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Introduction

Disorders of sex development (DSDs) represent a broad and heterogeneous group of congenital conditions, characterized by discordance between chromosomal, gonadal and anatomical sex (1). The clinical presentation of DSDs is highly variable, encompassing numerous conditions, such as hypospadias, ambiguous genitalia and complete sex reversal in 46, XX or 46, XY individuals (2). In 2006, the Chicago Consensus redefined DSD classifications into three major categories based on karyotype; namely, sex chromosome DSDs, 46, XY DSDs and 46, XX DSDs (3). Among these, 46, XY DSDs exhibit the greatest level of complexity, often involving atypical female genitalia, incomplete intrauterine masculinization and the absence of Müllerian structures (4,5). Androgen receptor dysfunction remains the most prevalent etiology in these cases. The psychological, physical and reproductive consequences of DSDs are profound, with patients facing an elevated risk of sex cord-stromal tumors, such as gonadoblastoma and experiencing considerable social and medical burdens (6).

The clinical heterogeneity of DSDs complicates the accuracy of diagnosis based solely on phenotypic assessments (6). Genetic factors underlying DSD pathogenesis remain to be elucidated, necessitating molecular diagnostics to complement clinical evaluations. The mitogen-activated protein 3 kinase 1 (MAP3K1) gene plays a crucial role in the genetic network associated with gonadal development (7). MAP3K1 mediates sex differentiation through modulating the balance between the pro-testicular SOX9/FGF9 pathway and the pro-ovarian WNT/β-catenin pathway (4,8,9). Variants in MAP3K1 have been identified in large families with 46, XY DSD, exhibiting an autosomal dominant, sex-limited mode of inheritance (7). In addition, MAP3K1 variants have been detected in 18% of sporadic cases of 46, XY gonadal dysgenesis, with phenotypic manifestations ranging from complete gonadal dysgenesis to milder presentations, such as hypospadias, micropenis and cryptorchidism (4,5). Results of a previous study demonstrated that gain-of-function variants in MAP3K1 enhance the phosphorylation of downstream targets, leading to reduced SOX9 expression levels and increased β-catenin levels (7). The shift in signaling pathways results in the disruption of normal testicular development, leading to various degrees of gonadal dysgenesis.

Gain-of-function variants in MAP3K1 include p.L189P, p.L189R and p.K246E and splice-site variants include c.634-8T>A and c.2180-2A>G. Notably, the aforementioned variants may enhance WNT/β-catenin signaling and suppress the testis-promoting pathway driven by SOX9 (7,8,10). These variants also increase phosphorylation events that skew the signaling cascade towards ovarian development in 46, XY individuals, leading to gonadal dysgenesis with partial or complete female characteristics. In addition, the existence of novel variants, such as c.3020A>G and c.2117T>G, highlight the role of MAP3K1 variants in human sex differentiation (5,11).

Results of previous studies reveal that Map3k1 null mutant mouse embryonic stem (ES) cells exhibit increased apoptosis under hyperosmotic stress, low-temperature shock and microtubule disruption (12,13). Moreover, cardiac myocytes derived from Map3k1-mutant ES cells display heightened susceptibility to oxidative stress-induced apoptosis (14), underscoring the critical role of Map3k1 in protecting mammalian cells from cell death. However, whether MAP3K1 variants contribute to apoptosis in patients with DSD remains unclear. Previous studies have reported increased DNA damage in individuals with DSD (15) and elevated DNA damage and chronic inflammation in male patients with idiopathic germ cell aplasia (16). Collectively, these results highlight the potential association between DNA damage and gonadal disorders. Notably, excessive DNA damage triggers apoptosis in various cell types, including germ cells (17). Thus, the present study hypothesized that increased DNA damage leads to abnormal apoptosis, ultimately contributing to the clinical phenotype observed in patients with DSD.

In the present study, a 3-year-old male presenting with an abnormal urethral opening was found to have a recurved penis with the urethral opening located at the penile-scrotal junction. Family history suggested a potential familial pattern of inheritance, and cytogenetic analysis revealed a 46, XY karyotype. Genetic analysis was conducted to identify potential pathogenic variants, followed by in vitro experiments to explore the underlying regulatory mechanisms. These findings contribute to a deeper understanding of the pathogenesis of 46, XY DSD.

Materials and methods

Patients

A family with 46, XY DSD was recruited from the Prenatal Diagnosis Center of Guizhou Medical University in December 2020. A total of five family members were enrolled in this study, including four males and one female: the proband, his brother, father, mother and cousin. The affected individuals were between 3 and 5 years of age. Ethics approval was obtained from the Ethics Committee of the Affiliated Hospital of Guizhou Medical University (approval no. 2020-325) and all individuals provided written informed consent.

Karyotype analysis and sex hormone examination

Peripheral blood samples were collected from the proband and the younger sibling and parents of the proband for karyotype analysis and C-banding. Peripheral blood chromosomes were analyzed as previously described (18). Briefly, venous blood anticoagulated with sodium heparin was inoculated into a medium containing phytohemagglutinin and incubated at 37°C for 66–72 h. Subsequently, 40 µg/ml colchicine was added to arrest cells at metaphase and cells were incubated for 1 h at 37°C. Chromosome harvesting was performed using an automated chromosome harvester. Cells were resuspended and dispersed at 25°C with 50% humidity. In total, 1–2 drops of each sample were deposited onto a slide and incubated at 80°C for 3 h.

For G-banding, slides were digested with 0.025% trypsin at 37°C for 30 sec, rinsed twice in saline, stained with Giemsa at 37°C for 5 min and rinsed with water. Images of chromosomes were obtained using the GSL-120 automated chromosome scanner and subsequently analyzed due to the clinical significance of abnormal sexual development.

For C-banding, slides were treated with a 5% Ba(OH)2 solution at 60°C for 10–20 min. Following treatment, slides were rinsed and incubated in 2X SSC solution at 60°C for 90 min. For the observation of chromosomal heterochromatin regions, slides were stained with Giemsa at 37°C for 50% less of the Ba(OH)2 exposure time. An additional 5 ml of venous blood was collected and serum was used for sex hormone profile analysis.

Gene sequencing and bioinformatics analysis

Genomic DNA was extracted from peripheral blood samples obtained from the proband and the younger sibling and parents of the proband using the Qiagen DNA Blood Mini kit (cat. no. 51104; Qiagen GmbH) following the manufacturer's protocol. The genomic DNA of the proband underwent whole exome sequencing (WES) and copy number variation sequencing (CNV-Seq), following the manufacturer's protocols (Tiangen Biotech Co., Ltd.) (19). Sequencing was performed using the Illumina NextSeq 2000 platform (Illumina, Inc.; Berry Genomics Co Ltd. with the reference genome, GRCh37/hg19. Variant filtering utilized multiple databases, including the 1,000 Genomes Project (https://www.internationalgenome.org/), GnomAD (https://gnomad.broadinstitute.org), ESP 6500 (https://esp.gs.washington.edu/drupal/) and ExAC (http://exac.broadinstitute.org/). Sanger sequencing (Sangon Biotech Co., Ltd.; http://www.sangon.com/) was used to validate identified variants in family members, including the proband, his brother, father and mother. Interpretation and pathogenicity were assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines (20). For variants of uncertain significance, databases were used to predict pathogenicity, conservation and protein structural effect of missense variants (https://www.internationalgenome.org/; https:/gnomad broadinstitute.org/; http://evs.gs.washington.edu/EVS/; http://exac.broadinstitute.org/). Data analysis was conducted using the Verita Trekker® variant detection system and the Enliven® variant annotation and interpretation system (Berry Genomics; http://www.berrygenomics.com/). The pathogenicity analysis was conducted using PolyPhen (version 2, http://genetics.bwh.harvard.edu/pph2/index.shtml). The genetic conservation of the variant site was assessed using the MEGA tool (version 7.0.14, http://www.megasoftware.net/). SWISS-MODEL (https://swissmodel.expasy.org/) was used to predict potential structural changes in the protein due to the amino acid substitution and PyMOL software (version 3.1, http://www.pymol.org/) was used for visualization of the alterations. The 1,000 Genomes Project database (https://www.internationalgenome.org/) was used to assess the novelty of the variant.

Cell culturing

293T cells were obtained from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences and cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM; cat. no. PM150210; Pricella; Elabscience Bionovation Inc.) with 10% fetal bovine serum (FBS; cat. no. 164210-50; Pricella; Elabscience Bionovation Inc.) at 37°C in a 5% CO2 incubator (21).

Construction of the heterozygous variant cell line using CRISPR/Cas9

To investigate the pathogenicity of the variant, a heterozygous variant cell line with MAP3K1 gene c.4445G>A was established in 293T cells through electroporation (22). The site of guide RNA (gRNA) was designed using CRISPOR (https://crispor.gi.ucsc.edu/). The target sequence, 5′-AGAGCCACATCTCGTAAACC-3′, is located within exon 20 of the MAP3K1 gene. Genome editing at this site affects a region corresponding to the protein kinase domain. Cas9 protein (NLS-Cas9-NLS Nuclease, cat. no. Z03469, GenScript, Inc., http://www.genscript.com/) was incubated with gRNA and subsequently co-electroporated into cells using oligonucleotides for monoclonal culture. A point mutation, c.4445G>A, was introduced, resulting in an amino acid substitution from arginine to glutamine at position 1482 (p.Arg1482Gln) of the MAP3K1 protein, which may alter the function of the kinase domain. Reverse transcription-quantitative (RT-q) PCR and gel electrophoresis were used to confirm cell genotypes. The MAP3K1 c.4445 G>A heterozygous variant cell line was obtained from Cyagen Biosciences, Inc.

Cell counting kit (CCK)-8 assay

The CCK-8 assay (cat. no. HY-K0301; MedChemExpress) was used to assess cell viability (23). Following culturing at 37°C for 48 h, cells were terminated with 2 ml of standard medium and resuspended in 2 ml of medium without antibiotics. Cells were incubated at 37°C for an additional 24 h and analyzed using an inverted microscope. CCK-8 reagent was added to cells and incubated for 2 h and the absorbance was measured at 450 nm using an enzyme marker.

Flow cytometry

Following culturing at 37°C for 48 h, cells were digested, resuspended and stained with 5 µl Annexin V-FITC and propidium iodide (PI) in the dark at room temperature for 15 min. Apoptosis was analyzed using a flow cytometer (Navios; Beckman Coulter, Inc.) within 1 h. For cell cycle analysis, cells were cultured at 37°C for 48 h and 1.5×106 cells were digested and fixed with 75% ethanol at 4°C for 24 h. Subsequently, cells were treated with 100 µl of RNase A solution and incubated at 37°C for 30 min. In total, 400 µl of PI dye was added and cells were incubated for 30 min at 4°C in the dark. Samples were filtered through a 400-mesh cell strainer and the cell cycle was assessed via flow cytometry. FlowJo (version 10.10; FlowJo LLC) was used to analyze the data.

Western blot analysis

Total protein was extracted from cells on ice using a protein extraction kit (Beijing Solarbio Science and Technology Co., Ltd.). Total protein was quantified using a bicinchoninic acid assay and samples were separated by SDS-PAGE on a 10% gel (25 µg/lane). The separated proteins were transferred onto 0.45-µm thick PVDF membranes via wet transfer. Membranes were blocked with 5% skimmed milk for 2 h at room temperature, followed by overnight incubation at 4°C with primary antibodies, including anti-GAPDH (1:5,000; cat. no. HRP-6004; Proteintech Group, Inc.), anti-Bax (1:2,000; cat. no. 50599-2-Ig; Proteintech Group, Inc.), anti-Bcl-2 (1:2,000; cat. no. 12789-1-AP; Proteintech Group, Inc.), anti-Caspase 3 (1:1,000; cat. no. WL02117; Wanleibio Group, Inc.), anti-cleaved Caspase 3 (1:1,000; cat. no. WL01992; Wanleibio Co., Ltd.), anti-SOX9 (1:5,000; cat. no. 67439-1-Ig; Proteintech Group, Inc.), anti-FOXL2 (1:5,000; cat. no. 84144-1-RR; Proteintech Group, Inc.), anti-ERK (1:2,000; cat. no. 16443-1-AP; Proteintech Group, Inc.), anti-phosphorylated (p)-ERK (1:2,000; cat. no. 28733-1-AP; Proteintech Group, Inc.), anti-p38 (1:2,000; cat. no. 14064-1-AP; Proteintech Group, Inc.) and anti-p-p38 (1:2,000; cat. no. 28796-1-AP; Proteintech Group, Inc.). Following primary incubation, membranes were washed three times with TBS-0.1% Tween-20 and incubated with HRP-conjugated goat anti-rabbit IgG (1:20,000; cat. no. BS22357; Bioworld Technology, Inc.) or HRP-conjugated goat anti-mouse IgG (1:20,000; cat. no. BS22356; Bioworld Technology, Inc.) for 2 h at room temperature. Protein bands were visualized using ECL (cat. no. WBKLS0050, MilliporeSigma). Images were obtained using an exposure meter (Universal Hood II; Bio-Rad Laboratories, Inc.) and protein expression was quantified using ImageJ software (version 1.6.0; National Institutes of Health).

RT-qPCR

Following culturing for 48 h, total RNA was extracted from cells (1×106 cells) using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA was reverse-transcribed into cDNA using the SYBR Fluorescence Quantification kit (Takara Bio, Inc.), according to the manufacturer's protocol. Thermocycling conditions: Pre-denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 35 sec and extension at 72°C for 10 sec. The expression levels were determined using the 2−ΔΔCq method (24). The following primer pairs were used for qPCR: SOX9 forward, 5′-GAGGAAGTCGGTGAAGAACGG-3′ and reverse, 5′-CCCTCTCGCTTCAGGTCAG-3′; FOXL2 forward, 5′-GAGAAGAGGCTCACGCTGTC-3′ and reverse, 5′-CTCGTTGAGGCTGAGGTTGT-3′; and GAPDH forward, 5′-GGTCTCCTCTGACTTCAACA-3′ and reverse, 5′-GTGAGGGTCTCTCTCTTCCT-3′. All experiments were repeated at least three times.

Statistical analysis

Data are presented as the mean ± standard deviation using at least three independent experiments. Analyses were performed using SPSS statistical software (version 19.0; IBM Corp.). Differences between groups were analyzed using independent Student's t-tests (unpaired). P<0.05 was considered to indicate a statistically significant difference.

Results

Clinical evaluation of a family with the DSD phenotype

The proband investigated in the present study was a 3-year-old male who presented with abnormal urethral opening that had been observed since birth. Physical examination revealed a recurved penis with the urethral opening located at the penile-scrotal junction. A review of the family history indicated that the proband's brother presented with the same clinical features and the son of a maternal aunt also exhibited comparable manifestations of hypospadias, suggesting a potential familial pattern. A pedigree chart illustrating the family lineage was constructed based on the genetic data (Fig. 1A). Pelvic and scrotal ultrasound examinations were conducted for both the proband and the brother of the proband. Imaging revealed bilateral kidneys and testes in both individuals, with no obvious solid masses detected posterior to the bladder (Fig. 1B). The proband underwent ultrasound examination and the results indicated right testicular hydrocele with no evidence of female reproductive structures, such as a uterus or ovaries (Fig. 1B). Notably, these results were indicative of the DSD phenotype. To rule out hormonal causes of this case, venous blood samples were collected from both the proband and the brother of the proband for sex hormone analysis. Results of the present study demonstrated that levels of luteinizing hormone, estradiol and testosterone were decreased, while levels of anti-Müllerian hormone and inhibin B remained unaltered (Table I), indicating that the phenotype may be not a result of hormones. Collectively, these results demonstrated that the pathogenesis of the patient may be attributed to a genetic variant.

Figure 1. Clinical evaluation of a family with the DSD phenotype. (A) Pedigree of the patient family. Squares and circles represent males and females, respectively. Roman numerals represent generations. (B) Images obtained using ultrasound examination of the proband and the parents and sibling of the proband revealed bilateral kidneys with no notable solid structures behind the bladder. Bilateral testes were observed in the proband and the sibling of the proband, with no visible female adnexa. Right testicular hydrocele was observed in the proband. DSD, disorder of sex development.

Table I. Levels of sex hormones.

Table I.

Levels of sex hormones.

Hormone	Proband	Proband's brother	Reference value range
Luteinizing hormone	0.18 ↓	0.22 ↓	1.70–8.60 IU/l
Follicle-stimulating hormone	1.59	2.40	1.50–12.40 IU/l
Prolactin	410.00	190.20	81.80–483.00 mlU/l
Estradiol (E2)	<18.35 ↓	<18.35 ↓	41.40–159.00 pmol/l
Progesterone (PRGE)	0.25	<0.16	0.00–0.47 nmol/l
Testosterone (T)	<0.09 ↓	<0.09 ↓	8.64–29.00 nmol/l
Deoxycorticosterone	<80	<80	≤350 pg/ml
17-Hydroxyprogesterone	239.7	<100	<1100 pg/ml
11-Deoxycortisol	310.0	209.7	<3440 pg/ml
Cortisol	0.83×105	0.96×105	(0.3–2.5)x105 pg/ml
Dehydroepiandrosterone	537.3	<500	<2300 pg/ml
Androstenedione	<50.0	<50.0	<510 pg/ml
anti-Müllerian hormone	>18	>18	2.04–19.22 ng/ml
Inhibin B	120.53	204.38	21–166 pg/ml

Identification of 46, XY DSD

Karyotype analysis of peripheral blood obtained from the proband and two other affected family members revealed a 46, XY chromosome pattern. In addition, C-band analysis identified a prominent heterochromatin region on the long arm of chromosome Y (q12) in all three individuals (Fig. 2A-C). CNV-Seq of peripheral blood obtained from the proband revealed no abnormalities (Fig. 2D). Collectively, these findings suggested that the disease affecting this family was consistent with 46, XY DSD.

Figure 2. Karyotype analysis and CNV-seq testing. Results of G-banding and C-banding karyotype analysis of peripheral blood at the level of 400 bands in (A) the proband, (B) the sibling of the proband and (C) the cousin of the proband. (D) CNV-seq testing results of ≥100 Kb without abnormal CNV. CNV, copy number variation.

Identification and bioinformatics analysis of the mutated gene

WES of peripheral blood obtained from the proband identified a heterozygous variant at the MAP3K1 gene locus c.4445G>A, associated with autosomal dominant 46, XY DSD type 6. Sanger sequencing was subsequently performed on the peripheral blood obtained from the brother and parents of the proband (Fig. 3A) and the results confirmed that the variant was inherited from the mother. Notably, these results were consistent with an autosomal dominant inheritance pattern. The MAP3K1 c.4445G>A variant is a missense variant, resulting in the substitution of arginine with glutamine at position 1482. This variant was not present in the 1,000 Genomes Project database and the frequencies observed in the ExAC and gnomAD databases were 8.30×106 and 2.90×105, respectively. Based on the ACMG guidelines (20), this variant was classified as a variant of unknown clinical significance (PM1 + PM2) and a search of existing databases did not identify relevant studies or case reports (Fig. 3B). Further pathogenicity analysis was conducted using PolyPhen and the results revealed a HumanDiv score of 0.98, indicative of disruption (Fig. 3C). Although the HumanVar score of 0.532 indicated a moderate likelihood of pathogenicity (Fig. 3C), it remained within the range of potentially damaging variants. Considering the complementary nature of these models, the results supported the classification of this variant as potentially disruptive. The MEGA tool was used to assess the genetic conservation of this variant site and the results revealed that the variant is highly conserved across species (Fig. 3D). In addition, SWISS-MODEL was used to predict potential structural changes in the protein due to the amino acid substitution and PyMOL software was used for visualization of the alterations. Results of the analysis revealed notable modifications in the tertiary structure of the protein (Fig. 3E). Collectively, these results suggested that the novel point variant c.4445G>A in the MAP3K1 gene may lead to protein dysfunction; thus, triggering the 46, XY DSD phenotype.

Figure 3. Identification and bioinformatics analysis of the mutated gene. (A) Sanger sequencing of the MAP3K1 gene in the family. (B) Location of the c.4445G>A variant on the MAP3K1 genome. (C) Polyphen (version 2) was used to predict the pathogenicity of the MAP3K1 c.4445G>A variant. (D) Analysis of the variant locus using MEGA software. (E) Prediction of protein tertiary structure alteration using PyMOL software. MAP3K1, mitogen-activated protein 3 kinase 1; WT, wild-type.

The heterozygous c.4445G>A variant in MAP3K1 gene reduces cell viability in vitro

To validate whether the MAP3K1 c.4445G>A variant was the causative factor of disease in the family investigated in the present study, the heterozygous variant was established in 293T cells using the CRISPR/Cas9-mediated gene editing system. Single clones were selected following electro-transformation and verified using RT-qPCR and sequencing. Results of the present study revealed that heterozygous MAP3K1 variant cells were successfully generated (Fig. 4A and B). To determine whether the variant affected the translation or stability of the MAP3K1 protein, MAP3K1 expression levels were assessed. Results of the western blot analysis revealed that MAP3K1 protein expression was not affected by the presence of the variant (Fig. 4C and D). However, the viability of variant cells was markedly reduced, with levels at 80.15% of that of wild-type cells (Fig. 4E). Collectively, these results indicated that the variant site may be required for MAP3K1 function; however, it is not essential for protein expression.

Figure 4. Generating heterozygous variant cells using the CRISPR/Cas9 gene editing system. (A) Reverse transcription-quantitative PCR and sanger sequencing were used to verify successful genomic editing. (B) Synonymous variants were designed to prevent gRNA from splicing the sequence following homologous recombination. (C) Protein expression levels of MAP3K1 in WT and variant cells. (D) Quantification of the relative expression of the MAP3K1 protein. (E) Viability of WT and variant cells. Error bars represent standard deviation. ns, no significance, ***P<0.001. gRNA, guide RNA; MAP3K1, mitogen-activated protein 3 kinase 1; WT, wild-type.

The heterozygous variant activates pathways associated with apoptosis and induces cell cycle arrest in vitro

To investigate the potential effects of the MAP3K1 c.4445G>A point variant on cell proliferation and apoptosis, the expression of proteins associated with apoptosis was investigated. Results of the western blot analysis revealed that expression levels of pro-apoptotic proteins in the variant group; namely, Bax and cleaved Caspase 3, were markedly increased following 48 h incubation, while expression levels of the anti-apoptotic protein, Bcl-2, were markedly reduced, compared with the control group (Fig. 5A-E). These results indicated that apoptosis may be associated with the MAP3K1 c.4445G>A point variant. Results of flow cytometry also revealed a significant increase in the rate of variant cell apoptosis (Fig. 5F and G) and results of the cell cycle analysis revealed a significant reduction in the number of cells in S phase following 48 h incubation. These results highlighted that the variant cells were predominantly arrested in the G0/G1 phase (Fig. 5H and I). Collectively, these findings suggested that the MAP3K1 c.4445G>A variant may induce abnormal activation of apoptosis and cell cycle arrest in the G0/G1 phase.

Figure 5. The heterozygous variant activates pathways associated with apoptosis and induces cell cycle arrest in vitro. (A) Expression levels of Bax and Bcl-2. (B) Quantification of the relative expression of Bax and Bcl-2. (C) Expression levels of Caspase 3 and Cleaved Caspase 3. (D) Quantification of the relative expression of Cleaved Caspase 3. (E) Quantification of the relative expression of Cleaved Caspase 3/Caspase 3. (F) Flow cytometry was used to determine the rate of cell apoptosis. (G) Quantification of the rates of cell apoptosis. (H) Flow cytometry was used to determine the number of cells in G0/G1 phase, S phase and G2/M phase. (I) Quantification of the number of cells in G0/G1 phase, S phase and G2/M phase. Error bars represent standard deviation. *P<0.05, **P<0.01, ***P<0.001. WT, wild-type.

MAP3K1 c.4445 G>A point variant disrupts the expression of sexual developmental factors

To investigate the underlying mechanism by which the MAP3K1 c.4445G>A variant contributes to 46, XY DSD, the expression levels of sexual developmental factors were investigated. Results of the western blot analysis revealed no significant difference in the protein expression of total ERK1/2 and p38 between the wild-type and variant groups. However, results of the present study demonstrated that the expression levels of p-ERK1/2 and p-p38 were markedly elevated in the variant group (Fig. 6A and B). These results highlighted that the MAP3K1 c.4445G>A variant may activate downstream signaling pathways, leading to a gain-of-function phenotype. To assess the impact of the variant on the expression levels of sexual developmental factors, RT-qPCR was performed in the present study. Compared with the wild-type group, the relative mRNA and protein expression levels of SOX9 were markedly reduced in the variant group (Fig. 6C and E), while the expression levels of FOXL2 were notably increased (Fig. 6D and F). These results suggested that the MAP3K1 c.4445G>A variant may activate the MAPK pathway, leading to ERK1/2 and p38 hyperphosphorylation. This may in turn modulate the expression of key sex-determining genes; namely, SOX9 and FOXL2, ultimately contributing to the pathogenesis of 46, XY DSD.

Figure 6. MAP3K1 c.4445 G>A point variant disrupts the expression of sexual developmental factors. (A) Protein expression levels of ERK1/2 and p-ERK1/2. (B) Protein expression levels of p38 and p-p38. (mRNA expression levels of (C) SOX9 and (D) FOXL2. Protein expression levels of (E) SOX9 and (F) FOXL2. Error bars represent standard deviation. *P<0.05, **P<0.01, ***P<0.001. MAP3K1, mitogen-activated protein 3 kinase 1; p-/Phospho, phosphorylated.

Discussion

The present study investigated a family with 46, XY DSD following identification of a novel MAP3K1 gene variant (c.4445G>A) in a 3-year-old male proband who presented with clinical features of hypospadias and gonadal dysgenesis. This variant resulted in a shift from testis to ovarian differentiation and was associated with apoptotic dysfunction, cell cycle arrest and hyperphosphorylation of key signaling molecules, such as ERK and p38. Notably, these molecules are crucial for sex determination and gonadal development. Results of the present study revealed that the mother of the proband also carried the MAP3K1 variant; however, this individual presented with no abnormalities in phenotype. Therefore, it was hypothesized that the unaffected 46, XX carrier may have transmitted a pathogenic variant in an autosomal gene to the affected 46, XY proband. In addition, both the proband and the sibling of the proband presented with the DSD phenotype, supporting the notion that MAP3K1-associated 46, XY DSD follows a sex-limited, autosomal dominant inheritance pattern. These results were comparable with those of a previous study (25).

Gain-of-function variants in the MAP3K1 gene have been increasingly recognized as key contributors to the development of 46, XY DSD (25). These variants, such as p.L189P and p.P153L, lead to abnormal activation of downstream signaling pathways, such as the WNT/β-catenin and MAPK pathways, which are crucial for sex determination (8). In healthy individuals, MAP3K1 acts as a regulator of cell signaling through modulating the balance between testis-promoting SOX9/FGF9 signaling and ovary-promoting WNT/β-catenin signaling (26,27). However, gain-of-function variants in MAP3K1 may result in enhanced activation of the WNT/β-catenin pathway, leading to a shift towards ovarian differentiation and suppression of testis formation. These variants cause an imbalance in the expression of critical sex-determining genes, such as SOX9 and FOXL2, contributing to gonadal dysgenesis in 46, XY individuals (6,26,27). Notably, the MAP3K1 c.4445G>A variant is a representative example of how hyperactivation of MAP3K1 may disrupt healthy testicular development, through increased phosphorylation of downstream kinases, such as ERK1/2 and p38. Results of the present study are comparable with those of previous studies, which demonstrated that gain-of-function variants in MAP3K1 not only alter gonadal development, but also contribute to the phenotypic heterogeneity observed in patients with 46, XY DSD (8,10). Collectively, these findings highlight the critical role of MAP3K1 in regulating sex differentiation and emphasize the importance of understanding MAP3K1 variants for the clinical management of DSD.

To the best of the authors' knowledge, the present study was the first to demonstrate that apoptosis may play a key role in the pathogenesis of 46, XY DSD. Previous studies assessed the expression of the downstream effector in vitro; however, cell viability was not investigated. Notably, the presence of apoptosis in heterozygous MAP3K1 variant cells remained to be fully elucidated (8,10). Results of the present study also revealed elevated levels of apoptosis and alterations in apoptosis-associated protein expression in cells harboring the MAP3K1 c.4445G>A variant. In addition, variant cell viability was markedly reduced, indicating that the MAP3K1 c.4445G>A variant may differ from variants previously described. Collectively, results of the present study highlighted that MAP3K1 variant-induced apoptosis and the associated cell cycle arrest may disrupt healthy testicular differentiation, promoting ovarian pathways in 46, XY individuals. Further investigations are required to determine whether apoptosis plays a role in cell lines with alternative genetic variants.

The c.4445G>A variant identified in present study is a novel MAP3K1 variant implicated in 46, XY DSD. Gain-of-function variants were described in a previous study (8) and these were comparable with the c.4445G>A variant, which resulted in hyperphosphorylation of downstream targets, such as ERK and p38. High levels of phosphorylation may promote ovarian differentiation through increased FOXL2 expression and reduced SOX9 expression. Notably, splice-site variants disrupt healthy splicing and promote aberrant protein function. By contrast, the missense variant observed in the present study may affect protein conformation independent of protein expression, leading to potential alterations in the interaction of MAP3K1 with key cofactors. These results highlighted that various variant types within MAP3K1 may exert comparable downstream effects; however, they may involve different molecular pathways and mechanisms.

The most notable clinical phenotype of patients with 46, XY DSD is abnormal external genitalia. The development of external genitalia occurs in three phases; namely, genital tubercle outgrowth, cloacal septation and urethral tubularization. In males, the external genitalia differentiate into the penis, with the urethral tube extending along its entire length. Incomplete urethral tubularization leads to hypospadias, characterized by an abnormally positioned urethral opening on the ventral side of the penis (28). Previous studies have reported increased apoptosis in the peri-cloacal, peri-urethral and urorectal septum mesenchyme of Pdgfra-cKO mutants, accompanied by p53 induction and Caspase 3 activation. Dysregulated Pdgfra signaling may be associated with apoptosis-mediated urorectal malformations, including anorectal defects and hypospadias (28). Moreover, excessive apoptosis and impaired mesenchymal growth in the peri-urethral region may disrupt urethral fold fusion, directly contributing to hypospadias (29). Failure of urethral fold fusion at the midline prevents the formation of a urethral groove, leading to urethral defects associated with hypospadias (30). Consistent with these findings, results of the present study demonstrated that the MAP3K1 variant observed in patients with 46, XY DSD may promote elevated levels of apoptosis, further supporting the role of apoptosis in hypospadias development. Collectively, these findings suggested that dysregulated apoptosis may act as a key mechanism in the pathogenesis of hypospadias in patients with 46, XY DSD.

Abnormalities in sex development are associated with an increased risk of tumor development, particularly during puberty (31). Malignancy of these tumors is associated with negative consequences, including the requirement for organ removal and complete loss of fertility (32). Gonadoblastoma typically arises from either embryonic or hypoplastic gonads and >90% of affected individuals carry a Y chromosome, with the 46, XY karyotype being the most common (33). Results of the in vitro analysis in the present study suggested that the c.4445G>A heterozygous variant in MAP3K1 may affect the cell cycle and cell viability and promote abnormal apoptosis. However, these results are not indicative of this variant being the cause of gonadoblastoma. Notably, the in vivo microenvironment is more complex and apoptosis in early phase may trigger apoptosis resistance, a feature of tumor formation (34). Thus, further investigations are required to explore whether this variant contributes to tumor development.

While the present study provides novel insights into the role of the MAP3K1 c.4445 G>A variant in 46, XY DSD, certain limitations should be acknowledged. For example, the experiments were conducted using 293T cells, which, while commonly used for studying gene function, do not fully replicate the physiological environment. Future studies utilizing more physiologically relevant cell models (for example, human granulosa KGN cells and PSC-derived human PGC-like cells) are required to further validate the findings of the present study. Moreover, results of the present study revealed that apoptosis may exhibit potential in the pathogenesis of the disease; however, the present study did not investigate whether similar apoptotic effects occur in other reported MAP3K1 variants. Examination of additional variants may provide a broader understanding of the role of MAP3K1 in 46, XY DSD. In addition, results of the present study revealed that reduced cell viability may be associated with the variant; however, the molecular pathways leading to this reduction, such as the potential involvement of mitochondrial dysfunction or intrinsic apoptosis pathways, were not examined. Therefore, future investigations should explore the aforementioned mechanisms to further elucidate the functional consequences of the variant.

In conclusion, the present study identified a novel MAP3K1 variant (c.4445G>A) associated with 46, XY DSD and demonstrated the associated impact on apoptosis, cell cycle regulation and disruption of key signaling pathways. These findings expand the current understanding of how gain-of-function variants in MAP3K1 contribute to gonadal dysgenesis, through shifting the balance between testis and ovary differentiation pathways. The present study provides novel insights for improved management and personalized treatment of patients with DSD.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Science and Technology Foundation of Guizhou Provincial Health and Construction Commission (grant no. gzwkj2021-300).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author. The data generated in the present study may be found in the Sequence Read Archive under accession number (BioProject no. PRJNA1250182) or at the following URL: https://www.ncbi.nlm.nih.gov/sra/PRJNA1250182.

Authors' contributions

ZW designed the study. YL and SW participated in all experiments. YX and CH analyzed the experimental data and drafted the manuscript. SW and JZ carried out the cell experiments. WP guided the operation of the flow cytometer. ZW, YL and SW confirmed the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of Guizhou Medical University [approval no. 2020 Ethics (No 325)]. Written informed consent was obtained from all participants.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References