No abstract

Campomelic dysplasia (CD) is a rare, neonatal human chondrodysplasia characterized by bowing of the long bones and often associated with male-to-female sexreversal. Patients present with either heterozygous mutations in the SOX9 gene or chromosome rearrangements mapping at least 50 kb upstream of SOX9. Whereas mutations in SOX9 ORF cause haploinsufficiency, the effects of translocations 5 to SOX9 are unclear. To test whether these rearrangements also cause haploinsufficiency by altering spatial and temporal expression of SOX9, we generated mice transgenic for human SOX9-lacZ yeast artificial chromosomes containing variable amounts of DNA sequences upstream of SOX9. We show that elements necessary for SOX9 expression during skeletal development are highly conserved between mouse and human and reveal that a rearrangement upstream of SOX9, similar to those observed in CD patients, leads to a substantial reduction of SOX9 expression, particularly in chondrogenic tissues. These data demonstrate that important regulatory elements are scattered over a large region upstream of SOX9 and explain how particular aspects of the CD phenotype are caused by chromosomal rearrangements 5 to SOX9.Major diagnostic criteria for the skeletal malformation syndrome, campomelic dysplasia (CD), are angulation of the tibiae and femura, hypoplastic scapulae, nonmineralization of the thoracic pedicles, 11 instead of 12 pairs of ribs, poor ossification of the pelvis, and bilateral talipes equinovaris (1-3). Other skeletal and nonskeletal defects are also associated with the disease, such as micrognathia, cleft palate, and low-set ears. Patients usually die soon after birth of respiratory distress, but the severity of the disease is variable and a few patients survive into adult life. Interestingly, male-to-female sex-reversal occurs in three-quarters of the XY CD patients, whose genitalia can be normal male, female, or ambiguous with various levels of male or female sexual differentiation (1, 3-5). The identification of de novo mutations in the SOX9 gene of sex-reversed CD patients implicated SOX9 as responsible for both skeletal and gonadal phenotypes. Only heterozygous mutations were detected in the patients, suggesting that CD has an autosomal dominant inheritance and haploinsufficiency is the probable cause of the CD and sex-reversal phenotypes (6, 7).The human SOX9 gene maps to chromosome 17q24 (6, 7) and belongs to the SRY-related HMG box (SOX) gene family (8,9). SOX genes encode proteins with greater than 60% similarity at the amino acid level with the SRY DNA-binding domain or HMG box. The SOX genes have been isolated from a variety of organisms, and their role as transcription factors during embryogenesis has been suggested (9-12). SOX9 encodes a putative 509 amino acid protein that contains an HMG box sharing 71% similarity with SRY HMG box and a transactivation domain at the C terminus, suggesting that SOX9 acts as a transcription activator (13,14). Mouse Sox9 gene shares 96% identity with its human homologue (15), indicat...