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Excretion of mannose-rich complex carbohydrates by a patient with α-mannosidase deficiency (mannosidosis)
Volume 84 Number 6
4. Von Steijnen, C.: Diastrophic dwarfism, Maandschr Kindergeneeskd 36: 188, 1968. 5. Langer, L. O.: Diastrophic dwarfism in early infancy, Am. J. Roentgenol. Radium Ther. Nucl. Med. 89: 399, 1965. 6. Stover, C. N., Hayes, J. %, and Hold, J. F.: Diastrophic
Excretion of mannose-rich complex carbohydrates by a patient with a-mannosidase deficiency (mannosidosis) Grace Chen Tsay, Ph.D., Glyn Dawson, Ph.D.,* and Reuben Matalon, M.D., Chicago, I11.
IN 1967 i3ckerman l described a case of mannosidosis characterized by a deficiency of a-mannosidase A and B activity and a greatly increased level of bound mannose in tissues and urine. Six cases, all European in origin, have now been reported. 2 The disorder is characterized clinically by progressive psychomotor retardation and increasing expression of the stigmas of mucopolysaccharidoses (coarse facial features, skeletal abnormalities, corneal clouding, hepatosplenomegaly, and vacuolated lymphocytes). The disease appears similar to one reported in cattle 3 and is of interest from the point of view of understanding normal giycoprotein metabolism in man. We have recently diagnosed a further case of mannosidosis on the basis Of an a-mannosidase A and B deficiency in cultured h u m a n skin fibroblasts.4 Urine from the homozygote, his parents, and an unaffected elder sibling was examined for glycopeptide and oligosaccharide content. Abnormal amounts of mannose-rich material were detected in the homozygote following fractionation on Sephadex G-50, providing a reliable method for diagnosis. In this report we wish to emphasize that direct measurement of urinary mannose levels may be unreliFrom the Departments of Pediatrics and Biochemistry, Joseph P. Kennedy, Jr., Mental Retardat&n Research Center, University of Chicago. Supported by United States Public Health Service Grants HD-06426, HD-04583, and AM-05996, and RRO0305 from the General Clinical Research Facilities and Resources. Drs. Dawsonand Matalon are Joseph P. Kennedy, Jr., Scholars. *Reprint address:Joseph P. Kennedy, ,It'.,MentalRetardation Research Centel, Universityof Chicago, 950 E. 59th St., Chicago, Ill. 60637.
B r i e f clinical and laboratory observations
865
dwarfism, Am. J. Roentgenol. Radium Ther. Nucl. Med. 89: 914, 1963. 7. Taybi, H.: Diastrophic dwarfism, Radiology 80: 1, 1963. 8. Wilson, D. W., Chrispin, and Carter, C. O.: Diastrophic dwarfism, Arch. Dis. Child. 44: 48, 1969.
able for diagnosis because of the wide variation in the level of excretion of glycoproteins both in normal individuals and in those with other inherited disorders of complex carbohydrate metabolism. MATERIALS
AND METHODS
Fresh samples of urine were collected from normal individuals, the patient and his immediate family, and patients with other inborn errors of carbohydrate metabolism, and frozen in aliquots of 100 ml. Small amounts (0.1 ml.) were evaporated to d r y n e s s and analyzed directly by gas-liquid chromatography of the derived trimethylsilylmethylgtycosides,s Initial purification of the urinary material was achieved by precipitation with Abbreviations used Man: D-rnannose GIcNAc: N-acetylglucosamine ethanol (5 volumes). This precipitate was redissolved in 0.1N acetic acid and subjected to gel filtration on Sephadex G-50 (1.1 by 147 cm.). The column effluent was assayed for hexose by the phenol-H2SO 4 technique and pooled into four major fractions. Aliquots of these fractions were subjected to sugar analysis by gas-liquid chromatography. 5 CASE REPORT The patient, a 10-1/2-month-old white boy, weight 9.5 kilograms, height 78 cm., was the product of a normal term pregnancy and delivery and was in good health. Slight hypertelorism and Hurler-like facial features were observed, but there were no corneal opacities and no psychomotor retardation. Liver and spleen were felt, but lymphocytes did not contain vacuoles; there was no hernia, but diastasis recti was present, and skeletal roentgenograms showed a minimal degree of longbone thickening with some widening of posterior ribs. Urinary mucopolysaccharide levels (6 mg. per 24 hr.) and species distribution were normal. RESULTS Lysosomal enzymes. Skin fibroblasts were cultured and a detailed study of lysosomal hydrolase levels was carried out as described by Matalon and associates. 4 c~Mannosidase activity, measured at pH 3.5, was almost
866
Brief clinical and laboratory observations
The Journal of Pediatrics June 1974
Table I. Monosaccharide composition of urinary complex carbohydrates Molar ratio
Patient
Controls (3) M. G. (proband) Jos. G. (fat her) Jo. G. (mother) J. G. (unaffected sibling) S. H. (lactosylceramidosis) C. F. (GMl-gangliosidosis, Type I) M; J. (GMl-gangliosidosis, Type II) Morquio's disease
Fucose
Mannose
Galactose*
GlcNAct
GalNAct
neuNAct
Total carbohydrates (izmoles/ml. urine)
0.17 0.35 0.15 0.20 0.10 0.25 0.04
0.49 125 0.70 0.80 0.53 0.50 0.60
1.00 1.00 1.00 1.00 1.00 1.00 1.00
1.03 1.22 1.15 1.00 1.40 0.90 1.11
0.10 0.41 0.22 0.10 0.40 0.35 0.16
0.67 0.85 0.78 0.74 0.91 0.40 0.10
0.05-0.10 0.207 0.054 0.118 0.421 0.251 0.517
0.06 0.01
0.44 0.02
1.00 1.00
0.92 0.94
0.1-2 0.06
0.22 0.02
0.463 0.357
*Galactose arbitrarily taken as 1.0; all samples contained varying amounts of glucose. "fGlcNAc, N-acetylglucosamine;GalNAc, N-acetylgalactosamine;neuNAc, N-acetylneuraminicacid (sialic acid).
Table II. Fractionation o f urinary carbohydrates on Sephadex G-50
Patient and Fr. No.
Fucose
I
Molar ratio *
Mannose
Galactose
GIcNAct
GalNAct
neuNAct
Total carbohydrate Ozmoles/l O0 ml. urine)
Proband (M. G.) I II Ilia Illb
0.28 0.46 0.76 0.10
0.84 0.92 3.60 4.10
2.46 2.15 1.16 0.10
1.00 1.00 1.00 1.00
0.77 0.15 Tr.~ Tr.
2.00 0.77 0.76 Tr.
8.42 5.68 1.46 5.12
Unaffected sibling (J. G.) I II Ilia IIIb
0.60 0.42 0.39 Tr.
1.66 0144 1.94 0.05
4.66 1.00 1.44 0.32
1.00 1.00 1.00 1.00
0.40 0.11 Tr. Tr.
0.8t 0.46 0.42 1.08
13.23 20.48 5.25 0.88
*All fractions contained some glucose. tGIcNAc, N-acetylglucosamine;GalNAc, N-acetylgalactosamine;neuNAc, N-acetylneuraminicacid. :~Tr.,only trace amounts detected by gas-liquidchromatographic analysis.
completely absent whereas other hydrolases involved in complex carbohydrate catabolism, such as /3-Na c e t y l h e x o s a m i n i d a s e s A a n d B, c~-N-acetyl-glucosaminidase, /3-galactosidase, a-fucosidase, aiduronidase, /3-glucuronidase, arylsulfatase A, and Nacetylglucosamine N-sulfatase, were normal. Complex carbohydrates in u r i n e , Most urine samples e x a m i n e d contained significant levels of carbohydrate, the complex nature of which has been reported by m a n y workers (e.g., Rosenfeld6). The level in n o r m a l individuals showed considerable variation and it can be seen from Table I that the actual level of urinary m a n -
nose (expressed as/z moles per milliliter) is equalled and sometimes exceeded in clinically n o r m a l individuals (such as J. G.) a n d those with certain other disorders of complex carbohydrate metabolism such as Gulgangliosidosis. However, the mannose:galactose ratio of 1.25 appears to be indicative of mannosidosis since only in the obligate heterozygotes have we detected a ratio greater than 0.6. No m a n n o s e - c o n t a i n i n g material could be detected in the s u p e r n a t a n t following precipitation with ethanol so all studies were carried out o n the precipitate. Chromatography on Sephadex G-50 yielded four fractions. Fractions (Frs.) I and II corresponded to
Volume 84 Number 6
Frs. I and II of Nordgn and colleagues,7 namely, excluded material and medium-sized molecules. The region corresponding to Fr. III of Nord~n and coworkers 7 was resolved into two components, Frs. Ilia (minor) and IIIb (major). Gas-liquid chromatographic analysis showed Frs. I and II to be normal urinary components and Fr. IIIb to be the major abnormal component associated with mannosidosis (Table II). The mannose:N-acetylglucosamine ratio of 4:1 is comparable to that reported by Nord6n and associates. 7 DISCUSSION The clinical symptoms of this case are compatible with the reports of Ockerman 1,2 and Norden and associates 7 since our patient is in the early stages of the disease. The clinical features of mannosidosis appear typical of many inborn errors of complex carbohydrate metabolism (sphingolipidoses and mucopolysaccharidoses) and biochemical studies are required in order to make a definitive diagnosis. T h i s ' must be based on the demonstration of ~-mannosidase A and B deficiency in either a liver biopsy, 2 leukocytes, 2 or fibroblasts cultured from a skin biopsy,4 or, providing that precautions are taken, the marked increase in the level of urinary mannose-rich complex carbohydrates. In this report we wish to emphasize that the diagnosis should be based on actual isolation of mannose-rich complex carbohydrates rather than simply on total urinary m a n n o s e content. T h e latter results must be interpreted with caution because of the comparably high levels of urinary glycopeptides sometimes found in normal individuals. The high level of mannose excretion in the elder, unaffected sibling of the proband could reflect his possible heterozygote status. Elevated urinary mannose is also found in patients with GMi-gangliosidosis and lactosylceramidosis, where the clinical symptoms are somewhat similar to the latter stages of mannosidosis,8, 9 but only in mannosidosis is the mannose:galactose greater than unity (Table I) and simple fractionation on Sephadex G-50 provides a definitive diagnosis. During the course of this work, Norden and associates 1~ reported the isolation of a trisaccharide (Man c~ (1---,3) Man/3(1---~4) GlcNAc) from the urine of patients with mannosidosis. We have been unable to characterize such a trisaccharide in this patient and this material may be characteristic of the latter stages of the disease. The m a n n o s e : N - a c e t y l g l u c o s a m i n e ratio of 4:1 suggests a structure similar to that proposed by Nord6n and associates. 1~Preliminary studies in this laboratory indicate the following possible structure:
Brief clinical and laboratory observations
867
Man a (l---2) Man a (1--3) Man/3 (1--,4) GIcNAc 3 Man for the pentasaccharide which is in broad agreement with the structures proposed by Norden and associates. 11 This material binds to concanavalin A and amino acid analysis suggests that approximately 30 per cent may be in the glycopeptide form. It seems likely that most of the mannose-rich material excreted by this and other patients with mannosidosis is derived from endogenous glycoprotein turnover (plasma proteins, renal glomerular basement membrane, etc.). The material is heterogeneous, consisting of tri-, tetra-, and pentasaccharide material. 2, 10, 11 A l t h o u g h the asparaginyl-N-acetylglucosylamine-cleavingenzyme appears active in mammalian tissue, some is excreted in the glycopeptide form and should be useful for the purification of a-mannosidase by affinity chromatography. However, exogenous contributions cannot be ruled out and may exacerbate the condition. Such factors, together with the possibility of enzyme replacement therapy by administering a-mannosidase to such patients, must be condidered in the management of this rare lysosomal storage disease.
REFERENCES
1. ~ckerman, P. A.: A generalized storage disorder resembling Hurler's syndrome, Lancet 2: 239, 1967. 2. ~ckerman, P. A.: Mannosidosis, in Hers, H. G., and Van Hoof, F., editors: Lysosomes and storage diseases, New York, 1973, Academic Press, Inc., Chap. 11. 3. Hocking, J. D., Jolly, R. D., and Batt, R. D.: Deficiency of ~-mannosidase in Angus cattle, Biochem. J. 128: 69, 1972. 4. Matalon, R., Aylsworth, A. S., and Dorfman, A.: Diagnosis of mannosidosis in cultut:ed skin fibroblasts.Submitted to Biochem. Biophys. Res. Commun. 5. Clamp, J. R., Dawson, G., and Hough, L.: Simultaneous estimation of 6-deoxy-L-galactose (L-fUcose), D-mannose, o-galactose, 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine) and N-acetylneuraminic acid (sialic acid) in glycoproteins and glycopeptides, Biochem. Biophys. Acta 148: 342, 1967. 6. Rosenfeld, L.: Quantitative levels of the constituents of acid mucopolysaccharides and other carbohydrate polymers in dialyzed normal human urine, Clin. Chim. Acta 31: 263, 1971. 7. Nord6n, N. E., Ockerman, P. -A., and Szabo, L.: Urinary mannose in mannosidosis, J. PEDIATR.82: 686, 1973. 8. O'Brien, J. S,: GMi-Gangliosidosis; in Stanbury, J. B., Wyngaarden, J. B., and Frederickson, D. S., editors: The metabolic basis of inherited disease, New York, 1972, McGraw-Hill Book Company, Inc., p. 639. 9. Dawson, G., Matalon, R., and Stein, A. O.: Lac-
868
B r i e f clinical and laboratory observations
tosylceramidosis: Lactosylceramide galactosyl hydrolase deficiency and accumulation of lactosylceramide in cultured skin fibrobtasts, J. PEDIATR.79: 423, 1971. 10. Norden, N. E., Lundblad, A., Svensson, S., iJckerman, P.A., and Autio, S.: A mannose-containing trisaccharide isolated from urines of three patients with mannosidosis, J. Biol. Chem. 248: 6210, 1973.
The Marshall syndrome: Report of a new family Hans Ze|lweger, M.D., Jeanne K. Smith, M.D.,* and Peter Griitzner, M.D.,** Iowa City, Iowa
T H E MARSHALL SYNDROME is v e r y r a r e ; to o u r k n o w l e d g e only two families with M a r s h a l l s y n d r o m e (Fig. 1, A, B ) h a v e b e e n reported, t,2 A t h i r d family with four affected m e m b e r s in two g e n e r a t i o n s (Fig. 1, C ) has b e e n followed by us o v e r a n u m b e r o f years a n d will be p r e s e n t e d here. CASE REPORTS Case 1. L. S. (Fig. 1, C, I1), a white woman, was first seen in the department of Ophthalmology, University of Iowa Hospitals, at the age of six years. She was a somewhat small, obese girl with a severely depressed nasal bridge and slightly everted and thickened lips. There was a high-grade myopia of 23 (right) and 21 (left) diopters, a myopic conus of the optic nerve head, alternating esotropia, and a sensorineural hearing deficit between 26 and 30 decibels. In subsequent years surgical correction of the saddle nose and esotropia was attempted without success. Bilateral cataracts were noted and surgically removed later. Intraocular pressure was always within the normal range. Her hearing loss proved to be progressive and is now at the 50 decibel level. In spite of her visual and auditory handicaps, this patient was and is leading a fairly normal life, first as a secretary, now as a housewife and mother of four children (Patients 2, 3, and 4, and a fourth child, a normal boy). Her parents and two siblings are normal. From the Departments of Ophthalmology, Otolaryngology an d Maxillofacial Surgery, an d Pediatrics o f the University o f lowa. In part supported by Grant 353 4360 HEW, spec. Res. M R 12. *Reprintaddress:Departmentof Otolaryngologyand Maxillofacial Surgery, Universityof lowa, Iowa City, Iowa 5224Z **Presentaddress: Universitaets-Augenklinik,Freiburgim Breisgau, Germany.
The Journal o f Pediatrics June 1974
11.
Norden, N. E., Lundblad, A., ~ckerman, P.-A., and Jolly, R. D.: Mannosidosis in Angus cattle: Partial characterization of two mannose-containing oligosaccharides, FEBS Lett. 35: 209, 1973.
A recent physical examination revealed a 36-year-old obese (94 kilogram) woman of small stature (149 cm.) with the above mentioned findings. Radiography of her skull showed hypertelorism, huge frontal sinuses, small and fiat nasal bones, small maxilla with slightly protruding upper incisors, a short ascending ramus of the mandible, widespread dural calcifications, and somewhat narrow internal auditory canals (about 60 per cent of normal). Electroencephalogram and echoencephalograms were normal. Case 2. R. S. is the first-born son. The pregnancy was uneventful and delivery was by cesarean section. His hearing acuity was questionable from early infancy. His latest examination (age 11) revealed a socially well-adjusted boy below the third percentile in height; the head circumference was normal; protruding frontal bones, saddle nose, slight hypertelorism, high-grade myopia, decreased visual acuity, mild esotropia and hypertropia, sensorineural hearing deficit of about 60 decibels, intelligence quotient of 101 (Wechsler Intelligence Scale for Children), Vineland Social Maturity Test score of 94, and average scholastic performance. His handicaps are partially compensated by glasses and a hearing aid. Cases 3 and 4. M. S. and B. S., two younger sisters, have essentially the same history, phenotypic appearance, and clinical
T a b l e I. E y e findings in M a r s h a l l s y n d r o m e Patient i Patient 2 Patient 3 Patient 4 Hypertelorism Extraorbital prominence* (mm): Right eye Left eye Normal f. age Myopia (diopters): Right eye Left eye Visual acuity: Right eye Left eye Esotropia Hypertropia
+
+
+
+
21 22 (17)
22 24 (15)
21 19 (14)
23 24 (13)
23 22
17 16.5
17 16
21.5 23
20/35 20/200
20/200 20/50
20/100 20/40
+
+
+
+
Left
Left
Right
Left
*Extraorbital prominence--distance from lateral margin of orbit to vertex of cornea. tRefraction measured before cataract surgery.
4. Von Steijnen, C.: Diastrophic dwarfism, Maandschr Kindergeneeskd 36: 188, 1968. 5. Langer, L. O.: Diastrophic dwarfism in early infancy, Am. J. Roentgenol. Radium Ther. Nucl. Med. 89: 399, 1965. 6. Stover, C. N., Hayes, J. %, and Hold, J. F.: Diastrophic
Excretion of mannose-rich complex carbohydrates by a patient with a-mannosidase deficiency (mannosidosis) Grace Chen Tsay, Ph.D., Glyn Dawson, Ph.D.,* and Reuben Matalon, M.D., Chicago, I11.
IN 1967 i3ckerman l described a case of mannosidosis characterized by a deficiency of a-mannosidase A and B activity and a greatly increased level of bound mannose in tissues and urine. Six cases, all European in origin, have now been reported. 2 The disorder is characterized clinically by progressive psychomotor retardation and increasing expression of the stigmas of mucopolysaccharidoses (coarse facial features, skeletal abnormalities, corneal clouding, hepatosplenomegaly, and vacuolated lymphocytes). The disease appears similar to one reported in cattle 3 and is of interest from the point of view of understanding normal giycoprotein metabolism in man. We have recently diagnosed a further case of mannosidosis on the basis Of an a-mannosidase A and B deficiency in cultured h u m a n skin fibroblasts.4 Urine from the homozygote, his parents, and an unaffected elder sibling was examined for glycopeptide and oligosaccharide content. Abnormal amounts of mannose-rich material were detected in the homozygote following fractionation on Sephadex G-50, providing a reliable method for diagnosis. In this report we wish to emphasize that direct measurement of urinary mannose levels may be unreliFrom the Departments of Pediatrics and Biochemistry, Joseph P. Kennedy, Jr., Mental Retardat&n Research Center, University of Chicago. Supported by United States Public Health Service Grants HD-06426, HD-04583, and AM-05996, and RRO0305 from the General Clinical Research Facilities and Resources. Drs. Dawsonand Matalon are Joseph P. Kennedy, Jr., Scholars. *Reprint address:Joseph P. Kennedy, ,It'.,MentalRetardation Research Centel, Universityof Chicago, 950 E. 59th St., Chicago, Ill. 60637.
B r i e f clinical and laboratory observations
865
dwarfism, Am. J. Roentgenol. Radium Ther. Nucl. Med. 89: 914, 1963. 7. Taybi, H.: Diastrophic dwarfism, Radiology 80: 1, 1963. 8. Wilson, D. W., Chrispin, and Carter, C. O.: Diastrophic dwarfism, Arch. Dis. Child. 44: 48, 1969.
able for diagnosis because of the wide variation in the level of excretion of glycoproteins both in normal individuals and in those with other inherited disorders of complex carbohydrate metabolism. MATERIALS
AND METHODS
Fresh samples of urine were collected from normal individuals, the patient and his immediate family, and patients with other inborn errors of carbohydrate metabolism, and frozen in aliquots of 100 ml. Small amounts (0.1 ml.) were evaporated to d r y n e s s and analyzed directly by gas-liquid chromatography of the derived trimethylsilylmethylgtycosides,s Initial purification of the urinary material was achieved by precipitation with Abbreviations used Man: D-rnannose GIcNAc: N-acetylglucosamine ethanol (5 volumes). This precipitate was redissolved in 0.1N acetic acid and subjected to gel filtration on Sephadex G-50 (1.1 by 147 cm.). The column effluent was assayed for hexose by the phenol-H2SO 4 technique and pooled into four major fractions. Aliquots of these fractions were subjected to sugar analysis by gas-liquid chromatography. 5 CASE REPORT The patient, a 10-1/2-month-old white boy, weight 9.5 kilograms, height 78 cm., was the product of a normal term pregnancy and delivery and was in good health. Slight hypertelorism and Hurler-like facial features were observed, but there were no corneal opacities and no psychomotor retardation. Liver and spleen were felt, but lymphocytes did not contain vacuoles; there was no hernia, but diastasis recti was present, and skeletal roentgenograms showed a minimal degree of longbone thickening with some widening of posterior ribs. Urinary mucopolysaccharide levels (6 mg. per 24 hr.) and species distribution were normal. RESULTS Lysosomal enzymes. Skin fibroblasts were cultured and a detailed study of lysosomal hydrolase levels was carried out as described by Matalon and associates. 4 c~Mannosidase activity, measured at pH 3.5, was almost
866
Brief clinical and laboratory observations
The Journal of Pediatrics June 1974
Table I. Monosaccharide composition of urinary complex carbohydrates Molar ratio
Patient
Controls (3) M. G. (proband) Jos. G. (fat her) Jo. G. (mother) J. G. (unaffected sibling) S. H. (lactosylceramidosis) C. F. (GMl-gangliosidosis, Type I) M; J. (GMl-gangliosidosis, Type II) Morquio's disease
Fucose
Mannose
Galactose*
GlcNAct
GalNAct
neuNAct
Total carbohydrates (izmoles/ml. urine)
0.17 0.35 0.15 0.20 0.10 0.25 0.04
0.49 125 0.70 0.80 0.53 0.50 0.60
1.00 1.00 1.00 1.00 1.00 1.00 1.00
1.03 1.22 1.15 1.00 1.40 0.90 1.11
0.10 0.41 0.22 0.10 0.40 0.35 0.16
0.67 0.85 0.78 0.74 0.91 0.40 0.10
0.05-0.10 0.207 0.054 0.118 0.421 0.251 0.517
0.06 0.01
0.44 0.02
1.00 1.00
0.92 0.94
0.1-2 0.06
0.22 0.02
0.463 0.357
*Galactose arbitrarily taken as 1.0; all samples contained varying amounts of glucose. "fGlcNAc, N-acetylglucosamine;GalNAc, N-acetylgalactosamine;neuNAc, N-acetylneuraminicacid (sialic acid).
Table II. Fractionation o f urinary carbohydrates on Sephadex G-50
Patient and Fr. No.
Fucose
I
Molar ratio *
Mannose
Galactose
GIcNAct
GalNAct
neuNAct
Total carbohydrate Ozmoles/l O0 ml. urine)
Proband (M. G.) I II Ilia Illb
0.28 0.46 0.76 0.10
0.84 0.92 3.60 4.10
2.46 2.15 1.16 0.10
1.00 1.00 1.00 1.00
0.77 0.15 Tr.~ Tr.
2.00 0.77 0.76 Tr.
8.42 5.68 1.46 5.12
Unaffected sibling (J. G.) I II Ilia IIIb
0.60 0.42 0.39 Tr.
1.66 0144 1.94 0.05
4.66 1.00 1.44 0.32
1.00 1.00 1.00 1.00
0.40 0.11 Tr. Tr.
0.8t 0.46 0.42 1.08
13.23 20.48 5.25 0.88
*All fractions contained some glucose. tGIcNAc, N-acetylglucosamine;GalNAc, N-acetylgalactosamine;neuNAc, N-acetylneuraminicacid. :~Tr.,only trace amounts detected by gas-liquidchromatographic analysis.
completely absent whereas other hydrolases involved in complex carbohydrate catabolism, such as /3-Na c e t y l h e x o s a m i n i d a s e s A a n d B, c~-N-acetyl-glucosaminidase, /3-galactosidase, a-fucosidase, aiduronidase, /3-glucuronidase, arylsulfatase A, and Nacetylglucosamine N-sulfatase, were normal. Complex carbohydrates in u r i n e , Most urine samples e x a m i n e d contained significant levels of carbohydrate, the complex nature of which has been reported by m a n y workers (e.g., Rosenfeld6). The level in n o r m a l individuals showed considerable variation and it can be seen from Table I that the actual level of urinary m a n -
nose (expressed as/z moles per milliliter) is equalled and sometimes exceeded in clinically n o r m a l individuals (such as J. G.) a n d those with certain other disorders of complex carbohydrate metabolism such as Gulgangliosidosis. However, the mannose:galactose ratio of 1.25 appears to be indicative of mannosidosis since only in the obligate heterozygotes have we detected a ratio greater than 0.6. No m a n n o s e - c o n t a i n i n g material could be detected in the s u p e r n a t a n t following precipitation with ethanol so all studies were carried out o n the precipitate. Chromatography on Sephadex G-50 yielded four fractions. Fractions (Frs.) I and II corresponded to
Volume 84 Number 6
Frs. I and II of Nordgn and colleagues,7 namely, excluded material and medium-sized molecules. The region corresponding to Fr. III of Nord~n and coworkers 7 was resolved into two components, Frs. Ilia (minor) and IIIb (major). Gas-liquid chromatographic analysis showed Frs. I and II to be normal urinary components and Fr. IIIb to be the major abnormal component associated with mannosidosis (Table II). The mannose:N-acetylglucosamine ratio of 4:1 is comparable to that reported by Nord6n and associates. 7 DISCUSSION The clinical symptoms of this case are compatible with the reports of Ockerman 1,2 and Norden and associates 7 since our patient is in the early stages of the disease. The clinical features of mannosidosis appear typical of many inborn errors of complex carbohydrate metabolism (sphingolipidoses and mucopolysaccharidoses) and biochemical studies are required in order to make a definitive diagnosis. T h i s ' must be based on the demonstration of ~-mannosidase A and B deficiency in either a liver biopsy, 2 leukocytes, 2 or fibroblasts cultured from a skin biopsy,4 or, providing that precautions are taken, the marked increase in the level of urinary mannose-rich complex carbohydrates. In this report we wish to emphasize that the diagnosis should be based on actual isolation of mannose-rich complex carbohydrates rather than simply on total urinary m a n n o s e content. T h e latter results must be interpreted with caution because of the comparably high levels of urinary glycopeptides sometimes found in normal individuals. The high level of mannose excretion in the elder, unaffected sibling of the proband could reflect his possible heterozygote status. Elevated urinary mannose is also found in patients with GMi-gangliosidosis and lactosylceramidosis, where the clinical symptoms are somewhat similar to the latter stages of mannosidosis,8, 9 but only in mannosidosis is the mannose:galactose greater than unity (Table I) and simple fractionation on Sephadex G-50 provides a definitive diagnosis. During the course of this work, Norden and associates 1~ reported the isolation of a trisaccharide (Man c~ (1---,3) Man/3(1---~4) GlcNAc) from the urine of patients with mannosidosis. We have been unable to characterize such a trisaccharide in this patient and this material may be characteristic of the latter stages of the disease. The m a n n o s e : N - a c e t y l g l u c o s a m i n e ratio of 4:1 suggests a structure similar to that proposed by Nord6n and associates. 1~Preliminary studies in this laboratory indicate the following possible structure:
Brief clinical and laboratory observations
867
Man a (l---2) Man a (1--3) Man/3 (1--,4) GIcNAc 3 Man for the pentasaccharide which is in broad agreement with the structures proposed by Norden and associates. 11 This material binds to concanavalin A and amino acid analysis suggests that approximately 30 per cent may be in the glycopeptide form. It seems likely that most of the mannose-rich material excreted by this and other patients with mannosidosis is derived from endogenous glycoprotein turnover (plasma proteins, renal glomerular basement membrane, etc.). The material is heterogeneous, consisting of tri-, tetra-, and pentasaccharide material. 2, 10, 11 A l t h o u g h the asparaginyl-N-acetylglucosylamine-cleavingenzyme appears active in mammalian tissue, some is excreted in the glycopeptide form and should be useful for the purification of a-mannosidase by affinity chromatography. However, exogenous contributions cannot be ruled out and may exacerbate the condition. Such factors, together with the possibility of enzyme replacement therapy by administering a-mannosidase to such patients, must be condidered in the management of this rare lysosomal storage disease.
REFERENCES
1. ~ckerman, P. A.: A generalized storage disorder resembling Hurler's syndrome, Lancet 2: 239, 1967. 2. ~ckerman, P. A.: Mannosidosis, in Hers, H. G., and Van Hoof, F., editors: Lysosomes and storage diseases, New York, 1973, Academic Press, Inc., Chap. 11. 3. Hocking, J. D., Jolly, R. D., and Batt, R. D.: Deficiency of ~-mannosidase in Angus cattle, Biochem. J. 128: 69, 1972. 4. Matalon, R., Aylsworth, A. S., and Dorfman, A.: Diagnosis of mannosidosis in cultut:ed skin fibroblasts.Submitted to Biochem. Biophys. Res. Commun. 5. Clamp, J. R., Dawson, G., and Hough, L.: Simultaneous estimation of 6-deoxy-L-galactose (L-fUcose), D-mannose, o-galactose, 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine) and N-acetylneuraminic acid (sialic acid) in glycoproteins and glycopeptides, Biochem. Biophys. Acta 148: 342, 1967. 6. Rosenfeld, L.: Quantitative levels of the constituents of acid mucopolysaccharides and other carbohydrate polymers in dialyzed normal human urine, Clin. Chim. Acta 31: 263, 1971. 7. Nord6n, N. E., Ockerman, P. -A., and Szabo, L.: Urinary mannose in mannosidosis, J. PEDIATR.82: 686, 1973. 8. O'Brien, J. S,: GMi-Gangliosidosis; in Stanbury, J. B., Wyngaarden, J. B., and Frederickson, D. S., editors: The metabolic basis of inherited disease, New York, 1972, McGraw-Hill Book Company, Inc., p. 639. 9. Dawson, G., Matalon, R., and Stein, A. O.: Lac-
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B r i e f clinical and laboratory observations
tosylceramidosis: Lactosylceramide galactosyl hydrolase deficiency and accumulation of lactosylceramide in cultured skin fibrobtasts, J. PEDIATR.79: 423, 1971. 10. Norden, N. E., Lundblad, A., Svensson, S., iJckerman, P.A., and Autio, S.: A mannose-containing trisaccharide isolated from urines of three patients with mannosidosis, J. Biol. Chem. 248: 6210, 1973.
The Marshall syndrome: Report of a new family Hans Ze|lweger, M.D., Jeanne K. Smith, M.D.,* and Peter Griitzner, M.D.,** Iowa City, Iowa
T H E MARSHALL SYNDROME is v e r y r a r e ; to o u r k n o w l e d g e only two families with M a r s h a l l s y n d r o m e (Fig. 1, A, B ) h a v e b e e n reported, t,2 A t h i r d family with four affected m e m b e r s in two g e n e r a t i o n s (Fig. 1, C ) has b e e n followed by us o v e r a n u m b e r o f years a n d will be p r e s e n t e d here. CASE REPORTS Case 1. L. S. (Fig. 1, C, I1), a white woman, was first seen in the department of Ophthalmology, University of Iowa Hospitals, at the age of six years. She was a somewhat small, obese girl with a severely depressed nasal bridge and slightly everted and thickened lips. There was a high-grade myopia of 23 (right) and 21 (left) diopters, a myopic conus of the optic nerve head, alternating esotropia, and a sensorineural hearing deficit between 26 and 30 decibels. In subsequent years surgical correction of the saddle nose and esotropia was attempted without success. Bilateral cataracts were noted and surgically removed later. Intraocular pressure was always within the normal range. Her hearing loss proved to be progressive and is now at the 50 decibel level. In spite of her visual and auditory handicaps, this patient was and is leading a fairly normal life, first as a secretary, now as a housewife and mother of four children (Patients 2, 3, and 4, and a fourth child, a normal boy). Her parents and two siblings are normal. From the Departments of Ophthalmology, Otolaryngology an d Maxillofacial Surgery, an d Pediatrics o f the University o f lowa. In part supported by Grant 353 4360 HEW, spec. Res. M R 12. *Reprintaddress:Departmentof Otolaryngologyand Maxillofacial Surgery, Universityof lowa, Iowa City, Iowa 5224Z **Presentaddress: Universitaets-Augenklinik,Freiburgim Breisgau, Germany.
The Journal o f Pediatrics June 1974
11.
Norden, N. E., Lundblad, A., ~ckerman, P.-A., and Jolly, R. D.: Mannosidosis in Angus cattle: Partial characterization of two mannose-containing oligosaccharides, FEBS Lett. 35: 209, 1973.
A recent physical examination revealed a 36-year-old obese (94 kilogram) woman of small stature (149 cm.) with the above mentioned findings. Radiography of her skull showed hypertelorism, huge frontal sinuses, small and fiat nasal bones, small maxilla with slightly protruding upper incisors, a short ascending ramus of the mandible, widespread dural calcifications, and somewhat narrow internal auditory canals (about 60 per cent of normal). Electroencephalogram and echoencephalograms were normal. Case 2. R. S. is the first-born son. The pregnancy was uneventful and delivery was by cesarean section. His hearing acuity was questionable from early infancy. His latest examination (age 11) revealed a socially well-adjusted boy below the third percentile in height; the head circumference was normal; protruding frontal bones, saddle nose, slight hypertelorism, high-grade myopia, decreased visual acuity, mild esotropia and hypertropia, sensorineural hearing deficit of about 60 decibels, intelligence quotient of 101 (Wechsler Intelligence Scale for Children), Vineland Social Maturity Test score of 94, and average scholastic performance. His handicaps are partially compensated by glasses and a hearing aid. Cases 3 and 4. M. S. and B. S., two younger sisters, have essentially the same history, phenotypic appearance, and clinical
T a b l e I. E y e findings in M a r s h a l l s y n d r o m e Patient i Patient 2 Patient 3 Patient 4 Hypertelorism Extraorbital prominence* (mm): Right eye Left eye Normal f. age Myopia (diopters): Right eye Left eye Visual acuity: Right eye Left eye Esotropia Hypertropia
+
+
+
+
21 22 (17)
22 24 (15)
21 19 (14)
23 24 (13)
23 22
17 16.5
17 16
21.5 23
20/35 20/200
20/200 20/50
20/100 20/40
+
+
+
+
Left
Left
Right
Left
*Extraorbital prominence--distance from lateral margin of orbit to vertex of cornea. tRefraction measured before cataract surgery.