MicroRNA expression changes in association with changes in interleukin-1ß/interleukin10 ratios produced by monocytes in autism spectrum disorders: their association with neuropsychiatric symptoms and comorbid conditions (observational study).

BACKGROUND: MicroRNAs (miRNAs) play a major role in regulating immune responses at post-transcriptional levels. Previously, we have reported fluctuating interlukine-1ß (IL-1ß)/IL-10 ratios produced by peripheral blood monocytes (PBMo) in some patients with autism spectrum disorders (ASD). This study examined whether changes in miRNA expression by PBMo are associated with changes in IL-1ß/IL-10 ratios and how such changes are associated with ASD clinical features. METHODS: miRNA expression by purified PBMo from ASD subjects (N = 69) and non-ASD controls (N = 27) were determined by high-throughput sequencing. Cytokine production by PBMo in responses to stimuli of innate immunity, and behavioral symptoms [assessed by aberrant behavioral checklist (ABC)] were also evaluated at the same time of sample obtainment. RESULTS: As a whole, there was no difference in miRNA expression between ASD and control non-ASD PBMo. However, when ASD cells were subdivided into 3 groups with high, normal, or low IL-1ß/IL-10 ratios as defined in the "Results" section, in comparison with the data obtained from non-ASD controls, we observed marked changes in miRNA expression. Namely, over 3-fold changes in expression of miR-181a, miR-93, miR-223, miR-342, and miR-1248 were observed in ASD PBMo with high or low IL-1ß/IL-10 ratios, but not in ASD PBMo with normal ratios. These miRNAs that had altered in expression are those closely associated with the regulation of key signaling pathways. With changes in IL-1ß/IL-10 ratios, we also observed changes in the production of cytokines (IL-6, TNF-α, and TGF-ß) other than IL-1ß/IL-10 by ASD PBMo. The association between behavioral symptoms and cytokine levels was different when ASD cells exhibit high/low IL-1ß/IL-10 ratios vs. when ASD cells exhibited normal ratios. Non-IgE-mediated food allergy was also observed at higher frequency in ASD subjects with high/low IL-1ß/IL-10 ratios than with normal ratios. CONCLUSIONS: Changes in cytokine profiles and miRNA expression by PBMo appear to be associated with changes in ASD behavioral symptoms. miRNAs that are altered in expression in ASD PBMo with high/low IL-1ß/IL-10 ratios are those associated with inflammatory responses. Changes in IL-1ß/IL-10 ratios along with changes in miRNA expression may serve as biomarkers for immune-mediated inflammation in ASD.

Noise filtering and nonparametric analysis of microarray data underscores discriminating markers of oral, prostate, lung, ovarian and breast cancer.

BACKGROUND: A major goal of cancer research is to identify discrete biomarkers that specifically characterize a given malignancy. These markers are useful in diagnosis, may identify potential targets for drug development, and can aid in evaluating treatment efficacy and predicting patient outcome. Microarray technology has enabled marker discovery from human cells by permitting measurement of steady-state mRNA levels derived from thousands of genes. However many challenging and unresolved issues regarding the acquisition and analysis of microarray data remain, such as accounting for both experimental and biological noise, transcripts whose expression profiles are not normally distributed, guidelines for statistical assessment of false positive/negative rates and comparing data derived from different research groups. This study addresses these issues using Affymetrix HG-U95A and HG-U133 GeneChip data derived from different research groups. RESULTS: We present here a simple non parametric approach coupled with noise filtering to identify sets of genes differentially expressed between the normal and cancer states in oral, breast, lung, prostate and ovarian tumors. An important feature of this study is the ability to integrate data from different laboratories, improving the analytical power of the individual results. One of the most interesting findings is the down regulation of genes involved in tissue differentiation. CONCLUSIONS: This study presents the development and application of a noise model that suppresses noise, limits false positives in the results, and allows integration of results from individual studies derived from different research groups.

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).

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.

Gene expression after crush injury of human saphenous vein: using microarrays to define the transcriptional profile.

BACKGROUND: Vein graft stenosis is believed to be the pathophysiologic response of vascular tissue to injury and is the major cause of vein graft failure. Therapeutic interventions might improve with knowledge of the physiologic pathways involved in the hyperplastic response to vascular injury. In this study, our purpose was to identify induced, early pathways that might be important in the human response to vascular injury. STUDY DESIGN: Human saphenous vein from 7 patients was organ cultured or crush injured and cultured for 48 or 72 hours after harvest. Gene expression was determined for syngeneic veins at harvest and at the experimental time points and compared to determine which genes were induced or repressed. Expressed genes (the transcriptional profile) were then assigned to functional physiologic classes. RESULTS: At 72 hours, in both organ-cultured and crush-injured vein, the gene for the Wnt ligand protein (WNT5A) was induced. At 48 hours in the organ-cultured vein only, the gene for the Frizzled protein (FZD2), a subunit of the Wnt receptor complex, was repressed. At 72 hours in injured vein only, the gene for the product of Wnt signaling (WISP1) was induced; the gene for the Wnt-binding, soluble Frizzled-related protein (FRZB) was repressed; and the gene for Dickkopf (DKK1) protein, which binds to the low density lipoprotein receptor-related protein subunit of the Wnt receptor complex, was induced. CONCLUSIONS: Early induction of WNT5A, coupled with the coordinated induction and repression of genes that modulate the Wnt signaling pathway, led to the early, selective induction of WISP1 and no other Wnt-inducible genes. This early, selective expression of a limited gene set might characterize the human vascular response to injury, and could enable development of therapies to treat the clinical sequelae of this response.

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.

Genome profiling of chondrosarcoma using oligonucleotide array-based comparative genomic hybridization.

Chondrosarcomas of the bone are malignant hyaline cartilage-forming tumors with an annual incidence rate of 3.6% of all primary bone malignancies in the United States. Specimens of 25 chondrosarcomas (10 grade I, 9 grade II, 1 grade III, and 5 dedifferentiated) from 23 patients were collected from the Department of Pathology at the University Hospital at UMDNJ-New Jersey Medical School from 1996 to 2007. Array-based comparative genomic hybridization (array-CGH) studies were performed on frozen tumor specimens. Recurrent deletions observed in at least in six tumors were 5q13.2, 5q14.2 approximately q21.3, 6q12 approximately q13, 6q16 approximately q25.3, 9p24.2 approximately q12, and 9p21.3. There was a statistically significant association between high-grade tumor (grade III and dedifferentiated) and the recurrent genetic deletions at 5q14.2 approximately q21.3, 6q16 approximately q25.3, 9p24.2 approximately q12, and 9p21.3. There is consistency between increased levels of aneuploidy and the progression of chondrosarcoma from lower to higher grades.

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.