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Atypical nonketotic hyperglycinemia confirmed by assay of the glycine cleavage system in lymphoblasts
10 0
Christodoulou et al.
The Journal of Pediatrics July 1993
Atypical nonketotic hyperglycinemia confirmed by assay of the glycine cleavage system in lymphoblasts John C h r i s t o d o u l o u , MB,BS, PhD, FRACP, S h i g e o Kure, MD, Kiyoshi H a y a s a k a , MD, a n d J o e T. R. C l a r k e , MD, PhD, FRCP(C) From the Division of Clinical Genetics, Hospital for Sick Children, Toronto, Ontario, Canada, the Department of Biochemical Genetics, Tohoku University School of Medicine, Sendal, Japan, and the Department of Pediatrics, Akita University School of Medicine, Hondo, Akita, Japan
A 13-year old girl with early-onset, mild, slowly progressive mental retardation caused by nonketotic hyperglycinemia is described. The plasma and cerebrospinal fluid glycine concentrations were elevated, but the cerebrospinal fluid/plasma glycine ratio was only mildly elevated. The diagnosis was confirmed by demonstration of a detect in the activity of the glycine c l e a v a g e system in cultured lymphoblasts. (J PEDIATR1993;123:100-2)
Nonketotic byperglycinemia is an inherited metabolic disorder of glycine catabolism caused by a defect of the glycine cleavage system] a mitochondrial enzyme system composed of four enzymes, the P-, H-, T-, and L-proteins. 2 Typically, the disease has its onset in the newborn period as a rapidly progressive disorder characterized by profound hypotonia, obtundation, seizures, and apnea, which is fatal without resuscitative treatment. 3 Survivors usually have profound intellectual handicap and seizures that are difficult to control. A late infantile form has been described with a rapidly progressive course similar to that of the early infantile form, but with onset later in the first year of life. 4, 5 Other variants with variable clinical features have been described, including an early-onset but slowly progressive neurologic disorder with moderate to severe intellectual handicap, with or without seizures, 6, 7 and a late-onset, slowly progressive course marked by spinocerebellar degeneration.8, 9 The diagnosis of variant forms of the disease is often difficult because the enzyme defect is not expressed in cultured skin fibroblasts. W e report here a patient with the early-onset, slowly progressive form of N K H in whom the diagnosis was con-
Submitted for publication Dec. 29, 1992; accepted March 5, 1993. Reprint requests: John Christodoulou, PhD, Department of Paediatrics and Child Health, Children's Hospital, Pyrmont Bridge Rd., Camperdown, New South Wales, 2050, Australia. Copyright | 1993 by Mosby-Year Book, Inc. 0022-3476/93/$1.00 + .10 9/22/46979
firmed by assay of the GCS in cultured transformed lymphoblasts. CASE REPORT
The patient is the second child of nonconsanguineous parents of Polish and British origin. She has an older brother who had seizures as an infant but is now clinically normal, and a younger brother who is also normal. The pregnancy was complicated by raised maternal blood pressure requiring no specific treatment. Labor was induced at term; the delivery was otherwise uncomplicated. Birth weight was 2900 gin. There were no problems in the perinatal period. She was described by her mother as a lethargic baby, although she fed well during infancy and childhood. Early developmental milestones were reported to be normal. At 12 months of age the patient required hospitalization for intravenous therapy because of gastroenteritis. At that time she was
[
GCS NKH
Glycine cleavage system Nonketotic hyperglycinemia
I
noted to have episodes of flaccidity and depressed level of consciousness, thought to be encephalitic in nature. She had hypotonia and poor fine and gross motor skills. A urine amino acid screen showed elevations of glycine and histidine; plasma amino acid measurements showed elevations of glycine, lysine, and alanine. The cerebrospinal fluid had no leukocytes, normal protein and glucose levels, and no pathogens on bacterial and viral culture. An electroencephalogram was normal. No diagnosis was made at that time. The patient's general health has been good. She had thelarche at age 10 years and menarche at age 11 years. Development was
The Journal o f Pediatrics Volume 123, Number 1
delayed, and she has had several episodes of visual hallucinations during illnesses associated with high fever. She also has had episodes of temper tantrums during which she shouted and acted irrationally. Currently, at the age of 13 years, she is in a special education class. She recognizes single words but does not read well. She can understand and carry out simple commands but has difficulty with verbal expression. She is unable to manage money or public transportation on her own. There is no history of developmental regression, nor have any seizures been recognized. When examined at 13 years of age, the patient was a healthylooking girl, with height and head circumference between the 25th and 50th percentiles and weight at the 50th percentile. There were no dysmorphic features. General physical examination findings were normal. Neurologic examination showed that she was alert and cooperative, though she had obvious difficulties with verbal expression. Cranial nerves and ophthalmoscopy findings were normal. Peripheral muscle tone was mildly increased. Muscle bulk and power were normal. Deep tendon reflexes were brisk, and the plantar reflexes were flexor bilaterally. There were two beats of ankle clonus bilaterally. Station, gait and results of cerebellar testing were normal. A complete blood cell count, venous blood gas values, serum electrolytes, and urea, creatinine, liver enzyme, calcium, magnesium, lactate, and ammonium concentrations were all normal. Analysis of urinary organic acids and screening tests for mucopolysacchariduria and oligosacchariduria showed normal results. Urinary amino acid analysis revealed an elevation of glycine (2524 mmol/mol creatinine; normal, <761). Quantitation of plasma amino acids on three occasions showed a consistent isolated elevation of glycine (991 to 1280 gmol/L; normal range, 120 to 553). In addition, the CSF glycine was elevated at 41 gmol/L (normal range, 4 to 18), giving a CSF/plasma glycine ratio of 0.041 (normal, <0.031~ The CSF glucose and protein values were normal. An electroencephalogram and computed tomographic scan of the brain were both normal. Analysis of the glycine cleavage system in Epstein-Barr virustransformed lymphoblasts was performed as previously described.11 Overall activity of the GCS, assayed by determining the rate of ~4CO2 from [ 1-14C]glycine, showed a total activity of the GCS of 0.22 nmol/mg per hour (normal range, 0.61 to 1.6). Activity of the P-protein was determined by the exchange reaction between [1-14C]glycine and 14CO2 in the presence of excess amounts of H-protein. The specific activity of the P-protein component was 0.19 nmol/mg per hour (normal, 0.87 to 1.5). DISCUSSION The clinical features of this patient are compatible with previously reported cases of early-onset, slowly progressive NKH.6, 7 In these patients the onset of symptoms was in early infancy, with developmental delay giving way to moderate to profound intellectual handicap, mild upper motor neuron signs, poor speech acquisition, aggressive or
Christodoulou et al.
10 1
self-mutilatory behavior, and, less consistently, with seizures having an onset in early childhood. The C S F / p l a s m a glycine ratio in our patient was lower (0.04) than in previously described patients (range, 0.07 to 0.09), or in patients with the classic infantile form (range, 0.2 to 0.33), 1~ but was higher than that found in patients with late onset (range, 0.022 to 0.039) 8, 9 or in normal adults (<0.03). l~ The diagnosis of N K H in an appropriate clinical setting is usually based on demonstration of an elevated C S F / plasma glycine ratio in an individual with normal urinary organic acids. Until recently, enzymatic confirmation of the diagnosis relied on assays of the GCS in liver samples, but it has been demonstrated recently that the GCS can be assayed reliably in Epstein-Barr virus-transformed lymphoblasts. 11 Moreover, patients with the classic infantile form of N K H have a profound deficiency of the GCS in cultured lymphoblasts, whereas their heterozygous parents have intermediate levels of activity. The demonstration in our patient of deficient activities of both the total G C S (24% of control mean) and the P-protein (17% of control mean) in cultured lymphoblasts confirmed that this girl has N K H . These activities are higher than those observed in infants with the severe infantile form of N K H but lower than the activities in these infants' heterozygous parents, 11 suggesting that residual activity in cultured lymphoblasts may be of prognostic value. To our knowledge, this is the first time that cultured lymphoblasts have been used to confirm the diagnosis of atypical N K H . We recommend that all individuals with intellectual handicap should have plasma amino acids quantified. If the plasma glycine concentration is elevated and urinary organic acids are normal, and if the C S F glycine level is also raised, the diagnosis of N K H can be confirmed noninvasively by using cultured lymphoblasts.
REFERENCES
1. Tada K, Narisawa K, Yoshida T, et al. Hyperglycinemia: a defect in glycine cleavage reaction. Tohoku J Exp Med 1969; 98:289-96. 2. Kikuchi G. The glycine cleavage system: composition, reaction mechanism, physiological significance. Mol Cell Biochem 1973; 1:169-87. 3. Nyhan WL. Nonketotic hyperglycinemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease. New York: McGraw-Hill, 1989;74353. 4. Trauner DA, Page T, Greco C, et al. Progressive neurodegenerative disorder in a patient with nonketotic hyperglycinemia. J PEDIATR 1981;98:272-5. 5. Haan EA, Kirby DM, Tada K, et al. Difficulties in assessing
10 2
Christodoulou et al.
The Journal of Pediatrics July 1993
the effect of strychnine on the outcome of nonketotic hyperglycinemia: observations on sisters with a mild T-protein defect. Eur J Pediatr 1986;145:267-70. 6. Flannery DB, Pellock J, Bousounis D, et al. Nonketotic hyperglycinemia in two retarded adults: a mild form of infantile nonketotic hyperglycinemia. Neurology 1983;33:1064-6. 7. Singer HS, Valle D, Hayasaka K, Tada K. Nonketotic hyperglycinemia: studies in an atypical variant. Neurology 1989; 39:286-8. 8. Bank W J, Morrow G. A familial spinal cord disorder with hyperglycinemia. Arch Neurol 1972;27:136-44.
9. Steiman GS, Yudkoff M, Bearman PH, et al. Late-onset nonketotic hyperglycinemia and spinocerebellar degeneration. J PEDIATR 1979;94:907-11. 10. Perry TL~ Uruquhart N, MacLean J, et aL Nonketotic hyperglycinemia: glycine accumulation due to absence of glycine cleavage in the brain. N Engl J Med 1975;292:126973. 11. Kure S, Narisawa K, Tada K. Enzymatic diagnosis of nonketotic hyperglycinemia with lymphoblasts. J PEDIA1"R 1992; 120:95-8.
BOUND VOLUMES AVAILABLE TO SUBSCRIBERS Bound volumes of the 1993 issues of ThE JOURNAL OF PEDIATRICS are available to subscribers (only) from the Publisher, at a cost of $63.50 for domestic, $83.96 for Canadian, and $79.50 for international subscribers, for Vol. 122 (January-June) and Vol. 123 (July-December), shipping charges included. Each bound volume contains subject and author indexes, and all advertising is removed. Copies are shipped within 60 days after publication of the last issue in the volume. The binding is durable buckram, with the Journal name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact Mosby, Subscription Services, 11830 Westline Industrial Dr., St. Louis, M O 63146-3318, USA/800-453-4351, or 314-453-4351. Subscriptions must be in force to qualify. Bound volumes are not available in place of a regular Journal subscription.
Christodoulou et al.
The Journal of Pediatrics July 1993
Atypical nonketotic hyperglycinemia confirmed by assay of the glycine cleavage system in lymphoblasts John C h r i s t o d o u l o u , MB,BS, PhD, FRACP, S h i g e o Kure, MD, Kiyoshi H a y a s a k a , MD, a n d J o e T. R. C l a r k e , MD, PhD, FRCP(C) From the Division of Clinical Genetics, Hospital for Sick Children, Toronto, Ontario, Canada, the Department of Biochemical Genetics, Tohoku University School of Medicine, Sendal, Japan, and the Department of Pediatrics, Akita University School of Medicine, Hondo, Akita, Japan
A 13-year old girl with early-onset, mild, slowly progressive mental retardation caused by nonketotic hyperglycinemia is described. The plasma and cerebrospinal fluid glycine concentrations were elevated, but the cerebrospinal fluid/plasma glycine ratio was only mildly elevated. The diagnosis was confirmed by demonstration of a detect in the activity of the glycine c l e a v a g e system in cultured lymphoblasts. (J PEDIATR1993;123:100-2)
Nonketotic byperglycinemia is an inherited metabolic disorder of glycine catabolism caused by a defect of the glycine cleavage system] a mitochondrial enzyme system composed of four enzymes, the P-, H-, T-, and L-proteins. 2 Typically, the disease has its onset in the newborn period as a rapidly progressive disorder characterized by profound hypotonia, obtundation, seizures, and apnea, which is fatal without resuscitative treatment. 3 Survivors usually have profound intellectual handicap and seizures that are difficult to control. A late infantile form has been described with a rapidly progressive course similar to that of the early infantile form, but with onset later in the first year of life. 4, 5 Other variants with variable clinical features have been described, including an early-onset but slowly progressive neurologic disorder with moderate to severe intellectual handicap, with or without seizures, 6, 7 and a late-onset, slowly progressive course marked by spinocerebellar degeneration.8, 9 The diagnosis of variant forms of the disease is often difficult because the enzyme defect is not expressed in cultured skin fibroblasts. W e report here a patient with the early-onset, slowly progressive form of N K H in whom the diagnosis was con-
Submitted for publication Dec. 29, 1992; accepted March 5, 1993. Reprint requests: John Christodoulou, PhD, Department of Paediatrics and Child Health, Children's Hospital, Pyrmont Bridge Rd., Camperdown, New South Wales, 2050, Australia. Copyright | 1993 by Mosby-Year Book, Inc. 0022-3476/93/$1.00 + .10 9/22/46979
firmed by assay of the GCS in cultured transformed lymphoblasts. CASE REPORT
The patient is the second child of nonconsanguineous parents of Polish and British origin. She has an older brother who had seizures as an infant but is now clinically normal, and a younger brother who is also normal. The pregnancy was complicated by raised maternal blood pressure requiring no specific treatment. Labor was induced at term; the delivery was otherwise uncomplicated. Birth weight was 2900 gin. There were no problems in the perinatal period. She was described by her mother as a lethargic baby, although she fed well during infancy and childhood. Early developmental milestones were reported to be normal. At 12 months of age the patient required hospitalization for intravenous therapy because of gastroenteritis. At that time she was
[
GCS NKH
Glycine cleavage system Nonketotic hyperglycinemia
I
noted to have episodes of flaccidity and depressed level of consciousness, thought to be encephalitic in nature. She had hypotonia and poor fine and gross motor skills. A urine amino acid screen showed elevations of glycine and histidine; plasma amino acid measurements showed elevations of glycine, lysine, and alanine. The cerebrospinal fluid had no leukocytes, normal protein and glucose levels, and no pathogens on bacterial and viral culture. An electroencephalogram was normal. No diagnosis was made at that time. The patient's general health has been good. She had thelarche at age 10 years and menarche at age 11 years. Development was
The Journal o f Pediatrics Volume 123, Number 1
delayed, and she has had several episodes of visual hallucinations during illnesses associated with high fever. She also has had episodes of temper tantrums during which she shouted and acted irrationally. Currently, at the age of 13 years, she is in a special education class. She recognizes single words but does not read well. She can understand and carry out simple commands but has difficulty with verbal expression. She is unable to manage money or public transportation on her own. There is no history of developmental regression, nor have any seizures been recognized. When examined at 13 years of age, the patient was a healthylooking girl, with height and head circumference between the 25th and 50th percentiles and weight at the 50th percentile. There were no dysmorphic features. General physical examination findings were normal. Neurologic examination showed that she was alert and cooperative, though she had obvious difficulties with verbal expression. Cranial nerves and ophthalmoscopy findings were normal. Peripheral muscle tone was mildly increased. Muscle bulk and power were normal. Deep tendon reflexes were brisk, and the plantar reflexes were flexor bilaterally. There were two beats of ankle clonus bilaterally. Station, gait and results of cerebellar testing were normal. A complete blood cell count, venous blood gas values, serum electrolytes, and urea, creatinine, liver enzyme, calcium, magnesium, lactate, and ammonium concentrations were all normal. Analysis of urinary organic acids and screening tests for mucopolysacchariduria and oligosacchariduria showed normal results. Urinary amino acid analysis revealed an elevation of glycine (2524 mmol/mol creatinine; normal, <761). Quantitation of plasma amino acids on three occasions showed a consistent isolated elevation of glycine (991 to 1280 gmol/L; normal range, 120 to 553). In addition, the CSF glycine was elevated at 41 gmol/L (normal range, 4 to 18), giving a CSF/plasma glycine ratio of 0.041 (normal, <0.031~ The CSF glucose and protein values were normal. An electroencephalogram and computed tomographic scan of the brain were both normal. Analysis of the glycine cleavage system in Epstein-Barr virustransformed lymphoblasts was performed as previously described.11 Overall activity of the GCS, assayed by determining the rate of ~4CO2 from [ 1-14C]glycine, showed a total activity of the GCS of 0.22 nmol/mg per hour (normal range, 0.61 to 1.6). Activity of the P-protein was determined by the exchange reaction between [1-14C]glycine and 14CO2 in the presence of excess amounts of H-protein. The specific activity of the P-protein component was 0.19 nmol/mg per hour (normal, 0.87 to 1.5). DISCUSSION The clinical features of this patient are compatible with previously reported cases of early-onset, slowly progressive NKH.6, 7 In these patients the onset of symptoms was in early infancy, with developmental delay giving way to moderate to profound intellectual handicap, mild upper motor neuron signs, poor speech acquisition, aggressive or
Christodoulou et al.
10 1
self-mutilatory behavior, and, less consistently, with seizures having an onset in early childhood. The C S F / p l a s m a glycine ratio in our patient was lower (0.04) than in previously described patients (range, 0.07 to 0.09), or in patients with the classic infantile form (range, 0.2 to 0.33), 1~ but was higher than that found in patients with late onset (range, 0.022 to 0.039) 8, 9 or in normal adults (<0.03). l~ The diagnosis of N K H in an appropriate clinical setting is usually based on demonstration of an elevated C S F / plasma glycine ratio in an individual with normal urinary organic acids. Until recently, enzymatic confirmation of the diagnosis relied on assays of the GCS in liver samples, but it has been demonstrated recently that the GCS can be assayed reliably in Epstein-Barr virus-transformed lymphoblasts. 11 Moreover, patients with the classic infantile form of N K H have a profound deficiency of the GCS in cultured lymphoblasts, whereas their heterozygous parents have intermediate levels of activity. The demonstration in our patient of deficient activities of both the total G C S (24% of control mean) and the P-protein (17% of control mean) in cultured lymphoblasts confirmed that this girl has N K H . These activities are higher than those observed in infants with the severe infantile form of N K H but lower than the activities in these infants' heterozygous parents, 11 suggesting that residual activity in cultured lymphoblasts may be of prognostic value. To our knowledge, this is the first time that cultured lymphoblasts have been used to confirm the diagnosis of atypical N K H . We recommend that all individuals with intellectual handicap should have plasma amino acids quantified. If the plasma glycine concentration is elevated and urinary organic acids are normal, and if the C S F glycine level is also raised, the diagnosis of N K H can be confirmed noninvasively by using cultured lymphoblasts.
REFERENCES
1. Tada K, Narisawa K, Yoshida T, et al. Hyperglycinemia: a defect in glycine cleavage reaction. Tohoku J Exp Med 1969; 98:289-96. 2. Kikuchi G. The glycine cleavage system: composition, reaction mechanism, physiological significance. Mol Cell Biochem 1973; 1:169-87. 3. Nyhan WL. Nonketotic hyperglycinemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease. New York: McGraw-Hill, 1989;74353. 4. Trauner DA, Page T, Greco C, et al. Progressive neurodegenerative disorder in a patient with nonketotic hyperglycinemia. J PEDIATR 1981;98:272-5. 5. Haan EA, Kirby DM, Tada K, et al. Difficulties in assessing
10 2
Christodoulou et al.
The Journal of Pediatrics July 1993
the effect of strychnine on the outcome of nonketotic hyperglycinemia: observations on sisters with a mild T-protein defect. Eur J Pediatr 1986;145:267-70. 6. Flannery DB, Pellock J, Bousounis D, et al. Nonketotic hyperglycinemia in two retarded adults: a mild form of infantile nonketotic hyperglycinemia. Neurology 1983;33:1064-6. 7. Singer HS, Valle D, Hayasaka K, Tada K. Nonketotic hyperglycinemia: studies in an atypical variant. Neurology 1989; 39:286-8. 8. Bank W J, Morrow G. A familial spinal cord disorder with hyperglycinemia. Arch Neurol 1972;27:136-44.
9. Steiman GS, Yudkoff M, Bearman PH, et al. Late-onset nonketotic hyperglycinemia and spinocerebellar degeneration. J PEDIATR 1979;94:907-11. 10. Perry TL~ Uruquhart N, MacLean J, et aL Nonketotic hyperglycinemia: glycine accumulation due to absence of glycine cleavage in the brain. N Engl J Med 1975;292:126973. 11. Kure S, Narisawa K, Tada K. Enzymatic diagnosis of nonketotic hyperglycinemia with lymphoblasts. J PEDIA1"R 1992; 120:95-8.
BOUND VOLUMES AVAILABLE TO SUBSCRIBERS Bound volumes of the 1993 issues of ThE JOURNAL OF PEDIATRICS are available to subscribers (only) from the Publisher, at a cost of $63.50 for domestic, $83.96 for Canadian, and $79.50 for international subscribers, for Vol. 122 (January-June) and Vol. 123 (July-December), shipping charges included. Each bound volume contains subject and author indexes, and all advertising is removed. Copies are shipped within 60 days after publication of the last issue in the volume. The binding is durable buckram, with the Journal name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact Mosby, Subscription Services, 11830 Westline Industrial Dr., St. Louis, M O 63146-3318, USA/800-453-4351, or 314-453-4351. Subscriptions must be in force to qualify. Bound volumes are not available in place of a regular Journal subscription.