IMPE Abstracts

Context: MIRAGE syndrome (OMIM #617053) is a multiorgan disorder including hypoplasia of the adrenal glands and gonads (Narumi S et al., Nat Genet 2016). The syndrome is caused by heterozygous pathogenic SAMD9 variants with potent growth-inhibitory activity. There is no effective treatment for the syndrome, and approximately 40% of documented cases die before age 3.

Objective: We aimed to establish a screening method for molecular targeted agents for the SAMD9 protein.

Methods and Results: We created various types of deletion-mutant expression vectors encoding for pathogenic SAMD9^E974K, and expressed them in HEK293 cells to identify domain(s) crucial for the growth-inhibitory activity. The results showed that removal of the SAM domain (1-90 aa) restored growth inhibitory activity, suggesting the role of the domain to exert the pathogenicity. In order to determine the crystal structural of the SAM domain, the SAMD9^12-90 protein was massively expressed in E. coli, purified, and the conditions for crystallization were explored. Using the crystallized SAMD9^12-90, X-ray diffraction data were collected to a resolution of 2.2 Å. In the crystal, SAMD9^12-90 molecules were found to form multimeric structure (SAM-SAM interaction) with a helical arrangement. We then performed structure-guided mutagenesis experiments to show that inhibiting the SAM-SAM interaction by V45R or G62D variants alleviated the growth inhibition of HEK293 cells. Finally, the SAMD9^1-90 protein was incorporated into the luciferase complementation assay with the NanoBiT technology (Promega), allowing SAM-SAM interactions to be quantified by luminescence in vitro.

Discussion: We have determined the crystal structure of the SAM domain of human SAMD9, which suggested its role as self-oligomerization domain. The fact that the pathogenicity of disease-causing SAMD9 variant was alleviated not only by removal of the entire SAM domain but also by point mutations (V45R and G62D) indicates that pharmacological inhibition of SAM self-oligomerization may be a feasible approach to molecular targeted therapy for SAMD9. Our method for quantifying SAM-SAM interaction based on the luciferase complementation assay is a scalable, low-cost and reproducible. This method may serve as a screening tool for candidate drugs for the molecularly targeted therapy of MIRAGE syndrome.