Biomedical subjects
Publications and source records attributed to K Hirschhorn.
A 12-year-old girl with small stature and a hypertrophic clitoris was found to be mosaic for 45,X/46,X.dic(Y)(qtertop11:p11toqter)/46,XX/47,XX,dic(Y)(qtertop11:p11toqter). The dicentric chromosome was identified using Q-banding. These findings indicate mitotic instability of the dicentric Y, as well as the presence of an X chromosome in this patient.
Using a reliable 2-step colorimetric assay, some catalytic properties of human erythrocyte ITPase (inosine triphosphate pyrophosphohydrolase) (EC 3.6.1.19) were determined. The enzyme hydrolyses ITP, dITP (deoxyinosine triphosphate) and XTP (xanthine triphosphate) but not other nucleotides. IDP is a competitive inhibitor. Subcellular fractionation of cultured lymphoid-line cells showed ITPase activity to be in the cytosol. ATP and GTP hydrolysing activities were not enriched in the cytosol fraction. The range of activity was investigated in erythrocytes, peripheral leukocytes, amniotic fluid cells, skin fibroblasts, long term lymphoid lines and bone marrow fibroblasts. The mean ITPase activity was found to vary in different cell types from 4.9 to 294 units per mg protein. Fractionation of red blood cells by age revealed that the enzyme decays exponentially with erythrocyte age with a half-life of 35 days. The enzyme had a wide range of specific activity in erythrocytes from 150 normal randomly selected individuals (15--722 pmol/h/g Hb). Within the low range of erythrocyte specific activities (from 15 to 200) a correlation was found between an individual's erythrocyte activity and the activity in peripheral leukocytes. This correlation was not found at higher erythrocyte ITPase activities. Two individuals were detected who had erythrocyte ITPase activities which were close to the lowest ITPase activities of all samples tested. Thus, low or absent ITPase activity is an individual characteristic which may be expressed both in erythrocytes and in other cell types.
Human alpha-L-fucosidase, purified from placenta, was taken up from the culture medium by skin fibroblasts from patients with fucosidosis (alpha-L-fucosidase deficiency). The rate of uptake was low (uptake coefficient = 6 X 10(-4) ml.mg-1.h-1). Intracellular alpha-L-fucosidase activity was directly proportional to enzyme in the medium up to an activity of at least 40 nmoles/min/ml. No evidence for saturation of specific cell-surface receptors was seen. However, uptake was reduced by 75% by 1 mM mannose-6-phosphate and by 50% by 1mM glucose-6-phosphate, suggesting that uptake may be mediated by a receptor recognising a phosphorylated sugar or an analagous compound. Enzyme taken up by the cells was most active in subcellular fractions enriched with lysosomes and had an isozyme pattern, by isoelectric focusing, identical to that of the original enzyme preparation. Fucosidosis fibroblasts were shown to accumulate low molecular-weight, fucose-containing compounds to a level several times greater than control cells. This stored material was eluted from Sephadex G-25 as an asymmetrical peak with an elution volume of approximately twice the void volume of the column. Addition of placental alpha-L-fucosidase to the culture medium of fucosidosis fibroblasts prevented excessive accumulation of fucose-containing material and accelerated the breakdown of material accumulated prior to enzyme uptake.
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The association of refractory anemia with partial deletion of the long arm of one No. 5 chromosome (5q-) has been reported in six patients within the last two years. We wish to report an additional patient with refractory anemia and an acquired chromosomal deletion, del(5)(q15), in a direct bone-moarrow preparation. The specificity of this deletion for refractory anemia is discussed. It remains to be seen whether patients with refractory anemia and 5q- tend to develop acute leukemia, as has been recently suggested.
Bone marrow fibroblasts were cultured from two patients with fucosidosis type 2, six control subjects, and three patients with other lysosomal disorders. Optimal conditions for measuring alpha-L-fucosidase activity in lysates of these cells with the fluorogenic substrate 4-methylumbelliferyl-alpha-L-fucoside were established. The pH profile of normal bone marrow fibroblasts showed three peaks and a shoulder of enzymatic activity, with maximum activity at pH 4.75. In cells derived from fucosidosis patients two peaks of apparent alpha-L-fucosidase activity were obtained; the pH optimum was 4.5. alpha-L-Fucosidase activity (mean +/- SD) in the fucosidosis and control bone marrow fibroblasts was 2.5 and 312.4 +/- 10.9 nmoles 4-methylumbelliferone per milligram protein per hour, respectively. A reduction in the apparent specific enzymatic activity in the fucosidosis cells was observed by using increasing concentrations of cellular protein in the assay system. Mixing experiments between normal and fucosidosis cells gave the expected activities. These findings indicate that cultured bone marrow fibroblasts can be used for the diagnosis and study of fucosidosis.
From February 1969 to August 1976, we studied 1,048 amniotic fluids. Of these, 958 (91.4%) were primarily for prenatal cytogenetic diagnosis. Cytogenetic studies were attempted in 1,021 cases; the diagnosis was successful in 1,000 of these. The failure rate of obtaining a diagnosis from the amniotic fluid cell culture of the first amniocentesis was 5% (50 cases); 29 cases had a repeat tap and successful diagnosis was achieved in all. In 21 cases, a repeat tap was refused. Thus, the overall failure rate of obtaining a final cytogenetic diagnosis was 2.06% (21/1,021). There were 32 fetal losses after amniocentesis including 16 spontaneous second trimester abortions, 7 fetal deaths in utero and 9 stillbirths. In two additional cases, fetal death had occurred before amniocentesis. This number of fetal losses does not exceed the number that would be expected in the same maternal age group without amniocentesis. In our series, the frequencies of trisomy in maternal age groups 40 years and over, 37-39 years, 35-36 years, and under 35 years were 4.5, 3.14, 0 and 0% respectively. These frequencies are comparable to those reported from other prospective prenatal studies and higher than those of retrospective live born studies. Various problems and pitfalls in prenatal cytogenetic diagnosis are discussed.
The alpha-L-fucosidases (EC 3.2.1.51) from human and mouse cells could be separated by isoelectric focusing of neuraminidase-treated cell extracts in acrylamide slab gels. Fourteen hybrid clones derived from the fusion of mouse and human cultured fibroblasts and 37 hybrid clones derived from the fusion of human long-term lymphoid lines with mouse RAG cells were tested for expression of human alpha-L-fucosidase. A strong correlation between the expression of the human enzyme and the presence or absence of human chromosome 1 was found. The presence of human alpha-L-fucosidase in clones scored as positive by isoelectric focusing was confirmed by Ouchterlony double immunodiffusion against IgG from rabbits immunized with purified human alpha-L-fucosidase. It is concluded that the structural gene locus for human alpha-L-fucosidase is located on chromosome 1.
A staining technique that detects sister-chromatid exchanges (SCEs) has been used to examine the response of human lymphocyte chromosomes to various dosages of X-irradiation. The SCE frequency was markedly increased following irradiation. However, the increase was of a significantly smaller magnitude when irradiation occurred in the presence of an antimutagenic agent. Scoring SCEs may provide a useful technique for assaying the mutagenic effects of environmental carcinogens as well as the protective effects of antimutagenic agents.
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Immunoglobulin synthesis was examined in 31 man-mouse hybrid clones produced by fusing RAG mouse cells with human lymphoid cells. Cells were grown in serum-free medium containing [(14)C]leucine and a (14)C-labeled amino acid mixture. Spent medium was dialyzed, concentrated, and subjected to radioimmunoelectrophoresis. Eighteen clones were found to produce material that gave a radiolabeled precipitin line with anti-human IgG (gamma-chain specific). Production of material which was indistinguishable on radioimmunoelectrophoresis from human Ig gamma heavy chain, was dependent on the presence in hybrid clones of human chromosome 6. The material was found to have the ion-exchange elution characteristics of human IgG. When radiolabeled spent medium from human lymphoid lines and from chromosome 6-positive hybrid clones was exposed to protein A-Sepharose and bound material eluted with 8 M urea was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, three radiolabeled peaks occurred with molecular weights of approximately 55,000 (coinciding with that of Ig gamma heavy chain), 40,000 and 25,000 (coinciding with that of Ig light chains). No similar peaks were detected in experiments where spent medium from RAG cells was treated identically. These studies lead us to conclude that certain RAG-human lymphoid hybrid clones produce human IgG and that the structural genes for gamma heavy chains are located on human chromosome 6. These results also imply that the locus coding for human alpha,-antitrypsin (Pi) is located on chromosome 6.
Monozygotic twins were born with the phenotypical appearance of the trisomy 8 syndrome. The first twin, a stillborn, had autopsy findings suggestive of trisomy 8 syndrome. Cultured lymphocytes and skin fibroblasts of the second, liveborn twin, showed trisomy 8. While the lymphocyte culture showed 46/47, +8 mosaicism, with normal cells predominating, skin fibroblasts yielded only cells with trisomy 8. Bone marrow preparation showed only normal cells, 46,XY. Repeat lymphocyte culture at age 14 months, showed a reduced number of trisomy 8 cells. We would like to emphasize the importance of cytogenetic studies in early infancy, using both peripheral lymphocyte and skin fibroblast cultures, to increase the chance of detecting chromosomal abnormalities.
With the combination of the various banding techniques (G,Q, and R), a small deletion of the short arm of a No.5 chromosome was detected prenatally in the pregnancy of a 39-year-old woman. The deletion appeared to be either intersitial in nature, involving part of p13 and p14, or the result of a translocation with deletion of p13 leads to pter. Both parents were found to have a normal chromosome constitution with normal banding patterns. Thus, this deletion was a de novo event. Repeat amniotic fluid cell chromosome analysis at the time of elective abortion, and postmortem examination of the fetus confirmed the prenatal cytogenetic diagnosis. We wish to emphasize that precise identification of a small deletion, as in this case, requires a combination of the various banding techniques.
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Different clinical expressions of acid alpha-glucosidase deficiency have been described. The present study was undertaken to investigate the basic metabolic defect in the infantile and adult forms of the disease. Acid alpha-glucosidase (EC 3.2.1.20) was purified from normal and from adult acid alpha-glucosidase deficiency fibroblasts. The pH optimum; Michaelis constant; electrophoretic mobility in starch; thermal denaturation at pH 4.0 and 7.0; and inhibition by turanose, alpha-methylglucoside and trehalose were the same in purified enzyme from normal and mutant cells. Placental acid alpha-glucosidase was purified to, or near, homogeneity. Monospecific antibodies raised against the enzyme in each of three enzyme peaks obtained from the last purification step were found to cross-react with the enzyme of all three peaks, and with purified, normal fibroblast enzyme. Cross-reacting material (CRM) also was identified in fibroblast lysates from normal subjects and from both forms of acid alpha-glucosidase deficiency. The amount of CRM in the adult form appeared to be significantly less than in normal cells or cells from the infantile form. Enzyme activity was demonstrated in the immune complexes of the normal and adult acid alpha-glucosidase deficiency fibroblasts, but not of the infantile form. Competition for antibody binding sites was observed between normal and both types of mutant enzymes. The findings indicate that this case of infantile acid alpha-glucosidase deficiency is the result of a structural gene mutation which causes the synthesis of a catalytically inactive (CRM-positive) enzyme protein. It appears that in the adult form, the mutation causes a reduction in the amount of the enzyme protein present in the cells.