Rapid prenatal diagnosis for chromosomal aneuploidy using cDNA microarray-based comparative genomic hybridization

OBJECTIVE: Prenatal cytogenetic diagnosis is limited to metaphase karyotype analysis of cultured cells obtained by amniocentesis or chorionic villus sampling. Moreover, genome wide analysis cannot be performed by FISH analysis using specific probe. Array comparative genomic hybridization (CGH) offers a number of advantages over conventional cytogenetic analysis and FISH. Microarray CGH can be highly comprehensive, amenable to very high resolution, sensitive and fast. The objective of this study was to determine the clinical use of cDNA microarray CGH for detection of fetal aneuploidy. METHODS: 21 amniotic fluid samples and 6 chorionic villi samples were obtained from 27 pregnant women in 9-19 gestational weeks. Genomic DNA was extracted from each sample and amplified. For cDNA microarray CGH analysis, test DNA sample and reference DNA sample were labeled with Cy3-dUTP and Cy5-dUTP, respectively. Each sample of labeled test and reference DNA was hybridized to microarray. The result was analysed with axon scanner and compared with cytogenetic analysis and FISH. RESULTS: In 27 cases, 3 cases with trisomy 21 and 1 case with trisomy 18 had increased hybridization signals on chromosome 21 and chromosome 18. One case with 45,X had decreased signals on chromosome X. One case with 46,X,i(Xq) had decreased signal on short arm of chromosome X and increased signal on long arm. And one case with 47,XYY had two fold increased signal on Y chromosome. cDNA microarray based CGH correctly identified fetal aneuploidy in all of the 7 cases with aneuploid fetuses. CONCLUSION: Prenatal genetic diagnosis by cDNA microarray-based CGH is an useful, innovative, rapid and accurate method. It is promising technique allowing rapid screening for whole chromosomal changes including aneuploidy, and may augment standard karyotyping techniques for prenatal genetic diagnosis by providing additional molecular information. This method may aid the discovery and description of minor genetic aberration, potentially enhancing future prenatal genetic diagnostic application.ope

Selection of efficacy predictive genes in locally

의과학과/석사[한글] 국소 진행성 직장암 치료는 항암 약물 방사선 병행 요법을 시행하여 종양의 크기를 최소화함으로써 수술을 가능하게 하고 있다. 그러나 항암 약물 방사선 병행 요법을 시행 받은 환자들의 치료 효과와 부작용은 환자에 따라 다양하게 나타나고 있다. 그러므로 환자들에게 항암 약물 방사선 병행 요법 시행 전에 치료의 효과와 부작용을 미리 예측하는 것은 개인 맞춤 치료를 하는데 중요한 정보를 제공해 줄 수 있다. 본 연구에서는 항암 약물 방사선 병행 요법에 대하여 효과와 부작용을 예측할 수 있는 생물학적 표지자를 찾기 위해 진단 당시 생검 조직 28예, 치료 후 정상 조직 15예, 종양 조직 14예와 항암 약물 방사선 병행 요법 시행 전의 림프구 20 예를 대상으로 7.5K cDNA Microarray를 이용하여 cDNA microarray-based comparative genomic hybridization (Microarray-CGH)를 수행하였다. 진단 당시 조직 생검의 Microarray-CGH 결과에서 치료 후에 반응군과 비 반응군을 비교하여 치료의 효과를 예측할 수 있는 유전자를 false discovery rate (FDR) 기준으로 103개 (FDR 24 %), 52개 (FDR 20 %), 29개 (FDR 14.9 %)유전자를 선별하였다. 림프구의 Microarray-CGH 결과에서 GⅣ neutropenia 를 예측하는 95 (FDR 17.1 %), 40 (FR 14.9 %), 31개 (FDR 11 %)의 유전자를 선별하였다. 본 연구에서는 Microarray-CGH 기법을 이용하여 항암 약물 방사선 병행 요법에 대하여 치료의 효과를 예측할 수 있는 29개의 후보 유전자군을, GⅣ neutropenia를 예측할 수 있는 31개의 후보 유전자군을 탐색하였다. 선별된 유전자군들의 유전학적 수준의 관찰은 항암 약물 방사선 병행 요법에 대한 효과와 부작용을 예측하여 개인 맞춤 치료에 있어 중요한 기반을 제공할 것으로 생각된다. [영문]In locally advanced rectal cancer, preoperative concurrent chemoradiotherapy (CCRT) is performed to reduce tumor size and improve operability. As the patients showed various responses to CCRT, the prediction of efficacy and toxicity of CCRT is very important in clinical practice to maximize the efficacy and minimize the toxicity of each patient. To identify the predictive markers of response and toxicity of CCRT in genome scale, we performed cDNA microarray-based comparative genomic hybridization (Microarray-CGH). 7.5K cDNA microarray was used with 28 biopsy tumor tissues and 20 peripheral mononuclear cells of locally advanced rectal cancer patients at the time of diagnosis and normal, tumor tissues after CCRT. Micorarray-CGH was performed in indirect design using sex-matched placenta tissue as a reference. After the preprocessing and normalization, we used 2-class SAM (Significance Analysis of Microarray, Stanford Univ.) for selection of differentially expressed genes between 2 groups. Then we performed the hierarchical cluster using the selected genes, and searched for ontological information of these genes using DATABASE (SOURCE and DAVID). We selected the efficacy predictive genes of 103 genes (FDR 24 %), 52 genes (FDR 20 %), 29 genes (FDR 14.9 %)and using 2-class SAM analysis are between 8 responders and 8 non-responders after CCRT using the the of biopsied tissues. we could identify 95 (FDR 17.1 %), 40 (FDR 14.9 %), 31 (FDR 11 %) predictive genes for grade Ⅳ neutropenia from the CCRT with peripheral blood mononuclear cell DNAs. In conclusion, Microarray-CGH could be used as an investigative tool for searching candidate predictive genes of efficacy and toxicity in locally advanced rectal cancer patient treated with CCRT.ope

Biomarker detection for the diagnosis of lymph node metastasis from oral squamous cell carcinoma

Lymph node metastasis is an important prognostic factor in oral squamous cell carcinoma. However, the lack of significant biomarkers for lymph node metastasis can cause patients to be inappropriately treated and produce a poor prognosis. Therefore, there is a need to identify gene sets that are associated with lymph node metastasis. In this study, we used three expression datasets obtained from a public database and selected candidate gene sets that were related with lymph node metastasis from two datasets and a combined dataset. We evaluated the selected gene set using OOB error rates in a validation dataset. The gene set detected from the combined dataset classified the lymph node status more accurately in the validation dataset and clear expression patterns classifying the lymph node status based on chromosomal location were observed. The combined dataset holds promise for use as a more accurate candidate gene set for the diagnosis of lymph node metastasis and the selected gene set could be used for biological validation in further studies.ope

Identification of significant regional genetic variations using continuous CNV values in aCGH data

Array comparative genomic hybridization (aCGH) provides a technique to survey the human genome for chromosomal aberrations in disease. The identification of genomic regions with aberrations may clarify the initiation and progression of cancer, improve diagnostic and prognostic accuracy, and guide therapy. The analysis of variance (ANOVA) model is widely used to detect differentially expressed genes after accounting for common sources of variation in microarray analysis. In this study, we propose a method, shifted ANOVA, to detect significantly altered regions. This method, based on the standard ANOVA, analyzes changes in copy number variation for regions. The selected regions have the group effect only, but no effect within samples and no interactive effects. The performance of the proposed method is evaluated from the homogeneity and classification accuracies of the selected regions. Shifted ANOVA may identify new candidate genes neighboring known because it detects significantly altered chromosomal regions, rather than independent probes.ope

Whole genome analysis for liver metastasis gene signatures in colorectal cancer

Liver metastasis is one of the major causes of death in colorectal cancer (CRC) patients. To understand this process, we investigated whether the gene expression profiling of matched colorectal carcinomas and liver metastases could reveal key molecular events involved in tumor progression and metastasis. We performed experiments using a cDNA microarray containing 17,104 genes with the following tissue samples: paired tissues of 25 normal colorectal mucosa, 27 primary colorectal tumors, 13 normal liver and 27 liver metastasis, and 20 primary colorectal tumors without liver metastasis. To remove the effect of normal cell contamination, we selected 4,583 organ-specific genes with a false discovery rate (FDR) of 0.0067% by comparing normal colon and liver tissues using significant analysis of microarray, and these genes were excluded from further analysis. We then identified and validated 46 liver metastasis-specific genes with an accuracy of 83.3% by comparing the expression of paired primary colorectal tumors and liver metastases using prediction analysis of microarray. The 46 selected genes contained several known oncogenes and 2 ESTs. To confirm that the results correlated with the microarray expression patterns, we performed RT-PCR with WNT5A and carbonic anhydrase II. Additionally, we observed that 21 of the 46 genes were differentially expressed (FDR = 2.27%) in primary tumors with synchronous liver metastasis compared with primary tumors without liver metastasis. We scanned the human genome using a cDNA microarray and identified 46 genes that may play an important role in the progression of liver metastasis in CRC.ope

The pattern of gene copy number changes in bilateral breast cancer surveyed by cDNA microarray-based comparative genomic hybridization.

The nature of genetic alterations in bilateral breast cancer (BBC) associated with the distinctive development of a second primary tumor or a metastatic lesion is not clearly established. In this study, patterns of promoter methylation and gene copy number changes were assessed for their utility in the distinction of two types of BBC (synchronous and metachronous). Seven cases of synchronous and five cases of metachronous breast cancer tissues were used in X chromosome inactivation assay to assess the methylation pattern of human androgen receptor gene. X chromosome inactivation assay alone did not provide enough information to distinguish the genetic origins of synchronous and metachronous BBC. When four pairs of paraffin-embedded BBC tissues were used in cDNA array-based CGH with placenta DNA as a reference, higher DNA copy number changes were observed from metachronous pairs (9.0%) than from synchronous pairs (3.1%). From the two cases of metachronous pairs tested, 44 genes were found to be commonly modulated in gene copy numbers in a cancer detected later.ope