Human NR5A1/SF-1 mutations show decreased activity on BDNF (brain-derived neurotrophic factor), an important regulator of energy balance: testing impact of novel SF-1 mutations beyond steroidogenesis

CONTEXT Human NR5A1/SF-1 mutations cause 46,XY disorder of sex development (DSD) with broad phenotypic variability, and rarely cause adrenal insufficiency although SF-1 is an important transcription factor for many genes involved in steroidogenesis. In addition, the Sf-1 knockout mouse develops obesity with age. Obesity might be mediated through Sf-1 regulating activity of brain-derived neurotrophic factor (BDNF), an important regulator of energy balance in the ventromedial hypothalamus. OBJECTIVE To characterize novel SF-1 gene variants in 4 families, clinical, genetic and functional studies were performed with respect to steroidogenesis and energy balance. PATIENTS 5 patients with 46,XY DSD were found to harbor NR5A1/SF-1 mutations including 2 novel variations. One patient harboring a novel mutation also suffered from adrenal insufficiency. METHODS SF-1 mutations were studied in cell systems (HEK293, JEG3) for impact on transcription of genes involved in steroidogenesis (CYP11A1, CYP17A1, HSD3B2) and in energy balance (BDNF). BDNF regulation by SF-1 was studied by promoter assays (JEG3). RESULTS Two novel NR5A1/SF-1 mutations (Glu7Stop, His408Profs*159) were confirmed. Glu7Stop is the 4th reported SF-1 mutation causing DSD and adrenal insufficiency. In vitro studies revealed that transcription of the BDNF gene is regulated by SF-1, and that mutant SF-1 decreased BDNF promoter activation (similar to steroid enzyme promoters). However, clinical data from 16 subjects carrying SF-1 mutations showed normal birth weight and BMI. CONCLUSIONS Glu7Stop and His408Profs*159 are novel SF-1 mutations identified in patients with 46,XY DSD and adrenal insufficiency (Glu7Stop). In vitro, SF-1 mutations affect not only steroidogenesis but also transcription of BDNF which is involved in energy balance. However, in contrast to mice, consequences on weight were not found in humans with SF-1 mutations

Genetic information on 5 subjects carrying <i>NR5A1</i> mutations.

<p>A. Family trees of 4 families and 7 affected individuals (5 patients and 2 parents) are shown. Scheme of the <i>NR5A1</i> gene showing the mutations identified in the reported patients (above the scheme) and all reported SF-1 mutations causing adrenal insufficiency (below the scheme). Electropherograms of novel mutations are also depicted. The <i>NR5A1</i> gene is composed of coding (<i>black</i>) and non-coding sequences (<i>gray</i>). Exons are indicated by <i>numbers</i>.</p

Weight related parameters of subjects carrying (heterozygote) <i>NR5A1</i> mutations.

<p>F: family; Fa: father; Mo: mother.</p

Promoter reporter studies for SF-1 regulating the human <i>BDNF</i> promoter I and comparative studies of specific <i>NR5A1</i> mutations on <i>BDNF</i> promoter activity.

<p>A consensus SF-1 transcription binding site was identified in the wild-type (WT) human BDNF promoter I. This <i>cis</i>-element was mutated to assess the role of SF-1 on BDNF transcription. Human placental JEG3 cells were transiently transfected with the WT or the SF-1 element mutant (Mt) BDNF promoter reporter with or without SF-1, and promoter activity was assessed by the Promega Dual Luciferase assay (A). After showing that SF-1 regulates the WT BDNF promoter I specifically (A), the ability of specific SF-1 mutations to <i>trans</i>-activate the <i>BDNF</i> promoter I was investigated (B). Results are expressed as percentage of WT SF-1 activity. Independent experiments were performed in duplicate 3 times. Error bars represent the mean and SEM. *<i>p</i><0.05; **<i>p</i><0.01.</p

Promoter reporter studies for reported <i>NR5A1</i> mutations

<p>. Human embryonic kidney HEK293 cells were transiently transfected with wild-type (WT) or mutant SF-1 and promoter luciferase reporter constructs of the genes for steroidogenic enzymes <i>HSD3B2</i> (A), <i>CYP11A1</i> (B), <i>CYP17A1</i> (C). Luciferase activity was measured with the Promega Dual Luciferase assay system. Results are expressed as percentage of WT SF-1 activity. Independent experiments were performed in duplicate at least 3 times. Error bars represent the mean and SEM. *<i>p</i><0.05; **<i>p</i><0.01.</p

Clinical and genetic characteristics of 5 patients harboring mutations in the <i>NR5A1</i> gene.

<p>YOB: year of birth; F: father; M: mother; WT: wild type; L: left; R: right; ND – not determined.</p