RESEARCH LETTER
Identification of the Fourth Duplication of Upstream IHH Regulatory Elements, in a Family with Craniosynostosis Philadelphia Type, Helps to Define the Phenotypic Characterization of These Regulatory Elements Eva Barroso,1,2 Julia Berges-Soria,1 Sara Benito-Sanz,1,2 Carlos Ivan Rivera-Pedroza,1 Marı´a Juliana Ballesta-Martı´nez,2,3 Vanesa Lo´pez-Gonza´lez,2,3 Encarna Guillen-Navarro,2,3,4 and Karen E Heath1,2* 1
Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Auto´noma de Madrid, IdiPAZ, Madrid, Spain 2 Centro de Investigacio´n Biome´dica en Enfermedades Raras (CIBERER), Instituto Carlos, Madrid, Spain 3 Medical Genetics Unit, Dept. of Pediatrics, Hospital Clı´nico Universitario Virgen de la Arrixaca, IMIB-Arrixaca, Murcia, Spain 4 Ca´tedra de Gene´tica Me´dica., UCAM-Universidad Cato´lica San Antonio de Murcia, Spain Manuscript Received: 22 July 2014; Manuscript Accepted: 15 September 2014
TO THE EDITOR: Craniosynostosis, caused by the premature fusion of one or more of the cranial sutures, can be classified into nonsyndromic or syndromic and by which sutures are affected. It affects one in 2,000– 2,500 children [Boulet et al., 2008]. Several craniosynostosis syndromes are associated with malformations of the digits, including craniosynostosis Philadelphia type (CP), a rare form of syndromic craniosynostosis with sagital craniosynostosis and syndactyly of the fingers and toes, with a relatively normal facial appearance [Robin et al., 1996]. Syndactyly is one of the most common abnormalities of the extremities, and occurs either as an isolated malformation or as part of a malformation syndrome. Syndactyly type 1 (SD1, OMIM 185900) is the most common type, with a prevalence of 2–3 in 10,000 newborns [Castilla et al., 1980]. Generally, SD1 involves complete or partial webbing between the third and fourth fingers and/or second and third toes, but other digits are occasionally involved. Bony fusion of the distal phalanges occurs in some cases. The syndactyly is not always bilateral or symmetrical, sometimes only affecting the hands or feet and incomplete penetrance is observed. Two multi-generation families with SD1 narrowed the candidate gene region to chromosome 2q34–36 [Bosse et al., 2000; Ghadami et al., 2001]. In 2008, linkage analysis delimited the implicated locus of CP to chromosome 2q25, suggesting that CP and SD1 shared a common gene defect [Jain et al., 2008]. Indeed, this was true, with the identification of variable sized duplications upstream of Indian hedgehog gene (IHH), localized on chromosome 2q35 in the three previously described families [Klopocki et al., 2011]. The two entities now share the OMIM code 185900. Subsequently, they identified and functionally characterized, using mouse models,
Ó 2015 Wiley Periodicals, Inc.
How to Cite this Article: Barroso E, Berges-Soria J, Benito-Sanz S, Rivera-Pedroza CI, Ballesta-Martı´nez MJ, Lo´pez-Gonza´lez V, Guillen-Navarro E, Heath KE. 2015. Identification of the fourth duplication of upstream IHH regulatory elements, in a family with craniosynostosis philadelphia type, helps to define the phenotypic characterization of these regulatory elements. Am J Med Genet Part A 167A:902–906.
three long-range enhancer elements of IHH, specifically regulating IHH expression during endochondral bone formation. IHH encodes a member of the Hedgehog family of secreted signalling proteins, essential regulators of a variety of developmental processes including growth, patterning and morphogenesis Conflict of interest: None. Grant sponsor: Ministerio de Innovacio´n y Ciencia; Grant number: SAF2012-30871. Correspondence to: Karen Heath, Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La Paz, P˚ Castellana 261, Madrid 28046, Spain E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 18 February 2015 DOI 10.1002/ajmg.a.36811
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BARROSO ET AL. [Briscoe and The´rond, 2013]. Its encoded protein, IHH, plays a specific role in bone growth and differentiation. Duplications in the regulatory regions result in a phenotype completely different from those caused by mutations within the gene itself. Mutations in IHH cause the autosomal dominant brachydactyly type A1 (OMIM 112500), characterized by shortening or malformation of the phalanges, and the autosomal recessive skeletal dysplasia, acrocapitofemoral dysplasia (OMIM 607778) characterized by cone-shaped epiphyses, short stature, and brachydactyly. A large 900 kb duplication of the IHH locus and 29 other contiguous genes was also observed in an individual with a phenotype resembling acrocallosal syndrome, characterized with polysyndactyly of the hands and feet, craniofacial abnormalities, severe hypertelorism, and profound psychomotor delay [Yuksel-Apak et al., 2012], mimicking the phenotype observed in the doublefoot mouse mutant (Dbf) with a 600 kb deletion 50 of IHH [Babbs et al., 2008]. These varying phenotypes demonstrate the importance of IHH during limb and cranial development. We describe a fourth family with the smallest duplication, identified to date, of the upstream IHH regulatory region and suggest a correlation between the phenotype and the implicated regulatory regions. The proband is a 6-year-old boy, first child of a nonconsanguineous couple (Fig. 1A). The pregnancy was uneventful and the child was born at term with a BW of 2650 g (6th centile), BL 49 cm (28th centile), and OFC 33.5 cm (25th centile). Cutaneous syndactyly between the third, fourth, and fifth digits of the hands and the second, third, and fourth toes was observed at birth. The child had normal psychomotor development and normal growth parameters. At 1 year of age sagital craniosynostosis was detected, which along with the syndactyly suggested a clinical diagnosis of CP. The proband, currently 6 years (Fig1B) underwent a TAC, which confirmed the sagittal suture fusion, with normal coronal, metopic, and lamboideal sutures (Fig. 1C and D). No surgical intervention for the craniosynostosis was undertaken. Images of the hand and foot syndactly are shown (Fig. 1I and K). Radiography showed that there was no osseous syndactyly (data not shown). He then underwent partial surgical reparation of cutaneous syndactyly. Karyotype analysis was normal. Family history revealed three other affected individuals (Fig. 1A). The mother, 28 years old, presented with asymmetrical cranial shape and the TAC revealed sagittal and right coronal craniosynostosis (Fig. 1E–H). She had no finger syndactyly (Fig. 1J) but bilateral syndactyly of the second and third toes (Fig. 1L). The maternal grandmother and uncle also had cranial abnormalities and hand and foot syndactyly. The study was approved by the Hospital Universitario La Paz Research Ethics Committee, and informed consent was obtained from all participants. DNA was collected from a total of 12 family members, four affected individuals and eight unaffected relatives. Genomic DNA was isolated from whole blood (Chemagic DNA extraction special, Perkin Elmer Chemagen). Chromosome 2 specific array-CGH (aCGH, Roche NimbleGen, Madison, WI) was performed in the proband and mother, according to the manufacturer. The average probe density was 575 bp. The data was analyzed using the SignalMap v1.0.0.0.3 software (Roche NimbleGen). All genomic positions given in the text are according
903 to human genome version hg18 (UCSC Genome browser, Mar. 2006; http://www.genome.ucsc.edu/). To identify the duplication breakpoint junction, oligonucleotides were designed according to the predicted duplicated region determined by the aCGH (available upon request). PCRs were undertaken in affected and unaffected individuals and the products were separated on an agarose gel PCR products. Direct sequencing of the products observed only in the affected individuals was undertaken. A custom designed MLPA was utilized to confirm duplications in the upstream regulatory region of IHH. The MLPA included nine probes spanning the 2q35 region including IHH and its upstream regulatory regions, located within NHEJ1, and six control probes (Supplementary Table I in supporting information online). The MLPA data were analyzed as previously described [Benito-Sanz et al., 2011]. Using chromosome 2 specific array CGH, an 31 kb duplication of chromosome 2q35 spanning 219,658,383–219,689,640 (hg18) was identified in both proband and mother (Fig. 2A). The duplication was confirmed, by a custom designed MLPA in all four affected family members (Supplementary Fig 1 in supporting information online) while absent in all other tested individuals. In order to delimit the duplication accurately and using the aCGH coordinates, oligonucleotides were designed to permit the identification of the amplified junction fragments in both directions, assuming that the duplication lay in direct tandem orientation. Using this approach, the junction breakpoint was successfully identified, indicating that the duplication was in tandem and spanned 31075 bp (Fig. 2B). Thus, we report the identification of the smallest tandem duplication including the IHH regulatory elements in a family with CP. Tandem duplications are thought to lead to an expansion in the distance between the regulatory element and the gene, causing changes in the complex time-dependent mechanism of the genes. In the mouse model in which the orthologous IHH regulatory region was inserted, the reporter gene expression was highly similar to that of wild type IHH expression, thus postulating that the observed duplications lead to a misexpression and/or overexpression of IHH [Klopocki et al., 2011]. In a finely dissected study of the HoxD locus [Montavon et al., 2012], it was shown that duplications of the proximal part of the regulatory region led to a partial loss of Hoxd expression, likely by interfering with the activity of more distally located elements, and their normal activities were not compensated for by the duplication of other regulatory islands, while a duplication of the entire regulatory interval had no detectable effect on Hox gene regulation. This indicated that it is the genomic organization of the regulatory elements relative to their target genes rather than their absolute number of copies that is of importance for the transcriptional output of the system. In this work they also showed that the HoxD expression depended on the precise duplicated sequence. A total of four families have now been observed to have duplications in the upstream IHH regulatory elements, three presented with craniosynostosis and cutaneous and distal osseous syndactyly while one family only had cutaneous syndactyly. We assessed the extensions and which CNEs were included for this and the three previously reported duplications (Fig. 3). Interesting the family which only presented syndactyly (Family 1, Fig. 3) only contains one of the CNEs, CNE1 and includes the IHH, whereas the duplications identified in the three families with CP, i.e., those
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FIG. 1. Pedigree and images of the proband and affected mother. A: Pedigree showing the presence of four affected members in three generations. Images of the proband (B–E, I and K), and mother (F–H, J and L). B: Photograph of the proband, aged 6, showing the dolichocephaly, frontal bossing and low-set ears; C and D: Computed axial tomographic images of the proband at 6 years old, with dolichocephaly and sagittal synostosis. Early fusion of the sagittal suture is shown by an arrow (D). E–H: Computed axial tomographic images of the skull of the affected mother, showing plagiocephaly, sagittal and right coronal synostosis (E and F). In the axial section, intracranial structures are conserved and asymmetric to the expense of the right side and liquid in subarachnoid space in left frontal region, shown by arrow (E). Craniofacial asymmetry to expense of the right side, nasal septum deviation to the right side, orbit left with upper edge flattened (G and H). Syndactyly of fingers (I) while the mother did not have cutaneous or osseous syndactyly (J). Syndactlyl of the toes of the proband, aged 6 (K) and affected mother (L).
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FIG. 2. Identification and characterization of the duplication of the upstream IHH regulatory region. A: Chromosome 2 specific aCGH showing the duplication of the 2q35 region in the proband (upper panel) and mother (lower panel). The y axis indicates the log2 ratio of each oligo on chromosome 2. The x axis indicates the physical position of the chromosome 2q35 (hg18). Genome positions are given according to hg18 assembly. B: Schematic overview of the identified tandem duplication and the sequence of the junction fragment. The genomic positions of the bases adjacent to the junction fragment are indicated by arrows (hg18). The hg18 coordinates correspond to 219,981,380 and 219,950,305 (hg19) or 219,116,658 and 219,085,583 (hg38).
with members affected with craniosynostosis and syndactyly (Families 2–4, Fig. 3) include all three identified CNEs, thus suggesting that CNEs 2 and 3 may control expression in the cranial bones while CNE1 controls expression in digit formation. Yet intrafamilial variability is also observed within the families. In the two previously reported families with CP with a duplication upstream of IHH, the sagittal craniosynostosis showed incomplete penetrance with 6/15 individuals only presenting with syndactyly and no craniosynostosis [Robin et al., 1996; Klopocki et al., 2011]. In contrast, all four affected individuals in this report showed the fully penetrant form. Thus, for now we can only propose that variation in temporal and spatial expression of IHH may explain the observed phenotypic heterogeneity. Human embryonic limb development begins at 32 days (week 5) with development of paddle like upper limb buds. At 35 days the handplate forms with the appearance of the digital rays within this handplate at 44 days (week 6). At 48 days (week 7) the fingers are short and slightly webbed, while at 56 days (week 8) the fingers and toes are clearly distinct and separated. Endochondral bone formation in the limbs is controlled by a large number of growth factors
such as BMPs (Bone morphogenic proteins, FGFs (Fibroblast growth factors), WNTs, IHH, and PTHrP (Parathyroid hormone-related protein). IHH is a member of the Hedgehog family of secreted signalling molecules. In mouse, IHH is first expressed in condensed mesechymal cells from at embryonic day (E) 12.0, in prechondrocytes at the center of the limb cartilage at E12.5 and in prehypertrophic and hypertrophic cho0ndrocytes in the developing growth plate from E14.5 [Bitgood and McMahon, 1995]. These mouse embryonic expression stages correspond to human embryo 40–52 days (weeks 5–7) when digital rays appear and the digits are formed and separated. Thus, defective IHH expression would be expected to cause defects in the formation and separation of the digits, such as syndactyly, observed in patients with CP. With respect to cranial development, IHH is expressed mainly on the osteogenic fronts of the calvarial bones, and functions to induce cell proliferation and differentiation [Reviewed by Pan et al., 2013]. Many morphologic abnormalities have been observed when misexpression of the Hedgehog signalling occurs, thus, highlighting its importance in cranial and limb development [reviewed by Pan et al., 2013].
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FIG. 3. Schematic representation of the four duplications of the upstream IHH regulatory regions. Family 1 presented with SD1 [Bosse et al., 2000] while Families 2–4 presented with craniosynostosis Philadelphia type [Robin et al., 1996; Klopocki et al., 2011, this study]. The duplication in Family 1 includes only CNE1 and also includes IHH, while the three families with both craniosynostosis and syndactyly have duplications of CNEs 2 and 3, suggesting that CNE1 is important for digit formation while CNEs 2 and 3 are important in cranial development. The diagram is not drawn to scale (hg18).
The characterization of the duplication extensions in these four families with either SD1 or CP suggests that CNEs 2 and 3 may be important in cranial development while CNE1 is important for digit formation. The identification of further cases and/or the analysis of the CNEs independently in mouse models may foment this hypothesis. Despite this, incomplete penetrance has been observed in two of the families showing variability of the pathological phenotype, thus demonstrating that the expression of IHH is finely regulated in a time and spatial manner.
REFERENCES Boulet SL, Rasmussen SA, Honein MA. 2008. A population-based study of craniosynostosis in metropolitan Atlanta 1989–2003. Am J Med Genet Part A 146:984–991. Babbs C, Furniss D, Morris-Kay GM, Wikie AO. 2008. Polydactyly in the mouse mutant Doublefoot involves Gli3 processing and is caused by a large deletion in cis to Indian hedgehog. Mech Dev 125:517–526. Benito-Sanz S, Aza-Carmona M, Rodriguez-Estevez A, Rica-Etxebarria I, Gracia R, Campos-Barros A, Heath KE. 2011. Clinical and molecular evaluation of SHOX/PAR1 duplications in Le´ri-Weill dyschondrosteosis (LWD) and idiopathic short stature (ISS). J Clin Endocrinol and Metab 96:E404–E412. Bitgood MJ, McMahon AP. 1995. Hedgehog and BMP genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev Biol 172:126–138.
Castilla EE, Paz JE, Orioli-Parreiras IM. 1980. Syndactyly: frequency of specific types. Am J Med Genet 5:357–364. Ghadami M, Majidzadeh-A K, Haerian BS, Damavandi E, Yamada K, Pasallar P, Najafi MT, Nishimura G, Tomita HA, Yoshiura KI, Niikawa N. 2001. Confirmation of genetic homogeneity of syndactyly type 1 in an Iranian family. Am J Med Genet 104:147–151. Jain M, Wallis D, Robin NH, De Vrieze FW, Hardy JA, Ghadami M, Bosse K, Betz RC, No¨then MM, Arcos-Burgos M, Muenke M. 2008. Locus homogeneity between syndactyly type 1A and craniosynostosis Philadelphia type?. Am J Med Genet Part A 146:2308–2311. Klopocki E, Lohan S, Brancati F, Koll R, Brehm A, Seemann P, Dathe K, Stricker S, Hecht J, Bosse K, Betz RC, Garaci FG, Dallapiccola B, Jain M, Muenke M, Ng VC, Chan W, Chan D, Mundlos S. 2011. Copy-number variations involving the IHH locus are associated with syndactyly and craniosynostosis. Am J Hum Genet 88:70–75. Montavon T, Thevenet L, Duboule D. 2012. Impact of copy number variations (CNVs) on long-range gene regulation at the HoxD locus. Proc Natl Acad Sci U S A 109:20204–20211. Pan A, Chang L, Nguyen A, James AW. 2013. A review of hedgehog signaling in cranial bone development. Front Physiol 4:-61. Robin NH, Segel B, Carpenter G, Muenke M. 1996. Craniosynostosis, Philadelphia type: a new autosomal dominant syndrome with sagittal craniosynososis and syndactyly of the fingers and toes. Am J Med Genet 62:184–191. Yuksel-Apak M, Bo¨gershausen N, Pawlik B, Li Y, Apak S, Uyguner O, Milz E, Nu¨rnberg G, Karaman B, Gu¨lgo¨ren A, Grzeschik KH, Nu¨rnberg P, Kayserili H, Wollnik B. 2012. A large duplication involving the IHH locus mimics acrocallosal syndrome. Eur J Hum Genet 20:639–644.
Bosse K, Betz RC, Lee YA, Wienker TF, Reis A, Kleen H, Propping P, Cichon S, No¨then MM. 2000. Localization of a gene for syndactyly type 1 to chromosome 2q34-q36. Am J Hum Genet 67:492–497.
SUPPORTING INFORMATION
Briscoe J, The´rond PP. 2013. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol 14:416–429.
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Identification of the Fourth Duplication of Upstream IHH Regulatory Elements, in a Family with Craniosynostosis Philadelphia Type, Helps to Define the Phenotypic Characterization of These Regulatory Elements Eva Barroso,1,2 Julia Berges-Soria,1 Sara Benito-Sanz,1,2 Carlos Ivan Rivera-Pedroza,1 Marı´a Juliana Ballesta-Martı´nez,2,3 Vanesa Lo´pez-Gonza´lez,2,3 Encarna Guillen-Navarro,2,3,4 and Karen E Heath1,2* 1
Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Auto´noma de Madrid, IdiPAZ, Madrid, Spain 2 Centro de Investigacio´n Biome´dica en Enfermedades Raras (CIBERER), Instituto Carlos, Madrid, Spain 3 Medical Genetics Unit, Dept. of Pediatrics, Hospital Clı´nico Universitario Virgen de la Arrixaca, IMIB-Arrixaca, Murcia, Spain 4 Ca´tedra de Gene´tica Me´dica., UCAM-Universidad Cato´lica San Antonio de Murcia, Spain Manuscript Received: 22 July 2014; Manuscript Accepted: 15 September 2014
TO THE EDITOR: Craniosynostosis, caused by the premature fusion of one or more of the cranial sutures, can be classified into nonsyndromic or syndromic and by which sutures are affected. It affects one in 2,000– 2,500 children [Boulet et al., 2008]. Several craniosynostosis syndromes are associated with malformations of the digits, including craniosynostosis Philadelphia type (CP), a rare form of syndromic craniosynostosis with sagital craniosynostosis and syndactyly of the fingers and toes, with a relatively normal facial appearance [Robin et al., 1996]. Syndactyly is one of the most common abnormalities of the extremities, and occurs either as an isolated malformation or as part of a malformation syndrome. Syndactyly type 1 (SD1, OMIM 185900) is the most common type, with a prevalence of 2–3 in 10,000 newborns [Castilla et al., 1980]. Generally, SD1 involves complete or partial webbing between the third and fourth fingers and/or second and third toes, but other digits are occasionally involved. Bony fusion of the distal phalanges occurs in some cases. The syndactyly is not always bilateral or symmetrical, sometimes only affecting the hands or feet and incomplete penetrance is observed. Two multi-generation families with SD1 narrowed the candidate gene region to chromosome 2q34–36 [Bosse et al., 2000; Ghadami et al., 2001]. In 2008, linkage analysis delimited the implicated locus of CP to chromosome 2q25, suggesting that CP and SD1 shared a common gene defect [Jain et al., 2008]. Indeed, this was true, with the identification of variable sized duplications upstream of Indian hedgehog gene (IHH), localized on chromosome 2q35 in the three previously described families [Klopocki et al., 2011]. The two entities now share the OMIM code 185900. Subsequently, they identified and functionally characterized, using mouse models,
Ó 2015 Wiley Periodicals, Inc.
How to Cite this Article: Barroso E, Berges-Soria J, Benito-Sanz S, Rivera-Pedroza CI, Ballesta-Martı´nez MJ, Lo´pez-Gonza´lez V, Guillen-Navarro E, Heath KE. 2015. Identification of the fourth duplication of upstream IHH regulatory elements, in a family with craniosynostosis philadelphia type, helps to define the phenotypic characterization of these regulatory elements. Am J Med Genet Part A 167A:902–906.
three long-range enhancer elements of IHH, specifically regulating IHH expression during endochondral bone formation. IHH encodes a member of the Hedgehog family of secreted signalling proteins, essential regulators of a variety of developmental processes including growth, patterning and morphogenesis Conflict of interest: None. Grant sponsor: Ministerio de Innovacio´n y Ciencia; Grant number: SAF2012-30871. Correspondence to: Karen Heath, Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La Paz, P˚ Castellana 261, Madrid 28046, Spain E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 18 February 2015 DOI 10.1002/ajmg.a.36811
902
BARROSO ET AL. [Briscoe and The´rond, 2013]. Its encoded protein, IHH, plays a specific role in bone growth and differentiation. Duplications in the regulatory regions result in a phenotype completely different from those caused by mutations within the gene itself. Mutations in IHH cause the autosomal dominant brachydactyly type A1 (OMIM 112500), characterized by shortening or malformation of the phalanges, and the autosomal recessive skeletal dysplasia, acrocapitofemoral dysplasia (OMIM 607778) characterized by cone-shaped epiphyses, short stature, and brachydactyly. A large 900 kb duplication of the IHH locus and 29 other contiguous genes was also observed in an individual with a phenotype resembling acrocallosal syndrome, characterized with polysyndactyly of the hands and feet, craniofacial abnormalities, severe hypertelorism, and profound psychomotor delay [Yuksel-Apak et al., 2012], mimicking the phenotype observed in the doublefoot mouse mutant (Dbf) with a 600 kb deletion 50 of IHH [Babbs et al., 2008]. These varying phenotypes demonstrate the importance of IHH during limb and cranial development. We describe a fourth family with the smallest duplication, identified to date, of the upstream IHH regulatory region and suggest a correlation between the phenotype and the implicated regulatory regions. The proband is a 6-year-old boy, first child of a nonconsanguineous couple (Fig. 1A). The pregnancy was uneventful and the child was born at term with a BW of 2650 g (6th centile), BL 49 cm (28th centile), and OFC 33.5 cm (25th centile). Cutaneous syndactyly between the third, fourth, and fifth digits of the hands and the second, third, and fourth toes was observed at birth. The child had normal psychomotor development and normal growth parameters. At 1 year of age sagital craniosynostosis was detected, which along with the syndactyly suggested a clinical diagnosis of CP. The proband, currently 6 years (Fig1B) underwent a TAC, which confirmed the sagittal suture fusion, with normal coronal, metopic, and lamboideal sutures (Fig. 1C and D). No surgical intervention for the craniosynostosis was undertaken. Images of the hand and foot syndactly are shown (Fig. 1I and K). Radiography showed that there was no osseous syndactyly (data not shown). He then underwent partial surgical reparation of cutaneous syndactyly. Karyotype analysis was normal. Family history revealed three other affected individuals (Fig. 1A). The mother, 28 years old, presented with asymmetrical cranial shape and the TAC revealed sagittal and right coronal craniosynostosis (Fig. 1E–H). She had no finger syndactyly (Fig. 1J) but bilateral syndactyly of the second and third toes (Fig. 1L). The maternal grandmother and uncle also had cranial abnormalities and hand and foot syndactyly. The study was approved by the Hospital Universitario La Paz Research Ethics Committee, and informed consent was obtained from all participants. DNA was collected from a total of 12 family members, four affected individuals and eight unaffected relatives. Genomic DNA was isolated from whole blood (Chemagic DNA extraction special, Perkin Elmer Chemagen). Chromosome 2 specific array-CGH (aCGH, Roche NimbleGen, Madison, WI) was performed in the proband and mother, according to the manufacturer. The average probe density was 575 bp. The data was analyzed using the SignalMap v1.0.0.0.3 software (Roche NimbleGen). All genomic positions given in the text are according
903 to human genome version hg18 (UCSC Genome browser, Mar. 2006; http://www.genome.ucsc.edu/). To identify the duplication breakpoint junction, oligonucleotides were designed according to the predicted duplicated region determined by the aCGH (available upon request). PCRs were undertaken in affected and unaffected individuals and the products were separated on an agarose gel PCR products. Direct sequencing of the products observed only in the affected individuals was undertaken. A custom designed MLPA was utilized to confirm duplications in the upstream regulatory region of IHH. The MLPA included nine probes spanning the 2q35 region including IHH and its upstream regulatory regions, located within NHEJ1, and six control probes (Supplementary Table I in supporting information online). The MLPA data were analyzed as previously described [Benito-Sanz et al., 2011]. Using chromosome 2 specific array CGH, an 31 kb duplication of chromosome 2q35 spanning 219,658,383–219,689,640 (hg18) was identified in both proband and mother (Fig. 2A). The duplication was confirmed, by a custom designed MLPA in all four affected family members (Supplementary Fig 1 in supporting information online) while absent in all other tested individuals. In order to delimit the duplication accurately and using the aCGH coordinates, oligonucleotides were designed to permit the identification of the amplified junction fragments in both directions, assuming that the duplication lay in direct tandem orientation. Using this approach, the junction breakpoint was successfully identified, indicating that the duplication was in tandem and spanned 31075 bp (Fig. 2B). Thus, we report the identification of the smallest tandem duplication including the IHH regulatory elements in a family with CP. Tandem duplications are thought to lead to an expansion in the distance between the regulatory element and the gene, causing changes in the complex time-dependent mechanism of the genes. In the mouse model in which the orthologous IHH regulatory region was inserted, the reporter gene expression was highly similar to that of wild type IHH expression, thus postulating that the observed duplications lead to a misexpression and/or overexpression of IHH [Klopocki et al., 2011]. In a finely dissected study of the HoxD locus [Montavon et al., 2012], it was shown that duplications of the proximal part of the regulatory region led to a partial loss of Hoxd expression, likely by interfering with the activity of more distally located elements, and their normal activities were not compensated for by the duplication of other regulatory islands, while a duplication of the entire regulatory interval had no detectable effect on Hox gene regulation. This indicated that it is the genomic organization of the regulatory elements relative to their target genes rather than their absolute number of copies that is of importance for the transcriptional output of the system. In this work they also showed that the HoxD expression depended on the precise duplicated sequence. A total of four families have now been observed to have duplications in the upstream IHH regulatory elements, three presented with craniosynostosis and cutaneous and distal osseous syndactyly while one family only had cutaneous syndactyly. We assessed the extensions and which CNEs were included for this and the three previously reported duplications (Fig. 3). Interesting the family which only presented syndactyly (Family 1, Fig. 3) only contains one of the CNEs, CNE1 and includes the IHH, whereas the duplications identified in the three families with CP, i.e., those
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FIG. 1. Pedigree and images of the proband and affected mother. A: Pedigree showing the presence of four affected members in three generations. Images of the proband (B–E, I and K), and mother (F–H, J and L). B: Photograph of the proband, aged 6, showing the dolichocephaly, frontal bossing and low-set ears; C and D: Computed axial tomographic images of the proband at 6 years old, with dolichocephaly and sagittal synostosis. Early fusion of the sagittal suture is shown by an arrow (D). E–H: Computed axial tomographic images of the skull of the affected mother, showing plagiocephaly, sagittal and right coronal synostosis (E and F). In the axial section, intracranial structures are conserved and asymmetric to the expense of the right side and liquid in subarachnoid space in left frontal region, shown by arrow (E). Craniofacial asymmetry to expense of the right side, nasal septum deviation to the right side, orbit left with upper edge flattened (G and H). Syndactyly of fingers (I) while the mother did not have cutaneous or osseous syndactyly (J). Syndactlyl of the toes of the proband, aged 6 (K) and affected mother (L).
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FIG. 2. Identification and characterization of the duplication of the upstream IHH regulatory region. A: Chromosome 2 specific aCGH showing the duplication of the 2q35 region in the proband (upper panel) and mother (lower panel). The y axis indicates the log2 ratio of each oligo on chromosome 2. The x axis indicates the physical position of the chromosome 2q35 (hg18). Genome positions are given according to hg18 assembly. B: Schematic overview of the identified tandem duplication and the sequence of the junction fragment. The genomic positions of the bases adjacent to the junction fragment are indicated by arrows (hg18). The hg18 coordinates correspond to 219,981,380 and 219,950,305 (hg19) or 219,116,658 and 219,085,583 (hg38).
with members affected with craniosynostosis and syndactyly (Families 2–4, Fig. 3) include all three identified CNEs, thus suggesting that CNEs 2 and 3 may control expression in the cranial bones while CNE1 controls expression in digit formation. Yet intrafamilial variability is also observed within the families. In the two previously reported families with CP with a duplication upstream of IHH, the sagittal craniosynostosis showed incomplete penetrance with 6/15 individuals only presenting with syndactyly and no craniosynostosis [Robin et al., 1996; Klopocki et al., 2011]. In contrast, all four affected individuals in this report showed the fully penetrant form. Thus, for now we can only propose that variation in temporal and spatial expression of IHH may explain the observed phenotypic heterogeneity. Human embryonic limb development begins at 32 days (week 5) with development of paddle like upper limb buds. At 35 days the handplate forms with the appearance of the digital rays within this handplate at 44 days (week 6). At 48 days (week 7) the fingers are short and slightly webbed, while at 56 days (week 8) the fingers and toes are clearly distinct and separated. Endochondral bone formation in the limbs is controlled by a large number of growth factors
such as BMPs (Bone morphogenic proteins, FGFs (Fibroblast growth factors), WNTs, IHH, and PTHrP (Parathyroid hormone-related protein). IHH is a member of the Hedgehog family of secreted signalling molecules. In mouse, IHH is first expressed in condensed mesechymal cells from at embryonic day (E) 12.0, in prechondrocytes at the center of the limb cartilage at E12.5 and in prehypertrophic and hypertrophic cho0ndrocytes in the developing growth plate from E14.5 [Bitgood and McMahon, 1995]. These mouse embryonic expression stages correspond to human embryo 40–52 days (weeks 5–7) when digital rays appear and the digits are formed and separated. Thus, defective IHH expression would be expected to cause defects in the formation and separation of the digits, such as syndactyly, observed in patients with CP. With respect to cranial development, IHH is expressed mainly on the osteogenic fronts of the calvarial bones, and functions to induce cell proliferation and differentiation [Reviewed by Pan et al., 2013]. Many morphologic abnormalities have been observed when misexpression of the Hedgehog signalling occurs, thus, highlighting its importance in cranial and limb development [reviewed by Pan et al., 2013].
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FIG. 3. Schematic representation of the four duplications of the upstream IHH regulatory regions. Family 1 presented with SD1 [Bosse et al., 2000] while Families 2–4 presented with craniosynostosis Philadelphia type [Robin et al., 1996; Klopocki et al., 2011, this study]. The duplication in Family 1 includes only CNE1 and also includes IHH, while the three families with both craniosynostosis and syndactyly have duplications of CNEs 2 and 3, suggesting that CNE1 is important for digit formation while CNEs 2 and 3 are important in cranial development. The diagram is not drawn to scale (hg18).
The characterization of the duplication extensions in these four families with either SD1 or CP suggests that CNEs 2 and 3 may be important in cranial development while CNE1 is important for digit formation. The identification of further cases and/or the analysis of the CNEs independently in mouse models may foment this hypothesis. Despite this, incomplete penetrance has been observed in two of the families showing variability of the pathological phenotype, thus demonstrating that the expression of IHH is finely regulated in a time and spatial manner.
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SUPPORTING INFORMATION
Briscoe J, The´rond PP. 2013. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol 14:416–429.
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
