Sclerostin deficiency in humans

BON-11153; No. of pages: 12; 4C: 2, 9 Bone xxx (2016) xxx–xxx

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Sclerostin deficiency in humans Antoon H van Lierop 1, Natasha M Appelman-Dijkstra, Socrates E Papapoulos ⁎ Center for Bone Quality, Leiden University Medical Center, Leiden, The Netherlands

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Article history: Received 12 June 2016 Revised 9 September 2016 Accepted 10 October 2016 Available online xxxx Keywords: Sclerostin Sclerosteosis Van Buchem disease Bone formation Bone resorption

a b s t r a c t Sclerosteosis and van Buchem disease are two rare bone sclerosing dysplasias caused by genetic defects in the synthesis of sclerostin. In this article we review the demographic, clinical, biochemical, radiological, and histological characteristics of patients with sclerosteosis and van Buchem disease that led to a better understanding of the role of sclerostin in bone metabolism in humans and we discuss the relevance of these findings for the development of new therapeutics for the treatment of patients with osteoporosis. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Sclerosteosis and van Buchem disease are two very rare bone sclerosing dysplasias belonging to the group of craniotubular hyperostoses. They were first described in the 1950s by Truswell and van Buchem et al. as “osteopetrosis with syndactyly”, and “hyperostosis corticalis generalisata familiaris”, respectively [1,2], and are characterized by endosteal hyperostosis and generalized osteosclerosis. The two disorders have very similar phenotypes caused by genetic deficiency of sclerostin. Osteocyte-produced sclerostin decreases bone formation by antagonizing the canonical Wnt signalling pathway in cells of the osteoblast lineage at the bone surface, an action facilitated by LRP4 [3]. In addition, sclerostin acts on neighbouring osteocytes and increases RANKL expression and the RANKL/OPG ratio increasing, thus, osteoclastic bone resorption [4,5]. Although osteocytes are the predominant source of sclerostin, the protein is also expressed by other terminally differentiated cells within mineralized matrices, such as cementocytes [6] and hypertrophic chondrocytes [7]. A detailed description of the control of the synthesis and mechanism of action of sclerostin is provided elsewhere [8,9]. We review here the clinical, biochemical, radiological, and histological characteristics of patients with sclerosteosis and van Buchem disease that led to a better understanding of the role of sclerostin in bone metabolism in humans and we discuss the relevance of these findings ⁎ Corresponding author at: Center for Bone Quality, Leiden University Medical Center, Albinusdreef 2, Leiden 2333 ZA, The Netherlands. E-mail address: [email protected] (S.E. Papapoulos). 1 Present address: Department of Internal Medicine, Amsterdam Medical Center, Amsterdam, The Netherlands.

for the development of new therapeutics for the treatment of patients with osteoporosis. For this, we reviewed the medical records of 66 South African patients with sclerosteosis and 15 Dutch patients with van Buchem disease and we performed a literature search of nine electronic databases (PubMed, Embase, Web of Science, Cohrane, Science Direct, CINAHL, Academic Search Premier, Wiley, HighWire) using the terms sclerosteosis and van Buchem disease, or combinations of the two, to identify additional cases. A total of 241 unique references were found and checked; we excluded abstracts of meetings. 2. Sclerosteosis We identified 96 cases of sclerosteosis born between 1896 and 2011 (Table 1). Sixty-six of these patients were members of the Afrikaner community of South Africa, descendants of Dutch settlers in this country in the 17th century; the remaining 30 patients were from the USA (n = 9), Brazil (n = 7), Germany (n = 2), Morocco (n = 1), Turkey (n = 3), Saudi Arabia (n = 1), Egypt (n = 2), Senegal (n = 1), India (n = 1), Japan (n = 1, and China (n = 2) [10–24]. The disorder is inherited as an autosomal recessive trait and is caused by loss-of-function mutations in the SOST gene on chromosome 17q12-q21, which encodes sclerostin. Eleven different mutations have been so far reported in patients with sclerosteosis [13,14,16–18,20,22,24–26]. Recently, patients with sclerosteosis phenotypes due to loss-of-function mutations of LRP4 were described [27–29]. In such patients the lack of a functional co-receptor (LRP4) impairs the action of sclerostin leading to upregulation of the Wnt pathway and to a phenotype similar to that of patients with sclerostin deficiency. Serum sclerostin levels are, however, increased rather than decreased and this condition is, therefore, not further discussed in this article.

http://dx.doi.org/10.1016/j.bone.2016.10.010 8756-3282/© 2016 Elsevier Inc. All rights reserved.

Please cite this article as: A.H. van Lierop, et al., Sclerostin deficiency in humans, Bone (2016), http://dx.doi.org/10.1016/j.bone.2016.10.010

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Table 1 Studies of patients with sclerosteosis. Origin

Number of patients

SOST gene mutation

South Africa USA Brazil Germany Morocco Turkey

66 9 7 2 1 2 1 2 1 1 2 1 1

c.69C N T Nonsense [26] c.376C N T Nonsense [25] c.373G N A Nonsense [13,25] IVS1 + 1G N C Spice site [14] c.79C N T Nonsense [16] c.449T N C Missense [17] c.371G N A Nonsense [18] c.87-88insC Frame shift [20] IVS1 + 3A N T Splice site [25] c.296_297insC Frame shift [22] c.444_445TC N AA Premature stop codon [24] No genetic confirmation [23] No genetic confirmation [19]

Egypt Senegal India China Japan Saudi Arabia

Mutation type

2.1. Clinical features In the majority of patients with sclerosteosis the first manifestation of the disorder is syndactyly. It was present in 66% of patients (56/85) and usually involved the 2nd and 3rd finger, although other fingers and toes could also be affected [16,20,22]. The severity of syndactyly ranged from soft tissue webbing to complete bony union of phalanges [10,18]. In patients without syndactyly minor deformities of digits such as radial deviation of the phalanges or nail dysplasias were often observed. Otherwise, prenatal skeletal development is normal and weight, length and facial proportions of affected individuals are within normal limits at birth. Postnatally, longitudinal growth is increased and patients with sclerosteosis are already taller than their peers at school age. Closure of the growth plates occurs normally and growth is arrested after puberty when patients are often exceptionally tall. The median height of 28 male adult patients was 190 cm (range 170–209 cm), and 179 cm of 23 adult female patients (range 151–194 cm). Interestingly, the height of patients from Brazil, China and India has been reported normal. In 19 South African patients and 26 related heterozygous gene carriers, the Z-score of height of the patients was significantly higher than that of carriers (+0.84 and −0.48 SD, respectively) [30]. Canonical Wnt signalling promotes differentiation and maturation of chondrocytes and sclerostin is expressed by terminally differentiated chondrocytes [7]. Sclerostin may, therefore, inhibit Wnt signalling in chondrocytes in the growth plate similarly to its action on osteoblasts in bone. In sclerosteosis, lack of sclerostin may lead to increased differentiation towards hypertrophic chondrocytes resulting in a larger hypertrophic

zone in the growth plate and, therefore, more new bone accrual and more longitudinal growth [30]. The clinical signs and symptoms of the disease are largely due to the increased growth of cranial bones caused by sclerostin deficiency (Fig. 1). To obtain a better insight in the clinical features of sclerosteosis (and van Buchem disease) we combined information reported in the literature with data from clinical records of studied patients. In the calculation, however, of the prevalence of complications we included only cases in which absence or presence of each complication was specifically mentioned. 2.2. Facial deformities Severe facial distortion due to excessive growth of the mandible and enlargement and bossing of the forehead starts in childhood and progresses in adulthood. Bossing of the forehead was present in 90% of patients (71/79) and the average head circumference of 20 adult patients was 61.1 cm (Z-score + 2.9). Mandibular overgrowth was present in 91% of patients (72/79); eight patients required corrective surgery and in one of them 2 kg of bone was removed from the mandible. In another patient, growth of the mandible progressed after corrective surgery at the age of 52 years requiring another operation 7 years later, after which the mandibular size remained stable. Other, less frequent facial deformities include proptosis, due to bone overgrowth in the orbitae, hypertelorism and midfacial hypoplasia [31]. 2.3. Entrapment of cranial nerves Complications result from thickening of the skull base and dome causing cranial nerve entrapment syndromes, of which facial palsy is very common and the first presenting sign. In total 93% of patients (77/83) experienced at least one attack of facial palsy at a median age of 3 years; this occurred within the first 3 months of life in a few patients (n = 6) but could also develop in early adulthood (n = 3, ages 19 to 21 years). During childhood patients may experience transient bilateral attacks of facial nerve palsy up to the age of 20 years, following which no more episodes generally occur. However, permanent paralysis of the facial muscles occurs frequently, and is accompanied by synkinesis, asymmetry, contractures and crocodile tears [15,32]. Facial palsies are caused by the narrowing of the neural foramina and fallopian canal, whereby the labyrinthine segment is most severely affected [15]. The impairment of the facial nerve is the result of direct compression by the thickened bone, venous occlusion leading to oedematous swelling with subsequent impingement of the facial nerve, or nerve ischemia resulting from the occlusion of the stylomastoid artery [15].

Fig. 1. Photos of the skull of a patient with sclerosteosis (A) and an individual without bone disease (B). From the collection of the University of Pretoria, South Africa.

Please cite this article as: A.H. van Lierop, et al., Sclerostin deficiency in humans, Bone (2016), http://dx.doi.org/10.1016/j.bone.2016.10.010

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Impingement syndromes of other cranial nerves are less common, and may include the trigeminal nerve associated with neuralgia, hypoesthesia or anosmia (13 cases), the acoustic nerve leading to sensory hearing loss (35 cases), and the optic nerve leading to visual impairment or complete blindness (11 cases) [11,12,17,21,22]. Although optic nerve atrophy was diagnosed in several cases this might have been the consequence of increased intracranial pressure in at least some of them [31]. In only one case impairment of nerves other than the cranial nerves was reported and involved the impingement of the cervical plexus by bone overgrowth of the intervertebral foramen, causing pain and paresis of the arms, which was relieved by unilateral decompression of the cervical nerve root [31]. 2.4. Hearing loss Similar to facial palsy, hearing loss is highly prevalent in sclerosteosis, with 94% of patients (73/78) affected. It becomes apparent in early childhood, at a median age of 6 years, and is often preceded by recurrent inner ear infections. In the beginning, hearing loss is purely conductive, but progresses into mixed conductive, sensorineural later in life [33]. Hearing loss appears to stabilize in adulthood but is severe in the majority of patients, requiring often hearing aids, and complete deafness was reported in two cases. The conductive component of hearing loss results from the narrowing of the outer hearing canal, and fixation of the auditory ossicles within the narrowed inner ear cavity. The sensorineural hearing loss is caused by closure of the round and oval window and impingement of the acoustic nerve due to concentric narrowing of internal acoustic meatus [10,33–35]. 2.5. Increased intracranial pressure The most severe and life-threatening complication of sclerosteosis is increased intracranial pressure. We used the following criteria to identify cases with increased intracranial pressure: need for decompressive interventions of the skull, radiological features of intracranial hypertension and severe recurring headaches. It occurred in 71% patients (48/68) and presented commonly in early adulthood with recurrent severe headaches, sometimes accompanied by visual impairment, dizziness or sudden death. The median age of first occurrence of this complication was 20 years, with a broad range (5 to 56 years). In patients with sclerosteosis the progressive overgrowth of the calvarium and skull base reduce the intracranial diameter; calvarial thickness, which is normally between 0.6 and 1.1 cm, can be increased to as much as 5 cm [11]. Besides the direct pressure on the brain, impaired drainage of cerebrospinal fluid by the compression of ventricles and aqueducts can also be contributory to intracranial hypertension [11,36]. Moreover, narrowing of the jugular canal at the skull base can lead to jugular vein occlusion, increasing intracranial pressure by reducing venous outflow from the brain [11]. When the brain loses compensatory mechanisms to absorb sudden increases in intracranial pressure, as it occurs during coughing or sneezing, death can result from the compression of the medulla oblongata within the narrowed foramen magnum [36]. This was considered to be the cause of death of at least 16 patients. 2.6. Fractures Fractures have not been reported in any of the patients described in the literature. Similarly, in the analysis of the South African patients there was no report of fractures except in one patient in whom a fracture of the dens of the second cervical vertebra was described and was attributed to the increased weight of the skull. 2.7. Mortality Life expectancy in sclerosteosis is reduced, with a large proportion of patients dying in early adulthood, mainly from complications of

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increased intracranial pressure, or following craniotomy (Fig. 2). Details of the causes of death in the South African patients are shown in Fig. 3. At least 29 of the 66 patients were alive during the last survey in 2010 with ages ranging from 10 to 72 years; four patients were lost to follow-up. Twelve patients died suddenly at a median age of 32.5 years (range 15 to 59 years) from coning of the brainstem, confirmed by post-mortem autopsy in 3 cases and radiography in another. Postoperative complications of craniotomy including meningitis, sepsis, and pulmonary embolism, were the cause of death in 5 patients while one patient died after anaesthesia for a dental operation. Median age of death in these patients was 28 years (range 16 to 56 years). In 8 cases the cause of death was unrelated to increased intracranial pressure while the cause of death was unknown, or not reported in 7 cases (see Fig. 3). Except for two patients who died from a heart condition at the ages of 56 and 70 years, respectively, no signs of impaired cardiac function, hypertension or arrhythmias were found in other elderly patients, and no abnormal cardiac or vascular findings have been reported in autopsies. It should be mentioned that these two patients were born in 1896 and 1920, when life expectancy was lower than today. 2.8. Disease course Within the cohort of South African patients with the same SOST mutation, clinical severity of the disease varied considerably. The more severely affected patients had evident facial disfiguration, recurrent facial palsies, even after surgical decompression, progression of hearing loss and regrowth of the calvarium after craniotomy, while in milder cases facial features were less prominent and secondary complications were rare or mild. In patients surviving into late adulthood, the disease tends to stabilize after the 3rd decade of life [10,30,31]. Although occurrence or recurrence of secondary complications have been observed up to the age of 56 years, in the majority of adult patients there is no progression of symptoms or signs of the disease. However, because a substantial number of patients died before the age of 30 years, it might be that the clinical course of sclerosteosis stabilizes only in the milder cases. On the other hand, as described below, cross-sectional data of biochemical markers of bone turnover, and histomorphometric bone studies suggest that bone formation rates decline with age, supporting the notion of stabilization of the disorder in adulthood. 3. van Buchem disease There are 31 known cases of van Buchem disease, born between 1900 and 2009, almost all inhabitants of the same village in the north of the Netherlands [2,37–43]. This village was an island, before land reclamation connected it to the mainland in 1940. In 1637 a plague epidemic reduced the population of the island to 151 inhabitants which remained up until recently professionally and religiously isolated. The ancestry of the majority of patients was traced back to a couple married in 1751 [39]. In addition to the Dutch patients two siblings of unknown genetic background were reported in Germany [44]. Before the discovery of the genetic defect of van Buchem disease, patients from other countries were diagnosed with the disease on the basis of their clinical and radiographic features. However, in some of these cases there was an autosomal dominant, rather that recessive inheritance, making it more likely that these patients suffered from diseases due to mutations of the LRP5 receptor such as High Bone Mass Disorder or Worth's disease [45–48]. Skeletal manifestations of van Buchem disease are similar to those of sclerosteosis and are caused by defective synthesis of sclerostin. Differently, however, from sclerosteosis, the SOST gene is not mutated in van Buchem disease. Instead, patients have a 52-kb homozygous noncoding deletion 35 kb downstream of the SOST gene [49,50] that is essential for the transcription of the gene in bone but is not required for its embryonic transcription [51]. This, may explain the similar phenotypes of van Buchem disease and sclerosteosis and the absence of digit

Please cite this article as: A.H. van Lierop, et al., Sclerostin deficiency in humans, Bone (2016), http://dx.doi.org/10.1016/j.bone.2016.10.010

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Proportion of patients without an event

1.0

0.8

0.6

0.4

0.2

0 0

20

40

60

80

100

Age (years) Mortality

Patients at risk

ICP

Mortality N= 89

84

71

41

23

14

7

4

2

0

0

ICP

59

38

16

8

7

5

3

2

0

0

N= 68

Fig. 2. Kaplan-Meier curves of mortality (continuous line) of patients with sclerosteosis and raised intracranial pressure (ICP, dashed line) of the South African cohort.

deficits included vestibular neuralgia (n = 2), hypoesthesia (n = 1), vertigo spells (n = 2), impaired vision (n = 5) and paralysis of the legs (n = 1). Increased intracranial pressure was reported in only five cases, of which only one patient required neurosurgical treatment [52]. Sudden death due to impaction of the brainstem has never been reported in patients with van Buchem disease, life expectancy appears to be normal and patients had no significant co-morbidities. The oldest patient studied was 81 year-old with type 2 diabetes mellitus, mild heart failure and non-metastasized prostate cancer, comorbidities frequent in elderly populations. One patient sustained a fracture of the wrist in puberty on two occasions, one at the age of 14 years after falling from a tree, and the other at the age of 17 years after a motorcycle accident. Other patients did not report any fractures despite involvement of 5 of them in major accidents such as collision with a tree while riding a motorcycle at very high speed or fall of a 200 kg weight on the back [43]. All patients reported difficulty

abnormalities, such as syndactyly, in patients with van Buchem disease. Whether the difference in genetic defect is also responsible for the milder clinical course of patients with van Buchem disease compared to that of patients with sclerosteosis is unknown. Enlargement of the forehead was present in 60% of patients (12/20), and overgrowth of the mandible in 68% (19/28) for which two patients underwent corrective surgery. Proptosis was reported in 4 cases. Patients with van Buchem disease are not tall, their height being comparable to that of the Dutch population [43]. This may be due to production of sclerostin by hypertrophic chondrocytes in which the regulatory element for the transcription of the SOST gene that is missing in patients with van Buchem disease is not essential. Facial palsies were present in 89% patients (23/26), with the first attack observed at a median age of 2.5 years being diagnosed at birth in two cases. Hearing loss was present in 78% patients (21/27) but was often mild, with 5/15 patients using hearing aids [43]. Other nerve

66 South African Patients with sclerosteosis

29

33

4

Alive

Deceased

Lost to follow-up

Age 10- 72 yrs

12

6

Coning brain stem

Peri-operative complications

Age 15-59 yrs

Age 16-56 yrs

7

8 Unrelated causes • • • • • •

Cause of death unknown Age 28-44 yrs

Old age (81, 85 yrs) Heart disease (56, 70 yrs) Hodgkin lymfoma (10 yr) Malaria (49 yr) Liver chirrosis (36 yr) Suicide (32 yr)

Fig. 3. Flow-chart of mortality and morbidity of South African patients with sclerosteosis.

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keeping afloat in water, only able to avoid sinking by continuous swimming. The clinical course of the disease differs markedly among patients, with some displaying limited facial distortion and minor complications, while in others the phenotype highly resembles that of sclerosteosis with extensive facial deformity, cranial nerve entrapment syndromes and increased intracranial pressure. The disease appears to stabilize in adulthood in most cases.

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may give the lower third an abnormal configuration. Radiographs of the hands may show bone union and malformations of phalanges, and there is prominent cortical thickening of the metacarpals and phalanges, with broadening of the diaphyses. While Beighton et al. reported sclerostic changes of the pedicles and laminae of the vertebrae, Nager et al. showed generalized sclerosis of the cervical and lumbar vertebrae [34,56]. 6. Bone mineral density

4. Carriers of sclerosteosis and van Buchem disease Although both diseases are rare, the carrier rate of sclerosteosis in the Afrikaner community of South Africa has been estimated as 1 in 140 individuals [53] while the carrier rate of van Buchem disease in not known. Heterozygous carriers of sclerosteosis have no signs or symptoms of the disease and do not report bone fractures but may show minor radiographic changes of the skull base [53,54] (Fig. 4). Similarly, carriers of van Buchem disease are symptom-free and have no clinical signs of the disease [43]. 5. Radiology Radiological features of sclerosteosis and van Buchem disease are similar and are characterized by endosteal hyperostosis already present at very young age. On plain X-rays hyperostosis of the skull was evident in children with sclerosteosis [18,54,55], while CT scans of 3 young children (3 to 6 year-old) with van Buchem disease revealed thickening of the calvarium, skull base and mandible, and narrowing of the facial nerve canals (Fig. 5) [42]. In adult patients the calvarial width exceeds 2 cm [43], there is complete obliteration of the diploic space [56], a prominent crista frontalis, and bony overgrowth of the posterior fossa [36]. The mandible is enlarged with prognathism and a wide angle [21,56,57]. Narrowing of the internal auditory meatus, facial canal and vascular foramina can be observed on CT scans of the mastoid as well as abnormalities of the inner ear such as obliteration of the middle ear space, fixation of ossicles and closure of the oval and round windows [33,40,43]. The clavicles are enlarged, and the ribs dense and widened. The pelvis has normal proportions, but is evidently sclerotic, especially on the medial portion of the iliac bones, the ischial and pubic rami [34,56]. There is extensive cortical thickening and relative widening of the tubular bones, with extension of the medullary cavity [41]. In the femurs the cortical thickening is most prominent in the upper two thirds, while widening of the metaphyseal region and lack of diaphyseal constriction

Patient

Bone mineral density measured by dual x-ray absorptiometry (DXA) was greatly increased with Z-scores ranging from + 7.7 to + 14.4 (mean: + 9.7) at the spine and +7.8 to + 11.5 (mean: + 10.2) at the hip in patients with sclerosteosis [14,17,24,30], and from + 5.4 to + 12.3 (mean: + 8.7) at the spine and + 5.2 to + 12.1 (mean:+ 9.5) at the hip, in patients with van Buchem disease [43]. It should be mentioned that in both diseases the high mineral density is evident already in early childhood [42,54] and increases further with age as in healthy subjects with minimal changes in Z-scores. Increased phalangeal bone mineral density using standard radiographic absorptiometry was also reported in 4 adult patients with van Buchem disease (T-score 3.2 ± 1.7) [41]. Importantly, BMD was high normal or increased in all heterozygous carriers of sclerosteosis [54] (Fig. 6). In carriers of van Buchem disease mean BMD Z-score at the spine and the hip was also increased (1.3 ± 1.5 and 0.9 ± 1.0, respectively) with a wider, however, distribution of values (ranges: − 2.2 to + 4.6 and − 1.1 to + 2.9 at the spine and the femoral neck, respectively) [43]. 7. Laboratory investigations 7.1. Serum sclerostin Circulating sclerostin measured with a very sensitive electrochemiluminescence assay was undetectable (b1 pg/ml) in all 19 patients with sclerosteosis tested [30] (Fig. 7). In 26 disease carriers the mean serum sclerostin value was 15.5 pg/ml (95% CI 13.4 to 16.9 pg/ml) which was about 60% lower than the values measured in healthy controls (40.0 pg/ml; 95% CI 37.2 to 42.9 pg/m; p b 0.001). These values most likely mirror the decreased synthesis of sclerostin by the osteocytes as a result of the affected SOST allele and are consistent with the higher BMD values of these individuals without, however, any of the clinical manifestations of patients with sclerosteosis.

Carrier

Fig. 4. Skull radiographs of a patient and a heterozygous carrier of sclerosteosis showing minimal changes of the skull base. Adapted from reference [54].

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Patient

Healthy

Fig. 5. Head CT scan of a 6 year-old patient with van Buchem disease and of a healthy child of the same age. From reference [42].

Despite existing uncertainties of the significance of serum sclerostin measurements in humans, the data obtained in patients and carriers with sclerostin deficiency demonstrate a clear gene-dose effect of the SOST mutation and the van Buchem deletion on immunologically detected circulating sclerostin.

In 15 patients with van Buchem disease circulating sclerostin was detectable in the serum of all but one patient; the mean value 8.0 pg/ml (95% CI 4.9 to 11.0 pg/ml) was significantly lower than that of 22 disease carriers (28.7 pg/ml; 95% CI 24.5 to 32.9 pg/ml) which was lower than the mean value of healthy controls (p b 0.001) [43]. Because van Buchem disease is caused by a defect in sclerostin synthesis, an explanation of the detectable sclerostin levels in patients with the disease is not readily available. It is likely that in van Buchem disease sclerostin production is due either to leaky transcription, the accidental transcription of a gene without prior activation of its regulatory element or to the production of sclerostin by cells other than osteocytes in which this regulatory element is not essential. In favor of the first hypothesis is the finding of a weak sclerostin signal by immunohistochemistry in a bone biopsy from a patient with van Buchem disease [6]. Alternatively, circulating sclerostin may originate from hypertrophic chondrocytes. As described above, patients with sclerosteosis are tall possibly due to lack of sclerostin at the growth plate while this is not the case in patients with van Buchem disease who have normal height and production of sclerostin by chondrocytes may be, at least partly, responsible for the measurable values in serum [43].

7.2. Serum Dickkopf 1 (Dkk1) Dkk1, a Wnt antagonist structurally unrelated to sclerostin, inhibits also bone formation by binding to LRP5/6 receptors. Because sclerostin and Dkk1 bind to the same receptors and inhibit bone formation, lack of sclerostin as it occurs in sclerosteosis and van Buchem disease might affect the production of Dkk1. We measured Dkk1 in serum of 19 patients with sclerosteosis and 13 patients with van Buchem disease and of 24 and 22 of their respective heterozygous carriers [58]. Compared with carriers, patients with sclerostin deficiency had significantly higher values of serum Dkk1 (mean 4.7 ng/ml vs 2.7 ng/ml); the latter values are comparable with those of healthy controls (2.8 ng/ml). Furthermore, serum Dkk1 levels were positively correlated with serum P1NP levels. It is likely that the increased Dkk1 levels in patients

LS-BM D

TH-BMD 16

16

12

Z-score

12 8

8

4

4

0

0

-2

-2 0

20

40 Age

60

80

0

20

40 Age

60

80

Fig. 6. Bone mineral density of the lumbar spine (LS-BMD) and total hip (TH-BMD) expressed as Z-scores of patients with sclerosteosis (closed circles) and heterozygous disease carriers (open circles). Adapted from reference [54].

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Serum sclerostin levels (pg/ml)

80

60

40

20

0 Controls

Carriers VBD

Patients SCL

VBD

SCL

Fig. 7. Serum sclerostin levels in healthy individuals (controls), patients with sclerosteosis (SCL) and van Buchem disease (VBD) and their respective heterozygous carriers of the diseases. Adapted from references [30] and [43].

represent an adaptive response to the increased bone formation that is, however, not sufficient to compensate the lack of sclerostin on bone metabolism. 7.3. Bone turnover markers In most studies of patients with sclerosteosis serum total alkaline phosphatase activity (ALP) values were reported normal for the age of the patients [14,16,24,30,41]. However, raised values were also reported in a few patients [22,59]. In our studies mean serum ALP value was 104 ± 36 SD μ/l in patients with sclerosteosis and 81 ± 32 μ/l in patients with van Buchem disease. In sclerosteosis serum P1NP values declined with age in both patients and carriers and reached a plateau after the age of 20 years (Fig. 8). The mean value of adult patients was 153.7 ng/ml (CI 95% 100.5 to 206.9 ng/ml) being significantly higher than the mean value of healthy controls (37.8 ng/ml; 95% CI 34.5 to 41 ng/ml, p = 0.006). Values of disease carriers were intermediate (mean 58.3 ng/ml; 95% CI 47.0 to 69.6 ng/ml). Thirteen of 14 patients (93%) older than 18 years and 7 of 22 disease carriers (32%) had serum P1NP values higher than 65 ng/ml (upper limit of the reference range). Similar to sclerosteosis serum P1NP values declined with age in patients with van Buchem disease [43]. In adult patients mean serum P1NP value was 96.0 (95% CI 4.6 to 137.4 ng/ml) and in disease carriers Patients Carriers

1400

Serum P1NP (ng/ml)

1200 1000 800 600 400 200 0 0

20

40 Age (years)

60

Fig. 8. Serum P1NP levels of patients with sclerosteosis (closed circles) and heterozygous carriers of the disease (open circles). Interrupted line represents the upper limit of the adult reference range. From reference [30].

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this was 47.8 (95% CI 39.4 to 56.2 ng/ml). In 67% patients and in 19% disease carriers serum P1NP values were higher than 65 ng/ml. Wergedal et al. reported significantly higher serum P1NP values in 6 adult patients with van Buchem disease compared with 9 disease-carriers (75.6 ± 13.7 vs 29.4 ± 5.0 ng/ml; p b 0.01) while in 3 young children values were significantly higher than age- and sex-matched reference values [41,42]. Serum osteocalcin but not bone specific AP levels were higher in patients than in disease carriers (25.1 ± 3.8 vs 13.6 ± 2.0 ng/ml, p b 0.05 and 13.6 ± 1.5 vs 10.0 ± 1.6, p = 0.12) and in the children both serum osteocalcin and bone specific AP were increased. In our studies, serum P1NP values of sclerosteosis disease-carriers and age-matched healthy controls were inversely related with serum sclerostin values (r = − 0.40, p = 0.008) [30]. This was also the case for patients and carriers of van Buchem disease (r = 0.39, p = 0.018) [43]. Taken together the data of bone formation markers in patients with sclerostin deficiency confirm the role of sclerostin on bone formation, help to explain the gene dose-response on bone mass and strengthen the notion that maximal bone accrual occurs in childhood. In line with the results of the bone formation marker serum P1NP, serum levels of the bone resorption marker CTX declined with age in patients with sclerosteosis and van Buchem disease and stabilized around the age of 20 years [30,43] However, absolute values in adult patients and disease-carriers were within the low reference range. Interestingly, serum CTX values of patients with sclerosteosis and lack of sclerostin were arithmetically lower than those of patients with van Buchem disease with sclerostin deficiency (213 pg/ml; 95% CI 103 to 323 pg/ml and 447 pg/ml; 95% CI 266 to 628 ng/ml, respectively). There is no apparent explanation for this finding that is not due to differences in the age of the patients. Wergedal et al. reported higher urinary NTX values in patients with van Buchem disease compared with carriers of the disease (73.0 ± 19.6 vs 36.9 ± 2.8 nmol BCE/mmol creat, p b 0.05) [41]. In the studies of van Lierop et al. serum P1NP and CTX values were significantly correlated both in patients with sclerosteosis (r = 0.86, p b 0.001) and van Buchem disease (r = 0.95, p b 0.001) [30,43]. A similar relationship was also observed in disease carriers (sclerosteosis r = 0.46, p = 0.025; van Buchem disease r = 0.71, p b 0.001). Taken together the data of bone turnover markers suggest that bone formation is increased while bone resorption is normal or decreased in patients with sclerostin deficiency. However, the term “uncoupling”, sometimes used to describe these changes, is not appropriate and the skeleton responds normally to local and systemic signals with changes, however, of bone formation and resorption of different magnitude. 7.4. Mineral metabolism Despite the profound changes of bone metabolism, serum calcium, phosphate, 25-hydroxyvitamin D and plasma PTH concentrations were within the reference ranges in patients with sclerosteosis and van Buchem disease and not different from those of their respective disease carriers [24,30,43,59]. An earlier report of calcium and phosphate balance studies revealed also no alterations in calcium and phosphate metabolism and normal plasma calcitonin levels in patients with sclerosteosis [59]. Notably, serum 1,25-dihydroxyvitamin D concentrations were higher in patients with sclerosteosis compared with those of disease carriers (147.8 ± 55.2 vs 92.0 ± 48.6 pmol/l; p b 0.05) and included some clearly elevated values while there were no differences between patients and carriers of van Buchem disease (115.5 ± 56.6 vs 117.1 ± 36.7 pmol/l). These increases in serum 1,25-dihydroxyvitamin D concentrations were not associated with any significant alterations of biochemical parameters of mineral metabolism. Elevated serum 1,25dihydroxyvitamin concentrations have been reported in sclerostin-deficient mice and were attributed either to a decrease of serum Fibroblast Growth Factor 23 (FGF23) levels or to a direct effect of sclerostin on renal 1α-hydroxylase [60]; 1,25-dihydroxyvitamin D was also shown to stimulate the expression of sclerostin mRNA in osteocyte-like cells [61]. In humans with sclerosteosis and van Buchem disease serum intact

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FGF23 values were within the reference range and not different from values of healthy control subjects or carriers of both diseases [58]. Whereas differences between sclerostin-deficient humans and mice might be due to the very young age of the mice (8 weeks) that had not yet reached a steady metabolic state, the mechanism underlying the increase in serum 1,25-dixydroxyvitamin D concentrations in humans with sclerosteosis remains to be elucidated. 7.5. Other laboratory findings Because the skull is always affected in patients with sclerosteosis and van Buchem disease, pituitary function might be altered by compression of the area of the pituitary fossa or by decreased pituitary blood flow due to increased intracranial pressure. There is no report of pituitary dysfunction in such patients in the literature and systematic evaluation of endocrine functions in a cohort of patients with sclerosteosis revealed no abnormalities [59]. Selected clinical features and laboratory findings of patients with sclerosteosis and van Buchem disease and their respective carriers are summarized in Table 2. 8. Bone tissue changes In contrast to the wealth of information on the clinical, radiographic and, more recently, genetic features of sclerosteosis and van Buchem disease, bone histology reports of affected individuals are limited and data have been mainly obtained from examination of surgically removed bone specimens. While this may reflect lack of specialized laboratories to study bone biopsies combined with the rarity of the diseases in different parts of the world, it should be also noted that performing iliac crest bone biopsies in such patients is technically difficult due to the hardness of the bone tissue and the marked thickness of the cortex of bone (Fig. 9). Furthermore, early data should be interpreted with caution because patients with phenotypic features similar to sclerosteosis, studied before the identification of the genetic defect of the disease, may have been misclassified. Initial reports of bone biopsy findings provided conflicting information. For example, Jackson et al. reported that a mandible specimen from a 7-year old boy with clinical and radiological features consistent with sclerosteosis showed lamellar bone with excessive formation of subperiosteal bone with marked osteoblastic activity in cortical and trabecular surfaces with no evidence of resorption [62]. In addition, Truswell et al. reported that tibial bone of an 8-year old girl with sclerosteosis was dense and lamellar with superabundance of osteocytes and no Haversian systems [1]. In contrast, Epstein et al. reported

Table 2 Selected clinical, densitometric and biochemical characteristics of patients with Sclerostin deficiency and disease carriers. Characteristic

Height Syndactyly Facial distortion Facial nerve palsy Hearing loss Increased ICP LS-BMD Z-score FN-BMD Z-score S-Scl (pg/ml) S-Dkk1 (ng/ml) S-P1NP (ng/ml) S-CTX (pg/ml)

Sclerosteosis

Van Buchem Disease

Patients

Carriers

Patients

Carriers

H Yes Yes (90%) Yes (93%) Yes (94%) Yes (71%) +7.8 to +14.4 +7.8 to +11.5 b0.5 ± 0.0 4.3 ± 1.8 154 ± 102 213 ± 207

N No No No No No +0.4 to +5.15 +0.4 to +3.7 15.5 ± 5.4 2.0 ± 0.6 58 ± 27 126 ± 100

N No Yes (68%) Yes (89%) Yes (78%) Yes (16%) +5.4 to +12.3 +5.2 to +12.1 8.0 ± 6.4 5.3 ± 2.6 96 ± 73 447 ± 320

N No No No No No 1.1 to +2.9 −2.2 to +4.6 28.7 ± 10.2 3.5 ± 1.2 48 ± 21 216 ± 118

H = high, N = normal, ICP = intracranial pressure, LS-BMD = lumbar spine BMD, FN-BMD = femoral neck BMD, S-Scl = serum sclerostin, S-Dkk1 = serum Dickkopf 1, S-P1NP = serum procollagen type 1 N-terminal propeptide; S-CTX = C-terminal telopeptide of type 1 collagen. Values given as mean ± standard deviation.

normal bone formation and osteoid width with increased resorption surfaces in an iliac crest bone biopsy from a 9-year old girl with sclerosteosis; interestingly, thickness of cortical and trabecular bone was increased, findings difficult to relate to the described changes of bone remodeling, as also recognized by the authors [59]. The first concise histological study was reported by Stein et al. who examined undecalcified sections of a craniotomy specimen from an 11-year old girl with sclerosteosis, later confirmed to be due to lossof-function mutation of SOST [11,25]. Bone had lamellar structure with predominant cuboidal active osteoblasts, osteoid of normal thickness and tetracycline uptake at all seams; trabecular bone volume, appositional and bone formation rates were greatly increased while osteoclast numbers and total resorption surfaces were within the low-normal range; osteoclasts examined by electonmicroscopy had normal structure. The authors concluded: “Although increased bone mass is primarily the result of increased bone formation and osteoblast hyperactivity in sclerosteosis, a minor contribution of decreased osteoclast activity or abnormal coupling cannot be ruled out”. This is, perhaps, the best demonstration of bone changes associated with sclerostin deficiency that conforms with current findings of exogenously inhibited sclerostin. Later studies of specimens of compact bone removed at surgery from patients with sclerosteosis, demonstrated lack of sclerostin in bone tissue by immunocytochemistry and abundant presence of active cuboidal osteoblasts with increased osteoid of normal thickness [63]. In addition, there was no difference in osteonal lacunar density between patients with sclerosteosis and healthy controls suggesting that sclerostin is not involved in the local recruitment of osteoblasts to become osteocytes and that the incorporation of new osteocytes in bone is not disturbed [64]. In a study of 6 patients with sclerosteosis van Lierop et al. showed that, compared with samples from healthy subjects, patients had increased bone formation while bone resorption did not differ between the two groups [30]. Notably, in these patients, bone formation rates had an age-related pattern (Fig. 10) increasing until puberty and decreasing thereafter being, however, higher than those of healthy individuals. This pattern is consistent with the observed changes of serum P1NP values in the larger group of subjects and strongly suggests that the skeleton of patients with sclerostin deficiency responds appropriately to hormonal changes associated with growth. Histological reports of patients with van Buchem disease are even scarcer and limited to a few individual patients. Similar to sclerosteosis, sclerostin was not expressed in the mandible of a 15-year old girl with van Buchem disease [6]. In this specimen osteoid surface was increased with many active osteoblasts laying down osteoid that was not thick (b4 lamellae); there was, therefore, evidence of increased bone formation without a mineralization defect. In contrast, in an iliac crest biopsy of a 21-year old patient with van Buchem disease on treatment with glucocorticoids, bone formation was dramatically decreased a finding consistent with the action of glucocorticoids on bone that reinforces the notion that the lack of sclerostin does not affect bone responsiveness to local and systemic factors [52]. Bone material properties were examined in chips of compact bone obtained during surgery from 4 children and 2 adults with sclerosteosis and were compared with similarly obtained specimens from subjects without history of a metabolic bone disease or use of medication that could affect bone metabolism [65]. Bone Mineral Density Distribution (BMDD), measured by quantitative backscattered electron imaging (qBEI), quantifies calcium concentration distribution in bone and determines local mineral content. The BMDD histogram is characterized by the parameter CaMean (mean Ca content), CaPeak (most frequent Ca content) and CaWidth (heterogeneity of mineralization) [66,67]. In bone from young patients with sclerosteosis there was a significant reduction of CaMean and CaPeak while CaWidth was significantly increased (Fig. 11). Reduction of CaMean and CaPeak and, therefore, lower mineralization was also seen in the specimens from the adults with the disease. Thus, the markedly increased mineral density of patients with sclerosteosis is not associated with increased bone matrix mineralization, as observed

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Fig. 9. Undecalcified sections of iliac crest bone biopsies of a 29 year-old healthy male (A) and a 29-year old patient with van Buchem disease illustrating the difference in cortical thickness between the two samples (healthy 1160 μm, patient 3235 μm). Magnification ×12.5, Goldner trichrome stain. Biopsies prepared and interpreted by Dr. Pascale Chavassieux.

in patients with osteopetrosis with high mineral density and increased bone fragility. Children and adults with sclerosteosis had also lower mineral maturity/crystallinity ratio measured by Raman spectrometry. This measurement has a direct bearing on crystal size with lower values indicative of younger, smaller crystals. The described studies of bone material properties demonstrated that bone mineralization kinetics are altered in sclerosteosis and result in lower bone matrix mineralization with smaller, younger crystals. These changes may be related to the high rates of bone formation in the presence of low-normal rates of bone resorption rather than to specific effects of sclerostin on bone composition. In sclerosteosis, the combination of high mineral density with the described bone tissue material properties are associated with increased biomechanical competence of bone and resistance to fractures. Results illustrate further that extrapolation of findings of isolated measurements of bone material properties to whole bone strength, as sometimes occurs in other conditions, is inappropriate and mass, turnover and material properties should be considered together before reaching conclusions about whole bone fragility. We also had the opportunity to measure tissue-level material properties of cortical bone by impact microindendation in vivo in a patient with van Buchem disease treated with glucocorticoids. Bone Material

Active:Total Canals (%)

25

Strength index (BMSi) was 93, clearly higher than the mean of 77.2 of 52 individuals measured within 4 weeks of starting glucocorticoid treatment with the same technique [68]. 9. Treatment of sclerosteosis and van Buchem disease There is no specific therapy for sclerosteosis and van Buchem disease and treatment is mainly surgical aiming at reducing the severity of complications. Decompressions of the facial nerve were performed in 32 patients with sclerosteosis, whereby a facial palsy recurred in 7 of them, and in 5 patients with van Buchem disease. Surgical interventions to alleviate hearing loss include tympanoplasty, repositioning of ossicles or widening of the internal acoustic meatus [69]. Positioning of hearing aids often necessitates the surgical widening of the external auditory canal, or the use of bone anchored hearing aids. Because of the inward pressure on the brain, the ventricles are often reduced in size in sclerosteosis patients suffering from intracranial hypertension. The use, therefore, of ventriculoperitoneal shunt is often futile [36]. Decompressive anterior and/or posterior craniectomies are preferred. The calvaria are replaced by a prosthetic cap, or the excessive bone is drilled away after which the calvaria is placed back in the skull [36]. In a young patient who underwent the latter procedure at the

Formation Resorption

20 15 10 5 0 0

10

20

30

40

50

Age (years) Fig. 10. Proportion of canals of compact bone undergoing formation or resorption in patients with sclerosteosis. Modified from reference [30].

Fig. 11. Bone Mineral Density Distribution (BMDD) of bone from a child with sclerosteosis (SC3, read line) compared with values of healthy children. Original data in reference [65].

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age of 5 years the width of the calvarium increased to the initial value of 2 cm within 4 years, requiring a second operation. Craniotomies were performed in 39 patients with sclerosteosis at a median age of 17 years (range 5 to 56 years). At least 13 patients were re-operated after a median period of 4 years (range 1 to 11 years) for recurrence of the complication. Two patients were operated three times (at the ages of: 5, 9, and 11 years, and 20, 22, and 23 years, respectively), while in one patient craniotomies had to be performed four times (at the ages of 7, 15, 18 and 29 years). In only one patient with van Buchem disease surgical interventions to decrease raised intracranial pressure were necessary [52]. In this patient a ventriculoperitoneal drain was implanted at the age of 23 years, but after recurrence of symptoms, the patient underwent a decompressive craniotomy at the age of 31 years. There are two reports in the literature of medical treatment of patients with sclerostin deficiency, all with van Buchem disease. The first, described an adult patient with severe and progressive disease and raised intracranial pressure who received long-term treatment with prednisone, an established inhibitor of bone formation. Treatment reduced bone formation, assessed biochemically and histologically, and prevented further increases in BMD (Fig. 12). Contrary, however, to the well-known action of glucocorticoids on bone resorption, in this patient the changes of the bone resorption marker serum CTX were tightly coupled to those of the bone formation marker serum P1NP. We previously suggested that in the absence of sclerostin glucocorticoids lose their ability to stimulate RANKL and decrease OPG and recent data of Sost −/− mice treated with prednisolone support this notion [70]. In the second report, two young children received short courses of prednisone to prevent recurrent facial palsies with no, however, success [42]. Biochemical parameters of bone turnover improved during prednisone treatment and returned to pretreatment values after cessation of treatment but no further details were provided. More studies are needed to examine the clinical efficacy of the only currently available medical intervention for the treatment of patients with sclerostin deficiency with serious complications. 10. Perspectives

30 20 10

250

2.5

200

2.0

150

1.5

100

1.0

50

0.5

0

0

50

100 TIME (WEEKS)

150

SERUM β-CTX (ng/ml)

SERUM P1NP (ng/ml)

PREDNISONE (mg/d)

Patients with sclerostin deficiency and their heterozygous carriers are unique models for the study of the role of sclerostin on bone metabolism. Findings provide compelling evidence that lack of sclerostin stimulates bone formation without concomitant increase and even decrease

0.0

Fig. 12. Biochemical markers of bone formation (serum P1NP, solid line) and bone resorption (serum β-CTX, interrupted line) of a patient with severe van Buchem disease during treatment with different doses of prednisone. From reference [52].

of bone resorption unravelling also the important role of sclerostin in the regulation of osteoclastic function. These findings were confirmed in studies of Sost-deficient mice and identified sclerostin as target for the development of a new bone forming treatment for osteoporosis with unique mechanism of action. Such mechanism is, admittedly, very important for osteoporosis therapy but it could have been of limited value if it were not accompanied by information on specific characteristics of sclerostin-deficient individuals. Firstly, the lack of clinical findings from organs other than the skeleton due to the highly selective expression of sclerostin in the target tissue of treatment; secondly, the normal phenotype with increased bone mass in individuals with haploinsufficiency of sclerostin indicating that non-excessive inhibition of sclerostin production can have favourable effects on the skeleton without causing any symptoms or complication of the disease. Such data are not available for other potential treatment targets within the Wnt signalling pathway. The pathway is abundant in the body and regulates essential processes related to growth and differentiation of cells and its activation might lead to uncontrolled cell growth and other disorders including malignancies. For example, mutations of LRP4, which facilitates the action of sclerostin on the Wnt pathway, cause a sclerostin phenotype. However, LRP4 mutations or malfunction have been implicated in serious neurological syndromes [71]. Therefore, potential targeting of LRP4 for the treatment of bone disease requires specific and exclusive delivery of the inhibitor to bone tissue. Another example of a very potent inhibitor of the Wnt pathway that is expressed in tissues other than bone is Dkk1. Apart from the rationale and the background for developing sclerostin inhibitors for clinical use, the studies of human sclerostin deficiency provided also some indication of the expected length of treatment with a sclerostin inhibitor. In patients, nearly maximal bone mass accrual occurs in early childhood with minimal changes thereafter. Moreover, the negative relationship between circulating P1NP and age and the stabilization of values with aging indicate that the absence of sclerostin does not act as a constant stimulus of bone formation. These findings raise questions whether prolonged treatment with a sclerostin inhibitor could be associated with a sustained anabolic effect on bone or whether the beneficial effect on bone formation will be mostly exerted early after the start of treatment and may be blunted with time. It should be also noted that sclerostin does not stimulate osteoblastogenesis but acts at later stages of osteoblast development and inhibits their activity and life span. Its action depends, therefore, on the presence of osteoblasts. In the young, osteoblastogenesis is increased to meet the needs for skeletal growth primarily by skeletal modelling and decreases after skeletal maturity when bone formation occurs as part of bone remodeling and osteoblast numbers and activity are far less than required for bone modelling. It may, therefore, be that in the presence of an increased pool of osteoblasts, as it occurs in the young, the lack of sclerostin leads to excessive bone formation which, however, decreases considerably when this pool is decreased later in life. Patients with osteoporosis are older and their osteoblastogenic capacity is reduced suggesting that the efficacy of an inhibitor of sclerostin on bone formation will be at some stage exhausted. The exact period of such occurrence cannot be predicted from the studies of human sclerostin deficiency and for optimal use of sclerostin inhibitors this needs to be determined in the ongoing clinical studies. Although patients with sclerostin deficiency have contributed greatly to our understanding of the role of sclerostin in bone metabolism and led to large clinical programs with sclerostin inhibitors in osteoporosis, there has been no action towards development of an efficacious treatment for sclerosteosis or van Buchem disease. We strongly believe that the pharmaceutical industry that has the necessary know-how together with the academic community owe that to these patients and we sincerely hope that such project will be soon implemented. We have an ethical obligation to patients who participated in studies around the world and efforts towards the development of an efficacious treatment of their disease are certainly warranted.

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Funding Studies performed at the Center for Bone Quality of the Leiden University Medical Center were funded by the EC (Grant Number: TALOS:Health-F2-2008-201099). Conflict of interest None. Acknowledgements We thank Dr. Pascale Chavassieux, Lyon, France for the preparation and interpretation of the bone biopsy specimens shown in Fig. 9, Dr. Herman Hamersma, Roodeport, South Africa for making available records of his patients and all patients and their relatives who participated in our studies. References [1] A.S. Truswell, Osteopetrosis with syndactyly; a morphological variant of Albers– Schonberg's disease, J. Bone Joint Surg. Br. 40-B (2) (1958) 209–218. [2] F.S. Van Buchem, H.N. Hadders, R. Ubbens, An uncommon familial systemic disease of the skeleton: hyperostosis corticalis generalisata familiaris, Acta Radiol. 44 (2) (1955) 109–120. [3] R. Baron, M. Kneissel, WNT signaling in bone homeostasis and disease: from human mutations to treatments, Nat. Med. 19 (2013) 179–192. [4] A.R. Wijenayaka, M. Kogawa, H.P. Lim, L.F. Bonewald, D.M. Findlay, G.J. Atkins, Sclerostin stimulates osteocyte support of osteoclast activity by a RANKL-dependent pathway, PLoS One 6 (2011), e25900. [5] X. Tu, J. Delgado-Calle, K.W. Condon, M. Maycas, H. Zhang, N. Carlesso, et al., Osteocytes mediate the anabolic actions of canonical Wnt/β-catenin signaling in bone, Proc. Natl. Acad. Sci. U. S. A. 112 (2015) E478–E486. [6] R.L. van Bezooijen, A.L. Bronckers, R.A. Gortzak, P.C. Hogendoorn, L. Wee-Pals, W. Balemans, et al., Sclerostin in mineralized matrices and van Buchem disease, J. Dent. Res. 88 (2009) 569–574. [7] D.G. Winkler, M.K. Sutherland, J.C. Geoghegan, C. Yu, T. Hayes, J.E. Skonier, et al., Osteocyte control of bone formation via sclerostin, a novel BMP antagonist, EMBO J. 22 (2003) 6267–6276. [8] A. Sebastian, G.G. Loots, Transcriptional control of sclerostin, Bone (2016) (Epub ahead of print). [9] T. Bellido, et al., Mechanism of action of sclerostin, Bone (2016) (Epub ahead of print). [10] H. Hamersma, J. Gardner, P. Beighton, The natural history of sclerosteosis, Clin. Genet. 63 (2003) 192–197 (Erratum in: Clin Genet 2003; 64:176). [11] S.A. Stein, C. Witkop, S. Hill, M.D. Fallon, L. Viernstein, G. Gucer, et al., Sclerosteosis: nemogenetic and pathophysiologic analysis of an American kinship, Neurology 33 (1983) 267–277. [12] A.F. Paes-Alves, J.L.C. Rubin, L. Cardoso, M.M. Rabelo, Sclerosteosis: a marker of Dutch ancestry? Rev. Bras. Genet. 4 (1982) 825–834. [13] C.A. Kim, R. Honjo, D. Bertola, L. Albano, L. Oliveira, S. Jales, et al., A known SOST gene mutation causes sclerosteosis in a familial and isolated case from Brazilian origin, Genet. Test. 4 (2008) 475. [14] W. Balemans, E. Cleiren, U. Siebers, J. Horst, W. Van Hul, A generalized skeletal hyperostosis in two siblings caused by a novel mutation in the SOST gene, Bone 36 (2005) 943–947. [15] J.C. Dort, A. Pollak, U. Fisch, The fallopian canal and facial nerve in sclerosteosis of the temporal bone: a histopathological study, Am. J. Otol. 5 (1990) 320–325. [16] M.R. Belkhribchia, C. Collet, J.L. Laplanche, R. Hassani, Novel SOST gene mutation in a sclerosteosis patient from Marocco: a case report, Eur. J. Med. Genet. 57 (2014) 133–137. [17] E. Piters, C. Culha, M. Moester, R. Van Bezooijen, D. Adriaensen, T. Mueller, et al., First missense mutation in the SOST gene causing sclerosteosis by loss of sclerostin function, Hum. Mutat. 31 (2010) E1526–E1543. [18] H. Yagi, M. Takagi, Y. Hasegawa, H. Kayserili, G. Nishimura, Sclerosteosis (craniotubular hyperostosis-syndactyly) with complex hyperphalangy of the index finger, Pediatr. Radiol. 45 (2015) 1239–1243. [19] S. Tholpady, Z.H. Dodd, R.J. Havlik, D.H. Fulkerson, Cranial reconstruction for treatment of intracranial hypertension from sclerosteosis: case-based update, World Neurosurg. 81 (2014) 442, e1-5. [20] A. Fayez, M. Aglan, N. Esmaiel, T. El Zanaty, M. Abdel Kader, M.A. El Ruby, Novel lossof-sclerostin function mutation in a first Egyptian family with sclerosteosis, Biomed. Res. Int. 2015 (2015) 517815. [21] P. Tacconi, P. Ferrigno, L. Cocco, A. Cannas, G. Tamburini, P. Bergonzi, et al., Sclerosteosis: report of a case in a black African man, Clin. Genet. 53 (1998) 497–501. [22] S.K. Bhadada, A. Rastogi, E. Steenackers, E. Boudin, A. Arya, V. Dhiman, et al., Novel SOST gene mutation in a sclerosteosis patient and her parents, Bone 52 (2013) 707–710. [23] Y. Sugiura, T. Yasuhara, Sclerosteosis. A case report, J. Bone Joint Surg. Am. 57 (1975) 273–277.

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