Morquio syndrome (mucopolysaccharidosis IV B) associated with β-galactosidase deficiency. Report of two cases

Am J Hum Genet 32:258 - 272, 1980

Morquio Syndrome (Mucopolysaccharidosis IV B) Associated with ,3-Galactosidase Deficiency. Report of Two Cases HELFRIED GROEBE,1 MICHAEL KRINS,1 HEINRICH SCHMIDBERGER,2 KURT VON FIGURA,3 KLAUS HARZER,4 HANS KRESSE,5 EDUARD PASCHKE,5 ADRIAN SEWELL,6 AND KURT ULLRICH3

SUMMARY Two male patients, aged 6 and 25, both with normal intelligence and absence

of neurological abnormalities, exhibited dysostosis multiplex, dwarfism, odontoid anomalies, cloudy corneas, excessive excretion of keratan sulfate, and abnormal urinary oligosaccharides. Leukocytes and fibroblasts of both patients were deficient in acid /8-galactosidase (,8-gal) and normal in N-acetylgalactosamine-6-sulfate sulfatase, the deficient enzyme in classical Morquio syndrome. The ,8-gal deficiency was not due to an endogenous inhibitor, and the parents exhibited intermediate activities. Deficient ,/-gal activity was observed towardp-nitrophenyl-f3-galactoside, 4-methylumbelliferyl-,8-galactoside (4 MU-,8-gal), lactose, GM, ganglioside, keratan sulfate, and asialofetuin (ASF). Under standard assay conditions, the residual activity was similar for all substrates tested. Toward p-nitrophenyl-,8-galactoside, the mutant enzyme behaved as a Km variant.

Received April 24, 1979; revised June 18, 1979. This study was supported by the Deutsche Forschungsgemeinschaft (SFB- 104) and the Fonds zur Forderung der wissenschaftlichen Forschung in Osterreich. ' Children's Hospital, University of Monster, Robert-Koch-Strasse 31, D-4400 Munster, West Germany. 2 Clinic for Radiology, University of Graz, Auenbruggerplatz 9, A-8036 Graz, Austria. 3Institute of Physiological Chemistry, University of Monster, Waldeyerstrasse 15, D-4400 Monster. 4Institute for Brain Research, University of Tubingen, Belthlestrasse 15, D-7400 Tubingen, West

Germany. sInstitute of Medical Chemistry and Pregl-Laboratory, University of Graz, Harrachgasse 21, A-8010 Graz. 6 Children's Hospital, University of Mainz, Langenbeckstrasse 1, D-6500 Mainz, West Germany. © 1980 by the American Society of Human Genetics. 0002-9297/80/3202-0002$1.65

258

MORQUIO SYNDROME

259

INTRODUCTION

The Morquio syndrome is a rare inherited disorder of mucopolysaccharide catabolism. It has been classified by McKusick as mucopolysaccharidosis IV (for review see [1- 3]). Classically, affected individuals are of normal intelligence and dwarfed, exhibiting characteristic facies and a number of skeletal anomalies, including universal platyspondyly, hypoplasia of the odontoid process, sternal protrusion, and obliquity of the lower ends of the radius and ulna, and they generally develop corneal clouding. Other characteristic features include enamel defects, joint laxity, and a variable increase in the urinary excretion of keratan sulfate and excessive accumulation of it in cartilage cells. This classical form of the Morquio syndrome is caused by the inactivity of a sulfatase, which is thought to cleave N-acetylgalactosamine-6-sulfate and galactose-6sulfate ester linkages that are present in chondroitin-6-sulfate and keratan sulfate, respectively [4- 7]. A mild variant of the syndrome can be related to different properties of the deficient sulfatase (unpublished observation). Storage and excretion of keratan sulfate is not unique for the Morquio syndrome. It is frequently observed in GM, gangliosidosis, an inherited lysosomal storage disorder which is caused by the deficiency of acid a-gal [8]. GM1 gangliosidosis exhibits phenotypically a great variance regarding the onset of symptoms, rapidity of central nervous system deterioration, severity of bone abnormalities, and development of hepatosplenomegaly (for review see [9]). However, all forms have in common a progressive psychomotor deterioration. Recently, O'Brien et al. [ 10] and Arbisser et al. [1 1 ] each described a patient with mild spondyloepiphyseal dysplasia, corneal clouding, absence of central nervous system abnormalities, and markedly reduced 8-gal activity. In one of the patients [11], excessive excretion of keratan sulfate was observed and the disorder designated as mucopolysaccharidosis IV B. The two patients we report appear to have a mild form of the Morquio syndrome: in leukocytes and cultured skin fibroblasts, normal N-acetylgalactosamine-6-sulfate sulfatase but markedly diminished ,8-gal activities were found when tested under the usual assay conditions. We show that it is the affinity to substrate and the catalytic property of the enzyme which are affected by mutation. MATERIALS AND METHODS

Fibroblast cultures were obtained from skin biopsies and maintained in culture as described [12]. Leukocytes were prepared according to [13]. For the isolation of urinary mucopolysaccharides, 500 ml of 24 hr urine specimens was loaded on a 100 ml Dowex I x 2 column, 200-400 mesh, equilibrated with 0.1 M NaCl. The column was washed with 500 ml 0.3 M NaCl and then developed with 300 ml 3 M NaCl. The latter fraction was dialyzed against water and made 75% (v/v) with ethanol and 1% (w/v) with potassium acetate. The precipitate was collected by centrifugation, washed with ethanol, dried with diethyl ether, and analyzed for uronic acids [14], hexosamine [15], sulfate [16], and galactose [17]. Chondroitin-4- and -6-sulfate were quantitated after digestion with chondroitin AC lyase and spectrophotometric analysis of unsaturated disaccharides separated by descending paper chromatography [18]. The amount of keratan sulfate was calculated from the analytical data of that material, which was obtained after digestion with chondroitin ABC lyase [18] and treatment with ethanol and potassium acetate as described above. Qualitative tests for urinary keratan sulfate and oligosaccharides were performed by thin-layer chromatography [19, 20].

260

GROEBE ET AL.

For enzyme activity determinations, fibroblasts were suspended in 0.15 M NaCI and leukocytes in 0.15 M NaCi, containing 0.2% (w/v) Triton X- 100. Homogenization was performed by ultrasonication. The reaction mixture for the assay of p-nitrophenyl-p-D-gal contained 0.04 M citratephosphate buffer, pH 4.4, 0.108 M NaCi, up to 0.04 M p-nitrophenyl-,8-D-galactopyranoside and 20 ,g- 165 gg cell protein; final volume, 120 ,ul. Following incubation for 90 min at 370C, the reaction was stopped by the addition of 1 ml 0.4 M glycine/NaOH-buffer, pH 10.4. Readings were taken at 405 nm. 4 MU-,8-D-gal was determined as reported [21]. GM, ganglioside-,8-gal [22] was assayed as follows. Appropriate stock solutions of ganglioside GM, chromatographically isolated from brain lipids of a case of Gm, gangliosidosis (1 year, 4 months at death) and of crude sodium taurocholate (Merck-Schuchardt, Darmstadt, West Germany, no. 12353) in chloroform/methanol (2:1, v/v) were prepared. Eleven nmoles ganglioside and 0.2 mg taurocholate were dried from the solution. Fifty jil fibroblast homogenate and 10 ,.l 0.25 M sodium acetate buffer, pH 4.5, containing 0.5 M NaCl were added. After shaking, the suspension was incubated for 2 hrs at 37°C. Thirty ,ul of the suspension (non-incubated assay for blank value) was dried with 1 ml acetone at 50°C. The residue was extracted with 0.3 ml chloroform/methanol 2:1, and the extract applied to silica gel thin-layer plates (MerckSchuchardt, no. 5721). Plates were run twice in chloroform/methanol/water ( 14:6: 1, v/v/v) and spots visualized by spraying with anisaldehyde/sulfuric acid/acetic acid (1:4:200, v/v/v) and heating (15 min, 1 20°C). The green ganglioside spots (GM, and, as the enzymic reaction product, GM2) were scanned by densitometry. From peak areas, the percentage of degraded ganglioside Gm, was calculated. Galactosyl ceramide-,3-gal was determined according to [23]. The incubation mixture for measurement of lactose-,/-D-gal contained 0.04 M citratephosphate buffer, pH 4.4, 0.108 M NaCI, 83 mM lactose, and protein; final volume, 120 ,lI. Liberated galactose was determined enzymatically with galactose dehydrogenase [17]. To determine the activity of a keratan sulfate-degrading A-gal, a radioactively labeled substrate was obtained as follows. Bovine corneas (10 g) were incubated in 40 ml complete tissue culture medium [12] containing 5 mCi D-[1-3H]galactose (specific radioactivity: 8 Ci/mmol) for 6 hrs at 37°C. A keratan-sulfate-rich fraction was obtained from the tissue by papain digestion and stepwise chromatography of ethanol-insoluble material on Dowex 1 x 2 [24]. Material desorbing from the resin between 1.3 M and 3.0 M NaCl was dialyzed against water, precipitated with ethanol, and digested with chondroitin ABC Iyase [18]. Proteins were removed by precipitation with trichloroacetic acid, and after dialysis against water, the polysaccharide was recovered by ethanol precipitation. It was free of uronic acids and contained, per mg, 1.42 ,umol hexosamine, 1.45 ,umol galactose, and 1.35 ,umol sulfate. The specific radioactivity was 0.6 ,uCi/mg. After acid hydrolysis (6 M HCl, 4 hrs, 105°C), no radioactive material migrating toward the cathode during high voltage electrophoresis at pH 3.9 [25] was found. The mixture for determination of keratan sulfate-,8-D-gal activity consisted of 0.04 M sodium acetate buffer, pH 4.25, 0.08 M NaCl, enzyme, and 50 ,ug keratan sulfate; final volume, 50 ,ul. Incubation was for 15 hrs at 37°C. Liberated galactose was measured after descending paper chromatography in ethyl acetate/pyridine/water (100:35:25, v/v/v). All the migrating radioactivity exhibited the same mobility as D-galactose. No radioactivity comigrated with D-mannose, L-fucose, or N-acetylneuraminic acid. To determine the ASF ,8-gal activity, ASF prepared from fetuin by acid hydrolysis [26] was digested for 48 hrs with galactose oxidase (Sigma, St. Louis, Mo.) under conditions as described [27]. The digest was dialyzed overnight against 0.1 M sodium borate, pH 8.0, and reduced with 32 nmoles NaB[3H]H4 (specific activity: 270 mCi/mmol) overnight. The reaction was quenched by an excess of unlabeled sodium borohydride. After adding 50 Il acetic acid, the sample was dialyzed for 48 hrs against 0.1 M sodium citrate, pH 4.5, containing 3 mM NaN3. ASF /-gal activity was assayed as follows. Thirty nmoles [3H]galactose bound to ASF was incubated with 5 ,ug- 40 ug cell homogenate in 70 mM sodium citrate, pH 4.6, containing 90 mM NaCl and 0.6 mM NaN3 in a final- volume of 50 ,ul for 10 hrs. The reaction was stopped by adding 10 ,u1 of 5% (w/v) bovine serum albumin, 60 p.l of 13.9 phosphotungstic acid in 2 M HCl

MORQUIO SYNDROME

261

and kept for 4 hrs at 40C. After centrifugation for 6 min at 8000 g, aliquots of the supernate were assayed for radioactivity. Blanks were incubated with boiled homogenate. Under the assay conditions described above, the reaction was linear with time for at least 12 hrs, and the split product comigrated with galactose on paper chromatography in ethyl acetate/pyridine/water (100:35:25, v/v/v). Activities of N-acetylgalactosamine-6-sulfate sulfatase [25] and of acid glycosidases [28] were determined as described. Protein was quantitated by staining with amido black [29]. The procedure for electrophoretic separation of 8-gal on cellulose acetate gel (Cellogel from Chemetron, Milano, Italy) followed closely the procedure of Fluharty et al. [30], except that the pH of the separation buffer was raised to pH 6.5 and incubation was carried out for 2 hrs. CASE REPORTS

Case I A. K., a male patient, born after an uneventful pregnancy on March 14, 1972, is the second child of Greek parents. The first child, an eight-year-old daughter, is clinically normal. There is no evidence of consanguinity nor history of bone disease in either family. The patient, A. K., had a birth weight of 3.8 kg. Postnatal development was normal, and psychomotor milestones were achieved at the normal times. Approximately at age 4, the parents noticed that the patient was small when compared with other children. At age 6, severe back pains occurred after a bicycle accident, and medical consultation was sought. An anomaly of the dorsal spine was found. At 6 years, 3 months, the boy was admitted to the Children's Hospital of Munster. We saw an alert child with normal behavior and normal intelligence. The stature was short (body length: 107 cm; below the 3rd percentile), the upper segment-lower segment ratio (0.80) below the one standard deviation, head circumference 52.5 cm, and the facies appeared normal (fig. IA and B). Physical examination revealed a scoliosis of the spine and a mild thoracolumbar kyphosis. Dorsal spine motion was moderately limited, with full range of motion in all other joints. There was no abnormal ligamentous laxity, and the gait was normal. Teeth were partially carious but without signs of hypoplasia. There were no cardiac murmurs. The liver was palpable 1 cm below the costal margin; the spleen was not enlarged. Neurologic findings and hearing were normal. In both corneas, subepithelial opacities were found by split lamp examination. Fundoscopy showed no retinal abnormalities. Lymphocyte vacuoles were not found in Pappenheim and Alcian blue-stained blood smears. The roentgenologic examination revealed signs of a dysostosis multiplex with hyperplasia of the odontoid process, platyspondyly, tonguelike protrusion of anterior portions, wedge shape of the third lumbar vertebra, ileal stenosis with factitious enlargement of acetabular cavities, partially resorbed femoral heads, widened femoral necks, coxa valga, hypoplasia of proximal carpals, and conical appearance of proximal metacarpal ends. Case 2 Patient R. E. was born at term on July 30, 1953, to a 32-year-old woman and a 34-year-old nonrelated man, both of Austrian origin. The mother reported increased amniotic fluid during pregnancy, but delivery was without complications. No bone diseases are known in either family. Three older siblings are healthy. Birth weight was 4.25 kg, and body length 53 cm. The neonatal period was uneventful. Significant retardation was noticed at 2- 3 years of age: the child fatigued rapidly, there was failure to thrive, and growth retardation became apparent. He attended school successfully and was trained at an institution for handicapped children. His occupation is that of a shoemaker. At age 18, a bilateral osteotomy was performed because of severe genua valga. Physical examination at 25 years, 5 months, revealed a white male with a poorly proportioned trunk, a height of 137 cm (below the 3rd percentile), and a weight of 41 kg. Head circumference and configuration were normal in accordance with age, but appeared too large with respect to body length. The neck was particulary shortened, and the sternum protruded (pectus carinatum).

GROEBE ET AL.

262

J.5

........

FIG. 1 A and B Patient A. K. at age 6 yrs 3 mos C and D patient R. E. at age 25 E and F, Patient R. E., upper cervical spine, lateral (E) and ap (F) tomogram; bilateral, parasagittal basilar impression with condylar hypoplasia; ossification defect in center of odontoid process. G, Lateral view of lumbar spine of patient R. E. irregular platyspondyly, wedge shape, of 11Ith thoracic vertebra. H, Hands of patient R. E. sloping of distal extremities of radius and ulna toward each other; irregular shape of carpals, squared ends of metacarpals, and shortening of metacarpal V and third and fourth distal phalanx. I, Pelvis of patient R. E. bilateral dysplasia of acetabular roof;, destruction of femoral heads, and shortening and thickening of femoral

necks; coxa valga.

The facial appearance was normal. The extremities appeared prolonged in comparison to the short trunk (fig. IC and D). The hands appeared clumsy because of shortened terminal phalanges. A mild hyperfiexion in the hip and knee joints was due to moderate muscular atrophy and hyperfiexibility of the wrist and toes. All other joint motion was normal, and there was no loose-jointedness. Genua valga and pedes plani were markedly pronounced. The patient exhibited a waddling gait. Heart examination showed no abnormalities. Liver was palpable 4 cm

MORQUIO SYNDROME

263

below the costal margin; spleen was not palpable. Liver scintigraphy revealed an increased size with regard to body surface of the patient; it corresponded to a body height of 170 cm and a weight of 68 kg. Spleen size was normal relative to the liver, but increased relative to the actual body surface. Split lamp examination showed diffuse clouding of both corneas. Hearing was normal. Psychological testing by two different methods revealed normal intelligence. In a Pappenheim-stained blood smear, lymphocyte vacuoles were not observed. X-ray examination revealed a bell-shaped thorax with protruding sternum. The signs of dysostosis multiplex included odontoid abnormality, platyspondyly, tonguelike protusions of the vertebral bodies, slight thoracolumbar kyphosis, dysplasia of the acetabular roofs and of the proximal and distal epiphyses of the humerus and femur, and moderate abnormalities of the hand bones (figs. IE, IF, IG, IH, and II).

LABORATORY FINDINGS

Excretion of Mucopolysaccharides and Oligosaccharides Both patients excreted abnormally high amounts of urinary mucopolysaccharides, although considerably less than patients with mucopolysaccharidoses I or II. The daily excretion was 58 mg for patient R. E. and 56 mg for patient A. K. (controls = 5 mg- 20 mg). Keratan sulfate showed the largest increase over the norm. In the urine of patient R. E., keratan sulfate accounted for 31% of total mucopolysaccharides; in patient A. K., for 26%. Interestingly, no relative increase of the excretion of chondroitin-6-sulfate was detected, which had frequently been observed in classical Morquio disease with N-acetylgalactosamine-6-sulfatase deficiency. It accounted for 15%-23% of total urinary mucopolysaccharides of patients and controls. Excessive keratan sulfaturia was detected by thin layer chromatography [19]. On thin layer chromatography of urinary oligosaccharides, two spots were detected which are not found in normal urine but are seen in the urine of patients with Gm, gangliosidosis (fig. 2).

Lysosomal Enzyme Activities in Leukocytes and Skin Fibroblasts A striking deficiency of p-nitrophenyl-18-gal was present in peripheral leukocytes from the affected patients. Normal N-acetylgalactosamine-6-sulfate sulfatase activities were found. Determination of acid ,8-gal in the leukocytes of one of the patients' parents revealed residual activities in the range of 49% - 72% of the controls (table 1). ,8-Gal deficiency was confirmed by the analysis of cultured fibroblasts. As shown in table 2, the enzyme deficiency could be detected by employing all principal types of substrates of the enzyme: aryl glycosides, glycolipids, glycoproteins, polysaccharides, and disaccharides. Only galactosylceramide-,8-D-gal activity was in the range of the controls. Depression of p-nitrophenyl-,8-D-gal activity was not due to the presence of excessive amounts of an enzyme inhibitor. Mixtures containing different proportions of normal and deficient homogenates gave the expected 3-gal activities (fig. 3). The activity of N-acetylgalactosamine-6-sulfate sulfatase, the deficient enzyme in classical Morquio disease, was normal (table 2) as well as the activities of B3-N-acetylglucosaminidase, ,8-glucosidase, a-N-acetylglucosaminidase, a-galactosidase, ,8-glucuronidase, and neuraminidase (not shown).

264

GROEBE ET AL.

FIG. 2. -Thin layer chromatography of urinary oligosaccharides: results of a control (Co), of patients A. K. and R. E., and of a patient affected with GM, gangliosidosis. Small arrows mark abnormal oligosaccharides in urine of patients A. K. and R. E. Migration of fructose (a), lactose (b), and raffinose (c) are indicated on left.

Characterization of Residual (3Gal Activity The influence of the substrate concentration on p-nitrophenyl-,/-n-gal activity was calculated from Hofstee-plots (plot of enzyme activity, v, vs. quotient of enzyme activity and substrate concentration, v/S, [31 ]) using the advantage of a rather uniform distribution of data along the curve. Normal fibroblasts showed regularly a complex kinetic behavior of acid (B-gal with a stimulation of enzyme activity at higher substrate concentration. Straight lines with different slopes were computed for high and low substrate concentrations, according to the least squares method. In accordance with previous findings [32], maximal activity seemed to be caused by an enzyme form with low affinity for the substrate. Conversely, at lower substrate concentrations, a "high affinity enzyme" seemed to be active (fig. 4A). Without being able to study the purified ,8-gal, a simple interpretation of these data was preferred, and Km values for both low and high substrate concentrations were calculated.

MORQUIO SYNDROME

265

TABLE 1 ENZYME ACTIVITIES IN PERIPHERAL LEUKOCYTES

ENZYME SOURCE

Patient R. E. ........................... Patient A. K. ........................... Mother of A. K. ............ ............. Father of A. K. Controls ................................

.........................

ENZYME ACTIVITY

(NMOL/MIN/MG PROTEIN)

f3-Gal

N-Acetylgalactosamine6-sulfate sulfatase

0.54 0.67 4.4 6.4 9.0 ± 1.8 (no. =5)

0.0046 0.0054

Not determined Not determined 0.006 ± 0.0015 (no. = 10)

NOTE. -,-Gal activity was measured with p-nitrophenyl-,-D-galactopyranoside as substrate at a concentration of 8.3 nmol/liter.

As for the normal enzyme, complex kinetics were also found for the fibroblast enzyme of the patients (fig. 4B). Especially at high substrate concentrations, however, the Km's were almost two orders of magnitude higher than the Km of the normal enzyme (table 3). On saturation with substrate, the maximal enzyme activity in the patients' fibroblasts would then amount to 19% - 33% of the mean normal a-gal

activity. Slight differences in the pH-dependence between normal and abnormal ,8-gal were observed; the patients' enzyme exhibited a broader and more alkaline pH optimum. Between pH 3.7-4.7, the patients' enzyme exhibited 80% of the maximal activity (controls from pH 3.5 to 4.0). Thermal stability at 70TC and electrophoretic mobility (fig. 5) did not differ from the controls.

0.5-

0.4-

0.3 uW03

XA

0.2-

0.1I

20

40

60

l 80

20 40 CONTROL FIBROBLASTS (% of homogenate mixture)

60

l 80

FIG. 3. -,-Ga1 activity in mixtures of homogenate of control fibroblasts and of fibroblasts derived from patient R. E. (A) and patient A. K. (v) and assayed forp-nitrophenyl-fl-D-gal activity.

GROEBE ET AL.

266

oco

C),-,

0

_1-

00o

11

6r.

11

0

*o

rr- cl, C14

8 66

0

94

Z

11

0

< _z

8 6

00 st

0

88 Oo

z .-

Gn

.D 0

m m 0

- o

oo O o

mA z_

z

F. Cw

Z,

1% Z w o

*

m oo

O

oo o o

oo o

v

8-1

* u: w

...... i......l ...... ....

-

w

-t

S

.Q

w

cn

b4$ Q

2 0

_o

._

[o w

*_

u:

w Q zv w c) w -

o ^ _ Q

c) Q

w