Lé ri-Weill Dyschondrosteosis (LWD) is a dominant skeletal disorder characterized by short stature and distinct bone anomalies. SHOX gene mutations and deletions of regulatory elements downstream of SHOX resulting in haploinsufficiency have been found in patients with LWD. SHOX encodes a homeodomain transcription factor and is known to be expressed in the developing limb. We have now analyzed the regulatory significance of the region upstream of the SHOX gene. By comparative genomic analyses, we identified several conserved non-coding elements, which subsequently were tested in an in ovo enhancer assay in both chicken limb bud and cornea, where SHOX is also expressed. In this assay, we found three enhancers to be active in the developing chicken limb, but none were functional in the developing cornea. A screening of 60 LWD patients with an intact SHOX coding and downstream region did not yield any deletion of the upstream enhancer region. Thus, we speculate that SHOX upstream deletions occur at a lower frequency because of the structural organization of this genomic region and/or that SHOX upstream deletions may cause a phenotype that differs from the one observed in LWD.

The short stature homeobox gene SHOX encodes a transcription factor which is important for normal limb development. In humans, SHOX deficiency has been associated with various short stature syndromes including Leri-Weill dyschondrosteosis (LWD), Langer mesomelic dysplasia and Turner syndrome as well as non-syndromic idiopathic short stature. A common feature of these syndromes is disproportionate short stature with a particular shortening of the forearms and lower legs. In our studies employing microarray analyses and cell culture experiments, we revealed a strong positive effect of SHOX on the expression of the fibroblast growth factor receptor gene FGFR3, another well-known factor for limb development. Luciferase reporter gene assays show that SHOX activates the extended FGFR3 promoter, and results from chromatin immunoprecipitation (ChIP)-sequencing, ChIP and electrophoretic mobility shift assay experiments suggest a direct binding of SHOX to multiple upstream sequences of FGFR3. To further investigate these regulations in a cellular system for limb development, the effect of viral overexpression of Shox in limb bud derived chicken micromass cultures was tested. We found that Fgfr3 was negatively regulated by Shox, as demonstrated by quantitative real-time polymerase chain reaction and in situ hybridization. This repressive effect might explain the almost mutually exclusive expression patterns of Fgfr3 and Shox in embryonic chicken limbs. A negative regulation that occurs mainly in the mesomelic segments, a region where SHOX is known to be strongly expressed, offers a possible explanation for the phenotypes seen in patients with FGFR3 (e.g. achondroplasia) and SHOX defects (e.g. LWD). In summary, these data present a link between two frequent short stature phenotypes.

Height is a classic polygenic quantitative trait with a high level of heritability. As it is a simple and stable parameter to measure, height is a model for both common, complex disorders and monogenic, Mendelian disease. In this Review, we examine height from the perspective of monogenic and complex genetics and discuss the lessons learned so far. We explore several examples of rare sequence variants with large effects on height and compare these variants to the common variants identified in genome-wide association studies that have small effects on height. We discuss how copy number changes or genetic interactions might contribute to the unidentified aspects of the heritability of height. We also ask whether information derived from genome-wide association studies on specific loci in the vicinity of genes can be used for further research in clinical paediatric endocrinology. Furthermore, we address key challenges that remain for gene discovery and for the transition of moving from genomic localization to mechanistic insights, with an emphasis on using next-generation sequencing to identify causative variants of people at the extremes of height distribution.

The human SHOX gene is composed of seven exons and encodes a paired-related homeodomain transcription factor. SHOX mutations or deletions have been associated with different short stature syndromes implying a role in growth and bone formation. During development, SHOX is expressed in a highly specific spatiotemporal expression pattern, the underlying regulatory mechanisms of which remain largely unknown. We have analysed SHOX expression in diverse embryonic, fetal and adult human tissues and detected expression in many tissues that were not known to express SHOX before, e.g. distinct brain regions. By using RT-PCR and comparing the results with RNA-Seq data, we have identified four novel exons (exon 2a, 7-1, 7-2 and 7-3) contributing to different SHOX isoforms, and also established an expression profile for the emerging new SHOX isoforms. Interestingly, we found the exon 7 variants to be exclusively expressed in fetal neural tissues, which could argue for a specific role of these variants during brain development. A bioinformatical analysis of the three novel 3′UTR exons yielded insights into the putative role of the different 3′UTRs as targets for miRNA binding. Functional analysis revealed that inclusion of exon 2a leads to nonsense-mediated RNA decay altering SHOX expression in a tissue and time specific manner. In conclusion, SHOX expression is regulated by different mechanisms and alternative splicing coupled with nonsense-mediated RNA decay constitutes a further component that can be used to fine tune the SHOX expression level.

The SHOX gene encodes for a transcription factor important for normal bone development. Mutations in the gene are associated with idiopathic short stature and are responsible for the growth failure and skeletal defects found in the majority of patients with Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia. SHOX is expressed in growth plate chondrocytes where it is supposed to modulate the proliferation, differentiation and cell death of these cells. Supporting this hypothesis, in vitro studies have shown that SHOX expression induces cell cycle arrest and apoptosis in both transformed and primary cells. In this study, we further characterized the cell death mechanisms triggered by SHOX and compared them with the effects induced by one clinically relevant mutant form of SHOX, detected in LWD patients (SHOX R153L) and a SHOX C-terminally truncated version (L185X). We show that SHOX expression in U2OS osteosarcoma cells leads to oxidative stress that, in turn, induces lysosomal membrane rupture with release of active cathepsin B to the cytosol and subsequent activation of the intrinsic apoptotic pathway characterized by mitochondrial membrane permeabilization and caspase activation. Importantly, cells expressing SHOX R153L or L185X did not display any of these features. Given the fact that many of the events observed in SHOX-expressing cells also characterize the complex cell death process occurring in the growth plate during endochondral ossification, our findings further support the hypothesis that SHOX may play a central role in the regulation of the cell death pathways activated during long bone development.