SfaNI polymorphism distinguishes the alleles of the glycophorin A locus that determine the MN blood group.
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Alleles↗
Biomedical subjects
R H Jensen
Publications and source records attributed to R H Jensen.
Biodosimetry of Chernobyl cleanup workers from Estonia and Latvia using the glycophorin A in vivo somatic cell mutation assay.
The reactor accident at Chernobyl in 1986 necessitated a massive environmental cleanup that involved over 600,000 workers from all 15 Republics of the former Soviet Union. To determine whether the whole-body radiation received by workers in the course of these decontamination activities resulted in a detectable biological response, over 1,500 blood samples were obtained from cleanup workers sent from two Baltic countries, Estonia and Latvia. Here we report the results of studies of biodosimetry using the glycophorin A (GPA) locus in vivo somatic cell mutation assay applied to 734 blood samples from these workers, to 51 control samples from unexposed Baltic populations and to 94 samples from historical U.S. controls. The data reveal inconsistent evidence that the protracted radiation exposures received by these workers resulted in a significant dose-associated increase in GPA locus mutations compared with the controls. Taken together, these data suggest that the average radiation exposure to these workers does not greatly exceed 10 cGy, the minimum levels at which radiation effects might be detectable by the assay. Although the protracted nature of the exposure may have reduced the efficiency of induction of GPA locus mutations, it is likely that the estimated physical doses for these cleanup worker populations (median reported dose 9.5 cGy) were too low to result in radiation damage to erythroid stem cells that can be detected reliably by this method.
Alleles↗
Glycophorin A as a biological dosimeter for radiation dose to the bone marrow from iodine-131.
The frequency of peripheral blood erythrocyte variants exhibiting allelic loss of glycophorin A (N/M antigen) has been used previously as a biological dosimeter to assess somatic mutations in bone marrow cells from external whole-body irradiation. The aim of the present study was to determine whether this marker could be used as a measure of bone marrow genotoxicity induced by 131I in the treatment of thyroid cancer. Flow cytometry of immunolabeled erythrocytes was performed to enumerate glycophorin A variants before and after eight therapy doses of 131I administered to five patients with differentiated thyroid carcinoma. Bone marrow radiation exposure from each dose was calculated from the integrated retention of 131I in the whole body and in the blood. In addition, the accumulated dose to the bone marrow received from earlier 131I therapy was calculated for each patient. Regression analysis was performed on the frequency of two glycophorin A variant cell types (N/O and N/N) as a function of accumulated dose to the bone marrow. Frequency of N/O variant cells showed a significant dose-related increase with a slope of 10.9 x 10(-6) per sievert. This dose effect is about one-half that previously observed after whole-body external irradiation at high dose rate. This decreased response could be explained by the low dose rate of the radiation to the bone marrow from 131I.
Bone Marrow↗
Cumulative genetic damage in hematopoietic stem cells in a patient with a 40-year exposure to alpha particles emitted by thorium dioxide.
Thorotrast, a colloidal suspension of the long-lived radionuclide, thorium-232, was widely used as a radiographic contrast medium for several decades. Due to the poor excretion of the sol, however, Thorotrast would deposit in the liver, bone marrow and other tissue, and patients would receive alpha-particle irradiation for life. To gauge the cumulative genetic damage to hematopoietic stem cells due to chronic exposure to alpha particles, we conducted a multi-end-point evaluation in a 72-year-old man who had been administered a 32-ml bolus of Thorotrast during cerebral angiography performed over 40 years ago in 1950. Peripheral T lymphocytes were cultured to quantify the frequencies and cellular distributions of asymmetrical and symmetrical types of chromosome aberrations in first-division metaphases and micronuclei in cytokinesis-arrested interphase II cells. Aberrations were scored using classical chromosome group analysis methods and chromosome painting techniques. Assays of glycophorin-A (GPA) mutations in red blood cells were also performed to obtain a relative measurement of damage sustained by the erythroid stem cell population. Results revealed that approximately 30% of the lymphocytes in this patient contained one or more chromosome aberrations, the majority of which were of the "stable" type. About one-third of the lymphocytes with chromosome damage carried multiple aberrations, suggesting that significant numbers of stem cells survive exposures to alpha-particle radiation that induce complex genomic alterations. Increased frequencies of GPA mutations were observed, demonstrating that genomic damage is also induced in erythroid progenitors. The numbers of micronuclei in lymphocytes were only moderately increased compared to expected values for persons of comparable age, and thus this end point was not useful for quantifying exposure level. Despite the relatively severe burden of somatic cell damage induced by 40 years of internal alpha-particle irradiation, the patient remains surprisingly free of any serious illness.
Alpha Particles↗
Biological dosimetry of radiation workers at the Sellafield nuclear facility.
The British Nuclear Fuels plc facility at Sellafield performs a range of nuclear-related activities. The site has been in operation since 1950 and has, in general, employed a stable work force, many of whom have accumulated relatively high occupational exposures to ionizing radiation. This paper compares the physical dosimetry with two biological end points for evaluating radiation exposure: fluorescence in situ hybridization with whole-chromosome painting probes to quantify stable chromosome aberrations (translocations and insertions), and glycophorin A (GPA) analysis of variant erythrocytes. For the cytogenetic analyses, 81 workers were evaluated in five dose categories, including 23 with minimal radiation exposure (< or = 50 mSv) and 58 with exposures ranging from 173 to 1108 mSv, all but 3 being > 500 mSv. In a univariate analysis, the mean stable chromosome aberration frequencies showed a significant increase with dose category (P = 0.032), and with cumulative dose when dose is treated as a continuous variable (P = 0.015). The slope of the dose response for stable aberrations is 0.79 +/- 0.22 aberrations per 100 cells per sievert (adjusted for smoking status), which is less than that observed among atomic bomb survivors, and suggests a dose and dose-rate effectiveness factor for chronic exposure of about 6. Analyses of the data for GPA N/O and N/N variants from 36 workers revealed no correlation with dose. Neither was there a correlation between the frequencies of N/O GPA variants and stable aberrations, although a weak negative association was observed between N/N variant frequency and stable aberrations (r = -0.38, P = 0.05). These results provide clear evidence for the accumulation of stable aberrations under conditions of chronic occupational exposure to ionizing radiation and show that stable chromosome aberrations are a more sensitive indicator for chronic radiation exposure than GPA variants. In comparison with human studies of brief exposure, chronic low-dose exposures appear substantially less effective for producing somatic effects as reflected by stable chromosome aberrations.
Cells, Cultured↗
Genetic alterations in untreated metastases and androgen-independent prostate cancer detected by comparative genomic hybridization and allelotyping.
A newly developed method of comparative genomic hybridization (CGH) employing quantitative statistical comparisons was applied to DNA from two different types of advanced prostate cancer tissue. Multiple CGH analyses were obtained for each chromosome in each tumor, and the results of point-by-point comparison of the mean tumor:normal color ratio to a control normal:normal color ratio in each of 1247 evenly distributed data channels constituting the entire human genome were interpreted as loss, gain, or no change in copy number in the tumor genome. Group I tissue was obtained from prostate cancer metastases from 20 patients, 19 of whom had received no prior prostate cancer treatment. This DNA also was analyzed by Southern and microsatellite allelotyping at 53 different loci on 20 different chromosome arms. CGH results agreed with allelotyping results at 92% of the informative loci studied. These samples, which contained highly enriched tumor DNA, showed the highest rates of alteration yet reported in several chromosomal regions known to be altered frequently in prostate cancer: 8q gain (85%), 8p loss (80%), 13q loss (75%), 16q loss (55%), 17p loss (50%), and 10q loss (50%). Group II tissue was obtained predominately from primary or recurrent tumor from 11 patients who had been treated with long-term androgen-deprivation therapy and developed androgen-independent metastatic disease. Quantitative CGH analysis on DNA from these tissues showed chromosomal alterations that were very similar to those found in group I, suggesting that untreated metastatic tumors contain the bulk of chromosomal alterations necessary for recurrence to occur during androgen deprivation. In the entire data set, a number of previously undetected regions of frequent loss or gain were identified, including losses of chromosomes 2q (42%), 5q (39%), 6q (39%), and 15q (39%) and gains of chromosomes 11p (52%), 1q (52%), 3q (52%), and 2p (45%). Chi-squared analysis showed a significantly higher frequency of gain of the 4q25-q28 region in tumors from African-American patients, indicating a possible oncogene whose activation may play a role in the higher rate of progression seen in this ethnic group. Additional study of these frequently altered regions may provide insight into the mechanism of prostate cancer progression and lead to important tools for tumor-specific prognosis and therapy.
Adult↗
Glycophorin A biodosimetry in Chernobyl cleanup workers from the Baltic countries.
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Estonia↗
Direct immunofluorescence labeling provides an improved method for the glycophorin A somatic cell mutation assay.
The glycophorin A (GPA) somatic cell mutation assay is being applied widely as an in vivo biomarker in molecular epidemiologic studies of human populations. The assay uses two-color immunolabeling and flow cytometry of peripheral blood samples to enumerate allele-loss variant erythrocytes that appear as a result of mutations at the GPA locus in bone marrow erythroid cells. We have developed an improved version of the assay in which both anti-GPA monoclonal antibodies are directly conjugated with distinguishable fluorophores, fluorescein and phycoerythrin. Parallel analyses of 77 blood samples using the existing BR6 assay and our new DB6 assay demonstrate that the DB6 assay produces cleaner bivariate flow histograms with generally lower variant cell frequencies and lower coefficients of variation on replicate analyses of individual blood samples. With the BR6 assay, an artifact was shown to exist that results in enumeration of high frequencies of variant erythrocytes in a small fraction of samples that have been subjected to poor shipping and/or storage conditions. Using DB6, these same samples display acceptable histograms and low frequency of variant cells. High speed cell sorting followed by immuno analysis indicates that the BR6 artifact results from inhibited binding of the very high molecular weight antibody plus secondary reagent, which is used for the BR6 assay. We therefore recommend that DB6 be adopted as standard protocol for the GPA assay, and to assist other researchers in standardization and comparison, we are making available a set of calibrated, fixed blood samples.
Flow Cytometry↗
Percutaneous dilatational tracheostomy.
METHODS: Fifteen patients electively selected for percutaneous dilatational tracheostomy as performed with the Ciaglia Percutaneous Tracheostomy Introducer Set are reported in this preliminary study. RESULTS: The median time for insertion of the tracheostomy tube was 13.9 min. Complications were cuff puncture of the translaryngeal tube at an early stage of the procedure in one case, and minor bleeding controllable by the compressible effect of the dilatational procedure in three cases. During the posttracheostomy period, complications were one case of minor bleeding controllable by digital compression and one case of minor infection, i.e. cellulitis in a few millimetres around the stoma without purulent secretion. CONCLUSIONS: Our preliminary results indicate the technique as performed with the Ciaglia Introducer Set to be effective and safe with only minor complications. Furthermore, we now use it as a bedside procedure in the ICU, thus avoiding the transportation of critically ill patients to the operating theatre. The conclusion of the superiority of percutaneous tracheostomy to dissectional tracheostomy has to await clinical randomized studies, including more patients.
Adolescent↗
Glycophorin A somatic cell mutation frequencies in Finnish reinforced plastics workers exposed to styrene.
We have used the glycophorin A (GPA) in vivo somatic cell mutation assay to assess the genotoxic potential of styrene exposure in 47 reinforced plastics workers occupationally exposed to styrene and 47 unexposed controls matched for age, gender, and active smoking status. GPA variant erythrocyte frequencies (Vf), reflecting GPA allele loss (phi/N) and allele loss and duplication (N/N) somatic mutations arising in vivo in the erythroid progenitor cells of individuals of GPA M/N heterozygous genotype, were flow cytometrically determined in peripheral blood samples from these subjects. Measurements of styrene exposure of the workers at the time of blood sampling showed a mean 8-h time-weighted average (TWA8-h) styrene concentration of 155 mg/m3 (37 ppm) in the breathing zone. Mean urinary concentrations of the styrene metabolites mandelic acid (MA) and mandelic acid plus phenyl glyoxylic acid (MA+PGA) were 4.4 mmol/liter (after workshift) and 2.1 mmol/liter (next morning), respectively. Multivariate analysis of covariance on log-transformed GPA Vf data with models allowing adjustment for age, gender, smoking status, and styrene exposure showed that N/N Vf were nearly significantly increased among all of the exposed workers (adjusted geometric mean, 6.3 per million versus 5.0 in the controls; P = 0.058) and were statistically significantly elevated (adjusted geometric mean, 6.8 versus 5.0 in the controls; P = 0.036) among workers classified into a high-exposure group according to personal TWA8-h concentration of styrene in the breathing zone of > or = 85 mg/m3 (20 ppm; Finnish threshold limit value). Women in this high exposure group showed especially elevated N/N Vf (adjusted geometric mean 8.5 versus 5.3 in control women; P = 0.020); this elevation was also significant if urinary MA+PGA of > or = 1.2 mmol/liter was used as the basis of classification (adjusted geometric mean, 8.3; P = 0.030). The occupational exposure could not be shown to influence phi/N Vf. Cigarette smoking was associated with significantly elevated GPA Vf among active smokers (P = 0.042 for phi/N and P = 0.020 for N/N) and among active and ex-smokers combined (P = 0.014 for N/N). Its influence on phi/N Vf was especially clear among active smokers in the control group (P = 0.005). An effect of smoking, nearly statistically significant, was also observed for the phi/N Vf of control ex-smokers (P = 0.055) and of all active and ex-smokers combined (P = 0.050). Thus, the two characterized chemical exposures experienced by this group of workers and controls appear to produce differential effects on the two independent classes of GPA variants enumerated in the assay. This result suggests that the genotoxicity of these agents is mediated, at least in part, by different genetic mechanisms. Styrene exposure is associated with a specific increase in GPA N/N Vf; these allele loss and duplication variants reflect predominantly somatic recombination mechanisms in erythroid progenitor cells. Tobacco smoke exposure in active and ex-smokers is also associated not only with an increase in N/N Vf but also with an increase in phi/N Vf, reflecting the induction of GPA gene-inactivating mutations, including point mutations and deletions. This finding is consistent with a broad mechanistic spectrum of tobacco smoke genotoxicity associated with this complex mixture of chemical mutagens. Finally, there was no detectable effect of age on phi/N Vf; however, a highly significant (P = 0.0002) increase in N/N Vf with age, even after adjustment for other variables, was observed.
Adult↗
Sequences of cDNAs encoding immunoglobulin heavy- and light-chain variable regions from two anti-dioxin monoclonal antibodies [Gene 149 (1994) 385-386].
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Amino Acid Sequence↗
Sensitivity of somatic mutations in human umbilical cord blood to maternal environments.
To assess the potential effect of maternal environments on human embryonic/fetal somatic mutation, we measured the frequencies of hypoxanthine-guanine phosphoribosyltransferase (HPRT, hprt gene), mutant T lymphocytes (Mf), and glycophorin A (GPA) variant erythrocytes (Vf) of both allele-loss (phi/N) and allele-loss-and-duplication (N/N) phenotypes in umbilical cord blood. The mean hprt Mf (1.40 +/- 1.11 x 10(-6), N = 66) and GPA Vf (phi/N 4.0 +/- 2.2 x 10(-6), N = 114; N/N 2.7 +/- 2.0 x 10(-6), N = 91) were significantly lower than those previously reported for adult populations. In addition, the hprt Mf was significantly higher than that of a published study of newborn cord blood samples from a geographically distant population (0.64 +/- 0.41 x 10(-6), N = 45, P < 0.01; t test, P < 0.01, Mann-Whitney U test). An examination of the demographic data from these two populations led to the sampling of 10 additional newborns specifically matched to the published study for maternal socioeconomic status. The hprt Mf (0.70 +/- 0.49 x 10(-6)) of this selected population was consistent with the published report and significantly lower than that of our initial population (P < 0.03, t test; P < 0.01, Mann-Whitney U test). These results indicate that there is an environmental effect related to maternal socioeconomic status on the frequency of embryonic/fetal somatic mutations. Molecular analyses of hprt mutants from this cohort with elevated Mf revealed a significant decrease in the relative contribution of gross structural mutations to the overall Mf (25 of 38, 66% vs. 34 of 41, 83%, P = 0.024, chi 2 test), suggesting that the higher Mf resulted from an elevated level of "point" mutations. No individual maternal demographic or environmental factor was identified as contributing more significantly than other any factor to the observed variability in hprt Mf or GPA Vf.
Adult↗
Mapping of regions of physical deletion on chromosome 16q in prostate cancer cells by fluorescence in situ hybridization (FISH).
Recent evidence suggests that a tumor suppressor gene important in the progression of prostatic carcinoma may reside on chromosome 16q. The exact location and identity of this gene are unknown. We used fluorescence in situ hybridization in a novel manner to define more clearly the location of this gene. Region-specific chromosome 16 cosmid contig probes were hybridized directly to interphase prostatic carcinoma nuclei in order to measure physical deletion of chromosomal loci. Fifteen of 30 tumors (50%) showed evidence of physical deletion of 16q24. Other probes were used to test for regions of chromosome 16 deletions in the same specimens, and a map showing a region of common deletion was created. This map showed the proximal terminus of the region of common deletion to be located distal to 16q23.1. These data correlate well with loss of heterozygosity data in the literature and provide further evidence for the presence of a prostatic carcinoma tumor suppressor gene on chromosome 16q.
Chromosome Deletion↗
Molecular characterization of glycophorin A transcripts in human erythroid cells using RT-PCR, allele-specific restriction, and sequencing.
Glycophorin A (GPA) is an erythroid-lineage-specific membrane sialoglycoprotein which occurs in two allelic forms, M and N, which form the antigens of the MN blood group. Purified cDNAs and RNAs isolated from peripheral blood and erythroleukemia cell lines, HEL and K562, were used to develop an RT-PCR technique for amplifying GPA gene transcripts (GYPA). The relative expression of transcripts from the M and N alleles was determined using restriction analysis of these amplified products with four allele-specific restriction endonucleases. The use of this method permits the sensitive identification of GYPA transcripts in these cells and confirms GPA protein expression in the erythroleukemia cell lines and the MN phenotypes of individuals determined by immunolabeling with GPA allele-specific monoclonal antibodies. A novel restriction pattern was obtained using peripheral blood RNA from two individuals with a rare inherited variant allele, GPA Mg. Sequencing of the cDNA obtained using this method revealed a single C to A transversion in the fourth codon in the mature GYPA N coding sequence is responsible for the difference between GYPA Mg and GYPA N.
Alleles↗
Impaired neutrophil chemotaxis after cardiac surgery.
Chemotaxis of circulating peripheral neutrophils was studied in 12 patients undergoing elective cardiac surgery. Tests of neutrophil chemotaxis were made preoperatively and 16-20 hours postoperatively, using a multipore filter assay. Statistically significant postoperative decrease was demonstrated in both stimulated and random neutrophil migration (p = 0.012 and p = 0.009, respectively). Although the peripheral neutrophil count showed a statistically significant postoperative increase (p = 0.001), the increase did not correlate to the decrease in neutrophil chemotaxis. Nor could correlation be demonstrated between the altered neutrophil chemotaxis and any per- or postoperative variables.
Anesthesia, General↗
Flow-dependent properties of positive expiratory pressure devices.
Valves for positive expiratory pressure (PEP) can be characterized as threshold resistors, ideally providing pressure independent of the expiratory flow, or as flow-dependent resistors. The aim of the study was to evaluate the flow-dependence properties of PEP devices of the flow resistor type compared to threshold resistor devices. Pressures were measured on three different flow resistor valves: the PEP-mask, the Pari-PEP-System and the System 22-PEP with orifice diameters of 1.5-5.0 mm; and on three threshold resistors, the underwater seal, the Ambu Positive End-Expiratory Pressure (PEEP) valve and the Vital Signs PEEP valve with pressures of 0, 5, 10, 15 and 20 cmH2O. All devices were studied with constant flows of 3, 6, 9, 12, 15, 18, 30, 60, 90, 120 and 150 l.min-1. The PEP-mask, the Pari-PEP and the System 22-PEP showed the typical pattern of flow resistors, i.e. a pressure increasing with flow, dependent on the diameter of the orifice. The underwater seal and the Vital Signs PEEP valves acted as almost ideal threshold resistors. The Ambu PEEP valves acted as threshold resistors at the lower flows, but showed flow-dependency at higher flows. The Vital Signs PEEP valves gave lower pressures and Ambu PEEP valves gave higher pressures compared with indicated values, whereas the underwater seal gave the intended pressure. In clinical use of PEP treatment the actual pressure should be measured to ensure the intended pressure, no matter which type of resistor is used.
Equipment Design↗
Elevated frequency of glycophorin A mutations in erythrocytes from Chernobyl accident victims.
In 1986, when an explosion accident occurred at the Chernobyl, Ukraine nuclear power plant, a large number of people were exposed to significant amounts of ionizing radiation. During the time between 1986 and 1992, peripheral blood samples were obtained from 102 people who either were on site during the emergency or were brought to Chernobyl shortly thereafter to assist in the cleanup of radioactive contaminants and isolate the damaged reactor from the environment. These blood samples plus samples from 13 unexposed Soviet individuals were analyzed by flow cytometry using the allele-loss somatic mutation assay for glycophorin A. Results of these assays show that the frequency of N/O variant red cells increased in proportion to the estimated radiation exposure of each individual. The radiation dose-response function derived from this population closely resembles that determined previously for atomic bomb survivors whose blood samples were obtained and analyzed 40 years after their exposure. This suggests comparable mutation induction per unit dose for these two populations and long-term persistence of the mutational damage. In addition, measurements on multiple blood samples from each of 10 donors taken over a 7-year period showed no significant changes in N/O variant cell frequencies, confirming the persistence of radiation-induced somatic mutations in long-lived bone marrow stem cells.
Dose-Response Relationship, Radiation↗
Sequences of cDNAs encoding immunoglobulin heavy- and light-chain variable regions from two anti-dioxin monoclonal antibodies.
We report the cDNA cloning of the gamma- and kappa-chain-encoding genes for two mouse monoclonal antibodies (mAb) which recognize dioxins. The nucleotide sequences encoding the variable regions of these mAb were also determined. Although the mAb have similar dioxin-binding characteristics, their deduced variable region amino-acid sequences are very different.
Amino Acid Sequence↗