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A girl with bone sclerosis and fracture.


A previously fit and well 5-year-old girl presented to the emergency department with right hip pain and limp following a minor fall from play equipment. X-ray imaging confirmed the presence of a femoral fracture as well as diffuse sclerosis in the sacrum, pelvis, and bilateral proximal femurs (Fig. 1). MRI scan revealed mild stenosis of auditory canals, optic canals, and foramen magnum with no neural compression. DEXA (dual energy X-ray absorptiometry) scan showed markedly increased bone density, with a total body age-matched z score of + 14. Bone marrow aspirate was normal. The patient had no other significant medical history and she was not on any regular medications. There was no family history of note but her mother also has increased bone mineral density.

Results for serum electrolytes, urea, creatinine, total calcium, phosphate, parathyroid hormone, vitamin D, alkaline phosphatase, and alanine aminotransferase were all within reference intervals. However, other biochemistry investigations revealed aspartate aminotransferase (AST)7 89 U/L (reference interval, 20-80 U/L); lactate dehydrogenase (LD) 667 U/L (reference interval, 100-200 U/L); acid phosphatase (ACP) 32.1 U/L (reference interval, <6.5 U/L), with tartrate-resistant ACP 29.9 U/L (reference interval, 1.2-4.4 U/L); creatine kinase (CK) 615 U/L (reference interval, <190 U/L); CK-MB activity by immunoinhibition technique 760 U/L (reference interval, <10 U/L); CK isoenzymes by electrophoresis, CK-MB <3% (reference interval, <6%) and CK-BB 87% (reference interval, < 1%). Complete blood examination was normal except mildly low hemoglobin of 10.1 g/dL (reference interval, 11.5-15.5 g/dL).


Bone is a dynamic tissue that is under continuous turnover or remodeling. It consists of specialized bone cells, mineralized and unmineralized connective tissue matrix, and spaces including the bone marrow cavity, vascular canals, canaliculi, and lacunae that contain osteocytes. The 2 main types of bone cells are osteoblasts and osteoclasts (1).

Osteoblasts originate from multipotent mesenchymal stem cells and are responsible for bone formation. Osteoclasts are multinucleated cells derived from the mononuclear precursors in the myeloid lineage of hematopoietic cells. The receptor activator of nuclear factor [kappa] B ligand (RANKL) and the macrophage colony-stimulating factor (M-CSF) are essential for the development, function, and survival of osteoclasts (1). Fully differentiated osteoclasts dissolve bone mineral and degrade bone matrix by acidification and proteolytic digestion (1, 2). The bone density is dependent on the relative function of osteoblasts and osteoclasts (1-3). High or low rates of remodeling with an imbalance between resorption and formation can be associated with decreased or increased bone mass.


Remodeling imbalance owing to failure of the resorptive process can result in dense (sclerotic) bones. Several causes for diffuse sclerotic bone lesions have been reported. These include hematological conditions (sickle cell disease), malignancy (leukemia, myeloproliferative diseases, metastatic bone disease), chemical poisoning (fluoride, lead), and congenital conditions (osteopetrosis, pyknodysostosis) (4).


Hematological diseases and malignancies were excluded on the basis of the clinical, hematological, and radiological findings. There was no obvious history suggestive of chemical poisoning, and blood lead concentration was undetectable. The presence of an increased CK-BB fraction distinguishes osteopetrosis from other sclerosing bone disorders (5). The presentation in early childhood, fracture with sclerosis, and stenotic auditory canal strongly suggest a diagnosis of the intermediate form of osteopetrosis. However, precise differentiation between intermediate- and late-onset autosomal dominant (AD) osteopetrosis (Albers-Schonberg disease) is not possible. Although genetic studies have not been performed, the biochemical changes with increased LD and AST are highly suggestive of a chloride channel, voltage-sensitive 7 (CLCN7) (8) mutation (5).


Osteopetrosis is a rare clinically and genetically heterogeneous group of inherited disorders characterized by a marked increase in bone density. It is also known as "marble bone disease" or Albers-Schonberg disease after the German radiologist who first described the condition and radiological findings in 1904 (2-4).

This disease is caused by the defective differentiation or function of osteoclasts. A number of mutations have been identified as causative of osteopetrosis in humans. Many forms of osteoclast-rich osteopetrosis are caused by mutations in genes expressing proteins involved in the acidification process of bone resorption (2). The main genes are the osteoclast-specific proton pump subunit, T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 sub-unit A3 (TCIRG1) (encodes the a3 subunit of vacuolar ATPase), CLCN7 (encodes the osteoclast-specific chloride channel), and carbonic anhydrase II (CA2) (1-3). Rare osteoclast-poor forms have been described in patients with tumor necrosis factor superfamily member 11 (TNFSF11, also known as RANK!) gene mutations (2). Osteopetrosis can be inherited in an AD, autosomal recessive (AR), or X-linked recessive (XR) manner (2).

The incidence of the disease is estimated to be around 1:100 000-1:500 000 (4). Some forms are more common than the others and a high incidence of AR form has been reported in Costa Rica (3.4:100 000) (2, 3).

Clinical presentation of osteopetrosis is very variable, ranging from asymptomatic to fatal in infancy. Based on age and clinical features, there are 3 main types: AR infantile or "malignant," AR intermediate, and AD adult (2, 4). Several other rare forms of osteopetrosis have been described in the literature (2, 4).

Infantile osteopetrosis is a rare, life-threatening condition. Patients present during the first few months of life with fractures, osteomyelitis, macrocephaly, frontal bossing, short stature (due to impaired longitudinal bone growth), and nasal stuffiness due to mastoid and paranasal sinus malformations (4). Sclerosis of the bone causes narrowing of the marrow cavity and bone marrow failure with consequent life-threatening pancytopenia and hepatosplenomegaly due to extramedullary hematopoiesis (2, 4). Delayed dentition and cranial nerve compressions can also occur (4). Patients are at risk of developing hypocalcemia and secondary hyperparathyroidism (2).

Patients with intermediate osteopetrosis are often asymptomatic at birth and bone marrow failure is rare. They may present during childhood with frequent fractures, osteomyelitis, mild-to-moderate anemia, tooth eruption defects, and occasional optic nerve compression (2, 4).

Patients with the adult form of osteopetrosis often present in late adolescence or adulthood with complications confined mainly to the skeletal system. The clinical presentation can be highly variable due to reduced penetrance of the AD phenotype (6). Their bone marrow function is usually not compromised. They can present with radiological findings such as "bone within bone" appearance and focal sclerosis of the skull base, pelvis, and vertebral end plates--"sandwich" vertebrae and "Ruggerjersey" spine (2, 4, 6, 7). Two distinct types of AD osteopetrosis have been described, type I and type II, on the basis of clinical, biochemical, and radiological features (4).

The diagnosis of osteopetrosis is mainly based on clinical and radiological findings. Increased serum CK-BB isoenzyme and tartrate-resistant acid phosphatase (TRAP) can be used in the absence of typical radiological findings to confirm the diagnosis in certain subtypes (e.g., AD type II) (2, 8). The exact mechanism for the increases in TRAP and CK-BB is yet to be confirmed. It is postulated that these enzymes are released from osteoclasts in osteopetrosis. However, this could be due to increased osteoclast size and number (in AD type II osteopetrosis) or overexpression by the osteoclast in response to the defect in bone resorption or derived from other tissues affected by the underlying gene mutations (5, 8). Increased serum LD and AST have also been reported, especially in the presence of CLCN7mutations (5). Usefulness of other bone turnover markers such as C-telopeptide and procollagen type 1 N-terminal propeptide in the diagnosis of osteopetrosis is yet to be confirmed.

Age of onset, pattern of inheritance, and presence of associated features may be helpful to identify the subtype of osteopetrosis. Genetic testing can be used to confirm the diagnosis and subtype in most patients. Identification of subtype is important for management and genetic counselling as the subtypes have differences in prognosis (2).

At present, there is no definitive treatment for osteopetrosis. Management is mainly supportive and depends on the type of the disease (2). Prognosis is poor with the infantile form in contrast to the intermediate and AD adult forms of osteopetrosis. Hemopoeitic stem cell transplantation, interferon 7, and calcitriol and steroids have been used only in selected forms of autosomal recessive infantile osteopetrosis (2, 9). However, multidisciplinary surveillance and symptomatic treatment of fractures, arthritis, and dental problems may be required in other forms of the disease.


CK is a dimeric cytosolic enzyme composed of 2 subunits (B and M) that catalyzes the reversible phosphorylation of creatine by ATP. There are 3 main isoenzymes of CK: CK-MM, CK-MB, and CK-BB, with characteristic distribution in different tissues. CK-MM is mainly distributed in skeletal muscle and heart muscle; CK-MB is mainly found in heart muscle (around 20%) and a very small percentage in skeletal muscle. The main tissue distribution of CK-BB is brain and smooth muscle but it is also present in neuronal cells, retina, kidney, and bone. In a healthy person, CK activity in the serum is due almost exclusively to CK-MM with a small amount of CK-MB.

There are several analytical methods for the identification and quantification of CK isoenzymes. Electrophoresis separates all forms of CK isoenzymes. The CK-BB isoenzyme migrates toward the anode at pH 8.6, in contrast to CK-MM, which remains cathodic to the application point (Fig. 2). Measurement of CK-MB can be done by an immunoinhibition technique ("activity") or by an immunochemical method using monoclonal antibodies ("direct" or "mass") (10).

Immunoinhibition technique measures the catalytic activity of the B-subunit using an antibody to inhibit the CK-M activity. This technique assumes that the CK-BB isoenzyme is absent in normal serum and the measured CK-B activity is from the CK-MB isoenzyme. Furthermore, because the CK-B subunit accounts for only one-half of CK-MB activity, the result obtained is multiplied by 2 to give a total "MB" activity.

In patients with osteopetrosis, the amount of CK-BB in serum is no longer negligible. In contrast to this activity measurement, the mass assay recognizes only the MB dimer because neither CK-MM nor CK-BB react with both antibodies (10).

In this patient, CK-MB activity was measured using an immunoinhibition technique (SENTINEL diagnostics reagents on a Beckman Coulter DxC analyzer). In the presence of increased CK-BB associated with osteopetrosis, this technique gave a falsely high CK-MB activity. This can be overcome by using an immunochemical method to measure CK-MB concentration or CK isoenzyme electrophoresis.


1. What conditions may cause diffuse sclerotic bone lesions?

2. What is the most probable diagnosis for this girl?

3. How do you explain the discrepancy between the CK-MB activity measured by the immunoinhibition method and the CK isoenzyme electrophoresis results?


* Hematological conditions (sickle cell disease), malignancy (leukemia, myeloproliferative diseases, metastatic bone disease), chemical poisoning (fluoride, lead), and congenital diseases (osteopetrosis, pyknodysostosis) can cause sclerotic bone lesions.

* Osteopetrosis is a rare but important condition, especially in the pediatric population.

* Proper understanding of analytical principle is important in correct interpretation of laboratory results.

* It is important to be aware of uncommon sources of common enzymes for the correct diagnosis of rare conditions.

* In the presence of increased CK-BB associated with osteopetrosis, immunoinhibition can result in falsely high CK-MB activity.

* Supplementation of biochemical markers with genetic markers is important to make more definitive diagnoses in many human diseases.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Acknowledgments: We thank Jennifer Burns, Senior Hospital Scientist, Department of Clinical Biochemistry, Royal Prince Alfred Hospital, Camperdown, NSW, Australia, for providing us with the image of CK isoenzyme electrophoresis gel.


(1.) Manolagas SC. Normal skeletal development and regulation of bone formation and resorption. UpToDate. (Accessed April 2015).

(2.) Stark Z, Savarirayan R. Osteopetrosis. Orphanet J Rare Dis.2009;4:5.

(3.) Tolar J, Teitelbaum SL, Orchard PJ. Osteopetrosis. NEJM 2004;351:2839-49.

(4.) Blank R, Bhargava A. Osteopetrosis. Medscape. (Accessed March 2015).

(5.) Whyte MP, Kempa LG, McAlister WH, Zhang F, Mumm S, Wenkert D. Elevated serum lactate dehydrogenase isoenzymes and aspartate transaminase distinguish Albers-Schonberg disease (chloride channel 7 deficiency osteopetrosis) among the sclerosing bone disorders. J Bone Miner Res 2010;25:2515-26.

(6.) Thomas A, Francis L, James BR. Osteopetrosis. Postgrad Med J 2009;85:250.

(7.) Waguespack SG, Hui SL, DiMeglio LA, Econs MJ. Autosomal Dominant osteopetrosis: clinical severity and natural history of 94 subjects with a chloride channel 7 gene mutation. J Clin Endocrinol Metab 2007;92:771-8.

(8.) Waguespack SG, Hui SL, White KE, Buckwalter KA, Econs MJ. Measurement of tartarate-resistant acid phosphatase and the brain isoenzyme of creatine kinase accurately diagnoses type II autosomal dominant osteopetrosis but does not identify gene carriers. J Clin Endocrinol Metab 2002;87:2212-7.

(9.) Nour M, Ward LM. Infantile malignant osteopetrosis. J Pediatr 2013;163:1230.

(10.) Panteghini M, Bais R. Serum enzymes. In: Burtis CA, Ashwood ER, Bruns DE, editors. Tietz textbookof clinical chemistry and molecular diagnostics. 5th ed. St. Louis (MO): Elsevier Saunders; 2012. p 565-98.


Matthew T. Drake *

This case highlights the significance of altered bone metabolism on bone mass and quality. In this patient, the increased CK-BB fraction strongly suggests osteopetrosis. Clinically, her presentation is most consistent with the intermediate disease form whereby affected persons have short stature, increased risk for cranial nerve deficits due to passage through narrowed skull foramina, increased risk for low-trauma fractures owing to poor bone quality, dental/tooth abnormalities, and anemia from crowding of the bone marrow cavity. At a molecular level, the increases in both LD and AST are most consistent with an osteoclast-specific chloride channel 7 (CLCN7) gene mutation.

Despite all patients with osteopetrosis having gene defects that affect osteoclast function and activity, most mutations result in normal or increased numbers of osteoclasts (albeit non- or only poorly-functional), a fact which may partially explain the observed high-bone mass phenotype. Normally, osteoclast-mediated bone resorption is well-matched temporally and spatially with osteoblast-mediated bone formation. To maintain this balance, osteoblasts produce factors including RANKL and M-CSF, which directly mediate osteoclastogenesis and osteoclast activity. Recent data suggest that osteoclasts also produce multiple factors to directly regulate osteoblast function and activity.

Consistent with this bidirectional osteoblast-osteoclast coupling are data showing that inhibition of cathepsin K, an osteoclastic cysteine protease, results in osteoclasts which attach to bone but are incapable of bone resorption. Intriguingly, humans with cathepsin K mutations have skeletal hypermineralization and increased fracture risk, with bone biopsies demonstrating normal or increased osteoclast numbers. Although bone histomorphometry is not provided here, if confirmed a CLCN7 mutation would be expected to diminish osteoclast function but not osteoclast numbers. Accordingly, the presence of non-/poorly functional osteoclasts incapable of resorbing bone, yet capable of signaling to neighboring osteoblasts to support bone anabolism, would suggest the osteoblast may be a viable therapeutic target for future treatments in patients with osteopetrosis.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Matthew T. Drake *

Division of Endocrinology, Diabetes, Metabolism, and Nutrition, Mayo Clinic College of Medicine, Rochester, MN.

* Address correspondence to the author at: Division of Endocrinology, Diabetes, Metabolism, and Nutrition, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905. Fax 507-293-3853; e-mail

Received December 22, 2015; accepted January 4, 2016.

DOI: 10.1373/clinchem.2015.248757


Michael P. Whyte [1, 2] *

The present case provides a good review of osteopetrosis, and from this disorder more than a "pearl" for those who assay the MB isoenzyme of CK (CK-MB).

Osteopetrosis reflects the consequences from failure of osteoclasts (OCs) to resorb skeletal tissue during growth. Complications can be brittle bones, cranial nerve palsies, and myelophthisic anemia from compromised marrow spaces. Traditionally, an autosomal recessive infantile ("malignant") type and an autosomal dominant adult ("benign") type are discussed. The diagnosis can be made from clinical, radiographic, and histopathological findings; however, a precise diagnosis of osteopetrosis now involves mutation analysis from among a dozen causal genes. This effort helps to define the etiology and recurrence risks and to further understand the prognosis. Importantly, for most but not all forms of osteopetrosis, bone marrow transplantation to replace ineffective with functional OCs can be curative. Nevertheless, an especially rare "OC-poor" osteopetrosis reflects mutations within TNFSF11 encoding RANKL, a key promoter of osteoclastogenesis, and is not treatable by bone marrow transplantation. What has been called autosomal dominant osteopetrosis type I results instead from enhanced osteoblast function.

For this girl, mutation analysis was not performed, but she likely has osteopetrosis from loss-of-function mutation^) within the gene for chloride channel 7 (CLCN7). Why can this be said? First, the studies from the clinical laboratory revealed increases in serum CK-BB and TRAP. This "duo" seems specific for osteopetrosis among the disorders of high bone mass, likely explained by excessive numbers of, and/or sick, OCs. Additionally, the increases in her serum LD (isoenzymes 3, 4, and 5) and sometimes AST seem specific for Albers-Schonberg disease, the autosomal dominant osteopetrosis involving CLCN7.

These findings from one rare patient teach us that inaccuracies can come from immuno-inhibition quantification of serum CK-MB when CK-BB is increased. This should be an important "heads-up" for those who encounter increased serum CK-BB from osteopetrosis or other disorders.

Author Contributions: The author confirmed that he contributed to the intellectual content of this paper and has met the following3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Author's Disclosures or Potential Conflicts of Interest: The author declared no potential conflicts of interest.

Michael P. Whyte [1,2] *

[1] Center for Metabolic Bone Disease and Molecular Research, Shriners Hospital for Children, and [2] Division of Bone and Mineral Diseases, Department of Internal Medicine, Washington University School of Medicine; St. Louis, MO.

* Address correspondence to the author at: Shriners Hospitals for Children, 4400 Clayton Ave., St. Louis, MO 63110. Fax314-872-7844; e-mail

Received December 4,2015; accepted December 10, 2015.

DOI: 10.1373/clinchem.2015.248765

Nilika Wijeratne, [1,2,3] * Kay Weng Choy, [1] Zhong Xian Lu, [1,2,4] Justin Brown, [5,6] James C.G. Doery [1,2]

[1] Department of Pathology and [5] Monash Children's Hospital, Monash Health, Clayton, Victoria, Australia; Departments of [2] Medicine and [6] Pediatrics, Monash University, Clayton, Victoria, Australia; [3] Dorevitch Pathology, Heidelberg, Victoria, Australia; [4] Melbourne Pathology, Collingwood, Victoria, Australia.

* Address correspondence to this author at: Dorevitch Pathology, Department of Biochemistry, 18 Banksia St. Heidelberg, Vic, 3084 Australia. Fax +61-3-9244-0207; e-mail

Received June 2,2015; accepted August 31,2015.

DOI: 10.1373/clinchem.2015.244616

[7] Nonstandard abbreviations: AST, aspartate aminotransferase; LD, lactate dehydrogenase; ACP, acid phosphatase; CK, creatine kinase; RANKL, receptor activator of nuclear factor [kappa] B ligand; M-CSF, macrophage colony-stimulating factor; AD, autosomal dominant; AR, autosomal recessive; XR, X-linked recessive; TRAP, tartrate-resistant acid phosphatase.

[8] Human genes: CLCN7, chloride channel, voltage-sensitive 7; TCIRG1, T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 subunit A3; CA2, carbonicanhydrase II; TNFSF11, tumor necrosis factor superfamily member 11 (also known as RANKL).

Caption: Fig. 1. Hip X-ray. Diffuse sclerosis of the sacrum, pelvis, proximal femurs bilaterally, and distal right femur. Right femoral neck fracture is also identified, although alignment of the right hip is maintained.

Caption: Fig. 2. CK isoenzyme electrophoresis pattern. (A), control; (B-D), healthy patients; (E), the case patient.
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Title Annotation:Clinical Case Study
Author:Wijeratne, Nilika; Choy, Kay Weng; Lu, Zhong Xian; Brown, Justin; Doery, James C.G.
Publication:Clinical Chemistry
Article Type:Clinical report
Geographic Code:8AUST
Date:May 1, 2016
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