Pathogenic MAST3 Variants in the STK Domain Are Associated with Epilepsy

Abstract

Objective: The MAST family of microtubule-associated serine-threonine kinases (STKs) have distinct expression patterns in the developing and mature human and mouse brain. To date, only MAST1 has been conclusively associated with neurological disease, with de novo variants in individuals with a neurodevelopmental disorder, including a mega corpus callosum.

Methods: Using exome sequencing, we identify MAST3 missense variants in individuals with epilepsy. We also assess the effect of these variants on the ability of MAST3 to phosphorylate the target gene product ARPP-16 in HEK293T cells.

Results: We identify de novo missense variants in the STK domain in 11 individuals, including 2 recurrent variants p.G510S (n = 5) and p.G515S (n = 3). All 11 individuals had developmental and epileptic encephalopathy, with 8 having normal development prior to seizure onset at <2 years of age. All patients developed multiple seizure types, 9 of 11 patients had seizures triggered by fever and 9 of 11 patients had drug-resistant seizures. In vitro analysis of HEK293T cells transfected with MAST3 cDNA carrying a subset of these patient-specific missense variants demonstrated variable but generally lower expression, with concomitant increased phosphorylation of the MAST3 target, ARPP-16, compared to wild-type. These findings suggest the patient-specific variants may confer MAST3 gain-of-function. Moreover, single-nuclei RNA sequencing and immunohistochemistry shows that MAST3 expression is restricted to excitatory neurons in the cortex late in prenatal development and postnatally.

Interpretation: In summary, we describe MAST3 as a novel epilepsy-associated gene with a potential gain-of-function pathogenic mechanism that may be primarily restricted to excitatory neurons in the cortex. ANN NEUROL 2021;90:274-284.

Figure 1.. MAST3 patient-specific variants occur at conserved sites that are intolerant to genetic variation.

(A) MAST3 patient-specific variants (number of individuals in parenthesis) in relation to ST kinase (amino acids 367–640) and PDZ domains (amino acids 958–1038). (B) Corresponding graph with missense tolerance ratio (MTR, y-axis) and protein position (x-axis). The tolerance ratio measures cDNA intolerance to missense variants with 5% representing the cutoff for more intolerant segments. Regions of the ST kinase domain which are intolerant to variations portions of the ST kinase domain overlap with the distribution of pathogenic variants. (C) Multispecies alignment of the STK domain harboring the MAST3 patient variants (highlighted in green) show all pathogenic variants are highly conserved across 11 species (identical/similar amino acids in red). (D) Alignment of the MAST1 and MAST3 protein sequences. The serine threonine kinase (STK) domain has high homology and the MAST1 p. G517S and MAST3 p.G510S (highlighted in green) occur at the same position in the STK domain. Overall, these protein sequences have 61% identity.

Figure 2.. MRI changes in 5 individuals with MAST3 variants.

(A-C) - Sagittal FLAIR images () Patient 7 (p.G515S) 4 years old, normal corpus callosum and small pituitary. (B) Patient 11, 15 years old, thing corpus callosum and normal pituitary. (C) Patient 3, 2 years 9 months old, thin corpus callosum and small pituitary. D. Table summarizing changes found in corpus callosum and pituitary gland in the 5 individuals in whom we had access to MRIs. All patients had either a thin corpus callosum and/or a small pituitary gland, but not the extensive abnormalities observed in in individuals with MAST1 pathogenic variants.

Figure 3:. Expression of MAST3 and MAST1 in whole brain throughout prenatal development and postnatally.

(A) Heatmap of bulk RNA-seq data from the Brainspan atlas of the developing human brain (see list of URLs). MAST3 expression begins at about 26 weeks in prenatal development. In contrast, expression of MAST1 is highest early in pretnatal development (9 weeks) and then is reduced throughout the lifespan. This pattern is similar in other DEE-associated genes, SCN1A and SCN3A, where only the latter is associated with malformation of cortical development. Key neuronal and developmental markers NESTIN, EOMES, SOX2 and TUBB3) are also shown. FPKM = Fragments Per Kilobase of transcript per Million mapped reads. (B) Heatmap of single-nucleus RNA-seq data from post-mortem adult human motor cortex for MAST3 and MAST1 (see list of URLs). MAST3 is expressed exclusively in excitatory neurons, while MAST1 is expressed in both inhibitory and excitatory neurons in the cortex. (C) Immunofluorescence in 11-week-old cerebral organoids shows the presence of MAST3 in a fraction of excitatory CTIP2-positive neurons (upper panels, arrows) and in the majority of CUX1-positive neurons (lower panels, arrowheads). (D) Immunofluorescence performed in E14.5 (left panels) and E16.5 (right panels) mouse coronal brain sections show MAST3 is mainly cytosolic, surrounding Tbr1+ cell nuclei (arrowheads). And some cells do present some MAST3 nuclear speckles (arrows). Scale bar=20 microns.(E) Immunofluorescence performed in E14.5 (left panels) and E16.5 (right panels) mouse coronal brain sections confirm MAST3 expression in postmitotic upper layer neurons, co-markers evolving as cerebral development progresses. TBR1 and CTIP2 are neuronal markers specific to deep cortical layers. SATB2 is a neuronal marker for upper cortical layers. Scale bars = 50 µm.

Figure 4.. Phosphorylation of ARPP-16 at Ser46 by MAST3 kinase.

(A) HA-tagged ARPP-16 alone, or together with MAST3, were expressed in HEK293T cells. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting for MAST3 protein, phospho-Ser46-ARPP-16, and HA-ARPP-16. Representative blots show that WT and all MAST3 mutant phosphorylate ARPP-16. There was minimal, background, phosphorylation of ARPP-16, as shown by the expression of ARPP-16 alone (lane 2) or when co-expressed with the dead-kinase mutant K396H (lane 4). Lanes: 1) GFP Only, 2) ARPP-16 Only, 3) Wild-Type MAST3 + ARPP-16, 4) K396H dead kinase control + ARPP-16, 5) G510S MAST3 + ARPP-16, 6) G515S MAST3 + ARPP-16, 7) L516P MAST3 + ARPP-16, 8) V551L MAST3 + ARPP-16. (B) Quantification of immunoblots revealed an increase in ARPP-16 phosphorylation when co-expressed with the G510S mutant compared to WT MAST3 (*p = 0.0159, one-way ANOVA with Dunnett’s posthoc test). Note that the GFP control, ARPP-16 alone control, and the dead kinase control quantifications were omitted from the graph due to the presence of zero values