Oligonucleotide Chip Research Articles

Outcome Prediction in CLL Patients Based on a Disease-Specific Oligonucleotide Microarray.

Chronic Lymphocytic Leukemia (CLL) is characterized by a extreme variability in the clinical course; some patients survive only a few months whereas others have stable disease for more than 10 years. Individual outcome prediction in CLL is still a challenge, in spite of the existence of multiple newly described prognostic factors: cytogenetic alterations, mutational IgVH status, and expression of surrogate markers such as ZAP-70. The accuracy of outcome prediction in CLL may improve thanks to the inclusion of new additional biological variables identified by high-throughput molecular techniques. This more reliable risk-stratification of patients could allow to assign a risk-adapted individualized therapy. With this aim, we have analysed with oligonucleotide microarrays three different series of CLL patients in a three-step process.In a first-step, RNA extracted from tumour samples of a series of 26 CLL patients, was hybridised using commercial 22K oligonucleotide chips (Agilent Technologies). A total of 135 genes whose expression was associated with changes in survival probability were identified. An additional review of published data yielded a total number of 497 genes whose expression has been related with outcome in CLL. A CLL specific chip was then built in a second-step including, besides these 632 genes, control genes until a total of 1023 genes. This comprehensive CLL-chip was then hybridised with RNA extracted from peripheral-blood samples in a new series of consecutively diagnosed 100 CLL patients. Cox's univariate analysis was performed to look for molecular variables associated with Progression Free Survival (PFS) as endpoint. FDR significance levels were also used when appropriate, for adjusting the associated p-value. Expression of 94 genes was significantly associated with PFS. Profile patterns of the 94 resulting genes were clustered, and allow to identify 12 clusters. Based on biological function, we selected a representing gene of each cluster, to perform a multivariate Cox's analysis. This approach generated a model of 4 genes, implicated in BCR signalling, signal transduction and transcription regulation, that distinguishes four quartiles of CLL patients with PFS at 5 years of 95%, 70%, 44% and 8% respectively.These results have been validated in a new independent series of 60 consecutively diagnosed cases of CLL. If confirmed in additional studies, the availability of this CLL-specific chip could allow a more accurate prediction of the clinical course in CLL patients, this giving support to a stratification of patients into different risk-groups

Logistic Broken Adaptive Ridge Procedure for Colon Cancer Data Analysis

Background: Colon cancer is the leading cause of cancer-related deaths in the world in both man and women. Knowing the causes and risk factors for colon cancer can help you understanding the importance of routine screening for colon cancer, as well as learn if you are one of the people who should begin screening at the earlier age. Due to the limitation of clinical diagnose, management and treatment outcomes, it is of great necessity to develop effective methods for colon cancer detection and prediction especially cDNA Microarrays and high- density oligonucleotide chips are increasingly used in cancer research. Methods: Here we propose a novel logistic broken adaptive ridge procedure to address the problem of colon cancer results prediction through selecting effective few variables or genes from 2000 candidate genes. Results: In total 62 cases with 40 colon cancer patients and 22 healthy patients were included in our analysis. Each case consists of 2000 genes which challenged all the competitive method. From the results, we are so surprised that our proposed method outperforms the classical variable selection approaches in error rate of training data and extra testing data. Conclusions: Logistic adaptive ridge procedure is very effective for colon cancer predictions, either in terms of prognosis or diagnose. It may benefit patients by guiding therapeutic options. We hope it will contribute to the wider biology and related communities.

Treatment of allergic rhinitis with acupoint herbal plaster: an oligonucleotide chip analysis.

BackgroundAllergic rhinitis is regarded as an imbalanced Th1/Th2 cell-mediated response. The present study used microarray analysis to compare gene expression levels between allergic rhinitis patients before and after a series of acupoint herbal plaster applications.MethodsIn this experimental pilot study, volunteers experiencing sneezing, runny nose, and congestion for more than 9 months in the year following initial diagnoses were included after diagnostic confirmation by otolaryngologists to exclude patients with sinusitis and nasal polyps. Patients with persistent allergic rhinitis each received four acupoint herbal plaster treatments applied using the moxibustion technique. Clinical outcomes were evaluated using the Rhinitis Quality of Life Questionnaire (RQLQ). Peripheral blood samples were analyzed using an ImmunoCAP Phadiatop test, and patients were classified as phadiatop (Ph)-positive or -negative. Microarray results were analyzed for genes that were differentially expressed between (1) Ph-positive and -negative patients treated with herbal plaster; and (2) before and after herbal plaster treatment in the Ph-positive patient group. Unsupervised and supervised methods were used for gene-expression data analysis.ResultsNineteen Ph-positive and four Ph-negative participants with persistent allergic rhinitis were included in the study. RQLQ results indicated that the 19 Ph-positive volunteers experienced improvement in six of seven categories following acupoint herbal plaster treatments, whereas the four Ph-negative participants reported improvement in only two categories. Hierarchical clustering and principle component analysis of the gene expression profiles of Ph-positive and –negative participants indicated the groups exhibited distinct physiological responses to acupoint herbal treatment. Evaluation of gene networks using MetaCore identified that the “Immune response_IL-13 signaling via JAK-STAT” and the “Inflammation_Interferon signaling” were down- and up-regulated, respectively, among Ph-positive subjects.ConclusionsIn this preliminary study, we find that the IL-13 immune response via JAK-STAT signaling and interferon inflammation signaling were down- and upregulated, respectively, in the Ph-positive group. Further studies are required to verify these pathways in Ph-positive patients, and to determine the mechanism of such pathway dysregulation.Trial registrationClinicalTrials.gov: NCT02486159. Registered 30 Jun 2015.

Genome-wide analysis of transcription and photosynthesis inhibition in the harmful dinoflagellate Prorocentrum minimum in response to the biocide copper sulfate

Copper is an essential trace metal for organisms; however, excess copper may damage cellular processes. Their efficiency and physiological effects of biocides have been well documented; however, molecular transcriptome responses to biocides are insufficiently studied. In the present study, a 6.0K oligonucleotide chip was developed to investigate the molecular responses of the harmful dinoflagellate Prorocentrum minimum to copper sulfate (CuSO4) treatment. The results revealed that 515 genes (approximately 8.6%) responded to CuSO4, defined as being within a 2-fold change. Further, KEGG pathway analysis showed that differentially expressed genes (DEGs) were involved in ribosomal function, RNA transport, carbon metabolism, biosynthesis of amino acids, photosystem maintenance, and other cellular processes. Among the DEGs, 49 genes were related to chloroplasts and mitochondria. Furthermore, the genes involved in the RAS signaling pathway, MAPK signaling pathway, and transport pathways were identified. An additional experiment showed that the photosynthesis efficiency decreased considerably, and reactive oxygen species (ROS) production increased in P. minimum after CuSO4 exposure. These results suggest that CuSO4 caused cellular oxidative stress in P. minimum, affecting the ribosome and mitochondria, and severely damaged the photosystem. These effects may potentially lead to cell death, although the dinoflagellate has developed a complex signal transduction process to combat copper toxicity.

Development of aptamer-modified SERS nanosensor and oligonucleotide chip to quantitatively detect melamine in milk with high sensitivity

Herein, we developed a novel nanosenor to detect trace amount of melamine (M) in the milk using aptamer-modified surface-enhanced Raman scattering (SERS) nanosensor and oligonucleotide chip. The SERS nanosensor was prepared by attaching Raman tag and ploy-thymine (T) aptamer to the gold nanoparticles, respectively. SERS nanosensor can be absorbed to the oligonucleotide chip with a high specificity where a “T–M–T” structure via multi-hydrogen-bond could be formed between the melamine molecule and the thymine molecule. In the measurement, the Raman signal intensities obtained from the chip increase when the concentrations of melamine in solutions increase. The proposed method allows us to detect melamine with a limit of 1.0ppt (1.0pgmL−1), which is fall far below the strictest safety limit. Compared with the previous methods, the sensitivity of this sensor shows about 100-fold improvement. The advantages of the present sensor are its low detection cost and the simplified sample pretreatment. Furthermore, the reliable and enough accurate results have been obtained by the using of the proposed nanosensor in the assay of trace melamine in real milk sample.

First Report of Clover yellow vein virus on White Clover (Trifolium repens) in South Korea.

White clover (Trifolium repens L.) is a herbaceous, perennial plant that has become one of the most widely distributed legumes in the world. It is extensively used in grass-legume pastures, but also has the potential to invade agricultural lands and natural ecosystems. White clover is a well-known natural host for Alfalfa mosaic virus (AMV), Clover yellow vein virus (ClYVV), Soybean dwarf virus (SbDV), Beet western virus (BWYV), Tomato spotted wilt virus (TSWV), Zucchini yellow mosaic virus (ZYMV), etc (1). In July 2013, during a survey to determine the presence of different viruses infecting weed plants in South Korea, three white clover leaf samples showing yellow mosaic symptoms were collected from Taean County, South Chungcheong Do Province, South Korea. In order to identify the infecting virus, total RNA from three leaf samples was extracted using the Tri-reagent (MRC Reagent, Inc., OH) as described by the manufacturer, and was applied to the large-scale oligonucleotide (LSON) chip (3), wherein probes specific to a ClYVV isolate produced a positive reaction. All three samples tested were positive for ClYVV. To confirm this result, ClYVV-specific primers were designed using the sequences of four ClYVV isolates from NCBI (GenBank Accession Nos. AF185959, AF203536, DQ333346, and NC003536). Total RNA was extracted from symptomatic white clover samples using Easy-Spin Total RNA Extraction Kit (iNtRon, Daejeon, Korea) and used as template for RT-PCR. The positive control RNA was used from ClYVV GM isolate (KF975894) and negative control RNA used symptomless white clover plants. The ClYVV coat protein (CP) gene was amplified by RT-PCR using the specific primer pairs ClYVV-CP-F / ClYVV-CP-R (5'-CAAGAGCAGCACGATGAG-3' and 5'-CTCGCTCTATAAAGATCAGAT-3'). DNA fragments of the expected size (1,042 bp) were obtained from the white clover Korea isolate (AB930132), and the PCR product was cloned into a T&A cloning vector (RBC Bioscience, Taipei, Taiwan) and sequenced directly in both directions. BLAST analyses of the nucleotide sequence CP gene fragments revealed the highest identity with 98% with other ClYVV isolates (AF203536). To determine the experimental host range of the ClYVV Korea isolate, we inoculated five species (Chenopodium amaranticolor, C. quinoa, Nicotiana clevelandii, N. benthamiana, and Trifolium repens) in three families using this isolate. All test plants were mechanically inoculated with 0.1 M phosphate buffered saline (Takara, Tokyo, Japan). Each test plant was inoculated nine times and grown in a greenhouse maintained at 27 to 33°C. Necrotic local lesions were produced on inoculated leaves of C. amaranticolor, C. quinoa, and N. clevelandii 4 to 6 days post-inoculation. After 10 to 14 days, C. amaranticolor and C. quinoa showed systemic chlorotic spot symptoms, and N. clevelandii, N. benthamiana, and T. repens showed chlorotic spot, mild mosaic, and mosaic in the upper leaves, respectively. Up to now, in South Korea, ClYVV has been detected in gladiolus (Gladiolus gandavensis) (3) and soybean (Glycine max) (4). ClYVV can be easily transmitted by insect, aphid, or mechanical inoculation and has a host range including tobacco, soybean, etc. The presence of ClYVV could become an important threat to crop production in South Korea. To our knowledge, this is the first report of a ClYVV infection of the white clover plant in South Korea.

Analysis of dose-response to hexanal-induced gene expression in A549 human alveolar cells

The problems of analyzing dose effects on gene expression are gaining attention in toxicological research. Determining how gene expression profiles change with toxicant dose will improve the utility of arrays in identifying biomarkers and elucidating their modes of toxic action. In the present study, we focused on determining the dose-dependent alterations of gene expression profiles with hexanal exposure and we identified the possible biomarkers of hexanal in A549 human alveolar cells. A549 cells were exposed to a 5% inhibitory concentration (IC5) and a 20% inhibitory concentration (IC20) of hexanal for 48 h. Through microarray analysis using an oligonucleotide chip, we identified that the gene expression patterns were differentially shown in the control group and the hexanalexposed groups. The hexanal-exposed groups are more sensitive to gene alteration than the control group, and gene expressions are more significantly altered in the IC20 exposure group than in the IC5 exposure group. With clustering analysis of gene expression profiles, we identified 2,929 IC5− and 3,678 IC20-specific genes, and 302 dose-dependently expressed genes. Gene ontology (GO) analysis with 246 annotated genes of the 302 dosedependent expressed genes showed correlation with the key biological processes involved in neurological system processes, immune system development, cell activation, and cell-cell signaling. In conclusion, current study describes alterations in gene expression profiles in response to exposure to different doses of hexanal and related toxic pathways induced by significantly expressed genes. Moreover, novel genes and pathways that could potentially play a role in the prevention of respiratory disease due to aldehydes are identified.

Microarray-Based Gene Expression Profiling to Elucidate Effectiveness of Fermented Codonopsis lanceolata in Mice

In this study, the effect of Codonopsis lanceolata fermented by lactic acid on controlling gene expression levels related to obesity was observed in an oligonucleotide chip microarray. Among 8170 genes, 393 genes were up regulated and 760 genes were down regulated in feeding the fermented C. lanceolata (FCL). Another 374 genes were up regulated and 527 genes down regulated without feeding the sample. The genes were not affected by the FCL sample. It was interesting that among those genes, Chytochrome P450, Dmbt1, LOC76487, and thyroid hormones, etc., were mostly up or down regulated. These genes are more related to lipid synthesis. We could conclude that the FCL possibly controlled the gene expression levels related to lipid synthesis, which resulted in reducing obesity. However, more detailed protein expression experiments should be carried out.

Development of the large-scale oligonucleotide chip for the diagnosis of plant viruses and its practical use.

A large-scale oligonucleotide (LSON) chip was developed for the detection of the plant viruses with known genetic information. The LSON chip contains two sets of 3,978 probes for 538 species of targets including plant viruses, satellite RNAs and viroids. A hundred forty thousand probes, consisting of isolate-, species- and genus-specific probes respectively, are designed from 20,000 of independent nucleotide sequence of plant viruses. Based on the economic importance, the amount of genome information, and the number of strains and/or isolates, one to fifty-one probes for each target virus are selected and spotted on the chip. The standard and field samples for the analysis of the LSON chip have been prepared and tested by RT-PCR. The probe’s specific and/or nonspecific reaction patterns by LSON chip allow us to diagnose the unidentified viruses. Thus, the LSON chip in this study could be highly useful for the detection of unexpected plant viruses, the monitoring of emerging viruses and the fluctuation of the population of major viruses in each plant.

Human Krüppel-related 3 (HKR3) Is a Novel Transcription Activator of Alternate Reading Frame (ARF) Gene

HKR3 (Human Krüppel-related 3) is a novel POK (POZ-domain Krüppel-like zinc-finger) family transcription factor. Recently, some of the POK (POZ-domain Krüppel-like zinc finger) family proteins have been shown to play roles in cell cycle arrest, apoptosis, cell proliferation, and oncogenesis. We investigated whether HKR3, an inhibitor of cell proliferation and an uncharacterized POK family protein, could regulate the cell cycle by controlling expression of genes within the p53 pathway (ARF-MDM2-TP53-p21WAF/CDKN1A). HKR3 potently activated the transcription of the tumor suppressor gene ARF by acting on the proximal promoter region (bp, -149∼+53), which contains Sp1 and FBI-1 binding elements (FREs). HKR3 interacted with the co-activator p300 to activate ARF transcription, which increased the acetylation of histones H3 and H4 within the proximal promoter. Oligonucleotide pull-down assays and ChIP assays revealed that HKR3 interferes with the binding of the proto-oncogenic transcription repressor FBI-1 to proximal FREs, thus derepressing ARF transcription.

Massively Parallel Patterning of Complex 2D and 3D Functional Polymer Brushes by Polymer Pen Lithography

We report the first demonstration of centimeter-area serial patterning of complex 2D and 3D functional polymer brushes by high-throughput polymer pen lithography. Arbitrary 2D and 3D structures of poly(glycidyl methacrylate) (PGMA) brushes are fabricated over areas as large as 2 cm × 1 cm, with a remarkable throughput being 3 orders of magnitudes higher than the state-of-the-arts. Patterned PGMA brushes are further employed as resist for fabricating Au micro/nanostructures and hard molds for the subsequent replica molding of soft stamps. On the other hand, these 2D and 3D PGMA brushes are also utilized as robust and versatile platforms for the immobilization of bioactive molecules to form 2D and 3D patterned DNA oligonucleotide and protein chips. Therefore, this low-cost, yet high-throughput "bench-top" serial fabrication method can be readily applied to a wide range of fields including micro/nanofabrication, optics and electronics, smart surfaces, and biorelated studies.

DepthTools: an R package for a robust analysis of gene expression data

BackgroundThe use of DNA microarrays and oligonucleotide chips of high density in modern biomedical research provides complex, high dimensional data which have been proven to convey crucial information about gene expression levels and to play an important role in disease diagnosis. Therefore, there is a need for developing new, robust statistical techniques to analyze these data.ResultsdepthTools is an R package for a robust statistical analysis of gene expression data, based on an efficient implementation of a feasible notion of depth, the Modified Band Depth. This software includes several visualization and inference tools successfully applied to high dimensional gene expression data. A user-friendly interface is also provided via an R-commander plugin.ConclusionWe illustrate the utility of the depthTools package, that could be used, for instance, to achieve a better understanding of genome-level variation between tumors and to facilitate the development of personalized treatments.

Decreased defense gene expression in tolerance versus resistance to Verticillium dahliae in potato

Verticillium dahliae Kleb., a soil-borne fungus that colonizes vascular tissues, induces wilting, chlorosis and early senescence in potato. Difference in senescence timing found in two diploid potato clones, 07506-01 and 12120-03, was studied and genetic variation in response to V. dahliae infection was identified as a causal factor. The clone, 07506-01, was infected with V. dahliae but did not develop symptoms, indicating tolerance to the pathogen. The other diploid clone, 12120-03 had low levels of pathogen with infection and moderate symptoms indicating partial resistance. 07506-01 was found to carry two susceptible alleles of the Ve2 gene and 12120-03 carried one Ve2 resistant and one susceptible allele. Infected leaves of the two clones were compared using gene expression profiling with the Potato Oligonucleotide Chip Initiative (POCI) microrarray. The results provide further evidence for differences in response of the two clones to infection with V. dahliae. Chlorophyll biosynthesis was higher in the tolerant 07506-01 compared to partially resistant 12120-03. On the other hand, expression of fungal defense genes, Ve resistance genes and defense phytohormone biosynthetic enzyme genes was decreased in 07506-01 compared to 12120-03 suggesting defense responses were suppressed in tolerance compared to resistance. Transcription factor gene expression differences pointed to the WRKY family as potential regulators of V. dahliae responses in potato.

Analysis of mRNA expression profiles highlights alterations in modulation of the DNA damage-related genes under butanal exposure in A549 human alveolar epithelial cells

Butanal is a lower-molecular-weight saturated aliphatic aldehyde (LSAA) that is known as one of the important causes of indoor pollution. Although a few studies have been reported, the toxicity of butanal and its underlying mechanisms remain unclear. This study focused on toxicity of butanal with microarray analysis relationships of gene expression patterns between vehicle control and two different concentrations of butanal-treated groups. For a comprehensive examination of butanal effects on gene expression, we exposed human alveolar epithelial (A549) cells to IC5 and IC20 for 48 h and compared the gene expression of treated cells with that of vehicle controls using a human oligonucleotide chip. We identified genes that were changed by more than 1.5-fold through gene expression profile analysis. Transcriptomic profiling indicated different gene expression patterns in vehicle control and butanal-exposed groups and showed that the butanal-exposure group had a higher sensitivity to gene alteration than the control group. Through gene expression profile cluster analysis, we identified 2,998 (1,379 up- and 1,619 down-regulated) in the butanal IC5 groups and 3,200 genes (1,369 up- and 1,831 downregulated) in the butanal IC20 groups, with 2,710 (1,281 up- and 1,429 down-regulated) specific genes in common. Through Gene Ontology (GO) analysis with these commonly expressed genes reported the several key biological processes which are known as related to DNA damage responses such as “regulation of apoptosis”, “regulation of cell proliferation”, “immune response”, “inflammatory response”. We confirmed the functional changes using a cellular assay.

Gene expression profiling of low dose exposure of saturated aliphatic aldehydes in A549 human alveolar epithelial cells

Low-molecular weight saturated aliphatic aldehydes (LSAAs), which include propanal, butanal, pentanal, hexanal, octanal, nonanal and heptanal, are volatile organic compounds (VOCs). They are ubiquitous in the environment of our daily life. Although LSAAs are harmful, with mutagenic and carcinogenic effects, the mechanisms underlying the toxicity of volatile aldehydes are still unclear. Therefore, in this study, we performed genome-wide expression profile analysis of A549 human alveolar epithelial cells exposed to seven LSSAs. We selected genes whose expression was changed more than 1.5-fold in A549 cells exposed to LSAAs by analysis of gene expression profiles using human oligonucleotide chips. Through gene expression profiling, we showed that LSSAs are related to the key biological processes “defense response”, “inflammatory response” and “immune response” in gene ontology (GO) analysis. In addition, we identified two genes that were up-regulated (GREB1, BC009808) and four that were down-regulated (UCP1, TCP11, FNDC3A, LOC645206) by all the tested LSAAs. Our data suggest that LSAAs exert toxic effects on A549 cells by modulating mRNA expression. Moreover, we suggest that genes expressed in response to LSAAs represent a molecular signature that can be widely used, in combination with more traditional techniques, to assess and predict the toxicity caused by exposure to LSAAs.

Comprehensive genome characterization of solitary fibrous tumors using high‐resolution array‐based comparative genomic hybridization

Solitary fibrous tumors (SFTs) are rare spindle cell tumors with limited therapeutic options. Their molecular basis is poorly known. No consistent cytogenetic abnormality has been reported. We used high-resolution whole-genome array-based comparative genomic hybridization (Agilent 244K oligonucleotide chips) to profile 47 samples, meningeal in >75% of cases. Few copy number aberrations (CNAs) were observed. Sixty-eight percent of samples did not show any gene CNA after exclusion of probes located in regions with referenced copy number variation (CNV). Only low-level CNAs were observed. The genomic profiles were very homogeneous among samples. No molecular class was revealed by clustering of DNA copy numbers. All cases displayed a "simplex" profile. No recurrent CNA was identified. Imbalances occurring in >20%, such as the gain of 8p11.23-11.22 region, contained known CNVs. The 13q14.11-13q31.1 region (lost in 4% of cases) was the largest altered region and contained the lowest percentage of genes with referenced CNVs. A total of 425 genes without CNV showed copy number transition in at least one sample, but only but only 1 in at least 10% of samples. The genomic profiles of meningeal and extra-meningeal cases did not show any differences.

Benzo[k]fluoranthene-induced changes in miRNA-mRNA interactions in human hepatocytes

Toxicology studies assessing the risk of environmental toxicants in humans frequently use in vitro systems in combination with transcriptomics to characterize toxic responses. Thus far, changes have mostly been investigated at the mRNA level. Recently, microRNAs (miRNAs) have attracted attention because they are powerful negative regulators of mRNA levels and thus may be responsible for the modulation of important mRNA networks implicated in toxicity. This study aimed to identify possible miRNA-mRNA networks as novel interactions at the gene expression level after exposure to environmental toxicants. Benzo[k]fluoranthene (BF), a polycyclic aromatic hydrocarbon that is ubiquitously distributed throughout the environment, was used. We analyzed mRNA and miRNA profiles in HepG2 cells, a human liver cell line, using a human oligonucleotide chip. Changes in miRNA expression in response to BF, in combination with multiple alterations of mRNA levels, were observed. Many of the altered mRNAs were targets of altered miRNAs. Using gene ontology (GO) and KEGG pathway analysis, we determined the relevance of such miRNA deregulation to carcinogenicity. This revealed five miRNAs that appear to participate in specific BFresponsive pathways relevant to genotoxicity and carcinogenicity, such as DNA damage repair, apoptosis, cancer, VEGF signaling, and Jak-STAT signaling. Our results indicate that miR-146a, miR-365, let-7f, miR-199b-5p, and miR-30c-1* are novel players in the BF response. Therefore, this study demonstrates the added value of an integrated miRNA-mRNA approach for identification of the molecular mechanisms induced by BF in an in vitro human model.

SEMA3A deletion in a family with Kallmann syndrome validates the role of semaphorin 3A in human puberty and olfactory system development

Kallmann syndrome (KS) is a genetic disorder associating pubertal failure with congenitally absent or impaired sense of smell. KS is related to defective neuronal development affecting both the migration of olfactory nerve endings and GnRH neurons. The discovery of several genetic mutations responsible for KS led to the identification of signaling pathways involved in these processes, but the mutations so far identified account for only 30% of cases of KS. Here, we attempted to identify new genes responsible for KS by using a pan-genomic approach. From a cohort of 120 KS patients, we selected 48 propositi with no mutations in known KS genes. They were analyzed by comparative genomic hybridization array, using Agilent 105K oligonucleotide chips with a mean resolution of 50 kb. One propositus was found to have a heterozygous deletion of 213 kb at locus 7q21.11, confirmed by real-time qPCR, deleting 11 of the 17 SEMA3A exons. This deletion cosegregated in the propositus' family with the KS phenotype, that was transmitted in autosomal dominant fashion and was not associated with other neurological or non-neurological clinical disorders. SEMA3A codes for semaphorin 3A, a protein that interacts with neuropilins. Mice lacking semaphorin 3A expression have been showed to have a Kallmann-like phenotype. SEMA3A is therefore a new gene whose loss-of-function is involved in KS. These findings validate the specific role of semaphorin 3A in the development of the olfactory system and in neuronal control of puberty in humans.

Gene expression analysis identifies DNA damage-related markers of benzo[a]pyrene exposure in HepG2 human hepatocytes

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) that is carcinogenic to humans. Although the environmental distribution and metabolism of BaP have been reported and many researchers are performing risk-assessment and toxicological studies of BaP by means of physical and chemical measurements, only a few studies have examined the expression of mRNAs and their functions in BaP-induced toxicity. Toxicogenomic technology, a new paradigm in toxicity screening, is a useful approach for evaluating the toxic properties of environmental pollutants. We analyzed gene expression profiles using human oligonucleotide chips and identified genes in human hepatocellular carcinoma (HepG2) cells whose expression changed > 1.5-fold after exposure to BaP. The expression of 4,048 and 3,926 genes was up-and down-regulated > 1.5-fold (P < 0.01), respectively, after exposure. Gene ontology (GO) analysis of these genes revealed significant enrichment in several key biological processes related to DNA damage, including DNA repair, cell cycle arrest, and apoptosis. We also performed a contrastive study of cellular effects in HepG2 cells exposed to BaP, and identified increased expression of related genes, cell cycle arrest, and apoptotic cells. These results suggest that genetic markers of BaP-induced toxicity may be molecular blueprints that can be more widely implemented in combination with more traditional techniques for assessment and prediction.

Dose-response functional gene analysis by exposure to 3 different polycyclic aromatic hydrocarbons in human hepatocytes

Polycyclic aromatic hydrocarbons (PAHs) are known as carcinogen and have been studied to show modulation of gene expression by exposure to various PAHs. However, few studies have been reported on microarray analysis of dose-response relationships of gene expression patterns. For comprehensive examination of dose-response effects of PAHs on gene expression, we elicited the genes which were changed more than 1.5-fold by analysis of gene expression profiles in human hepatocellular carcinoma (HepG2) cells, exposed for 48 h to nontoxic (NT) and IC20 doses of 3 different PAHs (benzo[a]anthracene (B[a]A), benzo[k]fluoranthene (B[k]F) and indeno[1,2,3-c,d]pyrene (IND)) by using human oligonucleotide chip. Transcriptomic profiling shows different gene expression patterns in NT and IC20 exposure groups and shows higher sensitivity to gene alteration in IC20 exposure group than NT group. Through the clustering analysis of gene expression profiles, we identified 7 up- and 3 down-regulated NT dose specific genes and 401 up- and 562 downregulated IC20 dose specific genes. After Gene Ontology (GO) analysis on IC20 dose specific genes, we determined several key pathways which are known as related to increase hepatotoxicity such as metabolism of xenobiotics by cytochrome P450, Jak-STAT signaling pathway, cytokine-cytokine receptor interaction and complement and coagulation cascade. But we did not find hepatotoxicity-related pathways through GO analysis on NT dose-specific genes. Genes that are expressed in only IC20 exposure group were regarded as biomarker of PAHs-induced hepatotoxicity. In conclusion, this study describes changes in gene expression profiles in hepatocytes in response to exposure to 3 PAHs with different doses and relates these gene expression changes to hepatotoxicity related pathways. Moreover, potential new leads to genes and pathways that could play a role in liver disease prevention by PAHs were identified.