Structural and functional analysis of SOX9 mutations in disorders of sex development (DSD): Integration of clinical data and in silico modeling

Abstract

The SOX9 gene, located on chromosome 17q24, belongs to the SOX family of transcription factors and shares over 70 % homology with SRY. It plays a central role in testis differentiation and cartilage formation, notably by regulating key genes such as AMH. Mutations in SOX9 are known to cause Disorders of Sex Development (DSD) and skeletal malformations such as campomelic dysplasia.

Objective

This study aimed to analyze the structural and functional impact of SOX9 mutations identified in DSD patients, using in silico predictive tools including IntFOLD, to evaluate changes in protein conformation and correlate them with observed phenotypes.

Methods

Twenty-eight patients with DSD (46,XX or 46,XY karyotypes) were enrolled. The SRY and SOX9 genes were amplified by PCR and sequenced. Four 46,XX patients were found to be SRY-positive, and two 46,XY patients were SRY-negative. Ten SOX9 variants were identified in 12 patients, including two novel intronic variants, two in the 3′UTR region, three synonymous, and three non-synonymous coding variants. All variants were found in the heterozygous state, and the presence of a normal allele was used to assess its functional implications.

Results

Non-synonymous mutations located within the HMG and dimerization domains were predicted to be deleterious. 3D modeling using IntFOLD revealed conformational changes, altered protein stability, and disrupted ligand-binding residues. These structural alterations correlated with the DSD phenotypes observed. The overall SOX9 structure showed a largely disordered organization, with ordered segments within key functional domains.

Conclusion

Our findings confirm the role of SOX9 in the etiology of DSD and highlight the relevance of structural modeling for interpreting rare variants. The integration of clinical, genetic, and in silico data contributes to a better understanding of sex differentiation mechanisms and may support improved molecular diagnosis of DSD.