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ANTENATAL DIAGNOSIS OF MAPLE-SYRUP-URINE DISEASE
212 Evidence continues to accumulate about the involvement of the central nervous system in all shock states and suggesting that the resulting pathophysiological changes involve endocrine and reticuloendothelial as well as the cardiovascular and respiratory systems. Possibly more success would follow treatment directed to the initiating sources rather than to where the end-products of the resulting changes appear. 11 Chelsea Embankment, London SW3 4LE
PRENATAL DIAGNOSIS OF M.S.U.D. IN AMNIOTIC-CELL CULTURE
D. A. BUXTON HOPKIN
ANTENATAL DIAGNOSIS OF MAPLE-SYRUP-URINE DISEASE
SIR,—Maple-syrup-urine disease (M.S.U.D.) is an autosomal recessive disease affecting the degradation of the three branched-chain aminoacids leucine, isoleucine, and valine. The classical form of the disease, if untreated, is rapidly lethal. Treatment is difficult, requiring life-long restriction of the three aminoacids. Parents who have had a child with M.S.U.D. often seek antenatal diagnosis in subsequent pregnancies. The demonstration of the branched-chain ketoacid decarboxylase in cultured amniotic cells suggested the possibility of antenatal diagnosis’ and this was confirmed in a patient from this laboratory’ and by Wendel et al.2 We have now studied eighteen pregnancies at risk for M.S.U.D. The diagnosis can be made with accuracy and confidence, but care is needed with cultures of epithelial cells and with slow-growing cultures. Twelve of the
eighteen specimens investigated were sent from
other
institutions, including four from other countries. Methods of subculhave been previously described.3 Cells were harvested for assay either with a rubber policeman or with a solution of Puck’s saline A containing 0-02% (w/v) edetic acid (E.D.T.A.) and 0.05% (w/v) trypsin (two patients). The E.D.T.A.-trypsin cell suspension was immediately added to one-quarter its volume of calf serum and centrifuged. The cell button was resuspended in buffered saline containing 20% serum and transferred to reaction flasks and centrifuged. The supernatant was ’replaced with reaction mixture and decarboxylase activity was assayed.4 Approximately 4x105 cells are required for assay, but reliable results are obtained with fewer actively growing cells. Most specimens were assayed 3-5 weeks after amniocentesis. ture
Eighteen amniotic-cell cultures were investigated and a diagnosis was made in sixteen, of which four had M.S.U.D., giving the frequency of 25% expected for an autosomal recessive disease. The other twelve assays were normal and in all twelve the postnatal diagnosis was also normal. Details of the remaining six cases (A-F) are given in the table. The diagnosis was confirmed in case B by post-abortion culture and assay of fetal skin fibroblasts. In case A a type-n variant was confirmed after birth by enzyme assay of leucocytes and by clinical course. In case C a macerated fetus was delivered at another institution after abortion with prostaglandin and urea. Culture of fetal tissues failed. In case F the baby was delivered at term; symptoms of M.S.U.D. developed and the infant died in a week. No diagnosis could be made in cases D and E. In both the cells were predominantly epithelial, as they were in 5 other successful cultures. One culture (E) was received from France. Cells grew poorly and an assay was attempted with few cells. No radioactive CO2 was released, but there was also no incorporation of radioactive aminoacids into protein, indicating that the cells were no longer metabolically active. The outcome of this pregnancy is not known. In D the ratio of the ’4C02to radioactivity in protein suggested variant M.S.U.D., but the incorporation of radioactivity into protein was too low to provide confidence in the results. No diagnosis was made and the fetus was aborted at another institution. Cultures were not established from fetal tissue. 1. Dancis, J. in Antenatal Diagnosis (edited by A. Dorfman); p. 123. Chicago, 1972. 2. Wendel, U., Rudiger, H. W., Passarge, E. Humangenetik, 1973, 19, 127. 3. Cox, R. P., Macleod, C. M.J. gen. Physiol. 1962, 45, 439. 4. Dancis, J., Hutzler, J., Cox, R. P. in Tissue Culture, Methods and Applications (edited by P. F. Kruse, Jr. and M. K. Patterson, Jr.) p. 639. New York, 1973.
Amniotic cell cultures that are predominantly fibroblastic have presented no problems with the technique devised for skin fibroblasts.s However, when cultures are predominantly
epithelial they multiply slowly, delaying analysis. Harvesting cells with a rubber policeman, as we have usually done, is associated with a variable, and frequently serious, loss of enzyme activity. The use of E.D.T.A.-trypsin to remove cells from glass surfaces seems to preserve enzyme activity in epithelial cells. We now assay cell suspensions prepared by E.D.T.A.-trypsin treatment of confluent cell monolayers. Intact cells are used because disruption of cells greatly reduces enzyme activity.’ The cells are incubated for 120 min with L-valine-1-14C (specific activity 0-5 µCi/µmol). The evolution of 14C02 provides a measure of the decarboxylase activity and is expressed per mg protein. The simultaneous incorporation of the radioactive aminoacid into protein provides an indication of the number of cells that are assayed and their metabolic activity. The ratio of the 14CO2to 14C-incorporation into protein is the most satis-
factory approach to diagnosis, provided 14C-incorporation per mg protein indicates active cell metabolism. This Service Fund.
study (AM
was
supported by grants
14528 and HD
from the U.S. Public Health
04526) and by the Samuel
Division of Human Genetics, Departments of Medicine and Pediatrics, New York University Medical Center, New York, N.Y. 10016, U.S.A.
A.
Berger
RODY P. Cox
JOEL HUTZLER JOSEPH DANCIS
MATERNAL HYPERTHERMIA AND NEURAL-TUBE DEFECTS
SIR,-Retrospective studies in the north-western United a significant correlation between maternal hyperthermia during the early part of pregnancy and the incidence of anencephalyl and meningomyelocele.2 Our similar study in the mid-southern United States has detected 3 instances of maternal hyperthermia in 45 pregnancies which resulted in children with neural-tube defects (anencephaly, meningomyelocele, spina bifida, or encephalocele). Only births during the past 11 months were included. States have revealed
In
man
the neural tube closes between the 22nd and 28th
gestation. The mothers in this study were questioned particularly about hyperthermia due to illness or prolonged sauna usage during the third, fourth, and fifth weeks of ges-
day
of
tation. This interval
was
chosen because most of the dates of
conception were only approximate. 1 mother reported "quite high" fever lasting 2 or 3 days and caused by streptococcal Elsas, L. J., Priest, J. H. Wheeler, F. B., Danner, D. J., Pask, B. A., Metabolism, 1974, 23, 569. 1. Miller, P., Smith, D. W., Shepard, T. H. Lancet, 1978, i, 519. 2. Chance, P. F., Smith, D. W. ibid. p. 769. 5.
PRENATAL DIAGNOSIS OF M.S.U.D. IN AMNIOTIC-CELL CULTURE
D. A. BUXTON HOPKIN
ANTENATAL DIAGNOSIS OF MAPLE-SYRUP-URINE DISEASE
SIR,—Maple-syrup-urine disease (M.S.U.D.) is an autosomal recessive disease affecting the degradation of the three branched-chain aminoacids leucine, isoleucine, and valine. The classical form of the disease, if untreated, is rapidly lethal. Treatment is difficult, requiring life-long restriction of the three aminoacids. Parents who have had a child with M.S.U.D. often seek antenatal diagnosis in subsequent pregnancies. The demonstration of the branched-chain ketoacid decarboxylase in cultured amniotic cells suggested the possibility of antenatal diagnosis’ and this was confirmed in a patient from this laboratory’ and by Wendel et al.2 We have now studied eighteen pregnancies at risk for M.S.U.D. The diagnosis can be made with accuracy and confidence, but care is needed with cultures of epithelial cells and with slow-growing cultures. Twelve of the
eighteen specimens investigated were sent from
other
institutions, including four from other countries. Methods of subculhave been previously described.3 Cells were harvested for assay either with a rubber policeman or with a solution of Puck’s saline A containing 0-02% (w/v) edetic acid (E.D.T.A.) and 0.05% (w/v) trypsin (two patients). The E.D.T.A.-trypsin cell suspension was immediately added to one-quarter its volume of calf serum and centrifuged. The cell button was resuspended in buffered saline containing 20% serum and transferred to reaction flasks and centrifuged. The supernatant was ’replaced with reaction mixture and decarboxylase activity was assayed.4 Approximately 4x105 cells are required for assay, but reliable results are obtained with fewer actively growing cells. Most specimens were assayed 3-5 weeks after amniocentesis. ture
Eighteen amniotic-cell cultures were investigated and a diagnosis was made in sixteen, of which four had M.S.U.D., giving the frequency of 25% expected for an autosomal recessive disease. The other twelve assays were normal and in all twelve the postnatal diagnosis was also normal. Details of the remaining six cases (A-F) are given in the table. The diagnosis was confirmed in case B by post-abortion culture and assay of fetal skin fibroblasts. In case A a type-n variant was confirmed after birth by enzyme assay of leucocytes and by clinical course. In case C a macerated fetus was delivered at another institution after abortion with prostaglandin and urea. Culture of fetal tissues failed. In case F the baby was delivered at term; symptoms of M.S.U.D. developed and the infant died in a week. No diagnosis could be made in cases D and E. In both the cells were predominantly epithelial, as they were in 5 other successful cultures. One culture (E) was received from France. Cells grew poorly and an assay was attempted with few cells. No radioactive CO2 was released, but there was also no incorporation of radioactive aminoacids into protein, indicating that the cells were no longer metabolically active. The outcome of this pregnancy is not known. In D the ratio of the ’4C02to radioactivity in protein suggested variant M.S.U.D., but the incorporation of radioactivity into protein was too low to provide confidence in the results. No diagnosis was made and the fetus was aborted at another institution. Cultures were not established from fetal tissue. 1. Dancis, J. in Antenatal Diagnosis (edited by A. Dorfman); p. 123. Chicago, 1972. 2. Wendel, U., Rudiger, H. W., Passarge, E. Humangenetik, 1973, 19, 127. 3. Cox, R. P., Macleod, C. M.J. gen. Physiol. 1962, 45, 439. 4. Dancis, J., Hutzler, J., Cox, R. P. in Tissue Culture, Methods and Applications (edited by P. F. Kruse, Jr. and M. K. Patterson, Jr.) p. 639. New York, 1973.
Amniotic cell cultures that are predominantly fibroblastic have presented no problems with the technique devised for skin fibroblasts.s However, when cultures are predominantly
epithelial they multiply slowly, delaying analysis. Harvesting cells with a rubber policeman, as we have usually done, is associated with a variable, and frequently serious, loss of enzyme activity. The use of E.D.T.A.-trypsin to remove cells from glass surfaces seems to preserve enzyme activity in epithelial cells. We now assay cell suspensions prepared by E.D.T.A.-trypsin treatment of confluent cell monolayers. Intact cells are used because disruption of cells greatly reduces enzyme activity.’ The cells are incubated for 120 min with L-valine-1-14C (specific activity 0-5 µCi/µmol). The evolution of 14C02 provides a measure of the decarboxylase activity and is expressed per mg protein. The simultaneous incorporation of the radioactive aminoacid into protein provides an indication of the number of cells that are assayed and their metabolic activity. The ratio of the 14CO2to 14C-incorporation into protein is the most satis-
factory approach to diagnosis, provided 14C-incorporation per mg protein indicates active cell metabolism. This Service Fund.
study (AM
was
supported by grants
14528 and HD
from the U.S. Public Health
04526) and by the Samuel
Division of Human Genetics, Departments of Medicine and Pediatrics, New York University Medical Center, New York, N.Y. 10016, U.S.A.
A.
Berger
RODY P. Cox
JOEL HUTZLER JOSEPH DANCIS
MATERNAL HYPERTHERMIA AND NEURAL-TUBE DEFECTS
SIR,-Retrospective studies in the north-western United a significant correlation between maternal hyperthermia during the early part of pregnancy and the incidence of anencephalyl and meningomyelocele.2 Our similar study in the mid-southern United States has detected 3 instances of maternal hyperthermia in 45 pregnancies which resulted in children with neural-tube defects (anencephaly, meningomyelocele, spina bifida, or encephalocele). Only births during the past 11 months were included. States have revealed
In
man
the neural tube closes between the 22nd and 28th
gestation. The mothers in this study were questioned particularly about hyperthermia due to illness or prolonged sauna usage during the third, fourth, and fifth weeks of ges-
day
of
tation. This interval
was
chosen because most of the dates of
conception were only approximate. 1 mother reported "quite high" fever lasting 2 or 3 days and caused by streptococcal Elsas, L. J., Priest, J. H. Wheeler, F. B., Danner, D. J., Pask, B. A., Metabolism, 1974, 23, 569. 1. Miller, P., Smith, D. W., Shepard, T. H. Lancet, 1978, i, 519. 2. Chance, P. F., Smith, D. W. ibid. p. 769. 5.