mTORC1 functional assay reveals SZT2 loss-of-function variants and a founder in-frame deletion

Abstract

Biallelic pathogenic variants in SZT2 result in a neurodevelopmental disorder with shared features, including early-onset epilepsy, developmental delay, macrocephaly, and corpus callosum abnormalities. SZT2 is as a critical scaffolding protein in the amino acid sensing arm of the mTORC1 signalling pathway. Due to its large size (3432 amino acids), lack of crystal structure, and absence of functional domains, it is difficult to determine the pathogenicity of SZT2 missense and in-frame deletions, but these variants are increasingly detected and reported by clinical genetic testing in individuals with epilepsy. To exemplify this latter point, here we describe a cohort of 12 individuals with biallelic SZT2 variants and phenotypic overlap with SZT2-related neurodevelopmental disorders. However, the majority of individuals carried one or more SZT2 variants of uncertain significance (VUS), highlighting the need for functional characterization to determine, which, if any, of these VUS were pathogenic. Thus, we developed a novel individualized platform to identify SZT2 loss-of-function variants in the context of mTORC1 signalling and reclassify VUS. Using this platform, we identified a recurrent in-frame deletion (SZT2 p.Val1984del) which was determined to be a loss-of-function variant and therefore likely pathogenic. Haplotype analysis revealed that this single in-frame deletion is a founder variant in those of Ashkenazi Jewish ancestry. Moreover, this approach allowed us to tentatively reclassify all of the VUS in our cohort of 12 individuals, identifying five individuals with biallelic pathogenic or likely pathogenic variants. Clinical features of these five individuals consisted of early-onset seizures (median 24 months), focal seizures, developmental delay and macrocephaly similar to previous reports. However, we also show a widening of the phenotypic spectrum, as none of the five individuals had corpus callosum abnormalities, in contrast to previous reports. Overall, we present a rapid assay to resolve VUS in SZT2, identify a founder variant in individuals of Ashkenazi Jewish ancestry, and demonstrate that corpus callosum abnormalities is not a hallmark feature of this condition. Our approach is widely applicable to other mTORopathies including the most common causes of the focal genetic epilepsies, DEPDC5, TSC1/2, MTOR and NPRL2/3.

Keywords: SZT2; epilepsy; genetics; mTOR; variant.

Figure 1

Development of gene editing approach for functional characterization of SZT2 VUSs. (A) Individual cell clones either homozygous or heterozygous (+ loss-of-function on other allele) for an individual SZT2 variant were generated by gene editing followed by limiting dilution cloning. Immunoblot for amino acid sensitive mTORC1 activity by P-S6K levels was used to determine whether individual variants caused SZT2 loss-of-function. Homology-directed repair rate of cells generated by transfection of px459 encoding targeting gRNAs was analysed using amplicon sequencing and varied between 12% and 25%. (B) Immunoblot of mTORC1 activity in control HEK cells (i.e. unedited) and homozygous HEK SZT2^{p.Val1984del/p.Val1984del} clone. (C) Densitometric quantification of (B). At least three individual replicates were performed. *P < 0.05 (t-test).

Figure 2

Development of medium throughput assay for functional characterization of SZT2 VUSs. (A) HEK293T cells were co-transfected with px459 encoding targeting gRNAs and repair oligonucleotides, followed by puromycin selection. Cells were starved of amino acids and then fixed for immunolabelling of phosphorylated S6 and FACS sorting. gDNA was isolated from unsorted and sorted cells followed by amplicon sequencing to confirm CRISPR/Cas9 targeting and to calculate CMAS. (B) Representative FACS plots. Dashed line represents the boundary for sorting P-S6^HIGH and P-S6^LOW cell populations. (C) CMAS scores derived from amplicon sequencing of sorted and unsorted cells. For all but p.Arg1948Gln (n = 2) and p.Pro466Ser (n = 2), at least three individual replicates were performed. *P < 0.05 (one-way ANOVA with Tukey’s post hoc test).

Figure 3

Shared haplotype suggests SZT2 p.Val1984del is a founder variant in those of Ashkenazi Jewish ancestry. Using exome sequencing data from three study trios in combination with allelic frequencies in the gnomAD population database, all individuals carry the same rare (MAF ranging from 0.00007–0.2) variants spanning a 4 Mb interval. Ref|Alt alleles for SNVs (1 and 5 flanking; 2-4 in haplotype block): (1) rs12406524 = G|A; (2) rs142849148 = G|C; (3) rs1126742 = A|G; (4) rs13376679 = T|C; (5) rs75538709 = G|A.