RESUMEN
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.
Asunto(s)
Anomalías Múltiples/genética , Secuencia Conservada/genética , ADN Intergénico/genética , Regulación de la Expresión Génica , Miembro Posterior/metabolismo , Proteínas de Homeodominio/genética , Osteocondrodisplasias/genética , Eliminación de Secuencia/genética , Adolescente , Adulto , Anciano , Animales , Secuencia de Bases , Estatura/genética , Embrión de Pollo , Niño , Mapeo Cromosómico , Análisis Mutacional de ADN , Cartilla de ADN , Electroporación , Femenino , Componentes del Gen , Genómica/métodos , Miembro Posterior/embriología , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , Polimorfismo de Nucleótido Simple/genética , Proteína de la Caja Homeótica de Baja Estatura , SíndromeRESUMEN
Human growth is influenced not only by environmental and internal factors but also by a large number of different genes. One of these genes, SHOX, is believed to play a major role in growth, since defects in this homeobox-containing gene on the sex chromosomes lead to syndromal short stature (Leri-Weill dyschondrosteosis, Langer mesomelic dysplasia, and Turner syndrome) as well as to idiopathic short stature. We have analyzed 118 unrelated patients with Leri-Weill dyschondrosteosis and >1,500 patients with idiopathic short stature for deletions encompassing SHOX. Deletions were detected in 34% of the patients with Leri-Weill dyschondrosteosis and in 2% of the patients with idiopathic short stature. For 27 patients with Leri-Weill dyschondrosteosis and for 6 with idiopathic short stature, detailed deletion mapping was performed. Analysis was performed by polymerase chain reaction with the use of pseudoautosomal polymorphic markers and by fluorescence in situ hybridization with the use of cosmid clones. Here, we show that, although the identified deletions vary in size, the vast majority (73%) of patients tested share a distinct proximal deletion breakpoint. We propose that the sequence present within this proximal deletion breakpoint "hotspot" region predisposes to recurrent breaks.