Abstract

Introduction: Sexual differentiation involves numerous genetic factors, including NR5A1 (Nuclear Receptor Subfamily 5 group A member 1), also known as SF1 (Steroidogenic Factor 1) or A4BP (Adrenal 4 Binding Protein), which is expressed very early in the undifferentiated gonad. Variants of this gene can cause Disorders of Sexual Development (DSD). The aim of this study was to identify variants of the NR5A1 gene in a cohort of patients with DSD. Materials and Methods: Ten patients registered as female at birth were selected from those referred for genetic tests to diagnose a sexual anomaly. After obtaining informed consent, we performed DNA (Deoxyribonucleic Acid) extraction in EDTA tubes, followed by polymerase chain reaction (PCR) amplification and Sanger sequencing of exon 4 of the NR5A1 gene. Results: The entire cohort (100%) presented at least one variant in exon 4 of the NR5A1 gene. In total, seven (7) different positions within this exon exhibited variants, including the c.437G>C (p.Gly146Ala) variant, which was present in the entire cohort (100%). Conclusion: For the first time in Senegal, variants of the NR5A1 gene have been identified in patients with disorders of sexual development. The c.437G>C (p.Gly146Ala) variant can be found in a variety of phenotypes.

Keywords

NR5A1, Disorders of Sex Development, Sanger Sequencing, pGly146Ala

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1. Introduction

Disorders of Sex Development (DSD) are congenital conditions in which chromosomal, gonadal, and anatomical sex are atypical [1]-[5]. Genetic sex (primary sex differentiation) induces the determination of gonadal sex (ovaries or testes), which itself determines phenotypic sex (secondary sex differentiation), defined by internal genital tracts and external genitalia [6]. During the undifferentiated phase, many genes are expressed in both sexes at a low and similar level [7]-[10]. Thus, they are all necessary for the establishment of the gonad regardless of sex. NR5A1 (SF1, A4BP), WT1 (Wilms Tumor suppressor gene 1), WNT4 (Wingless-type gene 4) are expressed in the urogenital ridge and play a role in the formation of gonads, kidneys, and adrenal cortex [11]-[16]. NR5A1, located at 9q33, encodes the transcription factor steroidogenic factor-1 (SF-1), which is involved in gonad development as well as adrenal gland development [17]. In 2001, De Santa Barbara et al. showed that NR5A1 is expressed in the genital ridge and then in somatic cells of the undifferentiated gonad before SRY (Sex-Determining Region of Y Chromosome) expression [18]. It has also been shown to participate in the regulation of SRY expression during male gonad differentiation. Once the gonad is differentiated, its expression persists in Sertoli cells and Leydig cells during testicular development. NR5A1 is secondarily involved in the development of the genital tract, as it regulates AMH levels [19] in association with other genes involved in gonad development, such as WT1 and SOX9 (SRY Homebox-Like Gene 9) [20]. The objective of this study was to search for variants of the NR5A1 gene in a cohort of patients with disorders of sex development.

2. Materials and Methods

Ten (10) patients were selected from those referred for disorders of sex development (21, 59, 72, 73, 75, 95, 99, 119, AD, AN). In addition, two controls were included in the study: a male control with normal sex differentiation and fertility (T.H) and a female control with normal sex differentiation and fertility (T.F).

After informed consent, we proceeded to collect blood in an EDTA tube for DNA extraction using the “Quick DNA Miniprep” kit protocol. Polymerase chain reaction (PCR) amplification was then performed using specific forward and reverse primers (Table 1).

Table 1. NR5A1 exon 4 primers used.

We focused on exon 4 because, during the period of our study, the majority of variants were listed in this region (Figure 1).

Figure 1. NCBI cited variations on different NR5A1 exons.

The PCR mix for a sufficient quantity for 25 µL contained: 1 µL of primer (forward + reverse) + 12.5 µL of Master Mix + 9.5 µL of milliQ water + 2 µL of DNA.

The amplification program for the NR5A1 gene exon 4 was as follows: 95˚C (5 min), 95˚C (30 s), 58˚C (30 sec), 72˚C (1 min), 72˚C (10 min).

The quality of amplification was verified by migration on a 1.5% agarose gel.

The PCR products were then sequenced according to the Sanger method.

The obtained sequences were processed using the following software: Mutation Surveyor, DNA-Baser, Chromaspro, Geneious, Mega X, BioEdit, and Vision Pro. For the search for variants and their pathogenicity, databases such as NCBI, UCSC, HGVS, ClinVar, dbSNP, Ensembl, OMIM, Orphanet, and GeneCards were used.

3. Results

The 10 patients were registered as female in the civil registry.

The age ranged from 4 to 32 years.

Clinical and paraclinical parameters were collected (Table 2).

Table 2. Parameters collected.

Table 3. NR5A1 exon 4 variants.

After sequencing of exon 4 of the NR5A1 gene, the entire cohort (100%) presented at least one variant at this exon 4, including the controls (T.H and T.F). (Table 3).

In total, seven (7) different positions of this exon showed variants, among which the variant c.437G>C (p.Gly146Ala) was shared by the entire cohort (Figure 2).

Figure 2. Chromatogram of the variant c.437G>C (p.Gly146Ala).

4. Discussion

NR5A1 variations are among the most frequently identified genetic causes of gonadal development disorders and are associated with a wide phenotypic spectrum [21].

Loss of function of the NR5A1 gene causes several different phenotypes, including some associated with disease in additional organs [22].

Indeed, all individuals in our cohort presented at least one variant of this gene.

100% of individuals presented the same variant position, g.11898, leading to a change in the produced protein p.Gly146Ala. This variant was also found in controls, raising questions about its pathogenicity.

According to Martinez de Lapiscinaet al. [23], NR5A1/SF-1 variants can lead to mild to severe DSD or can be found in healthy carriers (as the controls in our study).

The NR5A1/SF-1 c.437G>C (p.Gly146Ala) variant is frequent in individuals with DSD and has been suggested to act as a susceptibility factor for adrenal disease [23] (like the patient 72) or cryptorchidism.

At position p11898, two (2) patients (75 and AD) and the female control (TF) were heterozygous, and their phenotype was less ambiguous (breast development stage 5 of Tanner); moreover, these individuals are the only ones to present only one variant on this exon 4. Thus, Martinez de Lapiscina et al. state that given the high allele frequency in the general population and the non-conclusive functional tests of the p.Gly146Ala variant, the pathogenic effect of this allele variant has not been judged satisfactory [23].

In addition to the c.437G>C (p.Gly146Ala) variant, 50% of individuals (59, 73, 95, 119, AN) shared another variant position g11836, leading to a synonymous variant where the produced protein remains Proline (p.Pro125=), however, only patient AN, who was found to have an ovotestis, was homozygous (p.11.836G>A), the others were heterozygous (p.11836G>R).

It would appear that the phenotype varies depending on whether the karyotype is 46,XX or 46,XY. According to Dominice et al., in 46,XY individuals, NR5A1-related phenotypes can range from disorders of sex development (DSD) to oligo/azoospermia, and in 46,XX individuals, from 46,XX ovotesticular and testicular DSD to primary ovarian insufficiency (POI) [21]. In an international study in 2024, Kouri et al. confirmed the phenotype’s diversity among 197 individuals with NR5A1/SF-1 variants, and found that over 70% of 46,XY individuals had a severe DSD phenotype, while 90% of 46,XX individuals had female-typical sex development [24].

The most common 46,XY phenotype is the presence of atypical or female external genitalia with clitoromegaly, palpable gonad, and absence of Müllerian derivatives [20]. Notably, undervirilization of external genitalia is frequently observed at birth, while spontaneous virilization may occur later, at puberty: this is the case of patient 59, who has 3 variants observed at different positions compared to the reference genome.

In 46,XX individuals, NR5A1 mutations are a rare genetic cause of POI, manifesting as primary or secondary amenorrhea, as evidenced by almost the entire cohort (patients 72, 75, 95, 99, 119, AN, AD), infertility, hypoestrogenism, and elevated gonadotropin levels.

Indeed, it is suggested that NR5A1 gene mutations could be mainly associated with amenorrhea, ovarian failure, hypogonadism, and infertility during puberty [21].

Furthermore, as mentioned earlier, NR5A1 is involved in the formation of the adrenal gland, and its variants may explain the condition of patient 72, who has been experiencing congenital adrenal hyperplasia since birth and carries a specific variant in the cohort (g.12055G>R). This variant is synonymous with p.Pro198=, considered benign or potentially causing 46,XY or infertility in ClinVar. Patient 21, who had a naked glans surrounded by asymmetric labioscrotal structures and hypospadias, is the only one to have 4 variants on this exon, she carries an isolated variant (g.11847C>Y) which the homozugous form can lead to a missense variant (p.Pro129Leu), and reported as a cause of primary ovarian insufficiency (POI) or infertility in clinVar. She also carries another variant at position g.11977C>Y, leading to a synonymous variant (p.Ala172Ala), which she shared with the patient 73, who had a similar phenotype, and patient 119.

Thus, as our results demonstrate and Faienza et al. [25] affirm, the data confirm that NR5A1 gene mutations can present variable genital phenotypes. Regardless, the reproductive function has always been altered.

According to Luppino et al., Clinical phenotypes may vary, even among patients carrying the same NR5A1 variant, indicating that there is no specific genotype-phenotype correlation [22].

Naamneh Elzenaty et al. hypothesized that the broad phenotype of DSD associated with NR5A1/SF-1 variants may be caused by an oligogenic mechanism [26].

The observed phenotypic variability in individuals and families with NR5A1/SF-1 variants is large and remains unpredictable. It may often not be solely explained by the monogenic pathogenicity of the NR5A1/SF-1 variants but is likely influenced by additional genetic variants and as yet unknown factors.

5. Conclusion

Our study is the first in our sub-region to investigate NR5A1 gene mutations in patients with disorders of sex development. This gene is involved early in sex differentiation, and its variants can explain these conditions. In this study, we focused on exon 4, which suggests the most pathogenic variants, and found 7 different variant positions. As suggested by our patients’ conditions, NR5A1 gene mutations can present variable genital phenotypes.

Ethical Statement

This study was approved by the Doctoral School of Sciences of Life, Health, and Environment (ED/SEV) of Cheikh Anta Diop University and was conducted as part of a Ph.D. thesis (No. 202348, 2023). All procedures were conducted in accordance with the Declaration of Helsinki and adhered to national and institutional ethical guidelines. Written informed consent was obtained from all participants (regarding minors, parental consent was required) after a detailed explanation of the study objectives, procedures, and potential risks.

NOTES

*Corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

References