Novel calcineurin A (PPP3CA) variant associated with epilepsy, constitutive enzyme activation and downregulation of protein expression

Abstract

PPP3CA encodes calmodulin-binding catalytic subunit of calcineurin, a ubiquitously expressed calcium/calmodulin-regulated protein phosphatase. Recently de novo PPP3CA variants were reported as a cause of disease in 12 subjects presenting with epileptic encephalopathy and dysmorphic features. We describe a boy with similar phenotype and severe early onset epileptic encephalopathy in whom a novel de novo c.1324C>T (p.(Gln442Ter)) PPP3CA variant was found by whole exome sequencing. Western blot experiments in patient's cells (EBV transformed lymphocytes and neuronal cells derived through reprogramming) indicate that despite normal mRNA abundance the protein expression level is strongly reduced both for the mutated and wild-type protein. By in vitro studies with recombinant protein expressed in E. coli we show that c.1324C>T (p.(Gln442Ter)) results in constitutive activation of the enzyme. Our results confirm the role of PPP3CA defects in pathogenesis of a distinct neurodevelopmental disorder including severe epilepsy and dysmorphism and provide further functional clues regarding the pathogenic mechanism.

Fig. 1

Facial features of proband. Slight dysmorphia consisting mainly of microretrognathia at age 2 weeks (a), and then evolving into slightly turricephalic skull, hypertelorism, wide nasal alae, relative macrostomia with open mouth and full lips as well as microretrognathia at ages of 6 months (b) and 7 years (c)

Fig. 2

Analysis of Calcineurin Aα protein levels cells from patient with identified variant in PPP3CA gene and control subjects using western blotting (WB). Specific antibody recognizing both normal and mutant form (truncated protein, lacking autoinhibitory domain) of calcineurin Aα was used (Abcam #52761, 1:1000 in 1% milk). A Representative example of analysis in immortalized B-lymphocytes (left-protein loading shown by TGX stain-free SDS-PAGE gel). B Quantification of WB shown in A done for WT CaN protein, data presented as mean fold change relative to control subject, three independent cultures of each cell line were analyzed. Error bar indicates SEM. C Analysis in induced neuronal cells (lymphoblasts are shown for comparison). Arrows indicate truncated protein.

Fig. 3

Phosphatase activity for wild-type CaN (solid lines) and CaN∆442 (dashed lines). Kinetics for dephosphorylation of pRII in the a absence and b presence of CaM

Fig. 4

Structure of human calcineurin, isoform (PDBID 1AUI) [23]. CnA (from PPP3CA) is shown in dark blue and CnB (from PPP3R1) in grey, with the catalytic iron ion as an orange sphere and the catalytic zinc ion as a pale blue sphere. Calcium ions bound to CnB are shown as grey spheres. The intrinsically disordered regulatory domain (RD) was generated using MODELLER [24]. The variant identified in this work, c.1324C>T (p.(Gln442Ter)), is shown in red, variants identified by Myers et al. [7] and Deciphering Developmental Disorders Study [20] are purple, those identified by Mizuguchi et al. [8] in yellow, and c.275A>G (p.(His92Arg)), reported by both the latter two groups, in green. The top inset shows variants that occur in the autoinhibitory domain (AID) and the bottom inset shows those clustered around the active site