Computational Network Analysis for Drug Toxicity Prediction.

The computational prediction of compound effects from molecular data is an important task in hazard and risk assessment and pivotal for judging the safety of any drug, chemical or cosmetic compound. In particular, the identification of such compound effects at the level of molecular interaction networks can be helpful for the construction of adverse outcome pathways (AOPs). AOPs emerged as a guiding concept for toxicity prediction, because of the inherent mechanistic information of such networks. In fact, integrating molecular interactions in transcriptome analysis and observing expression changes in closely interacting genes might allow identifying the key molecular initiating events of compound toxicity.In this work we describe a computational approach that is suitable for the identification of such network modules from transcriptomics data, which is the major molecular readout of toxicogenomics studies. The approach is composed of different tools (1) for primary data analysis, i.e., the biostatistical quantification of the gene expression changes, (2) for functional annotation and prioritization of genes using literature mining, as well as (3) for the construction of an interaction network that consists of interactions with high confidence and the identification of predictive modules from these networks. We describe the different steps of the approach and demonstrate its performance with public data on drugs that induce hepatic and cardiac toxicity.

ToxDB: pathway-level interpretation of drug-treatment data.

MOTIVATION: Extensive drug treatment gene expression data have been generated in order to identify biomarkers that are predictive for toxicity or to classify compounds. However, such patterns are often highly variable across compounds and lack robustness. We and others have previously shown that supervised expression patterns based on pathway concepts rather than unsupervised patterns are more robust and can be used to assess toxicity for entire classes of drugs more reliably. RESULTS: We have developed a database, ToxDB, for the analysis of the functional consequences of drug treatment at the pathway level. We have collected 2694 pathway concepts and computed numerical response scores of these pathways for 437 drugs and chemicals and 7464 different experimental conditions. ToxDB provides functionalities for exploring these pathway responses by offering tools for visualization and differential analysis allowing for comparisons of different treatment parameters and for linking this data with toxicity annotation and chemical information.Database URL:http://toxdb.molgen.mpg.de.

[Teaching non-technical skills for critical incidents: Crisis resource management training for medical students]. / Vermittlung von "soft skills" für Belastungssituationen: "Crisis-resource-management"--Kurs für Studierende.

BACKGROUND: Physicians have to demonstrate non-technical skills, such as communication and team leading skills, while coping with critical incidents. These skills are not taught during medical education. A crisis resource management (CRM) training was established for 4th to 6th year medical students using a full-scale simulator mannikin (Emergency Care Simulator, ECS, METI). PATIENTS AND METHODS: The learning objectives of the course were defined according to the key points of Gaba's CRM concept. The training consisted of theoretical and practical parts (3 simulation scenarios with debriefing). Students' self-assessment before and after the training provided the data for evaluation of the training outcome. RESULTS: A total of 65 students took part in the training. The course was well received in terms of overall course quality, debriefings and didactic presentation, the mean overall mark being 1.4 (1: best, 6: worst). After the course students felt significantly more confident when facing incidents in clinical practice. The main learning objectives were achieved. CONCLUSION: The effectiveness of applying the widely used ECS full-scale simulator in interdisciplinary teaching has been demonstrated. The training exposes students to crisis resource management issues and motivates them to develop non-technical skills.

Sequence-based typing reveals a novel DLA-88 allele, DLA-88*04501, in a beagle family.

The dog is an important animal model for solid organ as well as stem cell allo transplantation. Methods such as cellular and serological typing and more recently sequence-based typing (SBT) have been used to discriminate tissue antigen disparity of donor and recipient. We applied SBT for the canine class I (DLA-88) and class II (DLA-DRB1) genes in beagle families prior stem cell transplantation. A novel DLA-88 (DLA-88*04501) allele in combination with a DLA-DRB1*01901 allele was found. Sequence comparison of exons 2 and 3 of the novel allele revealed most sequence identity to the DLA-88*01301 allele (96.15% identity at the nucleotide and 90.65% identity at the protein level).

Identification of a new HLA-B allele, HLA-B*4443, in a German family.

A novel human leukocyte antigen (HLA)-B allele is described. The allele was identified in a German blood donor of Caucasian origin. Because high-resolution HLA-typing using sequence-specific primers gave inconclusive results, sequence-based typing was performed. Nucleotide sequences of exons 2 and 3 most closely match with HLA-B*4417 and HLA-B*440301 (99.5% identity). The predicted protein sequence revealed a single amino acid substitution (D156L) compared with the HLA-B*4417 allele but two substitutions (Y113H, D116S) compared with the HLA-B*440301 allele. Therefore, the novel allele has been officially assigned HLA-B*4443 by the WHO Nomenclature Committee. The HLA-B*4443 allele was found with the A*2301, Cw*0401, B*4443, DRB1*0701, DRB4*0107, and DQB1*0202 haplotype.

[Quality of life in patients with remitting-relapsing multiple sclerosis in Germany]. / Lebensqualität bei Patienten mit schubförmiger MS in Deutschland.

The concept of health-related quality of life (QoL) includes physical, psychological,and social aspects. This pertains to consequences of chronic diseases and their therapies beyond biological or pharmacological relations. A considerable amount of literature concerning QoL has been published with regard to neurological and non-neurological entities. This study summarizes data from a study in 717 persons with remitting-relapsing multiple sclerosis (MS). Of them,576 could be reevaluated longitudinally after 1 year of treatment with interferon-beta 1a (44 microg subcutaneous Rebif once weekly). Compared to populations of healthy controls or other patients, considerable reductions in the eight subscales and both physical and emotional sum scales of the German version of the short form of the Rand Health Questionnaire (SF-36) questionnaire were assessed. These reductions were even more pronounced in persons with gait impairments. Most SF-36 scales only modestly correlated to physical disability. This indicates that QoL as reported by patients does not depend solely on the physical symptoms of MS. Most findings remained stable for the study population as a whole during 1 year of therapy, while statistically significant improvements were found in clinical responders as defined in this study (relapse-free, physically stable, stable or improved in physician's judgement). Side effects of therapy were not reflected in lower QoL scale values. Implications of findings for future concepts in MS therapy are discussed.

A null mutation within the ciliary neurotrophic factor (CNTF)-gene: implications for susceptibility and disease severity in patients with multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Impaired remyelination and axonal degeneration may account for progressive disability in MS patients. As ciliary neurotrophic factor (CNTF) takes part in myelogenesis, we examined the frequency of a CNTF-null mutation in 349 MS patients with respect to their clinical presentation and in comparison with 434 healthy controls. Similar genotype frequencies for the CNTF mutation were obtained in MS patients (genotype 0101=74.8%, 0102=22.3%, 0202=2.9%) and controls (genotype 0101=71.7%, 0102=26.5%, 0202=1.8%) even after stratification for the HLA-DRB1*15 allele. In addition, there was no significant correlation of CNTF genotypes to age at onset, course or severity of the disease. We therefore conclude, that the requirement for CNTF in myelogenesis or cell survival may be bypassed by a second ligand or redundancy of functional activity of other neurotrophic factors.

A complex containing at least one zinc dependent HeLa nuclear protein binds to the intronic (gaa)(n) block of the frataxin gene.

We analyzed HeLa nuclear proteins binding to the (gaa)(n) harbouring intron 1 of nine frataxin alleles and characterized the structures of the repeats. Fragments with blocks longer than (gaa)(9) form spontaneously different intramolecular H-y topoisomeres in linear state. The observed triplexes depend on the length of the repeat. Interruption of the perfectly repeated (gaa)(n) block entails two structural regions. At least two HeLa nuclear proteins bind to the (gaa)(n) fragments resulting in a distinct major retarded complex as revealed by EMSA. One of these proteins is zinc dependent. Importantly, the fragment harbouring (gan)(121) binds additional proteins. Protein binding appears to be locus specific, and the binding affinity was found to be not random. The affinities of the different target fragments varied by a factor of four. Binding affinities of the fragments were not obviously correlated to differences in the composition of the repeats. DNase I footprinting revealed only weakly protected binding regions, but multiple HS sites in the repeat regions of the fragments. These findings and the fact, that DNA conformers observed in EMSA and electron microscopical experiments bind proteins, lead to the assumption that the proteins recognize, both, B-DNA and triple helical structures, but with different affinity. Possible functions of the proteins are discussed in the context of transformation of triple helical structures into B-DNA and the pathogenesis of FRDA.

The GAA repeat expansion in intron 1 of the frataxin gene is related to the severity of cardiac manifestation in patients with Friedreich's ataxia.

Friedreich's ataxia is an autosomal recessive disorder characterized by spinocerebellar degeneration. It is caused by an unstable GAA trinucleotide repeat expansion (>120 repeats) in the first intron of the frataxin gene on chromosome 9 (9q13) in both alleles. Concentric left ventricular hypertrophy has been recognized as the major cardiac manifestation of Friedreich's ataxia. Our aim was to investigate the influence of the frataxin repeat length on cardiac hypertrophy in patients with Friedreich's ataxia and in patients with hypertrophic and dilated cardiomyopathy. Thirty-one patients with Friedreich's ataxia, 86 patients with hypertrophic cardiomyopathy, 134 patients with dilated cardiomyopathy, and 32 healthy individuals without cardiac disease were analysed by electrocardiography and 2D-M-mode echocardiography. Then, the size of the frataxin repeat was determined by polymerase chain reaction (PCR) and agarose gel electrophoresis. The number of GAA repeats in patients with hypertrophic and dilated cardiomyopathy was not different from the length in patients without cardiac disease (hypertrophic cardiomyopathy, 8+/-2 repeats on GAA 1 allele and 11+/-5 repeats on GAA 2 allele; dilated cardiomyopathy, 7+/-2 repeats on GAA 1 allele and 11+/-5 repeats on GAA 2 allele; Control, 9+/-1 repeats on GAA 1 allele and 12+/-6 repeats on GAA 2 allele). The septal and posterior wall thickness of these patients was not related to the GAA repeat length. All patients with Friedreich's ataxia had two enlarged alleles with a mean GAA repeat length of 757+/-316 and 1012+/-231, respectively. The lengths of both alleles were significantly greater than the lengths in the controls (P<0.0001), patients with hypertrophic cardiomyopathy (P<0.0001) and dilated cardiomyopathy (P<0.0001). A significant correlation was revealed between interventricular septal hypertrophy and frataxin repeat length in the smaller allele. Furthermore, the ratio of septal to posterior wall thickness was significantly correlated to GAA repeat size on the smaller allele. In conclusion, the size of the GAA repeat on the smaller allele in the frataxin gene is associated with the degree of left ventricular hypertrophy in patients with Friedreich's ataxia but is not related to the severity of hypertrophic cardiomyopathy.

Oxidative stress in patients with Friedreich ataxia.

Increased generation of reactive oxygen species may underlie the pathophysiology of Friedreich ataxia (FRDA). The authors measured concentrations of 8-hydroxy-2'-deoxyguanosine (8OH2'dG), a marker of oxidative DNA damage, in urine and of dihydroxybenzoic acid (DHBA), a marker of hydroxyl radical attack, in plasma of 33 patients with FRDA. They found a 2.6-fold increase in normalized urinary 8OH2'dG but no change in plasma DHBA as compared with controls. Oral treatment with 5 mg/kg/day of the antioxidant idebenone for 8 weeks significantly decreased urinary 8OH2'dG concentrations, indicating that 8OH2'dG may be useful in monitoring therapeutic interventions in patients with FRDA.-1721

Genetic analysis of immunomodulating factors in sporadic Parkinson's disease.

Immunomodulating factors have been shown to play a role in the pathogenesis of Parkinson's disease (PD) by biochemical methods. In order to investigate functionally important genes of the tumor necrosis factor alpha (TNFalpha) pathway we studied the frequency of DNA polymorphisms in the interleukin 6 (IL6), the TNFalpha, and the TNFalpha receptor 1 (TNFR1) genes in 264 sporadic German PD patients and in 183 age and sex matched German healthy controls. Analyzing the TNFalpha-308 polymorphism we found heterozygous individuals carrying alleles 1 and 2 more frequently in patients with a relative risk of 1.56 (p = 0.046, p(c) = 0.13, chi2 = 3.98). In contrast, the frequency of the B/2 haplotype described by the TNFR1-609 and TNFRI+36 polymorphisms was significantly decreased in our PD patients group (p = 0.0097, p(c) = 0.048, chi2 = 6.69) with a relative risk reduced to 0.52. Our results suggest an involvement of immunomodulating factors in the pathogenesis of sporadic PD as revealed by a molecular genetic approach.

Genetic background of apparently idiopathic sporadic cerebellar ataxia.

Disease-causing mutations have been identified in various entities of autosomal dominant ataxia and in Friedreich's ataxia. However, no molecular pathogenic factor is known to cause idiopathic cerebellar ataxias. We investigated the CAG/CTG trinucleotide repeats causing spinocerebellar ataxia types 1, 2, 3, 6, 7, 8 and 12, and the GAA repeat of the frataxin gene in 124 patients apparently suffering from idiopathic sporadic ataxia, including 20 patients with the clinical diagnosis of multiple system atrophy. Patients with a positive family history, a typical Friedreich phenotype, or symptomatic ataxia were excluded. Genetic analyses uncovered the most common Friedreich mutation in 10 patients with an age at onset between 13 and 36 years. The SCA6 mutation was present in nine patients with disease onset between 47 and 68 years of age. The CTG repeat associated with SCA8 was expanded in three patients. One patient had SCA2 attributable to a de novo mutation from a paternally transmitted, intermediate allele. We did not identify the SCA1, SCA3, SCA7 or SCA12 mutation in idiopathic sporadic ataxia patients. No trinucleotide repeat expansion was detected in the MSA subgroup. This study has revealed the genetic basis in 19% of apparently idiopathic ataxia patients. SCA6 is the most frequent mutation in late onset cerebellar ataxia. The frataxin trinucleotide expansion should be investigated in all sporadic ataxia patients with onset before age 40, even when the phenotype is atypical for Friedreich's ataxia.

Extrapyramidal motor signs in degenerative ataxias.

BACKGROUND: Extrapyramidal motor signs (EPS) are well-known symptoms of degenerative ataxia. However, little is known about frequency and appearance of EPS in subtypes of ataxia. METHODS: We characterized 311 patients with ataxia clinically and genetically. Course of the disease and EPS were investigated according to a standardized protocol. Diagnostic and prognostic impact of EPS in subtypes of ataxia was analyzed by Kaplan-Meier plots. RESULTS: Extrapyramidal motor signs occurred in all forms of ataxia, but frequency and type of EPS varied between genetically and clinically defined subtypes. Postural tremor in hereditary ataxias was typical for spinocerebellar ataxia type 2 (SCA2). Dystonia was generally rare in ataxias, but, if present, suggested SCA3. We observed a parkinsonian variant of SCA3 in which parkinsonism was present in the beginning of the disease and responded well to levodopa therapy, leading to diagnostic confusion. Parkinsonism in SCA3 was independent of CAG repeat length but ran in families, suggesting modifying genes. In idiopathic sporadic cerebellar ataxia (ISCA), EPS are more frequent in late-onset than in early-onset forms. In 50% of ISCA patients with parkinsonism, the diagnosis of multiple system atrophy remained questionable because of normal autonomic function. CONCLUSIONS: Extrapyramidal motor signs can help to predict the genetic subtype of ataxia. Extrapyramidal motor signs were more frequent in genetic subtypes in which basal ganglia affection has been demonstrated by postmortem studies. However, no type of EPS was specific for an underlying mutation. In ISCA, EPS are an adverse prognostic factor. Parkinsonism is especially associated with a more rapid course of the disease. Arch Neurol. 2000;57:1495-1500

The nonfunctional allele TCRBV6S1B is strongly associated with Helicobacter pylori infection.

To determine genetic susceptibility factors for Helicobacter pylori infection, polymorphic T-cell receptor gene elements were investigated in 203 H. pylori-infected individuals and 180 uninfected individuals (controls). H. pylori infection is highly associated with individuals homozygous for the nonfunctional TCRBV6S1B element (odds ratio = 5.9; chi(2) = 13; P = 0.00032; P value corrected for multiple comparisons [Bonferroni correction] = 0. 00063).

Heterozygous expansion of the GAA tract of the X25/frataxin gene is associated with insulin resistance in humans.

Friedreich's ataxia (FA) is an autosomal recessive disease that has been attributed to a GAA triplet repeat expansion in the first intron of the X25/frataxin gene. Impaired glucose tolerance is present in up to 39% of FA patients, and clinically apparent diabetes is seen in approximately 18% of the affected individuals. Subjects carrying the X25/frataxin GAA repeat in a heterozygous state do not develop FA and, therefore, represent an ideal model to study the underlying metabolic defects that contribute to the diabetes associated with this disorder. In the present study, we have compared 11 first-degree relatives of FA patients (i.e., parents or heterozygous siblings of FA patients) with matched normal control subjects to study the parameters of glucose metabolism. An oral glucose tolerance test revealed diabetes in one of the heterozygous subjects who was excluded from further analyses. Using an octreotide-based quantification of insulin sensitivity, 8 of the remaining 10 study subjects showed pronounced insulin resistance, reflecting a significant difference from the control group (P = 0.001). In conclusion, a heterozygous expansion of the X25/frataxin GAA repeat in healthy individuals is associated with insulin resistance and might be considered a genetic co-factor in the pathogenesis of mitochondrial subtypes of diabetes.

Mitochondrial impairment of human muscle in Friedreich ataxia in vivo.

Friedreich ataxia occurs due to mutations in the gene encoding the mitochondrial protein frataxin. This (31)P magnetic resonance spectroscopy study on the calf muscle of Friedreich ataxia patients provides in vivo evidence of a severe impairment of mitochondrial function. Mitochondrial adenosine triphosphate resynthesis was studied by means of the post-exercise recovery of phosphocreatine. After ischemic exercise in calf muscles of all patients, phosphocreatine recovery was dramatically delayed. Time constants of recovery correlated with mutations of the frataxin gene, the age of the patients, and disease duration. (31)P magnetic resonance spectroscopy represents the first expedient tool for monitoring therapeutic trials in Friedreich ataxia non-invasively.