Volume 88 Number 5
2.
3.
4. 5.
6.
Brief clinical and laboratory observations
Bloom SR, Polak JM, and Pearse AGE: Vasoactive intestinal peptide and watery-diarrhea syndrome, Lancet 2:14, 1973. Said SI, and Faloona GR: Elevated plasma and tissue levels of vasoactive intestinal polypeptide in the watery-diarrhea syndrome due to pancreatic, bronchogenic and other tumors, N Engl J Med 293:155, 1975. Verner JV, and Morrison AB: Endocrine pancreatic islet disease with diarrhea, Arch Intern Med 133:492, 1974. Pabst K, Kummerle F, Hennekeuser HH, and Mappes G: Beitrag zum Krankheitsbild des Verner-Morrison-Syndroms, Dtsch Med Wochenschr 94:9, 1969. Miettinen TA: Clinical implications of bile acid metabolism in man, in Nair PP, and Kritchevsky D, editors: The bile acids, chemistry, physiology and metabolism, vol. 2, New York, 1973, Plenum Publishing Corp., p. 191.
Mannosidosis." Clinical and biochemical studies in a family of affected adolescents and adults Carol W. Booth, M.D.,* Kathy K. Chen, and Henry L. Nadler, M.D., Chicago, HI. I N 19 6 7, a new lysosomal storage disorder was described by Ockerman. 1 Clinical manifestations in his patient and in four other children reported since that time were typical of the "Hurler syndrome" and included facial coarsening, corneal opacities, hepatomegaly, l u m b a r gibbus, and early death. ~-~ In two other young boys, mental retardation and deafness have been the only From the Division of Genetics, Children's Memorial Hospital, Department of Pediatrics, Northwestern University Medical School. Supported in part by grants from The National Institutes of Health (HD 00036 and RR 00199), The National Foundation-March of Dimes, and The Kroc Foundation. Presented in part at the annual meeting of The Society for Pediatric Research, Denver, Colorado, April 16-19, 1975. Henry L. Nadler is the Irene Heinz Given and John La Porte Given Research Professor of Pediatrics. Carol W. Booth is the recipient of The National Foundation-March of Dimesfellowship in memory of David Yi-Yung Hsia. *Reprint address: Lutheran General Hospital, 1775 Dempster, Park Ridge, Ill.
821
7.
Elias E, Bloom SR, Welbourn RB, Polak JM, Pearse AGE, Booth CC, and Brown JC: Pancreatic cholera due to production of gastric inhibitory peptide, Lancet 2:791, 1972. 8. Schwartz C J, Kimberg DV, Sheerin HE, Field M, and Said SI: Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa, J Clin Invest 54:536, 1974. 9. Amin K, Walia BNS, and Ghai OP: Small bowel functions and structure in malnourished children, Indian Pediatr 6:67, 1969. 10. Viteri FE, Flores JM, Alvarado J, and Behar M: Intestinal malabsorption in malnourished children before and during recovery: Relation between severity of protein deficiency and the malabsorption process, Am J Digest Dis 18:20I, 1973.
manifestations of this disorder? Biochemically, the disease is characterized by increased urinary excretion ~ ' and tissue accumulation of mannose-containing oligosaccharides, 6 and by deficiency of the lysosomal enzymes, a-mannosidases A and B. 7 On the basis of these findings, the disease has been classified as a mucolipidosis. We have recently seen three siblings, ages 13, 16, and 26 years, with mental retardation, coarse facial features (Fig. 1), and hearing problems. Deficient a-mannosidase activity was demonstrated in white blood cells and cultivated skin fibroblasts from all three patients. This sibship constitutes the oldest patients yet to be described with mannosidosis.
See related article, p. 814. CASE REPORTS Patients H. D., J. D., and E. D. were seen at ages 26, 16, and 13 years, respectively, for evaluation of familial mental retardation. Patient H. D., the oldest in a sibship of five, was the product of a term, uncomplicated pregnancy. She was considered slow from late infancy-walking at age 2V2 and talking at age 3V2. Chronic serous otitis media was treated in childhood with repeated adenoidectomies, with only minimal improvement. A chronic, sterile, lyric lesion in the left lateral femoral condyle has prevented weight bearing since age 15. At age 18, the patient experienced visual and auditory hallucinations, marked swings in affect, and sleeping disturbances. These symptoms were relieved by phenothiazine therapyl She has lived in a home for the retarded since age 20. Her IQ is estimated at 50. Patient J. D., the fourth in the sibship, was also born after an uncomplicated pregnancy. Congenital dislocation of the left hip was corrected by splinting. She walked at 18 months of age and talked at age 3. An umbilical hernia was repaired at age 2. Audiometry has shown persistent combined conductive and
822
Brief clinical and laboratory observations
The Journal of Pediatrics May 1976
galactosidase, fi-glucuronidase, arylsulfatase A, fi-Nacetylglucosaminidase, and a-glucosidase activities were determined as previously described?. Alpha-mannosidase and a-fucosidase were assayed in 0.1M sodium acetate buffer, pH 4.2, with 1 m M 4-methyl-umbelliferyl m a n n o pyranoside and p-nitrophenyl a-fucopyranoside~as substrates; 15 to 100/zg of protein were used in each assay in a final volume of 0.200 ml. Reactions were stopped by the addition of 0.8 (a-fucosidase) or 1.3 ml 0.2M glycine buffer, pH 10.7. Electrophoresis was performed in 16% starch in 0.1M phosphate buffer, p H 6.0. Voltage was maintained at 125 volts for 16 hours at 4 ~ C. Gels were stained with 1 m M 4-methylumbelliferyl a - m a n n o p y r a noside in sodium acetate buffer, p H 4.5, and developed with 0.2M glycine buffer, p H 10.7. Fluorescence was observed with ultraviolet light. Substrates were obtained from Koch-Light, Ltd. RESULTS
Fig. 1. Patients J, D., H. D., and E. D. described in this report. The facial appearance is coarsened in each patient.
sensorineural hearing loss. She presently hears loud speech with the help of a hearing aid. She has lived in a home for the retarded since age 14. Her IQ is estimated at 65. Patient E. D. was born at term after an internal version and extraction for a transverse lie. He sat at age 15 months and walked at age 4- He has combined conductive and sensorineural hearing loss which is not helped significantly by hearing aids. He has never spoken. Umbilical and inguinal hernias were repaired at age 6. He has lived in a home for the retarded since age 12. His IQ is estimated at 35. All three patients were tall and lax-jointed. The facial appearances were somewhat coarse (Fig. 1). None had corneal or lenficular opacities or abnormalities ot" the fundi. None had hepatosplenomegaly, gibbus formation, or cardiac murmurs. All had long, slender hands. Urinary mucopolysaccharide spot tests were normal in all three. Skull films from all three patients demonstrated a lack of pneumatization of the mastoids and sinuses. In addition, roentgenograms of the two older siblings demonstrated a thickening of the cranial vault with obliteration of the diploic spaces. There was demineralization of the long bones with loss of the trabecular pattern of the medullary cavities. Spine films were not remarkable. MATERIALS
AND METHODS
Skin biopsies were obtained and fibroblasts cultivated from the patients, unaffected siblings, and control patients as previously described. ~ White cell pellets were prepared after 6% dextran sedimentation of whole blood. Cells were lysed in water by five cycles of freezing and thawing. Beta-
Cultivated skin fibroblasts from all three patients showed deficient a-mannosidase activity averaging 3% to 7% of control activity (normal: 73 _+ 20 nmoles product liberated/hr/mg protein) and n o r m a l amounts of all other lysosomal enzymes assayed. Skin fibroblasts from both parents and both unaffected sibs had less than 50% of control a-mannosidase activity with no overlap with control subjects or affected patients. White blood cells obtained from the patients had 2, 4, and 2 units of amannosidase activity/mg protein (control: 65 to 120 units) and 188, 212, and 196 units offl-galactosidase activity/rag protein (control: 92 to 214 units). Starch gel electrophoresis revealed two b a n d s of amannosidase activity from fibroblasts which were similar to those found in normal liver. TM Alpha-mannosidase A obtained by DEAE-cellulose chromatography 7 from either liver or fibroblasts corresponded to the fast band; and a-mannosidase B to the slow band. Lysate from the mutant fibroblasts applied at five times the protein concentration of the control also displayed two bands of activity moving at similar rates to control enzymes (Fig. 2). Presumably, this represents low residual activity of both a-mannosidase isozymes. DISCUSSION The first?five patients described as having mannosidosis demonstrated overt signs of mucopolysaccharide storage and the condition was considered to represent a "Hurler variant." This report adds three patients with few physical findings of mucopolysaccharide or glycolipid storage and no evidence of neurologic deterioration. Our patients' physical app6arances are typical of m a n y persons with
Volume 88 Number 5
Brief clinical and laborato~7 observations
823
Fig. 2. Starch gel electrophoresis of crude lysates of control (slots 1 and 3) and mutant (slots 2 and 4) fibroblasts. Both preparations showed two bands of activity when stained with 4-methylumbelliferyl e~-mannopyranoside at pH 4.5. Similar results were obtained when a normal liver homogenate was applied to the gel.
simple unclassified retardation. Coarsening of the facies is a common finding in the retarded which has been attributed in the past to anticonvulsant therapy, 11 trauma, a n d u n k n o w n factors as well as to specific disorders. Mannosidosis, as represented by this sibship, may be more common than the paucity of reported cases would suggest. Although the previously reported cases are all children, the relatively benign course in our patients a n d others reported to date ~ should make this disorder a diagnostic consideration in the retarded adult. Skull films from all three patients demonstrated abnormal findings. Pneumatization of the mastoids and sinuses was not seen in any of our patients. In addition, roentgenograms of the two older siblings showed thickening of the cranial vault with obliteration of the diploic spaces. Thickening of the cranial vault is an u n u s u a l finding which has, however, been reported in one other patient with mannosidosis? Acid a-mannosidase activity could be separated into two bands when subjected to electrophoresis on starch. These bands had mobilities identical to h u m a n liver amannosidases A and B separated by D E A E cellulose chromatography. Taylor and associates 12 have recently reported a deficiency Of a single a-mannosidase isozyme
in cultivated skin fibroblasts from two patients with mannosidosis. The differences in electrophoretic patterns between these reports could be explained in at least two ways. The systems used for electrophoresis were different and may not separate mannosidase isozymes in a similar manner. Alternatively, Taylor and associates 1'-' did not describe the patients from whom skin biopsies were derived; the description of the disease as "Hurler-like" suggests that they were children. The precise gene defect and the pattern of residual enzyme activity may vary in phenotypically different patients with this disorder and most probably represents another example of genetic heterogeneity. REFERENCES
1. ()ckerman PA: A generalized storage disease resembling Hurler's syndrome, Lancet 2:239, 1967. 2. Kjellman B, Gamstorp I, Brun I, Ockerman PA, and Palmgren B: Mannosidosis: A clinical and taistopa~hologic study, J PEDIATR.75:366, 1969. 3. Norden NE, Ockerman PA, and Szabo L: Urinary mannosc in mannosidosis, J PEDIATR82:686, 1973. 4. Tsay GC, Dawson G, and Matalon R: Excretion of mannose-rich complex carbohydrates by a patient with c~mannosidase deficiency (mannosidosis), J PeDIATR84:865, 1974.
824
Brief clinical and laboratory observations
5. Autio S, Norden N, Ockerman PA, Riekkinen P, Rapola J, and Louhimo T: Mannosidosis: Clinical, fine-structural and biochemical findings in three cases, Acta Paediatr Scand 62:555, 1973. 6. Ockerman PA: Mannosidosis: Isolation of oligosaccharide storage material from brain, J PEDIATR 75:360, 1969. 7. Carroll N, Dance N, Masson PK, Robinson D, and Winchester BG: Human mannosidosis--the enzymic defect, Biochem Biophys Res Commun 49:579, 1972. 8. Nadler HL, Monteleone PL, Inouye T, and Hsia DY-Y: Enzymes in cultivated fibroblasts derived from patients with Down's syndrome (mongolism), Nature 213:1261, 1967. 9. Melancon SB, and Nadler HL: Prenatal diagnosis of genetic
A variant form of citrullinemia Ichiro Matsuda, M.D., Michiya Anakura, M.D., Sinichiro Arashima, M.D., Yoshiro Saito, M.D., and Yogo Oka, M.D., Sapporo,
Japan M U L T I P L E GENETIC HETEROGENEITY is seen in citrullinemia. T h e clinical variants include: (1) onset in infancy b e t w e e n two a n d 18 m o n t h s o f age with clinical s y m p t o m s o f periodic vomiting, seizure, a n d m e n t a l retardationS; (2) a disease which runs a rapid a n d fatal n e o n a t a l course-' (patients in each of these two groups have elevated s e r u m citrulline a n d a m m o n i a levels); (3) a v a r i a n t w h i c h has lesser elevations of s e r u m citrulline w i t h o u t clinical symptoms3; (4) the variant form r e p o r t e d by E m a k p o r a n d associates with elevated c o n c e n t r a t i o n o f citrulline (100-fold values over these of control subjects) a n d a n argininosuccinic acid synthetase deficiency of less t h a n 1% o f controls, but with no h y p e r a m m o n e m i a ? W e describe here a patient w h o h a d clinical s y m p t o m s o f h y p e r a m m o n e m i a , b u t with a lesser increase in serum c o n c e n t r a t i o n of citrulline similar to the third type m e n t i o n e d above. CASE REPORT A female infant, the third offspring of unrelated healthy parents, was born after an uncomplicated pregnancy of 40 weeks with a birth weight of 3,000 gm. Two siblings were healthy. The patient had recurrent vomiting and delayed physical development since early childhood. At seven years of age height was 99 cm (below 0.1 percentile); weight was 13.2 kg (below 0.1
From the Department of Pediatrics, Department of Psychiatry and Neurology, Hokkaido University School of Medicine.
The Journal of Pediatrics May 1976 disorders leading to mental retardation, in Fried R, editor: Methods of neurochemistry, New York, 1973, Marcel Dekker, Inc., pp 1-57. 10. Booth CW, and Nadler HL: Unpublished data. 11. Lefebvre EB, Haining RG, and Labb6 RF: Coarse facies, calvarial thickening and hyperphosphatasia associated with long-term anticonvulsant therapy, N Engl J Med 286:1301, 1972. 12. Taylor HA, Thomas GH, Aylsworth A, Stevenson RE, and Reynolds LN: Mannosidosis: Deficiency of a specific amannosidase component in cultivated fibroblasts, Clin Chim Acta 59:93, 1975.
percentile). She was brought to the hospital at age 13 years because of mental retardation and episodic ataxia, tremor, night terrors, and phychosis. The symptoms had become more pronounced after I0 years of age. Height and weight were 135 cm (below 0.1 percentile) and 21 kg (below 0. i percentile), respectively. Her IQ was 58. Laboratory data included: red blood cell count, 3,860,000/mm ~, white blood cell count, 5,900/mm3; and concentration of serum protein 6.2 gm/dl (albumin 67.2%, al, globulin 2.47%, a2, 14.9%, fi 10.7%, ~', 2.4%); blood urea concentration of 19.2 mg/dl; serum CCLF test negative; SGOT, 38-47.4 units; SGPT, 30 units; total serum bilirubin concentration of 0.4 mg/dl. HB~Ag and antibody were negative. Concentrations of ceruloplasmin and a-fetoprotein in serum were the same as those of a control. Blood ammonia concentration ranged from 420 to 610/~g/dl. Electroencephalogram revealed high-voltage slow waves ranging from 2 to 2.5 cycles per second in all areas. Abbreviations used ASA: argininosuccinic acid CCLF: cephalin-cholesterol-lecithin flocculation test Km: Michaelis constant SGOT: serum glutamic oxaloacetic transaminase SGPT: serum glutamic pyruvic transaminase During hospitalization from 13 to 21 years of age episodes of coma and tremor with elevated concentration of blood ammonia were observed frequently. With restriction of protein in her diet and after administration of lactulose, these symptoms became less frequent and the blood ammonia level was reduced to as low as 180/~g/dl. Recent laboratory data have included SGOT of 32 units, SGPT of 31 units, blood urea nitregen of 13 mg/dl and a negative CCLF. Quantitative estimation of amino acids in plasma by an automatic amino acid analyzer, ~ Hitachi Model KLA 3, demonstrated an increase in concentration of citrulline, with values approximately ten times that of the control (Table I). A portal-systemic shunt was excluded by angiography during laparotomy, and the liver was biopsied for histologic study and assay of enzyme activity. Histology of the liver. There were no severe necrotic lesions in the liver tissue. There was an irregular increase of connective
2.
3.
4. 5.
6.
Brief clinical and laboratory observations
Bloom SR, Polak JM, and Pearse AGE: Vasoactive intestinal peptide and watery-diarrhea syndrome, Lancet 2:14, 1973. Said SI, and Faloona GR: Elevated plasma and tissue levels of vasoactive intestinal polypeptide in the watery-diarrhea syndrome due to pancreatic, bronchogenic and other tumors, N Engl J Med 293:155, 1975. Verner JV, and Morrison AB: Endocrine pancreatic islet disease with diarrhea, Arch Intern Med 133:492, 1974. Pabst K, Kummerle F, Hennekeuser HH, and Mappes G: Beitrag zum Krankheitsbild des Verner-Morrison-Syndroms, Dtsch Med Wochenschr 94:9, 1969. Miettinen TA: Clinical implications of bile acid metabolism in man, in Nair PP, and Kritchevsky D, editors: The bile acids, chemistry, physiology and metabolism, vol. 2, New York, 1973, Plenum Publishing Corp., p. 191.
Mannosidosis." Clinical and biochemical studies in a family of affected adolescents and adults Carol W. Booth, M.D.,* Kathy K. Chen, and Henry L. Nadler, M.D., Chicago, HI. I N 19 6 7, a new lysosomal storage disorder was described by Ockerman. 1 Clinical manifestations in his patient and in four other children reported since that time were typical of the "Hurler syndrome" and included facial coarsening, corneal opacities, hepatomegaly, l u m b a r gibbus, and early death. ~-~ In two other young boys, mental retardation and deafness have been the only From the Division of Genetics, Children's Memorial Hospital, Department of Pediatrics, Northwestern University Medical School. Supported in part by grants from The National Institutes of Health (HD 00036 and RR 00199), The National Foundation-March of Dimes, and The Kroc Foundation. Presented in part at the annual meeting of The Society for Pediatric Research, Denver, Colorado, April 16-19, 1975. Henry L. Nadler is the Irene Heinz Given and John La Porte Given Research Professor of Pediatrics. Carol W. Booth is the recipient of The National Foundation-March of Dimesfellowship in memory of David Yi-Yung Hsia. *Reprint address: Lutheran General Hospital, 1775 Dempster, Park Ridge, Ill.
821
7.
Elias E, Bloom SR, Welbourn RB, Polak JM, Pearse AGE, Booth CC, and Brown JC: Pancreatic cholera due to production of gastric inhibitory peptide, Lancet 2:791, 1972. 8. Schwartz C J, Kimberg DV, Sheerin HE, Field M, and Said SI: Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa, J Clin Invest 54:536, 1974. 9. Amin K, Walia BNS, and Ghai OP: Small bowel functions and structure in malnourished children, Indian Pediatr 6:67, 1969. 10. Viteri FE, Flores JM, Alvarado J, and Behar M: Intestinal malabsorption in malnourished children before and during recovery: Relation between severity of protein deficiency and the malabsorption process, Am J Digest Dis 18:20I, 1973.
manifestations of this disorder? Biochemically, the disease is characterized by increased urinary excretion ~ ' and tissue accumulation of mannose-containing oligosaccharides, 6 and by deficiency of the lysosomal enzymes, a-mannosidases A and B. 7 On the basis of these findings, the disease has been classified as a mucolipidosis. We have recently seen three siblings, ages 13, 16, and 26 years, with mental retardation, coarse facial features (Fig. 1), and hearing problems. Deficient a-mannosidase activity was demonstrated in white blood cells and cultivated skin fibroblasts from all three patients. This sibship constitutes the oldest patients yet to be described with mannosidosis.
See related article, p. 814. CASE REPORTS Patients H. D., J. D., and E. D. were seen at ages 26, 16, and 13 years, respectively, for evaluation of familial mental retardation. Patient H. D., the oldest in a sibship of five, was the product of a term, uncomplicated pregnancy. She was considered slow from late infancy-walking at age 2V2 and talking at age 3V2. Chronic serous otitis media was treated in childhood with repeated adenoidectomies, with only minimal improvement. A chronic, sterile, lyric lesion in the left lateral femoral condyle has prevented weight bearing since age 15. At age 18, the patient experienced visual and auditory hallucinations, marked swings in affect, and sleeping disturbances. These symptoms were relieved by phenothiazine therapyl She has lived in a home for the retarded since age 20. Her IQ is estimated at 50. Patient J. D., the fourth in the sibship, was also born after an uncomplicated pregnancy. Congenital dislocation of the left hip was corrected by splinting. She walked at 18 months of age and talked at age 3. An umbilical hernia was repaired at age 2. Audiometry has shown persistent combined conductive and
822
Brief clinical and laboratory observations
The Journal of Pediatrics May 1976
galactosidase, fi-glucuronidase, arylsulfatase A, fi-Nacetylglucosaminidase, and a-glucosidase activities were determined as previously described?. Alpha-mannosidase and a-fucosidase were assayed in 0.1M sodium acetate buffer, pH 4.2, with 1 m M 4-methyl-umbelliferyl m a n n o pyranoside and p-nitrophenyl a-fucopyranoside~as substrates; 15 to 100/zg of protein were used in each assay in a final volume of 0.200 ml. Reactions were stopped by the addition of 0.8 (a-fucosidase) or 1.3 ml 0.2M glycine buffer, pH 10.7. Electrophoresis was performed in 16% starch in 0.1M phosphate buffer, p H 6.0. Voltage was maintained at 125 volts for 16 hours at 4 ~ C. Gels were stained with 1 m M 4-methylumbelliferyl a - m a n n o p y r a noside in sodium acetate buffer, p H 4.5, and developed with 0.2M glycine buffer, p H 10.7. Fluorescence was observed with ultraviolet light. Substrates were obtained from Koch-Light, Ltd. RESULTS
Fig. 1. Patients J, D., H. D., and E. D. described in this report. The facial appearance is coarsened in each patient.
sensorineural hearing loss. She presently hears loud speech with the help of a hearing aid. She has lived in a home for the retarded since age 14. Her IQ is estimated at 65. Patient E. D. was born at term after an internal version and extraction for a transverse lie. He sat at age 15 months and walked at age 4- He has combined conductive and sensorineural hearing loss which is not helped significantly by hearing aids. He has never spoken. Umbilical and inguinal hernias were repaired at age 6. He has lived in a home for the retarded since age 12. His IQ is estimated at 35. All three patients were tall and lax-jointed. The facial appearances were somewhat coarse (Fig. 1). None had corneal or lenficular opacities or abnormalities ot" the fundi. None had hepatosplenomegaly, gibbus formation, or cardiac murmurs. All had long, slender hands. Urinary mucopolysaccharide spot tests were normal in all three. Skull films from all three patients demonstrated a lack of pneumatization of the mastoids and sinuses. In addition, roentgenograms of the two older siblings demonstrated a thickening of the cranial vault with obliteration of the diploic spaces. There was demineralization of the long bones with loss of the trabecular pattern of the medullary cavities. Spine films were not remarkable. MATERIALS
AND METHODS
Skin biopsies were obtained and fibroblasts cultivated from the patients, unaffected siblings, and control patients as previously described. ~ White cell pellets were prepared after 6% dextran sedimentation of whole blood. Cells were lysed in water by five cycles of freezing and thawing. Beta-
Cultivated skin fibroblasts from all three patients showed deficient a-mannosidase activity averaging 3% to 7% of control activity (normal: 73 _+ 20 nmoles product liberated/hr/mg protein) and n o r m a l amounts of all other lysosomal enzymes assayed. Skin fibroblasts from both parents and both unaffected sibs had less than 50% of control a-mannosidase activity with no overlap with control subjects or affected patients. White blood cells obtained from the patients had 2, 4, and 2 units of amannosidase activity/mg protein (control: 65 to 120 units) and 188, 212, and 196 units offl-galactosidase activity/rag protein (control: 92 to 214 units). Starch gel electrophoresis revealed two b a n d s of amannosidase activity from fibroblasts which were similar to those found in normal liver. TM Alpha-mannosidase A obtained by DEAE-cellulose chromatography 7 from either liver or fibroblasts corresponded to the fast band; and a-mannosidase B to the slow band. Lysate from the mutant fibroblasts applied at five times the protein concentration of the control also displayed two bands of activity moving at similar rates to control enzymes (Fig. 2). Presumably, this represents low residual activity of both a-mannosidase isozymes. DISCUSSION The first?five patients described as having mannosidosis demonstrated overt signs of mucopolysaccharide storage and the condition was considered to represent a "Hurler variant." This report adds three patients with few physical findings of mucopolysaccharide or glycolipid storage and no evidence of neurologic deterioration. Our patients' physical app6arances are typical of m a n y persons with
Volume 88 Number 5
Brief clinical and laborato~7 observations
823
Fig. 2. Starch gel electrophoresis of crude lysates of control (slots 1 and 3) and mutant (slots 2 and 4) fibroblasts. Both preparations showed two bands of activity when stained with 4-methylumbelliferyl e~-mannopyranoside at pH 4.5. Similar results were obtained when a normal liver homogenate was applied to the gel.
simple unclassified retardation. Coarsening of the facies is a common finding in the retarded which has been attributed in the past to anticonvulsant therapy, 11 trauma, a n d u n k n o w n factors as well as to specific disorders. Mannosidosis, as represented by this sibship, may be more common than the paucity of reported cases would suggest. Although the previously reported cases are all children, the relatively benign course in our patients a n d others reported to date ~ should make this disorder a diagnostic consideration in the retarded adult. Skull films from all three patients demonstrated abnormal findings. Pneumatization of the mastoids and sinuses was not seen in any of our patients. In addition, roentgenograms of the two older siblings showed thickening of the cranial vault with obliteration of the diploic spaces. Thickening of the cranial vault is an u n u s u a l finding which has, however, been reported in one other patient with mannosidosis? Acid a-mannosidase activity could be separated into two bands when subjected to electrophoresis on starch. These bands had mobilities identical to h u m a n liver amannosidases A and B separated by D E A E cellulose chromatography. Taylor and associates 12 have recently reported a deficiency Of a single a-mannosidase isozyme
in cultivated skin fibroblasts from two patients with mannosidosis. The differences in electrophoretic patterns between these reports could be explained in at least two ways. The systems used for electrophoresis were different and may not separate mannosidase isozymes in a similar manner. Alternatively, Taylor and associates 1'-' did not describe the patients from whom skin biopsies were derived; the description of the disease as "Hurler-like" suggests that they were children. The precise gene defect and the pattern of residual enzyme activity may vary in phenotypically different patients with this disorder and most probably represents another example of genetic heterogeneity. REFERENCES
1. ()ckerman PA: A generalized storage disease resembling Hurler's syndrome, Lancet 2:239, 1967. 2. Kjellman B, Gamstorp I, Brun I, Ockerman PA, and Palmgren B: Mannosidosis: A clinical and taistopa~hologic study, J PEDIATR.75:366, 1969. 3. Norden NE, Ockerman PA, and Szabo L: Urinary mannosc in mannosidosis, J PEDIATR82:686, 1973. 4. Tsay GC, Dawson G, and Matalon R: Excretion of mannose-rich complex carbohydrates by a patient with c~mannosidase deficiency (mannosidosis), J PeDIATR84:865, 1974.
824
Brief clinical and laboratory observations
5. Autio S, Norden N, Ockerman PA, Riekkinen P, Rapola J, and Louhimo T: Mannosidosis: Clinical, fine-structural and biochemical findings in three cases, Acta Paediatr Scand 62:555, 1973. 6. Ockerman PA: Mannosidosis: Isolation of oligosaccharide storage material from brain, J PEDIATR 75:360, 1969. 7. Carroll N, Dance N, Masson PK, Robinson D, and Winchester BG: Human mannosidosis--the enzymic defect, Biochem Biophys Res Commun 49:579, 1972. 8. Nadler HL, Monteleone PL, Inouye T, and Hsia DY-Y: Enzymes in cultivated fibroblasts derived from patients with Down's syndrome (mongolism), Nature 213:1261, 1967. 9. Melancon SB, and Nadler HL: Prenatal diagnosis of genetic
A variant form of citrullinemia Ichiro Matsuda, M.D., Michiya Anakura, M.D., Sinichiro Arashima, M.D., Yoshiro Saito, M.D., and Yogo Oka, M.D., Sapporo,
Japan M U L T I P L E GENETIC HETEROGENEITY is seen in citrullinemia. T h e clinical variants include: (1) onset in infancy b e t w e e n two a n d 18 m o n t h s o f age with clinical s y m p t o m s o f periodic vomiting, seizure, a n d m e n t a l retardationS; (2) a disease which runs a rapid a n d fatal n e o n a t a l course-' (patients in each of these two groups have elevated s e r u m citrulline a n d a m m o n i a levels); (3) a v a r i a n t w h i c h has lesser elevations of s e r u m citrulline w i t h o u t clinical symptoms3; (4) the variant form r e p o r t e d by E m a k p o r a n d associates with elevated c o n c e n t r a t i o n o f citrulline (100-fold values over these of control subjects) a n d a n argininosuccinic acid synthetase deficiency of less t h a n 1% o f controls, but with no h y p e r a m m o n e m i a ? W e describe here a patient w h o h a d clinical s y m p t o m s o f h y p e r a m m o n e m i a , b u t with a lesser increase in serum c o n c e n t r a t i o n of citrulline similar to the third type m e n t i o n e d above. CASE REPORT A female infant, the third offspring of unrelated healthy parents, was born after an uncomplicated pregnancy of 40 weeks with a birth weight of 3,000 gm. Two siblings were healthy. The patient had recurrent vomiting and delayed physical development since early childhood. At seven years of age height was 99 cm (below 0.1 percentile); weight was 13.2 kg (below 0.1
From the Department of Pediatrics, Department of Psychiatry and Neurology, Hokkaido University School of Medicine.
The Journal of Pediatrics May 1976 disorders leading to mental retardation, in Fried R, editor: Methods of neurochemistry, New York, 1973, Marcel Dekker, Inc., pp 1-57. 10. Booth CW, and Nadler HL: Unpublished data. 11. Lefebvre EB, Haining RG, and Labb6 RF: Coarse facies, calvarial thickening and hyperphosphatasia associated with long-term anticonvulsant therapy, N Engl J Med 286:1301, 1972. 12. Taylor HA, Thomas GH, Aylsworth A, Stevenson RE, and Reynolds LN: Mannosidosis: Deficiency of a specific amannosidase component in cultivated fibroblasts, Clin Chim Acta 59:93, 1975.
percentile). She was brought to the hospital at age 13 years because of mental retardation and episodic ataxia, tremor, night terrors, and phychosis. The symptoms had become more pronounced after I0 years of age. Height and weight were 135 cm (below 0.1 percentile) and 21 kg (below 0. i percentile), respectively. Her IQ was 58. Laboratory data included: red blood cell count, 3,860,000/mm ~, white blood cell count, 5,900/mm3; and concentration of serum protein 6.2 gm/dl (albumin 67.2%, al, globulin 2.47%, a2, 14.9%, fi 10.7%, ~', 2.4%); blood urea concentration of 19.2 mg/dl; serum CCLF test negative; SGOT, 38-47.4 units; SGPT, 30 units; total serum bilirubin concentration of 0.4 mg/dl. HB~Ag and antibody were negative. Concentrations of ceruloplasmin and a-fetoprotein in serum were the same as those of a control. Blood ammonia concentration ranged from 420 to 610/~g/dl. Electroencephalogram revealed high-voltage slow waves ranging from 2 to 2.5 cycles per second in all areas. Abbreviations used ASA: argininosuccinic acid CCLF: cephalin-cholesterol-lecithin flocculation test Km: Michaelis constant SGOT: serum glutamic oxaloacetic transaminase SGPT: serum glutamic pyruvic transaminase During hospitalization from 13 to 21 years of age episodes of coma and tremor with elevated concentration of blood ammonia were observed frequently. With restriction of protein in her diet and after administration of lactulose, these symptoms became less frequent and the blood ammonia level was reduced to as low as 180/~g/dl. Recent laboratory data have included SGOT of 32 units, SGPT of 31 units, blood urea nitregen of 13 mg/dl and a negative CCLF. Quantitative estimation of amino acids in plasma by an automatic amino acid analyzer, ~ Hitachi Model KLA 3, demonstrated an increase in concentration of citrulline, with values approximately ten times that of the control (Table I). A portal-systemic shunt was excluded by angiography during laparotomy, and the liver was biopsied for histologic study and assay of enzyme activity. Histology of the liver. There were no severe necrotic lesions in the liver tissue. There was an irregular increase of connective
