Patient’s genomic DNA was extracted from 200 μl peripheral blood leukocytes using the Qiagen DNA Blood kit (Qiagen, Dusseldorf, Germany). First, 50 ng DNA was broken into a size of about 200bp by fragmentation enzymes. The DNA fragments were then end-repaired, and the 3’end was added with one A base. Second, the DNA fragments were ligated with barcoded sequencing adaptors, and fragments of about 320bp were collected by XP beads. After PCR amplification, the DNA fragments were hybridized and captured by NanoWES according to the manufacturer’s protocol. The hybrid products were eluted and collected and then subjected to PCR amplification and purification. Next, the libraries were quantified by qPCR, and size distribution was determined using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, United States). Finally, the Novaseq6000 platform (Illumina, San Diego, United States), with 150 bp pair-end sequencing mode, was used for sequencing the genomic DNA of the patient. Raw image files were processed using CASAVA v1.82 for base calling and generating raw data. The sequencing reads were aligned to the human reference genome (hg19/GRCh37) using the Burrows–Wheeler Aligner tool. GATK software was employed for variant calling. Variant annotation and interpretation were conducted by ANNOVAR.

Exon 7 and exon 13 of the PPP2R3C gene were amplified by PCR, and then the PCR products of exon 7 and exon 13 were purified and underwent Sanger sequencing. Primers were designed using Primer3 software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). Primer sequences were as follows: PPP2R3C-E7-F: 5′-ACT​ACA​TAT​TGG​AAC​TTA​TCC​CTA​CG-3′, PPP2R3C-E7-R: 5′-GGCAG TGAC AGG​AAA​ATA​AAT​GAT​A-3′; PPP2R3C-E13-F: 5′-ATT​TTA​TTC​CTC​GTA​CTT​CAG TGGA-3′, PPP2R3C-E13-R: 5′-GGTCCTATT GCTTCTAAACTATT GC -3′.

TA clone of exon 7 was performed for a small deletion existing in one allele of the patient. The Taq polymerase-amplified PCR products of exon 7 were inserted into the pMD19T vector, and then the constructs were transformed into DH5α-competent cells and grew in the X-Gal and IPTG plates. The white clones were selected and amplified, and their DNA was extracted and sequenced.

Sequencing of PCR products and TA clone was performed using a Taq big dye terminator sequencing kit and an ABI-3730 automated sequencer (Applied Biosystems, Foster City, CA, United States). DNAs from both parents were sequenced to ascertain the inheritance. We read the sequencing results by 4Peaks (Nucleobytes, Netherlands) and then blasted each in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) taking the following sequence as references: cDNA: NM_017917.4; protein: NP_060387.2.

Structural models of wild PPP2R3C protein and mutant protein were built by inputting protein sequences in the SWISS-MODEL server (https://swissmodel.expasy.org). Then, we downloaded the PDB file and uploaded it into UCSF ChimeraX (version: 1.2.5), a molecular visualization program. By comparing the changes in the contacts and hydrogen bonds with other amino acids in a spatial model, we can predict the potential pathogenic effects the specific variant brings to the protein.

Skeletal deformity and external genital development were assessed by physical examination. Uterine and gonadal development was assessed by laparoscopy. Bone age was assessed by X-rays of hands. In addition, we evaluated the muscular and lymphocytic systems using electromyography and lymphocyte counts. Hormone test results included levels of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T), which were measured by chemiluminescent immunoassays (Bayer Diagnostics Corporation, United States).

We searched for reports about PPP2R3C variations in PubMed and MEDLINE, and the keywords for searching included “PPP2R3C” or “G5PR” in combination with “46,XY disorders of sex development”. Available data on clinical evaluations and genetic findings were extracted and summarized.

By WES, we identified two variants (c.684_686delTTC/p.F229del in exon 7 and c.1250G > A/p. G417E in exon 13) of PPP2R3C and confirmed by Sanger sequencing (Figure 1) in the patient, which came from his father and mother, respectively. Potentially pathogenic variants in other genes associated with syndromic 46, XY DSD had not been found. The two wild-type amino acids involved in both variants are highly conserved among ortholog proteins (Figure 2). The deletion of Phe229 will cause an incomplete alpha helix structure and change the condition of four repeated phenylalanines. The Gly417Glu does not affect a significant protein domain, but the number of hydrogen bonds and contacts formed within residues is changed (Figure 2).

A 24-year-old female of Chinese Han descent was diagnosed with 46, XY gonadal dysgenesis at age 18, when she was presented with poor development of secondary sexual characteristics and primary amenorrhea. She was born of a non-consanguineous marriage at term by vaginal delivery without any perinatal complications and was raised female gender. She was born with a birth weight of around 1 kg and normal body length. There was no history of feeding difficulties or developmental retardation in childhood. At ten years of age, she presented retardation of bone age about two years, and the closure of bone epiphysis did not appear till over 20 years old. The patient was 148 cm tall (-3SD) at 18 years old and was diagnosed with short stature. She received growth hormone therapy for three months and gained the height increase of about 2.5 cm in the following two years. The patient did not receive other therapy till presentation.

On physical examination, she had a height of 155 cm, a weight of 48 kg, and a blood pressure of 124/75 mmHg. Tanner staging was B1P2. External genitalia revealed the clitoris of normal and puerile female genitalia. There were two distinct openings in the urethra and vagina. No gonads or lumps were found in the inguinal region. The patient did not have facial hair, acne, male torso, body hairs, and deepening of voice. Systemic examination revealed remarkable facial features including unruly scalp hair, thin eyebrows, thin lips, and long and smooth philtrum. The patient had hypoplastic ala nasi and beaked nose, low-set ears, and facial pigmented nevus which were non-ignorable facial characteristics as well. The systemic examination also revealed abnormalities of the musculoskeletal system including a short fifth phalanx, webbing of the neck, and cubitus valgus (Figures 3A–C).

On investigation, the hormone profile showed LH 23.22IU/L (normal range 1.2–8.6), FSH 70.88IU/L (normal range 1.2–19.2), T 0.34 ng/ml (normal range 1.75–7.81), E2 11 pg/ml (normal range <39), 17-OHP 1.19 ng/ml (-), FT4 1.19 ng/dl (normal range 0.81–1.89), FT3 2.95 pg/ml (normal range 1.80–4.10), and TSH1.2μIU/ml (normal range 0.38–4.34) (Table.1). Lab evaluation also included a normal white blood cell count, normal hemoglobin concentration, and normal lymphocyte count and proportion. PPP2R3C gene is highly expressed in lymphocytes and plays a role in the survival of CD19⁺ B cells. Gene knockout (PPP2R3C-/-) mice by conditional targeting in CD19⁺ B cells showed a deficit in B-cell survival and a reduced number of mature B cells. After we identified the PPP2R3C variants of our patient, we tested her T/B cell subsets. The analysis of T/B cell subsets revealed a decreased number of CD19⁺ B cells (1.6%, normal range: 8.5%–14.5%) and CD4⁺ T cells (21.5%, normal range: 30.0–46.0%) (Table.2). The karyotype was 46, XY. Ultrasound and laparoscopy revealed the dysgenesis of the uterus and bilateral oviduct-like structure (Figure 3D).

Considering the risk of gonadal malignancy, she underwent bilateral gonadectomy at twenty years of age, and streak gonads and bilateral oviducts were found during the operation. Histopathology of gonadectomy material showed an oviduct-like structure bilaterally. After gonadectomy, she was treated with Femoston (estradiol/dydrogesterone: 1/10 mg), with a dose of one tablet daily for almost four years.

Thirteen 46, XY DSD patients had been reported in three articles previously (Guran et al., 2019; Cicek et al., 2021; Altunoglu et al., 2022). All the patients came from the consanguineous marriage and had been identified as homozygous PPP2R3C variants. Eleven out of thirteen 46, XY DSD patients were presented with complete gonadal dysgenesis, complete female vulva with hypoplastic labium major, and hypoplastic uterus. Sex hormone levels were compatible with complete gonadal dysgenesis. One patient presented with partial testicular dysgenesis, and he had ambiguous genitalia, micropenis, and left cryptorchidism. PPP2R3C variants are also reported to be associated with syndromic 46, XX DSD. Three such patients were presented with complete gonadal dysgenesis, recognizable facial features, and multiple extragenital malformations. All patients with PPP2R3C mutations had some similar extragonadal system manifestations, such as facial deformity (16 of 16,100%), retardation of bone age (15 of 16, 93.7%), and delayed development of the nervous system (14 of 16, 87.5%). The typical facial deformity includes abnormal eyebrows, low small-ears, and dysplasia of the alar nose. Among sixteen patients, more than half of the cases had special clinical manifestations, such as impaired vision (10 of 16, 62.5%), low birth weight (9 of 16, 56.2%), and myopathy (8 of 16, 50%). Symptoms including, renal agenesis (6 of 16, 37.5%), gastrointestinal dysfunction (6 of 16, 37.5%), sensorineural hearing loss (4 of 16, 25%), and cardiac defect (3 of 16, 18.7%) also appeared in some patients (Table 3).

A total of four PPP2R3C variants had been identified in DSD patients. Specifically, 10 patients had homozygous p.Leu193Ser variant, and 2 of the other 6 patients had homozygous p.Leu103Pro, p.Phe350Ser, or p.Ser216_Tyr218dup variants, respectively. The association between the genotype and phenotype cannot be found based on the current data (Table 3).