Long-range conserved non-coding SHOX sequences regulate expression in developing chicken limb and are associated with short stature phenotypes in human patients

Abstract

Defects in long-range regulatory elements have recently emerged as previously underestimated factors in the genesis of human congenital disorders. Léri-Weill dyschondrosteosis is a dominant skeletal malformation syndrome caused by mutations in the short stature homeobox gene SHOX. We have analysed four families with Léri-Weill dyschondrosteosis with deletions in the pseudoautosomal region but still with an intact SHOX coding region. Using fluorescence in situ hybridization and single nucleotide polymorphism studies, we identified an interval of approximately 200 kb that was deleted in all tested affected family members but retained in the unaffected members and in 100 control individuals. Comparative genomic analysis of this interval revealed eight highly conserved non-genic elements between 48 and 215 kb downstream of the SHOX gene. As mice do not have a Shox gene, we analysed the enhancer potential in chicken embryos using a green fluorescent protein reporter construct driven by the beta-globin promoter, by in ovo electroporation of the limb bud. We observed cis-regulatory activity in three of the eight non-genic elements in the developing limbs arguing for an extensive control region of this gene. These findings are consistent with the idea that the deleted region in the affected families contains several distinct elements that regulate Shox expression in the developing limb. Furthermore, the deletion of these elements in humans generates a phenotype apparently undistinguishable to those patients identified with mutations in the SHOX coding region and, for the first time, demonstrates the potential of an in vivo assay in chicken to monitor putative enhancer activity in relation to human disease.

Identification and characterization of cryptic SHOX intragenic deletions in three Japanese patients with Léri–Weill dyschondrosteosis

“…Furthermore, while a short segment common to distal and proximal breakpoint sequences was identified at the deletion junction in case 1, the segment appears to be too short to permit an aberrant recombination, and NHEJ associated with such a tiny overlapping segment has been reported previously (Kozak et al 2006). In addition, the scattered distribution of the microdeletion breakpoints around SHOX (Kosho et al 1999;Benito-Sanz et al 2005, 2006aFukami et al 2006;Huber et al 2006;Sabherwal et al 2007) may primarily reflect genomic rearrangements caused by NHEJ, and NHEJ may be facilitated by the high recombination frequency in the PAR1 and by the abundant presence of repeat sequences (e.g., Alu elements) (Shaw et al 2004;Blaschke and Rappold 2006). Collectively, the present study implies that the microdeletions affecting SHOX can be caused by both homologous and nonhomologous rearrangements.…”

“…It is caused by haploinsufficiency of the short-stature homeobox-containing gene (SHOX) on the short arm pseudoautosomal region (PAR1) of the human sex chromosomes (Ogata 2002;Blaschke and Rappold 2006). To date, extensive studies have been performed, identifying multiple intragenic mutations (Niesler et al 2007) and various submicroscopic deletions encompassing the entire SHOX coding region and/or the putative downstream enhancer region(s) (Kosho et al 1999;Ogata 2002;BenitoSanz et al 2005BenitoSanz et al , 2006aFukami et al 2006;Huber et al 2006;Sabherwal et al 2007). Submicroscopic deletions are more frequent than intragenic mutations (Ogata 2002), and this would be consistent with repeat sequences being abundantly present around SHOX, because aberrant intrachromosomal or interchromosomal recombinations are prone to occur between such sequences (Ogata 2002;Blaschke and Rappold 2006).…”

Identification and characterization of new long conserved noncoding sequences in vertebrates

“…Even intronic LCNS are often separated from neighboring coding exons by more than 10 kb. As yet, we cannot explain why they are separated from coding sequences, but the distance may be important for their mechanism of action, such as long-range regulation of gene expression (Kleinjan and van Heyningen 2005;Loots et al 2000Loots et al , 2005Masuya et al 2007;Nobrega et al 2003;Sabherwal et al 2007). (3) In addition to sequence conservation, the distances and orientations between LCNS and neighboring coding sequences (genes) are also conserved among multiple species, i.e., the syntenic relationship is conserved.…”

“…On the other hand, several lines of genetic evidence have indicated that deletions of CNS can lead to specific phenotypes. For example, patients with Leri-Weill dyschondrosteosis have an intact SHOX coding gene, but a region located downstream of the gene, including the CNS, is deleted (Sabherwal et al 2007). A patient with Van Buchem disease has a deletion of a large noncoding region, including seven CNS, located downstream of the SOST coding gene (Loots et al 2005).…”

“…Indeed, some CNS have tissue-specific enhancer activity (Bailey et al 2006;Nobrega et al 2003;Pennacchio et al 2006;Prabhakar et al 2006;Shin et al 2005;Visel et al 2008;Woolfe et al 2005). CNS have also been associated with the long-range regulation of gene expression (Loots et al 2000;Nobrega et al 2003;Sabherwal et al 2007;Sagai et al 2005). Some studies have even provided genetic evidence that CNS have biological functions; for example, point mutations in a CNS are responsible for mouse and human preaxial polydactyly with mirror-image digit duplications (Masuya et al 2007;Sagai et al 2004).…”