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.
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 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.