Identification and validation of putative pathogenic variants in a patient with intellectual disability, epilepsy, autism and macrocephaly

Whole exome sequencing was performed to identify putative disease-causing variants in a girl with macrocephaly, epilepsy and severe mental retardation, who had an unknown molecular diagnosis. Compound heterozygous SZT2 variants were detected in the girl: The maternally inherited SZT2 Chr1: g.43902997 A>T splice site variant was shown to cause skipping of the exon 42, and the missense variant Chr1: g.43905659 G>A NP_056099.3p: Asp2327Asn was paternally inherited. Previously 13 patients have been described with mutations in the SZT2 gene with phenotypes overlapping with the patient in this study. The SZT2 protein is a component of the KICSTOR complex which has an important role in the inactivation of MTORC1. Under absence of nutrients, MTORC1 will be activated and translocated to the lysosomal membrane. Deficiency in the SZT2 protein may result in the constitutive localization of an active MTORC1 on the lysosomal membrane. In order to establish tools to study the impact of the SZT2 variants on the localization of MTOR in patient cells, specific siRNAs and antibodies were assessed for functionality. The MTOR and SZT2 mRNA expression level was shown to be reduced in HEK293T cells transfected with the MTOR or SZT2 siRNAs, and Western blotting detected reduction in the MTOR protein level, while with the SZT2 antibody gave no specific bands. Therefore, patient fibroblasts were studied only with the MTOR antibody using immunofluorescence staining to assess the cellular localization of the MTOR protein in the presence and absence of amino acids. Transcriptome analyses were also performed on mRNA extracted from fibroblast patient and her parents to identify differentially expressed genes, and pathway enrichments investigation was carried out to retrieve enriched pathways in the data from patient cells. These analyses indicated down regulation of EREG, FGF7 and SHC3 genes which play a role in the transduction of cell signaling leading to the RAS/RAF/MEK/ERK pathway activation, which is up stream of MTORC1. We identified compound heterozygous SZT2 variants Chr1: g.43902997 A>T, and Chr1: g.43905659 G>A. Further transcriptome analyses with samples from additional patients are required to reveal details on the molecular disease mechanisms. Furthermore, the MTOR antibody has been validated for future study on the localization of MTOR in nutrient deprived cells