Frequency of 23 human papillomavirus types using DNA microarray in women with and without cytological anomalies.

OBJECTIVE: To evaluate the frequency of single and multiple human papillomavirus (HPV) infections in women with and without cervical dysplasia. MATERIALS AND METHODS: An oligonucleotide microarray system was used to detect 19 types of high-risk HPV (HPV-16/-18/-311-33/-35/-39/-45/-51/ -52/-53/-56/-58/-59/-66/-68/-73/-821-16 variant E-E6-G350/-16 variant E-E6-T350) and 4 types of low-risk HPV (HPV-6/-11/ -42/-44) in 122 consecutive women visiting our colposcopy outpatient clinic classified into controls (normal epithelium, nonspecific cervicitis, metaplasia; n = 56) and cervical intraepithelial neoplasia (CIN) (n = 66). RESULTS: In 78/122 (64%) cervical samples, HPV DNA was detected. Compared to controls, HPV infection was significantly more prevalent among women with CIN (8/56 [14%] versus 49/66 [74%]; p = 0.001). HPV-18 and HPV-16 were the most common HPV types in all specimens (25% [31/122] and 25% [31/122], respectively). Of note, HPV-16 was significantly more frequent in women with CIN compared to controls (35% [23/66] vs. 14% [8/56], respectively; p = 0.02). Double HPV infections were detected in 16/122 (13%) and multiple infections in 43/122 (35%) women. Multiple HPV infections were found significantly more often among women with CIN compared to controls (30/66 [45%] vs. 13/56 [23%], respectively; p = 0.002). Using a univariate and multivariate logistic regression model to estimate the relative risk of CIN vs. HPV type, HPV-16-positive cases were found to have the highest risk of CIN (odds ratio [OR] 3.2; 95% confidence interval [CI] 1.3-7.9; p = 0.002). CONCLUSION: Multiple HPV infections are common in women with and without CIN, but significantly more prevalent among women with CIN compared to controls.

Evidence for an association between mannose-binding lectin 2 (MBL2) gene polymorphisms and pre-term birth.

PURPOSE: Human mannose-binding lectin, encoded by the MBL2 gene, is an important component of innate immunity and an important regulator of inflammatory processes. MBL2 gene polymorphisms are associated with an increased risk of neonatal infections and some data suggest a relation between the maternal MBL2 genotype and the risk of premature delivery. In this study, we evaluated whether there is an association between the fetal MBL2 genotype and prematurity. METHODS: A microarray-based on-chip PCR method was used to simultaneously detect five common MBL2 polymorphisms (codon 52, 54, 57; promoter -550, -221) in 204 DNA samples isolated from archival blood cards. MBL2 genotypes of infants born before the 36th week of pregnancy (N = 102) were compared to a control group of infants born at term after the 37th week (N = 102). RESULTS: The frequency of the codon 52 polymorphism was significantly higher in the pre-term group compared to the term group (10.8% versus 4.9%, P = 0.04), while the frequency of the codon 54 polymorphism was equal in both groups (11.3% versus 11.8%). Interestingly, carriers of genotypes (O/O) likely conferring deficient MBL plasma levels were more common in the group of premature birth (9.8% versus 2.9%, P = 0.05), while the promoter -550 C/C genotype was underrepresented in the pre-term birth group (24.5% versus 39.2%, P = 0.03). CONCLUSION: Our data add to the knowledge about genetic predisposition to prematurity and suggest that the fetal MBL2 genotype might be an additional genetic factor contributing to the risk of premature delivery.

Microarray-based detection of mannose-binding lectin 2 (MBL2) polymorphisms in a routine clinical setting.

The assessment of allelic variants in the human mannose-binding lectin 2 (MBL2) gene is of great clinical importance in newborns or immune-suppressed patients at high risk for a variety of infections. Here, we present a study on the genotyping accuracy of a DNA microarray-based on-chip PCR method suited for the detection of five different polymorphisms in the MBL2 gene. We tested 153 genomic DNA samples, prepared from archival blood spots on Guthrie cards, for the presence of allelic variants in the human MBL2 gene by the on-chip PCR method and compared the obtained results of three variants to standard DNA capillary sequencing. The genotyping power of the described assay was readily comparable to DNA sequencing (453/459 correct genotype calls in 153 DNA samples; 98.7% accuracy), mainly due to intrinsic technical benefits of microarrays such as high number of test replicates and automated data analysis. This study demonstrates, for the first time, the accuracy and reliability of a microarray-based on-chip PCR genotyping assay for measuring allelic variants in a routine clinical setting.

Dissecting progressive stages of 5-fluorouracil resistance in vitro using RNA expression profiling.

Resistance to anticancer drugs such as the widely used antimetabolite 5-fluorouracil (FU) is one of the most important obstacles to cancer chemotherapy. Using GeneChip arrays, we compared the expression profile of different stages of FU resistance in colon cancer cells after in vitro selection of low-, intermediate- and high-resistance phenotypes. Drug resistance was associated with significant changes in expression of 330 genes, mainly during early or intermediate stage. Functional annotation revealed a majority of genes involved in signal transduction, cell adhesion and cytoskeleton with subsequent alterations in apoptotic response, cell cycle control, drug transport, fluoropyrimidine metabolism and DNA repair. A set of 33 genes distinguished all resistant subclones from sensitive progenitor cells. In the early stage, downregulation of collagens and keratins, together with upregulation of profilin 2 and ICAM-2, suggested cytoskeletal changes and cell adhesion remodeling. Interestingly, 6 members of the S100 calcium-binding protein family were suppressed. Acquisition of the intermediate-resistance phenotype included upregulation of the well-known drug resistance gene ABCC6 (ATP-binding cassette subfamily C member 6). The very small number of genes affected during transition to high resistance included the primary FU target thymidylate synthase. Although limited to an in vitro model, our data suggest that resistance to FU cannot be explained by known mechanisms alone and substantially involves a wide molecular repertoire. This study emphasizes the understanding of resistance as a time-depending process: the cell is particularly challenged at the beginning of this process, while acquisition of the high-resistance phenotype seems to be less demanding.

Estrogen-metabolizing gene polymorphisms in the assessment of breast carcinoma risk and fibroadenoma risk in Caucasian women.

BACKGROUND: Genes encoding enzymes involved in estrogen metabolism are held to be candidate genes for associations with breast disease. In these candidate genes, no critical combination of single-nucleotide polymorphisms (SNPs) for assessing breast carcinoma risk has been reported to date. METHODS: In a large case-control study, the authors investigated 10 estrogen-metabolizing SNPs in 396 patients with breast carcinoma, 154 patients with fibroadenoma, and 1936 healthy control patients without breast carcinoma in their personal history. The following 10 SNPs were analyzed using sequencing-on-chip technology via a solid-phase polymerase chain reaction assay performed on oligonucleotide microarrays: catechol-O-methyltransferase Val158Met G-->A, 17-beta-hydroxysteroid dehydrogenase type 1 vIV A-->C, cytochrome P-450 (CYP) family 17 A2 allele T-->C, CYP1A1-1 MspI restriction fragment length polymorphism (RFLP) T-->C, CYP1A1-2 Ile462Val A-->G, CYP19-1 Trp39Arg T-->C, CYP19-2 Arg264Cys C-->T, CYP19-3 Cys1558Thr C-->T, steroid-5-alpha reductase type 2 Val89Leu G-->C, and vitamin D receptor BsmI RFLP. A total of 21,350 genotypes were evaluated. Associations and two-way interaction models were calculated using stepwise logistic regression. RESULTS: In a multiple model, CYP1A1-1 (P = 0.004) and CYP1A1-2 (P = 0.03) were found to be associated with significantly decreased and increased risks of breast carcinoma, respectively. When two-way interactions involving investigated SNPs were ascertained, no significant interactions among polymorphisms were noted. Comparison of patients with fibroadenoma with control patients revealed significantly increased and decreased risks of fibroadenoma when the mutant alleles of CYP17 (P = 0.02) and CYP1A1-1 (P = 0.04), respectively, were present. CONCLUSIONS: The authors obtained the first SNP data indicating that CYP17 and CYP1A1-1 play a role in the pathogenesis of fibroadenoma. Although the authors were not able to develop interaction models involving SNPs, they did provide evidence that CYP1A1 is a low-penetrance susceptibility gene with respect to breast carcinoma in a large series of Caucasian women.

Microarray-based identification of bacteria in clinical samples by solid-phase PCR amplification of 23S ribosomal DNA sequences.

The rapid identification of the bacteria in clinical samples is important for patient management and antimicrobial therapy. We describe a DNA microarray-based PCR approach for the quick detection and identification of bacteria from cervical swab specimens from mares. This on-chip PCR method combines the amplification of a variable region of bacterial 23S ribosomal DNA and the simultaneous sequence-specific detection on a solid phase. The solid phase contains bacterial species-specific primers covalently bound to a glass support. During the solid-phase amplification reaction the polymerase elongates perfectly matched primers and incorporates biotin-labeled nucleotides. The reaction products are visualized by streptavidin-cyanine 5 staining, followed by fluorescence scanning. This procedure successfully identified from pure cultures 22 bacteria that are common causes of abortion and sterility in mares. Using the on-chip PCR method, we also tested 21 cervical swab specimens from mares for the presence of pathogenic bacteria and compared the results with those of conventional bacteriological culture methods. Our method correctly identified the bacteria in 12 cervical swab samples, 8 of which contained more than one bacterial species. Due to the higher sensitivity of the on-chip PCR, this method identified bacteria in five cervical swab samples which were not detected by the conventional identification procedure. Our results show that this method will have great potential to be incorporated into the routine microbiology laboratory.

Microarrayed allergens for IgE profiling.

Diagnosis of type I allergy is based on anamnesis, provocation testing, and serological determination of total and specific IgE. Currently, in vivo and in vitro diagnostic tests employ allergen extracts prepared from various allergen sources (e.g., pollen, mites, animal dander, moulds, foods, venoms, etc.). The application of recombinant DNA technology to the field of allergen characterization has allowed to reveal the molecular nature of the most common allergens. To date a continuously increasing number of allergen sequences has become available and panels of recombinant allergens-assembling the epitope complexity of natural allergens sources-can be produced. The use of recombinant allergens instead of crude, natural extracts for allergy diagnosis allows us to determine the individual IgE reactivity profile of each patient. To enable a comprehensive analysis of the patient's IgE binding pattern to a large number of individual allergens, a new type of serological test is required. In this paper, we applied microarray technology to create a multi-allergen test system, based on microarrayed recombinant allergens.

Accessing single nucleotide polymorphisms in genomic DNA by direct multiplex polymerase chain reaction amplification on oligonucleotide microarrays.

This study introduces a DNA microarray-based genotyping system for accessing single nucleotide polymorphisms (SNPs) directly from a genomic DNA sample. The described one-step approach combines multiplex amplification and allele-specific solid-phase PCR into an on-chip reaction platform. The multiplex amplification of genomic DNA and the genotyping reaction are both performed directly on the microarray in a single reaction. Oligonucleotides that interrogate single nucleotide positions within multiple genomic regions of interest are covalently tethered to a glass chip, allowing quick analysis of reaction products by fluorescence scanning. Due to a fourfold SNP detection approach employing simultaneous probing of sense and antisense strand information, genotypes can be automatically assigned and validated using a simple computer algorithm. We used the described procedure for parallel genotyping of 10 different polymorphisms in a single reaction and successfully analyzed more than 100 human DNA samples. More than 99% of genotype data were in agreement with data obtained in control experiments with allele-specific oligonucleotide hybridization and capillary sequencing. Our results suggest that this approach might constitute a powerful tool for the analysis of genetic variation.

Inhibition of nuclear pre-mRNA splicing by antibiotics in vitro.

A number of antibiotics have been reported to disturb the decoding process in prokaryotic translation and to inhibit the function of various natural ribozymes. We investigated the effect of several antibiotics on in vitro splicing of a eukaryotic nuclear pre-mRNA (beta-globin). Of the eight antibiotics studied, erythromycin, Cl-tetracycline and streptomycin were identified as splicing inhibitors in nuclear HeLa cell extract. The K(i) values were 160, 180 and 230 microm, respectively. Cl-tetracycline-mediated and streptomycin-mediated splicing inhibition were in the molar inhibition range for hammerhead and human hepatitis delta virus ribozyme self-cleavage (tetracycline), of group-I intron self-splicing (streptomycin) and inhibition of RNase P cleavage by some aminoglycosides. Cl-tetracycline and the aminocyclitol glycoside streptomycin were found to have an indirect effect on splicing by unspecific binding to the pre-mRNA, suggesting that the inhibition is the result of disturbance of the correct folding of the pre-mRNA into the splicing-compatible tertiary structure by the charged groups of these antibiotics. The macrolide, erythromycin, the strongest inhibitor, had only a slight effect on formation of the presplicing complexes A and B, but almost completely inhibited formation of the splicing-active C complex by binding to nuclear extract component(s). This results in direct inhibition of the second step of pre-mRNA splicing. To our knowledge, this is the first report on specific inhibition of nuclear splicing by an antibiotic. The functional groups involved in the interaction of erythromycin with snRNAs and/or splicing factors require further investigation.