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Argininosuccinic aciduria: Investigation of an affected family
January 1974
The Journal o f P E D I A T R I C S
85
Argininosuccinic aciduria: Investigation of an affected family Two siblings with argininosuccinic aciduria were studied by an interdisciplinary team. Considerable variability in the clinical expression of this disorder was observe d in comparison with previously reported cases. Detection o f the heterozygous state in the parents and in a fetal sibling was demonstrated by tissue assay for argininosuccinase acitivity. Mental retardation and neurologic deficits in the affected children were irreversible with dietotherapy but growth was progressive and the general course was relatively benign.
Gerard J. Billmeier, Jr., M.D.,* Samuel V. Molinary, Ph.D.,
R. S. Wilroy, Jr.,M.D., Danilo A. Duenas, M.D.~ and M. Elizabeth Brannon, M.S., Memphis, Tenn.
AMONG THE disorders known as inbtgrn errors o f metabolism are seVeral entities involving enzymatic defects in urea synthesis. At least five enzymes are known to be essential in the Krebs-Hensleit urea cycle; these enzymatic .activities are measurable biochemically. 1 o n e such enzyme, argininosuccinase [Largininosuccinate arginine-lyase (EC No. 4.3.2.1)] acts as a cleavage e n z y m e splitting a r g i n i n o s u c c i n a t e to arginine and fumarate. A deficiency of ASase results in argininosuccinic aciduria, a rare inborn error of metabolism inherited in an autosomal recessive manner. Mental retardation, coarse a n d brittle hair, convulsions, ataxia, and liver disease comprise the usual clinical manifestations, and characteristically large amounts of ASA are found in the plasma, urine, and cerebrospinal fluid. However, 22 previously reported cases (Table I) display variable clinical manifestations a n d degrees of morbidity. 2-4 The present study deals with the investigation of two
From the University o f Tennessee Child Development Center. Supported by Special Project Grant No. 900, Division o f Health Seevices, Maternal and Child Health Service, HSMHA, Department o f Health, Education and Welfare, andNational Institute of Health Gran t No. RR-00211. *Reprint address: 332 N. Lauderdale. Memphis. Tenn. 38101.
affected siblings and their family including prenatal diagnosi~ of a third sibling, who p r o v e d to be heterozygous for the disorder. METHODS Urinary amino acid screening studies were carried out by use of high-voltage paper electrophoresis. 5 Quantitative argininosuccinic acid assays of urine, plasma, and cerebrospinal fluid were accomplished according to the method of Berry and associates: 6 Assays for argiAbbreviations used: ASase: argininosuccinase ASA: argininosuccini c acid nihosuccinic activity in e r y t h r o c y t e s and hepatic cells were done according to the method of Tomlinson and Westall. 7 The method for argininosuccinase essay using amniotic and skin fibroblasts has been described by Jacoby and associates. 8 CASE R E P O R T S Case 1 (index ease). W. T., a Caucasian boy, was referred to the University of Tennessee Child Development Center at age 4 years and 5 months because of delayed development of speech and language. He was the first born of healthy unrelated parents. The pregnancy was complicated by first trimester nausea and vomiting and an 18 kilogram gain in weight. At 40
VoL 84, No. 1, pp 85-89
86
Billmeier et al.
The Journal of Pediatrics January 1974
Table I. C o m p a r a t i v e data: Previously r e p o r t e d cases a n d p r e s e n t study
LO. Cases 22
Reference 2-4
Sex 13 ~
Range
Median
20-92
51
Abnormal hair 12/20"
Hepatic dysfunction 10/15"
w.T.
9~ ~
57
+
-
T.T.
~
72
+
--
Convulsive Abnormal disorder EEG
A taxia
Hypotonia
Growth retardation
15/22"
9/13"
8/15"
4/12"
3/10"
--
+
--
+
+
+, Present; --, absent. * = Number positive/reporting.
Table II, C o n c e n t r a t i o n s o f argininosuccinic acid
Subject
Urine (mg./mL)
Plasma (mg./mL)
Cerebrospinal fluid (mg./ml.)
W.T. T.T. M.T. Father Mother
6.0 18.1 0 0 0
5.0 6,4 0 0 0
14.6 11.4
weeks gestation, labor and delivery were uncomplicated. The birth weight was 3,500 grams. Vomiting and diarrhea necessitated frequent formula changes during the first nine months of life. Developmental milestones were normal except for late acquisition of speech and language. The mother noted that the patient's hair had always been coarse and subject to breaking. He had always shown dislike for meats and was said to be hyperactive and clumsy. Family history revealed two maternal uncles who had "slow speech" and were thought-to be retarded. A younger female sibling (Case 2) had a similar lag in acquisition of speech and language. Physical examination revealed a very active and distractible Caucasian boy who had limited verbal language. Height was 100.6 cm. (third percentile), weight, 17.4 kilograms (fiftieth percentile), and head circumference, 50.5 cm. (fiftieth percentile). The child's blond hair was coarse and friable. The remainder of the physical examination revealed no abnormalities with the exception of neuromotor incoordination and generalized muscular hypotonia. A battery of psychometric tests showed mildly retarded intellectual capacity. A urinary amino acid chromatogram was strongly positive for argininosuccinic acid. Further biochemical studies included quantitation of ASA in urine, plasma, and cerebrospinal fluid (Table II). ASase activity in erythrocytes and hepatic tissue was also measured (Table III). An ammonia tolerance test showed a fasting blood ammonia level of 53 p,g per 100 ml. wi[h a level of 105/zg per 100 ml. two
hours after a 10 Gm. protein meal. A 5 Gm. protein meal resulted in two-hour postprandial blood ammonia level of 86 p,g per cent. An arginine loading test produced no elevation of blood ammonia but a transient rise in plasma and urinary ASA levels was observed. Serial urinary ASA determinations revealed a mean excretion of 8.4 • 1.1 mg. per milliliter or approximately 4 Gm. per 24 hours. Radiographic bone age was compatible with the patient's chronologic age. An electroencephalogram ( E E G ) w a s interpreted as abnormal with poor regulation and diffuse slow activity. A pneumoencephalogram revealed no cortical atrophy. Hair was grossly abnormal in its coarse texture and friability, yet not compatible microscopically with trichorrhexis nodosa. Liver tissue was normal microscopically with abundant glycogen and no detectable fat. Chromosomal analysis of peripheral blood lymphoeytes revealed a normal karyotype (46, XY). Case 2. T. T., the product of her mother's second pregnancy, was evaluated at age 3 years 2 months after her older brother (Case 1) was found to have argininosuccinic aciduria. Her mother had an uncomplicated pregnancy and delivered a female infant weighing 3,500 grams at term. Language skills were delayed although motor milestones were attained normally. T. T. also disliked meat and had had occipital loss of hair since early infancy. Physical examination revealed a quiet Caucasian girl whose height was 95.7 cm., weight 14.4 kilograms, and head circumference 50 cm. (all fiftieth percentile). Pertinent findings included coarse, friable, blond hair with a bald occiput (Fig. 1) and a firm liver margin, palpable 3 cm. below the right costal margin. The neurologic examination revealed no abnormalities except for a mild degree of generalized muscular hypotonia. A battery of psychometric tests revealed a mental age of 2 to 2 years, representing borderline intelligence. Urinary, plasma, and cerebrospinal fluid levels of ASA were elevated (Table II) and tissue ASase activity was reduced (Table liD. An ammonia tolerance test showed a fasting blood ammonia level of 100/xg per 100 ml. with an increase to 110/xg per 100 ml., two hours after a 10 Gm. protein meal. Citrulline and arginine loading tests produced no elevation of blood ammonia
Volume84 Number 1
A t~ininosuccinic aciduria
87
Table III. Erythrocyte and liver e n z y m e activity
Liver
Erythrocyte Subject
W. T. (ASA) T. T. (ASA) M.T.* Father Mother Control
ASase (Izmole urea/Gm. Hb./hr.)
A rginase (txmole urea/Gm. Hb./hr.)
0 0 2.32 5.23 5.93 11.7
231 293 344 423 381 447
ASase (tzmole urea/mg. protein~hr.) 0
0
(I-tmole urea~rag. protein~hr.) 0.111 0,0137
*Assays performed on cord blood.
levels in this subject. Serial urinary ASA determinations revelaed mean excretion of 10.1 • 1.9 rag. per milliliter, approximating 4 Gin. daily. An EEG was abnormal with diffuse irregular and slow activity indicative of widespread encephalopathy. Chromosomal analysis of peripheral blood lymphocytes revealed a normal karyotype (46, XX). Case 3, The mother of these two children conceived for the third time, although she and her husband had been counseled as to the mode of inheritance. An amniocentesis performed during the fifteenth week of gestation yielded fetal ceils which revealed an apparently normal karyotype, 46, XX, and female sex was predicted. ASase values using fetal cells were compatible with enzyme activity of the heterozygous state. 8 This pregnancy was Uncomplicated and terminated in the birth of a healthy female infant, M. T., weighing 3,750 grams. Arginase and ASase activity in the cord blood confirmed the prenatal diagnosis of the heterozygous state for this child (Table liD. Subsequent determinations on blood and urine were negative for ASA (Table II). FAMILY
INVESTIGATION
A family survey for additional affected m e m b e r s yielded n o n e 'with urine positive for ASA. Both parents were studied for erythrocyte ASase activity; each was heterozygous for the disorder (Fig. 2 and Table III). Subs e q u e n t assays o f skin fibroblast ASase in W. T., T. T., and the parents have revealed activity comparable with that o f their red blood cells. 8 DISCUSSION T h e initial report of argininosuccinic aciduria by Allan and colleagues 9 in 1958 described two mentally retarded siblings with similar facies, friable hair, and abnormal E E G ' s . The older sibling also exhibited a severe
Fig. 1. Case 2 (T. T.). Note balding occiput. convulsive disorder and m a r k e d ataxia. Laboratory investigation revealed urinary excretion of an u n k n o w n c o m p o u n d later identified as argininosuccinic acid (ASA). T h e concentration of A S A in cerebrospinal fluid was found to be three times that of the plasma. 10 At least twenty cases o f argininosuccinic aciduria h a v e ,. b e e n r e p o r t e d s u b s e q u e n t l y with considerable variability in clinical and biochemical manifestations.
88
The Journal of Pediatrics January 1974
Billmeier et al.
II
Argininosuccinic aciduria ASA heterozygote
~]
"Slow speech"
?
I.
II.
III. WT
TT
MT
Fig. 2. Pedigree of T. family. Clinical manifestations. Nearly all such patients exhibited mental subnormality with a mean I.Q. of 51 in those subjects testable or moderate mental retardation according to standard intelligence levels. An exception to this clinical feature is reported by Carson and Neill H who described a 4-year-old girl with argininosuccinic aciduria and a measured I.Q of 92. Coarse, friable hair was the only physical abnormality. Psychometric testing in our patients revealed mild to borderline mental subnormality, which seems to parallel their relatively benign courses. As in o u r patients, delayed acquisition of speech and language was a major presenting problem in several cases reported earlier. This seems to represent language delay consistent with over-all intellectual deficit, however, rather than a specific language disorder. Abnormal hair, described as coarse, brittle, and easily broken, was noted in the majority of these patients. However, several reported patients had normal hair texture and distribution. The classical trichorrhexis nodosa associated with argininosuccinic aciduria is thought to be due to depletion of arginine from the usual components of normal hair keratin. In support of this hypothesis is the improvement of hair appearance in these patients after arginine dietary supplementation. Our patients have grossly abnormal hair patterns with occipital balding and very coarse, brittle texture, yet microscopic studies are not compatible with trichorrhexis nodosa.
Generalized seizures were present in most previously reported patients and E E G patterns typically showed diffuse, marked slowing with two to three per second spike and slow wave discharge. This pattern has been described in phenylketonuria 12and appears to represent a diffuse.metabolic encephatopathy. Our patients had c o m p a r a b l e E E G p a t t e r n s , although neither has developed a convulsive disorder. Both, however, exhibit generalized hypotonia which appears to be of cerebral origin. Hepatic dysfunction as reflected in elevated serum alkaline phosphatase and hepatic enzyme occurred in two thirds of previously reported cases. Jaundice has not been described in this disorder and hepatomegaly, which occurred in one fourth o f prior cases, was often transient. Hepatomegaly in T. T. was not associated with other clinical signs of liver disease, and liver architecture appeared normal microscopically, except for vascular congestion in our patients as well as those previously studied. Biochemisty. All affected patients excrete large amounts of argininosuccinic acid in urine, the mean excretion rate approximating 3.5 to 4.0 Gm. per day. The previous observation of heterozygous individuals excreting trace amounts of ASA in urinO ~ was not demonstrated in the present study. The enzyme defect in this disorder has been demonstrated in blood cell hemolysates which typically lack ASase activity in homozygous i n d i v i d f l a l s a n d s h o w r e d u c e d a c t i v i t y in t h e heterozygous state] Such a defect was confirmed in the erythrocytes and hepatic cells of our patients. Reduced ASase activity was also demonstrated in the erythrocytes of their parents. Another human cell type, the skin fibroblast, has been shown to be deficient in ASase activity in this disease despite the presence of essential enzymes such as arginase. 8 Absent or extremely low ASase activity in erythrocytes, liver cells, and skin fibroblasts, as well as brain tissue, L3,~4casts serious doubt upon the existence of isozymes in the disorder. G e n e t i c s . T h e e v i d e n c e suggests an a u t o s o m a l recessive mode of inheritance; consanguineous marriages in such pedigrees plus single generations with affected siblings support this assumption. Ethnic background in this disease is variable, with cases reported in Jewish, Sicilian and Irish people. Detection of the heterozygous state is not practical by urine screening tests but is possible by erythrocyte ASase determination as demonstrated in this report. D i e t o t h e r a p y . Basic a p p r o a c h e s to t h e r a p y in argininosuccinic aciduria are: (1) to minimize hyperammonemia, (2) to prevent arginine deficiency, and (3) to p r o v i d e sufficient nutrients for n o r m a l growth.
Volume 84 Number 1
Arginine supplementation has failed to improve the over-all clinical course but normal hair texture may be restored. 15 The administration of citric acid has been shown to reduce the degree of hyperammonernia following a protein meal, apparently through the enhancement of the tricarboxylic acid cycle to accomodate excess ammoniaJ 5In the present study an effort was made to provide sufficient protein for growth yet not induce hyperammonemia by excess of protein. A diet consisting of 1 Gin. of protein per kilogram of body weight daily divided into four feedings was prescribed in addition to arginine and citric acid supplementation. It is interesting that calculation of the children's pretreatment diets revealed relatively low protein content, possibly a reflection of their strong dislike for meats, which may be a self-regulatory mechanism. The children are being followed by an interdisciplinary team for analysis of growth and development. The third sibling in this report, M. T., is thriving on a regular diet and exhibits normal development at the time of this writing. In her case, amniocentesis and subsequent identification as a heterozygote for argininosuccinic aciduria underscore the reality of prenatal diagnosis in the area of inborn errors of metabolism. This useful tool in conjunction with knowledgeable genetic counseling is essential for proper management of such families. We are indebted to Arthur Fort, M.D., University of Tennessee Department of Obstetrics and Gynecology for performing the amniocentesis and to J. W. Littlefield, M.D., and associates, Boston, Mass., for enzyme determinations in fetal cells and skin fibroblasts. We also thank George McCormick, M.D., University of Tennessee Department of Pathology, and E. W. Rosenberg, M.D., University of Tennessee Department of Dermatology, for their consultations, and Mrs. Connie Gray for her technical assistance. Psychometric data were provided by members of the University of Tennessee Child Development Center Physchology Department, to whom we are grateful. REFERENCES 1. Brown, G. W., Jr., and Cohen, P. O.: Comparative biochemistry of urea synthesis. I. Methods for the quan-
,4 t~ininosuccinic aciduria
2.
3. 4. 5.
6. 7.
8.
9.
10.
11.
12. 13.
14.
15.
89
titative assay of urea cycle enzymes in liver, J. Biol. Chem. 234: 1769, 1959. Shih, V. E., and Et'ron, M. L.: Urea cycle disorders, in Stanbury,vJ. B., Wyngaarden, J. B., and Fredrickson, D. F., editors: The metabolic basis of inherited disease, ed. 3, New York, 1971, McGraw-Hill Book Company, Inc., pp. 370-392. Maagoe,H.: Argininosuccinic aciduria, Danish Med. Bull. 16: 308, 1969. Shih, V. E.: Early dietary management in an infant with argininosuccinase dieficiency, J. PED1ATR.80: 645, 1972. Howell, R. R, Holtzman, N. A., and Thomas G: H.: Selected screening tests for genetic metabolic diseases, Baltimore, Md., 1969, Johns Hopkins University School of Medicine. Berry, H. K., Leonard, C., Peters, H., Granger, M., and Chunekamrai, N.: Detection of metabolic disorders, Clin. Chem. 14: 1033;1968. Tomlinson, S., a n d Westall R. G.: Argininosuccinic aciduria: Argininosuccinase and arginase in human blood cells, Clin. Sci. 26: 261, 1964. Jacoby,L. B., Littlefield, J., Milunsky, A., Shih, V. E., and. Wilroy, R. S., Jr.: A microassay for argininosuccinase in cultured cells, Am. J. Hum. Genet. 24: 321, 1972. Allan, J. D., Cusworth, D. C., Dent, C. E., and Wilson, V. D.: A disease, probably hereditary, characterized by severe mental deficiency and a constant gross abnormality of amino acid metabolism, Lancet 1: 182, 1958. Westall, R. G.: Argininosaccinic aciduria: identification and reactions of the abnormal metabolites in a newly described form of mental disease, with some preliminary metabolic studies, Biochem J. 77: 135, 1960. Carson, N. A. J., and Neill, D. W.: Metabolic abnormalities detected in a survey of mentally backward children in Northern Ireland, Arch. Dis. Child. 37: 505, 1962. Fois, A.: The electroencephalogram in phenylpyruvic oligophrenia, Electroencephalography 7: 569, 1955. Kint, J., and Carton, D.: Deficient argininosuccinase activity in brain in argininosuccinic aciduria, Lancet 2: 635, 1968. Solitare, G., Shih V. E., Nelligan, D., and Dolan, T.: Argininosuccinic aciduria: Clinical, biochemical, anatomical and neuropathological observations, J. Ment. Def. Res. 13: 153, 1969. Levin, B:: Argininosuccinic aciduria, Am. J. Dis. Child. 113: 162, 1967.
The Journal o f P E D I A T R I C S
85
Argininosuccinic aciduria: Investigation of an affected family Two siblings with argininosuccinic aciduria were studied by an interdisciplinary team. Considerable variability in the clinical expression of this disorder was observe d in comparison with previously reported cases. Detection o f the heterozygous state in the parents and in a fetal sibling was demonstrated by tissue assay for argininosuccinase acitivity. Mental retardation and neurologic deficits in the affected children were irreversible with dietotherapy but growth was progressive and the general course was relatively benign.
Gerard J. Billmeier, Jr., M.D.,* Samuel V. Molinary, Ph.D.,
R. S. Wilroy, Jr.,M.D., Danilo A. Duenas, M.D.~ and M. Elizabeth Brannon, M.S., Memphis, Tenn.
AMONG THE disorders known as inbtgrn errors o f metabolism are seVeral entities involving enzymatic defects in urea synthesis. At least five enzymes are known to be essential in the Krebs-Hensleit urea cycle; these enzymatic .activities are measurable biochemically. 1 o n e such enzyme, argininosuccinase [Largininosuccinate arginine-lyase (EC No. 4.3.2.1)] acts as a cleavage e n z y m e splitting a r g i n i n o s u c c i n a t e to arginine and fumarate. A deficiency of ASase results in argininosuccinic aciduria, a rare inborn error of metabolism inherited in an autosomal recessive manner. Mental retardation, coarse a n d brittle hair, convulsions, ataxia, and liver disease comprise the usual clinical manifestations, and characteristically large amounts of ASA are found in the plasma, urine, and cerebrospinal fluid. However, 22 previously reported cases (Table I) display variable clinical manifestations a n d degrees of morbidity. 2-4 The present study deals with the investigation of two
From the University o f Tennessee Child Development Center. Supported by Special Project Grant No. 900, Division o f Health Seevices, Maternal and Child Health Service, HSMHA, Department o f Health, Education and Welfare, andNational Institute of Health Gran t No. RR-00211. *Reprint address: 332 N. Lauderdale. Memphis. Tenn. 38101.
affected siblings and their family including prenatal diagnosi~ of a third sibling, who p r o v e d to be heterozygous for the disorder. METHODS Urinary amino acid screening studies were carried out by use of high-voltage paper electrophoresis. 5 Quantitative argininosuccinic acid assays of urine, plasma, and cerebrospinal fluid were accomplished according to the method of Berry and associates: 6 Assays for argiAbbreviations used: ASase: argininosuccinase ASA: argininosuccini c acid nihosuccinic activity in e r y t h r o c y t e s and hepatic cells were done according to the method of Tomlinson and Westall. 7 The method for argininosuccinase essay using amniotic and skin fibroblasts has been described by Jacoby and associates. 8 CASE R E P O R T S Case 1 (index ease). W. T., a Caucasian boy, was referred to the University of Tennessee Child Development Center at age 4 years and 5 months because of delayed development of speech and language. He was the first born of healthy unrelated parents. The pregnancy was complicated by first trimester nausea and vomiting and an 18 kilogram gain in weight. At 40
VoL 84, No. 1, pp 85-89
86
Billmeier et al.
The Journal of Pediatrics January 1974
Table I. C o m p a r a t i v e data: Previously r e p o r t e d cases a n d p r e s e n t study
LO. Cases 22
Reference 2-4
Sex 13 ~
Range
Median
20-92
51
Abnormal hair 12/20"
Hepatic dysfunction 10/15"
w.T.
9~ ~
57
+
-
T.T.
~
72
+
--
Convulsive Abnormal disorder EEG
A taxia
Hypotonia
Growth retardation
15/22"
9/13"
8/15"
4/12"
3/10"
--
+
--
+
+
+, Present; --, absent. * = Number positive/reporting.
Table II, C o n c e n t r a t i o n s o f argininosuccinic acid
Subject
Urine (mg./mL)
Plasma (mg./mL)
Cerebrospinal fluid (mg./ml.)
W.T. T.T. M.T. Father Mother
6.0 18.1 0 0 0
5.0 6,4 0 0 0
14.6 11.4
weeks gestation, labor and delivery were uncomplicated. The birth weight was 3,500 grams. Vomiting and diarrhea necessitated frequent formula changes during the first nine months of life. Developmental milestones were normal except for late acquisition of speech and language. The mother noted that the patient's hair had always been coarse and subject to breaking. He had always shown dislike for meats and was said to be hyperactive and clumsy. Family history revealed two maternal uncles who had "slow speech" and were thought-to be retarded. A younger female sibling (Case 2) had a similar lag in acquisition of speech and language. Physical examination revealed a very active and distractible Caucasian boy who had limited verbal language. Height was 100.6 cm. (third percentile), weight, 17.4 kilograms (fiftieth percentile), and head circumference, 50.5 cm. (fiftieth percentile). The child's blond hair was coarse and friable. The remainder of the physical examination revealed no abnormalities with the exception of neuromotor incoordination and generalized muscular hypotonia. A battery of psychometric tests showed mildly retarded intellectual capacity. A urinary amino acid chromatogram was strongly positive for argininosuccinic acid. Further biochemical studies included quantitation of ASA in urine, plasma, and cerebrospinal fluid (Table II). ASase activity in erythrocytes and hepatic tissue was also measured (Table III). An ammonia tolerance test showed a fasting blood ammonia level of 53 p,g per 100 ml. wi[h a level of 105/zg per 100 ml. two
hours after a 10 Gm. protein meal. A 5 Gm. protein meal resulted in two-hour postprandial blood ammonia level of 86 p,g per cent. An arginine loading test produced no elevation of blood ammonia but a transient rise in plasma and urinary ASA levels was observed. Serial urinary ASA determinations revealed a mean excretion of 8.4 • 1.1 mg. per milliliter or approximately 4 Gm. per 24 hours. Radiographic bone age was compatible with the patient's chronologic age. An electroencephalogram ( E E G ) w a s interpreted as abnormal with poor regulation and diffuse slow activity. A pneumoencephalogram revealed no cortical atrophy. Hair was grossly abnormal in its coarse texture and friability, yet not compatible microscopically with trichorrhexis nodosa. Liver tissue was normal microscopically with abundant glycogen and no detectable fat. Chromosomal analysis of peripheral blood lymphoeytes revealed a normal karyotype (46, XY). Case 2. T. T., the product of her mother's second pregnancy, was evaluated at age 3 years 2 months after her older brother (Case 1) was found to have argininosuccinic aciduria. Her mother had an uncomplicated pregnancy and delivered a female infant weighing 3,500 grams at term. Language skills were delayed although motor milestones were attained normally. T. T. also disliked meat and had had occipital loss of hair since early infancy. Physical examination revealed a quiet Caucasian girl whose height was 95.7 cm., weight 14.4 kilograms, and head circumference 50 cm. (all fiftieth percentile). Pertinent findings included coarse, friable, blond hair with a bald occiput (Fig. 1) and a firm liver margin, palpable 3 cm. below the right costal margin. The neurologic examination revealed no abnormalities except for a mild degree of generalized muscular hypotonia. A battery of psychometric tests revealed a mental age of 2 to 2 years, representing borderline intelligence. Urinary, plasma, and cerebrospinal fluid levels of ASA were elevated (Table II) and tissue ASase activity was reduced (Table liD. An ammonia tolerance test showed a fasting blood ammonia level of 100/xg per 100 ml. with an increase to 110/xg per 100 ml., two hours after a 10 Gm. protein meal. Citrulline and arginine loading tests produced no elevation of blood ammonia
Volume84 Number 1
A t~ininosuccinic aciduria
87
Table III. Erythrocyte and liver e n z y m e activity
Liver
Erythrocyte Subject
W. T. (ASA) T. T. (ASA) M.T.* Father Mother Control
ASase (Izmole urea/Gm. Hb./hr.)
A rginase (txmole urea/Gm. Hb./hr.)
0 0 2.32 5.23 5.93 11.7
231 293 344 423 381 447
ASase (tzmole urea/mg. protein~hr.) 0
0
(I-tmole urea~rag. protein~hr.) 0.111 0,0137
*Assays performed on cord blood.
levels in this subject. Serial urinary ASA determinations revelaed mean excretion of 10.1 • 1.9 rag. per milliliter, approximating 4 Gin. daily. An EEG was abnormal with diffuse irregular and slow activity indicative of widespread encephalopathy. Chromosomal analysis of peripheral blood lymphocytes revealed a normal karyotype (46, XX). Case 3, The mother of these two children conceived for the third time, although she and her husband had been counseled as to the mode of inheritance. An amniocentesis performed during the fifteenth week of gestation yielded fetal ceils which revealed an apparently normal karyotype, 46, XX, and female sex was predicted. ASase values using fetal cells were compatible with enzyme activity of the heterozygous state. 8 This pregnancy was Uncomplicated and terminated in the birth of a healthy female infant, M. T., weighing 3,750 grams. Arginase and ASase activity in the cord blood confirmed the prenatal diagnosis of the heterozygous state for this child (Table liD. Subsequent determinations on blood and urine were negative for ASA (Table II). FAMILY
INVESTIGATION
A family survey for additional affected m e m b e r s yielded n o n e 'with urine positive for ASA. Both parents were studied for erythrocyte ASase activity; each was heterozygous for the disorder (Fig. 2 and Table III). Subs e q u e n t assays o f skin fibroblast ASase in W. T., T. T., and the parents have revealed activity comparable with that o f their red blood cells. 8 DISCUSSION T h e initial report of argininosuccinic aciduria by Allan and colleagues 9 in 1958 described two mentally retarded siblings with similar facies, friable hair, and abnormal E E G ' s . The older sibling also exhibited a severe
Fig. 1. Case 2 (T. T.). Note balding occiput. convulsive disorder and m a r k e d ataxia. Laboratory investigation revealed urinary excretion of an u n k n o w n c o m p o u n d later identified as argininosuccinic acid (ASA). T h e concentration of A S A in cerebrospinal fluid was found to be three times that of the plasma. 10 At least twenty cases o f argininosuccinic aciduria h a v e ,. b e e n r e p o r t e d s u b s e q u e n t l y with considerable variability in clinical and biochemical manifestations.
88
The Journal of Pediatrics January 1974
Billmeier et al.
II
Argininosuccinic aciduria ASA heterozygote
~]
"Slow speech"
?
I.
II.
III. WT
TT
MT
Fig. 2. Pedigree of T. family. Clinical manifestations. Nearly all such patients exhibited mental subnormality with a mean I.Q. of 51 in those subjects testable or moderate mental retardation according to standard intelligence levels. An exception to this clinical feature is reported by Carson and Neill H who described a 4-year-old girl with argininosuccinic aciduria and a measured I.Q of 92. Coarse, friable hair was the only physical abnormality. Psychometric testing in our patients revealed mild to borderline mental subnormality, which seems to parallel their relatively benign courses. As in o u r patients, delayed acquisition of speech and language was a major presenting problem in several cases reported earlier. This seems to represent language delay consistent with over-all intellectual deficit, however, rather than a specific language disorder. Abnormal hair, described as coarse, brittle, and easily broken, was noted in the majority of these patients. However, several reported patients had normal hair texture and distribution. The classical trichorrhexis nodosa associated with argininosuccinic aciduria is thought to be due to depletion of arginine from the usual components of normal hair keratin. In support of this hypothesis is the improvement of hair appearance in these patients after arginine dietary supplementation. Our patients have grossly abnormal hair patterns with occipital balding and very coarse, brittle texture, yet microscopic studies are not compatible with trichorrhexis nodosa.
Generalized seizures were present in most previously reported patients and E E G patterns typically showed diffuse, marked slowing with two to three per second spike and slow wave discharge. This pattern has been described in phenylketonuria 12and appears to represent a diffuse.metabolic encephatopathy. Our patients had c o m p a r a b l e E E G p a t t e r n s , although neither has developed a convulsive disorder. Both, however, exhibit generalized hypotonia which appears to be of cerebral origin. Hepatic dysfunction as reflected in elevated serum alkaline phosphatase and hepatic enzyme occurred in two thirds of previously reported cases. Jaundice has not been described in this disorder and hepatomegaly, which occurred in one fourth o f prior cases, was often transient. Hepatomegaly in T. T. was not associated with other clinical signs of liver disease, and liver architecture appeared normal microscopically, except for vascular congestion in our patients as well as those previously studied. Biochemisty. All affected patients excrete large amounts of argininosuccinic acid in urine, the mean excretion rate approximating 3.5 to 4.0 Gm. per day. The previous observation of heterozygous individuals excreting trace amounts of ASA in urinO ~ was not demonstrated in the present study. The enzyme defect in this disorder has been demonstrated in blood cell hemolysates which typically lack ASase activity in homozygous i n d i v i d f l a l s a n d s h o w r e d u c e d a c t i v i t y in t h e heterozygous state] Such a defect was confirmed in the erythrocytes and hepatic cells of our patients. Reduced ASase activity was also demonstrated in the erythrocytes of their parents. Another human cell type, the skin fibroblast, has been shown to be deficient in ASase activity in this disease despite the presence of essential enzymes such as arginase. 8 Absent or extremely low ASase activity in erythrocytes, liver cells, and skin fibroblasts, as well as brain tissue, L3,~4casts serious doubt upon the existence of isozymes in the disorder. G e n e t i c s . T h e e v i d e n c e suggests an a u t o s o m a l recessive mode of inheritance; consanguineous marriages in such pedigrees plus single generations with affected siblings support this assumption. Ethnic background in this disease is variable, with cases reported in Jewish, Sicilian and Irish people. Detection of the heterozygous state is not practical by urine screening tests but is possible by erythrocyte ASase determination as demonstrated in this report. D i e t o t h e r a p y . Basic a p p r o a c h e s to t h e r a p y in argininosuccinic aciduria are: (1) to minimize hyperammonemia, (2) to prevent arginine deficiency, and (3) to p r o v i d e sufficient nutrients for n o r m a l growth.
Volume 84 Number 1
Arginine supplementation has failed to improve the over-all clinical course but normal hair texture may be restored. 15 The administration of citric acid has been shown to reduce the degree of hyperammonernia following a protein meal, apparently through the enhancement of the tricarboxylic acid cycle to accomodate excess ammoniaJ 5In the present study an effort was made to provide sufficient protein for growth yet not induce hyperammonemia by excess of protein. A diet consisting of 1 Gin. of protein per kilogram of body weight daily divided into four feedings was prescribed in addition to arginine and citric acid supplementation. It is interesting that calculation of the children's pretreatment diets revealed relatively low protein content, possibly a reflection of their strong dislike for meats, which may be a self-regulatory mechanism. The children are being followed by an interdisciplinary team for analysis of growth and development. The third sibling in this report, M. T., is thriving on a regular diet and exhibits normal development at the time of this writing. In her case, amniocentesis and subsequent identification as a heterozygote for argininosuccinic aciduria underscore the reality of prenatal diagnosis in the area of inborn errors of metabolism. This useful tool in conjunction with knowledgeable genetic counseling is essential for proper management of such families. We are indebted to Arthur Fort, M.D., University of Tennessee Department of Obstetrics and Gynecology for performing the amniocentesis and to J. W. Littlefield, M.D., and associates, Boston, Mass., for enzyme determinations in fetal cells and skin fibroblasts. We also thank George McCormick, M.D., University of Tennessee Department of Pathology, and E. W. Rosenberg, M.D., University of Tennessee Department of Dermatology, for their consultations, and Mrs. Connie Gray for her technical assistance. Psychometric data were provided by members of the University of Tennessee Child Development Center Physchology Department, to whom we are grateful. REFERENCES 1. Brown, G. W., Jr., and Cohen, P. O.: Comparative biochemistry of urea synthesis. I. Methods for the quan-
,4 t~ininosuccinic aciduria
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