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Localization of the gene for X-linked calvarial hyperostosis to chromosome Xq27.3–Xqter
Bone 58 (2014) 67–71
Contents lists available at ScienceDirect
Bone journal homepage: www.elsevier.com/locate/bone
Original Full Length Article
Localization of the gene for X-linked calvarial hyperostosis to chromosome Xq27.3–Xqter V.M. Borra a, E. Steenackers a, F. de Freitas a, E. Van Hul a,1, I. Glass b, W. Van Hul a,⁎ a b
Department of Medical Genetics, University of Antwerp, Belgium Division of Genetic Medicine, Department of Pediatrics, University of Washington, Seattle, Children's Hospital, Seattle, WA, USA
a r t i c l e
i n f o
Article history: Received 26 July 2013 Revised 23 September 2013 Accepted 14 October 2013 Available online 18 October 2013 Edited by: Mark Johnson Keywords: Calvarial hyperostosis Xq27.3–Xqter Calvarium Skull
a b s t r a c t X-linked calvarial hyperostosis is a rare disorder characterized by isolated calvarial thickening. Symptoms are prominent frontoparietal bones, a flat nasal root and a short upturned nose, a high forehead with ridging of the metopic and sagittal sutures, and lateral frontal prominences. The mandible is normal, as are the clavicles, pelvis and long bones. The thickened bone in the skull appears to be softer than normal bone. Despite calvarial hyperostosis, increased intracranial pressure and cranial nerve entrapment do not occur. The major disability seems to be cosmetic. The disease segregates with an X-linked recessive mode of inheritance. Female carriers do not show any clinical symptoms. To date, only one family has been described with X-linked calvarial hyperostosis including three affected individuals. In order to localize the disease causing gene, 31 polymorphic microsatellite markers that spread across the X-chromosome were analyzed. Genotypes were combined in haplotypes to delineate the region. A chromosomal region spanning from Xq27.3 to Xqter cosegregates with the disorder. This region encompasses 23.53 cM or 8.2 Mb according to the deCODE map and contains 165 genes. CNV-analysis did not show small duplications or deletions in this region. Exome sequencing was performed on a male patient in this family. However, this did not reveal any putative mutation. These results indicate that a non-coding regulatory sequence might be involved in the pathogenesis of this disorder. © 2013 Elsevier Inc. All rights reserved.
Introduction Bone is a dynamic tissue that is continuously remodeled by a balanced process of bone formation and bone resorption. Disturbance of this balance can lead to a wide variety of bone disorders, characterized by either low or high bone mineral density [1]. In the last years, several genes have been identified underlying monogenic diseases with an abnormal bone mineral density [2–5]. These studies provide new insights into bone homeostasis and can be important in the understanding of the pathogenesis of complex diseases, like osteoporosis; they might even lead to therapeutical applications for this condition. Calvarial hyperostosis is a benign X-linked disorder that affects only the skull. Symptoms are prominent frontoparietal bones, a flat nasal root and a short upturned nose, a high forehead with ridging of the metopic and sagittal sutures, and lateral frontal prominences. The mandible is normal, as are the clavicles, pelvis and long bones. Radiographs of the skull show increased bone thickness at the sagittal suture line and prominent lateral frontal horns. The thickened bone in the skull appears
⁎ Corresponding author at: Department of Medical Genetics, University of Antwerp, Prins Boudewijnlaan 43, B-2650 Edegem, Belgium. Fax: +32 32759722. E-mail address: [email protected] (W. Van Hul). 1 Current affiliation: Department of Medical Genetics, University Hospital Antwerp, Belgium. 8756-3282/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bone.2013.10.011
to be softer than normal bone. Despite calvarial hyperostosis, increased intracranial pressure and cranial nerve entrapment do not occur. The disorder appears to be benign and the major disability seems to be cosmetic. The disease segregates with an X-linked recessive mode of inheritance. Family and methods Case reports To date, only one family including three affected individuals with calvarial hyperostosis (Fig. 1) has been described in 1986 by Pagon et al. [6]. The proband (IV.1, Fig. 1) was diagnosed with calvarial hyperostosis at approximately 3 years of age because he showed prominence of the frontoparietal bones (Fig. 2a). He had a flat nasal root, a short upturned nose and a high forehead with ridging of the metopic and sagittal sutures and lateral prominences. His neurologic exam was normal as were his vision and hearing. Radiographs showed thickened diploic spaces, prominent trabeculae and closure of all but the lambdoidal sutures. There was thickening of the diploic spaces and prominent maxillary sinuses. He had normal radiographs of the pelvis and one leg. At age twenty-four he returned to the genetics clinic for follow-up. His only health complaints at that time were headaches. They were pounding
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Fig. 1. Pedigree of a family with X-linked calvarial hyperostosis. Black symbols indicate affected individuals, white symbols indicate unaffected individuals, white symbols with a dot are carriers. The haplotypes are zoomed in on the long arm of chromosome X. The black haplotype is present in all carriers and affected individuals. Arrow: proband.
in nature and appeared to be in the mid occipital region. They only occurred during waking hours and were not associated with nausea or vomiting, visual disturbance, weakness or paralysis. His facial appearance showed fullness to the right face and in mid-to-lower aspects. However, the eyes themselves were not distorted. There was bony thickening overlying the lateral right frontal prominence and supraorbital region. The mid part of the frontal skull was depressed and there was a flat region overlying the formal sagittal suture. The extremities were normal, there was no sign of nerve entrapment. The disease showed no obvious progression. A maternal cousin (III.3, Fig. 2b) and a maternal second cousin (IV.3) were also diagnosed with calvarial hyperostosis. Patient III.12 was diagnosed at the age of one. Skull radiographs showed calvarial hyperostosis which involved bones that originate from membranous bone. At age two 8/12 radiographs showed prominent lateral horns (Fig. 3) and increased thickness of the bone in the region of the closed sagittal suture line. There was presumed intracranial pressure with slight Luckenschadel appearance to the entire skull. The mandible was normal, as were the clavicles, the pelvis and the long bones. A craniectomy and morcellation of the coronal, lambdoidal and sagittal sutures with excision of the frontal protuberances were performed. At the age of eighteen his face had a normal appearance [6]. Patient IV.3 was diagnosed at eighteen months of age when unusual frontoparietal bony prominences were noted and skull radiographs
showed thickening of the diploic space, lateral and frontal horns and a Luckenschadel appearance in the frontal and occipital areas [6]. Obligate gene carriers (II.1, II.7, and III.1) show no unusual cranial configuration. However, radiographs were not obtained. Genotyping Peripheral blood was collected from 7 family members (two patients, four obligate carriers and one with unknown status). Genomic DNA was isolated from these blood samples using standard procedures. 31 polymorphic markers that spread across the X chromosome were analyzed. These markers were selected from the deCODE genetic map and were analyzed by a Taq DNA polymerase mediated PCR, using fluorescently labeled primers [7]. Fragment analysis of amplified products was performed by an ABI PRISM® 3130 XL Genetic Analyzer (Applied Biosystems). Allele identification was done with Gene mapper v3.7 software (Applied Biosystems). CNV analysis The Illumina iScan system was used with the Human OmniExpress12 v1.0 DNA Analysis BeadChip (Illumina, San Diego, CA) for CNV analysis. Resulting data were analyzed using the CNV-Webstore tool [8].
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Fig. 3. Radiograph of III.3 showing the prominent lateral frontal horn (arrow). From: “Calvarial Hyperostosis: a benign X-linked recessive disorder”. Clin Genet 29:73–78 © Wiley-Liss, Inc. This material is reproduced with permission of John Wiley & Sons, Inc.
Exome sequencing Exome sequencing was performed on the proband (IV.1; Fig. 1) with the NimbleGenV2 enrichment panel on the HiSeq2000. Raw sequencing data were analyzed with Galaxy [9–11] and the variants were filtered in VariantDB. The different filters are shown in Fig. 4. Unknown variants
Fig. 2. a. patient IV.1, b. patient III.3. Note the prominent lateral horns. From: “Calvarial Hyperostosis: a benign X-linked recessive disorder”. Clin Genet 29:73–78 © Wiley-Liss, Inc. This material is reproduced with permission of John Wiley & Sons, Inc.
Fig. 4. Filters used in VariantDB to select variants in the co-segregating region on chromosome X.cy1.
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were further analyzed with the Integrative Genomics Viewer (IGV) 2.3 [12,13] and compared to a set of male controls. Direct sequencing of uncovered exons In one affected individual and an unrelated control sample uncovered regions were amplified by GoTaq DNA polymerase-mediated PCR with primers covering the exons and the intron–exon boundaries followed by direct sequencing. Sequencing reactions were performed using the ABI BigDye Terminator v1.1 Cycle sequencing Ready Reaction Kit (Applied Biosystems). Purification with the BigDye Terminator Purification Kit (Applied Biosystems) was carried out to remove unincorporated BigDye terminators, after which fragments were analyzed on an ABI 3130 Genetic Analyzer (Applied Biosystems). Results Delineation of the candidate region Pedigree and haplotypes were generated using the Cyrillic 2.1 software (CyrillicSoftware, Oxfordshire, UK). The black haplotype is seen only in patients and obligated carriers (Fig. 1). The region is delineated by a recombination proximal of marker DXS1193 in patient IV.3 and by the telomeres at the distal side. This region encompasses 23.53 cM or 8.2 Mb and contains 165 genes according to the deCODE map. CNV analysis Focusing on the X-chromosome, one region showed a duplication at the terminal side of the short arm. However, this region is consistent with the pseudoautosomal region PAR1, which explains the suggested duplication. Apart from this pseudoautosomal region, no copy number variations were identified on the X-chromosome. Whole exome sequencing Since the region contains 165 genes, whole exome sequencing was performed to analyze all genes at once. The average coverage throughout the whole exome was 40× and a total of 50,464 variants were found. Basic filtering was performed in VariantDB (Fig. 4). First, all known SNPs with a minor allele frequency (MAF) higher than 0.01 according to the dbSNP, 1000 genomes and ESP5400 databases, were filtered out. In the next step only non-synonymous, frameshift, nonsense and splice site mutations were maintained. The mapping quality was set at higher than 50 to ensure proper mapping of the reads to the reference genome, and a quality by depth of 4.8 was used to remove false positive results. The allelic ratio is the fraction of total reads called as an alternative read. By filtering on allelic ratio N 0.9 we searched for hemizygous variants. As a final filter step we looked at the candidate region on the X-chromosome. No unknown variants were found in this region. Of the 982 exons that were amplified in this region, seven exons remained completely uncovered. These exons were amplified by PCR amplification. Direct sequencing was performed without identification of any unknown variants. There is still 1.88% of the region that remains uncovered, i.e. exons that are only partly covered, mostly only a few bases per exon. Discussion Bone is a dynamic tissue that is constantly remodeled by a balanced process of bone formation and bone resorption. Disruptions in this balance can lead to pathologies characterized by either increased or decreased bone density. X-linked calvarial hyperostosis is such a condition characterized by increased bone density. Several other disorders involving the calvarium and the facial bones exist, such as craniometaphyseal dysplasia (CMD), craniodiaphyseal dysplasia (CDD), LRP5-related disorders, Van Buchem disease and
Sclerosteosis. In CMD, the mandible, maxilla and frontal bones are affected, leading to a dysmorphic appearance. Neurological symptoms associated with cranial nerve entrapment can occur. Besides the hyperostosis of cranial and facial bones, the metaphyses show club-shaped widening [14,15]. CMD is inherited in an autosomal dominant and an autosomal recessive manner. The autosomal dominant form is caused by a mutation in the ANKH gene [15], while the recessive form is mapped to chromosome 6q21–q22 [14]; the disease causing gene, however, has not yet been discovered. Craniodiaphyseal dysplasia is a severe autosomal dominant disorder with generalized sclerosis and hyperostosis of the skull and facial bones. Neurological symptoms occur due to cranial abnormalities. The long bones show diaphyseal sclerosis and hyperostosis [16,17]. CDD is caused by mutations in SOST [18]. The LRP5-related disorders are a group of different disorders all characterized by high bone mass and caused by mutations in the first β-propeller domain of the LDL-receptor related protein 5 (LRP5). The phenotypes are similar in all patients: dense bones and cortical hyperostosis in the skull and tubular bones. Further clinical findings range from an asymptomatic to square jaw and torus palatinus [4,5,19,20]. Van Buchem disease and Sclerosteosis are similar disorders characterized by hyperostosis of the skull, mandible, clavicles, ribs and tubular bones. The enlarged mandible is typical in most patients. The main difference between the two disorders is that Sclerosteosis patients show excessive height and hand abnormalities, which are absent in Van Buchem patients [21,22]. Van Buchem disease is caused by a 52-kb deletion downstream of SOST [2], while Sclerosteosis is caused by loss of function mutations in the same gene [23]. The difference between the above mentioned disorders and X-linked calvarial hyperostosis is that in all these cases not only the skull, but also the long bones are affected. Often neurological symptoms are present and the mode of inheritance is autosomal dominant or recessive. In this family with calvarial hyperostosis excessive bone growth is confined to the skull and presents as lateral frontal horns. The disease appears to be benign and is mainly cosmetic. It has an X-linked recessive inheritance pattern. Female carriers show no symptoms. We were able to localize the gene for calvarial hyperostosis in this family on a chromosomal region spanning from Xq28 to Xqter. This region encompasses 23.53 cM or 8.2 Mb and contains 165 genes. Located at the bottom of this region is the second pseudoautosomal region (PAR2). Human sex chromosomes share two short regions of homology, the pseudoautosomal regions (PAR). These regions behave like an autosome and are subject to recombination during meiosis. PAR1 comprises 2.6 Mb and is located at the tips of the short arms of the X and Y chromosomes, while PAR2 is located at the tips of the long arms and spans only 330 kb. PAR2 consists of 10 genes according to the HGNC database (AMDP1, DDX11L16, DPH3P, IL9R, SPRY3, TCEB1P24, TRPC6P, VAMP7, WASH6P and WASIR1) of which seven are pseudogenes and thus non-functional. Only three of these PAR2 genes, IL9R, SPRY3 and VAMP7, have a protein product. Biallelic expression is seen for IL9R, while VAMP7 and SPRY3 undergo both X- and Y-inactivation [24,25]. CNV analysis showed one duplicated region at the terminal side of the short arms. This region is consistent with PAR1, which explains the duplication signal. No additional duplications or deletions were found further on the X-chromosome. There is no duplication signal seen for PAR2, since that region is not covered by SNPs on the Human OmniExpress-12 v1.0 DNA Analysis BeadChip from Illumina. Exome sequencing revealed four unknown hemizygous variants on the X-chromosome, however, none of these variants are located in the delineated region. Uncovered coding exons were amplified by PCR and sequenced with direct Sanger sequencing, which did not reveal unknown variants. However, theoretically we cannot exclude the possibility that there might be a mutation in one of the short sequences that remain uncovered. Nevertheless, these results indicate that a non-coding regulatory sequence might be involved in the pathogenesis of this disorder. Whole genome sequencing of patient DNA will be considered.
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In this way, a number of genomic variants will be identified with the remaining challenge to identify the real causative variant. Acknowledgments This work was supported by grants from the FWO (Fund for scientific Research) Vlaanderen (G0197.12N) and the University of Antwerp (NOI-BOF and TOP-BOF) all to W. Van Hul and by a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT) to V.M. Borra. References [1] Perdu B, Van Hul W. Sclerosing bone disorders: too much of a good thing. Crit Rev Eukaryot Gene Expr 2010;20:195–212. [2] 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. [3] Balemans W, Van Hul W. Identification of the disease-causing gene in sclerosteosis — discovery of a novel bone anabolic target? J Musculoskelet Neuronal Interact 2004;4:139–42. [4] Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, Mitnick MA, et al. High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med 2002;346:1513–21. [5] Van Wesenbeeck L, Cleiren E, Gram J, Beals RK, Benichou O, Scopelliti D, et al. Six novel missense mutations in the LDL receptor-related protein 5 (LRP5) gene in different conditions with an increased bone density. Am J Hum Genet 2003;72:763–71. [6] Pagon RA, Beckwith JB, Ward BH. Calvarial hyperostosis: a benign X-linked recessive disorder. Clin Genet 1986;29:73–8. [7] Schuelke M. An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 2000;18:233–4. [8] Vandeweyer G, Reyniers E, Wuyts W, Rooms L, Kooy RF. CNV-WebStore: online CNV analysis, storage and interpretation. BMC Bioinformatics 2011;12:4. [9] Blankenberg D, Von Kuster G, Coraor N, Ananda G, Lazarus R, Mangan M, et al. Galaxy: a web-based genome analysis tool for experimentalists. Curr Protoc Mol Biol 2010;10:11–21 [Chapter 19:Unit 19]. [10] Giardine B, Riemer C, Hardison RC, Burhans R, Elnitski L, Shah P, et al. Galaxy: a platform for interactive large-scale genome analysis. Genome Res 2005;15:1451–5.
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[11] Goecks J, Nekrutenko A, Taylor J. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol 2010;11:R86. [12] Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol 2011;29:24–6. [13] Thorvaldsdottir H, Robinson JT, Mesirov JP. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 2013;14:178–92. [14] Prontera P, Rogaia D, Sobacchi C, Tavares VL, Mazzotta G, Passos-Bueno MR, et al. Craniometaphyseal dysplasia with severe craniofacial involvement shows homozygosity at 6q21–22.1 locus. Am J Med Genet A 2011;155A:1106–8. [15] Dutra EH, Chen IP, McGregor TL, Ranells JD, Reichenberger EJ. Two novel large ANKH deletion mutations in sporadic cases with craniometaphyseal dysplasia. Clin Genet 2012;81:93–5. [16] Brueton LA, Winter RM. Craniodiaphyseal dysplasia. J Med Genet 1990;27:701–6. [17] Schaefer B, Stein S, Oshman D, Rennert O, Thurnau G, Wall J, et al. Dominantly inherited craniodiaphyseal dysplasia: a new craniotubular dysplasia. Clin Genet 1986;30:381–91. [18] Kim SJ, Bieganski T, Sohn YB, Kozlowski K, Semenov M, Okamoto N, et al. Identification of signal peptide domain SOST mutations in autosomal dominant craniodiaphyseal dysplasia. Hum Genet 2011;129:497–502. [19] Whyte MP, Reinus WH, Mumm S. High-bone-mass disease and LRP5. N Engl J Med 2004;350:2096–9 [author reply 2096-2099]. [20] Little RD, Carulli JP, Del Mastro RG, Dupuis J, Osborne M, Folz C, et al. A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant highbone-mass trait. Am J Hum Genet 2002;70:11–9. [21] 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. [22] 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. [23] 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. [24] Graves JA, Wakefield MJ, Toder R. The origin and evolution of the pseudoautosomal regions of human sex chromosomes. Hum Mol Genet 1998;7:1991–6. [25] Ciccodicola A, D'Esposito M, Esposito T, Gianfrancesco F, Migliaccio C, Miano MG, et al. Differentially regulated and evolved genes in the fully sequenced Xq/Yq pseudoautosomal region. Hum Mol Genet 2000;9:395–401.
Contents lists available at ScienceDirect
Bone journal homepage: www.elsevier.com/locate/bone
Original Full Length Article
Localization of the gene for X-linked calvarial hyperostosis to chromosome Xq27.3–Xqter V.M. Borra a, E. Steenackers a, F. de Freitas a, E. Van Hul a,1, I. Glass b, W. Van Hul a,⁎ a b
Department of Medical Genetics, University of Antwerp, Belgium Division of Genetic Medicine, Department of Pediatrics, University of Washington, Seattle, Children's Hospital, Seattle, WA, USA
a r t i c l e
i n f o
Article history: Received 26 July 2013 Revised 23 September 2013 Accepted 14 October 2013 Available online 18 October 2013 Edited by: Mark Johnson Keywords: Calvarial hyperostosis Xq27.3–Xqter Calvarium Skull
a b s t r a c t X-linked calvarial hyperostosis is a rare disorder characterized by isolated calvarial thickening. Symptoms are prominent frontoparietal bones, a flat nasal root and a short upturned nose, a high forehead with ridging of the metopic and sagittal sutures, and lateral frontal prominences. The mandible is normal, as are the clavicles, pelvis and long bones. The thickened bone in the skull appears to be softer than normal bone. Despite calvarial hyperostosis, increased intracranial pressure and cranial nerve entrapment do not occur. The major disability seems to be cosmetic. The disease segregates with an X-linked recessive mode of inheritance. Female carriers do not show any clinical symptoms. To date, only one family has been described with X-linked calvarial hyperostosis including three affected individuals. In order to localize the disease causing gene, 31 polymorphic microsatellite markers that spread across the X-chromosome were analyzed. Genotypes were combined in haplotypes to delineate the region. A chromosomal region spanning from Xq27.3 to Xqter cosegregates with the disorder. This region encompasses 23.53 cM or 8.2 Mb according to the deCODE map and contains 165 genes. CNV-analysis did not show small duplications or deletions in this region. Exome sequencing was performed on a male patient in this family. However, this did not reveal any putative mutation. These results indicate that a non-coding regulatory sequence might be involved in the pathogenesis of this disorder. © 2013 Elsevier Inc. All rights reserved.
Introduction Bone is a dynamic tissue that is continuously remodeled by a balanced process of bone formation and bone resorption. Disturbance of this balance can lead to a wide variety of bone disorders, characterized by either low or high bone mineral density [1]. In the last years, several genes have been identified underlying monogenic diseases with an abnormal bone mineral density [2–5]. These studies provide new insights into bone homeostasis and can be important in the understanding of the pathogenesis of complex diseases, like osteoporosis; they might even lead to therapeutical applications for this condition. Calvarial hyperostosis is a benign X-linked disorder that affects only the skull. Symptoms are prominent frontoparietal bones, a flat nasal root and a short upturned nose, a high forehead with ridging of the metopic and sagittal sutures, and lateral frontal prominences. The mandible is normal, as are the clavicles, pelvis and long bones. Radiographs of the skull show increased bone thickness at the sagittal suture line and prominent lateral frontal horns. The thickened bone in the skull appears
⁎ Corresponding author at: Department of Medical Genetics, University of Antwerp, Prins Boudewijnlaan 43, B-2650 Edegem, Belgium. Fax: +32 32759722. E-mail address: [email protected] (W. Van Hul). 1 Current affiliation: Department of Medical Genetics, University Hospital Antwerp, Belgium. 8756-3282/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bone.2013.10.011
to be softer than normal bone. Despite calvarial hyperostosis, increased intracranial pressure and cranial nerve entrapment do not occur. The disorder appears to be benign and the major disability seems to be cosmetic. The disease segregates with an X-linked recessive mode of inheritance. Family and methods Case reports To date, only one family including three affected individuals with calvarial hyperostosis (Fig. 1) has been described in 1986 by Pagon et al. [6]. The proband (IV.1, Fig. 1) was diagnosed with calvarial hyperostosis at approximately 3 years of age because he showed prominence of the frontoparietal bones (Fig. 2a). He had a flat nasal root, a short upturned nose and a high forehead with ridging of the metopic and sagittal sutures and lateral prominences. His neurologic exam was normal as were his vision and hearing. Radiographs showed thickened diploic spaces, prominent trabeculae and closure of all but the lambdoidal sutures. There was thickening of the diploic spaces and prominent maxillary sinuses. He had normal radiographs of the pelvis and one leg. At age twenty-four he returned to the genetics clinic for follow-up. His only health complaints at that time were headaches. They were pounding
68
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Fig. 1. Pedigree of a family with X-linked calvarial hyperostosis. Black symbols indicate affected individuals, white symbols indicate unaffected individuals, white symbols with a dot are carriers. The haplotypes are zoomed in on the long arm of chromosome X. The black haplotype is present in all carriers and affected individuals. Arrow: proband.
in nature and appeared to be in the mid occipital region. They only occurred during waking hours and were not associated with nausea or vomiting, visual disturbance, weakness or paralysis. His facial appearance showed fullness to the right face and in mid-to-lower aspects. However, the eyes themselves were not distorted. There was bony thickening overlying the lateral right frontal prominence and supraorbital region. The mid part of the frontal skull was depressed and there was a flat region overlying the formal sagittal suture. The extremities were normal, there was no sign of nerve entrapment. The disease showed no obvious progression. A maternal cousin (III.3, Fig. 2b) and a maternal second cousin (IV.3) were also diagnosed with calvarial hyperostosis. Patient III.12 was diagnosed at the age of one. Skull radiographs showed calvarial hyperostosis which involved bones that originate from membranous bone. At age two 8/12 radiographs showed prominent lateral horns (Fig. 3) and increased thickness of the bone in the region of the closed sagittal suture line. There was presumed intracranial pressure with slight Luckenschadel appearance to the entire skull. The mandible was normal, as were the clavicles, the pelvis and the long bones. A craniectomy and morcellation of the coronal, lambdoidal and sagittal sutures with excision of the frontal protuberances were performed. At the age of eighteen his face had a normal appearance [6]. Patient IV.3 was diagnosed at eighteen months of age when unusual frontoparietal bony prominences were noted and skull radiographs
showed thickening of the diploic space, lateral and frontal horns and a Luckenschadel appearance in the frontal and occipital areas [6]. Obligate gene carriers (II.1, II.7, and III.1) show no unusual cranial configuration. However, radiographs were not obtained. Genotyping Peripheral blood was collected from 7 family members (two patients, four obligate carriers and one with unknown status). Genomic DNA was isolated from these blood samples using standard procedures. 31 polymorphic markers that spread across the X chromosome were analyzed. These markers were selected from the deCODE genetic map and were analyzed by a Taq DNA polymerase mediated PCR, using fluorescently labeled primers [7]. Fragment analysis of amplified products was performed by an ABI PRISM® 3130 XL Genetic Analyzer (Applied Biosystems). Allele identification was done with Gene mapper v3.7 software (Applied Biosystems). CNV analysis The Illumina iScan system was used with the Human OmniExpress12 v1.0 DNA Analysis BeadChip (Illumina, San Diego, CA) for CNV analysis. Resulting data were analyzed using the CNV-Webstore tool [8].
V.M. Borra et al. / Bone 58 (2014) 67–71
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Fig. 3. Radiograph of III.3 showing the prominent lateral frontal horn (arrow). From: “Calvarial Hyperostosis: a benign X-linked recessive disorder”. Clin Genet 29:73–78 © Wiley-Liss, Inc. This material is reproduced with permission of John Wiley & Sons, Inc.
Exome sequencing Exome sequencing was performed on the proband (IV.1; Fig. 1) with the NimbleGenV2 enrichment panel on the HiSeq2000. Raw sequencing data were analyzed with Galaxy [9–11] and the variants were filtered in VariantDB. The different filters are shown in Fig. 4. Unknown variants
Fig. 2. a. patient IV.1, b. patient III.3. Note the prominent lateral horns. From: “Calvarial Hyperostosis: a benign X-linked recessive disorder”. Clin Genet 29:73–78 © Wiley-Liss, Inc. This material is reproduced with permission of John Wiley & Sons, Inc.
Fig. 4. Filters used in VariantDB to select variants in the co-segregating region on chromosome X.cy1.
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were further analyzed with the Integrative Genomics Viewer (IGV) 2.3 [12,13] and compared to a set of male controls. Direct sequencing of uncovered exons In one affected individual and an unrelated control sample uncovered regions were amplified by GoTaq DNA polymerase-mediated PCR with primers covering the exons and the intron–exon boundaries followed by direct sequencing. Sequencing reactions were performed using the ABI BigDye Terminator v1.1 Cycle sequencing Ready Reaction Kit (Applied Biosystems). Purification with the BigDye Terminator Purification Kit (Applied Biosystems) was carried out to remove unincorporated BigDye terminators, after which fragments were analyzed on an ABI 3130 Genetic Analyzer (Applied Biosystems). Results Delineation of the candidate region Pedigree and haplotypes were generated using the Cyrillic 2.1 software (CyrillicSoftware, Oxfordshire, UK). The black haplotype is seen only in patients and obligated carriers (Fig. 1). The region is delineated by a recombination proximal of marker DXS1193 in patient IV.3 and by the telomeres at the distal side. This region encompasses 23.53 cM or 8.2 Mb and contains 165 genes according to the deCODE map. CNV analysis Focusing on the X-chromosome, one region showed a duplication at the terminal side of the short arm. However, this region is consistent with the pseudoautosomal region PAR1, which explains the suggested duplication. Apart from this pseudoautosomal region, no copy number variations were identified on the X-chromosome. Whole exome sequencing Since the region contains 165 genes, whole exome sequencing was performed to analyze all genes at once. The average coverage throughout the whole exome was 40× and a total of 50,464 variants were found. Basic filtering was performed in VariantDB (Fig. 4). First, all known SNPs with a minor allele frequency (MAF) higher than 0.01 according to the dbSNP, 1000 genomes and ESP5400 databases, were filtered out. In the next step only non-synonymous, frameshift, nonsense and splice site mutations were maintained. The mapping quality was set at higher than 50 to ensure proper mapping of the reads to the reference genome, and a quality by depth of 4.8 was used to remove false positive results. The allelic ratio is the fraction of total reads called as an alternative read. By filtering on allelic ratio N 0.9 we searched for hemizygous variants. As a final filter step we looked at the candidate region on the X-chromosome. No unknown variants were found in this region. Of the 982 exons that were amplified in this region, seven exons remained completely uncovered. These exons were amplified by PCR amplification. Direct sequencing was performed without identification of any unknown variants. There is still 1.88% of the region that remains uncovered, i.e. exons that are only partly covered, mostly only a few bases per exon. Discussion Bone is a dynamic tissue that is constantly remodeled by a balanced process of bone formation and bone resorption. Disruptions in this balance can lead to pathologies characterized by either increased or decreased bone density. X-linked calvarial hyperostosis is such a condition characterized by increased bone density. Several other disorders involving the calvarium and the facial bones exist, such as craniometaphyseal dysplasia (CMD), craniodiaphyseal dysplasia (CDD), LRP5-related disorders, Van Buchem disease and
Sclerosteosis. In CMD, the mandible, maxilla and frontal bones are affected, leading to a dysmorphic appearance. Neurological symptoms associated with cranial nerve entrapment can occur. Besides the hyperostosis of cranial and facial bones, the metaphyses show club-shaped widening [14,15]. CMD is inherited in an autosomal dominant and an autosomal recessive manner. The autosomal dominant form is caused by a mutation in the ANKH gene [15], while the recessive form is mapped to chromosome 6q21–q22 [14]; the disease causing gene, however, has not yet been discovered. Craniodiaphyseal dysplasia is a severe autosomal dominant disorder with generalized sclerosis and hyperostosis of the skull and facial bones. Neurological symptoms occur due to cranial abnormalities. The long bones show diaphyseal sclerosis and hyperostosis [16,17]. CDD is caused by mutations in SOST [18]. The LRP5-related disorders are a group of different disorders all characterized by high bone mass and caused by mutations in the first β-propeller domain of the LDL-receptor related protein 5 (LRP5). The phenotypes are similar in all patients: dense bones and cortical hyperostosis in the skull and tubular bones. Further clinical findings range from an asymptomatic to square jaw and torus palatinus [4,5,19,20]. Van Buchem disease and Sclerosteosis are similar disorders characterized by hyperostosis of the skull, mandible, clavicles, ribs and tubular bones. The enlarged mandible is typical in most patients. The main difference between the two disorders is that Sclerosteosis patients show excessive height and hand abnormalities, which are absent in Van Buchem patients [21,22]. Van Buchem disease is caused by a 52-kb deletion downstream of SOST [2], while Sclerosteosis is caused by loss of function mutations in the same gene [23]. The difference between the above mentioned disorders and X-linked calvarial hyperostosis is that in all these cases not only the skull, but also the long bones are affected. Often neurological symptoms are present and the mode of inheritance is autosomal dominant or recessive. In this family with calvarial hyperostosis excessive bone growth is confined to the skull and presents as lateral frontal horns. The disease appears to be benign and is mainly cosmetic. It has an X-linked recessive inheritance pattern. Female carriers show no symptoms. We were able to localize the gene for calvarial hyperostosis in this family on a chromosomal region spanning from Xq28 to Xqter. This region encompasses 23.53 cM or 8.2 Mb and contains 165 genes. Located at the bottom of this region is the second pseudoautosomal region (PAR2). Human sex chromosomes share two short regions of homology, the pseudoautosomal regions (PAR). These regions behave like an autosome and are subject to recombination during meiosis. PAR1 comprises 2.6 Mb and is located at the tips of the short arms of the X and Y chromosomes, while PAR2 is located at the tips of the long arms and spans only 330 kb. PAR2 consists of 10 genes according to the HGNC database (AMDP1, DDX11L16, DPH3P, IL9R, SPRY3, TCEB1P24, TRPC6P, VAMP7, WASH6P and WASIR1) of which seven are pseudogenes and thus non-functional. Only three of these PAR2 genes, IL9R, SPRY3 and VAMP7, have a protein product. Biallelic expression is seen for IL9R, while VAMP7 and SPRY3 undergo both X- and Y-inactivation [24,25]. CNV analysis showed one duplicated region at the terminal side of the short arms. This region is consistent with PAR1, which explains the duplication signal. No additional duplications or deletions were found further on the X-chromosome. There is no duplication signal seen for PAR2, since that region is not covered by SNPs on the Human OmniExpress-12 v1.0 DNA Analysis BeadChip from Illumina. Exome sequencing revealed four unknown hemizygous variants on the X-chromosome, however, none of these variants are located in the delineated region. Uncovered coding exons were amplified by PCR and sequenced with direct Sanger sequencing, which did not reveal unknown variants. However, theoretically we cannot exclude the possibility that there might be a mutation in one of the short sequences that remain uncovered. Nevertheless, these results indicate that a non-coding regulatory sequence might be involved in the pathogenesis of this disorder. Whole genome sequencing of patient DNA will be considered.
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In this way, a number of genomic variants will be identified with the remaining challenge to identify the real causative variant. Acknowledgments This work was supported by grants from the FWO (Fund for scientific Research) Vlaanderen (G0197.12N) and the University of Antwerp (NOI-BOF and TOP-BOF) all to W. Van Hul and by a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT) to V.M. Borra. References [1] Perdu B, Van Hul W. Sclerosing bone disorders: too much of a good thing. Crit Rev Eukaryot Gene Expr 2010;20:195–212. [2] 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. [3] Balemans W, Van Hul W. Identification of the disease-causing gene in sclerosteosis — discovery of a novel bone anabolic target? J Musculoskelet Neuronal Interact 2004;4:139–42. [4] Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, Mitnick MA, et al. High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med 2002;346:1513–21. [5] Van Wesenbeeck L, Cleiren E, Gram J, Beals RK, Benichou O, Scopelliti D, et al. Six novel missense mutations in the LDL receptor-related protein 5 (LRP5) gene in different conditions with an increased bone density. Am J Hum Genet 2003;72:763–71. [6] Pagon RA, Beckwith JB, Ward BH. Calvarial hyperostosis: a benign X-linked recessive disorder. Clin Genet 1986;29:73–8. [7] Schuelke M. An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 2000;18:233–4. [8] Vandeweyer G, Reyniers E, Wuyts W, Rooms L, Kooy RF. CNV-WebStore: online CNV analysis, storage and interpretation. BMC Bioinformatics 2011;12:4. [9] Blankenberg D, Von Kuster G, Coraor N, Ananda G, Lazarus R, Mangan M, et al. Galaxy: a web-based genome analysis tool for experimentalists. Curr Protoc Mol Biol 2010;10:11–21 [Chapter 19:Unit 19]. [10] Giardine B, Riemer C, Hardison RC, Burhans R, Elnitski L, Shah P, et al. Galaxy: a platform for interactive large-scale genome analysis. Genome Res 2005;15:1451–5.
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