De novo missense variants in exon 9 of SEPHS1 cause a neurodevelopmental condition with developmental delay, poor growth, hypotonia, and dysmorphic features

Abstract

Selenophosphate synthetase (SEPHS) plays an essential role in selenium metabolism. Two mammalian SEPHS paralogues, SEPHS1 and SEPHS2, share high sequence identity and structural homology with SEPHS. Here, we report nine individuals from eight families with developmental delay, growth and feeding problems, hypotonia, and dysmorphic features, all with heterozygous missense variants in SEPHS1. Eight of these individuals had a recurrent variant at amino acid position 371 of SEPHS1 (p.Arg371Trp, p.Arg371Gln, and p.Arg371Gly); seven of these variants were known to be de novo. Structural modeling and biochemical assays were used to understand the effect of these variants on SEPHS1 function. We found that a variant at residue Trp352 results in local structural changes of the C-terminal region of SEPHS1 that decrease the overall thermal stability of the enzyme. In contrast, variants of a solvent-exposed residue Arg371 do not impact enzyme stability and folding but could modulate direct protein-protein interactions of SEPSH1 with cellular factors in promoting cell proliferation and development. In neuronal SH-SY5Y cells, we assessed the impact of SEPHS1 variants on cell proliferation and ROS production and investigated the mRNA expression levels of genes encoding stress-related selenoproteins. Our findings provided evidence that the identified SEPHS1 variants enhance cell proliferation by modulating ROS homeostasis. Our study supports the hypothesis that SEPHS1 plays a critical role during human development and provides a basis for further investigation into the molecular mechanisms employed by SEPHS1. Furthermore, our data suggest that variants in SEPHS1 are associated with a neurodevelopmental disorder.

Keywords: ROS production; SEPHS1; clinical exome sequencing; developmental delay; hypotonia; neurodevelopmental disorder; selenium metabolism; selenophosphate synthetase.

Figure 1

Identification of missense SEPHS1 variants in nine individuals (A) Schematic of SEPHS1 organization. The blue boxes reflect exons. The yellow box depicts SEPHS1 variant position. (B) Evolutionary conservation of missense variants observed in SEPHS1. Protein alignment of SEPHS1 orthologs was performed to determine protein sequence conservation of the region of interests. Residues impacted by variants within our cohort are highlighted in red. The Trp352 and Arg371 amino acids are highly conserved from human to frog. (C) Tolerance Landscape of SEPHS1. The tolerance landscape of SEPHS1 was generated using Metadome (https://stuart.radboudumc.nl/metadome/). The color in the plot is an indication for the tolerance (red, intolerant; blue, tolerant). Graphical representation of the linear protein structure of SEPHS1 with two functional Air Synthase Related Protein domains is below the plot. Missense variants observed in this study are labeled, p.Arg371 and p.Trp352G. The missense variants in SEPHS1 are located in regions that are intolerable for variation (red).

Figure 2

Structural modeling of SEPHS1 (A) Overall structural organization of SEPHS1 (PDB: 3FD6). Interactions between the N-terminal regions of monomers 1 and 2 form a β-barrel-like structure to stabilize the homodimer and active sites. ADP is shown in yellow. Residues Trp352 and Arg371 are situated in the C-terminal β-sheet and are highlighted in green. (B) Trp352 resides in a hydrophobic pocket stabilized by interactions with neighboring hydrophobic residues. (C) Arg371 is located on the solvent-exposed face of β11 and does not appear to form any significant interactions with surrounding residues. All modeling was performed using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).

Figure 3

Analysis of SEPHS1 stability and enzymatic activity (A) Protein thermal stability was measured using the Tycho NT.6 instrument. Intrinsic protein fluorescence was monitored as a thermal ramp was applied to each sample. The resulting curves are plotted as the first derivative and used to calculate the inflection temperature (Ti) for each sample. (B) Calculated Ti values. (C) Initial fluorescence values measured for each sample using the Tycho NT.6. All Ti and initial fluorescence values are reported with ±SD from 4 independent runs. (D) SEPHS1-mediated ATP hydrolysis monitored using the CellTiter-Glo Assay 2.0 assay kit. Relative ATP consumption was measured after 18 h of incubation at +37°C. Assays were performed in triplicates and reported with ±SD. (E) Trypsin or chymotrypsin was added to SEPHS1 samples and allowed to digest the protein for 30 min. Time points were taken at 0, 1, 5, 15, and 30 min. Digested products were analyzed on 4%–20% TGX gels. Representative gels are shown from 2 to 3 independent cleavage reactions. (F) Densitometric quantification performed with ImageJ of total fraction cleaved after 30 min. Statistical significance for all panels was determined by one-way ANOVA with the Bonferroni correction, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 4

Analysis of SEPHS1 cellular functions in SH-SY5Y cells (A) Cell proliferation analysis of SH-SY5Y cells overexpressing SEPHS1 variants at the indicated days. The % live cell values were normalized to the day 1 cells (considered as 100% viable). (B) Immunoblotting for immunoprecipitation of FLAG-tagged SEPHS1 variants. Arrows indicate the endogenous SEPHS1. (C) Fluorescence-activated cell sorting (FACS) analysis of CellROX Deep Red fluorescence in SH-SY5Y cells with or without treatment with ROS inducer tBHP, showing the quantitative bar graphs and statistical analysis of the median fluorescence intensity (MFI). Error bars derived from three independent measurements. (D) FACS histograms showing ROS production as described in (C). The numerical values accompanying each histogram signify the percentage of MFI for SEPHS1 variants relative to the wild-type (WT). (E) Quantitative real-time PCR for genes encoding stress-related selenoproteins and ROS-scavenging enzymes in SH-SY5Y cells harboring SEPHS1 variants. mRNA levels were normalized to PolB. Data are represented as mean ± SEM, n = 3. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 and ns, not statistically significant.