A case of mucolipidosis II: Biochemical, nutritional, and immunological studies

A Case of Mucolipidosis II: Biochemical, Nutritional, and Immunological Studies Shigeo Kojima, MD, Shintaro Okada, MD, Hiroshi Kai, MD, Kyungsae Ha, MD, Osamu Nose, MD, Teruo Ikeda, MD, Tort! Yutaka, MD, Masahiro Kato, MD, and Hyakuji Yabuuchi, MD

A case of mucolipidosis II was studied biochemically, nutritionally and immunologically. A possible functional deficiency of T cells was observed, but discrepancy between B cells wid immunoglobulin content was not reasonably explained at this moment. There was no basic nutritional problem in this case and it is more likely that his growth retardation was due to frequent episodes of severe respiratory infection because he received adequate calorie intake with low normal basal metabolic rate. Results of enzymatic assays were also presented. Kojima S, Okada S, Kai H, Ha K, Nose 0, Ikeda T, Yutaka T, Kato M, Yabuuchi H: A case of mucolipidoses II: Biochemical, nutritional and immunological studies. Brain Dev 1:26-30,1979

Mucolipidosis II (ML-II, I-cell disease) was first described by Leroy and Demars 6 as a single genetic entity among Hurler-like syndromes. Its clinical features resemble those of the Hurler syndrome, but can be distinguished by the following features: early evidence of generalized skeletal dysplasia, lack of striking corneal clouding, lack of excessive mucopolysacchariduria. 8 Besides fibroblasts cultured from patients with ML-II bear numerous inclusions visible on phase contrast microscopy.6 ML-I1 is inherited as an autosomal recessive disorder which is characterized by coarse facies,

From the Pediatric Clinic, Meiwa Hospital, Hyogo (SK), Department of Pediatrics, Osaka University Hospital, Osaka (SO, HK, KH, ON, TI, TY, HY), Pediatric Clinic, Kansai Rosai Hospital, Hyogo (MK). Accepted for publication; April 2, 1979.

Key words: Mucolipidosis II, neuraminidase, urinary oligosaccharides, energy metabolism, malnutrition, lymphocyte subpopulation, delayed type skin test. Correspondence address: Dr. Shin taro Okada, Department of Pediatrics, Osaka University Hospital, Fukushima 1-1-50, Fukushima-ku, Osaka 553, Japan.

severe growth and mental retardation, severe bony dysplasia, and susceptibility to infection. It has been generally reported that patients with ML-I1 die between 3 and 8 years of age. 8 In this report we describe some aspects of the causes of important clinical problems; growth retardation and susceptibility to infection. Understanding of these matters will greatly benefit clinicians who wish to maintain a good condition of ML-II patients. Case Report K. K., a 5-year-and-ll-month-old Japanese boy, was referred to our clinic because of developmental retardation with a suspicion of mucopolysaccharidosis. He was the second child from healthy parents who were the first cousins. Although pregnancy and delivery were uneventful, feeding and crying were found to be very weak soon after birth. At 3 months of age congenital bilateral dislocation of the hip joints and bilateral inguinal hernias were recognized. Head control was possible after 7 months of age. He had to be hospitalized very frequently because of severe pneumonia. At 3 years of age craniostenosis was noticed and cranial H-tomy was performed.

When he first visited Osaka University Hospital, he was a very small and undernourished child (height 81.0 cm, -6.0 S.D.; weight 10.0 Kg, -4.0 S.D.; head circumference 45 .7 cm). His face was Hurler-like with saddle nose, thick lips, hypertrophic gingiva and tongue, and hypoplastic teeth. Low set ears were also noted. Respiratory stridor was so striking that cardiac sound was not investigated completely. Liver and spleen were palpable 5 cm below the costal margin. Kyphoscoliosis and contracture of each joint were severe. He could stand alone but could not walk. He spoke only a few single words. Roentogenograms revealed generalized skeletal dysplasia which was compatible with the changes of ML-II. Laboratory investigations revealed mild anemia due to iron deficien cy (hemoglobin 9.4 gldl, hematocrit 30%, plasma iron 34 pg/dl, VIBC 469 pg/dl), but other routine data were within normal limits. Endocrinological data including urinary 17-0HCS, plasma T 3, T 4, TSH were normal. The LE test, RA test and antinuclear factor were negative. Urinary excretion of mucopolysac&aride was not increased. Ultrastructural investigation of skin and conjunctiva revealed that fibroblasts and endothelial cells contained numerous vacuoles. 16 Methods Lysosomal enzymes were measured fluorometrically with 4-methylumbelliferyl glycosides as described previously. I 0 Neuraminidase toward fetuin was assayed by the method of Thomas et al l3 and toward 2 -+ 3 and 2 -+ 6 sialyllactose by the method of Cantz et al. I The method of 'investigation of urinary oligosaccharides was reported elsewhere. I 0

Nutritional Investigations Fat absorption rate was determined by the method of van de Kamer. ls Measurement of standard energy metabolism (BMR) was performed in the morning after overnight fasting, under the light sedation with chloral hydrate. Oxygen consumption and CO 2 production were measured using an open-circuit method, as modified Kipp and Noyon's diaferometer 2 connected to the respiratory plastic box covered over a child head. Room air was passed at the rate of 7,576 ml/min through the respiratory box and the O2 content of the outflow measured with a paramagnetic oxygen analyser (Beckman G3). The rate of O2 consumption is the product of the rate of flow and the difference in O2 concentration in the inflow

and outflow gas. Measurement was begun 15-30 minutes after the child was seted in the respiratory box, and the measurement was made over a period of 30-60 minutes when the record was stable. The resting O2 consumption was calculated in ml/h, corrected to standard temperature and pressure, and converted to Cal/h. Energy metabolic rate was referred to s the standard energy metabolism of Karlberg.

Immunological Investigations Serum immunoglobulins were measured by inlmunoplate by the method of Mancini et al.? Lymphocyte subpopulations were detected through their EN and EAC receptors according to Tachibana et a1. 12 For delayed type skin reaction candida, PPD, PHA and varicella were tested as allergens. The response to PHA was measured after 24 hOUTS, those to PPD and varicella were measured after 48 hOUTS . PHA blast formation was measured by 3 H-thymidine uptake . Results Biochemically several lysosomal enzymes of the patient were elevated in the plasms and within the normal range in the white blood cells although they were normal in the parents (Fig 1). Cultured skin fibroblasts of the patient showed deficient neuraminidase activity toward fetuin, 2 -+ 3 sialyllactose and 2 -+ 6 sialyllactose (Table 1). Urinary excretion of oligosaccharides showed about 12-fold and 8-fold increase above the control values, in neutral sugar and bound sialic acid content, respectively (Table 2). As can be seen in Table 3, food intake and components during three days for nutritional tests were not abnormal. Fat absorption and BMR were slightly below the normal range. As shown in Table 4, the B subpopulation of lymphocytes was very small. Nevertheless the patient had serum immunoglobulin that fell within normal range, and the 19A might be considered higher than normal value. He also had a slightly low T subpopulation with a normal response to PHA mitogen. However, the results of the skin tests were all negative. Discussion Biochemical Studies Biochemically ML-II is a particularly interesting / Kojima et al: Studies on mucolipidosis II 27

Plasma (n mol 4 MU liberated/ml/hour)

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Fig 1 Lysosomal hydrolases in plasma and white blood cells. Shaded area represents normal activity range.

28 Brain & Development, Vall, No 1, 1979

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disease because it is thought to be a model for a receptor-medicated transfer of lysosomal hydrolases . Lysosomal enzymes are elevated in the plasma of patients with ML-II (Fig 1) and also are in the medium of cultured fibroblasts. Hickman and Neufeld 4 observed that hydrolases excreted by ML-II fibroblasts were altered so that they could not enter fibroblasts. Valantiu and Ratazzi 14 reported that extracellular and intracellular hydrolases in ML-I1 are different in electric charges from control en-

zymes. Recently there have been reports of increased amount of urinary sialyloligosaccharides 9 - 11 and elevated levels of sialic acid-rich materials in skin fibroblasts l3 in ML-II . The results shown in Table 1 are in good agreement with data reported previously. Thomas et al 13 suggested that the storage or excretion of sialyl oligosaccharides in ML-II is probably related to fetuin o:-neuraminidase deficiency in skin fibroblasts. Our results indicated that o:-neuramini-

Table I Neuraminidase activities in cultured fibroblasts

Table 2 Urinary oligosaccharide content

Substrate Fetuin 2-3 Sialyllactose 2-6 Sialyllactose

Patient

Control

0.03 0.03 0.02

1.85 ± 0.41 13.3 ± 4.8 5.3 ± 0.7

(J.l.g/mg creatinine)

Patient

Control

639 163.5

54 ± 20 19.9±7.5

Neutral sugar Bound sialic acid

Note Activities are expressed as nanom oles/mg protein/h.

Table 3 Nutritional tests of our case Intake Water (g) Protein (g) Fat (g) Carbohydrate (g) Calories

1st day (Oct. 29)

2nd day (Oct. 30)

3rdday (Oct. 31)

756 40 30 160

550 33 22 147 979

941 34 24 187 1183

1077

3.5 g/kg/day 2.5 g/kg/day 108 cal/kg/day

Fat absorption

86.5%

BMR

484 cal/day(-IO%)

Table 4 Immunological findings of our case Lymphocyte sUbpopulation

T cell

B cell

55% 2%

4864

total 8844

177

Response to PHA mitogen

Normal (Stimulation index 47.0)

Delayed hypersensitivity skin test

Candida PPD

0

PHA

_0_ 6x5

Varicella Serum immunoglobulin levels

_0_ 4x3

IgG IgA IgM

0 1,100 mg/dl 315 mg/dl 150 mg/dl

Kojima et al: Studies on mucolipidosis II 29

dase was deficient for not only fetuin, but also 2 -+ 3 and 2 -+ 6 neuramine lactose in ML-If fibroblasts (Table 2). However, it is very likely that these enzyme deficiencies in this disease are a secondary phenomenon caused by another lysosomal enzyme deficiency. And the fundamental cause of ML-I1 remains uncertain at this moment.

Nutritional Studies Food intake and composition of this patient in three days for nutritional tests were adequate for his age although he was very undernourished. Thus he was an infant who received adequate calorie intake with unsatisfactory gain in weight. Steatorrhea or unusually high energy needs may be common cause for this situation. 3 As shown in Table 3, there was no steatorrhea, and basal metabolic rate was within the normal limits. Recurrent respiratory infection throughout his childhood might have required extra calorie other than basal metabolism. Immunological Investigations It is interesting to note that the patient had a normal response to PHA blast formation but all the skin tests proved to be negative (even when PHA was used as allergen). There might be some partial disturbance of T-cell functions. The elevated IgA might be related to the fact that the patient was constantly suffered from respiratory tract infections. The easiest explanation for the discrepancy between a low percentage for B cells and a normal titer of immunoglobulins is technical error since surface immunoglobulin was not checked through surface recognition. Another possibility was a lapse between two investigations. The subpopulations of lymphocytes was investigated three months later. However, the most fascinating. explanation lies on that the receptors on lymphocytes might be covered by mucopolysaccharides and made insensitive to complement. This argument needs further studies. References

1. Cantz M, Gehler J, Spranger J: Mucolipidosis I: Increased sialic acid content and deficiency of an

30 Brain & Development, Vol 1, No 1, 1979

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~N-acetyl-neuraminidase in cultured fibroblasts. Biochem Biophys Res Commun 74: 732-738, 1977. Carpenter TM, Coropatchinsky VS: A modified Noyon's thermic diaferometer for respiratory gas analysis. Industr Engin Chem 14: 159-163,1942. Fomon SH: Infant Nutrition. 2nd ed., WB Saunders, Philadelphia, 1974. Hickman S, Neufeld EF: A hypothesis for I-cell disease: Defective hydrolase that do not enter Iysosomes. Biochem Biophys Res Commun 49: 992-999,1974. Karlberg P: Determination of standard energy metabolism in normal infants. Acta Paediatr 41: supp!. 89,1952. Leroy JG, DeMars RI: Mutant enzymatic and cytological phenotypes in cultured human fibroblasts. Science 157: 804-805, 1967. Mancini G, Carbonara AD, Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2: 235-254, 1965. McKusick VA, Neufeld EF, Kelly TE: The mucopolysaccharide storage diseases. In Stanbury JS, Wyngaarden JB, Fredrickson OS (eds): Metabolic Basis of Inherited Disease. McGraw-Hill, New York, 1978, pp 1282-1307. Michalski JC, Strecker G, Fournet B, et al: Structures of sialyl-oligosaccharides excreted in the urine of a patient with mucolipidosis I. FEBS Lett 79: 101-104,1977. Okada S, Kato T, Miura S, et al: Hypersialyloligosacchariduria in mucolipidoses: a method for diagnosis. CZin Chim Acta 86: 159-167,1978. Strecker G, Peers MC, Michalski JC, et al: Structure of nine sialyl-oligosaccharides accumulated in urine of eleven patients with three different types of sialidosis. Eur J Biochem 75: 391-403,1977. Tachibana T, Yoshida A: Micromethod to detect for human T & B cell. In Migita S (ed): Methodology of Immunological Experiments(A). Japanese Immunological Research Committee, Kanazawa, 1976 (in Japanese). Thomas GH, Tiller GE Jr, Reynolds LW, et al: Increased levels of sialic acid associated with a sialidase deficiency in I-cell disease (mucolipidosis II) fibroblasts. Biochem Biophys Res Commun 71: 188-195,1976. Valantiu GO, Ratazzi MC: Abnormal lysosomal hydrolases excreted by cultured fibroblasts in I-cell disease (Mucolipidosis 11). Biochem Biophys Res Commun 67: 956-964, 1975. Van de Kamer JH, ten Bokkel Huinink H, et al: Rapid method for the determination of fat in the feces. JBiol Chem 177: 343-355,1949. Yamano T, Shimada M, Okada S, et al: Electromicroscopic examination of skin and conjunctival biopsy specimens in neural storage diseases. Brain Dev 1:16-25, 1979.