73
A survey has shown that the frequency of antibody rises steadily through childhood to reach about 30% in adult life (table 11). It has not been possible to associate the new antigen with a specific illness. Paired sera from 22 hepatitis-A patients and 14 hepatitis-B patients have been tested; none contained the antigen. 2 of the hepatitis-A and 1 of the hepatitisB patients showed conversion from antibody negative Both acute and convalescent sera were to positive. antibody-positive in 5 patients from each group. The new antigen closely resembles the particles described in faeces of patients with acute gastroenteritis by Paver et al.,2 but it is smaller than the spherical particles described by Feinstone et al.’ in faeces of hepatitis patients or the core antigen of
HBAg. While the main interest of the new antigen is as possible infective agent, it is also of some immediate practical importance, since it can readily be confused with HBAg. The size of the small particles is very similar; the diameter of HBAg particles ranges between 16 and 21 nm. and that of the new antigen between 20-5 and 25 nm. In addition, many of the human antisera used for detecting HBAg also contain antibodies to the new antigen.
a
Requests for reprints should be addressed to Y. E. C., Virus Laboratory, Central Public Health Laboratory, Colindale Avenue, London NW9.
Reference
REFERENCES
Vandervelde, E. M., Goffin, G., Megson, B., Mahmood, N., Porter, A., Cossart, Y. E. Lancet, 1974, ii, 1066. 2. Paver, W. K., Caul, E. O., Ashley, C. R., Clarke, S. K. R. ibid. 1973, i, 237. 3. Feinstone, S. M., Kapikian, A. Z., Purcell, R. H. Science, 1974, 182, 1.
1026. 4.
Cossart, Y. E., Field, A. M., Hargreaves, F. D., Porter, A. A. Microbios, 1971, 3, 5.
ADENOSINE-DEAMINASE DEFICIENCY IN A CHILD DIAGNOSED PRENATALLY ROCHELLE HIRSCHHORN
Department of Medicine, New York University School of Medicine, New York, N.Y., U.S.A. NICHOLAS BERATIS Department of Pediatrics, Mount Sinai School of Medicine, New York
FRED S. ROSEN
ROBERTSON PARKMAN
contained less than 1.5 ’it> of A.D.A. activity of normal amniotic cultures. The prenatal diagnosis of A.D.A. deficiency was confirmed at birth by the absence of Clinical A.D.A. activity in the child’s red blood-cells. and laboratory findings in this child are similar to those of the sibling who had died with S.C.I.D. INTRODUCTION
SEVERE combined immunodeficiency (S.C.I.D.) is a childhood disease which is fatal if untreated. Lifethreatening infections result from impairment of both cellular and humoral immunity. The disorder occurs in X-linked and autosomal recessive forms.1 A deficiency of the erythrocyte enzyme adenosine deaminase (R.B.C.-A.D.A.) has been reported in some, but not all, patients with the autosomal recessive form of S.C.I.D .2 The deficiency of R.B.C.-A.D.A. is transmitted as an autosomal recessive trait, with the parents and onehalf the grandparents showing diminished levels of enzyme activity.3 Besides lacking the low-molecularweight R.B.c. form of the enzyme, the affected children also have deficiencies in the various highermolecular-weight, tissue-specific forms of A.D.A.4 These tissue-specific isozymes can be generated by coincubation of R.B.C.-A.D.A. with different tissue extracts.5 These two findings indicate that the gene affected in s.c.I.D. with A.D.A. deficiency codes for a catalytic unit common to the tissue and R.B.c. isozymes of A.D.A. As we have suggested,6 it should, therefore, be possible to diagnose R.B.C.-A.D.A. deficiency in cultured amniotic-fluid cells. These cells, like fibroblasts, can express the R.B.C. and tissue isozyme, but may express the tissue isozyme alone. We now report the in-utero diagnosis of A.D.A. deficiency in a child subsequently shown to have a deficiency of A.D.A. activity in his red blood-cells. FAMILY HI STORY
The family was investigated during a survey of kindreds in which a previous child had died with S.C.I.D. Both parents and their 6-year-old son are healthy. The parents are apparently not related. A girl (K. W.) had died 3 years earlier at 9 months of age. She had been well until the age of 3 months. She then failed to continue to gain weight and developed respiratory congestion. Subsequently oral and perineal candidiasis, diarrhoea, and pneumonitis were diagnosed. A thymic shadow was never seen on several chest X-rays. Leukopenia (2000-7000 per c.mm) and lymphopenia (0-17%) were prominent. There was no
Department of Pediatrics, Children’s Hospital Medical Center, Harvard Medical School, Boston,
delayed hypersensitivity to common antigens, including candida, and the child could not be sensitised to 2,4-dini-
Massachusetts
trochlorobenzene. Lymphocytes showed reduced stimulation with phytohsemagglutinin (stimulation index 8, normal approximately 40). A small amount of thymic tissue obtained at mediastinoscopy had the characteristic histology seen in s.c.I.D. Serum-immunoglobulins (mg. per 100 ml.) at 6 months were: IgG 380, IgM 100, and IgA 28. Schick test was negative, anti-A titre was 1/2, and only a twofold rise in antitetanus titre was observed after immunisation. This child died after an unsuccessful bonemarrow transplantation from the father. A.D.A. studies were not done.
STEPHEN POLMAR Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, Ohio ROBERT STERN
of red-blood-cell adenosine
Summary Deficiency deaminase (R.B.C.-A.D.A.) has been rein a ported proportion of patients with the autosomal recessive form of severe combined immunodeficiency in which a child had died with S.C.I.D., R.B.C.-A.D.A. levels in the parents and other members of the family were compatible with a heterozygous state for A.D.A. deficiency. Cultured amnioticfluid cells obtained from a subsequent pregnancy
(S.C.I.D.).
In
a
family
METHODS
Enzyme Assays A.D.A. activity in a
linked assay
at
R.B.c. haemolysates was determined by 37’C (method 1) using 0-4 mg. adeno-
74 sine per ml. 0’05M phosphate buffer pH 7’5 as substrate. In the presence of endogenous nucleoside phosphorylase and exogenous xanthine oxidase (0-2 units per ml.), uric acid is produced and measured at 293 nmo.7 A modification of this method (method 2) was also used.8 The two methods showed a correlation coefficient of 0-93. Values for R.B.c. activity were expressed as nmol per mg. Hb per hour. A log transformation of all values was performed because of a skewed distribution towards higher R.B.C.S were also typed for the genetically A.D.A. values.3 determined polymorphism demonstrated by R.B.C.-A.D.A.9 A.D.A. activity of amniotic cells and mononuclear-cell/ platelet cell preparations was determined at 37°C using 50-200 g. protein per ml. assay containing 0’4 mg. adenosine in 0-05M phosphate buffer pH 7’5 in the presence of exogenous nucleoside phosphorylase (0’2S units per ml.) as well as xanthine oxidase (0’2 units per ml.) and the amount of uric acid produced was measured at 293 nm.
Amniocentesis Amniocentesis
done during the 28th week of gestation, immediately after ascertainment of the family. The mother was told that her pregnancy was too far advanced for a therapeutic abortion to be undertaken, but she nevertheless agreed to amniocentesis for investigative purposes after being informed of all potential risks. The TABLE
was
I-R.B.C.-A.D.A. ACTIVITIES AND PROPOSED IN A FAMILY WITH S.C.I.D.
GENOTYPES
A.D.A. genotypes in S.C.I.D.
a
family with
two children affected with
Numbers in ( ) represent proposed genotypes based on electrophoretic phenotypes. The arrow indicates the who was diagnosed prenatally as A.D.A. deficient.
A.D.A.
child
instead he expressed only the A.D.A. 1 phenotype. This indicates that the paternal grandmother was not homozygous for 4D but heterozygous for ADA2 and silent gene 4D4 transmitting the silent gene to her son who was then ADAl-o. He and his wife then transmitted silent genes to their affected children
(see figure). Activity in Amniotic Cells activity in fifteen normal amniotic-cell cultures averaged 17 19 ±7 79 nmol per mg. protein per minute by method 1. Similar values have been reported by Chen et al,u No difference could be observed between cultures which were epithelioid or fibroblastic, and A.D.A. activity was constant for a given line over several passages. A.D.A. activity in amriiotic A.D.A.
A.D.A.
* By method 2, mean in 44 controls now 99-2 (95% confidence interval 55-1-178). By method 1, normal mean was 83-8 (58-1-113-8).
amniotic-fluid cells were grown by standard procedures and harvested along with normal amniotic-fluid cell cultures. ·
RESULTS
Detection of Heterozygotes; A.D.A. Activity and A.D.A. Phenotypes of R.B.C.s Diminished levels of R.B.C.-A.D.A. activity were found in the mother and the father, in one maternal and one paternal grandparent, and in an unaffected sibling (table I). Two presumably normal members of the kindred had A.D.A. levels of 107 and 100. Similar levels of activity were found with method 1. Whereas the paternal grandmother had A.D.A. activity within the 95% confidence limits of normals when activity was determined by method 1, activity was below the lower level of these limits when method 2 was employed. Furthermore, she could be shown to carry a silent gene for A.D.A. on the basis of typing for the genetically determined A.D.A. polymorphism-i.e., the paternal grandmother appeared to be homozygous for the rare A.D.A. polymorphic gene ADA2, while her husband appeared to be homozygous for D. Their son should have been heterozygous ADA3-’, since the two gene products are codominantly expressed, but
cells from the pregnancy at risk was 0 208 and 0’192 nmol per mg. protein per minute on two separate occasions or 1-2% of normal (table II).
Clinical A 3-0
Findings kg. male (C. W.)
was born to the 24-year-old mother at 41 weeks’ gestation. para 2, and delivery were uncomplicated except for
gravida 3, Pregnancy a urinary-tract infection.
Physical examination at birth was entirely normal. A chest X-ray failed to reveal a thymic shadow. At 1 day of age the child was placed in protective isolation. During the first 6 weeks of life the child has been feeding well, gaining weight, and has been free of infection. However, leukopenia has been observed in TABLE II-A.D.A. ACTIVITY IN NORMAL AMNIOTIC CELL CULTURES AND IN AMNIOTIC CELL CULTURES FROM A FETUS WITH R.B.C.-
A.D.A. DEFICIENCY AND S.C.I.D.
75
complete blood-counts. White-blood-cell counts have ranged from 1350 to 5620 per c.mm. and absolute lymphocyte-counts from 200 to 2248 per T and B cell proportions were studied using c.mm. rosette techniques at 3 and 9 days of age: T cells were 44% and 46%, respectively (control 50% on
four of six
both occasions), and B cells were 43 % and 19 % (control 32% and 35%). In-vitro lymphocyte stimulation with concanavalin A (con A) and P.H.A. at 3 days of age was diminished with stimulation indices of 6-3 and 6-4 for con A and P.H.A., respectively, as compared with control stimulation of 23-4 and 23-3. Immunoglobulin levels at 20 days of age were within normal limits (mg. per 100 ml.): IgG 750, IgA 0, IgM 5-4. Karyotype analysis of P.H.A.-stimulated peripheral blood performed on the tenth day revealed only XY cells. R.B.C. and lymphocyte A.D.A. studies were performed on samples taken at 9 days of age. No A.D.A. activity could be detected in R.B.C.S in the first week of life. A.D.A. activity could also not be detected using a prolonged incubation assay which can detect as little as 1-5 nmol per mg. Hb per hour or 1-5% of normal R.B.C. activity. In contrast, residual A.D.A. activity could be detected in mononuclear-cell/platelet preparations obtained after centrifugation of blood on a’HypaqueFicoll’ gradient. The child’s mononuclear-cell/platelet preparation contained only 0-865 nmol per mg. protein per minute as compared with 150±42 in normal
mononuclear-cell/platelet preparations.
finding of residual A.D.A. activity in the mononuclear-cell/platelet preparation has been previously The
observed in another child with classical S.C.I.D. and R.B.C.-A.D.A. deficiency 13 In the present case this residual activity did not represent chimxrism with maternal cells because all of the cells responding to P.H.A. had The presence early in life of both a male karyotype. peripheral-blood lymphocytes responsive to P.H.A. and con A as well as maternal antibodies probably accounts for the relative normality of this and similar children during the early weeks of life. We have demonstrated in this report the ability accurately to identify heterozygotes for A.D.A. deficiency and to diagnose the affected fetus in utero. These techniques will lead to more satisfactory genetic counselling in these families and will offer the option of selective abortion of affected fetuses or adequate preparation for therapy of an affected child. This work was aided by grants from the National Institutes of Health (Al 10343 and GM 19443) and the National Foundation. R. H. is a recipient of N.I.H. research career development
The ability to detect heterozygotes with 90% confidence has been reported in a study of a large kindred in which a child had previously been born with R.B.c.A.D.A. deficiency and S.C.I.D.3,l1 In the family reported here, in which a child had died with S.C.I.D. but whose A.D.A. status was unknown, we have identified the parents and other members as heterozygotes for A.D.A. deficiency. Accurate identification of carriers was made possible by the study of three generations, the use of two different methods for assay of R.B.C.-A.D.A. activity, and determination of the phenotypes of the electro-
phoretic A.D.A. polymorphism. The prenatal diagnosis of the homozygous state of A.D.A. deficiency was unambiguous and was confirmed by findings in the child after birth. The amniotic cells of the child at risk contained less than 15% of the A.D.A. activity seen in normal amniotic cells. The range of A.D.A. activity in the normal amniotic cells was large but appeared to be characteristic for the individual amniotic-cell culture. It will be of importance to compare the A.D.A. activity in fibroblasts derived from this child with that found in the amniotic cells, since we have found that fibroblasts of occasional affected children may have as much as 20% residual A.D.A. activity.12 Electrophoresis of fibroblast extracts containing such residual activity has revealed an electrophoretically altered tissue isozyme, probably representing a mutant enzyme 13 Variation in the amount of residual fibroblast A.D.A. activity may represent genetic heterogeneity which, if reflected in amniotic-fluid cell cultures, could lead to difficulty in distinguishing affected from carrier fetuses.
to
R. H.
REFERENCES
Rosen, F. S., Merler, E. in The Metabolic Basis of Inherited Disease (edited by J. B. Stanbury, J. B. Wyngaarden, and D. S. Frederickson); p. 1643. New York, 1972. 2. Giblett, E. R., Anderson, J. E., Cohen, F., Pollara, B., Meuwissen, H. J. Lancet, 1972, ii, 1067. 3. Scott, C. R., Chen, S.-H., Giblett, E. R. J. clin. Invest. 1974, 53, 1.
1194. 4.
5. DISCUSSION
award Al 70254.
Requests for reprints should be addressed
6. 7.
8. 9. 10. 11. 12.
Hirschhorn, R., Levytska, V., Meuwissen, H. J., Pollara, B. Nature new Biol. 1973, 246, 200. Hirschhorn, R. J. clin. Invest. (in the press). Hirschhorn, R., Beratis, N. G. Lancet, 1973, ii, 1217. Hopkinson, D. A., Cook, P. J. L., Harris, H. Ann hum. Genet. 1969, 32, 361. Hirschhorn, R., Levytska, V. Cell. Immun. 1974, 12, 387. Spencer, N., Hopkinson, D., Harris, H. Ann. hum. Genet. 1968, 32, 9. Chen, S.-H., Scott, C. R. Am. J. hum. Genet. 1973, 25, 21a. Chen, S.-H., Scott, C. R., Giblett, E. R. ibid. 1974, 26, 103. Hirschhorn, R., Levytska, V., Parkman, R. J. clin. Invest. 1974, 53, 33a.
13.
Parkman, R., Rosen, F. S., Gelfand, E., Sanderson, A., Hirschhorn, R. Unpublished.
Hypothesis LOW-RENIN HYPERTENSION : NEPHROSCLEROSIS ?
J. D. SWALES Department of Medicine, General Hospital, Leicester LE5 4PW
A substantial group of patients with essential hypertension have abnorrenin levels which respond poorly to stimulow mally lation. Important differences in response to therapy and in prognosis have been described between these and other hypertensive patients. It is suggested that the vascular changes of nephrosclerosis, which may be seen in both hypertensive and normal subjects, result in a reduction of afferent arteriolar distensibility, with impairment of basal renin secretion and responsiveness. This hypothesis accords with both the known clinical characteristics of low-renin hypertension and with the known effect of arterial changes upon the activity of other baroreceptors.
Summary
A survey has shown that the frequency of antibody rises steadily through childhood to reach about 30% in adult life (table 11). It has not been possible to associate the new antigen with a specific illness. Paired sera from 22 hepatitis-A patients and 14 hepatitis-B patients have been tested; none contained the antigen. 2 of the hepatitis-A and 1 of the hepatitisB patients showed conversion from antibody negative Both acute and convalescent sera were to positive. antibody-positive in 5 patients from each group. The new antigen closely resembles the particles described in faeces of patients with acute gastroenteritis by Paver et al.,2 but it is smaller than the spherical particles described by Feinstone et al.’ in faeces of hepatitis patients or the core antigen of
HBAg. While the main interest of the new antigen is as possible infective agent, it is also of some immediate practical importance, since it can readily be confused with HBAg. The size of the small particles is very similar; the diameter of HBAg particles ranges between 16 and 21 nm. and that of the new antigen between 20-5 and 25 nm. In addition, many of the human antisera used for detecting HBAg also contain antibodies to the new antigen.
a
Requests for reprints should be addressed to Y. E. C., Virus Laboratory, Central Public Health Laboratory, Colindale Avenue, London NW9.
Reference
REFERENCES
Vandervelde, E. M., Goffin, G., Megson, B., Mahmood, N., Porter, A., Cossart, Y. E. Lancet, 1974, ii, 1066. 2. Paver, W. K., Caul, E. O., Ashley, C. R., Clarke, S. K. R. ibid. 1973, i, 237. 3. Feinstone, S. M., Kapikian, A. Z., Purcell, R. H. Science, 1974, 182, 1.
1026. 4.
Cossart, Y. E., Field, A. M., Hargreaves, F. D., Porter, A. A. Microbios, 1971, 3, 5.
ADENOSINE-DEAMINASE DEFICIENCY IN A CHILD DIAGNOSED PRENATALLY ROCHELLE HIRSCHHORN
Department of Medicine, New York University School of Medicine, New York, N.Y., U.S.A. NICHOLAS BERATIS Department of Pediatrics, Mount Sinai School of Medicine, New York
FRED S. ROSEN
ROBERTSON PARKMAN
contained less than 1.5 ’it> of A.D.A. activity of normal amniotic cultures. The prenatal diagnosis of A.D.A. deficiency was confirmed at birth by the absence of Clinical A.D.A. activity in the child’s red blood-cells. and laboratory findings in this child are similar to those of the sibling who had died with S.C.I.D. INTRODUCTION
SEVERE combined immunodeficiency (S.C.I.D.) is a childhood disease which is fatal if untreated. Lifethreatening infections result from impairment of both cellular and humoral immunity. The disorder occurs in X-linked and autosomal recessive forms.1 A deficiency of the erythrocyte enzyme adenosine deaminase (R.B.C.-A.D.A.) has been reported in some, but not all, patients with the autosomal recessive form of S.C.I.D .2 The deficiency of R.B.C.-A.D.A. is transmitted as an autosomal recessive trait, with the parents and onehalf the grandparents showing diminished levels of enzyme activity.3 Besides lacking the low-molecularweight R.B.c. form of the enzyme, the affected children also have deficiencies in the various highermolecular-weight, tissue-specific forms of A.D.A.4 These tissue-specific isozymes can be generated by coincubation of R.B.C.-A.D.A. with different tissue extracts.5 These two findings indicate that the gene affected in s.c.I.D. with A.D.A. deficiency codes for a catalytic unit common to the tissue and R.B.c. isozymes of A.D.A. As we have suggested,6 it should, therefore, be possible to diagnose R.B.C.-A.D.A. deficiency in cultured amniotic-fluid cells. These cells, like fibroblasts, can express the R.B.C. and tissue isozyme, but may express the tissue isozyme alone. We now report the in-utero diagnosis of A.D.A. deficiency in a child subsequently shown to have a deficiency of A.D.A. activity in his red blood-cells. FAMILY HI STORY
The family was investigated during a survey of kindreds in which a previous child had died with S.C.I.D. Both parents and their 6-year-old son are healthy. The parents are apparently not related. A girl (K. W.) had died 3 years earlier at 9 months of age. She had been well until the age of 3 months. She then failed to continue to gain weight and developed respiratory congestion. Subsequently oral and perineal candidiasis, diarrhoea, and pneumonitis were diagnosed. A thymic shadow was never seen on several chest X-rays. Leukopenia (2000-7000 per c.mm) and lymphopenia (0-17%) were prominent. There was no
Department of Pediatrics, Children’s Hospital Medical Center, Harvard Medical School, Boston,
delayed hypersensitivity to common antigens, including candida, and the child could not be sensitised to 2,4-dini-
Massachusetts
trochlorobenzene. Lymphocytes showed reduced stimulation with phytohsemagglutinin (stimulation index 8, normal approximately 40). A small amount of thymic tissue obtained at mediastinoscopy had the characteristic histology seen in s.c.I.D. Serum-immunoglobulins (mg. per 100 ml.) at 6 months were: IgG 380, IgM 100, and IgA 28. Schick test was negative, anti-A titre was 1/2, and only a twofold rise in antitetanus titre was observed after immunisation. This child died after an unsuccessful bonemarrow transplantation from the father. A.D.A. studies were not done.
STEPHEN POLMAR Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, Ohio ROBERT STERN
of red-blood-cell adenosine
Summary Deficiency deaminase (R.B.C.-A.D.A.) has been rein a ported proportion of patients with the autosomal recessive form of severe combined immunodeficiency in which a child had died with S.C.I.D., R.B.C.-A.D.A. levels in the parents and other members of the family were compatible with a heterozygous state for A.D.A. deficiency. Cultured amnioticfluid cells obtained from a subsequent pregnancy
(S.C.I.D.).
In
a
family
METHODS
Enzyme Assays A.D.A. activity in a
linked assay
at
R.B.c. haemolysates was determined by 37’C (method 1) using 0-4 mg. adeno-
74 sine per ml. 0’05M phosphate buffer pH 7’5 as substrate. In the presence of endogenous nucleoside phosphorylase and exogenous xanthine oxidase (0-2 units per ml.), uric acid is produced and measured at 293 nmo.7 A modification of this method (method 2) was also used.8 The two methods showed a correlation coefficient of 0-93. Values for R.B.c. activity were expressed as nmol per mg. Hb per hour. A log transformation of all values was performed because of a skewed distribution towards higher R.B.C.S were also typed for the genetically A.D.A. values.3 determined polymorphism demonstrated by R.B.C.-A.D.A.9 A.D.A. activity of amniotic cells and mononuclear-cell/ platelet cell preparations was determined at 37°C using 50-200 g. protein per ml. assay containing 0’4 mg. adenosine in 0-05M phosphate buffer pH 7’5 in the presence of exogenous nucleoside phosphorylase (0’2S units per ml.) as well as xanthine oxidase (0’2 units per ml.) and the amount of uric acid produced was measured at 293 nm.
Amniocentesis Amniocentesis
done during the 28th week of gestation, immediately after ascertainment of the family. The mother was told that her pregnancy was too far advanced for a therapeutic abortion to be undertaken, but she nevertheless agreed to amniocentesis for investigative purposes after being informed of all potential risks. The TABLE
was
I-R.B.C.-A.D.A. ACTIVITIES AND PROPOSED IN A FAMILY WITH S.C.I.D.
GENOTYPES
A.D.A. genotypes in S.C.I.D.
a
family with
two children affected with
Numbers in ( ) represent proposed genotypes based on electrophoretic phenotypes. The arrow indicates the who was diagnosed prenatally as A.D.A. deficient.
A.D.A.
child
instead he expressed only the A.D.A. 1 phenotype. This indicates that the paternal grandmother was not homozygous for 4D but heterozygous for ADA2 and silent gene 4D4 transmitting the silent gene to her son who was then ADAl-o. He and his wife then transmitted silent genes to their affected children
(see figure). Activity in Amniotic Cells activity in fifteen normal amniotic-cell cultures averaged 17 19 ±7 79 nmol per mg. protein per minute by method 1. Similar values have been reported by Chen et al,u No difference could be observed between cultures which were epithelioid or fibroblastic, and A.D.A. activity was constant for a given line over several passages. A.D.A. activity in amriiotic A.D.A.
A.D.A.
* By method 2, mean in 44 controls now 99-2 (95% confidence interval 55-1-178). By method 1, normal mean was 83-8 (58-1-113-8).
amniotic-fluid cells were grown by standard procedures and harvested along with normal amniotic-fluid cell cultures. ·
RESULTS
Detection of Heterozygotes; A.D.A. Activity and A.D.A. Phenotypes of R.B.C.s Diminished levels of R.B.C.-A.D.A. activity were found in the mother and the father, in one maternal and one paternal grandparent, and in an unaffected sibling (table I). Two presumably normal members of the kindred had A.D.A. levels of 107 and 100. Similar levels of activity were found with method 1. Whereas the paternal grandmother had A.D.A. activity within the 95% confidence limits of normals when activity was determined by method 1, activity was below the lower level of these limits when method 2 was employed. Furthermore, she could be shown to carry a silent gene for A.D.A. on the basis of typing for the genetically determined A.D.A. polymorphism-i.e., the paternal grandmother appeared to be homozygous for the rare A.D.A. polymorphic gene ADA2, while her husband appeared to be homozygous for D. Their son should have been heterozygous ADA3-’, since the two gene products are codominantly expressed, but
cells from the pregnancy at risk was 0 208 and 0’192 nmol per mg. protein per minute on two separate occasions or 1-2% of normal (table II).
Clinical A 3-0
Findings kg. male (C. W.)
was born to the 24-year-old mother at 41 weeks’ gestation. para 2, and delivery were uncomplicated except for
gravida 3, Pregnancy a urinary-tract infection.
Physical examination at birth was entirely normal. A chest X-ray failed to reveal a thymic shadow. At 1 day of age the child was placed in protective isolation. During the first 6 weeks of life the child has been feeding well, gaining weight, and has been free of infection. However, leukopenia has been observed in TABLE II-A.D.A. ACTIVITY IN NORMAL AMNIOTIC CELL CULTURES AND IN AMNIOTIC CELL CULTURES FROM A FETUS WITH R.B.C.-
A.D.A. DEFICIENCY AND S.C.I.D.
75
complete blood-counts. White-blood-cell counts have ranged from 1350 to 5620 per c.mm. and absolute lymphocyte-counts from 200 to 2248 per T and B cell proportions were studied using c.mm. rosette techniques at 3 and 9 days of age: T cells were 44% and 46%, respectively (control 50% on
four of six
both occasions), and B cells were 43 % and 19 % (control 32% and 35%). In-vitro lymphocyte stimulation with concanavalin A (con A) and P.H.A. at 3 days of age was diminished with stimulation indices of 6-3 and 6-4 for con A and P.H.A., respectively, as compared with control stimulation of 23-4 and 23-3. Immunoglobulin levels at 20 days of age were within normal limits (mg. per 100 ml.): IgG 750, IgA 0, IgM 5-4. Karyotype analysis of P.H.A.-stimulated peripheral blood performed on the tenth day revealed only XY cells. R.B.C. and lymphocyte A.D.A. studies were performed on samples taken at 9 days of age. No A.D.A. activity could be detected in R.B.C.S in the first week of life. A.D.A. activity could also not be detected using a prolonged incubation assay which can detect as little as 1-5 nmol per mg. Hb per hour or 1-5% of normal R.B.C. activity. In contrast, residual A.D.A. activity could be detected in mononuclear-cell/platelet preparations obtained after centrifugation of blood on a’HypaqueFicoll’ gradient. The child’s mononuclear-cell/platelet preparation contained only 0-865 nmol per mg. protein per minute as compared with 150±42 in normal
mononuclear-cell/platelet preparations.
finding of residual A.D.A. activity in the mononuclear-cell/platelet preparation has been previously The
observed in another child with classical S.C.I.D. and R.B.C.-A.D.A. deficiency 13 In the present case this residual activity did not represent chimxrism with maternal cells because all of the cells responding to P.H.A. had The presence early in life of both a male karyotype. peripheral-blood lymphocytes responsive to P.H.A. and con A as well as maternal antibodies probably accounts for the relative normality of this and similar children during the early weeks of life. We have demonstrated in this report the ability accurately to identify heterozygotes for A.D.A. deficiency and to diagnose the affected fetus in utero. These techniques will lead to more satisfactory genetic counselling in these families and will offer the option of selective abortion of affected fetuses or adequate preparation for therapy of an affected child. This work was aided by grants from the National Institutes of Health (Al 10343 and GM 19443) and the National Foundation. R. H. is a recipient of N.I.H. research career development
The ability to detect heterozygotes with 90% confidence has been reported in a study of a large kindred in which a child had previously been born with R.B.c.A.D.A. deficiency and S.C.I.D.3,l1 In the family reported here, in which a child had died with S.C.I.D. but whose A.D.A. status was unknown, we have identified the parents and other members as heterozygotes for A.D.A. deficiency. Accurate identification of carriers was made possible by the study of three generations, the use of two different methods for assay of R.B.C.-A.D.A. activity, and determination of the phenotypes of the electro-
phoretic A.D.A. polymorphism. The prenatal diagnosis of the homozygous state of A.D.A. deficiency was unambiguous and was confirmed by findings in the child after birth. The amniotic cells of the child at risk contained less than 15% of the A.D.A. activity seen in normal amniotic cells. The range of A.D.A. activity in the normal amniotic cells was large but appeared to be characteristic for the individual amniotic-cell culture. It will be of importance to compare the A.D.A. activity in fibroblasts derived from this child with that found in the amniotic cells, since we have found that fibroblasts of occasional affected children may have as much as 20% residual A.D.A. activity.12 Electrophoresis of fibroblast extracts containing such residual activity has revealed an electrophoretically altered tissue isozyme, probably representing a mutant enzyme 13 Variation in the amount of residual fibroblast A.D.A. activity may represent genetic heterogeneity which, if reflected in amniotic-fluid cell cultures, could lead to difficulty in distinguishing affected from carrier fetuses.
to
R. H.
REFERENCES
Rosen, F. S., Merler, E. in The Metabolic Basis of Inherited Disease (edited by J. B. Stanbury, J. B. Wyngaarden, and D. S. Frederickson); p. 1643. New York, 1972. 2. Giblett, E. R., Anderson, J. E., Cohen, F., Pollara, B., Meuwissen, H. J. Lancet, 1972, ii, 1067. 3. Scott, C. R., Chen, S.-H., Giblett, E. R. J. clin. Invest. 1974, 53, 1.
1194. 4.
5. DISCUSSION
award Al 70254.
Requests for reprints should be addressed
6. 7.
8. 9. 10. 11. 12.
Hirschhorn, R., Levytska, V., Meuwissen, H. J., Pollara, B. Nature new Biol. 1973, 246, 200. Hirschhorn, R. J. clin. Invest. (in the press). Hirschhorn, R., Beratis, N. G. Lancet, 1973, ii, 1217. Hopkinson, D. A., Cook, P. J. L., Harris, H. Ann hum. Genet. 1969, 32, 361. Hirschhorn, R., Levytska, V. Cell. Immun. 1974, 12, 387. Spencer, N., Hopkinson, D., Harris, H. Ann. hum. Genet. 1968, 32, 9. Chen, S.-H., Scott, C. R. Am. J. hum. Genet. 1973, 25, 21a. Chen, S.-H., Scott, C. R., Giblett, E. R. ibid. 1974, 26, 103. Hirschhorn, R., Levytska, V., Parkman, R. J. clin. Invest. 1974, 53, 33a.
13.
Parkman, R., Rosen, F. S., Gelfand, E., Sanderson, A., Hirschhorn, R. Unpublished.
Hypothesis LOW-RENIN HYPERTENSION : NEPHROSCLEROSIS ?
J. D. SWALES Department of Medicine, General Hospital, Leicester LE5 4PW
A substantial group of patients with essential hypertension have abnorrenin levels which respond poorly to stimulow mally lation. Important differences in response to therapy and in prognosis have been described between these and other hypertensive patients. It is suggested that the vascular changes of nephrosclerosis, which may be seen in both hypertensive and normal subjects, result in a reduction of afferent arteriolar distensibility, with impairment of basal renin secretion and responsiveness. This hypothesis accords with both the known clinical characteristics of low-renin hypertension and with the known effect of arterial changes upon the activity of other baroreceptors.
Summary
