Linkage analysis of susceptibility to ozone-induced lung inflammation in inbred mice.

Exposures to the common air pollutant ozone (O3) cause decrements in pulmonary function and induce airway inflammation that is characterized by infiltration of polymorphonuclear neutrophils (PMNs; refs 1-4). Because of the impact that O3 may have on public health, it is critical to identify susceptibility factors. Highly reproducible, significant inter-individual variations in human pulmonary function responses to O3 support the hypothesis that genetic background is an important determinant. Initial analysis of PMN responses to O3 exposure in segregant populations derived from inflammation-prone (susceptible) C57BL/6J (B6) and inflammation-resistant C3H/HeJ (C3) inbred mice indicated that susceptibility was controlled by a locus we termed Inf2 (ref. 7). Subsequent analyses with recombinant inbred strains suggested that a more complex interaction of genes is involved. In this report, we identify a quantitative trait locus (QTL) for O3 susceptibility on chromosome 17. Candidate genes for the locus include Tnf, the gene encoding the pro-inflammatory cytokine tumour necrosis factor-alpha (Tnf). Antibody neutralization of the protein product of this putative candidate gene significantly protected against O3 injury in susceptible mice. These results strongly support linkage of O3 susceptibility to a QTL on chromosome 17 and Tnf as a candidate gene.

Concordant methylation of the ER and N33 genes in glioblastoma multiforme.

Methylation of promoter-associated CpG islands appears to be a potential way by which tumor suppressor genes are inactivated in cancer. Using Southern blot analysis, we have studied the methylation of several genes in glioblastoma multiforme (GBM), trying to determine their contribution to tumorigenesis. Genes studied included the estrogen receptor (ER), N33, the candidate tumor-suppressors P15, P16 and HIC1 and a control gene, c-abl. Hypermethylation of N33, ER, HIC1, P16, P15 and c-abl were found in 61%, 59%, 60%, 5%, 2% and 0% of GBM respectively. HIC1 methylation was detected in normal brain as well, but appeared to be more extensive in tumors. ER and N33 methylation were significantly more frequent in tumors from individuals over the age of 40 (70% and 88% vs 36% and 14%). In addition, there was a strong association between ER and N33 methylation, which were concordant in 81% of the cases (P<0.01). ER and N33 methylation in GBM may therefore appear as a result of shared etiologic factors, which may relate in part to aging cell populations in the brain.

Effect of TNF and LTA polymorphisms on biological markers of response to oxidative stimuli in coal miners: a model of gene-environment interaction. Tumour necrosis factor and lymphotoxin alpha.

INTRODUCTION: Interaction between genetic background and oxidative environmental stimuli in the pathogenesis of human lung disease has been largely unexplored. METHODS: A prospective epidemiological study was undertaken in 253 coal miners. Intermediate quantitative phenotypes of response to oxidant exposure, including erythrocyte glutathione peroxidase (GSH-Px) and catalase activities, were studied. Oxidant exposures studied were smoking habits and cumulative dust exposure assessed by job history and ambient measures. Disease phenotypes included subclinical computed tomography score at the first survey and x ray profusion grades twice, five years apart, to assess established coal workers' pneumoconiosis (CWP). Miners were genotyped for common functional polymorphisms in the gene for tumour necrosis factor alpha (TNF) and lymphotoxin alpha (LTA), two proinflammatory cytokines that have been implicated in the pathogenesis of chronic lung diseases. RESULTS: Regarding gene-environment interaction on intermediate phenotypes, results showed interaction of a promoter polymorphism at the -308 position in TNF with occupational exposure on erythrocyte GSH-Px activity with a significant association in those with high exposure (p=0.003), whereas no association was observed among those with low exposure (interaction p=0.06). Regarding gene intermediate phenotype interaction on clinical outcome, results showed an association of CWP prevalence with an NcoI polymorphism in LTA in those with low catalase activity (p=0.05), whereas no association was observed in those with high activity (interaction p=0.03). No other significant association was observed. CONCLUSION: The results suggest that interactions of genetic background with environmental exposure and intermediate response phenotypes are important components in the pathogenesis of CWP.

Differential inspiratory timing is genetically linked to mouse chromosome 3.

Genetic control of differential inspiratory timing (TI) at baseline has been previously demonstrated among inbred mouse strains. The inheritance pattern for TI between C3H/HeJ (C3; 188 +/- 3 ms) and C57BL/6J (B6; 111 +/- 2 ms) progenitors was consistent with a two-gene model. By using the strain distribution pattern for recombinant inbred strains derived from C3 and B6 progenitors, 100% concordance was established between TI phenotypes and DNA markers on mouse chromosome 3. This genotype-phenotype hypothesis was tested by typing 52 B6C3F2 (F2) progeny by using simple sequence repeat DNA markers (n = 21) polymorphic between C3 and B6 strains on mouse chromosome 3. Linkage analysis compared marker genotypes to baseline ventilatory phenotypes by computing log-likelihood values. A putative quantitative trait locus located in proximity to D3Mit119 was significantly associated with baseline TI phenotypes. At the peak (log-likelihood = 3.3), the putative quantitative trait locus determined 25% of the phenotypic variance in TI among F2 progeny. In conclusion, this genetic model of ventilatory characteristics demonstrated an important linkage between differential baseline TI and a candidate genomic region on mouse chromosome 3.

HPLC method with UV detection for evaluation of digoxin tablet dissolution in acidic medium after solid-phase extraction.

A simple and reliable method for the evaluation of dissolution of digoxin tablets in 0.01 M hydrochloric acid was developed. Digoxin and its degradation products after solid-phase extraction using C18 Sep-Pak cartridges were evaluated. Analyses were performed on C18 column (LiChrospher RP-18e, 5 microm, 125 x 4.0 mm), as mobile phase water and acetonitrile (72:28, v/v) were used. Detection wavelength was 218 nm. Identity of digoxin degradation products was confirmed by HPLC-MS.

Reversed-phase HPLC methods for purity test and assay of pioglitazone hydrochloride in tablets.

A reversed-phase gradient HPLC method was developed for the evaluation of pioglitazone hydrochloride (PG-HCl) in tablets. Limit of detection for PG-HCl was found to be 42 ng/ml. Analyses were performed on a C18 column (Symmetry C18, 5 microm, 250 x 4.6 mm), mobile phase was a mixture of ammonium formate buffer adjusted with formic acid to pH 4.1 and acetonitrile. Shortened purity method was used as the assay method. Methods were validated.

[Evaluation of water-soluble vitamins in the vitamin preparation Spofavit dragée using reversed-phase HPLC]. / Stanovení ve vode rozpustných vitaminu ve vitaminovém prípravku Spofavit drazé metodou HPLC na reverzní fázi.

A reversed-phase HPLC gradient method with good separation efficiency was developed and validated for the evaluation of water-soluble vitamins in the pharmaceutical preparation Spofavit dragee. Analyses were performed on a C8 column (LiChrospher RP-select B), the mobile phase was a mixture of phosphate buffer with sodium hexanesulphonate and acetonitrile. Detection was at 280 and 210 nm. The method is capable within 7 minutes to evaluate 6 water-soluble vitamins (ascorbic acid, nicotinamide, panthotenate.Ca, pyridoxine.HCl, thiamine.HCl, and riboflavin). All validated parameters of this method are discussed; the developed method appeared robust and suitable for evaluation of water-soluble vitamins in vitamin preparations.

IL18 and IL18R1 polymorphisms, lung CT and fibrosis: A longitudinal study in coal miners.

It has been suggested that interleukin (IL)-18 plays a role in the development of inflammatory and fibrosing lung diseases. Associations of polymorphisms in the genes coding for IL-18 (IL18 /G-656T, C-607A, G-137C, T113G, C127T) and its receptor (IL18R1 /C-69T) with coal workers' pneumoconiosis (CWP) were studied in 200 miners who were examined in 1990, 1994 and 1999. Coal-dust exposure was assessed according to job history and ambient measures. The main health outcome was lung computed tomography (CT) score in 1990. Internal coherence was assessed by studying CT score in 1994, 4-yr change in CT score and CWP incidence and prevalence. CT score in 1990 was a good predictor of radiographic grade in 1999 and, therefore, an appropriate subclinical quantitative trait. The IL18 -137C allele was associated with lower CT score in 1990 and 1994 (1.24 versus 1.69 and 1.57 versus 2.46, respectively), slower progression of CT score between 1990 and 1994 and lower pneumoconiosis prevalence in 1999 relative to the G allele (0.33 versus 0.77 and 8.2 versus 19.6%, respectively). Smoking- or dust-adjustment, and stratification on IL18R1 genotype and adjustment for haplotype effects did not change the conclusions. In conclusion, the results of the present study suggest a role for IL18 in reducing the development of this fibrosing lung disease.

Localization of the highly polymorphic locus D19S120 to 19p13.3 by linkage.

To obtain the regional localization of a dinucleotide repeat D19S120 (formerly designated LIPE) with 7 alleles and an observed heterozygosity of 73% (Levitt et al., 1992) we typed 40 CEPH families. Given the high heterozygosity and ease of typing, this marker represents a useful addition to the index map of chromosome 19.

Genetic susceptibility to ozone-induced lung hyperpermeability: role of toll-like receptor 4.

The pollutant ozone (O(3)) induces lung hyperpermeability and inflammation in humans and animal models. Among inbred strains of mice, there is a 3-fold difference in total protein (a marker of permeability) recovered in bronchoalveolar lavage (BAL) fluid after a 72-h exposure to 0.3 ppm O(3). To determine the chromosomal locations of susceptibility genes, we performed a genome screen using recombinant inbred (RI) strains of mice derived from O(3)-susceptible C57BL/6J (B6) and O(3)-resistant C3H/HeJ (HeJ) progenitors. Each RI strain was phenotyped for O(3)-induced hyperpermeability, and linkage was assessed for 558 markers using Map Manager QTb27. A significant quantitative trait locus (QTL) was identified on chromosome 4. The likelihood ratio chi(2) statistic (16.6) for the peak of the QTL was greater than the significance threshold (16.3) determined empirically by permutation test. This QTL contains a candidate gene, Toll-like receptor 4 (Tlr4 ), that recently has been implicated in innate immunity and endotoxin susceptibility. The amount of the total trait variance explained by the QTL at Tlr4, the gene with the highest likelihood ratio statistic in the QTL, was approximately 70%. To test the role of Tlr4 in O(3)-induced hyperpermeability, BAL protein responses to O(3) were compared in C3H/HeOuJ (OuJ) and HeJ mice that differ only at a polymorphism in the coding region of Tlr4. Significantly greater protein concentrations (430 +/- 35 microg/ml) were found in OuJ mice compared with HeJ mice (258 +/- 18 microg/ml) after exposure to O(3). Furthermore, reverse transcriptase/polymerase chain reaction analysis demonstrated differential expression of Tlr4 message levels between HeJ and OuJ mice after O(3) exposure. Together, results indicate that a QTL on mouse chromosome 4 explains a significant portion of the genetic variance in O(3)-induced hyperpermeability, and support a role for Tlr4 as a strong candidate susceptibility gene.

Toll-like receptor 4 mediates ozone-induced murine lung hyperpermeability via inducible nitric oxide synthase.

We tested the hypotheses that 1) inducible nitric oxide synthase (iNOS) mediates ozone (O3)-induced lung hyperpermeability and 2) mRNA levels of the gene for iNOS (Nos2) are modulated by Toll-like receptor 4 (Tlr4) during O3 exposure. Pretreatment of O3-susceptible C57BL/6J mice with a specific inhibitor of total NOS (N(G)-monomethyl-L-arginine) significantly decreased the mean lavageable protein concentration (a marker of lung permeability) induced by O3 (0.3 parts/million for 72 h) compared with vehicle control mice. Furthermore, lavageable protein in C57BL/B6 mice with targeted disruption of Nos2 [Nos2(-/-)] was 50% less than the protein in wild-type [Nos2(+/+)] mice after O3. To determine whether Tlr4 modulates Nos2 mRNA levels, we studied C3H/HeJ (HeJ) and C3H/HeOuJ mice that differ only at a missense mutation in Tlr4 that confers resistance to O3-induced lung hyperpermeability in the HeJ strain. Nos2 and Tlr4 mRNA levels were significantly reduced and correlated in resistant HeJ mice after O3 relative to those in susceptible C3H/HeOuJ mice. Together, the results are consistent with an important role for iNOS in O3-induced lung hyperpermeability and suggest that Nos2 mRNA levels are mediated through Tlr4.

Comparison of fluorescence-based semi-automated genotyping of multiple microsatellite loci with autoradiographic techniques.

The practical application of highly efficient fluorescence-based methods for the semi-automated genotyping of polymerase chain reaction-based microsatellite markers will depend on the development of robust protocols that provide accurate and reproducible data. In the present report we compare the accuracy of a fluorescence-based protocol with a benchmark radiolabeling method that depends on a known sequence ladder or amplified DNA from reference individuals for sizing by autoradiography. Three microsatellite markers, IGF1 (mfd 1), D4S174 (mfd 59), and D5S211 (mfd 154), with products overlapping in size were each labeled with a different fluorophore and run simultaneously with an internal size standard in a single electrophoretic lane. The size of each allele was compared for these markers by using both techniques for five larger CEPH families (884, 1331, 1332, 1333, and 1362). Of 462 possible alleles, four discrepancies (0.8%) were identified when the two approaches were compared. We conclude that the fluorescence-based protocol is at least as accurate as the standard radiolabeling technique since none of the sizing errors arose as a result of the fluorescence-based technique. We describe the adaptation of this fluorescence-based protocol to the simultaneous analysis of up to 24 microsatellite loci per electrophoretic lane. These highly accurate and efficient semi-automated techniques will be useful in high-resolution genomic analyses.

Glioblastoma-related gene mutations and over-expression of functional epidermal growth factor receptors in SKMG-3 glioma cells.

Amplification of the epidermal growth factor receptor (EGFR) gene is found in about 40% of glioblastomas (GBMs) but is rarely detected in GBM cell lines. We confirmed that the exceptional SKMG-3 GBM cell line retained amplified EGFR genes in vitro, and found that these sequences were concentrated on extra-chromosomal DNA particles similar to double-minute chromosomes. The cells contained two other gene mutations that are associated with high-grade astrocytic tumors: extra-chromosomal amplification of the cyclin-dependent kinase-4 (CDK4) gene and a homozygous mutation within the PTEN tumor suppressor gene. Immunoblots revealed very high levels of EGFR, moderately increased expression of CDK4, and no detectable PTEN protein. The overexpressed SKMG-3 EGFRs responded to exogenous ligand and resembled normal rather than mutant receptors. A heterozygous mutation of the p53 gene (p53R282W) correlated with failure of radiation to induce the expression of cyclin-dependent kinase inhibitor p21waf1 or an early G1 cell cycle arrest. Although each of these gene mutations occurs in GBMs, SKMG-3 cells had an unusual genotype in that a p53 gene mutation co-existed with amplified EGFR genes. Nonetheless, the SKMG-3 cell line can be exploited as a model to study how oncogenic EGFR signals in GBM cells interact with over-expressed CDK4 and loss of PTEN to confer the malignant phenotype.

Evidence for genetic heterogeneity in malignant hyperthermia susceptibility.

Malignant hyperthermia susceptibility (MHS) is a clinically heterogeneous pharmacogenetic disorder characterized by accelerated metabolism, hyperthermia, and frequently muscle rigidity. MHS is elicited by all commonly used potent inhalation anesthetics and depolarizing neuromuscular blockers and remains an important cause of death due to anesthesia. Recent linkage studies suggest a single genetic locus for this disorder on chromosome 19q13.1. The results of our linkage analyses exclude several loci on 19q13.1 as a site for the gene(s) that produces the MHS phenotype in three unrelated families and clearly establish genetic heterogeneity in this disorder. These results are consistent with the hypothesis that the genetic defect that alters thermoregulation may vary in MHS and that clinical variability in the expression of MHS may be explained by genetic heterogeneity.

Genetic linkage analysis of susceptibility to particle exposure in mice.

Particle-induced increases in respiratory morbidity and mortality have been observed worldwide in industrialized cities but the toxicologic mechanisms have not been elucidated. It is hypothesized that subpopulations including the elderly and individuals with cardiopulmonary disease are particularly at risk to the effects of exposure. Genetic background is another important host factor that may contribute to interindividual responsivity to particulate exposure. This study was designed to identify susceptibility loci for alveolar macrophage (AM) immune dysfunction induced by inhalation of sulfate-associated carbon particles in susceptible C57BL/6J and resistant C3H/ HeJ inbred mice. AMs were chosen for study because they represent an important component of host defense, and compromised host defense has been hypothesized to be an important factor in particle-induced respiratory morbidity. The quantitative phenotype for these studies was Fc receptor-mediated phagocytic function, an index of AM integrity. Analyses of macrophage dysfunction phenotypes of segregant and nonsegregant populations derived from these two strains indicate that two unlinked genes control susceptibility. A genome-wide linkage analysis of an intercross (F(2)) cohort identified significant and suggestive quantitative trait loci (QTLs) on chromosomes 17 and 11, respectively. Candidate susceptibility genes were identified for mice and humans by comparative mapping. Importantly, both QTLs overlap previously identified QTLs for susceptibility to another common pollutant, ozone. This is the first demonstration that genetic background is an important determinant of responsiveness to particle-induced immune dysfunction, and it has important implications for understanding the epidemiologic associations between particulates and morbidity and mortality.

Fluorescence-based resource for semiautomated genomic analyses using microsatellite markers.

To facilitate the practical application of highly efficient semiautomated methods for general application in genomic analyses, we have developed a fluorescence-based microsatellite marker resource. Ninety highly polymorphic microsatellite markers were combined to provide a rapid, accurate, and highly efficient initial genome-wide screening system. These markers are spaced on average every 33 cM, with a mean heterozygosity of 81% (range 65-94%), covering 22 autosomes and the X and Y chromosomes. Less than 10% of the genome lies beyond 20 cM of the nearest marker. Since this genomic analysis system is fully compatible with automated fragment analyzers using simultaneous four-color fluorescence-based detection systems, the 5 groups of 18 markers can be detected concurrently. This multiplex detection provides a through-put of 1944 genotypes daily per instrument. This system will be highly beneficial in a number of clinical and research applications including linkage, cancer genetics, forensics, and cytogenetics.

Masseter muscle rigidity associated with glycine1306-to-alanine mutation in the adult muscle sodium channel alpha-subunit gene.

BACKGROUND: Succinylcholine-induced masseter muscle rigidity (MMR) is a potentially life-threatening complication of anesthesia and is closely correlated with the heterogeneous disorder malignant hyperthermia (MH) susceptibility. MMR also is identified with a variety of neuromuscular disorders, including the myotonias, that are associated with abnormal in vitro contracture test (IVCT) results. Recently, mutations in the adult skeletal muscle sodium channel alpha-subunit gene (SCN4A) have been shown to cause generalized nondystrophic myotonias, some of which are associated with mild nonspecific symptoms. The purpose of the current investigation was to begin to evaluate the molecular genetic relationship between known mutations in the SCN4A gene, MMR, and the results of the IVCT used to diagnose MH-susceptibility. METHODS: A single extended pedigree of 16 individuals was ascertained through a proband who experienced MMR and whole-body rigidity after succinylcholine administration. Subsequently, four individuals were shown to have a mild form of myotonia on clinical and laboratory examination. IVCT was carried out according to standardized protocols. Mutations in the SCN4A gene were sought in exons 22 and 24 using single-strand conformational analyses. Variability in the SCN4A gene sequence was confirmed by direct DNA sequence analyses. RESULTS: Four individuals with myotonia were shown to carry a guanine-to-cytosine mutation at nucleotide position 3917 of the reported SCN4A sequence. This DNA mutation was coinherited with MMR and an abnormal IVCT result in this family. Previous studies have demonstrated that the glycine1306-to-alanine substitution is associated with a mild clinical syndrome referred to as myotonia fluctuans. CONCLUSIONS: The current report provides direct evidence that succinylcholine-induced MMR, whole-body rigidity, and an abnormal IVCT result are associated with a mutation in the SCN4A gene.

Molecular genetic alterations in radiation-induced astrocytomas.

Astrocytic tumors occasionally arise in the central nervous system following radiotherapy. It is not clear if these gliomas represent a unique molecular genetic subset. We identified nine cases in which an astrocytoma arose within ports of previous radiation therapy, with total doses ranging from 2400 to 5500 cGy. Irradiated primary lesions included craniopharyngioma, pituitary adenoma, Hodgkin's lymphoma, ependymoma, pineal neoplasm, rhabdomyosarcoma, and three cases of lymphoblastic malignancies. Patients ranged from 9 to 60 years of age and developed secondary tumors 5 to 23 years after radiotherapy. The 9 postradiation neoplasms presented as either anaplastic astrocytoma (3 cases) or glioblastoma multiforme (6 cases). Two of the latter contained malignant mesenchymal components. We performed DNA sequence analysis, differential polymerase chain reaction (PCR), and quantitative PCR on DNA from formalin-fixed, paraffin-embedded tumors to evaluate possible alterations of p53, PTEN, K-ras, EGFR, MTAP, and p16 (MTS1/CDKN2) genes. By quantitative PCR, we found EGFR gene amplification in 2 of 8 tumors. One of these demonstrated strong immunoreactivity for EGFR. Quantitative PCR showed chromosome 9p deletions including p16 tumor suppressor gene (2 of 7 tumors) and MTAP gene (3 of 7). Five of 9 tumors demonstrated diffuse nuclear immunoreactivity for p53 protein. Sequencing of the p53 gene in these 9 cases revealed a mutation in only one of these cases, a G-to-A substitution in codon 285 (exon 8). Somewhat unexpectedly, no mutations were identified in PTEN, a commonly altered tumor suppressor gene in de novo glioblastoma multiformes. Unlike some radiation-induced tumors, no activating point mutations of the K-ras proto-oncogene or base pair deletions of tumor suppressor genes were noted. These radiation-induced tumors are distinctive in their high histological grade at clinical presentation. The spectrum of molecular genetic alterations appears to be similar to that described in spontaneous high grade astrocytomas, especially those of the de novo type.

Losses of chromosomal arms 1p and 19q in the diagnosis of oligodendroglioma. A study of paraffin-embedded sections.

Comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), polymerase chain reaction-based microsatellite analysis, and p53 sequencing were performed in paraffin-embedded material from 18 oligodendrogliomas and histologically similar astrocytomas. The study was undertaken because of evidence that concurrent loss of both the 1p and 19q chromosome arms is a specific marker for oligodendrogliomas. Of the six lesions with a review diagnosis of oligodendroglioma, all had the predicted loss of 1p and 19q seen by CGH, FISH, and polymerase chain reaction. Other lesions, including some considered oligodendroglioma or mixed glioma by the submitting institution, did not. There were no p53 mutations in any of the six oligodendrogliomas, whereas 5 of the 10 remaining, successfully studied cases did have p53 mutations. The results suggest that CGH and FISH performed on current or archival tissue can aid in classification of infiltrating gliomas such as oligodendrogliomas and astrocytomas. The results of the p53 studies are consistent with findings of previous investigations that such mutations are less common in oligodendrogliomas than they are in astrocytomas.