S. Simsek¹ , K. Janssens², M. L. Kwee³, W. Van Hul², J. Veenstra⁴ and J. C. Netelenbos¹

(1)	Department of Endocrinology/Diabetes Center, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands

(2)	Department of Medical Genetics, University of Antwerp, Antwerp, Belgium

(3)	Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands

(4)	Department of Internal Medicine, St. Lucas Andreas Hospital, Amsterdam, The Netherlands

Received: 29 April 2004  Accepted: 15 September 2004  Published online: 16 June 2005

Abstract  We report on a 46-year-old mother of Moroccan origin, suffering mainly from painful, swollen legs, and her 26-year-old son who had experienced intense pain in his legs, without fever, for approximately 3 years. They did not have dysmorphic features or abnormal gaits. Radiographic studies of the mother revealed diaphyseal sclerosis of the tibia and spondylosis of the thoracal and lumbar vertebrae. The son had sclerosis of the diaphyses of the metacarpalia of the left hand, the femur and the fibula. The other parts of the skeleton were normal. Several osteosclerotic/hyperostotic disorders, such as melorheostosis (present mostly in sporadic cases and affecting lower extremities) and van Buchem s disease (autosomal recessive and commonly affecting the mandible) were considered as a diagnosis in the proposita. However, similar symptoms in the son of the proposita suggested an autosomal dominant inheritance pattern. This brought us to the diagnosis of progressive diaphyseal dysplasia (PDD) or Camurati–Engelmann disease (CED), an autosomal dominant disorder characterized by limb pain, reduced muscle mass, weakness, a waddling gait, progressive periosteal and endosteal sclerosis of the diaphyses of the long bones and sclerosis of the skull base. Mutations in the transforming growth factor (TGF)- 1 gene on chromosome 19q13.1 have been reported to cause this disorder. The diagnosis of PDD/CED in this family was confirmed at the molecular level by detection of a C-to-T transition at position 466, leading to an arginine-to-cysteine amino acid change (position 156) in exon 2 of the transforming growth factor- 1 (TGFB1) gene.

Keywords  Camurati–Engelmann disease - Progressive diaphyseal dysplasia - Transforming growth factor  -1

Introduction

Camurati–Engelmann Disease (CED) was first described in 1920 as a symmetrical, hereditary bone disease involving the long bones of the lower limbs [1]. In 1922, Camurati was the first to recognize the hereditary nature of the disorder [2]. A few years later an isolated case of a bone disease in combination with muscular wasting was described, designated as osteopathica hyperostotica (sclerotisans) multiplex infantilis [3]. This led to the recognition of CED as a distinct entity. This disease is also known as progressive diaphyseal dysplasia (PDD).

Patients with CED usually develop severe, bilateral, and symmetrical pain in the limbs, muscular weakness due to muscular hypoplasia and a waddling gait (claudication) from early childhood onwards. Other observed clinical symptoms are easy fatiguability, exophthalmus, hearing problems and loss of vision. Sometimes, systemic manifestations such as hepatosplenomegaly, bone marrow dysfunction (anemia, leukopenia) and delayed sexual development can occur [4, 5]. Biochemically, elevation of alkaline phosphatase and erythrocyte sedimentation rate may be present, although only in a minority of the patients. Radiographically, CED is characterized by endosteal and periosteal thickening of the diaphyses of the long bones, causing narrowing of the medullary cavity. The metaphyses can be affected as well, but the epiphyses are typically spared. The most commonly affected bones are the tibiae and femora [4, 5]. In approximately 50% of affected individuals sclerosis of the skull base is present as well.

CED is inherited in an autosomal dominant manner and has a variable penetrance [4]. Alterations in the sequence of the transforming growth factor- 1 (TGFB1) gene on the chromosomal region 19q13.1 have been found to be associated with this disorder [6, 7, 8, 9]. At the protein level, most mutations were found to negatively affect the capacity of the so-called latency associated peptide to keep the mature TGF- 1 inactive [10]. This leads to premature/facilitated activation of the mature peptide. As a result the balance of bone turnover is disrupted, causing an increase in bone formation. We describe a mutation in the coding sequence of the TGFB1 gene in a Moroccan family living in the Netherlands.

Case report

A 28-year-old woman was admitted to her district hospital in 1985 with a painful and swollen left leg. Histological examination of a biopsy was recognized by the Committee for Bone Tumors as an osteoid osteoma, for which she underwent an operation with an osteosynthesis. The complaints disappeared and the osteosynthesis material could be removed.

In March 2000 she was seen again, this time with a painful and swollen right leg. The patient had periods of feeling feverish, without objective measurement of her body temperature. She had no complaints of night sweating. There was no history of trauma. On physical examination she had a body-mass index (BMI) of 27.0 kg/m² (normal range 18.0–24.0 kg/m²). Except for the swollen, red right leg, no other abnormalities could be observed. Laboratory tests showed a slightly elevated erythrocyte sedimentation rate and C-reactive protein. Kidney function, liver enzymes, alkaline phosphatase, hemoglobin, and thyroid function were all normal. No elevated bone turnover could be detected by urine analysis (normal hydroxyproline). Serology for Brucella spp., Coxiella burnetii and hepatitis-C was normal. Total-body technetium bone scintigraphy, however, showed increased uptake in the right tibia and also some uptake in the left tibia (Fig. 1a).

Fig. 1  Total-body scan of the proposita showing a focal uptake in the right lower extremity (A). A radiograph of the right lower extremity shows hyperostotic lesions/bone thickening of the diaphyses (periosteal and endosteal) (B)

The patient underwent a bone biopsy for microbiological culture as well as for histological examination. The microbiological culture of the bone biopsy did not yield any growth of bacteria. The Committee for Bone Tumors proposed the diagnosis chronic osteomyelitis, after histological examination of the bone biopsy. This was based on the following findings in the biopsy: reactive changes with elevated bone turnover and new bone formation in the periosteum, focally some lymphocytes and plasma cells and fields of marrow fibrosis. The Committee for Bone Tumors suggested that the osteomyelitis could have been caused by bone infarction, as can be seen, for example, in sickle-cell anemia (not present in our patient). Since the proposed diagnosis of (chronic) osteomyelitis was not completely consistent with the patient s complaints, she was referred to our institute for re-assessment. At examination she showed no dysmorphic features and her gait was normal. X-ray examination showed cortical hyperostosis of the right tibia (Fig. 1b). On the basis of this finding, we considered melorheostosis and van Buchem s disease as possible diagnoses.

The son of the proposita presented with a painful and swollen right leg and was easily fatigued. At examination he showed no dysmorphic features and had a normal gait. Initially, it was thought that his symptoms were caused by traumas, as he practiced kickboxing as a hobby. Radiologically, in retrospect, the typical, symmetrical, cortical thickening of the diaphysis of the lower extremities was observed (Fig. 2). He also showed sclerosis of the metacarpalia of the left hand. The presence of radiologically confirmed bone abnormalities in the proposita (Fig. 1) as well as in her son (Fig. 2) suggested an autosomal dominant inheritance pattern. Consequently, we could rule out melorheostosis (which are mostly sporadic cases) as well as van Buchem s disease (autosomal recessive inheritance) as possible diagnoses. This led us to the diagnosis of Camurati–Engelmann Disease (CED). Both the proposita and her son are on low-dose corticosteroids, which has resulted in some improvement in the quality of life of both patients.

Fig. 2  Hyperostosis of the right fibula (A) and the distal right femur (B) of the son

Since the molecular genetic basis of CED is established [6, 7], the coding sequence of the TGFB1 gene in the proposita and her son was investigated.

Molecular analysis of the TGFB1 gene

Genomic DNA was isolated from blood leukocytes in accordance with standard methods. Primers were used to amplify the entire coding region of the TGFB1 gene. Direct sequencing was carried out in both orientations of genomic DNA PCR products on an ABI 3100 automated sequencer (Applied Biosystems, USA) using the DYEnamic ET terminator cycle sequencing kit (Amersham Biosciences, UK).

Results and discussion

Sequence analysis of the entire coding region and intron–exon boundaries of TGFB1 revealed a heterozygous C-to-T transition in exon 2 (position 466) in the proband and her son. The resulting Arg156Cys transition is a rather dramatic change, substituting a large, charged, amino acid for a small, uncharged one with a thiol-group. Moreover, the presence of the same mutation in a CED family of Colombian origin [11] established that this mutation in the TGFB1 gene was responsible for CED in the family described in this study.

The clinical history of the proposita shows that it was difficult for one to come to the correct diagnosis. This has several reasons: (1) the unilateral symptomatology (first the left leg of the patient and, later, her right leg); (2) initially, the recognition of the alternative diagnosis of osteoid osteoma; (3) a relatively long disease-free interval (15 years); (4) a seemingly sporadic occurrence, until the manifestation of the disease in another family member. Therefore, other hyperostotic disorders were considered in the differential diagnosis, namely melorheostosis and van Buchem s disease (hyperostosis corticalis generalisata).

Van Buchem s disease is an autosomal recessive disease, and, although painful long bones can be present, they are not the hallmark of this disorder. The predominant symptom is progressive asymmetrical enlargement of the mandible and cranium due to endosteal hyperostosis [4, 5]. Because of the absence of the typical involvement of the mandible, van Buchem s disease was not the most likely diagnosis in the proposita. The autosomal dominant form of endosteal hyperostosis, the so-called Worth type, is a more benign form [5] and would have been possible as the causative disorder. However, since the associated mandible enlargement was not present in the woman and her affected son, this diagnosis was excluded as well.

Melorheostosis is characterized by osteosclerosis and soft tissue involvement. This disorder usually affects the long bones, especially the lower extremities. Radiologically, the bone abnormalities resemble the dripping of a candle, the so-called candle-wax phenomenon, although an osteoma-like pattern of melorheostosis has been described as well. Melorheostosis typically occurs sporadically and has a segmental distribution. A distinctive clinical finding in melorheostosis is a joint restriction. Although both the candle-wax phenomenon and the joint restriction were absent, it was believed that the woman suffered from melorheostosis because of the unilateral location and the seemingly sporadic case.

Once the son s complaints had been recognized, an autosomal dominant inheritance pattern seemed more likely. This prompted us to consider CED as the diagnosis, in spite of the presence of asymmetrical lesions in our patient, because CED usually leads to symmetrical bony abnormalities. Another interesting finding is that CED had presented after childhood in this family, although usually this disease manifests itself during childhood. In addition, after retrospective analysis of the medical history of the patients, no (minor) signs/symptoms in earlier years could be recalled by the patients that could be attributed to this disease.

Until now eight different mutations in the TGFB1 gene have been characterized in almost 30 families in Europe, Australia, Israel, Japan, South America (Colombia) and the USA. The Colombian mutation (R156C) [11] is the same genetic defect as the one identified in this study, confirming the disease-causing nature of the base change identified in our family. The family in our study is the first family with CED characterized at the molecular genetic level from northern Africa.

Five of the eight mutations (R218C, R218H, H222D, C223S and C225R) are located in exon 4 at the C-terminal region of the latency-associated peptide close to or within the two cysteine residues (cysteine 223 and cysteine 225) that form the intra-chain disulfide bridges. Experimental data have shown that these mutations do not lead to overproduction of TGF- 1, but destabilize the precursor protein, thus facilitating activation of TGF- 1 [10, 12]. The pathogenetic mechanism caused by the mutations in exon 1 (Y81H and the L10-L12 duplication in the signal peptide) is different. The mutations cause a defective secretion of TGF- 1, leading to intracellular accumulation of the protein. Despite this fact, signaling by the (mutant) TGF- 1 is increased, possibly through a novel, intracrine loop with intracellular ligand–receptor interaction [10].

The mutation found in our study and in the family from Colombia has not been examined at the functional level yet. It creates a putative alternative disulfide bridging partner, possibly leading to a conformational change, thus causing disruption of the association of the LAP with the mature TGF- 1. Subsequently, the mature TGF- 1 might be activated more easily, classifying this mutation in the same group as the exon 4 mutations.

In conclusion, this paper describes the first Moroccan family with molecular genetically confirmed CED. This case shows that reaching the correct diagnosis in sporadic cases of small families may be difficult. A thorough family history is required and may help to circumvent these problems. Moreover, the unraveling of the molecular genetic basis of diseases is/will be of major help in confirming and/or establishing the correct diagnosis.

References

1.	Cockayne EA (1920) Case for diagnosis. Proc R Soc Med 13:132–136
2.	Camurati M (1922) Di uno raro caso di osteite simmetrica ereditaria degli arti inferiori. Chir Organi Mov 6:662–665
3.	Engelmann G (1929) Ein fall von osteopathia hyperostotica (sclerotisans) multiplex infantilis. Rofo Fortschr Geb Roentgenstr Neuen Bildgeb Verfahr 39:1101–1106
4.	Whyte MP (2003) Primer on the metabolic bone diseases and disorders of mineral metabolism, sect VIII. Genetic, developmental, and dysplastic skeletal disorders, fifth edn. Published by the American Society for Bone and Mineral Research, pp 449–478
5.	Vanhoenacker FM, De Beuckeleer LH, Van Hul W, Balemans W, Tan GJ, Hill SC, De Schepper AM (2000) Sclerosing bone dysplasias: genetic and radioclinical features. Eur Radiol 10:1423–1433
6.	Kinoshita A, Saito T, Tomita H, Makita Y, Yoshida K, Ghadami M, Yamada K, Kondo S, Ikegawa S, Nishimura G, Fukushima Y, Nakagomi T, Saito H, Sugimoto T, Kamegaya M, Hisa K, Murray JC, Taniguchi N, Niikawa N, Yoshiura K (2000) Domain-specific mutations in TGFB1 result in Camurati–Engelmann disease. Nat Genet 26:19–20
7.	Janssens K, Gershoni-Baruch R, Guanabens N, Migone N, Ralston S, Bonduelle M, Lissens W, Van Maldergem L, Vanhoenacker F, Verbruggen L, Van Hul W (2000) Mutations in the gene encoding the latency-associated peptide of TGF-beta1 cause Camurati–Engelmann disease. Nat Genet 26:273–275
8.	Campos-Xavier B, Saraiva JM, Savarirayan R, Verloes A, Feingold J, Faivre L, Munnich A, Le Merrer M, Cormier-Daire V (2001) Phenotypic variability at the TGF-beta1 locus in Camurati–Engelmann disease. Hum Genet 109:653–658
9.	Mumm SR, Obrecht S, Podgornik MN, Whyte MP (2001) Camurati–Engelmann disease: new mutations in the latency-associated peptide of the transforming growth factor beta-1 gene. J Bone Miner Res 16:S223
10.	Janssens K, ten Dijke P, Ralston SH, Bergmann C, Van Hul W (2003) Transforming growth factor-beta1 mutations in Camurati–Engelmann disease lead to increased signaling by altering either activation or secretion of the mutant protein. J Biol Chem 278:7718–7724
11.	Hecht JT, Blanton SH, Broussard S, Scott A, Rhoades Hall C, Milunsky JM (2001) Evidence for locus heterogeneity in the Camurati–Engelmann (DPD1) syndrome. Clin Genet 59:198–200
12.	Saito T, Kinoshita A, Yoshiura Ki, Makita Y, Wakui K, Honke K, Niikawa N, Taniguchi N (2001) Domain-specific mutations of a transforming growth factor (TGF)-beta1 latency-associated peptide cause Camurati–Engelmann disease because of the formation of a constitutively active form of TGF-beta1. J Biol Chem 276:11469–11472

Camurati–Engelmann disease (progressive diaphyseal dysplasia) in a Moroccan family(摩洛哥家庭中的Camurati–Engelmann疾病（进行性骨干发育异常）)
Coronary Calcium Screening Seen Useful Beginning Between Age 40 and 50(40岁至50岁开始冠状动脉钙筛查有益)
Healthcare System Approach May Reduce Disability Associated With Musculoskeletal Disorders(医疗保健的系统方法可以降低肌与骨骼疾病相关的残疾)