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

PEREZ G N; SARACO N; MARINO R; RAMIREZ P; CIACCIO M; CONSTANZO M,; GUERCIO G; WARMAN D M,; MININI L; PORTILLO-LEDESMA S; RIVAROLA M A; COITIÑO EL; BELGOROSKY A

HORMONES-INTERNATIONAL JOURNAL OF ENDOCRINOLOGY AND METABOLISM

HELLENIC ENDOCRINE SOC

Año: 2015

1109-3099

AbstractPurpose: 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 sexdevelopment (DSD) index patients.Methods: Clinical characteristics, genomic DNA sequencing analysis, protein prediction software study and protein structure analysis, andfunctional 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 heterozygousmissense point mutation (p.Ser303Arg) was detected. Both mutations were predicted to be as ?probably damaging?. The transcriptional activityof SF1 mutants p.Arg313His and p.Ser303Arg, studied using two different promoters in two cell lines, exhibited significant reductions oftransactivation activity. Structural analysis showed differences between both mutants, such as changes in the flexibility of the receptor backboneand 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 bothvariations 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 clinicalphenotype in affected 46,XY DSD patients.Keywords: Steroidogenic factor-1; SF1; NR5A1 gene; LBD mutations; 46,XY DSDIntroductionSteroidogenic factor-1 (SF1/AD4BP/FTZF1) plays a key role in theregulation of adrenal and reproductive organs differentiation. The SF1gene (NR5A1) is an autosomal gene located on chromosome 9q33 and it isthe member 1 of the nuclear receptor subfamily 5, group A [1]. It expandsover 30 kb of genomic DNA and it is comprised of one non-coding exon(I) followed by six coding exons (II to VII).The protein consists of 461 amino acids that include a DNA-bindingdomain (DBD) containing two zinc fingers, a hinge region containinga first functional activation domain (AF-1), a ligand-binding domain(LBD) of 12 helices (H1-H12) that includes a second functionalactivation domain (AF-2), and an accessory region [2]. It is expressedin undifferentiated gonads even before SRY and it is necessary for testisdetermination and differentiation. SF1 protein is highly expressed insteroidogenic tissues, such as gonads, adrenals, and placenta, and regulatesalmost all the enzymes related to this process. It is also expressed in theventromedial hypothalamic nucleus and pituitary gonadotropes withrelevant physiological roles in the central nervous system [3].Homozygous null mice (Nr5a1−/−) have adrenal and gonadal agenesis,persistent Müllerian structures in XY karyotype, partial hypogonadotropichypogonadism, and other features such as hypospl