzed by MLPA and array CGH. Results： All patients exhibited the core clinical symptoms of WHS, including severe growth delay, a Greek warrior helmet facial appearance, differing degrees of intellectual disability, and epilepsy or electroencephalogram anomalies. The 4p deletions ranged from 2.62 Mb to 17.25 Mb in size and included LETM1, WHSC1, and FGFR3. Conclusions： The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype.

Identification of metastasis-associated genes in colorectal cancer through an integrated genomic and transcriptomic analysis

Objective： Identification of colorectal cancer （CRC） metastasis genes is one of the most important issues in CRC research. For the purpose of mining CRC metastasis-associated genes, an integrated analysis of mJcroarray data was presented, by combined with evidence acquired from comparative genornic hybridization （CGH） data. Methods： Gene expression profile data of CRC samples were obtained at Gene Expression Omnibus （GEO） website. The 15 important chromosomal aberration sites detected by using CGH technology were used for integrated genomic and transcriptomic analysis. Significant Analysis of Microarray （SAM） was used to detect significantly differentially expressed genes across the whole genome. The overlapping genes were selected in their corresponding chromosomal aberration regions, and analyzed by using the Database for Annotation, Visualization and Integrated Discovery （DAVID）. Finally, SVM-T-RFE gene selection algorithm was applied to identify ted genes in CRC. Results： A minimum gene set was obtained with the minimum number [14] of genes, and the highest classification accuracy （100%） in both PRI and META datasets. A fraction of selected genes are associated with CRC or its metastasis. Conclusions- Our results demonstrated that integration analysis is an effective strategy for mining cancer- associated genes.

Loss of fragile histidine triad and amplification of 1p36.22 and 11p15.5 in primary gastric adenocarcinomas

AIM: To investigate the genomic copy number alterations that may harbor key driver genes in gastric tumorigenesis. METHODS: Using high-resolution array comparative genomic hybridization (CGH), we investigated the genomic alterations of 20 advanced primary gastric adenocarcinomas (seventeen tubular and three mucinous) of Chinese patients from the Jilin province. Ten matching adjacent normal regions from the same patients were also studied. RESULTS: The most frequent imbalances detected in these cancer samples were gains of 3q26.31-q27.2, 5p, 8q, 11p, 18p, 19q and 20q and losses of 3p, 4p,18q and 21q. The use of high-resolution array CGH increased the resolution and sensitivity of the observed genomic changes and identified focal genetic imbalances, which included 54 gains and 16 losses that were smaller than 1 Mb in size. The most interesting focal imbalances were the intergenic loss/homozygous deletion of the fragile histidine triad gene and the amplicons 11q13, 18q11.2 and 19q12, as well as the novel amplicons 1p36.22 and 11p15.5. CONCLUSION: These regions, especially the focal amplicons, may harbor key driver genes that will serve as biomarkers for either the diagnosis or the prognosis of gastric cancer, and therefore, a large-scale investigation is recommended.

Clinical cytogenetics and molecular cytogenetics

The short report will be focused on helping our students to understand commonly used conventional and cutting edge cytogenetic techniques and their clinical applications, the advances and drawbacks of each technique, and how to pick the right test(s) for a specific patient in order to achieve a proper diagnosis efficiently and economically.

Human cancer genetics

The short report will be focused on the genetic basis and possible mechanisms of tumorigenesis, common types of cancer, the importance of genetic diagnosis of cancer, and the methodology of cancer genetic diagnosis. They will also review presymptomatic testing of hereditary cancers, and the application of expression profiling to identify patients likely to benefit from particular therapeutic approaches.

Genomic landscape of pancreatic neuroendocrine tumors

AIM: To investigate the prognostic role of genomic stability and copy number alterations (CNAs) pancreatic neuroendocrine tumors (PanNETs).

Identification of p63 expression in human lung cancer: analysis by complementary DNA and tissue microarray

Objective: To evaluate p63 expression at mRNA transcripts and protein levels in lung squamous cell cancer (SCC), adenocarcinoma, large cell lung cancer (LCLC) and small cell lung cancer (SCLC) and their matched metastatic tumors. The association between p63 expression and p63 locus at chromosomal 3q27 q29 was also investigated. Methods: p63 mRNA expression levels in a large series of lung cancers including SCC, adenocarcinoma, LCLC, SCLC and their matched metastatic tumors were analyzed by cDNA microarray technology. A tissue microarray from 150 primary lung cancer specimens was constructed and used for immunohistochemical detection of p63 protein expression. Chromosomal imbalances at the p63 locus in 70 primary lung cancers samples were studied by comparative genomic hybridization (CGH) technology. Results: mRNA levels were 10 fold in SCC compared to LCLC, SCLC, and adenocarcinoma. Interestingly, the mRNA expression of p63 in metastatic carcinomas was significantly higher than that in their matched primary tumors ( P <0 001). Immunohistochemistry demonstrated that p63 expression was 94.64% in SCC but only 1 79% in lung adenocarcinoma and 2 of 4 LCLC were positive staining. All the results in of SCLC were negative. There was a statistically significant difference for p63 positivity between pT1 tumors and those of higher stage ( P =0 035). The CGH results indicated that p63 locus at chromosomal 3q27 q29 was overrepresented in SCC. p63 immunopositivity correlated significantly with pronounced gains of the p63 locus at chromosomal 3q27 q29 (P=0.0001), indicating that strong expression of p63 in lung SCC correlated with increased gene amplification. Conclusion: p63 might play an important role not only in squamous differentiation of lung cancer but also in tumor development and progression.

DNA COPY PROFILE IN NASOPHARYNGEAL CARCINOMA AND ITS CORRELATION WITH CLINICAL STAGING

Objective: To detect genetic alterations in nasopharyngeal carcinoma (NPC) in Cantonese, the population with the highest incidence of NPC, and to correlate the findings with clinical staging. Methods: Comparative genomic hybridization (CGH) was performed on 35 primary nasopharyngeal carcinomas and a nonparametric x2 test was used to analyze relationship between chromosome changes and clinical staging. Results: The identified common chromosomal alterations in NPC included gain of chromosomes 12q (21 cases, 60%), 4q (19cases, 43%), 3q (18 cases, 51%), 1q (15 cases, 43%),8q (14 cases, 40%), and 2q (12 cases, 30%). The most frequently detected loss of chromosomal materials involved chromosome 1p (24 cases, 69%), chromosome 3p (21 cases, 60%), 11q (20 cases, 57%), 14q (18 cases, 51%), 16q (14 cases, 40%), 13(12 cases, 34%), and 9p(11 cases, 31%). The high frequency (>50%) 4q gain and 1p loss were novel findings. Compared by nonparametric x2 test, gains on 12q and 8q were found mainly in stages III/IV and there were significant differences between two clinical stage groups (stages I/II vs stages III/IV). Conclusions: Current analysis has revealed a comprehensive profile of the chromosomal regions showing DNA copy number changes, which may harbor oncogenes or tumor suppressor genes involved in the development of primary NPC.

A Microarray Based Genomic Hybridization Method for Identification of New Genes in Plants：Case Analyses of Arabidopsis and Oryza

To systematically estimate the gene duplication events In closely related species, we have to use comparative genomlc approaches, either through genomlc sequence comparison or comparative genomlc hybridization （CGH）. Given the scarcity of complete genomlc sequences of plant species, in the present study we adopted an array based CGH to Investigate gene duplications In the genus Arabldopsls. Fragment genomlc DNA from four species, namely Arabidopsls thallana, A. lyrata subsp, lyrata, A. lyrata subsp, petraea, and A. halleri, was hybridized to Affymetrlx （Santa Clara, CA, USA） tiling arrays that are designed from the genomlc sequences of A. thallana. Pairwlse comparisons of signal intensity were made to infer the potential duplicated candidates along each phylogenetic branch. Ninety-four potential candidates of gene duplication along the genus were Identified. Among them, the majority （69 of 94） were A. thallana lineage specific. This result indicates that the array based CGH approach may be used to Identify candidates of duplication In other plant genera containing closely related species, such as Oryza, particularly for the AA genome species. We compared the degree of gene duplication through retrotransposon between O. satlva and A. thallana and found a strikingly higher number of chimera retroposed genes In rice. The higher rate of gene duplication through retroposltlon and other mechanisms may Indicate that the grass species Is able to adapt to more diverse environments.

Advances in preimplantation genetic diagnosis/screening

Preimplantation genetic diagnosis(PGD)gives couples who have a high risk of transmitting genetic disorders to their baby the chance to have a healthy offspring through embryo genetic analysis and selection.Preimplantation genetic screening(PGS)is an effective method to select euploid embryos that may prevent repeated implantation failure or miscarriage.However,how and to whom PGS should be provided is a controversial topic.The first successful case of PGD of a human being was reported in 1990,and there have been tremendous improvements in this technology since then.Both embryo biopsy and genetic technologies have been improved dramatically,which increase the accuracy and expand the indications of PGD/PGS.