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A generalized skeletal hyperostosis in two siblings caused by a novel mutation in the SOST gene
Bone 36 (2005) 943 – 947 www.elsevier.com/locate/bone
A generalized skeletal hyperostosis in two siblings caused by a novel mutation in the SOST gene Wendy Balemansa, Erna Cleirena, Ulrike Siebersb, Ju¨rgen Horstb, Wim Van Hula,* a
Department of Medical Genetics, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium b Institut fu¨r Humangenetik, Universita¨t Mu¨nster, Mu¨nster, Germany Received 23 November 2004; revised 10 February 2005; accepted 17 February 2005
Abstract In this study, a brother and sister of German origin are described with a possible diagnosis of van Buchem disease, a rare autosomal recessive sclerosing bone dysplasia characterized by a generalized hyperostosis of the skeleton mainly affecting the cranial bones. Clinically, patients suffer from cranial nerve entrapment potentially resulting in facial paresis, hearing disturbances, and visual loss. The radiological picture of van Buchem disease closely resembles sclerosteosis, although in the latter patients, syndactyly, tall stature, and raised intracranial pressure are frequently observed, allowing a differential diagnosis with van Buchem disease. Previous molecular studies demonstrated homozygous loss-of-function mutations in the SOST gene in sclerosteosis patients while a chromosomal rearrangement creating a 52-kb deletion downstream of this gene was found in Dutch patients with van Buchem disease. This deletion most likely suppresses SOST expression. Sclerostin, the SOST gene product, has been shown to play a role in bone metabolism. The two siblings reported here were evaluated at the molecular level by carrying out a mutation analysis of the SOST gene. This resulted in the identification of a novel putative disease-causing splice site mutation (IVS1 + 1 GYC) homozygously present in both siblings. D 2005 Elsevier Inc. All rights reserved. Keywords: Craniotubular hyperostosis; Sclerostin; Splice site mutation; Osteopathia striata
Introduction van Buchem disease and sclerosteosis, two autosomal recessive disorders, belong to the heterogeneous group of craniotubular hyperostosis [1]. In both conditions, radiographic examination reveals a generalized, progressive overgrowth and sclerosis of the skeleton. Predominantly, the skull (both the calvarium and the cranial base) and mandible are affected but a general enlargement with thickening of the cortex and an increase in density is also observed in the ribs, clavicles, pelvis, diaphysis of the long bones, as well as the tubular bones of hands and feet [2 –
* Corresponding author. Fax: +32 3 820 2566. E-mail addresses: [email protected] (W. Balemans), [email protected] (E. Cleiren), [email protected] (U. Siebers), [email protected] (J. Horst), [email protected] (W. Van Hul). 8756-3282/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2005.02.019
12]. Clinical consequences of a thickened skull might be deafness, visual disturbances, facial nerve palsy, and neurological pain due to the narrowing of the cranial foramina [4– 6,8,9,13]. Despite the radiographic resemblances of van Buchem disease and sclerosteosis, these conditions were classified as distinct entities whereas sclerosteosis displays with a number of clinical characteristics which were never observed in patients diagnosed with van Buchem disease. Sclerosteosis presents with a more severe phenotype including hand malformations, with syndactyly and radial deviation of terminal phalanges, a tall stature and increased cranial pressure which sometimes leads to a sudden death of the patient [1,8]. Previous linkage studies co-localized the sclerosteosis and van Buchem disease-causing genes to a region on the long arm of chromosome 17 between genetic markers D17S1787 and D17S934 [14,15]. A subsequent positional cloning strategy resulted in the identification of loss-of-
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function mutations homozygously present in patients with sclerosteosis in a previously unknown two-exon gene called SOST [16,17]. This SOST gene encodes sclerostin, a secreted protein harboring a cysteine-knot domain and is predominantly found in areas of the bone tissue where osteogenesis is actively taking place. The major sites of sclerostin expression are osteocytes and osteocytic canaliculi [18]. In vitro studies demonstrated a modulating effect of sclerostin on downstream events in the BMP signaling cascade [18,19], and an in vivo transgenic mouse model in which human SOST is specifically overexpressed in bone showed a reduction in bone formation and more fragile bones [18]. Interestingly, recent studies in primary human mesenchymal cells suggested a possible involvement of sclerostin in survival of osteoblasts whereas sclerostin selectively initiated their apoptosis [20]. These functional data strongly indicate that sclerostin might function as an important mediator in bone homeostasis. To date, only four mutations have been identified in SOST, three of which were nonsense mutations and one was a splice site mutation affecting transcript processing [16,17]. These mutations were found in patients diagnosed with sclerosteosis. Within the genomic sequence of the SOST gene, no disease-causing mutations have been identified thus far in patients with van Buchem disease. However, 35 kb downstream of the gene, a homozygous deletion of 52 kb was found in the genomic DNA of van Buchem patients all belonging to an extended, highly consanguineous family from the Netherlands [21,22]. The presence of this deletion most likely suppresses SOST gene expression. Here, we describe the identification of a novel splice site mutation in the SOST gene in a brother and sister of German origin with radiographic and clinical characteristics resembling van Buchem disease.
Materials and methods Patients We report on a brother and sister, 40 and 42 years old, respectively, of German origin suffering from a hyperostotic bone disorder. The first symptoms occurred around the age of 4 years when progressive thickening of the cranial bones with a predominant mandibular overgrowth became noticable (Figs. 1A – E). Both patients display with a generalized hyperostosis also affecting the long bones (Figs. 1F –G), lumbar spine and ribs (Fig. 1H), ulna, radius, and metacarpal bones (Fig. 1I). Vertical dense striations at the diaphyses and metaphyses of the long bones, resembling an osteopathia striata pattern, could also be observed (Figs. 1F – H). The sibs developed peripheral facial nerve palsy which was first unilateral but later on during life became bilateral (Figs. 1A,C). Additionally, they suffer from hearing loss due to a mechanical alteration of the nervus acusticus. Residual hearing capacity is about 50% in the woman. Her
brother is nearly deaf on one side and shows only little residual hearing in the other ear although repeated decompression surgery was performed. He is wearing a hearing aid. Seizures never occurred in both patients, but recurrent headaches are documented. The frequency and intensity are not unusually high and no regular pain medication is necessary. Syndactyly and other hand malformations related to the sclerosteosis phenotype are not present (Fig. 1J) and both patients are of normal height (sister 1.68 m and brother 1.75 m). Teeth were normal in the woman but were irregular in shape and position in the man (Fig. 1K). Both siblings never experienced a peripheral bone fracture and no impairment of bone marrow function is reported. Biochemistries of mineral homeostasis in both patients revealed normal values. Serum alkaline phosphatase activity was measured once in the man and was in the upper normal range (173 Ag/L). No biochemical data are available for the woman. The patient’s parents, two additional sibs, and the daughter of the affected woman do not show any similar symptoms. Absence of radiological and clinical features in the parents suggests an autosomal recessive form of a genetic hyperostotic disorder. There is no evidence of consanguinity of the parents who are both of German origin. Sequence analysis of the SOST gene Genomic DNA was isolated from peripheral blood leukocytes by standard techniques and direct sequence analysis was carried out. We designed primers for PCR amplification using the sequence surrounding both exons 1 and 2 of the human SOST gene (Genbank ID NT_010783) which allowed mutational analysis of the complete coding sequence with inclusion of the intron– exon boundaries. For exon 1, the forward primer was 5V-CTAGAGGAGAAGTCTTTGGG-3V which corresponds to bases 190 to 209 upstream of exon 1 and the reverse primer was 5VGACTGTTCCTCGACCAGTGC-3V corresponding to bases 116 –135 within intron 1. For exon 2, the forward primer was 5V-CTTTCCACCAGCTCTAGAGC-3V and the reverse primer was 5V-CGCAGAGGACAGAAATGTGG-3V, which corresponds to bases 170 to 190 uptream of exon 2 and bases 54 to 73 downstream of exon 2, respectively. PCR products were enzymatically purified in 1 CIAP buffer using 4 units of ExoI (New England Biolabs Inc.) and 2.5 units CIAP (Amersham Biosciences). Reaction conditions were 70 min at 37-C followed by a 20-min heat inactivation step at 72-C. Sequencing was carried out using the DYEnamic ET terminator cycle sequencing kit (Amersham Biosciences) and analyzed on a ABI3100 automated sequencer according to the manufacturer’s protocol. Controls We screened a panel of genomic DNA from fifty control individuals for the presence of the splice site mutation by
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Fig. 1. (A – D) Clinical pictures (frontal and lateral view) of both patients showing a square face appearance primarily due to symmetrical enlargement of the jaw. Protruding chin, high forehead, slight proptosis of the eyes, and bilateral paralysis of the facial nerves are also evident. (E) Lateral scout view of a cerebral CT scan of the man clearly illustrating hyperostosis and thickening of the skull and mandible. (F) Radiograph (AP view) of both knees of the brother and (G) sister demonstrating cortical thickening with narrowing of the medullary cavity, especially at the diaphyses of the long bones (femora, tibiae, and fibulae). Vertical dense striations are seen at the diaphyses and metaphysis of the long bones, resembling osteopathia striata. (H) Radiograph (PA view) of the lumbar spine of the male patient, showing thickening and sclerosis of the spinous processes of the vertebra. Note also mild thickening and hyperostosis of the ribs. (I) Standard radiograph (PA view) of the left wrist of the female patient. There is thickening of the cortices of the distal ulna and radius and the metacarpal bones. Apart from the cortical hyperostosis, there is a diaphyseal modeling defect. (J) Clinical picture of the sister’s hand without syndactyly, radial deviation of terminal phalanges, and dysplastic nails, and (K) severe malalignment of the dentition with irregular shape of the individual teeth in the male patient.
PCR analysis followed by digestion with AluI. PCR amplification was carried out using a forward and reversed primer with respective sequences 5V-AGCTGGAGAACAACAAGACC-3V and 5V-GACTGTTCCTCGACCAGTGC-3V which results in a PCR fragment of 210 bp. This PCR
fragment was digested using AluI (Fermentas) for 2 h at 37-C followed by heat inactivation at 65-C for 20 min. Digestions were analyzed on 2% agarose gels. Wild type alleles show a fragment of 210 bp while a mutated allele is cut by AluI resulting in two fragments of, respectively, 75 and 135 bp.
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Results Mutation analysis of the SOST gene Genomic DNA from both patients was isolated from peripheral blood leukocytes by standard techniques and direct sequence analysis of the SOST gene was carried out. Using primers designed for the amplification of the first exon of SOST including the exon – intron boundary, we were able to identify a homozygous basepair substitution on position +1 of the splice donor site (IVS1 + 1 GYC) (Fig. 2). The patients’ parents, unaffected siblings, and daughter of the affected woman were not studied at the DNA level. This splice site variation could not be found in fifty genomic DNA samples from a random control panel. In silico calculations of the Shapiro –Senapathy splice scores (http://home.snafu.de/probins/Splice/SpliceApplet. html) [23] for the wild type versus the mutant allele, a drop in the splice donor consensus score from 84.12 for the wild type allele to 65.88 for the mutant allele was demonstrated (ratio wild type versus mutant is 0.78). Previous studies on other genes have already demonstrated that splice site mutations located at the +1 position of the splice donor site with similar drops in the splice scores are disease-causing [24] and might lead to exon skipping [25] and/or usage of a cryptic splice site [26]. Additionally, according to the matrix of Shapiro and Senapathy (1987) [23], the G at position +1 has a frequency of occurrence of 100% while the frequency of C on position +1 is 0%.
Discussion In this study, two patients –a brother and sister –with a generalized skeletal hyperostosis are described in whom mutation analysis has been carried out. Radiological and clinical examination of the siblings suggested the diagnosis of van Buchem disease. X-rays of both patients revealed a hyperostosis predominantly noticable in the skull and mandible but other parts of the skeleton are also affected. The increased bone thickness in the skull in both patients resulted in the entrapment of cranial nerves which in turn
Fig. 2. Sequence of the splice site mutation (IVS1 + 1 GYC) found in the two affected siblings. The arrow indicates the position of the mutation. The wild type DNA sequence is shown at the right. Capital and small letters, respectively, represent the 3V-part of the first exon and the splice donor site of intron 1.
caused a bilateral facial palsy and hearing loss. In the man, irregular shape and position of the teeth are also observed. For both siblings, a diagnosis of sclerosteosis is very unlikely since none of the additional clinical symptoms characterizing sclerosteosis –hand malformations, tall stature, and elevated intracranial pressure – are present. Although dental malocclusion, partial anodontia, and delayed tooth eruption have been reported in patients with sclerosteosis [27,28], irregular teeth shape and position have not been described in patients with sclerosteosis or van Buchem disease. Since the diagnosis of both patients is most likely van Buchem disease, SOST was considered to be a strong candidate disease-causing gene. A molecular genetic study of the SOST gene demonstrated a novel splice site mutation (IVS1 + 1 GYC) homozygously present in both patients and absent in a random control panel. Although no evidence of consanguinity has been found in the family, analysis of genetic markers in a region of approximately 1.2 Mb surrounding the SOST gene revealed a homozygous haplotype for both siblings (data not shown), suggesting the existence of one common ancestor who passed the disease-causing mutation to the next generations. Another splice site mutation (IVS1 + 3 AYT) in SOST has been previously described by us and others in an isolated Senegalese case with sclerosteosis [16,17]. Brunkow et al. [17] carried out an in vitro splicing assay in which they were able to demonstrate that this IVS1 + 3 AYT mutation results in the creation of a cryptic splice site located 214 bp downstream of the authentic site. The abundance of this aberrantly spliced transcript is more than 10-fold lower than the wild type transcript which was explained by the introduction of an in-frame nonsense codon resulting in nonsense-mediated mRNA decay. The functional effect of the IVS1 + 1 GYC splice site mutation reported here in the two German siblings is most likely similar to that of the Senegalese SOST mutation. Thus far, five different homozygous mutations in the SOST gene, three nonsense and two splice site mutations, have been identified in patients with radiological and clinical characteristics of sclerosteosis and van Buchem disease [16,17]. Additionally, a deletion mutation located 52 kb downstream of SOST which most likely down-regulates SOST transcription has been identified in Dutch patients diagnosed with van Buchem disease [21,22]. Since sclerosteosis presents with additional clinical characteristics not described in patients with van Buchem disease, the question raises whether a specific SOST mutation correlates with the presence or absence of these additional features. Although nonsense mutations in SOST (Gln24X, Trp124X, and Arg126X) seem to result in the sclerosteosis phenotype [16,17] and a regulatory mutation leads to van Buchem disease [21,22], no genotype – phenotype correlation could be observed for the splice site mutations. The homozygous IVS1 + 3 AYT mutation results in a clear sclerosteosis
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phenotype with syndactyly, nail hypoplasia, tall stature, and facial distortion as reported in a Senegalese patient with sclerosteosis [12], while patients carrying a homozygous IVS1 + 1 GYC mutation present without any of the additional features observed in sclerosteosis. With regard to these genotype – phenotype correlations, it should be remarked that the presence of the additional features is not observed in all sclerosteosis patients as illustrated in papers by Paes-Alves et al. [10], Stein et al. [11], and Hamersma et al. [13]. Moreover, Beighton and colleagues already stated that a mild form of sclerosteosis is indistinguishable from a severe case of van Buchem disease [29]. To conclude, we report here on two novel cases presenting with a generalized skeletal hyperostosis mainly affecting the skull and mandible. Mutation analysis revealed a new splice site mutation in the SOST gene. Although the number of SOST mutations described thus far is low, the identification of additional disease-causing SOST mutations could aid in understanding the phenotypical differences between sclerosteosis and van Buchem disease.
Acknowledgments We would like to thank Dr. Filip Vanhoenacker, radiologist at the University Hospital of Antwerp (Antwerp, Belgium) for his critical evaluation of the patients’ X-rays. This study is supported by the Fund for Scientific Research Flanders (FWO), with a research project grant (G.0404.00), by the Interuniversity Attraction Poles program P5/19 of the Belgian Federal Science Policy Office (IUAP) and by the European grant ANABONOS (LSHM-CT-2003-503020) all to WVH.
References [1] Beighton P. Craniotubular hyperostosis. In: Emery AEH, Emery AEHs, editors. Inherited Disorders of the Skeleton. New York’ Churchill Livingstone Inc; 1984. p. 191 – 211. [2] van Buchem FSP, Hadders HN, Ubbens R. An uncommon familial systemic disease of the skeleton: hyperostosis corticalis generalisata familiaris. Acta Radiol 1955;44:109 – 20. [3] van Buchem FSP, Hadders HN, Fix HJ, Woldring MG. Hyperostosis corticalis generalisata. Am J Med 1962;33:387 – 97. [4] van Buchem FSP. Hyperostosis corticalis generalisata. Eight new cases. Acta Med Scand 1971;189:257 – 67. [5] van Buchem FSP, Prick JJG, Jaspar H.H.J.. Hyperostosis corticalis generalisata familiaris (van Buchem’s disease). Amsterdam’ Excerpta Medica; 1976. p. 1 – 201. [6] Beighton P, Durr L, Hamersma H. The clinical features of sclerosteosis. A review of the manifestations in twenty-five affected individuals. Ann Intern Med 1976;84:393 – 7. [7] Beighton P, Davidson J, Durr L, Hamersma H. Sclerosteosis—An autosomal recessive disorder. Clin Genet 1977;11:1 – 7. [8] Beighton P, Hamersma H. Sclerosteosis in South Africa. S Afr Med J 1979;55:783 – 8.
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[9] Nager GT, Stein SA, Dorst JP, Holliday MJ, Kennedy DW, Diehn KW, et al. Sclerosteosis involving the temporal bone: clinical and radiologic aspects. Am J Otolaryngol 1983;4:1 – 17. [10] Paes-Alves AF, Rubin JLC, Cardoso L, Rabelo MM. Sclerosteosis: a marker of Dutch ancestry? Rev Bras Genet 1982;4:825 – 34. [11] Stein SA, Witkop C, Hill S, Fallon MD, Viernstein L, Gucer G, et al. Sclerosteosis: neurogenetic and pathophysiologic analysis of an American kinship. Neurology 1983;33:267 – 77. [12] Tacconi P, Ferrigno P, Cocco L, Cannas A, Tamburini G, Bergonzi P, et al. Sclerosteosis: report of a case in a black African man. Clin Genet 1998;53:497 – 501. [13] Hamersma H, Gardner J, Beighton P. The natural history of sclerosteosis. Clin Genet 2003;63:192 – 7. [14] Van Hul W, Balemans W, Van Hul E, Dikkers FG, Obee H, Stokroos RJ, et al. Van Buchem disease (hyperostosis corticalis generalisata) maps to chromosome 17q12 – q21. Am J Hum Genet 1998;62:391 – 9. [15] Balemans W, Van Den Ende J, Freire Paes-Alves A, Dikkers FG, Willems PJ, Vanhoenacker F, et al. Localization of the gene for sclerosteosis to the van Buchem disease-gene region on chromosome 17q12 – q21. Am J Hum Genet 1999;64:1661 – 9. [16] Balemans W, Ebeling M, Patel N, Van Hul E, Olson P, Dioszegi M, et al. Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum Mol Genet 2001;10:537 – 43. [17] Brunkow ME, Gardner JC, Van Ness J, Paeper BW, Kovacevich BR, Proll S, et al. Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein. Am J Hum Genet 2001;68:577 – 89. [18] Winkler DG, Sutherland MK, Geoghegan JC, Yu C, Hayes T, Skonier JE, et al. Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J 2003;22:6267 – 76. [19] van Bezooijen RL, Roelen BA, Visser A, van der Wee-Pals L, de Wilt E, Karperien M, et al. Sclerostin is an osteocyte-expressed negative regulator of bone formation, but not a classical BMP antagonist. J Exp Med 2004;199:805 – 14. [20] Sutherland MK, Geoghegan JC, Yu C, Turcott E, Skonier JE, Winkler DG, et al. Sclerostin promotes the apoptosis of human osteoblastic cells: a novel regulation of bone formation. Bone 2004;35:828 – 35. [21] Balemans W, Patel N, Ebeling M, Van Hul E, Wuyts W, Lacza C, et al. Identification of a 52 kb deletion downstream of the SOST gene in patients with van Buchem disease. J Med Genet 2002; 39:91 – 7. [22] Staehling-Hampton K, Proll S, Paeper BW, Zhao L, Charmley P, Brown A, et al. A 52-kb deletion in the SOST-MEOX1 intergenic region on 17q12 – q21 is associated with van Buchem disease in the Dutch population. Am J Med Genet 2002;110:144 – 52. [23] Shapiro MB, Senapathy P. RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 1987;15:7155 – 74. [24] Krawczak M, Reiss J, Cooper DN. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet 1992;90:41 – 54. [25] Garey JR, Harrison LM, Franklin KF, Metcalf KM, Radisky ES, Kushner JP. Uroporphyrinogen decarboxylase: a splice site mutation causes the deletion of exon 6 in multiple families with porphyria cutanea tarda. J Clin Invest 1990;86:1416 – 22. [26] Ohno K, Suzuki K. Multiple abnormal beta-hexosaminidase alpha chain mRNAs in a compound-heterozygous Ashkenazi Jewish patient with Tay – Sachs disease. J Biol Chem 1988;263:18563 – 7. [27] Beighton P. Sclerosteosis. J Med Genet 1988;25:200 – 3. [28] Stephen LX, Hamersma H, Gardner J, Beighton P. Dental and oral manifestations of sclerosteosis. Int Dent J 2001;51:287 – 90. [29] Beighton P, Barnard A, Hamersma H, van der Wouden A. The syndromic status of sclerosteosis and van Buchem disease. Clin Genet 1984;25:175 – 81.
A generalized skeletal hyperostosis in two siblings caused by a novel mutation in the SOST gene Wendy Balemansa, Erna Cleirena, Ulrike Siebersb, Ju¨rgen Horstb, Wim Van Hula,* a
Department of Medical Genetics, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium b Institut fu¨r Humangenetik, Universita¨t Mu¨nster, Mu¨nster, Germany Received 23 November 2004; revised 10 February 2005; accepted 17 February 2005
Abstract In this study, a brother and sister of German origin are described with a possible diagnosis of van Buchem disease, a rare autosomal recessive sclerosing bone dysplasia characterized by a generalized hyperostosis of the skeleton mainly affecting the cranial bones. Clinically, patients suffer from cranial nerve entrapment potentially resulting in facial paresis, hearing disturbances, and visual loss. The radiological picture of van Buchem disease closely resembles sclerosteosis, although in the latter patients, syndactyly, tall stature, and raised intracranial pressure are frequently observed, allowing a differential diagnosis with van Buchem disease. Previous molecular studies demonstrated homozygous loss-of-function mutations in the SOST gene in sclerosteosis patients while a chromosomal rearrangement creating a 52-kb deletion downstream of this gene was found in Dutch patients with van Buchem disease. This deletion most likely suppresses SOST expression. Sclerostin, the SOST gene product, has been shown to play a role in bone metabolism. The two siblings reported here were evaluated at the molecular level by carrying out a mutation analysis of the SOST gene. This resulted in the identification of a novel putative disease-causing splice site mutation (IVS1 + 1 GYC) homozygously present in both siblings. D 2005 Elsevier Inc. All rights reserved. Keywords: Craniotubular hyperostosis; Sclerostin; Splice site mutation; Osteopathia striata
Introduction van Buchem disease and sclerosteosis, two autosomal recessive disorders, belong to the heterogeneous group of craniotubular hyperostosis [1]. In both conditions, radiographic examination reveals a generalized, progressive overgrowth and sclerosis of the skeleton. Predominantly, the skull (both the calvarium and the cranial base) and mandible are affected but a general enlargement with thickening of the cortex and an increase in density is also observed in the ribs, clavicles, pelvis, diaphysis of the long bones, as well as the tubular bones of hands and feet [2 –
* Corresponding author. Fax: +32 3 820 2566. E-mail addresses: [email protected] (W. Balemans), [email protected] (E. Cleiren), [email protected] (U. Siebers), [email protected] (J. Horst), [email protected] (W. Van Hul). 8756-3282/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2005.02.019
12]. Clinical consequences of a thickened skull might be deafness, visual disturbances, facial nerve palsy, and neurological pain due to the narrowing of the cranial foramina [4– 6,8,9,13]. Despite the radiographic resemblances of van Buchem disease and sclerosteosis, these conditions were classified as distinct entities whereas sclerosteosis displays with a number of clinical characteristics which were never observed in patients diagnosed with van Buchem disease. Sclerosteosis presents with a more severe phenotype including hand malformations, with syndactyly and radial deviation of terminal phalanges, a tall stature and increased cranial pressure which sometimes leads to a sudden death of the patient [1,8]. Previous linkage studies co-localized the sclerosteosis and van Buchem disease-causing genes to a region on the long arm of chromosome 17 between genetic markers D17S1787 and D17S934 [14,15]. A subsequent positional cloning strategy resulted in the identification of loss-of-
944
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function mutations homozygously present in patients with sclerosteosis in a previously unknown two-exon gene called SOST [16,17]. This SOST gene encodes sclerostin, a secreted protein harboring a cysteine-knot domain and is predominantly found in areas of the bone tissue where osteogenesis is actively taking place. The major sites of sclerostin expression are osteocytes and osteocytic canaliculi [18]. In vitro studies demonstrated a modulating effect of sclerostin on downstream events in the BMP signaling cascade [18,19], and an in vivo transgenic mouse model in which human SOST is specifically overexpressed in bone showed a reduction in bone formation and more fragile bones [18]. Interestingly, recent studies in primary human mesenchymal cells suggested a possible involvement of sclerostin in survival of osteoblasts whereas sclerostin selectively initiated their apoptosis [20]. These functional data strongly indicate that sclerostin might function as an important mediator in bone homeostasis. To date, only four mutations have been identified in SOST, three of which were nonsense mutations and one was a splice site mutation affecting transcript processing [16,17]. These mutations were found in patients diagnosed with sclerosteosis. Within the genomic sequence of the SOST gene, no disease-causing mutations have been identified thus far in patients with van Buchem disease. However, 35 kb downstream of the gene, a homozygous deletion of 52 kb was found in the genomic DNA of van Buchem patients all belonging to an extended, highly consanguineous family from the Netherlands [21,22]. The presence of this deletion most likely suppresses SOST gene expression. Here, we describe the identification of a novel splice site mutation in the SOST gene in a brother and sister of German origin with radiographic and clinical characteristics resembling van Buchem disease.
Materials and methods Patients We report on a brother and sister, 40 and 42 years old, respectively, of German origin suffering from a hyperostotic bone disorder. The first symptoms occurred around the age of 4 years when progressive thickening of the cranial bones with a predominant mandibular overgrowth became noticable (Figs. 1A – E). Both patients display with a generalized hyperostosis also affecting the long bones (Figs. 1F –G), lumbar spine and ribs (Fig. 1H), ulna, radius, and metacarpal bones (Fig. 1I). Vertical dense striations at the diaphyses and metaphyses of the long bones, resembling an osteopathia striata pattern, could also be observed (Figs. 1F – H). The sibs developed peripheral facial nerve palsy which was first unilateral but later on during life became bilateral (Figs. 1A,C). Additionally, they suffer from hearing loss due to a mechanical alteration of the nervus acusticus. Residual hearing capacity is about 50% in the woman. Her
brother is nearly deaf on one side and shows only little residual hearing in the other ear although repeated decompression surgery was performed. He is wearing a hearing aid. Seizures never occurred in both patients, but recurrent headaches are documented. The frequency and intensity are not unusually high and no regular pain medication is necessary. Syndactyly and other hand malformations related to the sclerosteosis phenotype are not present (Fig. 1J) and both patients are of normal height (sister 1.68 m and brother 1.75 m). Teeth were normal in the woman but were irregular in shape and position in the man (Fig. 1K). Both siblings never experienced a peripheral bone fracture and no impairment of bone marrow function is reported. Biochemistries of mineral homeostasis in both patients revealed normal values. Serum alkaline phosphatase activity was measured once in the man and was in the upper normal range (173 Ag/L). No biochemical data are available for the woman. The patient’s parents, two additional sibs, and the daughter of the affected woman do not show any similar symptoms. Absence of radiological and clinical features in the parents suggests an autosomal recessive form of a genetic hyperostotic disorder. There is no evidence of consanguinity of the parents who are both of German origin. Sequence analysis of the SOST gene Genomic DNA was isolated from peripheral blood leukocytes by standard techniques and direct sequence analysis was carried out. We designed primers for PCR amplification using the sequence surrounding both exons 1 and 2 of the human SOST gene (Genbank ID NT_010783) which allowed mutational analysis of the complete coding sequence with inclusion of the intron– exon boundaries. For exon 1, the forward primer was 5V-CTAGAGGAGAAGTCTTTGGG-3V which corresponds to bases 190 to 209 upstream of exon 1 and the reverse primer was 5VGACTGTTCCTCGACCAGTGC-3V corresponding to bases 116 –135 within intron 1. For exon 2, the forward primer was 5V-CTTTCCACCAGCTCTAGAGC-3V and the reverse primer was 5V-CGCAGAGGACAGAAATGTGG-3V, which corresponds to bases 170 to 190 uptream of exon 2 and bases 54 to 73 downstream of exon 2, respectively. PCR products were enzymatically purified in 1 CIAP buffer using 4 units of ExoI (New England Biolabs Inc.) and 2.5 units CIAP (Amersham Biosciences). Reaction conditions were 70 min at 37-C followed by a 20-min heat inactivation step at 72-C. Sequencing was carried out using the DYEnamic ET terminator cycle sequencing kit (Amersham Biosciences) and analyzed on a ABI3100 automated sequencer according to the manufacturer’s protocol. Controls We screened a panel of genomic DNA from fifty control individuals for the presence of the splice site mutation by
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Fig. 1. (A – D) Clinical pictures (frontal and lateral view) of both patients showing a square face appearance primarily due to symmetrical enlargement of the jaw. Protruding chin, high forehead, slight proptosis of the eyes, and bilateral paralysis of the facial nerves are also evident. (E) Lateral scout view of a cerebral CT scan of the man clearly illustrating hyperostosis and thickening of the skull and mandible. (F) Radiograph (AP view) of both knees of the brother and (G) sister demonstrating cortical thickening with narrowing of the medullary cavity, especially at the diaphyses of the long bones (femora, tibiae, and fibulae). Vertical dense striations are seen at the diaphyses and metaphysis of the long bones, resembling osteopathia striata. (H) Radiograph (PA view) of the lumbar spine of the male patient, showing thickening and sclerosis of the spinous processes of the vertebra. Note also mild thickening and hyperostosis of the ribs. (I) Standard radiograph (PA view) of the left wrist of the female patient. There is thickening of the cortices of the distal ulna and radius and the metacarpal bones. Apart from the cortical hyperostosis, there is a diaphyseal modeling defect. (J) Clinical picture of the sister’s hand without syndactyly, radial deviation of terminal phalanges, and dysplastic nails, and (K) severe malalignment of the dentition with irregular shape of the individual teeth in the male patient.
PCR analysis followed by digestion with AluI. PCR amplification was carried out using a forward and reversed primer with respective sequences 5V-AGCTGGAGAACAACAAGACC-3V and 5V-GACTGTTCCTCGACCAGTGC-3V which results in a PCR fragment of 210 bp. This PCR
fragment was digested using AluI (Fermentas) for 2 h at 37-C followed by heat inactivation at 65-C for 20 min. Digestions were analyzed on 2% agarose gels. Wild type alleles show a fragment of 210 bp while a mutated allele is cut by AluI resulting in two fragments of, respectively, 75 and 135 bp.
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Results Mutation analysis of the SOST gene Genomic DNA from both patients was isolated from peripheral blood leukocytes by standard techniques and direct sequence analysis of the SOST gene was carried out. Using primers designed for the amplification of the first exon of SOST including the exon – intron boundary, we were able to identify a homozygous basepair substitution on position +1 of the splice donor site (IVS1 + 1 GYC) (Fig. 2). The patients’ parents, unaffected siblings, and daughter of the affected woman were not studied at the DNA level. This splice site variation could not be found in fifty genomic DNA samples from a random control panel. In silico calculations of the Shapiro –Senapathy splice scores (http://home.snafu.de/probins/Splice/SpliceApplet. html) [23] for the wild type versus the mutant allele, a drop in the splice donor consensus score from 84.12 for the wild type allele to 65.88 for the mutant allele was demonstrated (ratio wild type versus mutant is 0.78). Previous studies on other genes have already demonstrated that splice site mutations located at the +1 position of the splice donor site with similar drops in the splice scores are disease-causing [24] and might lead to exon skipping [25] and/or usage of a cryptic splice site [26]. Additionally, according to the matrix of Shapiro and Senapathy (1987) [23], the G at position +1 has a frequency of occurrence of 100% while the frequency of C on position +1 is 0%.
Discussion In this study, two patients –a brother and sister –with a generalized skeletal hyperostosis are described in whom mutation analysis has been carried out. Radiological and clinical examination of the siblings suggested the diagnosis of van Buchem disease. X-rays of both patients revealed a hyperostosis predominantly noticable in the skull and mandible but other parts of the skeleton are also affected. The increased bone thickness in the skull in both patients resulted in the entrapment of cranial nerves which in turn
Fig. 2. Sequence of the splice site mutation (IVS1 + 1 GYC) found in the two affected siblings. The arrow indicates the position of the mutation. The wild type DNA sequence is shown at the right. Capital and small letters, respectively, represent the 3V-part of the first exon and the splice donor site of intron 1.
caused a bilateral facial palsy and hearing loss. In the man, irregular shape and position of the teeth are also observed. For both siblings, a diagnosis of sclerosteosis is very unlikely since none of the additional clinical symptoms characterizing sclerosteosis –hand malformations, tall stature, and elevated intracranial pressure – are present. Although dental malocclusion, partial anodontia, and delayed tooth eruption have been reported in patients with sclerosteosis [27,28], irregular teeth shape and position have not been described in patients with sclerosteosis or van Buchem disease. Since the diagnosis of both patients is most likely van Buchem disease, SOST was considered to be a strong candidate disease-causing gene. A molecular genetic study of the SOST gene demonstrated a novel splice site mutation (IVS1 + 1 GYC) homozygously present in both patients and absent in a random control panel. Although no evidence of consanguinity has been found in the family, analysis of genetic markers in a region of approximately 1.2 Mb surrounding the SOST gene revealed a homozygous haplotype for both siblings (data not shown), suggesting the existence of one common ancestor who passed the disease-causing mutation to the next generations. Another splice site mutation (IVS1 + 3 AYT) in SOST has been previously described by us and others in an isolated Senegalese case with sclerosteosis [16,17]. Brunkow et al. [17] carried out an in vitro splicing assay in which they were able to demonstrate that this IVS1 + 3 AYT mutation results in the creation of a cryptic splice site located 214 bp downstream of the authentic site. The abundance of this aberrantly spliced transcript is more than 10-fold lower than the wild type transcript which was explained by the introduction of an in-frame nonsense codon resulting in nonsense-mediated mRNA decay. The functional effect of the IVS1 + 1 GYC splice site mutation reported here in the two German siblings is most likely similar to that of the Senegalese SOST mutation. Thus far, five different homozygous mutations in the SOST gene, three nonsense and two splice site mutations, have been identified in patients with radiological and clinical characteristics of sclerosteosis and van Buchem disease [16,17]. Additionally, a deletion mutation located 52 kb downstream of SOST which most likely down-regulates SOST transcription has been identified in Dutch patients diagnosed with van Buchem disease [21,22]. Since sclerosteosis presents with additional clinical characteristics not described in patients with van Buchem disease, the question raises whether a specific SOST mutation correlates with the presence or absence of these additional features. Although nonsense mutations in SOST (Gln24X, Trp124X, and Arg126X) seem to result in the sclerosteosis phenotype [16,17] and a regulatory mutation leads to van Buchem disease [21,22], no genotype – phenotype correlation could be observed for the splice site mutations. The homozygous IVS1 + 3 AYT mutation results in a clear sclerosteosis
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phenotype with syndactyly, nail hypoplasia, tall stature, and facial distortion as reported in a Senegalese patient with sclerosteosis [12], while patients carrying a homozygous IVS1 + 1 GYC mutation present without any of the additional features observed in sclerosteosis. With regard to these genotype – phenotype correlations, it should be remarked that the presence of the additional features is not observed in all sclerosteosis patients as illustrated in papers by Paes-Alves et al. [10], Stein et al. [11], and Hamersma et al. [13]. Moreover, Beighton and colleagues already stated that a mild form of sclerosteosis is indistinguishable from a severe case of van Buchem disease [29]. To conclude, we report here on two novel cases presenting with a generalized skeletal hyperostosis mainly affecting the skull and mandible. Mutation analysis revealed a new splice site mutation in the SOST gene. Although the number of SOST mutations described thus far is low, the identification of additional disease-causing SOST mutations could aid in understanding the phenotypical differences between sclerosteosis and van Buchem disease.
Acknowledgments We would like to thank Dr. Filip Vanhoenacker, radiologist at the University Hospital of Antwerp (Antwerp, Belgium) for his critical evaluation of the patients’ X-rays. This study is supported by the Fund for Scientific Research Flanders (FWO), with a research project grant (G.0404.00), by the Interuniversity Attraction Poles program P5/19 of the Belgian Federal Science Policy Office (IUAP) and by the European grant ANABONOS (LSHM-CT-2003-503020) all to WVH.
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