An oligonucleotide based array-CGH system for detection of genome wide copy number changes including subtelomeric regions for genetic evaluation of mental retardation.

Developmental delay (DD) and mental retardation (MR) are important child heath issues with a one percent prevalence. Karyotyping with or without subtelomeric FISH (fluorescent in situ hybridization), unless the phenotype of the patient suggests a specific aberration for a specific FISH assay, is the most common procedure in cytogenetic evaluation of MR/DD. In addition, there are several platforms utilizing microarray based comparative genomic hybridization technology (array-CGH) for genetic testing. Array-CGH can detect deletions or duplications in very small segments of chromosomes and the use of this technology is expected to increase the diagnostic yield. The major limitation of the current BAC based array technologies is the low resolution ( approximately 1 Mb) of the chip and suboptimal coverage particularly in the subtelomeric regions. Our aim was to design a novel array-CGH chip with high-density of probes in the subtelomeric regions as well as to maintain sufficient density in other regions of the genome to provide comprehensive coverage for DD/MR. For this purpose, we used Human Genome CGH Microarray 44B chip (Agilent) as the template for the novel design. Using e-array 4.0 (Agilent), one third of the probes were randomly removed from the array and replaced by 14,000 subtelomeric probes. The average density of the probe coverage is 125 kb and 250-400 probes interrogate subtelomeric regions. To evaluate the array, we tested 15 samples (including subtelomeric aberrations and other microdeletion syndromes), which were previously analyzed by karyotyping and/or FISH. The concordance rate between array results and previous results is 100%. In addition we detected two novel aberrations that were not detected by karyotyping. These results demonstrate the utility of this format of array-CGH in detecting genome wide submicroscopic copy number changes as well as providing comprehensive coverage of all subteleomeric regions.

Association between gene expression profile and tumor invasion in oral squamous cell carcinoma.

There are limited studies attempting to correlate the expression changes in oral squamous cell carcinoma with clinically relevant variables. We determined the gene expression profile of 16 tumor and 4 normal tissues from 16 patients by means of Affymetrix Hu133A GeneChips. The hybridized RNA was isolated from cells obtained with laser capture microdissection, then was amplified and labeled using T7 polymerase-based in vitro transcription. The expression of 53 genes was found to differ significantly (33 upregulated, 20 downregulated) in normal versus tumor tissues under two independent statistical methods. The expression changes in four selected genes (LGALS1, MMP1, LAGY, and KRT4) were confirmed with reverse transcriptase polymerase chain reaction. Two-dimensional hierarchical clustering of the 53 genes resulted in the samples clustering according to the extent of tumor infiltration: normal epithelial tissue, tumors less than or equal to 4 cm in dimension, and tumors more than 4 cm in dimension (P = 0.0014). The same pattern of clustering was also observed for the 20 downregulated genes. We did not observe any associations with lymph node metastasis (P = 0.097).

High incidence of two methylenetetrahydrofolate reductase mutations (C677T and A1298C) in Hispanics.

Mutations in the 5,10-methylenetetrahydrofolate reductase (MTHFR) and coagulation factors II and V genes have been found at high frequencies in European and American Caucasian populations and are associated with increased risk for thrombophilia, premature coronary artery disease, and a variety of adverse pregnancy outcomes. Hispanic populations in the United States exhibit high levels of some of these conditions, so we initiated a population-based study to determine the frequency of these mutations (MTHFR C677T and A1298C, Factor II G20210A and Factor V G1691A) in this group. We find comparable frequencies of the Factors II and V mutations, but a high incidence of the two MTHFR mutations in a diverse sample of American Hispanics compared to those reported in Caucasians. Prospective studies of Hispanic women with these mutations and pregnancy outcomes will establish if there is a causal relationship.

Comprehensive genome-wide comparison of DNA and RNA level scan using microarray technology for identification of candidate cancer-related genes in the HL-60 cell line.

Genome-wide scans for DNA and RNA changes in the HL-60 cell line relative to normal leukocytes were conducted. Microarray-based comparative genome hybridization (CGH) studies were performed with the Spectral Genomics Human Bacterial Artificial Chromosome (BAC) 3MB system. Transcriptional measurements of approximately 12,500 human genes were monitored using Affymetrix U95A GeneChips. In HL-60, genomic DNA amplification of the 8q24 locus, trisomy 18, and deletions at loci 5q11.2 approximately q31, 6q12, 9p21.3 approximately p22, 10p12 approximately p15, 14q22 approximately q31, 17p12 approximately p13.3, and monosomy X were detected. After obtaining locus information about the RNA transcripts from the Affymetrix database, 4368 genes were stratified both according to status of RNA expression and the DNA copy number of their designated loci. The expression level of 2326 (53.25%) of 4368 transcripts is concordant with DNA copy number. Examples of specific, highly expressed, cancer-associated genes in amplified loci include SERPINB10, MYC, TYMS, HEC, and EPB41L3, while CD14, GZMK, TCF7, FOS, MLH3, CTNNA1, IRF1, VIM, CRK, MAP3K1, STAM, MAX, SFRG5, ENC1, PURA, MNT, RASA1, GLRX, UBE2B, NR3C1, PTENP1, BS69, COPEB, SKIP, PIM2, and MIC2 represent cancer-associated genes in deleted loci with decreased expression. The complementary usage of genome-wide DNA and RNA scans should enhance the identification of candidate genes in the neoplastic process.