- Email: [email protected]
DEOXYRIBOSE-NUCLEIC-ACID REPLICATION PATTERN OF TRISOMY 18
286 These results demonstrate the reproducibility of the plasmavolume method used, and suggest that the deviations from predicted blood-volume in the control group reflect physiological variation. Among the eleven treated patients, five showed a greater change of blood-volume, four a greater change in red-cell volume, and six a greater change in circulating haemoglobin mass than the normal subjects. Only one patient (case 14) showed a reduction in red-cell volume and haemoglobin mass. This patient had acute bronchitis with cardiac failure, and he was receiving continuous oxygen when the second estimation was done. The changes in haemoglobin mass which were greater than those found in the control subjects varied from +- 8.7 to --35.7’o
Serurrr-iron and Iron-binding Capacity Seven of the control subjects investigated in the present series were within the normal range; one (subject 5) showed the findings of iron deficiency. Of the seventeen patients, five had serum-iron levels below the normal range in this laboratory, but in none of these was the binding capacity high enough to suggest iron deficiency. Four of these low-serum-iron subjects were in the treated group, and all four showed normal serumiron levels when retested after treatment.
with chronic obstructive airway disease and the in the blood which may accompany them.
changes
Summary Seventeen patients with severe chronic obstructive airway disease had haemoglobin concentrations, red-cell counts, and haematocrit values within accepted limits. Five had low serum-iron levels, but none of these had an unsaturated-iron-binding capacity above the normal range. Eleven of these patients were treated with intramuscular iron-sorbitol. Five patients subsequently showed a significant increase in blood-volume, four an increase in red-cell volume, and six an increase in circulating haemoglobin mass.
It is
suggested that availability of iron for haemoglobin synthesis may be a factor which limits the physiological polycythxmic response to anoxia in this disorder. We are grateful to Dr. Lynne Reid and Dr. George Simon for their help in assessing chest radiographs and for advice, and to Dr. E. E. Keal for permission to include his patients. The work was aided by a grant from the Research Committee of the North West Metropolitan Regional Hospital Board. REFERENCES
Discussion
Beutler, E., Robson, M. J., Buttenwierser, E. (1960) Ann. intern. Med. 52,
observed that standard harmatological investigations on venous blood do not accurately mirror changes in red-cell volume in the chronic anoxia of pulmonary emphysema. Absolute increases in red-cell volume are often masked by similar increases in plasmavolume. This may not be the only explanation for a failure to detect polycythaemia, since many patients are not polycythxmic even when this is measured as red-cell volume per unit body-weight. No patient in this study showed all the findings of iron deficiency, although five had serum-iron levels below the normal range. Nevertheless latent iron deficiency was demonstrated in five of eleven patients, because after intramuscular iron there was an increase in red-cell volume and circulating haemoglobin mass. Three treated cases with low initial serum-iron, and two cases with normal initial serum-iron, produced this response to treatment; one case with low initial serum-iron showed no alteration in red-cell volume. All four low serum-iron levels became normal after treatment. It appears, therefore, that inadequate availability of iron may be a factor which limits the haemopoietic response to anoxia in emphysema. Clearly lack of iron is not the only factor restricting such a response. One patient, for instance, lived on the very margin of functional lung capacity, with severe anoxia continuously present, yet his blood and red-cell volumes and haemoglobin mass were normal and remained exactly the same after iron therapy. The factors preventing a hxmatological response in such
Haskins, D., Stevens, A. R., Jr., Finch, S. C., Finch, C. A. (1952) J. clin. Invest. 31, 543. MacKenzie, A., Tindle, J. (1959) Lancet, i, 333. Mollison, P. L. (1961) Blood Transfusion in Clinical Medicine. Oxford. Morgan, E. H., Walters, M. N. I. (1963) J. clin. Path. 16, 101. Nadler, S. B., Hidalgo, J. U., Bloch, T. H. (1962) Surgery, 51, 224. Ratto, O., Briscoe, W. A., Morton, J. W., Comroe, J. H. (1955) Amer. J. Med. 19, 958. Reid, L., Millard, F. J. C. (1964) Clin. Radiol. (in the press). Shaw, D. B., Simpson, T. (1961) Quart. J. Med. 30, 135. Simon, G. (1964) Clin. Radiol. (in the press). Simpson, T. (1957) Lancet, ii, 105. Stafford, J. L., Kemp, N. H. (1963) Proceedings of the IXth Congress of the European Society of Hæmatology, Lisbon. Stuart-Harris, C. H., Hanley, T. (1957) Chronic Bronchitis, Emphysema
It has
previously been
need further investigation. We have hesitated to record symptomatic changes after treatment since daily variation is common in this condition. But four patients with hxmatological improvement reported some lessening of dyspnoea after the course of iron injections. One patient without haematological change said he felt better after iron. Even a marginal change means much to such handicapped patients. We suggest that accepted normal values for red-cell volume and circulating haemoglobin mass require revision for anoxic conditions. Patients with " normal " hxmoglobin may in this sense be considered anaemic. Parenteral iron therapy may remove one factor limiting the physiological polycythaemic response. In particular these results show the need for closer analysis of the progress of cases cases
378.
and Cor Pulmonale. Bristol.
Waldenstrom, J. (1938) Acta med. scand. suppl. 90, p. 380.
DEOXYRIBOSE-NUCLEIC-ACID REPLICATION PATTERN OF TRISOMY 18
JORGE J. YUNIS M.D. Madrid
ERNEST B. HOOK M.D. New York OF THE HUMAN GENETICS STUDY
MEDICINE
AND
MARY MAYER B.A. Nebraska GROUP, DEPARTMENTS
OF LABORATORY
PEDIATRICS, UNIVERSITY OF MINNESOTA MEDICAL SCHOOL, MINNEAPOLIS
THERE is as yet no agreement as to the identification of the extra chromosome present in the trisomy 17-18 (E) syndrome. Edwards et al. (1960) in their original report identified it as a no. 17 chromosome. Subsequently, Gottleib et al. (1962), because they could not distinguish morphologically the extra autosome as either 17 or 18, suggested that the extra chromosome associated with the syndrome be defined as a no. 17, in deference to the original report of Edwards et al. On the other hand, Patau et al. (1961) reported unequivocally that the extra chromosome was a no. 18. Most subsequent workers have referred to the syndrome as trisomy 17-18 or trisomy E, although some seem to agree with Patau et al. in identifying it as trisomy 18 (Uchida et al. 1962). We report here the results of autoradiographic studies, using tritiated thymidine, which show that the deoxyribose-nucleic-acid (D.N.A.) replication pattern of the extra autosome in trisomy 17-18 (E) is that of the eighteenth chromosome. The Patient, Material, and Methods Blood was drawn from a female infant with the clinical stigmata and dermatoglyphic pattern of the trisomy 17-18 (E)
syndrome.
287 The patient was a 31/2 month-old baby, borr cxsarean section after a 39-week uncomplicated gestation, to a 44-year-old mother anc a 52-year-old father. There were three othet daughters and one son in the family, and no history of stillbirths or abortions. Her birthweight was 3 lb. 7 oz.; by 31/2 months of age she weighed 2980 g. and was 53 cm. long. She appeared to be retarded for her age, and she had generalised hypertonicity. She had multi-
by
ple congenital anomalies, including an asymmetrical head with underdevelopment of the left side, a prominent occiput, mild micrognathia, a high arched palate, slightly low-set ears, index fingers that were flexed in a beckoning position, overlapping of the 5th over the 4th finger bilaterally, slight ulnar deviation of the hands, short metacarpals, and small thumbs. In addition, there were thin ribs, a short sternum, a small pelvis, forward rotation of the ilia, diastasis of the recti abdominis, small dorsiflexed great toes, and rocker-bottom heels. A ventricular septal defect and pulmonary hypertension were diagnosed on the basis of the auscultatory, radiographic, and Metaphase plate of a blood-cell from a patient with trisomy 18. electrocardiographic findings. An intravenous The cell was exposed to tritiated thymidine for 5’/2 hours, and photographed pyelogram demonstrated a tilted and low-set before (left) and after (right) the autoradiogram was made. There are three late left kidney. The bases of her fingernails were no. 18 chromosomes and two earlier replicating no. 17 chromosomes. replicating deeply set, but the nails were not atrophic. Dermatoglyphic analysis demonstrated ten simple arches on her fingers and nine on her toes, and a fibular of the material after initial examination or were not loop on her left 3rd toe. There were no raised palmar triradii labelled. In 25 of the suitable plates, 3 late replicating or palmar simian creases, nor were the distal flexion creases no. 18 chromosomes and two early replicating no. 17 of the digits absent. She was still alive 7 months after birth. chromosomes were found. The remaining 10 mitoses did Chromosome analysis was carried out as previously described not give a consistent D.N.A. replication pattern of the (Yunis and Gorlin 1963) and in addition an autoradiographic 17-18 chromosomes. study, using tritiated thymidine and a continuous labelling no. Previous studies with tritiated thymidine of chromotechnique, was performed. The autoradiographic technique somes from normal subjects have also demonstrated two used was that of Schmid (1963), with minor modifications. In brief, peripheral blood-leucocytes were grown in Eagle’s distinct types of labelling pattern of the no. 17-18 chromomedium, pH 7.0, for 72 hours at 36°C. After this time, somes. When the seventeenth and eighteenth chromotritiated thymidine (specific activity 1.9C. per millimol) was could be morphologically distinguished, the somes added to the cultures to make a final concentration of 1 (J.C seventeenth chromosomes were found to replicate earlier per ml. After 31/2 hours, colchicine (final concentration of than the eighteenth chromosomes (Schmid 1963, Yunis 0001 mg. per ml.) was added for 2 more hours, the total time personal observations). in thymidine being 51/2 hours. The cultures were terminated These observations confirm the contention of Patau et 10 the with citrate 36°C for cells sodium at 1% by treating al. (1961) that the syndrome originally described by the cells in Aceto-orcein and acid. acetic minutes, 50% fixing et al. (1960) is indeed due to trisomy of the Edwards were made and suitable plates squash preparations metaphase
photographed under bright microscopy. The coverslips then removed by the dry-ice freezing technique, and the slides were coated with Kodak nuclear track emulsion NTB2, and diluted 1 to 2 with distilled water. The prepared sample slides were exposed for 6 days at 4°C, and the emulsion was then developed with D19b Kodak for 5 minutes at 4°C. The material was counterstained with Wright’s stain and coverslipped, and the metaphase plates were re-examined. were
eighteenth chromosome.
were
Results and Conclusions
Karyotype analysis showed
a modal number of fortychromosomes. A total of 117 metaphase plates were counted, of which 110 contained forty-seven 6 contained forty-six chromosomes, and chromosomes, 1 contained forty-four chromosomes. Cells which were obviously broken were not analysed. The extra chromosome involved was in the group E, and it had the morphological characteristics of the no. 18 chromosome in approximately 70% of the metaphase plates. In the remaining nitoses the extra chromosome could not be definitely identified as a no. 17 or 18. seven
Of 68 plates examined in the autoradiographic study, 35 were found suitable for D.N.A. replication study. The remaining ones were either lost during manipulation
only
REFERENCES
Edwards, J. N., Harnden, D. G., Cameron, A. H., Crosse, V. M., Wolff, O. H. (1960) Lancet, i, 787. Gottleib, M. I., Hirschhorn, K., Cooper, H. L., Luskin, N., Moloshok, R. E., Hodes, H. L. (1962) Amer. J. Med. 33, 763. Patau, K., Therman, E., Smith, D. W., DeMars, R. I. (1961) Chromosoma, 12, 280. Schmid, W. (1963) Cytogenetics, 2, 175. Uchida, I. A., Lewis, A. J., Bowman, J. M., Wang, H. C. (1962) J. Pediat. 60, 498. Yunis, J. J., Gorlin, R. J. (1963) Chromosoma, 14, 140.
"... The role of the
responsible
citizen thus
urgently
requires recognition and active underpinning. Political parties, trade unions, local government authorities, churches, and indeed universities should give heed to this, and it may well be that one of the important problems, if not the most important problem of all, which will have to be faced in the new society we are now creating will be to find a secure place for those who possess their own vision of a better world, some understanding of how it might be created, and an urgent sense of obligation to defend the weak from oppression. Without such people, social science can never be married to social purpose; without them, the element which gives our society its meaning and its vitality will be gone from it."-Prof. T. S. SIMEY, Social Purpose and Social Science, p. 29. Liverpool: University Press.
1964.
Serurrr-iron and Iron-binding Capacity Seven of the control subjects investigated in the present series were within the normal range; one (subject 5) showed the findings of iron deficiency. Of the seventeen patients, five had serum-iron levels below the normal range in this laboratory, but in none of these was the binding capacity high enough to suggest iron deficiency. Four of these low-serum-iron subjects were in the treated group, and all four showed normal serumiron levels when retested after treatment.
with chronic obstructive airway disease and the in the blood which may accompany them.
changes
Summary Seventeen patients with severe chronic obstructive airway disease had haemoglobin concentrations, red-cell counts, and haematocrit values within accepted limits. Five had low serum-iron levels, but none of these had an unsaturated-iron-binding capacity above the normal range. Eleven of these patients were treated with intramuscular iron-sorbitol. Five patients subsequently showed a significant increase in blood-volume, four an increase in red-cell volume, and six an increase in circulating haemoglobin mass.
It is
suggested that availability of iron for haemoglobin synthesis may be a factor which limits the physiological polycythxmic response to anoxia in this disorder. We are grateful to Dr. Lynne Reid and Dr. George Simon for their help in assessing chest radiographs and for advice, and to Dr. E. E. Keal for permission to include his patients. The work was aided by a grant from the Research Committee of the North West Metropolitan Regional Hospital Board. REFERENCES
Discussion
Beutler, E., Robson, M. J., Buttenwierser, E. (1960) Ann. intern. Med. 52,
observed that standard harmatological investigations on venous blood do not accurately mirror changes in red-cell volume in the chronic anoxia of pulmonary emphysema. Absolute increases in red-cell volume are often masked by similar increases in plasmavolume. This may not be the only explanation for a failure to detect polycythaemia, since many patients are not polycythxmic even when this is measured as red-cell volume per unit body-weight. No patient in this study showed all the findings of iron deficiency, although five had serum-iron levels below the normal range. Nevertheless latent iron deficiency was demonstrated in five of eleven patients, because after intramuscular iron there was an increase in red-cell volume and circulating haemoglobin mass. Three treated cases with low initial serum-iron, and two cases with normal initial serum-iron, produced this response to treatment; one case with low initial serum-iron showed no alteration in red-cell volume. All four low serum-iron levels became normal after treatment. It appears, therefore, that inadequate availability of iron may be a factor which limits the haemopoietic response to anoxia in emphysema. Clearly lack of iron is not the only factor restricting such a response. One patient, for instance, lived on the very margin of functional lung capacity, with severe anoxia continuously present, yet his blood and red-cell volumes and haemoglobin mass were normal and remained exactly the same after iron therapy. The factors preventing a hxmatological response in such
Haskins, D., Stevens, A. R., Jr., Finch, S. C., Finch, C. A. (1952) J. clin. Invest. 31, 543. MacKenzie, A., Tindle, J. (1959) Lancet, i, 333. Mollison, P. L. (1961) Blood Transfusion in Clinical Medicine. Oxford. Morgan, E. H., Walters, M. N. I. (1963) J. clin. Path. 16, 101. Nadler, S. B., Hidalgo, J. U., Bloch, T. H. (1962) Surgery, 51, 224. Ratto, O., Briscoe, W. A., Morton, J. W., Comroe, J. H. (1955) Amer. J. Med. 19, 958. Reid, L., Millard, F. J. C. (1964) Clin. Radiol. (in the press). Shaw, D. B., Simpson, T. (1961) Quart. J. Med. 30, 135. Simon, G. (1964) Clin. Radiol. (in the press). Simpson, T. (1957) Lancet, ii, 105. Stafford, J. L., Kemp, N. H. (1963) Proceedings of the IXth Congress of the European Society of Hæmatology, Lisbon. Stuart-Harris, C. H., Hanley, T. (1957) Chronic Bronchitis, Emphysema
It has
previously been
need further investigation. We have hesitated to record symptomatic changes after treatment since daily variation is common in this condition. But four patients with hxmatological improvement reported some lessening of dyspnoea after the course of iron injections. One patient without haematological change said he felt better after iron. Even a marginal change means much to such handicapped patients. We suggest that accepted normal values for red-cell volume and circulating haemoglobin mass require revision for anoxic conditions. Patients with " normal " hxmoglobin may in this sense be considered anaemic. Parenteral iron therapy may remove one factor limiting the physiological polycythaemic response. In particular these results show the need for closer analysis of the progress of cases cases
378.
and Cor Pulmonale. Bristol.
Waldenstrom, J. (1938) Acta med. scand. suppl. 90, p. 380.
DEOXYRIBOSE-NUCLEIC-ACID REPLICATION PATTERN OF TRISOMY 18
JORGE J. YUNIS M.D. Madrid
ERNEST B. HOOK M.D. New York OF THE HUMAN GENETICS STUDY
MEDICINE
AND
MARY MAYER B.A. Nebraska GROUP, DEPARTMENTS
OF LABORATORY
PEDIATRICS, UNIVERSITY OF MINNESOTA MEDICAL SCHOOL, MINNEAPOLIS
THERE is as yet no agreement as to the identification of the extra chromosome present in the trisomy 17-18 (E) syndrome. Edwards et al. (1960) in their original report identified it as a no. 17 chromosome. Subsequently, Gottleib et al. (1962), because they could not distinguish morphologically the extra autosome as either 17 or 18, suggested that the extra chromosome associated with the syndrome be defined as a no. 17, in deference to the original report of Edwards et al. On the other hand, Patau et al. (1961) reported unequivocally that the extra chromosome was a no. 18. Most subsequent workers have referred to the syndrome as trisomy 17-18 or trisomy E, although some seem to agree with Patau et al. in identifying it as trisomy 18 (Uchida et al. 1962). We report here the results of autoradiographic studies, using tritiated thymidine, which show that the deoxyribose-nucleic-acid (D.N.A.) replication pattern of the extra autosome in trisomy 17-18 (E) is that of the eighteenth chromosome. The Patient, Material, and Methods Blood was drawn from a female infant with the clinical stigmata and dermatoglyphic pattern of the trisomy 17-18 (E)
syndrome.
287 The patient was a 31/2 month-old baby, borr cxsarean section after a 39-week uncomplicated gestation, to a 44-year-old mother anc a 52-year-old father. There were three othet daughters and one son in the family, and no history of stillbirths or abortions. Her birthweight was 3 lb. 7 oz.; by 31/2 months of age she weighed 2980 g. and was 53 cm. long. She appeared to be retarded for her age, and she had generalised hypertonicity. She had multi-
by
ple congenital anomalies, including an asymmetrical head with underdevelopment of the left side, a prominent occiput, mild micrognathia, a high arched palate, slightly low-set ears, index fingers that were flexed in a beckoning position, overlapping of the 5th over the 4th finger bilaterally, slight ulnar deviation of the hands, short metacarpals, and small thumbs. In addition, there were thin ribs, a short sternum, a small pelvis, forward rotation of the ilia, diastasis of the recti abdominis, small dorsiflexed great toes, and rocker-bottom heels. A ventricular septal defect and pulmonary hypertension were diagnosed on the basis of the auscultatory, radiographic, and Metaphase plate of a blood-cell from a patient with trisomy 18. electrocardiographic findings. An intravenous The cell was exposed to tritiated thymidine for 5’/2 hours, and photographed pyelogram demonstrated a tilted and low-set before (left) and after (right) the autoradiogram was made. There are three late left kidney. The bases of her fingernails were no. 18 chromosomes and two earlier replicating no. 17 chromosomes. replicating deeply set, but the nails were not atrophic. Dermatoglyphic analysis demonstrated ten simple arches on her fingers and nine on her toes, and a fibular of the material after initial examination or were not loop on her left 3rd toe. There were no raised palmar triradii labelled. In 25 of the suitable plates, 3 late replicating or palmar simian creases, nor were the distal flexion creases no. 18 chromosomes and two early replicating no. 17 of the digits absent. She was still alive 7 months after birth. chromosomes were found. The remaining 10 mitoses did Chromosome analysis was carried out as previously described not give a consistent D.N.A. replication pattern of the (Yunis and Gorlin 1963) and in addition an autoradiographic 17-18 chromosomes. study, using tritiated thymidine and a continuous labelling no. Previous studies with tritiated thymidine of chromotechnique, was performed. The autoradiographic technique somes from normal subjects have also demonstrated two used was that of Schmid (1963), with minor modifications. In brief, peripheral blood-leucocytes were grown in Eagle’s distinct types of labelling pattern of the no. 17-18 chromomedium, pH 7.0, for 72 hours at 36°C. After this time, somes. When the seventeenth and eighteenth chromotritiated thymidine (specific activity 1.9C. per millimol) was could be morphologically distinguished, the somes added to the cultures to make a final concentration of 1 (J.C seventeenth chromosomes were found to replicate earlier per ml. After 31/2 hours, colchicine (final concentration of than the eighteenth chromosomes (Schmid 1963, Yunis 0001 mg. per ml.) was added for 2 more hours, the total time personal observations). in thymidine being 51/2 hours. The cultures were terminated These observations confirm the contention of Patau et 10 the with citrate 36°C for cells sodium at 1% by treating al. (1961) that the syndrome originally described by the cells in Aceto-orcein and acid. acetic minutes, 50% fixing et al. (1960) is indeed due to trisomy of the Edwards were made and suitable plates squash preparations metaphase
photographed under bright microscopy. The coverslips then removed by the dry-ice freezing technique, and the slides were coated with Kodak nuclear track emulsion NTB2, and diluted 1 to 2 with distilled water. The prepared sample slides were exposed for 6 days at 4°C, and the emulsion was then developed with D19b Kodak for 5 minutes at 4°C. The material was counterstained with Wright’s stain and coverslipped, and the metaphase plates were re-examined. were
eighteenth chromosome.
were
Results and Conclusions
Karyotype analysis showed
a modal number of fortychromosomes. A total of 117 metaphase plates were counted, of which 110 contained forty-seven 6 contained forty-six chromosomes, and chromosomes, 1 contained forty-four chromosomes. Cells which were obviously broken were not analysed. The extra chromosome involved was in the group E, and it had the morphological characteristics of the no. 18 chromosome in approximately 70% of the metaphase plates. In the remaining nitoses the extra chromosome could not be definitely identified as a no. 17 or 18. seven
Of 68 plates examined in the autoradiographic study, 35 were found suitable for D.N.A. replication study. The remaining ones were either lost during manipulation
only
REFERENCES
Edwards, J. N., Harnden, D. G., Cameron, A. H., Crosse, V. M., Wolff, O. H. (1960) Lancet, i, 787. Gottleib, M. I., Hirschhorn, K., Cooper, H. L., Luskin, N., Moloshok, R. E., Hodes, H. L. (1962) Amer. J. Med. 33, 763. Patau, K., Therman, E., Smith, D. W., DeMars, R. I. (1961) Chromosoma, 12, 280. Schmid, W. (1963) Cytogenetics, 2, 175. Uchida, I. A., Lewis, A. J., Bowman, J. M., Wang, H. C. (1962) J. Pediat. 60, 498. Yunis, J. J., Gorlin, R. J. (1963) Chromosoma, 14, 140.
"... The role of the
responsible
citizen thus
urgently
requires recognition and active underpinning. Political parties, trade unions, local government authorities, churches, and indeed universities should give heed to this, and it may well be that one of the important problems, if not the most important problem of all, which will have to be faced in the new society we are now creating will be to find a secure place for those who possess their own vision of a better world, some understanding of how it might be created, and an urgent sense of obligation to defend the weak from oppression. Without such people, social science can never be married to social purpose; without them, the element which gives our society its meaning and its vitality will be gone from it."-Prof. T. S. SIMEY, Social Purpose and Social Science, p. 29. Liverpool: University Press.
1964.