PPP2R3C gene variants cause syndromic 46,XY gonadal dysgenesis and impaired spermatogenesis in humans

in European Journal of Endocrinology
Correspondence should be addressed to T Guran; Email: tulayguran@yahoo.com
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Most of the knowledge on the factors involved in human sexual development stems from studies of rare cases with disorders of sex development. Here, we have described a novel 46, XY complete gonadal dysgenesis syndrome caused by homozygous variants in PPP2R3C gene. This gene encodes B″gamma regulatory subunit of the protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase involved in the phospho-regulation processes of most mammalian cell types. PPP2R3C gene is most abundantly expressed in testis in humans, while its function was hitherto unknown.

Patients and methods

Four girls from four unrelated families with 46, XY complete gonadal dysgenesis were studied using exome or Sanger sequencing of PPP2R3C gene. In total, four patients and their heterozygous parents were investigated for clinical, laboratory, immunohistochemical and molecular characteristics.


We have identified three different homozygous PPP2R3C variants, c.308T>C (p.L103P), c.578T>C (p.L193S) and c.1049T>C (p.F350S), in four girls with 46, XY complete gonadal dysgenesis. Patients also manifested a unique syndrome of extragonadal anomalies, including typical facial gestalt, low birth weight, myopathy, rod and cone dystrophy, anal atresia, omphalocele, sensorineural hearing loss, dry and scaly skin, skeletal abnormalities, renal agenesis and neuromotor delay. We have shown a decreased SOX9-Phospho protein expression in the dysgenetic gonads of the patients with homozygous PPP2R3C variants suggesting impaired SOX9 signaling in the pathogenesis of gonadal dysgenesis. Heterozygous males presented with abnormal sperm morphology and impaired fertility.


Our findings suggest that PPP2R3C protein is involved in the ontogeny of multiple organs, especially critical for testis development and spermatogenesis. PPPR3C provides insight into pathophysiology, as well as emerging as a potential therapeutic target for male infertility.

Downloadable materials

  • Supplemental table 1. The gonadal and adrenal function tests and clinically significant laboratory results of patients with syndromic 46,XY gonadal dysgenesis due to PPP2R3C gene variants.
  • Supplemental table 2. Whole-exome sequencing variant filtration.
  • Supplemental figure 1. The skeletal and some pathognomonic clinical characteristics of the unique syndrome due to homozygous PPP2R3C variants. (A) Anteroposterior and lateral views of the spine, thorax and hip (Patient 3) showing kyphoscoliosis, narrow thorax and corrected hip dysplasia. (B) Unusual scalp hair whorls, unruly scalp hair (Patient 1). (C) The characteristic “squashed down” appearance of the nose and the typical posteriorly rotated low set ear with the overfolded helix, narrowed and bilaterally elongated intertragic notch with a sinus (Patient 3). (D) Short, broad hands especially in the thumb (and toe), a simian line, interphalangeal webbing in hands (Patient 3). (E, F) Bone age is markedly delayed particularly in the carpal bones (Bone age/chronological age is 13/12/58/12 years old for Patient 2 and 116/12/1510/12 years old for Patient 3). Short and notched distal phalanx of the thumb (and in toe) and clinodactyly (Patient 2), short middle phalanx of 5th finger and the short distal phalanx of the thumb (Patient 3). (G) Muscular habitus, thick and stiff muscles particularly in the neck (Patient 3).
  • Supplemental figure 2: Overview of protein phosphatases. A protein phosphatase (PP) is an enzyme that removes a phosphate moiety from the phosphorylated amino acid residue of its target protein. Protein phosphorylation is one of the most common forms of reversible protein posttranslational modification. Proteins are phosphorylated predominantly on Ser, Thr and Tyr residues. The largest class of PPs is the phosphoprotein phosphatase (PPP) family. Protein Ser/Thr phosphatases are classified as, type 1 (PP1) or type 2 (PP2), and are subdivided based on metal-ion requirement (PP2A, no metal ion; PP2B, Ca2+ stimulated; PP2C, Mg2+ dependent) (15). The PP2A is a heterotrimeric holoenzyme which is ubiquitously expressed, accounting for a large fraction of phosphatase activity in eukaryotic cells. PP2A consists of a dimeric core enzyme composed of the structural A and catalytic C subunits, and a regulatory B subunit. Subunit C and A sequences show remarkable conservation throughout eukaryotes, regulatory B subunits are more heterogeneous and are believed to play key roles in controlling the localization and specific activity of different holoenzymes. Multicellular eukaryotes express four classes of variable regulatory subunits: B (PR55), B′ (B56 or PR61), B″ (PR72), and B‴ (PR93/PR110), with at least 16 members in these subfamilies. The B'' family has been further divided into four subfamilies including PPP2R3C (5).
  • Supplemental figure 3. Schematic presentation of the possible signaling pathways regulated by PPP2R3C in testicular development, androgen biosynthesis and spermatogenesis. Heterotrimeric holoenzyme protein phosphatase 2A (PP2A) dephosphorylates various phospho-proteins generated by the action of protein kinase A (PKA) and protein kinase C (PKC). G protein-coupled receptor (GPCR) activation leads to the increase of intracellular cAMP, which activates PKA. The canonical downstream targets of PKA and PKC (encircled in the figure) include SOX9 (involved in testis development), P38α, Tau and ERK1/2 (involved in spermatogenesis), P38α (involved in the sex steroid biosynthesis by the activation of 17, 20 lyase activity of CYP17A1). Homozygous variants in PPP2R3C are suggested to cause an increased PP2A activity disturbing the balance between phosphorylation and dephosphorylation processes in favor of increased dephosphorylation of downstream target molecules. These multi-hit alterations impair the testicular development, androgen biosynthesis and spermatogenesis at the posttranscriptional level. Furthermore, impaired SOX9 signaling due to PPP2R3C variants is associated with abnormal chondrogenesis and bone formation, which may be responsible for the abnormal cartilage development of the ears and nose of the patients with homozygous PPP2R3C variants contributing to the characteristic appearance.


     European Society of Endocrinology

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    Pedigrees and clinical characteristics of patients with syndromic 46, XY gonadal dysgenesis. (A) Pedigrees of families 1–4 where all affected individuals manifested syndromic 46, XY complete gonadal dysgenesis, and were positive for mutations in PPP2R3C. Black symbols indicate individuals with syndromic 46, XY complete gonadal dysgenesis, gray symbols indicate those who had been stated to be infertile. All affected individuals were homozygous for the indicated variants (patients sequenced have been numbered from 1 to 4), and parents were heterozygous. Variants for patients 1 and 2 were identified by WES and the remainder by Sanger sequencing of PPP2R3C. The symbols with ‘?’ indicate suggestive clinical phenotype but no further biochemical or genetic data about that individual. (B) Phenotypic features of the patients with syndromic 46, XY gonadal dysgenesis due to homozygous variants in PPP2R3C. Pictures at each row belong to the respective patient numbered at upper left corner of each row. The characteristic facial gestalt includes flat face, flat vertex, unusual scalp hair whorls, unruly scalp hair, frontal upsweep, arched and sparse eyebrows, bilateral epicanthal folds, thin lips, hypoplastic ala nasi and beaked nose, long and smooth philtrum, hypodontia, posteriorly rotated and low set ears, overfolded helix, narrowed and elongated intertragic notch with or without a sinus under the tip of the intertragal angle common to all four patients. Muscular habitus with thick and stiff muscles, short and broad hands, a horizontal single palmar crease and interphalangeal webbing are apparent in each patient.

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    Molecular analysis of patients with the PPP2R3C variants. (A) Electropherogram showing the homozygous PPP2R3C variants (the black arrows and yellow boxes indicates the nucleotide peak of interest) in probands and respective heterozygous variants in their parents. (B) Partial alignment of PPP2R3C protein sequences, generated by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/), showing conservation of leucine (Leu;L) at position 103, leucine (Leu;L) at position 193 and phenylalanine (Phe;F) at position at 350 highlighted in gray, with numbering relative to human sequence. These amino acids are highly conserved among ortholog proteins. (C) Levels of PPP2R3C mRNA were quantified by RT-qPCR and were unchanged between control (wt/wt), parents (wt/m) and patients (m/m). The column and whisker graph represents the mean and standard errors of mean (s.e.m.) of the measurements. NS, not significant. (D) Schematic domains and ligand-binding sites of PPP2R3C (UniprotKB: Q969Q6) are illustrated. These include a EF-hand 1 (residues 273-308), EF-hand 2 (341-376) domain and calcium-binding site (286-297). The location of the domains is marked by the red arrows. Locations of pLeu103, pLeu193 and pPhe350 of wild-type PPP2R3C and pPro103, pSer193 and pSer350 identified in our patients are presented in magnified frame for viewing at higher quality. The models are visualized by PyMol, with rainbow painting starting from dark blue at ‘N terminal’ and ending red at ‘C terminal’. Elements of residues are painted in stick format and view zoomed to 20 Å below for each model. (E) Secondary structure and the solvent accessibility scores of the investigated wild type and mutation sites are presented in I-Tasser simulated models. Predicted secondary structure is scored from zero to nine; the higher the score, the more confident is the prediction. The predicted secondary structure suggests that this protein is an alpha-beta protein. ‘H’ and ‘C’ indicate helix and coil, respectively. The predicted solvent accessibility at the bottom is presented in ten levels, from buried (zero) to higher exposed (eight). pLeu103 residues are pointed with red, pSer193 with green and pSer350 with blue arrows.

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    Overview of histology and, PPP2R3C, SOX9 and SOX9-Phospho protein expressions in the dysgenetic gonads from Patient 2, Patient 4, and the developing normal fetal testis and adult testis tissues. The representative images of the gonadal tissues of Patient 2 and Patient 4 are shown in the upper 1st and the 2nd panels (A1–5, B1–5). The control tissues were selected from a normal fetal testis (C1–5) and a normal adult testis (D1–5). H&E staining revealed normal Leydig cells and seminiferous tubular structures in the control fetal (C1) and adult testis (D1). The gonadal tissue of Patient 2 revealed only few clusters of Leydig cells and peripherally located Müllerian-like tubular structures in a loose connective tissue containing vessels and peripheral nerve bundles (A1, A2). The gonadal biopsy of Patient 2 revealed no Leydig cells but peripherally located Müllerian-like tubular structures in a loose connective tissue containing vessels and peripheral nerve bundles (B1, B2). Inhibin immunohistochemical staining indicates Leydig cells (A2, C2, D2). The gonad tissue of Patient 4 was negative for inhibin immunostaining due to the absence of Leydig cells (B2). PPP2R3C expression was seen in Patients 2 and 4 similar to control tissues (A3, B3, C3, D3). PPP2R3C immunoreactivity in Sertoli cells of seminiferous tubules of control testis tissues is indicated by black arrows in C3, D3. Note that the Müllerian-like tubules were positive for SOX9 (A4, B4), but are negative for SOX9-Phospho protein expression (A5, B5) in the Patient 2 and Patient 4 with homozygous PPP2R3C variants. However, the seminiferous tubules of the control testis sections were positive for both SOX9 (C4, D4) and SOX9-Phospho (C5, D5) immunostaining (images are at ×40 magnification).

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    Representative light micrographs of semen smears from the fathers of Patient 3 (A) and Patient 4 (B). *, midpiece defect; m, mixed defect (head and tail); n, normal spermatozoa; g, globozoospermatozoa; p, pinhead spermatozoa; t, tail defect (dag or double tail). Diff-Quick staining. The father of Patient 3 showed a few number of normal spermatozoa. Head defects with globozoospermatozoa and pinhead were observed in both patients. Moreover, spermatozoa with mixed defects (head, midpiece and tail) were present. There were severe tail defects in both patients. However, tail defects with double tail were more prominent in the father of Patient 4.

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    Evaluation of CD19+ B lymphocytes for viability, apoptosis and cell-cycle characteristics in patients with homozygous PPP2R3C variants. (A) Dot plots of three patients’ Annexin V/PI staining. (B, C, D and E) The viability, early apoptosis, late apoptosis and necrosis ratios of B lymphocytes were compared at 0, 24 and 48 h after blood sampling from patients with PPP2R3C variants. (F, G, H and I) Comparison between patients’ and control samples’ for viability, early apoptosis, late apoptosis and necrosis ratios of B lymphocytes. (J) Histogram plots of three patients’ DNA content analysis. (K) Statistical analysis of cell-cycle distribution (G2/M, S, G1/G0) between three time points. P < 0.05 is considered significant (*P = 0.01; ***P < 0.001; ****P < 0.0001).


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