Elevated s e m levels of creatine b a s e BB in autosomal dominant osteoDetrosis Type I1- a family study I

Yoneyama T, Fowler HL, Pendleton JW, Sforza PP, Gerard RD, Lui CY, Eldridge TH,Iranmanesh A. Elevated serum levels of creatine kinase BB in autosomal dominant osteopetrosis type I1 - a family study. Clin Genet 1992: 42: 39-42. A markedly elevated BB isoenzyme fraction of serum creatine kinase was noted in four male siblings and correlated with typical radiographic findings

of autosomal dominant osteopetrosis Type I1 (ADO Type 11). Patients with other sclerosing bone diseases had no elevation of CK-BB. The precision of the electrophoretic mobility patterns and correlation by 1-125 tagged radioimmunoassay method confirms that this is CK-BB. We postulate that the dysfunctional and/or immature osteoclasts in ADO are more dependent on CK-BB than on the usual tricarboxylic acid cycle for the production of energy. The correlation of marked elevation of serum CK-BB with radiographic evidence of ADO Type I1 may prove to be of value as a biologic marker in the early diagnosis of the illness and lead to better understanding of the metabolism of bone.

Tatsno Yonsyama, Hilton L Fowler, John W. Psndleton, Peter P. Sfona, Roy D. Gerard, Charles Y. Lui, Thomas H. Eldridge and All lranmanesh Department of Veteran Affairs Medical Center, Salem. Virginia, and University of Virginia, School of Medicine, Chadottesville, Virginia, USA

Key words: autosomal dominant osteopetrosis type II elevated serum CK-BB family study - radiographs

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Dr. Tatsuo Yoneyama, %term Health Administration, Oept, of %terns Affairs, VA Medical Center, Salem. Virginii 24153, USA Received 12 August 1991. revised version received 7 February, accepted for publication 12 February 1992

Osteopetrosis is a group of inherited bone disorders characterized by generalized osteosclerosis. The increased bone density is due to an underlying defect in the ability of osteoclasts to reabsorb bone (Marks 1987, Shapiro et al. 1980, Silvestrini et al. 1987). Of the various subgroups, two forms are most familiar (Beighton et al. 1977, McKusick 1990). In the malignant, autosomal recessive form of childhood, the bone sclerosis leads to fractures, bone marrow replacement, and neurologic complications, including cranial nerve palsies and hydrocephalus. In the more benign adult, autosomal dominant osteopetrosis (ADO), the bone sclerosis is less rapidly progressive, and it is a fracture that usually leads to the diagnosis in the otherwise asymptomatic adult. Since our discovery of the elevated serum BB isoenzyme of creatine kinase (CK-BB; EC 2.7.3.2) in patients with known ADO (Yoneyama et al. 1989), we have completed this study of five sibs. Four of the five sibs had typical radiographic findings of ADO Type I1 and elevation of the serum CK-BB to the 31% to 60% range, while the remaining sib had no radiographic evidence of osteopetrosis and no measurable serum CK-BB. This study also included patients with other sclerotic bone diseases who were found to have normal serum

CK-BB. We postulate that the dysfunctional osteoclasts in ADO Type I1 are more dependent on CKBB than on the usual tricarboxylic acid cycle for production of energy, and the high levels of serum CK-BB reflect this activity. Material and methods

After informed consent, the five survivors of this sibship of seven were evaluated by history, general physical and neurological examinations, and batteries of clinical laboratory tests, including hematological, chemical, and enzymo-chemical studies. Radiographs of the lumbar spine and pelvis were taken if adequate radiographs did not exist. Two of the male siblings had been subjects in our original report. A female child of one of the sibs underwent complete evaluation and two other children had serum CK-BB measured. One case each of Paget’s disease, metastatic prostatic carcinoma to vertebral bones, and progressive diaphysical dysplasia (Camurati-Englemann disease) were also completely studied. Total serum creatine kinase (CK) activity was measured by an automated chemistry analyzer (Dupont aca 11) as international units per liter (IU/ 1). The CK isoenzymes CK-MM, CK-MB, and 39

Yoneyama et al.

CK-BB were measured by agarose gel-electrophoresis (Helena Automated Isodot) and scanned by a densitometer (Helena edc) to obtain a percentage and IU/1 for each fraction.

Results This family consisted of seven sibs (Fig. 1). A male sib had been killed in an accident at age 10 and a sister had died in a fire at age 70. Neither of these sibs was known to have osteopetrosis. The father, who died at age 30 as a result of an accident, and the mother, who died at age 67 of unknown causes, were not known ever to have been evaluated for osteopetrosis. There was no known consanguinity. Patient 1, the 67-year-old, white male propositus, and all three of his surviving male sibs, Patients 2, 3 and 4, whose ages were 68, 72, and 76, respectively, had the diagnosis of osteopetrosis made in early adult life when they suffered fractures. The only surviving female sib, 79-year-old, Patient 5, had no history of osteopetrosis or fractures. Radiographs (Fig. 2) demonstrated the generalized increased bone density and typical “ruggerjersey spine” in Patients I through 4, and the “bone within a bone,” or endobone, of the ilium of Patient 3. Patient 5 had decreased bone density consistent with osteoporosis, without the lesions associated with osteopetrosis. Although all patients had normal values of total serum CK, there was marked elevation of CK-BB to the 3 1 4 0 % range in Patients 1 through 4 (Table 1). No CK-BB was detected in Patient 5. The 42year-old daughter of Patient 1 had normal radiographs and normal serum CK-BB, while the 31year-old daughter of Patient 3 and the 3 1-year-old son of Patient 4 had normal serum CK-BB, but did not undergo radiography because of their reproductive ages. The patients with Paget’s disease, metastatic prostate carcinoma to vertebral bones,

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Fig. 2. On the left, lateral views of the spine of Patients 1 (top) and 2 (bottom). On the right, anterior-posterior Views of the pelvis and lower lumbar spine of patients 3 (top), 4 (middle), and 5 (bottom). For discussion, see text.

and progressive diaphysial dysplasia (Camurati-Englemann disease) had normal serum CK-BB levels. To eliminate the possibility that our patients’ CKBB is an abnormal CK isoenzyme, such as CK-MIT, anodic CK, or adenylate kinase (AK, EC 2.7.4.3), the CK isoenzymecontrol pattern was overlaid onto the patients’ electrophoreticpattern, using the computer graphic capability of the Helena edc densitometer. This identified the electrophoretic mobility of the patients’ isoenzymes as being exactly the same

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II

Table 1. Normal values: total CK. 35 to 232 International Units per I; CK-MM, 97-100%; CK-MB, 0 4 % CK-BB. 0-1%

Serum creatine kinase isoenzymes Case

Fig. I . Pedigree of a family with adult osteopetrosis. Black

symbols represent subjects with disease confirmed by radiographs of the lumbar spine and pelvis, circles arc female subjects and squares are males. Symbols with dots are subjects who did not have radiographs because of their young age and those with diagonal lines had died prior to the study. Patient 1, the propositus, is identified by an arrow.

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M.

1 2

3 4

5

Total CK UA

CK-MM

CK-MB

CK-BB

%

%

%

128 44 114 42 50

40

0

69 40

0 0 0 0

60 31

67 100

60 33 0

Elevated CK-BB in ADO Type I1 as that of the CK-BB in the control. Further positive confirmation was obtained by the 1-125 tagged radioimmunoassay (RIA) method, using rabbit anti-human polyclonal an tibody against the B subunit of CK-BB. The comparison of the quantitation of the CK-BB by these two methods is not possible, because there is no conversion factor available to calculate from IU/l of the electrophoreticmethod to nmol/l of the RIA method. In all patients, however, elevation of identifiable CK-BB was confirmed by the second method. Thus, we demonstrated that we were dealing with true CK-BB in our patients. Discussion

Two major phenotypes, based on radiographic differences, have been defined in ADO (Andersen & Bollerslev 1987). In Type I, there is marked sclerosis of the cranial vault with density of vertebral arches as the only changes in the spine. In Type 11, the converse is true, with sclerosis of the skull limited to the base, while there is marked vertebral end-plate thickening, resulting in the rugger-jersey sign, and endobones are seen in the pelvis. As a rule, only one type is seen within a family. Members of the sibship described here have the Type I1 radiographicphenotypeWithout documentation of this illness in a female or in more than one generation, one cannot definitely conclude autosomal dominant inheritance and exclude sex-linkage or autosomal recessive inheritance. However, we believe the patients in this family have ADO Type I1 because of the classic phenotypic expression of their illness. This family does not have the intermediate form called autosoma1recessive osteopetrosis with renal tubular acidosis (Sly et al. 1985) where the phenotype is expressed in infancy or early childhood by hypotonia, psychomotor retardation, and fractures; and renal tubular acidosis develops in early adulthood. Nor does this family have the phenotypic expression of mild autosomal recessive osteopetrosis(Kahler et al. 1984),where short stature, macrocephaly, fractures, and mandibular prognathismcomplicated by osteomyelitis and dental abnormalities are evident in childhood. The marked elevation of the serum CK-BB to the range of 3140% of total CK correlated with the presence of bone lesions typical of ADO Type I1 in sibs 1 through 4. The only living female sib, Patient 5, showed absence of CK-BB and no evidence of osteopetrosison radiographs. Because ADO Type I1 was the only clinical abnormality common to these four sibs, we postulated that this illness was the cause of their markedly elevated CK-BB. The origin of this elevated CK-BB in ADO Type I1 is of importance. In addition to brain, for which the B subunit

was named, gastrointestinal tract, urinary bladder, and some malignancies have moderate amounts of CK-BB (Pesce 1982, Roberts et al. 1975). Body fluids that have been demonstrated to have high levels of CK-BB are vitreous humor (Pragay & Toppin 1981),seminal fluid (Asseo et al. 1981),and skin blister fluid (Paavonen et al. 1988). It is important to note that it has not been possible to assay bone for CK isoenzymesbecause its calcificationprevents application of the homogenized technique used on other tissue. Central nervous system (CNS) tissue destruction by cerebrovascular accidents, trauma, infection, and anoxia has been demonstrated to elevate serum CK-BB (Bell et al. 1978). The sibs of the present report had no evidence of CNS damage by neurologic examination and there was only evidence of a small, old lacunar infarction in Patient 4 by computerized tomography. The high levels of CK-BB were observed in repeated tests over a 3-year period. If destruction of CNS, or other tissue, were responsible for the constantly high levels of this isoenzyme, with a short half-life of 1 to 5 h (Pesce 1982), it would require massive and progressive damage that should be evident clinically. A more plausible explanation is that there is a constant source of CK-BB from the physiologic activity of some tissue. Creatine kinase normally catalyzes the reversible transfer of high energy phosphate from phosphocreatine to adenosine diphosphate to regenerate adenosine triphosphate. We have previously postulated that the relatively anaerobic environment of the cellular elements of osteopetrotic bone requires an increased dependence on phosphocreatine for energy, and the elevated serum CK-BB in these patients may reflect increased activity in the abnormally dense bones (Yoneyamaet al. 1989).However, our finding in this study of normal CK-BB levels in one case each of advanced osteosclerosis due to Paget's disease, prostatic carcinoma metastatic to bone, and progressive diaphysial dysplasia suggests that it is not due to the postulated anoxic environment. The more likely explanation for the elevation of CK-BB is that it is the consequence of the abnormality of the osteoclast itself. Electron microscopy has revealed that there is an increased number of osteoclasts in osteopetrosis and these osteoclasts have an absence of ruffled borders and clear zone which are necessary for bone reabsorption (Shapiro et al. 1980). It is possible that these dysfunctional osteoclasts are more dependent on CK-BB for the production of energy than on the tricarboxylic acid cycle. There may be an analogy to cardiac muscle where CK-BB predominates in the fetal state (Goto et al. 1969). Thus, the dysfunctional osteoclasts in 41

Yoneyama et al. osteopetrosis may have the metabolism of a more immature stage of development. The osteoclast is a multinucleated cell which is formed by fusion of precursor mononuclear cells (Marks 1987). The latter arise from stem cells in the hematopoietic reticuloendothelial system and migrate to bone where fusion occurs and is followed by differentiation of the multinucleated cell to become a mature osteoclast. Parabiosis created by a skin window from normal littermates to mice with inherited osteopetrosis has been curative (Walker 1972). It was suggested that the precursor mononuclear cells of the normal littermates migrated to the bone of osteopetrotic mice, where the above described fusion and differentiation to form mature osteoclasts at the surface of the bone occur. Predicated on this biology, bone marrow from a S-yearold brother was successfully transplanted to his 5month-old sister who had infantile osteopetrosis (Coccia et al. 1980). Following this transplantation, there was radiographic improvement and two types of osteoclasts were found in the girl’s bone, one with the expected female chromosome pattern and the other with the male chromosome pattern of her brother. This phenomenon of migrating cells is accepted as a basic characteristic of the macrophage system and opens a new approach to the consideration of bone pathology and future treatment. In a report of elevated serum CK-BB in a child with infantile osteopetrosis (Hiroyama et a]. 1987), it was postulated that the source of the elevated CKBB was hematopoietic cells which gained access to the peripheral blood by a breakdown of the endothelial lining of the bone marrow sinusoids. Since completion of the present study, there has been a report of a series of patients who had elevation of CK-BB in ADO Type 11, but not in ADO Type I (Gram et al. 1991). Those authors also postulated that the immature osteoclast was the source of the elevated CK-BB. The alternative postulation that the gene defect causing ADO Type I1 might cause a general defect in the production or degradation of CK-BB is not likely because autosomal dominant illnesses do not have the primary enzymatic defects which are often Seen in the autosomal recessive disorders. Elevated serum CK-BB, as a result of immature osteoclasts, offersexciting possibilities for diagnosis of ADO Type I1 and for research opportunities to further understand ADO and bone related metabolism.

Acknowledgements The authors appreciate the assistanceof Rosalie Layman, Linnie Skidmore, and the Medical Technologists of the VAMC, Salem, Virginia, and Robin Reichner of the VAMC, Hampton, Virginia.

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References Andersen PE Jr, Bollerslev J. Heterogeneity of autosomal dominant osteopetrosis. Radiology 1987: 164: 223-225. Asseo PP, Panidis DK. Papadimas JS, Ikkos DG. Creatine kinase in seminal plasma of infertile men: activity and isoenzymes. Int J Androl 1981: 4 431439. Beighton P, Horan F, Hamersma H. A review of the osteopetroWS. Postgrad Med J 1977 53: 507-516. Bell RD, Rosenberg RN, Ting R, Mukherjee A, Stone MJ, Willerson JT.Creatine kinase BB isoenzyme levels by radioimmunoassay in patients with neurologicaldisease. Ann Neurol 1978: 3: 52-59. Coccia PF, Krivit W,Cervenka J, Clawson C, Kersey JH, Kim TH, Nesbit ME, Ramsay NK, Warkentin PI, Teitelbaum SL, Kahn AJ, Brown DM. Successful bone-marrow transplantation for infantile malignant osteopetrosis. N Engl J Med 1980: 302: 701-708. Goto I, Nagamine M. Katsuki S.Creatine phosphokinase isozymes in muscles. Human fetus and patients. Arch Neuroll969: 2 0 422429. Gram J, Antonsen S, Horder M, Bollerslev J. Elevated serum levels of creatine kinase BB in autosomal dominant osteopetrosis type 11. Calcif Tissue Int 1991: 48: 438439. Hiroyama Y,Miike T,Sugino S,Taku K. Creatine kinase brain isoenzyme in infantile osteopetrosis. Pediatr Neurol 1987: 3: 54-57. Kahler SG, Bums JA, Aylsworth AS. A mild autosomal recessive form of osteopetrosis. Am J Med Genet 1984: 1 7 45 1-464. Marks SC. Osteopetrosis - multiple pathways for the interception of osteoclast function. Bone Pathol Appl Pathol 1987: 5: 172-183. McKusick VA. Osteopetrosis. In: Mendelian inheritance in man, 9th ed. Baltimore: The Johns Hopkins University Press, 1990 698: 1402. Paavonen T, Aronen H, Saarelainen I, Neittaanmaki H, Hjelm I, Kiistala U. The BB-isoenzyme is a major component of creatine kinase in skin blister fluid. Br J Dermatol 1988: 118: 753-757. Pesce MA. The CK isoenzymes: findings and their meaning. Laboratory Management 1982: Oct: 25-37. Pragay DA, Toppin M. Lactate dehydrogenase and creatine kinase isoenymes in human vitreous humor (letter). Clin Chem 1981: 27: 344. Roberts R. Gowda KS. Ludbrook PA, Sobel BE. Specificity of elevated serum MB creatine phosphokinase activity in the diagnosis of acute myocardial infarction. Am J Cardiol 1975: 3 6 433-437. Shapiro F, Glimcher MJ, Holtrop ME, Tashjian AH Jr, Brickley-Parsons D, Kenzora JE. Human osteopetrosis: a histological, ultrastructural, and biochemical study. J Bone Joint Surg (Am) 1980: 6 2 384399. Silvestrini G, Ferraccioli GF, Quaini F, Palummeri E, Bonucci E. Adult osteopetrosis: study of two brothers. Bone Pathol Appl Pathol 1987: 5: 184-189. Sly WS, Whyte MP, Sundaram V, Tashian R, Hewett-Emmett D, Guibaud P, Vainsel M, Baluarte HJ,Gruskin A, Al-Mosawi M. Sakati N, Ohlsson A. Carbonic anhydrase I1 deficiency in 12 families with autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. N En@ J Med 1985: 313: 139-145. Walker DG. Congenital osteopetrosis in mice cured by parabiotic union with normal siblings. Endocrinology 1972 91: 916-920. Yoneyama T, Fowler HL, Pendleton JW,Sforza PP, Lui CY, Iranmanesh A, Gerard RD. Elevated levels of creatine kinase BB isoenzyme in thm patients with adult osteopetrosis (letter). N Engl J Med 1989: 320 1284-1285.

Elevated serum levels of creatine kinase BB in autosomal dominant osteopetrosis type II--a family study.

A markedly elevated BB isoenzyme fraction of serum creatine kinase was noted in four male siblings and correlated with typical radiographic findings o...
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