April 1976 TheJournalofPEDIATRICS
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The acro-osteolysis syndrome: Morphologic and biochemical studies The acro-osteolysis syndrome consists of dissolution of terminal phalanges of the hands and feet, dolichocephaly with multiple wormian bones, delayed closure of cranial sutures, absence of frontal sinuses, a prominent occipital ridge, skeletal demineralization, vertebral and extremity fractures, joint laxity, and coarse hair. Studies of bone morphology reveal diminished bone density and bone formation. Osteoblasts have widely dilated smooth endoplasmic reticulum. It is postulated that an abnormality of a structuralprotein is the pathogenic basis of this disease.
David M. Brown, M.D.,* David S. Bradford, M.D., Robert J. Gorlin, D.D.S., Robert J. Desnick, M.D., Ph.D., Leonard O. Langer, Jr., M.D., Jenifer Jowsey, D.Phil., and John J. Sauk, Jr., D.D.S., Minneapolis, Minn.
AN UNUSUAL FORM of cranioskeletal dysplasia with peripheral dysostosis and spinal osteoporosis was d e scribed originally by Hajdu and Kauntze 1 in 1948. Subsequently 14 additional cases of this disorder, which has been called the acro-osteolysis syndrome, have b e e n described. 2-1~The prominent features consist of dissolution of terminal phalanges of the hands and feet, bizarreshaped dolichocephalic skull with multiple wormian bones, delayed closure of cranial sutures, absence of frontal sinuses, prominent occipital ridge, small mandible and maxilla and early loss of teeth, generalized skeletal demineralization particularly affecting the vertebra frequently resulting in fractures, laxity of joints, thick and coarse hair, conductive hearing loss, and speech impairment. The etiology and pathogenesis of this disorder are unknown. Two additional cases and results of relevant morphologic and biochemical studies will be presented. From the Departments of Pediatrics. Laboratory Medicine and Pathology, Orthopedic Surgery, Oral Pathology, and Human and Oral Genetics, University of Minnesota, and Mayo Medical School Supported by grants from the National Institutes of Health (AM-08658, DE-03686, and 5-MO1-RRO0400) and the National Foundation. *Reprint address: Mayo Box 491, University of Minnesota Hospital, Minneapolis, Minn. 55455.
METHODS The patients were hospitalized in the Clinical Research Center. By history each patient had ingested a minimum of 1,000 mg of calcium and 400 units of vitamin D per day for the previous several years. Serum and urine chemistry determinations were performed at the clinical laboratories of the University of Minnesota Hospitals. Total urinary hydroxyproline was determined by the method of Kivivikko and associates. 11 Urinary acid mucopolysaccharides were performed by the carbazole method of Teller and associates? ~ Serum parathyroid hormone levels were determined by radioimmunoassay. 13 Abbreviation used AMP: acid mucopolysaccharide
f
The femoral trabecular-pattern index of Singh and associates14 was determined as an indication of the loss of trabecular bone. Bone density was determined by a photon attenuation external scanning method. 15 The evaluation of bone morphology in biopsied tissue obtained from the iliac crest was performed by quantitative microradiography as previously described. 16' 17 Comparable bone samples were obtained from six persons for each control group between the ages of 6 and 8 and 15 and 17 years, who had died suddenly and w h o had not been
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Fig. lB. Case 1. Note short, broad hands and spade-fike distal phalanges. Fig. 1A. Case 1. Note broad face, maxillary and mandibular hypoplasia, thick eyebrows, and coarse hair.
hospitalized. Videodensitometry, a technique which is used to evaluate the percentage of a bone biopsy which contains bone, was performed by scanning a standardized photograph of a microradiograph of the specimen using a closed-circuit television camera which is digitized with a computerized, high-resolution videodensitometry system (details of this method are available from the authors). Skin-derived fibroblasts were grown in DulbeccoVogt's modification of Eagle's medium buffered with Hepes buffer containing penicillin, 100 U/ml, and streptomycin, 100 /Lg/ml, and 10% fetal calf serum using Falcon plastic flasks. Cells were subcultured using 0.125% trypsin. Bone and dentin, and skin, fibroblast culture, and hair bulbs were prepared for transmission electron microscopy after fixation in 6% or 3% (respectively) cacodylatebuffered glutaraldehyde, pH 7.0 at 4~ for two hours and postfixed in cacodylate-buffered 1% osmium for one hour. Fibroblasts were fixed in situ, removed from the surface of the flask by scraping with a rubber policeman, and sedimented by centrifugation. The cell button was resuspended in cacodylate buffer and again washed and centrifuged two times after which t h e button was postfixed and processed. All specimens were embedded in epon, and thin sections were obtained on an LKB ultramicrotome. The samples were then stained with lead
citrate and uranyl acetate and observed in a Phillips 301 electron microscope at 80 kv. Specimens for scanning electron microscopy were fixed as described above, dehydrated in alcohol and critical point dried from Freon in a Bomar critical point dryer. The specimens were mounted on coverslips and coated with carbon-gold. The samples were observed in a Cambridge scanning electron microscope at 20 kv. The scale structure of hair was described by measuring the distance between scale margins, the thickness of the scales, and the amplitudes of the scales. Plasma and isolated leukocytes for enzymatic analyses were obtained from the patients with acro-osteolysis and age- and sex-matched normal control subjects. 'Leukocytes were isolated from heparinized blood by modification of the method of Snyder and Brady? ~ Plasma and leukocyte enzyme assays. The activities of a-galactosidase A and B at pH 4.6, fi-hexosaminidase A and B at pH 4.4, a-mannosidase at pH 4.4 and 6.0, fl-galactosidase at pH 4.5, a-glucosidase at pH 4.0 and 6.0, and fi-glucuronidase at pH 3.6 were determined fluorometrically in plasma and leukocytes as previously described? .... Aryl sulfatase A and B activities were assayed by the method of Baum and associates. ~1 CASE REPORTS Case 1. Patient D.L. was a 789 girl who was referred for evaluation of unusual facies and nasal-like speech. She had
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Fig. 1C. Case 1. Note dolichocephaly, open sutures, prominent wormian bones, unusually shaped sella turcica, and prominent occipital ridge.
sustained a fracture of the left tibia and fibula at 6 years of age. She had difficulty walking and recurrent back pain. U p p e r and lower incisors had been lost at 4 to 5 years of age. There were five normal siblings and a 19-year-old brother with Down syndrome. Prominent features on physical examination included an unusual appearance characterized by a broad face with thick eyebrows and coarse and stiff hair (Fig. 1A). Her speech had a nasal character. Height was 110 cm and weight 16.3 kg, both o f which were greater than two standard deviations below the normal mean for age. The anterior fontanelle measured 3 x 3.5 cm. A prominent horizontal occipital ridge was palpable. The hard palate was high arched, and velopharyngeal incompetence was diagnosed. The digits of the hands and feet were short and broad, the distal portions appearing spade like (Fig. 1B). The skin appeared dry and coarse. Spinal extension and flexion were painful and limited all motions. There was general laxity of the joints of the extremities. There was diffuse tenderness over the lumbar spine. Her gait was somewhat shuffling with a poker back and obvious back discomfort. The neurologic evaluation was normal. The Wechsler intelligence scale was 81. Roentgenographic examination of the skull showed dolichocephaly, multiple wormian bones, and persistent anterior and posterior fontanelles with wide sutures (Fig. tC). The lambdoid suture was particularly wide. The sphenoid bone was malformed and had an abnormally shaped sella. The maxilla and mandible were small. There was generalized demineralization of the spine with compression fractures at several levels in the thoracic and lumbar regions (Fig. 1D). Mild scoliosis was noted. Osteolysis of the distal phalanges of both hands (Fig. 1E) and feet (Fig. 1F) was prominent. The long bones of the legs showed generalized demineralization and healing of the fractures of the left tibia and fibula.
Fig. 1D. Demineralized and compressed vertebra in Case 1.
Serum calcium, phosphorus, urea nitrogen, creatinine, sodium, potassium, chloride, bicarbonate, cholesterol, triglycerides, glutamic oxalacetic transaminase, total protein and electrophoretic pattern, thyroxine, triiodothyronine, parathyroid hormone, and 8 A.M. and 9 P.M. plasma cortisol were within normal limits. The serum alkaline phosphatase was 494 to 673 IU/1 (age-related normal = 225-380 IU/1, range of 2 SD). Urinary total acid mucopolysaccharide excretion was 6.4 mg/ 24 hr (normal for age = < 8 mg/24 hr) with an AMP/creatinine ratio of 1:38 (normal = < 1:4). While she was receiving a gelatin-free diet, the urinary total hydroxyproline excretion was 55.7 and 68.6 m g / m V d a y (normal for age = 37-95). During a 12day balance study (after a 3-day equilibration period) and while she was receiving an average daily intake of calcium of 723 rag/
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Fig. IF. Distal phalangeal resorption of feet in Case 1.
Fig. 1E, Distal phalangeal resorption of h a n d s in Case 1.
day (dietary Ca/P = 0.92), the patient excreted an average o f 21.4 mg/day of calcium in the urine (1.31 m g / k g / d a y ) and an average of 678 rag/day of calcium in the stool resulting in an apparent over-all calcium retention of 13 m g / d a y (sweat calcium not measured). The average percentage of tubular reabsorption of phosphate was 77.7 _+ 4.4 (SEM, n ~ 11) (N = < 82%). Case 2. Patient K.D. was a 16-year-old boy who presented with a six month history of persistent back pain and fractures of the fight and left tibia and fibula, respectively, at 6 and 15 years o f age. His hands had appeared "stubby" since early childhood. His hair had been noted to be coarse for most of his lifel There was no history of premature loss of teeth, although many of his permanent teeth were capped. No members of th e family were noted to have any of the physical characteristics of Patient K.D. or any history of fractures or back pain. Physical examination revealed a coarse facial appearance with a broad nose, coarse and bushy hair, and thick eyebrows (Fig. 2). He was 170 c m tall and weighed 56 k g . A prominent ridge was palpated horizontally over the occipital region. Speech was normal. He had a highly arched palate. Joint laxity of the extremities was prominent. He had prominent scoliosis with the
convexity in the lumbar spine. The distal digits of the hands and feet appeared spade like and shortened. No abnormalities of the skin were apparent. He had completed pubertal genital development. The neurologic examination was normal. Roentgenographic studies of the skull revealed several wormian bones in the lambdoid suture and marked dolichocephaly with a prominent occipital ridge. The mandible was small. There was marked demineralization of the spine with biconcave deformities and posterior scalloping particularly in the lumbar region. S-shaped scoliosis was noted in thoracic and lumbar regions. There was marked resorption o f the distal phalanges of the fingers and toes. Serum calcium, phosphorus, magnesium, urea nitrogen, sodium, potassium, chloride, cholesterol, triglycerides, glutamic oxalacetic transaminase, acid phosphatase, total protein and electrophoretic pattern, and parathyroid hormone levels were within normal limits. The serum alkaline phosphatase was 489 IU/1 (age- related normal = 120-450 IU/1). Urinary total acid mucopolysaccharide excretion was 11.2 m g / day (normal for age = < 12 mg/24 hr with an AMP/creatinine ratio of 1:12.6 (normal = < 1.4). While he was receiving a gelatin-free diet, the urinary total hydroxyproline excretion was 87.9 and 113 mg/m~/day (normal for age = 40-113). During a 12-day balance study (after a 3-day equilibrium period).and while lie was receiving an average daily intake o f calcium of 1,729 mg/day (dietary C a / P = 1.12), the patient excreted an average
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Fig. 2. Case 2. Note coarse facial appearance, coarse hair, and thick eyebrows. Fig. 3. Microradiograph of iliac crest biopsy. A, Patient with acroosteolysis. B, Normal 6-year-old girl. (x 16.) of 129 rag/day of calcium in the urine (2.3 mg/kg/day) and an average of 1,289 rag/day of calcium in the stool resulting in an apparent over-all calcium retention of 443 rag/day (sweat calcium not measured). The average percentage of tubular reabsorption of phosphate was 89.0 + 0.8 (SEM, n = 10). EXPERIMENTAL
RESULTS
Bone morphology. The appearance of the microradiograph of iliac crest bone of Case 1 is compared with those from a normal 6-year-old girl in Fig. 3, A and B. The thin cortex and reduced number of trabecula give an over-all appearance of reduced bone mass. In both patients, the percentage of the biopsy containing bone was diminished (24.2 and 13.9%; control subjects = 52.3 _+ 6.2 and 40.6 _+ 10.8%). Decreased trabecular pattern ratings in the proximal femur of both patients (III and IV; control subjects VI to VII) indicates the generalized nature of the diminished mineralization since more than 95% of normal persons below the age of 50 years have femoral trabecular patterns of at least grade V; individuals with grade II or III almost invariably have symptomatic osteoporosis and are generally over 60 years of age. 14 The bone density measured at the midshaft of the radius was normal in both children. In both biopsies, percentage of total endosteal bone surfaces typical of formation were diminished (2.4
and 1.0%; control subjects = 6.7 _ 2.5 and 6.2 _+ 3.2%) while the resorption surfaces were increased significantly in one patient (D.L.) (11.6 and 5.4%; control subjects = 6.0 _+ 2.4 and 7.0 _+ 1.0%). However, the osteoid width was normal in both children. Electron micrographs of iliac crest bone from patients revealed well-developed osteoblasts with distinctly developed rough endoplasmic reticulum and greatly dilated golgi zones (Fig. 4). Differentiation between golgi apparatus and smooth endoplasmic reticulum was indistinct. Osteoclasts were present and were normal. Active resorption by osteoclasts was characterized by the presence of numerous villous processes of osteoclast plasma membrane and resultant dissolution of the poorly mineralized bone matrix. Examination of the collagen fibers revealed normal diameters and periodicity. Calcium containing extracellular vesicles of bone appear normal in this disorder. Dentin from a freshly extracted tooth from one patient revealed an unaltered matrix and normal mineralization. Skin fibroblasts. Examination of skin-derived fibroblasts from both tissue culture and in fresh biopsy
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Fig. 4. Undecalcified section through iliac crest. Osteoblast from iliac crest of Case 2 with acro-osteolysis. The rough endoplasmic reticulum (R) is well developed. The central golgi zones (G) are extremely dilated and contain fine granular material. Matrix vesicles (my) can be noted near the cell periphery. Nucleus (N) and mitochondria (M) are present but unaltered. Extracellular matrices reveal an organizing organic matric (OM). However, apatite crystal are rare or not present. ( X 22,500.)
material of skin revealed normal morphology. Cellular organelles were well developed. No dilation of golgi apparatus or smooth endoplasmic reticulum were observed. Histochemical studies of these fibroblasts (not included in this report) using alcian blue and toluidine blue stains revealed no increased metachromatic staining as compared with control fibroblasts. Skin and hair. The epidermis was hyperkeratotic. Dermal collagen had normal periodicity and organization. Electron microscopy of the skin revealed normal melanocytes and well-developed keratinocytes, the latter containing numerous mitochondria and extensive tonofibrils. In addition, there was an abundance of keratohyalin granules in the stratum spinosum. The hairbulbs were normal morphologically. Keratinocytes appeared normal although keratohyalin granules were exceptionally large. Examination of the hair by scanning electron microscopy revealed two hair types, one which was distinctly thinner and contained light brown pigmentation and the other which had a larger diameter and was coarse and darker. Both types of hair had normal scale dimensions. The thinner a n d lighter hair shaft was approximately one-third the diameter of the thicker hair. Lysosomal hydrolase enzymes. The activities of WBC
and/or plasma (aryl sulfatase, a-glucosidase, fl-hexosaminidase, a-mannosidase, and fi-galactosidase) were normal or only slightly greater than the control range. However, the plasma activity of fl-glucuronidase was increased approximately 2.5 times the upper limit of the control range (812 vs control range, 90-312 nmoles/hr/ ml) while WBC fi-glucuronidase activity was normal. DISCUSSION The syndrome of idiopathic acro-osteolysis is a distinct entity involving the generalized skeleton with particularly unique features dearly indicating its identification. Its manifestations are reviewed in Table I. Most striking is the distal osteolysis but widespread cranio-skeletal dysplasia has been found in all of the cases. Acro-osteolysis may also be found in hyperparathyroidism, pycnodysostosis, progeria, epidermolysis bullosa, scleroderma, syringomyelia, leprosy, syphilis, psoriasis, trauma, neurogenic ulcerative acropathy, and p01yvinyl chloride toxicity. The etiology of the syndrome is not known. The studies of the two patients in this report revealed no abnormalities .suggesting a hormonal basis. In the two patients presented here, the serum alkaline phosphatase activities (obtained after an overnight fast) were slightly elevated. Few of the cases reported in the literature were described
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during childhood although, where reported, serum alkaline phosphatase has been normal. It has been suggested that alkaline phosphatase of bone may in large part represent pyrophosphatases which aid in the removal of inorganic pyrophosphates which in turn modify the conversion of amorphous to crystalline hydroxyapatite. 2~ The alkaline phosphatase and pyrophosphatase activity are found particularly within calcium-accumulating vesicles in the intercellular matrix of bone 2~and are associated with the process of bone formation. We do not have sufficient information on the patients reported herein to speculate as to the significance of the elevations of serum alkaline phosphatase in this disorder beyond the discussion based upon our morphologic observations. (The presence of subtle fractures may account for the elevations noted in these patients). Our studies of calcium balance suggest that there was diminished skeletal retention of calcium in one patient (D. L.) but do not reveal the basis. Although the percentage of tubular reabsorption of phosphorus was diminished in that patient, there is a significant degree of nonspecificity of that finding which precludes its use as a diagnostic test of hyperparathyroidism? 4 Morphologic studies of the bone (iliac crest) of each patient reported here show decreases in skeletal mass. The percentage of endosteal bone surfaces characteristic of bone formation was diminished in both patients, and in one resorptive surfaces were increased. The absence of excessive osteoid rules out an abnormality of mineral access to bone such as would be expected to occur in osteomalacia. Hence, these findings clearly differ from primary hyperparathyroidism and hyperthyroidism, in which increased bone resorption predominates. '~ Although in one patient (D. L.) formative surfaces were decreased and resorptive surfaces were increased, as is found in Cushing's syndrome, ~6 there was no clinical or laboratory evidence of glucocorticoid excess. The electron microscopic studies of osteoblasts in these patients demonstrated marked dilation of smooth endoplasmic reticulum channels. Several studies have pointed to the golgi apparatus as the site of elaboration of both matrix glycosaminoglycan synthesis~ as well as of glycoproteins. ~ Although in neither patient in our studies was there evidence of excessive urinary excretion of acid mucopolysaccharides (glycosaminoglycans) or of total hydroxyproline, these by no means exclude the possibility that an abnormality may exist in either glycoprotein, proteoglycan, or glycosaminoglycan metabolism. Although the unusual characteristics of the hair observed grossly and verified morphologically in terms of thickness of the hair are probably nonspecific, they do suggest the presence of a structural protein abnormality.
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Table I. Primary clinical features of idiopathic acro-
osteolysis
Feature Dolichocephaly Delayed closure of cranial sutures Prominent occipital ridge Absent frontal sinuses Small mandible/maxilla Early loss of teeth Distal phalangeal resorption Generafized skeletal detaineralization Fractures long bones Fractures/compression vertebra Kyphosis/scoliosis Joint laxity Thick, coarse hair Speech abnormalities Shortness of stature
Reported occurrence (15 patients)
Present report (2 patients)
9 4
2 2
10 9 12 13 15 7
2 1" 2 1 2 2
9 10
2 2
7 10
2 2 2 1 1
*Frontal sinuses development may not be expected in a 7-year-old child. The elevation of plasma /?-glucuronidase activity in association with normal WBC/3-glucuronidase activity is analagous to observations that have been made in other clinical circumstances. For example, I-cell disease is associated with increased activity of several plasma acid hydrolases, while the activities of the same hydrolases is decreased in fibroblasts but not in leukocytes or in other tissues. 2~ In that instance, leakage of enzymes from cells has been postulated to account for these observations.~" The significance of the observation reported in this patient with acro-osteolysis will be determined by further studies. We conclude that the idiopathic acro-osteolysis syndrome is a distinct dominantly inherited entity, the general skeletal demineralization representing a major impairment for these patients. The morphologic studies suggest an abnormality of osteoblast function and may be the basis for a postulated structural protein abnormality. We appreciate the excellent assistance of Mrs. Mary Jo Jansen in the preparation of the manuscript. REFERENCES
1. Hajdu N, and Kauntze R: Cranio-skeletal dysplasia, Br J Radiol 21:42, 1948. Harnasch H: Die Akroosteolysis,ein neues Krankheitsbild, Fortschr Rontgenstr 72:352, 1950. 3. Greenberg BE, and Street DM: Idiopathic non-familial acro-osteolysis, Radiology 69:259, 1957.
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5. 6. 7. 8.
9.
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11.
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16.
17.
18.
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Papavasiliou CG, Gargano FP, and Wails WL: Idiopathic non-familial acro-osteolysis associated with other bone abnormalities, Am J Roentgenol 83:687, 1960. Toglia JU: Hereditary dysostosis, Tex State Med J 62:36, 1966. Chaviola S: Cranio-skeletal dysplasia with acro-osteolysis, Br J Radiol 37:702, 1964. Cheney WD: Acro-osteolysis, Am J Roentgenol 94:595, 1965. Dorst JP, and McKusick VA: Acro-osteolysis (Cheney syndrome), Birth Defects Original Article Series 5:215, 1969. Janner VM, Rohde B, and Jannasch G: Zum Krankheitsbild der famili/iren Akroosteolyse, Z Haut Geschlechtski 34:65, 1963. Herrmann J, Zugibe FT, Gilbert EF, and Opitz JM: Arthrodento-osteodysplasia (Hajdu-Cheney syndrome), Z Kinderheilkd 114:93, 1973. Kivivikko KI, Laitinen O, and Prockop D J: Modification of a specific assay for hydroxyproline in the urine, Anal Biochem 19:249, 1967. Teller WM, Burke EC, Rosevear JW, and McKenzie BS: Urinary excretion of acid mucopolysaccharides in normal children and patients with gargoylism,-J Lab Clin Med 59:95, 1962. Wong ET, and Lindall AW: Preliminary evidence for a microsomal precursor to human parathyroid hormone, Proc Nail Acad Sci 70:229, 1973. Singh M, Riggs BL, Beabout JW, and Jowsey J: Femoral trabecular pattern index for evaluation of spinal osteoporosis, Ann Intern Med 77:63, 1972. Maress RB, and Cameron JR: Skeletal growth in school children: maturation and bone mass, Am J Phys Anthropol 35:399, 1971. Jowsey J, Kelly PJ, Riggs BL, Bianco AJ Jr, Schulz DA, and Gershon-Cohen J: Quantitative microradiographic studies of normal and osteoporotic bone, J Bone Joint Surg 47A:785, 1965. Johnson KA, Riggs BL, Kelly P J, and Jowsey J: Osteoid tissue in normal and osteoporotic individuals, J Clin Endocrinol Metab 33:745, 1971. Snyder RA, and Brady RO: The use of white cells as a
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19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
source of diagnostic material for lipid storage diseases, Clin Chim Acta 25:33I, 1969. Desnick RJ, Allen KY, Desnick SJ, Raman MK, Bernlohr RW, and Krivit W: Fabry's disease: enzymatic diagnosis of hemizygotes and heterozygotes, J Lab Clin Med 81:157, 1973. Ikonne JU, Rattazzi MC, and Desnick RJ: Characterization of hexs: The major residual fi-hexosaminadase activity in type O Gm2-gangliosidosis (Sandhoff-Jalzaewetz disease) Am J Hum Genet 27:639, 1975. Baum H, Dodgson KS, and Spencer B: The assay of aryl sulfatases A and B in human urine, Clin Chim Acta 4:453, 1959. Russell RGG, and Fleisch H: Inorganic pyrophosphate and pyrosphosphatases in calcification and calcium homeostasis, Clin Ortho 69:101, 1970. Anderson HC: Calcinm-accumulating vesicles in the intercellular matrix of bone, in: Hard Tissue Growth, Repair and Remineralization, Ciba Foundation Symposium 11, Amsterdam, 1973, Elsevier Publishing Company, p. 213. Strott CA, and Nugent CA: Laboratory tests in the diagnosis of hyperparathyroidism in hypercalcemic patients, Ann Intern Med 68:188~ 1968. Jowsey J: Microradiography: a morphologic approach to quantitating bone turnover, in Frame B, Parfitt AM, and Duncan H, editors: Clinical aspects of metabolic bone disease, Amsterdam, 1973, Excerpta Medica, p 114. Riggs BL, Jowsey J, and Kelly PJ: Quantitative microradiographic study of bone remodeling in Sushing's syndrome, Metabolism 15:773, 1966. Revel JP: Role of the golgi apparatus of cartilage cells in the elaboration of matrix glycosaminoglycans, in Balazs EA, editor: Chemistry and molecular biology of the intercellular matrix, New York, 1970, Academic Press, Inc, p 1485. Bennett G, LeBlond CP, and Haddad A: Migration of glycoprotein from the golgi apparatus to the surface of various cell types as shown by radioautography after labeled fucose injection into rats, J Celi Biol 60:258, 1974. Glaser JH, McAlister WH, and Sly WS: Genetic heterogeneity in multiple lysosomal hydrolase deficiency, J PEDIATR 85:192, 1974.
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The acro-osteolysis syndrome: Morphologic and biochemical studies The acro-osteolysis syndrome consists of dissolution of terminal phalanges of the hands and feet, dolichocephaly with multiple wormian bones, delayed closure of cranial sutures, absence of frontal sinuses, a prominent occipital ridge, skeletal demineralization, vertebral and extremity fractures, joint laxity, and coarse hair. Studies of bone morphology reveal diminished bone density and bone formation. Osteoblasts have widely dilated smooth endoplasmic reticulum. It is postulated that an abnormality of a structuralprotein is the pathogenic basis of this disease.
David M. Brown, M.D.,* David S. Bradford, M.D., Robert J. Gorlin, D.D.S., Robert J. Desnick, M.D., Ph.D., Leonard O. Langer, Jr., M.D., Jenifer Jowsey, D.Phil., and John J. Sauk, Jr., D.D.S., Minneapolis, Minn.
AN UNUSUAL FORM of cranioskeletal dysplasia with peripheral dysostosis and spinal osteoporosis was d e scribed originally by Hajdu and Kauntze 1 in 1948. Subsequently 14 additional cases of this disorder, which has been called the acro-osteolysis syndrome, have b e e n described. 2-1~The prominent features consist of dissolution of terminal phalanges of the hands and feet, bizarreshaped dolichocephalic skull with multiple wormian bones, delayed closure of cranial sutures, absence of frontal sinuses, prominent occipital ridge, small mandible and maxilla and early loss of teeth, generalized skeletal demineralization particularly affecting the vertebra frequently resulting in fractures, laxity of joints, thick and coarse hair, conductive hearing loss, and speech impairment. The etiology and pathogenesis of this disorder are unknown. Two additional cases and results of relevant morphologic and biochemical studies will be presented. From the Departments of Pediatrics. Laboratory Medicine and Pathology, Orthopedic Surgery, Oral Pathology, and Human and Oral Genetics, University of Minnesota, and Mayo Medical School Supported by grants from the National Institutes of Health (AM-08658, DE-03686, and 5-MO1-RRO0400) and the National Foundation. *Reprint address: Mayo Box 491, University of Minnesota Hospital, Minneapolis, Minn. 55455.
METHODS The patients were hospitalized in the Clinical Research Center. By history each patient had ingested a minimum of 1,000 mg of calcium and 400 units of vitamin D per day for the previous several years. Serum and urine chemistry determinations were performed at the clinical laboratories of the University of Minnesota Hospitals. Total urinary hydroxyproline was determined by the method of Kivivikko and associates. 11 Urinary acid mucopolysaccharides were performed by the carbazole method of Teller and associates? ~ Serum parathyroid hormone levels were determined by radioimmunoassay. 13 Abbreviation used AMP: acid mucopolysaccharide
f
The femoral trabecular-pattern index of Singh and associates14 was determined as an indication of the loss of trabecular bone. Bone density was determined by a photon attenuation external scanning method. 15 The evaluation of bone morphology in biopsied tissue obtained from the iliac crest was performed by quantitative microradiography as previously described. 16' 17 Comparable bone samples were obtained from six persons for each control group between the ages of 6 and 8 and 15 and 17 years, who had died suddenly and w h o had not been
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Fig. lB. Case 1. Note short, broad hands and spade-fike distal phalanges. Fig. 1A. Case 1. Note broad face, maxillary and mandibular hypoplasia, thick eyebrows, and coarse hair.
hospitalized. Videodensitometry, a technique which is used to evaluate the percentage of a bone biopsy which contains bone, was performed by scanning a standardized photograph of a microradiograph of the specimen using a closed-circuit television camera which is digitized with a computerized, high-resolution videodensitometry system (details of this method are available from the authors). Skin-derived fibroblasts were grown in DulbeccoVogt's modification of Eagle's medium buffered with Hepes buffer containing penicillin, 100 U/ml, and streptomycin, 100 /Lg/ml, and 10% fetal calf serum using Falcon plastic flasks. Cells were subcultured using 0.125% trypsin. Bone and dentin, and skin, fibroblast culture, and hair bulbs were prepared for transmission electron microscopy after fixation in 6% or 3% (respectively) cacodylatebuffered glutaraldehyde, pH 7.0 at 4~ for two hours and postfixed in cacodylate-buffered 1% osmium for one hour. Fibroblasts were fixed in situ, removed from the surface of the flask by scraping with a rubber policeman, and sedimented by centrifugation. The cell button was resuspended in cacodylate buffer and again washed and centrifuged two times after which t h e button was postfixed and processed. All specimens were embedded in epon, and thin sections were obtained on an LKB ultramicrotome. The samples were then stained with lead
citrate and uranyl acetate and observed in a Phillips 301 electron microscope at 80 kv. Specimens for scanning electron microscopy were fixed as described above, dehydrated in alcohol and critical point dried from Freon in a Bomar critical point dryer. The specimens were mounted on coverslips and coated with carbon-gold. The samples were observed in a Cambridge scanning electron microscope at 20 kv. The scale structure of hair was described by measuring the distance between scale margins, the thickness of the scales, and the amplitudes of the scales. Plasma and isolated leukocytes for enzymatic analyses were obtained from the patients with acro-osteolysis and age- and sex-matched normal control subjects. 'Leukocytes were isolated from heparinized blood by modification of the method of Snyder and Brady? ~ Plasma and leukocyte enzyme assays. The activities of a-galactosidase A and B at pH 4.6, fi-hexosaminidase A and B at pH 4.4, a-mannosidase at pH 4.4 and 6.0, fl-galactosidase at pH 4.5, a-glucosidase at pH 4.0 and 6.0, and fi-glucuronidase at pH 3.6 were determined fluorometrically in plasma and leukocytes as previously described? .... Aryl sulfatase A and B activities were assayed by the method of Baum and associates. ~1 CASE REPORTS Case 1. Patient D.L. was a 789 girl who was referred for evaluation of unusual facies and nasal-like speech. She had
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Fig. 1C. Case 1. Note dolichocephaly, open sutures, prominent wormian bones, unusually shaped sella turcica, and prominent occipital ridge.
sustained a fracture of the left tibia and fibula at 6 years of age. She had difficulty walking and recurrent back pain. U p p e r and lower incisors had been lost at 4 to 5 years of age. There were five normal siblings and a 19-year-old brother with Down syndrome. Prominent features on physical examination included an unusual appearance characterized by a broad face with thick eyebrows and coarse and stiff hair (Fig. 1A). Her speech had a nasal character. Height was 110 cm and weight 16.3 kg, both o f which were greater than two standard deviations below the normal mean for age. The anterior fontanelle measured 3 x 3.5 cm. A prominent horizontal occipital ridge was palpable. The hard palate was high arched, and velopharyngeal incompetence was diagnosed. The digits of the hands and feet were short and broad, the distal portions appearing spade like (Fig. 1B). The skin appeared dry and coarse. Spinal extension and flexion were painful and limited all motions. There was general laxity of the joints of the extremities. There was diffuse tenderness over the lumbar spine. Her gait was somewhat shuffling with a poker back and obvious back discomfort. The neurologic evaluation was normal. The Wechsler intelligence scale was 81. Roentgenographic examination of the skull showed dolichocephaly, multiple wormian bones, and persistent anterior and posterior fontanelles with wide sutures (Fig. tC). The lambdoid suture was particularly wide. The sphenoid bone was malformed and had an abnormally shaped sella. The maxilla and mandible were small. There was generalized demineralization of the spine with compression fractures at several levels in the thoracic and lumbar regions (Fig. 1D). Mild scoliosis was noted. Osteolysis of the distal phalanges of both hands (Fig. 1E) and feet (Fig. 1F) was prominent. The long bones of the legs showed generalized demineralization and healing of the fractures of the left tibia and fibula.
Fig. 1D. Demineralized and compressed vertebra in Case 1.
Serum calcium, phosphorus, urea nitrogen, creatinine, sodium, potassium, chloride, bicarbonate, cholesterol, triglycerides, glutamic oxalacetic transaminase, total protein and electrophoretic pattern, thyroxine, triiodothyronine, parathyroid hormone, and 8 A.M. and 9 P.M. plasma cortisol were within normal limits. The serum alkaline phosphatase was 494 to 673 IU/1 (age-related normal = 225-380 IU/1, range of 2 SD). Urinary total acid mucopolysaccharide excretion was 6.4 mg/ 24 hr (normal for age = < 8 mg/24 hr) with an AMP/creatinine ratio of 1:38 (normal = < 1:4). While she was receiving a gelatin-free diet, the urinary total hydroxyproline excretion was 55.7 and 68.6 m g / m V d a y (normal for age = 37-95). During a 12day balance study (after a 3-day equilibration period) and while she was receiving an average daily intake of calcium of 723 rag/
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Fig. IF. Distal phalangeal resorption of feet in Case 1.
Fig. 1E, Distal phalangeal resorption of h a n d s in Case 1.
day (dietary Ca/P = 0.92), the patient excreted an average o f 21.4 mg/day of calcium in the urine (1.31 m g / k g / d a y ) and an average of 678 rag/day of calcium in the stool resulting in an apparent over-all calcium retention of 13 m g / d a y (sweat calcium not measured). The average percentage of tubular reabsorption of phosphate was 77.7 _+ 4.4 (SEM, n ~ 11) (N = < 82%). Case 2. Patient K.D. was a 16-year-old boy who presented with a six month history of persistent back pain and fractures of the fight and left tibia and fibula, respectively, at 6 and 15 years o f age. His hands had appeared "stubby" since early childhood. His hair had been noted to be coarse for most of his lifel There was no history of premature loss of teeth, although many of his permanent teeth were capped. No members of th e family were noted to have any of the physical characteristics of Patient K.D. or any history of fractures or back pain. Physical examination revealed a coarse facial appearance with a broad nose, coarse and bushy hair, and thick eyebrows (Fig. 2). He was 170 c m tall and weighed 56 k g . A prominent ridge was palpated horizontally over the occipital region. Speech was normal. He had a highly arched palate. Joint laxity of the extremities was prominent. He had prominent scoliosis with the
convexity in the lumbar spine. The distal digits of the hands and feet appeared spade like and shortened. No abnormalities of the skin were apparent. He had completed pubertal genital development. The neurologic examination was normal. Roentgenographic studies of the skull revealed several wormian bones in the lambdoid suture and marked dolichocephaly with a prominent occipital ridge. The mandible was small. There was marked demineralization of the spine with biconcave deformities and posterior scalloping particularly in the lumbar region. S-shaped scoliosis was noted in thoracic and lumbar regions. There was marked resorption o f the distal phalanges of the fingers and toes. Serum calcium, phosphorus, magnesium, urea nitrogen, sodium, potassium, chloride, cholesterol, triglycerides, glutamic oxalacetic transaminase, acid phosphatase, total protein and electrophoretic pattern, and parathyroid hormone levels were within normal limits. The serum alkaline phosphatase was 489 IU/1 (age- related normal = 120-450 IU/1). Urinary total acid mucopolysaccharide excretion was 11.2 m g / day (normal for age = < 12 mg/24 hr with an AMP/creatinine ratio of 1:12.6 (normal = < 1.4). While he was receiving a gelatin-free diet, the urinary total hydroxyproline excretion was 87.9 and 113 mg/m~/day (normal for age = 40-113). During a 12-day balance study (after a 3-day equilibrium period).and while lie was receiving an average daily intake o f calcium of 1,729 mg/day (dietary C a / P = 1.12), the patient excreted an average
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Fig. 2. Case 2. Note coarse facial appearance, coarse hair, and thick eyebrows. Fig. 3. Microradiograph of iliac crest biopsy. A, Patient with acroosteolysis. B, Normal 6-year-old girl. (x 16.) of 129 rag/day of calcium in the urine (2.3 mg/kg/day) and an average of 1,289 rag/day of calcium in the stool resulting in an apparent over-all calcium retention of 443 rag/day (sweat calcium not measured). The average percentage of tubular reabsorption of phosphate was 89.0 + 0.8 (SEM, n = 10). EXPERIMENTAL
RESULTS
Bone morphology. The appearance of the microradiograph of iliac crest bone of Case 1 is compared with those from a normal 6-year-old girl in Fig. 3, A and B. The thin cortex and reduced number of trabecula give an over-all appearance of reduced bone mass. In both patients, the percentage of the biopsy containing bone was diminished (24.2 and 13.9%; control subjects = 52.3 _+ 6.2 and 40.6 _+ 10.8%). Decreased trabecular pattern ratings in the proximal femur of both patients (III and IV; control subjects VI to VII) indicates the generalized nature of the diminished mineralization since more than 95% of normal persons below the age of 50 years have femoral trabecular patterns of at least grade V; individuals with grade II or III almost invariably have symptomatic osteoporosis and are generally over 60 years of age. 14 The bone density measured at the midshaft of the radius was normal in both children. In both biopsies, percentage of total endosteal bone surfaces typical of formation were diminished (2.4
and 1.0%; control subjects = 6.7 _ 2.5 and 6.2 _+ 3.2%) while the resorption surfaces were increased significantly in one patient (D.L.) (11.6 and 5.4%; control subjects = 6.0 _+ 2.4 and 7.0 _+ 1.0%). However, the osteoid width was normal in both children. Electron micrographs of iliac crest bone from patients revealed well-developed osteoblasts with distinctly developed rough endoplasmic reticulum and greatly dilated golgi zones (Fig. 4). Differentiation between golgi apparatus and smooth endoplasmic reticulum was indistinct. Osteoclasts were present and were normal. Active resorption by osteoclasts was characterized by the presence of numerous villous processes of osteoclast plasma membrane and resultant dissolution of the poorly mineralized bone matrix. Examination of the collagen fibers revealed normal diameters and periodicity. Calcium containing extracellular vesicles of bone appear normal in this disorder. Dentin from a freshly extracted tooth from one patient revealed an unaltered matrix and normal mineralization. Skin fibroblasts. Examination of skin-derived fibroblasts from both tissue culture and in fresh biopsy
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Fig. 4. Undecalcified section through iliac crest. Osteoblast from iliac crest of Case 2 with acro-osteolysis. The rough endoplasmic reticulum (R) is well developed. The central golgi zones (G) are extremely dilated and contain fine granular material. Matrix vesicles (my) can be noted near the cell periphery. Nucleus (N) and mitochondria (M) are present but unaltered. Extracellular matrices reveal an organizing organic matric (OM). However, apatite crystal are rare or not present. ( X 22,500.)
material of skin revealed normal morphology. Cellular organelles were well developed. No dilation of golgi apparatus or smooth endoplasmic reticulum were observed. Histochemical studies of these fibroblasts (not included in this report) using alcian blue and toluidine blue stains revealed no increased metachromatic staining as compared with control fibroblasts. Skin and hair. The epidermis was hyperkeratotic. Dermal collagen had normal periodicity and organization. Electron microscopy of the skin revealed normal melanocytes and well-developed keratinocytes, the latter containing numerous mitochondria and extensive tonofibrils. In addition, there was an abundance of keratohyalin granules in the stratum spinosum. The hairbulbs were normal morphologically. Keratinocytes appeared normal although keratohyalin granules were exceptionally large. Examination of the hair by scanning electron microscopy revealed two hair types, one which was distinctly thinner and contained light brown pigmentation and the other which had a larger diameter and was coarse and darker. Both types of hair had normal scale dimensions. The thinner a n d lighter hair shaft was approximately one-third the diameter of the thicker hair. Lysosomal hydrolase enzymes. The activities of WBC
and/or plasma (aryl sulfatase, a-glucosidase, fl-hexosaminidase, a-mannosidase, and fi-galactosidase) were normal or only slightly greater than the control range. However, the plasma activity of fl-glucuronidase was increased approximately 2.5 times the upper limit of the control range (812 vs control range, 90-312 nmoles/hr/ ml) while WBC fi-glucuronidase activity was normal. DISCUSSION The syndrome of idiopathic acro-osteolysis is a distinct entity involving the generalized skeleton with particularly unique features dearly indicating its identification. Its manifestations are reviewed in Table I. Most striking is the distal osteolysis but widespread cranio-skeletal dysplasia has been found in all of the cases. Acro-osteolysis may also be found in hyperparathyroidism, pycnodysostosis, progeria, epidermolysis bullosa, scleroderma, syringomyelia, leprosy, syphilis, psoriasis, trauma, neurogenic ulcerative acropathy, and p01yvinyl chloride toxicity. The etiology of the syndrome is not known. The studies of the two patients in this report revealed no abnormalities .suggesting a hormonal basis. In the two patients presented here, the serum alkaline phosphatase activities (obtained after an overnight fast) were slightly elevated. Few of the cases reported in the literature were described
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during childhood although, where reported, serum alkaline phosphatase has been normal. It has been suggested that alkaline phosphatase of bone may in large part represent pyrophosphatases which aid in the removal of inorganic pyrophosphates which in turn modify the conversion of amorphous to crystalline hydroxyapatite. 2~ The alkaline phosphatase and pyrophosphatase activity are found particularly within calcium-accumulating vesicles in the intercellular matrix of bone 2~and are associated with the process of bone formation. We do not have sufficient information on the patients reported herein to speculate as to the significance of the elevations of serum alkaline phosphatase in this disorder beyond the discussion based upon our morphologic observations. (The presence of subtle fractures may account for the elevations noted in these patients). Our studies of calcium balance suggest that there was diminished skeletal retention of calcium in one patient (D. L.) but do not reveal the basis. Although the percentage of tubular reabsorption of phosphorus was diminished in that patient, there is a significant degree of nonspecificity of that finding which precludes its use as a diagnostic test of hyperparathyroidism? 4 Morphologic studies of the bone (iliac crest) of each patient reported here show decreases in skeletal mass. The percentage of endosteal bone surfaces characteristic of bone formation was diminished in both patients, and in one resorptive surfaces were increased. The absence of excessive osteoid rules out an abnormality of mineral access to bone such as would be expected to occur in osteomalacia. Hence, these findings clearly differ from primary hyperparathyroidism and hyperthyroidism, in which increased bone resorption predominates. '~ Although in one patient (D. L.) formative surfaces were decreased and resorptive surfaces were increased, as is found in Cushing's syndrome, ~6 there was no clinical or laboratory evidence of glucocorticoid excess. The electron microscopic studies of osteoblasts in these patients demonstrated marked dilation of smooth endoplasmic reticulum channels. Several studies have pointed to the golgi apparatus as the site of elaboration of both matrix glycosaminoglycan synthesis~ as well as of glycoproteins. ~ Although in neither patient in our studies was there evidence of excessive urinary excretion of acid mucopolysaccharides (glycosaminoglycans) or of total hydroxyproline, these by no means exclude the possibility that an abnormality may exist in either glycoprotein, proteoglycan, or glycosaminoglycan metabolism. Although the unusual characteristics of the hair observed grossly and verified morphologically in terms of thickness of the hair are probably nonspecific, they do suggest the presence of a structural protein abnormality.
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Table I. Primary clinical features of idiopathic acro-
osteolysis
Feature Dolichocephaly Delayed closure of cranial sutures Prominent occipital ridge Absent frontal sinuses Small mandible/maxilla Early loss of teeth Distal phalangeal resorption Generafized skeletal detaineralization Fractures long bones Fractures/compression vertebra Kyphosis/scoliosis Joint laxity Thick, coarse hair Speech abnormalities Shortness of stature
Reported occurrence (15 patients)
Present report (2 patients)
9 4
2 2
10 9 12 13 15 7
2 1" 2 1 2 2
9 10
2 2
7 10
2 2 2 1 1
*Frontal sinuses development may not be expected in a 7-year-old child. The elevation of plasma /?-glucuronidase activity in association with normal WBC/3-glucuronidase activity is analagous to observations that have been made in other clinical circumstances. For example, I-cell disease is associated with increased activity of several plasma acid hydrolases, while the activities of the same hydrolases is decreased in fibroblasts but not in leukocytes or in other tissues. 2~ In that instance, leakage of enzymes from cells has been postulated to account for these observations.~" The significance of the observation reported in this patient with acro-osteolysis will be determined by further studies. We conclude that the idiopathic acro-osteolysis syndrome is a distinct dominantly inherited entity, the general skeletal demineralization representing a major impairment for these patients. The morphologic studies suggest an abnormality of osteoblast function and may be the basis for a postulated structural protein abnormality. We appreciate the excellent assistance of Mrs. Mary Jo Jansen in the preparation of the manuscript. REFERENCES
1. Hajdu N, and Kauntze R: Cranio-skeletal dysplasia, Br J Radiol 21:42, 1948. Harnasch H: Die Akroosteolysis,ein neues Krankheitsbild, Fortschr Rontgenstr 72:352, 1950. 3. Greenberg BE, and Street DM: Idiopathic non-familial acro-osteolysis, Radiology 69:259, 1957.
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