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Homozygous Mutations in IHH Cause Acrocapitofemoral Dysplasia, an Autosomal Recessive Disorder with Cone-Shaped Epiphyses in Hands and Hips

2003; Elsevier BV; Volume: 72; Issue: 4 Linguagem: Inglês

10.1086/374318

1537-6605

Jan Hellemans, Paul Coucke, A Giedion, Anne De Paepe, Peter Krämer, Frits A. Beemer, Geert Mortier,

Epigenetics and DNA Methylation

Acrocapitofemoral dysplasia is a recently delineated autosomal recessive skeletal dysplasia, characterized clinically by short stature with short limbs and radiographically by cone-shaped epiphyses, mainly in hands and hips. Genomewide homozygosity mapping in two consanguineous families linked the locus to 2q35-q36 with a maximum two-point LOD score of 8.02 at marker D2S2248. Two recombination events defined the minimal critical region between markers D2S2248 and D2S2151 (3.74 cM). Using a candidate-gene approach, we identified two missense mutations in the amino-terminal signaling domain of the gene encoding Indian hedgehog (IHH). Both affected individuals of family 1 are homozygous for a 137C→T transition (P46L), and the three patients in family 2 are homozygous for a 569T→C transition (V190A). The two mutant amino acids are strongly conserved and predicted to be located outside the region where brachydactyly type A-1 mutations are clustered. Acrocapitofemoral dysplasia is a recently delineated autosomal recessive skeletal dysplasia, characterized clinically by short stature with short limbs and radiographically by cone-shaped epiphyses, mainly in hands and hips. Genomewide homozygosity mapping in two consanguineous families linked the locus to 2q35-q36 with a maximum two-point LOD score of 8.02 at marker D2S2248. Two recombination events defined the minimal critical region between markers D2S2248 and D2S2151 (3.74 cM). Using a candidate-gene approach, we identified two missense mutations in the amino-terminal signaling domain of the gene encoding Indian hedgehog (IHH). Both affected individuals of family 1 are homozygous for a 137C→T transition (P46L), and the three patients in family 2 are homozygous for a 569T→C transition (V190A). The two mutant amino acids are strongly conserved and predicted to be located outside the region where brachydactyly type A-1 mutations are clustered. Recently, we delineated a new autosomal recessive skeletal dysplasia in two consanguineous families of Belgian and Dutch origin (Mortier et al. Mortier et al., 2003Mortier GR Kramer PPG Giedion A Beemer FA Acrocapitofemoral dysplasia: an autosomal recessive skeletal dysplasia with cone-shaped epiphyses in hands and hips.J Med Genet. 2003; 40: 201-207Crossref PubMed Google Scholar). The clinical phenotype is characterized by short stature of variable degree with short limbs and brachydactyly, relatively large head, and narrow thorax with pectus deformities (fig. 1). Affected individuals do not exhibit associated congenital anomalies and are of normal intelligence. Radiographically, cone-shaped epiphyses (CSE) or a similar epiphyseal configuration are observed in the hands, the proximal part of the femur, and, to a variable degree, at the shoulders, knees, and ankles (fig. 2). These CSE appear early in childhood and disappear with the premature fusion of the growth plate before puberty, resulting in shortening of the involved skeletal components. This process leads, in the hips, to a characteristic egg-shaped femoral head attached to a very short femoral neck and, in the hands, to shortening of the tubular bones, especially the middle phalanges. Since all affected individuals show these striking and characteristic abnormalities in hands and hips, we named the disorder acrocapitofemoral dysplasia (ACFD).Figure 2Most characteristic radiographic abnormalities in the affected individuals. A–C, Radiographs of the left hand. A, Cone-shaped epiphyses in the thumbs and the middle and distal phalanges of digits II–V of patient VII-4 at age 9.5 years. B, Shortening of the tubular bones more pronounced in patient IX-2 at age 11 years. Note the remnants of cone-shaped epiphyses and the fused “tear-drop” metacarpal epiphyses. C, Severe brachydactyly in patient IX-1 at age 14 years. Note complete epimetaphyseal fusion of metacarpals and phalanges. D, Radiograph of the right hand of parent VI-1, showing no obvious signs of BDA1. E, Radiograph of the pelvis in patient VII-4 at age 6 years, showing coxa vara with egg-shaped femoral head and very short femoral neck after epimetaphyseal fusion. F, Normal radiograph of the pelvis in parent VI-1, with no evidence of a premature fusion of the proximal femoral growth plate (normal femoral head and neck). G, Radiograph of the lower limbs in patient IX-2 at age 11 years. Varus deformity of the tibiae with proximal and distal premature epimetaphyseal fusion is most likely the result of cone-shaped epiphyses. Note the voluminous distal femoral epiphyses and the proximal overgrowth of the fibulae.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To unravel the genetic basis of this skeletal dysplasia, we performed homozygosity mapping in both consanguineous families, using 400 genetic markers with an average spacing of 10 cM (ABI PRISM Linkage Mapping Set version 2) (Lander and Botstein Lander and Botstein, 1987Lander ES Botstein D Homozygosity mapping: a way to map human recessive traits with the DNA of inbred children.Science. 1987; 236: 1567-1570Crossref PubMed Scopus (661) Google Scholar). Initially, the genomewide screening was performed on four affected individuals (VII-1, VII-4, IX-2, and IX-5) from both families (fig. 3). A fifth patient (IX-1), later identified, was subsequently used in the analysis. Appropriate informed consent was obtained from all human subjects involved in the study. Alleles were scored using the Genescan and Genemapper software (Applied Biosystems). After the initial screening, the region on 2q35-q36 was further investigated, because four affected individuals were homozygous by descent for two consecutive markers, D2S325 and D2S2382. Analysis with additional, closely spaced markers refined the candidate region to between D2S2382 and D2S163, a distance of about 5 cM (fig. 3). A recombination event between loci D2S2382 and D2S2248 in patient IX-2 (fig. 3B) defined the proximal boundary of the genetic interval. A recombination event between loci D2S2151 and D2S163 in individual IX-4 (fig. 3B) defined the distal boundary of the candidate region. The MLINK program of the LINKAGE software package (Lathrop and Lalouel Lathrop and Lalouel, 1984Lathrop GM Lalouel JM Easy calculations of LOD scores and genetic risks on small computers.Am J Hum Genet. 1984; 36: 460-465PubMed Google Scholar) was used to calculate two-point LOD scores between the disease phenotype and each of the markers, assuming a recessive disease with an allele frequency of 0.001 and marker allele frequencies of 0.125. This resulted in a maximum LOD score of 8.02 at θ=0 for marker D2S2248 (table 1). A search on the human genome resources of the National Center for Biotechnology Information disclosed several candidate genes within the linked region on chromosome 2q35-q36: IGFBP2 and IGFBP5 (both encoding an insulin-like growth-factor–binding protein), IHH (encoding Indian hedgehog), and STK36 (human homologue of the Drosophila melanogaster gene fused). We selected IHH as the strongest candidate because this gene is expressed in the growth plate (Kindblom et al. Kindblom et al., 2002Kindblom JM Nilsson O Hurme T Ohlsson C Savendahl L Expression and localization of Indian hedgehog (Ihh) and parathyroid hormone related protein (PTHrP) in the human growth plate during pubertal development.J Endocrinol. 2002; 174: R1-R6Crossref PubMed Scopus (62) Google Scholar), dominant mutations have been identified in brachydactyly type A1 (BDA1 [MIM 112500]) (Gao et al. Gao et al., 2001Gao B Guo J She C Shu A Yang M Tan Z Yang X Guo S Feng G He L Mutations in IHH, encoding Indian hedgehog, cause brachydactyly type A-1.Nat Genet. 2001; 28: 386-388Crossref PubMed Scopus (183) Google Scholar; McCready et al. McCready et al., 2002McCready ME Sweeney E Fryer AE Donnai D Baig A Racacho L Warman ML Hunter AG Bulman DE A novel mutation in the IHH gene causes brachydactyly type A1: a 95-year-old mystery resolved.Hum Genet. 2002; 111: 368-375Crossref PubMed Scopus (33) Google Scholar), and Ihh-null mice exhibit a short-limb dwarfism phenotype (St-Jacques et al. St-Jacques et al., 1999St-Jacques B Hammerschmidt M McMahon AP Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation.Genes Dev. 1999; 13: 2072-2086Crossref PubMed Scopus (1246) Google Scholar).Table 1Two-Point LOD Scores for Linkage of the ACFD Locus to Chromosome 2q35-q36Combined LOD Score at θ=Family 1Family 2Families 1 and 2MarkerPositionaThe position is given in centiMorgans according to the Marshfield map..00.01.05.10.30ZmaxθZmaxθZmaxθD2S2382213.49−∞7.016.815.962.033.2403.89.057.09.02D2S2248214.718.027.806.925.7891.661.9006.1208.020D2S1371215.256.326.115.284.26.971.4004.9206.320D2S2151218.455.024.854.173.34.74.6304.4005.020D2S163218.45−∞10.8510.629.724.982.0208.89.0110.92.02a The position is given in centiMorgans according to the Marshfield map. Open table in a new tab Sequence analysis of Indian hedgehog (IHH [GenBank accession number Q14623]) in the affected individuals revealed two distinct nucleotide changes. In the two affected individuals in family 1, a homozygous 137C→T transition, predicted to result in a P46L substitution, was observed (fig. 4). In the three affected individuals in family 2, a homozygous 569T→C transition, predicted to result in a V190A substitution, was identified (fig. 4). Both substitutions are located in the amino-terminal domain of the Indian hedgehog protein, which is responsible for local and long-range signaling (Porter et al. Porter et al., 1995Porter JA von Kessler DP Ekker SC Young KE Lee JJ Moses K Beachy PA The product of hedgehog autoproteolytic cleavage active in local and long-range signalling.Nature. 1995; 374: 363-366Crossref PubMed Scopus (425) Google Scholar). Heterozygosity for the 137C→T and 569T→C mutation was observed in the unaffected parents in family 1 and 2, respectively. Both sequence changes were absent in 75 unrelated control subjects (150 chromosomes), confirming that they do not represent common polymorphisms. Finally, alignment of the protein sequence of homologues of Indian hedgehog in several species showed that both proline at position 46 and valine at position 190 are strongly conserved residues (fig. 5).Figure 5Cross-species alignment of the amino-terminal signaling domain of several Hedgehog homologues: HH Drome, IHH Mouse, IHH Brare, DHH Mouse, DHH2 Xenla, TWHH Brare, and AmphiHH (GenBank accession numbers Q02936, P97812, Q98862, Q61488, Q91611, Q90419, and CAA74169.1). Amino acids matching the HH Drome sequence are represented as dots. ACFD mutations are indicated with a gray bar and BDA1 mutations with an asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Indian hedgehog is one of the three mammalian homologues of the D. melanogaster segment polarity gene, Hedgehog (Jeong and McMahon Jeong and McMahon, 2002Jeong J McMahon AP Cholesterol modification of hedgehog family proteins.J Clin Invest. 2002; 110: 591-596Crossref PubMed Scopus (88) Google Scholar). The Hedgehog gene family encodes a group of secreted signaling molecules that are essential for growth and patterning of many different body parts of vertebrate and invertebrate embryos (Ingham and McMahon Ingham and McMahon, 2001Ingham PW McMahon AP Hedgehog signaling in animal development: paradigms and principles.Genes Dev. 2001; 15: 3059-3087Crossref PubMed Scopus (2325) Google Scholar). These molecules are processed after translation by internal cleavage to generate an amino-terminal fragment and a carboxy-terminal fragment with the subsequent addition of cholesterol at the C-terminus of the amino-terminal fragment. This amino-terminal fragment has all the known signaling activity, whereas the carboxy-terminal fragment is responsible for the autoproteolytic process (Lee et al. Lee et al., 1994Lee JJ Ekker SC von Kessler DP Porter JA Sun BI Beachy PA Autoproteolysis in hedgehog protein biogenesis.Science. 1994; 266: 1528-1537Crossref PubMed Scopus (438) Google Scholar; Porter et al. Porter et al., 1995Porter JA von Kessler DP Ekker SC Young KE Lee JJ Moses K Beachy PA The product of hedgehog autoproteolytic cleavage active in local and long-range signalling.Nature. 1995; 374: 363-366Crossref PubMed Scopus (425) Google Scholar). The precise regulation of proliferation and hypertrophic differentiation of chondrocytes in the growth plate is critical for balancing longitudinal growth and ossification of bones (Erlebacher et al. Erlebacher et al., 1995Erlebacher A Filvaroff EH Gitelman SE Derynck R Toward a molecular understanding of skeletal development.Cell. 1995; 80: 371-378Abstract Full Text PDF PubMed Scopus (600) Google Scholar). Several lines of evidence indicate that Ihh signaling controls growth of bones by coordinating chondrocyte proliferation and differentiation (Vortkamp et al. Vortkamp et al., 1996Vortkamp A Lee K Lanske B Segre GV Kronenberg HM Tabin CJBR Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein.Science. 1996; 273: 613-622Crossref PubMed Scopus (1573) Google Scholar; Long et al. Long et al., 2001Long F Zhang XM Karp S Yang Y McMahon AP Genetic manipulation of hedgehog signaling in the endochondral skeleton reveals a direct role in the regulation of chondrocyte proliferation.Development. 2001; 128: 5099-5108PubMed Google Scholar). The regulation of differentiation is indirectly mediated through the production of parathyroid hormone–related peptide (PTHrP). By stimulating PTHrP production in the periarticular growth plate, Ihh delays the switch from proliferation to differentiation. In this way, Ihh determines the rate and site in the growth plate at which hypertrophic differentiation occurs (Karp et al. Karp et al., 2000Karp SJ Schipani E St-Jacques B Hunzelman J Kronenberg H McMahon APBR Indian hedgehog coordinates endochondral bone growth and morphogenesis via parathyroid hormone related-protein-dependent and -independent pathways.Development. 2000; 127: 543-548Crossref PubMed Google Scholar). We postulate that the recessive mutations, identified in our patients with ACFD, cause an increased rate of chondrocyte differentiation by diminishing Indian hedgehog signaling in the growth plate. This increased rate of differentiation is radiographically reflected by the appearance of CSE and subsequent premature closure of the growth plate, with shortening of the bone as a consequence. The cone-shaped configuration of the epiphyses may indicate that this increased rate of differentiation is most pronounced or starts earlier at the center of the physis. There is no evidence for a concentration gradient of IHH and/or PTHrP within a certain zone of the growth plate that could explain an earlier fusion at the center. However, in normal conditions, the knee growth plates seem to close earlier in their central portion than in their peripheral portion (Sasaki et al. Sasaki et al., 2002Sasaki T Ishibashi Y Okamura Y Toh S Sasaki T MRI evaluation of growth plate closure rate and pattern in the normal knee joint.J Knee Surg. 2002; 15: 72-76PubMed Google Scholar). Of note are also the presence of CSE in other skeletal dysplasias affecting the balance between chondrocyte proliferation and differentiation in the growth plate (e.g., cartilage hair hypoplasia [MIM 250250]) and the appearance of CSE after infantile meningococcemia (Patriquin et al. Patriquin et al., 1981Patriquin HB Trias A Jecquier S Marton D Late sequelae of infantile meningococcemia in growing bones of children.Radiology. 1981; 141: 77-82Crossref PubMed Scopus (29) Google Scholar; Grogan et al. Grogan et al., 1989Grogan DP Love SM Ogden JA Millar EA Johnson LO Chondro-osseous growth abnormalities after meningococcemia: a clinical and histopathological study.J Bone Joint Surg Am. 1989; 71: 920-928PubMed Google Scholar; Giedion Giedion, 1998Giedion A Phalangeal cone-shaped epiphyses of the hand: their natural history, diagnostic sensitivity, and specificity in cartilage hair hypoplasia and the trichorhinophalangeal syndromes I and II.Pediatr Radiol. 1998; 28: 751-758Crossref PubMed Scopus (31) Google Scholar). Radiographic evaluation of the heterozygous parents of patients VII-1, VII-4 (family 1), and IX-2 (family 2) does not reveal obvious signs of BDA1 (fig. 2D). None of these parents show absent or rudimentary middle phalanges or middle phalanges fused with the distal phalanges. Mild shortening of middle phalanges II–IV (up to 2.5 SD below the mean) and severe shortening of the middle phalanx V (up to 5 SD below the mean) are observed only in the parents of patients VII-1 and VII-4. The parents of patient IX-2 show only relative shortening (>2 SD below the mean) of the metacarpals and proximal phalanges (data not shown). In addition, both parents of patients VII-1 and VII-4 (family 1) show normal radiographs of the pelvis (fig. 2F). A similar situation, where heterozygotes of the recessive mutations do not exhibit the dominant phenotype, has been observed in brachydactyly type B (BDB [MIM 113000]) and Robinow syndrome (MIM 268310). Heterozygous mutations flanking the intracellular tyrosine kinase domain of ROR2 cause BDB (Schwabe et al. Schwabe et al., 2000Schwabe GC Tinschert S Buschow C Meinecke P Wolff G Gillessen-Kaesbach G Oldridge M Wilkie AOM Kömec R Mundlos S Distinct mutations in the receptor tyrosine kinase gene ROR2 cause brachydactyly type B.Am J Hum Genet. 2000; 67: 822-831Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Homozygous ROR2 mutations scattered throughout the entire coding region cause Robinow syndrome. However, heterozygous carriers of Robinow syndrome do not seem to have BDB (Afzal et al. Afzal et al., 2000Afzal AR Rajab A Fenske CD Oldridge M Elanko N Ternes-Pereira E Tuysuz B Murday VA Patton MA Wilkie AO Jeffery S Recessive Robinow syndrome, allelic to dominant brachydactyly type B, is caused by mutation of ROR2.Nat Genet. 2000; 25: 419-422Crossref PubMed Scopus (225) Google Scholar; Schwabe et al. Schwabe et al., 2000Schwabe GC Tinschert S Buschow C Meinecke P Wolff G Gillessen-Kaesbach G Oldridge M Wilkie AOM Kömec R Mundlos S Distinct mutations in the receptor tyrosine kinase gene ROR2 cause brachydactyly type B.Am J Hum Genet. 2000; 67: 822-831Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar; van Bokhoven et al. van Bokhoven et al., 2000van Bokhoven H Celli J Kayserili H van Beusekom E Balci S Brussel W Skovby F Kerr B Percin EF Akarsu N Brunner HG Mutation of the gene encoding the ROR2 tyrosine kinase causes autosomal recessive Robinow syndrome.Nat Genet. 2000; 25: 423-426Crossref PubMed Scopus (200) Google Scholar). A possible explanation may be that Robinow mutations are loss-of-function mutations with no phenotypic consequences in carriers, whereas BDB mutations exert a dominant effect. In contrast to these observations, loss-of-function mutations of GDF5 cause brachydactyly type C (BDC [MIM 113100]) in the heterozygous state and Grebe dysplasia (MIM 200700), an autosomal recessive acromesomelic dysplasia, in the homozygous state, with radiographic features of BDC in the heterozygous carriers of Grebe mutations (Costa et al. Costa et al., 1998Costa T Ramsby G Cassia F Peters KR Soares J Correa J Quelce-Salgado A Tsipouras P Grebe syndrome: clinical and radiographic findings in affected individuals and heterozygous carriers.Am J Med Genet. 1998; 75: 523-529Crossref PubMed Scopus (44) Google Scholar; Everman et al. 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The mutations in IHH, causing either BDA1 or ACFD, are both missense mutations. However, their precise locations within the amino-terminal signaling domain differ. To compare the locations of different Hedgehog mutations, the crystal structure of the amino-terminal domain of mouse Shh (1VHH [Molecular Modeling DataBase accession number 3860]) can be used because of the high similarity between human IHH and mouse Shh (GenBank accession number Q62226) (Hall et al. Hall et al., 1995Hall TM Porter JA Beachy PA Leahy DJ A potential catalytic site revealed by the 1.7-A crystal structure of the amino-terminal signalling domain of Sonic hedgehog.Nature. 1995; 378: 212-216Crossref PubMed Scopus (165) Google Scholar) (fig. 6). The mutations reported in BDA1 (fig. 6, green) are predicted to be clustered together on one side of the protein, whereas the ACFD (fig. 6, red) mutations seem to be located at the other side, in regions where mutations in SHH (GenBank accession number Q15465) have been reported to cause holoprosencephaly (HPE3 [MIM 142945]) (fig. 6, yellow). In addition, the P46L mutation is located in a region important for binding of the sonic hedgehog protein to its receptor Patched (Fuse et al. Fuse et al., 1999Fuse N Maiti T Wang B Porter JA Hall TM Leahy DJ Beachy PA Sonic hedgehog protein signals not as a hydrolytic enzyme but as an apparent ligand for patched.Proc Natl Acad Sci USA. 1999; 96: 10992-10999Crossref PubMed Scopus (146) Google Scholar). In conclusion, this study indicates that mutations in IHH can cause not only BDA1 but also a more severe skeletal dysplasia with short-limb dwarfism (ACFD). Additional in vitro and in vivo experiments are necessary to investigate the consequences of the different (BDA1 and ACFD) mutations on Indian hedgehog signaling in the growth plate. We are indebted to the families for their interest and cooperation. Special thanks to M. L. Duyts for her excellent technical assistance in preparing the radiographic illustrations. We are grateful to Mike Briggs for critical review of the manuscript. This study is supported, in part, by the Fund for Scientific Research, Flanders, with a mandate “fundamental clinical research” to G.M., and by the Fifth Framework of the specific research and technological development program “Quality of Life and Management of Living Resources” of the European Commission (Contract QLG1-CT-2001-02188) to J.H., A.D.P., and G.M.