Functional Characterization of Two Mutations Located in the Ligand Binding Domain in the SF1

Abstract

Purpose: Since SF1 gene mutations located in the ligand binding domain are associated with a wide phenotypic spectrum in 46,XY subjects, the functional and structural characterization of these variations is of great interest. The aim of this study is to evaluate the clinical phenotype, hormonal pattern and molecular studies (genetic, functional data and protein structural analysis) in two non-related 46,XY disorder of sex development (DSD) index patients. Methods: Clinical characteristics, genomic DNA sequencing analysis, protein prediction software study and protein structure analysis, and functional characterization of the mutations was carried out. Results: Both index DSD patients showed a similar phenotype, however several affected members of Family 1 showed variable phenotypes. While in Family 1 a previously reported heterozygous missense point mutation (p.Arg313His) was found, in Family 2 a novel heterozygous missense point mutation (p.Ser303Arg) was detected. Both mutations were predicted to be as “probably damaging”. The transcriptional activity of SF1 mutants p.Arg313His and p.Ser303Arg, studied using two different promoters in two cell lines, exhibited significant reductions of transactivation activity. Structural analysis showed differences between both mutants, such as changes in the flexibility of the receptor backbone and in the tertiary structure around the ligand and in the AF-2 domain. Conclusions: One of these ligand binding domain mutations in SF1 showed phenotypic heterogeneity among family members, while both variations showed similarities in prepubertal phenotype, as well as in damage prediction and experimental decreases in transcriptional activity, but marked differences in structural consequence predictions. Finally the present study reinforces the concept of the wide variability in the clinical phenotype in affected 46,XY DSD patients.