Biomedical subjects

D Schindler

Publications and source records attributed to D Schindler.

At least 19 recordsLinked to original sources

PubMed2002

Reverse mosaicism in Fanconi anemia: natural gene therapy via molecular self-correction.

Fanconi anemia (FA) is a genetically and phenotypically heterogenous autosomal recessive disease associated with chromosomal instability and hypersensitivity to DNA crosslinkers. Prognosis is poor due to progressive bone marrow failure and increased risk of neoplasia, but revertant mosaicism may improve survival. Mechanisms of reversion include back mutation, intragenic crossover, gene conversion and compensating deletions/insertions. We describe the types of reversions found in five mosaic FA patients who are compound heterozygotes for single base mutations in FANCA or FANCC. Intragenic crossover could be shown as the mechanism of self-correction in the FANCC patient. Restoration to wildtype via back mutation or gene conversion of either the paternal or maternal allele was observed in the FANCA patients. The sequence environments of these mutations/reversions were indicative of high mutability, and selective advantage of bone marrow precursor cells carrying a completely restored FANCA allele might explain the surprisingly uniform pattern of these reversions. We also describe a first example of in vitro phenotypic reversion via the emergence of a compensating missense mutation 15 amino acids downstream of the constitutional mutation, which explains the reversion to MMC resistance of the respective lymphoblastoid cell line. With one exception, our mosaic patients showed improvement of their hematological status during a three- to six-year observation period, indicating a proliferative advantage of the reverted cell lineages. In patients with Fanconi anemia, genetic instability due to defective caretaker genes sharply increases the risk of neoplasia, but at the same time increases the chance for revertant mosaicism leading to improved bone marrow function.

Adolescent↗

PubMed2001

Forecasting agriculturally driven global environmental change.

During the next 50 years, which is likely to be the final period of rapid agricultural expansion, demand for food by a wealthier and 50% larger global population will be a major driver of global environmental change. Should past dependences of the global environmental impacts of agriculture on human population and consumption continue, 10(9) hectares of natural ecosystems would be converted to agriculture by 2050. This would be accompanied by 2.4- to 2.7-fold increases in nitrogen- and phosphorus-driven eutrophication of terrestrial, freshwater, and near-shore marine ecosystems, and comparable increases in pesticide use. This eutrophication and habitat destruction would cause unprecedented ecosystem simplification, loss of ecosystem services, and species extinctions. Significant scientific advances and regulatory, technological, and policy changes are needed to control the environmental impacts of agricultural expansion.

Agriculture↗

PubMed2001

Sources of variation in patient response to radiation treatment.

PURPOSE: To investigate sources of variation in radiosensitivity displayed by cancer patients and blood donors using the leukocyte apoptosis assay. METHODS AND MATERIALS: Probes were obtained from 105 healthy blood donors, 48 cancer patients displaying normal sensitivity to radiotherapy, 12 cancer patients displaying hypersensitivity to radiotherapy, 12 Ataxia telangiectasia blood donors, and 4 additional individuals with genetic diseases of potentially modified radiosensitivity; 2 neurofibromatosis (NF) donors, a Nijmegen breakage syndrome (NBS) donor, and an Immunodeficiency, Chromosome fragility, Facial anomaly syndrome (ICF) donor. Heparinized blood was diluted in medium, irradiated, and left to incubate for 48 h. CD4 and CD8 T-lymphocyte DNA was stained with propidium iodide and the cells were analyzed by flow cytometry. RESULTS: Radiation-induced apoptosis depended on age and cell type. Cohorts of hypersensitive cancer patients, NBS and AT donors displayed compromised apoptotic response. An asymmetric apoptotic response of T-lymphocytes was observed in an ICF donor and a cryptic hypersensitivity donor. Two NF donors displayed no abnormal sensitivity to radiotherapy but compromised apoptotic T-cell response to X-rays. CONCLUSION: Our studies reveal 4 physiologic sources of variation in radiation response-2 are genetic: cryptic hypersensitivity and hereditary disease, and 2 are epigenetic: cell type and donor age. They emphasize the important role of proteins involved in the recognition and repair of DNA double-strand breaks in determining the response of individuals to radiotherapy.

Adolescent↗

PubMed2001

The substrate for brain-stimulation reward in the lateral preoptic area: III. Connections to the lateral hypothalamic area.

Double-pulse tests were used to estimate the refractory periods and anatomical linkage of the reward-relevant fibers that course between the lateral preoptic and lateral hypothalamic areas. In the 1st study, pairs of conditioning and test pulses were delivered to each site, and the interval between pulses varied; recovery from refractoriness was similar at both sites, with the curves generally rising from 0.6 to 2.0 ms. In the 2nd study, the pairs of pulses were delivered to both sites. Six of 7 rats showed evidence of axonal collision, with estimates of conduction velocity that ranged from 0.48 to 8.95 m/s across rats. These results suggest that a wide spectrum of fiber types characterizes the reward-relevant axons that course uninterruptedly between these 2 regions.

Action Potentials↗

PubMed2001

Response to X-irradiation of Fanconi anemia homozygous and heterozygous cells assessed by the single-cell gel electrophoresis (comet) assay.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by bone marrow failure and cancer susceptibility. Patient cells are sensitive to a variety of clastogens, most prominently cross-linking agents. Although there is the long-standing clinical impression of radiosensitivity, in vitro studies have yielded conflicting results. We exposed peripheral blood mononuclear cells from FA patients and carriers to x-rays and determined their DNA damage and repair profiles using the alkaline single-cell gel electrophoresis (comet) assay. Studies were carried out in two independent series of experiments by two laboratories using different protocols. The cells of both FA patients and carriers showed uniformly high initial DNA damage rates as assessed by the total initial tail moment. In addition, the average residual tail moment at 30 to 50 minutes and the repair half-time parameters were significantly elevated. These findings suggest an increased release of fragmented DNA following x-ray exposure in cells that carry one or two mutations in one of the FA genes. The comet assay may be a useful adjunct for heterozygote detection in families of FA patients.

Adult↗

PubMed2001

The relation between well-being, impostor feelings, and gender role orientation among Canadian university students.

A theoretical model of well-being identifies 6 key components that have been examined primarily in older adults (e.g., C. D. Ryff, 1989c, 1991): self-acceptance, positive relations with others, autonomy, environmental mastery, purpose in life, and personal growth (C. D. Ryff, 1995; C. D. Ryff & C. L. M. Keyes, 1995; C. D. Ryff & B. Singer, 1996). The authors examined them in a sample of 379 Canadian university students to determine how well-being was correlated with endorsement of stereotypic gender roles and with the impostor phenomenon. The participants completed Ryff's Scales of Psychological Well-Being (Ryff, personal communication, March 1996), the Clance Impostor Phenomenon Scale (P. R. Clance & M. A. O'Toole, 1988), and the Extended Personal Attributes Questionnaire (J. T. Spence, R. L. Helmreich, & C. K. Holahan, 1979). The results supported the hypotheses that (a) people with higher scores for expressive traits score higher for well-being stemming from positive relations with others, (b) people with higher scores for instrumental traits score higher for well-being related to feelings of autonomy, (c) people with higher scores for impostor feelings (and lower scores for ability confidence) score lower for self-acceptance and (d) for environmental mastery.

Adolescent↗

PubMed2000

The substrate for brain-stimulation reward in the lateral preoptic area. II. Connections to the ventral tegmental area.

This experiment investigated the existence of a direct anatomical connection between lateral preoptic and ventral tegmental areas that mediate brain stimulation reward using the behavioral adaptation of the collision test. This test is a double-pulse, two-electrode technique based on the axonal conduction failure that occurs when two separate sites in the same axon bundle are concurrently stimulated. This anatomical arrangement is inferred from the shape of the function relating the effectiveness of double-pulse stimulation to the interval between pulses. In this study, nine rats with a total of 44 pairs of sites were examined. In two pairs only was there a profile suggestive of an axonal collision effect, while the double-pulse effectiveness curve consistent with the properties of transynaptic collision was apparent for a single pair of sites; the remaining 93% were associated with relatively flat effectiveness curves. While electrode misalignment could be responsible for these results, there was adequate sampling to suggest that the preponderance of first stage signals that give rise to the rewarding effects mediated by the lateral preoptic and ventral tegmental areas do not travel along the same fiber bundle. However, stimulation applied to both sites concurrently produces a summation that is roughly 40% greater than stimulation at either site alone, suggesting reasonable integration of the reward signals generated by lateral preoptic and ventral tegmental area stimulation.

Action Potentials↗

PubMed2000

Spectrum of mutations in the Fanconi anaemia group G gene, FANCG/XRCC9.

FANCG was the third Faconi anaemia gene identified and proved to be identical to the previously cloned XRCC9 gene. We present the pathogenic mutations and sequence variants we have so far identified in a panel of FA-G patients. Mutation screening was performed by PCR, single strand conformational polymorphism analysis and protein truncation tests. Altogether 18 mutations have been determined in 20 families - 97% of all expected mutant alleles. All mutation types have been found, with the exception of large deletions, the large majority is predicted to lead to shortened proteins. One stop codon mutation, E105X, has been found in several German patients and this founder mutation accounts for 44% of the mutant FANCG alleles in German FA-G patients. Comparison of clinical phenotypes shows that patients homozygous for this mutation have an earlier onset of the haematological disorder than most other FA-G patients. The mouse Fancg sequence was established in order to evaluate missense mutations. A putative missense mutation, L71P, in a possible leucine zipper motif may affect FANCG binding of FANCA and seems to be associated with a milder clinical phenotype.

Amino Acid Sequence↗

PubMed2000

Cell-surface expression of transrearranged Vgamma-cbeta T-cell receptor chains in healthy donors and in ataxia telangiectasia patients.

Transrearrangements between the T-cell receptor (TCR) VgammaI family and JbetaCbeta loci occur at increased frequencies in patients with ataxia telangiectasia (AT). We have used an optimized reverse transcriptase polymerase chain reaction (RT-PCR) approach to investigate the occurrence of TCRVgamma-JbetaCbeta transrearrangements involving the dominantly used Vgamma element in peripheral blood gammadelta T cells, i.e. Vgamma9. We detected in frame transcripts of Vgamma9-JbetaCbeta transrearrangements in 4/16 AT patients and in 3/13 healthy control donors. A panel of monoclonal antibodies (mAb) against all expressed TCRVgamma elements was used to monitor cell-surface expression of transrearranged TCR. A very low proportion (< 1%) of peripheral blood TCRalphabeta cells expressed Vgamma instead of Vbeta elements. For the first time, we have isolated and molecularly characterized alphabeta T cells with a Vgamma9-JbetaCbeta transrearrangement from two AT patients and we have shown that such TCR are functional. We conclude that functional TCR transrearrangements can also involve Vgamma9, the dominant Vgamma element in peripheral blood gammadelta T cells.

Ataxia Telangiectasia↗

PubMed2000

Spectral karyotyping of Werner syndrome fibroblast cultures.

Fibroblast cultures from two Werner syndrome patients were analyzed by spectral karyotyping. There were multiple, pseudodiploid clones in both cultures, mostly marked by random balanced reciprocal translocations. One of the cultures contained a clone with three-way exchanges involving chromosomes 2, 3, and 16. Duplication-deficiencies were exceptional, as were completely normal metaphases. The most frequent breakpoint occurred at 16q22 which corresponds to FRA16B, possibly reflecting difficulties of WS cells in replicating AT-rich repetitive DNA structures. Both cultures ceased proliferation after eight in vitro passages, but a single clone with exceptional growth potential emerged in one of the senescing cultures. Due to its identical translocations, the derivation of this near tetraploid clone (with tetrasomy for all autosomes except chromosomes 4 and 6) could be traced to the most prevalent pseudodiploid clone of the parental mass culture. Our study confirms the existence of variegated translocation mosaicism as the cytogenetic hallmark of WS fibroblast cultures and suggests that tetraploidization in combination with certain chromosome rearrangements and selective chromosome dosage may overcome the severely limited in vitro lifespan of WS fibroblasts.

AT Rich Sequence↗

PubMed1999

Sequence analysis of the ATM gene in 20 patients with RTOG grade 3 or 4 acute and/or late tissue radiation side effects.

PURPOSE: Patients with ataxia-telangiectasia (A-T) show greatly increased radiation sensitivity and cancer predisposition. Family studies imply that the otherwise clinically silent heterozygotes of this autosomal recessive disease run a 3.5 to 3.8 higher risk of developing cancer. In vitro studies suggest moderately increased cellular radiation sensitivity of A-T carriers. They may also show elevated clinical radiosensitivity. We retrospectively examined patients who presented with severe adverse reactions during or after standard radiation treatment for mutations in the gene responsible for A-T, ATM, considering a potential means of future identification of radiosensitive individuals prospectively to adjust dosage schedules. MATERIAL AND METHODS: We selected 20 cancer patients (breast, 11; rectum, 2; ENT, 2; bladder, 1; prostate, 1; anus, 1; astrocytoma, 1; Hodgkins lymphoma, 1) with Grade 3 to 4 (RTOG) acute and/or late tissue radiation side effects by reaction severity. DNA from the peripheral blood of patients was isolated. All 66 exons and adjacent intron regions of the ATM gene were PCR-amplified and examined for mutations by a combination of agarose gel electrophoresis, single-stranded conformational polymorphism (SSCP) analysis, and exon-scanning direct sequencing. RESULTS: Only 2 of the patients revealed altogether four heteroallelic sequence variants. The latter included two single-base deletions in different introns, a single-base change causing an amino acid substitution in an exon, and a large insertion in another intron. Both the single-base deletions and the single-base change represent known polymorphisms. The large insertion was an Alu repeat, shown not to give rise to altered gene product. CONCLUSIONS: Despite high technical efforts, no unequivocal ATM mutation was detected. Nevertheless, extension of similar studies to larger and differently composed cohorts of patients suffering severe adverse effects of radiotherapy, and application of new technologies for mutation detection may be worthwhile to assess the definite prevalence of significant ATM mutations within the group of radiotherapy patients with adverse reactions. To date, it must be recognized that our present results do not suggest that heterozygous ATM mutations are involved in clinically observed radiosensitivity but, rather, invoke different genetic predisposition or so far unknown exogenous factors.

Adult↗

PubMed1999

Characterization of ATM gene mutations in 66 ataxia telangiectasia families.

Ataxia telangiectasia (AT) is an autosomal recessive disease characterized by neurological and immunological symptoms, radiosensitivity and cancer predisposition. The gene mutated in AT, designated the ATM gene, encodes a large protein kinase with a PI-3 kinase-related domain. In this study, we investigated the mutational spectrum of the ATM gene in a cohort of AT patients living in Germany. We amplified and sequenced all 66 exons and the flanking untranslated regions from genomic DNA of 66 unrelated AT patients. We identified 46 different ATM mutations and 26 sequence polymorphisms and variants scattered throughout the gene. A total of 34 mutations have not been described in other populations. Seven mutations occurred in more than one family, but none of these accounted for more than five alleles in our patient group. The majority of the mutations were truncating, confirming that the absence of full-length ATM protein is the most common molecular basis of AT. Transcript analyses demonstrated single exon skipping as the consequence of most splice site substitutions, but a more complex pattern was observed for two mutations. Immunoblot studies of cell lines carrying ATM missense substitutions or in-frame deletions detected residual ATM protein in four cases. One of these mutations, a valine deletion proximal to the kinase domain, resulted in ATM protein levels >20% of normal in an AT lymphoblastoid cell line. In summary, our results survey and characterize a plethora of variations in the ATM gene identified by exon scanning sequencing and indicate a high diversity of mutations giving rise to AT in a non-isolated population.

Ataxia Telangiectasia↗

PubMed1999

Cerebral glucose metabolism in type I alpha-N-acetylgalactosaminidase deficiency: an infantile neuroaxonal dystrophy.

Cerebral glucose metabolism was investigated in a 4.8-year-old boy with alpha-N-acetylgalactosaminidase deficiency using 2-[18F]fluoro-2-deoxy-D-glucose and positron emission tomography (PET). In comparison to normal values for age, the overall cerebral glucose metabolism was reduced and the regional cerebral glucose metabolism was decreased in proportion to the degree of atrophy. In the supratentorial cortical regions, the hypometabolism was asymmetric. However, the level of regional cerebral glucose metabolism in all cortical regions excluded a persistent vegetative state. In the lentiform nucleus and the head of the caudate, comparatively increased regional cerebral glucose metabolism was documented, similar to findings in neurodegenerative disorders with active epilepsy. In contrast, the infratentorial structures (cerebellar hemispheres, brain stem, mesencephalon, and hypothalamus), which are predominantly affected by the atrophic process, showed distinct and symmetric hypometabolism. Thus, the 2-[18F]-fluoro-2-deoxy-D-glucose PET scans provided additional insight into and correlation of the functional and structural disturbances in type I alpha-N-acetylgalactosaminidase deficiency, in addition to documenting the hypometabolism due to brain atrophy.

Blood Glucose↗

PubMed1999

Identification of ataxia telangiectasia heterozygotes, a cancer-prone population, using the single-cell gel electrophoresis (Comet) assay.

Heterozygotes of ataxia telangiectasia (AT) may comprise up to 1% of the general population. Because these individuals have no clinical expression of AT but may be highly radiosensitive and strongly predisposed for several forms of cancer, identification of AT carriers represents a considerable interest in cancer epidemiology and radiotherapy. We report a new approach for the in vitro identification of AT-heterozygotes based on the evaluation of the radiosensitivity and DNA damage repair ability of peripheral blood mononuclear cells using the single-cell gel electrophoresis (Comet) assay. The assay was performed on cells isolated from four different groups of individuals: (1) apparently healthy donors (n = 10); (2) patients with breast cancer showing a normal reaction to radiotherapy (n = 10); (3) a group of obligate AT carriers (parents of AT-homozygotes, n = 20); and (4) AT-homozygotes (n = 4). Cells irradiated with 3 Gy of x-rays were assayed for three parameters: (1) the initial and (2) residual DNA damage and (3) the kinetics of DNA damage repair. Both AT-heterozygotes' and AT-homozygotes' cells were found to be highly sensitive to x-irradiation. Quantitative evaluation of the single-cell electrophoregrams revealed that the average initial DNA damage in AT-heterozygous and AT-homozygous cells was almost three times higher than that in control non-AT cells. In addition, the DNA repair process in irradiated AT carrier cells was almost three times slower, and the extent of irreparable DNA damage in these cells was three times greater than in controls. Simultaneous assessment of the three parameters enabled correct identification of all tested AT carriers. This method seems to be a sensitive and useful tool for populational studies as a rapid prescreening test for a mutated AT status. The approach can also be extended for prediction of the in vivo radiosensitivity, which would enable optimization of individual radiotherapy schedules.

Adolescent↗

PubMed1998

Fanconi's anaemia cells have normal steady-state levels and repair of oxidative DNA base modifications sensitive to Fpg protein.

Cells from Fanconi's anaemia (FA) patients are abnormally sensitive to oxygen. However, a distinct genetic defect in either the cellular defence against reactive oxygen species (ROS) or in their metabolic generation has not been identified to date. Recently, the gene for the human 8-hydroxyguanine (8-oxoG) glycosylase, which removes this oxidative base modification from the genome, has been localized on chromosome 3p25, i.e., in the same region as the FA complementation group D (FAD) gene. We therefore studied the removal of photosensitization-induced 8-oxoG residues from the DNA of FA cells, using Fpg protein, the bacterial 8-oxoG glycosylase, to quantify the lesions by alkaline elution. Similar repair kinetics (approx. 50% removal within 2 h) were observed in Epstein-Barr virus (EBV) immortalized lymphoid cells from FA complementation groups A, B, C and D and in control cells from normal donors, as well as in primary fetal lung fibroblasts not yet assigned to a specific complementation group. The susceptibility for the induction of oxidative DNA modifications by photosensitization was similar in all cells. In addition, the background (steady-state) levels of Fpg-sensitive oxidative DNA base modifications, which reflect the balance between generation and removal of the lesions, were similar in control and FA cells. It is concluded that both the generation and the overall removal of 8-oxoG residues in nuclear DNA is not impaired in FA cells.

Cell Division↗

PubMed1998

Molecular cloning, structural organization, sequence, chromosomal assignment, and expression of the mouse alpha-N-acetylgalactosaminidase gene.

Alpha-N-acetylgalactosaminidase (2-acetamido-2-deoxy-alpha-d-galactoside acetamidodeoxy-galactohydrolase, NAGA; EC 3.2.1.49) deficiency is a recently recognized autosomal recessive lysosomal disease. As a prerequisite for the generation of an animal model, the mouse NAGA gene was cloned and characterized. The NAGA gene was assigned to mouse chromosome 15 band E3, syntenic to the region encompassing the human gene, and NAGA-deficient mutant human cells transfected with the cosmid clone containing the mouse NAGA gene expressed NAGA activity. Comparison of the mouse NAGA nucleotide sequence with the human NAGA sequence predicted that the mouse NAGA gene contains an open reading frame of 1245bp, comprising nine coding exons and spanning a genomic region of 8258bp, and a 3' untranslated region of 0.5kb. The 5' untranslated region was determined in primer extension studies to be 235bp in length. Nucleotide identity between the human and mouse NAGA exons ranged from 67.4 to 89.5%, with better matches for exons 1-7 than for 8 and 9. The overall amino acid identity between the mouse and human deduced NAGA polypeptides was 82.0%, between those of mouse and chicken 72.9%. Homology was found to only one other mouse gene, i.e. the alpha-galactosidase A (GALA; EC 3.2.1. 22) gene. The amino acid identity ranged from 51.6 to 62.1% in the polypeptide regions corresponding to NAGA exons 2-7 and GALA exons 1-6, but little, if any, in the remainder. These analyses gave emphasis to the strong conservation of the NAGA gene and its origin from an ancestor common with the GALA gene, with NAGA exons 8 and 9 and GALA exon 7 being the most divergent regions in the evolution of the two genes.

Amino Acid Sequence↗

PubMed1998

Small deletions in the regulatory 3' UTR of the human alpha-tropomyosin gene identified by differential display.

The differential display technique (DDT) was used to compare Fanconi anaemia (FA) fibroblasts with those of normal controls in a screen for genes involved in DNA repair, recognizing and handling damage or indicating cell cycle abnormalities as a result of genetic changes. The DDT revealed two different deletions of 5 and 11 bp at a single locus in the 3' untranslated region (UTR) of a gene known to encode human alpha-tropomyosin (TPM1) in FA cells. These small deletions were detected by analysis of shifted 900-bp long cDNA fragments on polyacrylamide gels. They were characterized as loss of GTTTT or TGTTTTGTTTT, respectively, in a region with five GTTTT tandem repeats. Since it was postulated that the 3' UTR of the TPM1 gene plays a regulatory role in cell differentiation and tumour suppression, the existence and possible patterns of deletions in a variety of normal donors was investigated. The heterogenous distribution of non-deleted, 5- and 11-bp deleted 3' UTR regions indicate a polymorphism of the TPM1 gene in this tandem repeat motif. Therefore the expression pattern of these mutations among FA and non-FA cells rendered any direct relationship to the putative DNA repair defect in FA unlikely. Of note, however, the fact remains that such deletions reportedly facilitate mRNA degradation and may bear significance in the TPM1 gene action. Finally, of further interest is the finding that even small deletions can be identified by DDT in addition to the identification of the differential expression patterns of genes.

Amino Acid Sequence↗

PubMed1998

The Fanconi anaemia group G gene FANCG is identical with XRCC9.

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms including developmental anomalies, bone marrow failure and early occurrence of malignancies. In addition to spontaneous chromosome instability, FA cells exhibit cell cycle disturbances and hypersensitivity to cross-linking agents. Eight complementation groups (A-H) have been distinguished, each group possibly representing a distinct FA gene. The genes mutated in patients of complementation groups A (FANCA; refs 4,5) and C (FANCC; ref. 6) have been identified, and FANCD has been mapped to chromosome band 3p22-26 (ref. 7). An additional FA gene has recently been mapped to chromosome 9p (ref. 8). Here we report the identification of the gene mutated in group G, FANCG, on the basis of complementation of an FA-G cell line and the presence of pathogenic mutations in four FA-G patients. We identified the gene as human XRCC9, a gene which has been shown to complement the MMC-sensitive Chinese hamster mutant UV40, and is suspected to be involved in DNA post-replication repair or cell cycle checkpoint control. The gene is localized to chromosome band 9p13 (ref. 9), corresponding with a known localization of an FA gene.

5' Untranslated Regions↗