A Track Record on SHOX: From Basic Research to Complex Models and Therapy

Abstract

SHOX deficiency is the most frequent genetic growth disorder associated with isolated and syndromic forms of short stature. Caused by mutations in the homeobox gene SHOX, its varied clinical manifestations include isolated short stature, Léri-Weill dyschondrosteosis, and Langer mesomelic dysplasia. In addition, SHOX deficiency contributes to the skeletal features in Turner syndrome. Causative SHOX mutations have allowed downstream pathology to be linked to defined molecular lesions. Expression levels of SHOX are tightly regulated, and almost half of the pathogenic mutations have affected enhancers. Clinical severity of SHOX deficiency varies between genders and ranges from normal stature to profound mesomelic skeletal dysplasia. Treatment options for children with SHOX deficiency are available. Two decades of research support the concept of SHOX as a transcription factor that integrates diverse aspects of bone development, growth plate biology, and apoptosis. Due to its absence in mouse, the animal models of choice have become chicken and zebrafish. These models, therefore, together with micromass cultures and primary cell lines, have been used to address SHOX function. Pathway and network analyses have identified interactors, target genes, and regulators. Here, we summarize recent data and give insight into the critical molecular and cellular functions of SHOX in the etiopathogenesis of short stature and limb development.

Figure 1.

The SHOX gene. The SHOX gene maps to 505–527 kb from the telomere of the sex chromosomes on Xp22.33 and Yp11.32 and spans approximately 40 kb. It is composed of nine exons that produce two main transcripts, SHOXa and SHOXb, of different length. The two transcripts contain a DNA sequence called a homeobox that encodes the homeodomain, a conserved DNA-binding domain that characterizes the family of the homeodomain-containing transcription factors. Alternative SHOX isoforms are also formed by alternative splicing of the exons. The function of these isoforms is not entirely clear, but they may be involved in the spatiotemporal regulation of SHOX expression and activity (see also Section II C). Exon 7 variants are found to be exclusively expressed in fetal neuro-tissues arguing for a specific role of these variants during brain development. The different mRNAs are predicted to lead to peptides of different length. Tel, telomere.

Figure 2.

Mechanisms regulating SHOX gene expression. A, Alternative promoters. Two promoters, P1 and P2, control SHOX expression. These promoters produce transcripts that differ in their 5′-UTR: P1 produces transcripts containing seven untranslated AUG codons, whereas P2 transcripts lack these regulative elements. The two types of mRNA are translated with different efficiency, thereby contributing to the fine-tuned regulation of the levels and tissue specificity of SHOX expression. B, Enhancer elements. Evolutionarily conserved regions in PAR1 identified in different studies. CNE, highly evolutionarily conserved noncoding DNA elements (30, 31, 162); ECR, evolutionarily conserved sequence (29); ECS, evolutionarily conserved sequence (5, 49). The upper horizontal line indicates the physical distance from Xp/Yp telomere (Tel;hg19,build37). Genomic positions: SHOXa (NM_000451.3), chrX:585, 079-607558; CNE-2, chrX:516, 610-517229; CNE-3, chrX:460, 279-460664; CNE-5, chrX:398, 357-398906; CNE-4, chrX:714, 085-714740; CNE-5, chrX:750, 825-751850; ECR1, xhrX:780, 580-781235; and CNE-9/ECS4, chrX:834, 746-835548 (Tel;hg19,build37). C, Splice variants. Different isoforms are generated by the SHOX gene through alternative splicing. Addition of exon 7 can be either attached directly to exon 5 and therefore become a part of the open reading frame or elongates the 3′-UTR of the SHOX transcript. The different 3′-UTR may be subjected to alternative microRNA-mediated regulation. Insertion of exon 2a leads to a premature stop codon in exon 3, which could lead to mRNA degradation. Exon III and IV contain the homeobox. Light gray boxes indicate untranslated regions; dark gray boxes depict open reading frame.

Figure 3.

SHOX at a glance. A, Functional domains. Schematic view of the SHOX protein and its main functional domains. HD, homeodomain; NLS, nuclear localization signal; OAR, transactivation domain; N, N terminus; C, C terminus. B, SHOX interactome. The cellular factors known to interact with SHOX or mediate its cellular functions are depicted as a network.

Figure 4.

SHOX as a regulator of endochondral ossification. A, Expression pattern of cellular factors involved in growth plate regulation interacting with SHOX. A schematic representation of the mouse long bone growth plate at E15.5-E16.5 is displayed. The growth plate is subdivided into different zones that contain chondrocytes at different stages of maturation. Chondrocytes at the end of their differentiation process undergo cell death and are replaced by bone. The expression pattern of FGFR3, BNP, NPR2, NPR3, and RUNX2 is illustrated according to Kozhemyakina et al (82). SHOX is not expressed in mice, but the mouse genome contains the closely related SHOX2 gene. Here we speculate that SHOX2 and BNP in mouse growth plate have similar expression patterns to those found for SHOX and BNP in human growth plate specimens (98, 103). Expression patterns of SHOX/Shox2 target genes are depicted in green (up-regulated) or in red (down-regulated). B, Tentative model illustrating the involvement of SHOX in pathways regulating chondrocyte proliferation and maturation in the growth plate. SHOX-mediated down-regulation of FGFR3 may repress FGFR3 signaling, whereas up-regulation of the NPPB gene may stimulate the CNP/NPR2 pathway. This results in the repression of JAK-STAT and MAPK signaling pathways, which negatively regulate chondrocyte proliferation and maturation, respectively. Note that FGFR3 and CNP/NPR2 signaling pathways converge in the regulation of the levels of activated MAPK, which blocks the initiation of chondrocyte hypertrophy (FGFR3 signaling being an activator and CNP/NPR2 signaling being an inhibitor of the MAPK pathway). It has been shown that through the repression of BMP4 signaling, SHOX2 may regulate the levels of RUNX2, a master regulator of chondrocyte hypertrophy. Although a similar role of SHOX in activating RUNX2 remains to be demonstrated, this is hypothesized in the model, given the high homology and functional redundancy between the two homeodomain proteins. Depicted in green are pathways promoting chondrocyte hypertrophy, whereas in red are those having a negative impact. Plus and minus signs indicate the possible effects in response to SHOX/SHOX2 expression.

Figure 5.

Summary of 230 exonic SHOX mutations. Data were extracted by the SHOX database (www.shox.uni-hd.de). Exon 2 encloses part of the 5′-UTR, and exons 6a and 6b enclose part of the 3′-UTR; exon 1 only encloses 5′ untranslated sequences and is usually not screened in a diagnostic analysis. The homeobox resides in exon 3 and exon 4. The most frequent recurrent mutations are pArg147 in exon 3 (seven times) and pArg195 in exon 5 (10 times). Stops occurring in exons 2 to 6a are frameshift mutations. No stops were identified in exon 6b. Blue indicates unique mutations, and red indicates total mutations in the respective exon/region. Ex, exon; HB, homeobox region.

Figure 6.

Skeletal defects associated with SHOX deficiency. A, Madelung deformity in a patient with LWD. The 19-year-old female with a 46,XX karyotype harbors a paternally inherited heterozygous microdeletion involving the SHOX coding region and the 3′ enhancer region. She shows severe short stature (−3.7 SD) and full pubertal development with regular menses. B, Hypoplasia of the ulna and fibula and severe shortening of the radius and tibia in an individual with Langer mesomelic dysplasia. The 19-month-old girl has a 45,X[191]/46,X,r(X)(p22.3q24)[9] karyotype. The ring X chromosome missing SHOX is formed as a de novo event in the X chromosome of paternal origin, whereas the structurally normal X chromosome harboring a microdeletion involving the SHOX enhancer(s) at the 3′ region is derived from the mother with subtle but SHOX-haploinsufficiency compatible skeletal features. The upper, middle, and lower images represent the roentgenograms of the right arm, the left arm, and the lower legs, respectively. C, Short metacarpal in a patient with TS (lower panel) compared to normal metacarpal (upper panel). Shown in the upper panel is an apparently normal hand roentgenogram of a 14-year and 7-month-old female with 45,X[15]/46,X,idic(X)(p11.2)[15] TS. She has been placed on GH treatment since 8 years and 9 months of age and on sex steroid supplementation therapy since 13 years and 8 months of age. Shown in the lower panel is the hand roentgenogram of a 13-year-old female with 45,X TS, showing a short fourth metacarpal associated with premature fusion of the growth plate. D, Radial bowing with decreased carpal angle. Forearm roentgenograms in an 11-year and 6-month-old girl (proband) with apparent ISS and her parents. The proband has a 46,XX karyotype and a paternally derived microdeletion affecting the SHOX 3′ enhancer region. She exhibits mild mesomelic short stature (−2.3 SD) and Tanner 3 breast development. Radial bowing, epiphyseal hypoplasia of the medial side of the distal radius, and decreased carpal angle are observed. The father with the same microdeletion shows mildly decreased carpal angle as the sole recognizable abnormality. His height remains within the normal range (−1.9 SD). This indicates that SHOX haploinsufficiency can permit an apparently normal phenotype as well as an ISS phenotype. The mother is free from discernible genetic and clinical abnormality.

Figure 7.

Radiological indications for SHOX deficiency. Hand roentgenograms obtained at 8 years and 2 months of age (A and B) and at 16 years of age (C) in patient 1 and those obtained at 11 years and 9 months of age (D–F) in patient 2. Patient 1 has a 46,XY karyotype and a de novo microdeletion encompassing the SHOX coding and enhancer regions. Patient 2 has a 46,XX karyotype and a paternally derived microdeletion involving the SHOX-downstream enhancer region. Both patients show metaphyseal lucency of the medial side of the distal radius (arrows), epiphyseal hypoplasia of the medial side of the distal radius (arrowheads), and decreased carpal angle.