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Hyaluronan (hyaluronic acid) is one of the major glycosaminoglycans and has a vital role in many physiologic processes.1,2,3,4 Genetic deficiencies of most of the lysosomal enzymes that catalyze the degradation of glycosaminoglycans have been identified, with the exception of hyaluronidase, a lysosomal endoglycosidase that catalyzes the degradation of hyaluronan.5,6,7 We describe the clinical, pathological, and biochemical findings in a child with short stature and multiple periarticular soft-tissue masses who proved to have a storage disease of hyaluronan due to a genetic deficiency of hyaluronidase.
Case Report
A 14-year-old girl had a normal early medical history except for frequent episodes of otitis media and a "ganglion" that was excised from her left wrist when she was 6 months old. At the age of 7 1/2 years, she noted a soft-tissue mass over the lateral aspect of her left ankle. During the next year, additional periarticular masses developed involving the proximal right second finger, the left popliteal fossa, the left inferior patella, and the right and left lateral malleoli. Biopsies of the ankle and finger lesions and the skin were performed. At the age of 9 1/2 years the patient had the first of several episodes of transient, painful swelling of the periarticular masses and generalized cutaneous swelling. These episodes accompanied or followed exertion or a febrile illness and were self-limited, resolving spontaneously within a period of 72 hours.
Between the ages of 8 1/2 and 14 years, her growth rate declined from about the 15th percentile to below the 5th percentile, some of the periarticular masses enlarged, and new masses and right-foot pain developed. Otherwise, her general health was good. At the age of 14 years, her head circumference was 53 cm (25th percentile), she weighed 43.8 kg (25th percentile), and she was 145.5 cm tall (<5th percentile). She had mildly dysmorphic craniofacial features with a flattened nasal bridge, bifid uvula, and a submucosal cleft palate. There was full range of motion of all joints and no scoliosis, kyphosis, adenopathy, or organomegaly. Her ophthalmologic and neurologic examinations were normal, and her academic performance was good. The family history and the results of routine blood chemical measurements were unremarkable.
Radiographic Findings
Radiographs of the ankles and knees at the age of nine years showed prominent bilateral nodular, periarticular soft-tissue masses. The masses were not calcified, there were no bony erosions, and the joint spaces were preserved. Magnetic resonance imaging of the left knee at the age of 10 years revealed a nodular synovium, a popliteal (Baker's) cyst, and a large joint effusion. Apart from the recent development of mild chondrocalcinosis of the right talonavicular joint, the patient's ankle and knee joints had not changed radiographically since the age of 10.
Radiographs of the pelvis at 12 years of age revealed multiple bilateral nodular, intraarticular soft-tissue masses and acetabular erosions (Figure 1). Computed tomography and magnetic resonance imaging of the hip joints confirmed the presence of the masses and erosions and also revealed bilateral joint effusions and a possible cyst of the right trochanteric bursa. The results of other radiographic studies, including chest and spine x-ray films (at 10 and 14 years of age), radionuclide bone and liver–spleen scans (at 10 years of age), abdominal ultrasonography (at 10 years of age), and cranial magnetic resonance imaging (at 10 years of age), were normal.
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Histochemical and Electron-Microscopical Analyses
Fresh tissue specimens were processed routinely for light microscopy. Hematoxylin and eosin and special histochemical stains were applied according to standard procedures.8,9 Tissues for ultrastructural analyses were fixed and processed as described elsewhere.10
Biochemical Studies
Urinary mucopolysaccharides and oligosaccharides, lysosomal-enzyme activities, and the results of a fibroblast sulfatide-loading study were analyzed as described elsewhere.11,12,13,14,15,16,17,18 Plasma and serum hyaluronidase activity was determined with human umbilical-cord hyaluronan (Sigma Chemical, St. Louis) (250 mg per liter) as the substrate. The results were similar for either type of specimen.18 Plasma hyaluronidase was also assayed with chondroitin (Seikagaku, Tokyo, Japan) (250 mg per liter) as the substrate. The results of these assays were normalized to standard curves of N-acetylglucosamine and N-acetylgalactosamine, respectively, and were expressed as milliunits per liter, a unit of activity representing the production of a micromole of reaction product per minute at 37°C. Plasma hyaluronan concentrations were determined by a 125I-labeled hyaluronan-binding-protein assay (HA Test radiometric assay, Pharmacia Diagnostics, Uppsala, Sweden) according to the manufacturer's instructions.
Results
Histologic and Electron-Microscopical Findings
Histologic examination of the patient's ankle and finger masses showed similar pathologic changes. The ankle lesion consisted of synovium with marked villonodular transformation (Figure 2A, Figure 2B, Figure 2C, and Figure 2D). The villi were bulbous and contained many large histiocytes that had abundant cytoplasm filled with clear vacuoles. The vacuoles stained with Alcian blue and colloidal iron, indicating the presence of mucopolysaccharide, and the degree of staining was reduced by pretreatment with hyaluronidase.
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Ultrastructurally, the histiocytes from both the ankle and finger lesions were nearly filled with numerous large membrane-bound vacuoles whose appearance was consistent with that of lysosomes. The vacuoles contained a flocculent, medium-dense material, as well as a small number of dense secondary lysosomes (Figure 2A, Figure 2B, Figure 2C, and Figure 2D). Fibroblasts also contained the membrane-enclosed vacuoles with flocculent material but in lower quantities than the histiocytes. Endothelial cells and vascular smooth-muscle cells appeared normal. Fibroblasts in a separate skin-biopsy specimen also showed lysosomes filled with fibrillar material (data not shown).
Biochemical Findings
The results of analyses of urinary mucopolysaccharides and oligosaccharides and numerous lysosomal-enzyme activities were normal. The patient had no plasma hyaluronidase activity (Table 1); a mixing experiment that used various proportions of serum from the patient and pooled serum from control subjects provided no evidence of an inhibitor of enzyme activity (data not shown). The plasma hyaluronidase activity of the patient's father and mother was 30 and 53 percent of normal, respectively, and two of her grandparents also had low or intermediate levels of enzyme activity (Table 1). The patient also had no detectable plasma hyaluronidase activity in assays in which chondroitin was used as the substrate, whereas the activity in plasma from her parents was 38 and 58 percent of normal (Table 1). The plasma concentrations of hyaluronan ranged from 918 to 2118 µg per liter in three specimens from the patient and were 10.4 and 8.4 µg per liter in plasma from her father and mother, respectively (mean [±SD] value in normal subjects, 23.7±14.6 µg per liter). Plasma hyaluronidase activity was normal in 56 patients with 25 different lysosomal storage disorders, and there was no marked elevation of the plasma hyaluronan concentration in 16 patients with other storage diseases (data not shown).
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Hyaluronan, one of the principal glycosaminoglycans of the extracellular matrix, is a high-molecular-weight polymer of repeating units of [N-acetylglucosamine (1-
-4) d-glucuronic acid (1--3)]n that is synthesized in the plasma membrane of cells and translocated to the pericellular space. It is believed to have numerous important biologic functions, including the modulation of cell proliferation, migration, and differentiation and the regulation of extracellular water and protein homeostasis. It is also an integral structural component of cartilage and other tissues and acts as a lubricant in joints.1,2,3,4 Hyaluronan is catabolized locally or after clearance by the lymphatic system and hepatic endothelial cells. Intracellular degradation occurs after endocytosis and is followed by digestion by hyaluronidase, a lysosomal endoglycosidase that cleaves the 1--4 N-acetylhexosaminide linkages.1,4
We describe a patient with the mucopolysaccharidosis hyaluronidase deficiency. We based our conclusion on the following findings: pathologic storage of mucopolysaccharide in lysosomes of histiocytes and fibroblasts, the markedly elevated plasma hyaluronan concentration, and the complete deficiency of plasma hyaluronidase activity. The decreased plasma hyaluronidase activity in the patient's parents and in two of her grandparents, together with the other data, indicates that the enzyme deficiency is an autosomal recessive genetic disorder.
The principal abnormalities in our patient were periarticular soft-tissue masses composed of nodular aggregates of histiocytes, acquired short stature, and erosions of the acetabula. The first of these findings is unique for the mucopolysaccharidoses but not surprising. Hyaluronan concentrations are especially high in synovial fluid, and the highest concentrations of hyaluronan in solid tissues are in cartilage and skin.1 In addition, much of the catabolism of hyaluronan in joints occurs locally.1,19 Consequently, the loss of a major mechanism of hyaluronan catabolism, its degradation by hyaluronidase, could result in the development of periarticular soft-tissue masses. The histiocytic predominance at those sites might be the result of the hyaluronan-induced aggregation of macrophages.20 The marked intracellular storage of substrate in macrophages was presumably due to receptor-mediated internalization of hyaluronan by these cells, in conjunction with the enzymatic deficiency.4,21,22 The lesser involvement of fibroblasts (and of the skin) and, possibly, other tissues may be due to less efficient internalization of hyaluronan, the delivery of hyaluronan to the lymphatic system, or other enzymatic or nonenzymatic degradative pathways.23,24,25,26
The patient's short stature might be the result of impaired function of growth-plate chondrocytes or abnormal properties of the extracellular matrix. Hyaluronan is internalized by chondrocytes by a receptor-mediated process27 and might, if it accumulates, cause dysfunction of these cells; intracellular accumulation of macromolecular substrates and consequent chondrocytic abnormalities have been noted in other lysosomal storage disorders.28,29 Hyaluronan reduces the synthesis of constituents of the matrix of hyaline cartilage by chondrocytes and their precursors2,3 and interacts with other matrix components.1,4 Abnormal concentrations of hyaluronan could alter the normal biochemical and physical interactions between hyaluronan and other matrix components and thereby alter the assembly and stability of the skeletal matrix. Any of these pathophysiologic mechanisms could result in short stature.
It is curious that the periarticular aggregates of histiocytes caused bone erosions detected only in the acetabula. Activated macrophages secrete a variety of cytokines that can stimulate localized osteoclastic activity.30 Their accumulation in an anatomically tight joint such as the hip joint, in which the cells are in close proximity to bone, may explain why these joints are the only sites where bone erosions were present.
The lack of hyaluronidase activity, coupled with continued biosynthesis of its substrate, suggests that the patient may be at risk for slow progression of her condition. The success of exogenous enzyme replacement in patients with type 1 Gaucher's disease,31 another lysosomal storage disorder that predominantly involves the reticuloendothelial system, suggests that the administration of hyaluronidase may be an appropriate form of treatment.
We are indebted to Drs. James England, Richard I. Kelley, Elizabeth M. Prence, Jerry N. Thompson, and Barbara Triggs-Raine for their critical evaluations of this work; to the members of the Division of Medical Genetics, Shriver Center for Mental Retardation, for performing the assays of lysosomal enzymes and metabolites; and to Dr. Joe Alroy, New England Medical Center, for performing the electron-microscopical analysis of the skin-biopsy specimens.
Source Information
From the Division of Medical Genetics, Shriver Center for Mental Retardation, Waltham, Mass. (M.R.N., Y.W.); and the Departments of Neurology (M.R.N., M.P.S., K.B.S.), Pathology (M.R.N., M.P.S., G.R.D., A.E.R.), Radiology (D.I.R.), and Orthopedic Surgery (M.C.G.), Massachusetts General Hospital, Boston.
Address reprint requests to Dr. Natowicz at the Division of Medical Genetics, Shriver Center for Mental Retardation, 200 Trapelo Rd., Waltham, MA 02254.
References
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