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.

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.

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.

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).

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.