Mutations in SOX9, the gene responsible for Campomelic dysplasia and autosomal sex reversal

Campomelic dysplasia (CD) is a skeletal malformation syndrome frequently accompanied by 46,XY sex reversal. A mutation-screening strategy using SSCP was employed to identify mutations in SOX9, the chromosome 17q24 gene responsible for CD and autosomal sex reversal in man. We have screened seven CD patients with no cytologically detectable chromosomal aberrations and two CD patients with chromosome 17 rearrangements for mutations in the entire open reading frame of SOX9. Five different mutations have been identified in six CD patients: two missense mutations in the SOX9 putative DNA binding domain (high mobility group, or HMG, box); three frameshift mutations and a splice-acceptor mutation. An identical frameshift mutation is found in two unrelated 46,XY patients, one exhibiting a male phenotype and the other displaying a female phenotype (XY sex reversal). All mutations found affect a single allele, which is consistent with a dominant mode of inheritance. No mutations were found in the SOX9 open reading frame of two patients with chromosome 17q rearrangements, suggesting that the translocations affect SOX9 expression. These findings are consistent with the hypothesis that CD results from haploinsufficiency of SOX9.     

Rapid sequence evolution of the mammalian sex-determining gene SRY

In mammals, induction of male sex determination requires the Y-chromosome gene SRY. SRY encodes a protein with a central 'high mobility group' domain (HMG box) of about 78 amino acids. HMG boxes are found in a wide variety of proteins that bind to DNA with high affinity but differing degrees of sequence specificity. The human SRY protein binds to linear DNA with sequence specificity and to cruciform DNA structures without sequence specificity. The DNA-binding activity of the SRY protein resides in the HMG box and mutations in this region are associated with sex reversal in XY females. No function has been ascribed to the portions of the SRY protein outside the HMG box. SRY belongs to a family of genes that are related by sequence homology within the DNA-binding domain: the genes most similar to SRY (> 60%) have been named SOX genes (SRY box genes). None of the known SOX genes is homologous to SRY outside the HMG-box region. Although SRY is an important developmental regulator, its sequence is poorly conserved between species apart from the HMG-box domain. Here we investigate the coding sequence of SRY in primates and find that evolution has been rapid in the regions flanking the conserved domain. The high degree of sequence divergence and the frequency of non-synonymous mutations suggest either that the majority of the coding sequence has no functional significance or that directional selection has occurred.     

Mechanism of regulatory target selection by the SOX high-mobility-group domain proteins as revealed by comparison of SOX1/2/3 and SOX9

SOX proteins bind similar DNA motifs through their high-mobility-group (HMG) domains, but their action is highly specific with respect to target genes and cell type. We investigated the mechanism of target selection by comparing SOX1/2/3, which activate delta-crystallin minimal enhancer DC5, with SOX9, which activates Col2a1 minimal enhancer COL2C2. These enhancers depend on both the SOX binding site and the binding site of a putative partner factor. The DC5 site was equally bound and bent by the HMG domains of SOX1/2 and SOX9. The activation domains of these SOX proteins mapped at the distal portions of the C-terminal domains were not cell specific and were independent of the partner factor. Chimeric proteins produced between SOX1 and SOX9 showed that to activate the DC5 enhancer, the C-terminal domain must be that of SOX1, although the HMG domains were replaceable. The SOX2-VP16 fusion protein, in which the activation domain of SOX2 was replaced by that of VP16, activated the DC5 enhancer still in a partner factor-dependent manner. The results argue that the proximal portion of the C-terminal domain of SOX1/2 specifically interacts with the partner factor, and this interaction determines the specificity of the SOX1/2 action. Essentially the same results were obtained in the converse experiments in which COL2C2 activation by SOX9 was analyzed, except that specificity of SOX9-partner factor interaction also involved the SOX9 HMG domain. The highly selective SOX-partner factor interactions presumably stabilize the DNA binding of the SOX proteins and provide the mechanism for regulatory target selection.     

SOX20, a new member of the SOX gene family, is located on chromosome 17p13

SOX genes share a high sequence identity with the HMG box present in the testis determining gene SRY. We have identified a HMG box-like sequence motif on six contiguous cosmids, which cross-hybridize to a SOX9 cDNA probe. A data base search revealed a high similarity of the deduced amino acid sequence to the human SOX12 and the murine Sox16 HMG domains. The cosmids were assigned to chromosome 17p13 by FISH analysis.     

Optimizing form and function with the direct posterior composite resin: a case report

The human testis determining factor (SRY) has been cloned from the Y chromosome. This gene is a dominant inducer of male differentiation. Mutations in the SRY gene result in an XY individual developing as a sex reversed phenotypic female. Sex reversal in humans can also be caused by mutations located in autosomal or X-linked loci. One such sex-reversing locus (SRAI) is associated with the developmental disorder campomelic dysplasia (CD). Both these syndromes were mapped to human chromosome 17q by the identification of balanced reciprocal translocations in five unrelated patients. The translocation breakpoint of one such XY-female CD patient was mapped and the region surrounding it cloned. The closest distal marker used to map the translocation breakpoint was the SOX9 gene. Because of the close proximity of this gene to the breakpoint, it was subjected to mutation analysis in patients without overt chromosome rearrangements. Analysis of DNA from these patients and their parents identified de novo mutations in the SOX9 gene in patients with both autosomal sex reversal and CD. This showed that mutations in the SOX9 gene are responsible for both syndromes.     

SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene

The identification of mutations in the SRY-related SOX9 gene in patients with campomelic dysplasia, a severe skeletal malformation syndrome, and the abundant expression of Sox9 in mouse chondroprogenitor cells and fully differentiated chondrocytes during embryonic development have suggested the hypothesis that SOX9 might play a role in chondrogenesis. Our previous experiments with the gene (Col2a1) for collagen II, an early and abundant marker of chondrocyte differentiation, identified a minimal DNA element in intron 1 which directs chondrocyte-specific expression in transgenic mice. This element is also a strong chondrocyte-specific enhancer in transient transfection experiments. We show here that Col2a1 expression is closely correlated with high levels of SOX9 RNA and protein in chondrocytes. Our experiments indicate that the minimal Col2a1 enhancer is a direct target for Sox9. Indeed, SOX9 binds to a sequence of the minimal Col2a1 enhancer that is essential for activity in chondrocytes, and SOX9 acts as a potent activator of this enhancer in cotransfection experiments in nonchondrocytic cells. Mutations in the enhancer that prevent binding of SOX9 abolish enhancer activity in chondrocytes and suppress enhancer activation by SOX9 in nonchondrocytic cells. Other SOX family members are ineffective. Expression of a truncated SOX9 protein lacking the transactivation domain but retaining DNA-binding activity interferes with enhancer activation by full-length SOX9 in fibroblasts and inhibits enhancer activity in chondrocytes. Our results strongly suggest a model whereby SOX9 is involved in the control of the cell-specific activation of COL2A1 in chondrocytes, an essential component of the differentiation program of these cells. We speculate that in campomelic dysplasia a decrease in SOX9 activity would inhibit production of collagen II, and eventually other cartilage matrix proteins, leading to major skeletal anomalies.     

Deletion of long-range regulatory elements upstream of SOX9 causes campomelic dysplasia

Campomelic dysplasia (CD) is a rare, neonatal human chondrodysplasia characterized by bowing of the long bones and often associated with male-to-female sex-reversal. 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.     

A gene involved in XY sex reversal is located on chromosome 9, distal to marker D9S1779

The genetic mechanisms involved in sex differentiation are poorly understood, and progress in identification of the genes involved has been slow. The fortuitous finding of chromosomal rearrangements in association with a sex-reversed phenotype has led to the isolation of SRY and SOX9, both shown to be involved in the sex-determining pathway. In addition, duplications of the X chromosome, deletions of chromosomes 9 and 10, and translocations involving chromosome 17 have been reported to be associated with abnormal testicular differentiation, leading to male-to-female sex reversal in 46,XY individuals. We present the cytogenetic and molecular analyses of four sex-reversed XY females, each with gonadal dysgenesis and other variable malformations, and with terminal deletions of distal chromosome 9p, resulting from unbalanced autosomal translocations. PCR amplification and DNA sequence analysis of SRY revealed no mutations in the high-mobility-group domain (i.e., HMG box) in any of the four patients. Conventional and molecular cytogenetic analyses of metaphase chromosomes from each patient suggest that the smallest region of overlap (SRO) of deletions involves a very small region of distal band 9p24. Loss-of-heterozygosity studies using 17 highly polymorphic microsatellite markers, as well as FISH using YAC clones corresponding to the most distal markers on 9p, showed that the SRO lies distal to marker D9S1779. These results significantly narrow the putative sex-determining gene to the very terminal region of the short arm of chromosome 9.