EIF2S3 Mutations Associated with Severe X-Linked Intellectual Disability Syndrome MEHMO

Abstract

Impairment of translation initiation and its regulation within the integrated stress response (ISR) and related unfolded-protein response has been identified as a cause of several multisystemic syndromes. Here, we link MEHMO syndrome, whose genetic etiology was unknown, to this group of disorders. MEHMO is a rare X-linked syndrome characterized by profound intellectual disability, epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity. We have identified a C-terminal frameshift mutation (Ile465Serfs) in the EIF2S3 gene in three families with MEHMO syndrome and a novel maternally inherited missense EIF2S3 variant (c.324T>A; p.Ser108Arg) in another male patient with less severe clinical symptoms. The EIF2S3 gene encodes the γ subunit of eukaryotic translation initiation factor 2 (eIF2), crucial for initiation of protein synthesis and regulation of the ISR. Studies in patient fibroblasts confirm increased ISR activation due to the Ile465Serfs mutation and functional assays in yeast demonstrate that the Ile465Serfs mutation impairs eIF2γ function to a greater extent than tested missense mutations, consistent with the more severe clinical phenotype of the Ile465Serfs male mutation carriers. Thus, we propose that more severe EIF2S3 mutations cause the full MEHMO phenotype, while less deleterious mutations cause a milder form of the syndrome with only a subset of the symptoms.

Keywords: EIF2S3; MEHMO syndrome; XLID; integrated stress response; translation initiation; unfolded-protein response.

Figure 1.. Co-segregating Truncating and Missense Mutations in EIF2S3 Cause MEHMO Syndrome.

A: Pedigrees of families 1–4 with co-segregating truncating and missense mutations in EIF2S3. Open circles represent females, open squares represent unaffected males, closed squares represent affected males, * = mutation carrier, wt = wild-type. B: Physical appearance of index patient from family 1 at 10 months - obese with full cheeks, large ears, downturned corners of mouth, epiblepharon, long eyelashes and thick eyebrows, tapered fingers and talipes. Microgenitalism is visible. C: Cranial MRI - Patient 1 – (left) Sagittal T1W image at 4 months showing thin and flat corpus callosum (arrow), and (right) axial T2W image at 19 months showing cerebral atrophy, delay of myelination, acute demyelinating changes in the periventricular white matter, enlarged ventricular system and subarachnoid fronto-temporal space. Patient 2 – (left) T1W image at 10 months showing cerebral atrophy and thin corpus callosum (arrow), and (right) axial T2W image showing enlargement of ventricular system, delay of myelination, and acute demyelinating changes in the periventricular white matter. D: Electron microscopy of patient 1 muscle biopsy shows myofilament degradation (left) and presence of huge mitochondria with abnormal cristae arrangement (right, arrows). E: Clustal Omega analysis of eIF2γ C-terminus (left) and the mutated serine residue (right) in humans and other species. The C-terminus is conserved in vertebrates, the Ser108 is conserved down to Drosophila melanogaster. F: Western blot analysis of protein cell lysate from control and patient fibroblast cell lines immunoblotted with anti-eIF2γ antibody shows the presence of eIFγ protein. I = Input, C = concentrated protein.

Figure 2.. Mutations Map to Distinct Regions of The eIF2γ Structure

A: Ribbons representation of the yeast 48S preinitiation complex (PIC; pdb 3JAP). Only the 18S rRNA of the 40S subunit (gray), Met-tRNA_i^Met (green), eIF2α (N-terminal domains, light yellow; C-terminal domain, yellow), eIF2β (helix α1, blue; C-terminal domain, slate) and eIF2γ (cyan) are depicted. B: Image from panel A rotated as shown and zoomed to highlight the eIF2γ subunit. Sites of the four human mutations are highlighted: S108R (Sc D167; red), I222T (Sc V281; purple), I259M (Sc I318; blue), and I465Sfs*4 (Sc L523–C-terminus; orange). C: Zoom of eIF2γ from panel B. The other eIF2 subunits and 48S complex components have been removed for clarity. Sites of eIF2γ mutations are colored as in panel B and labeled.

Figure 3.. Impact of Mutations in Yeast eIF2γ on Cell Growth

A: Schematics of human (top, brown) and yeast (Saccharomyces cerevisiae, Sc, bottom, cyan) eIF2γ highlighting the N-terminal extensions (N), G domains and domains DII and DIII. Sites of mutations in human eIF2γ and the corresponding residues in yeast eIF2γ are labeled and colored as in Figure 2. B: Alignments (top) of the C-terminal sequences of human (Hs, brown) and yeast (Sc, cyan) eIF2γ and (bottom) of a yeast/human eIF2γ chimera with either the native human C-terminus (Sc/Hs) or with the C-terminal frameshift mutation (Sc/Hs-I465Sfs*4). C: Serial dilutions of yeast cells expressing, as the sole-source of eIF2γ, the indicated eIF2γ mutants were grown on minimal synthetic dextrose (SD) medium at 30 or 37 °C for 3 days.

Figure 4.. Mutations in Yeast eIF2γ Impair Translation Initiation

A, B: A GCN4-lacZ (A) or HIS4(AUG) or HIS4(UUG) (B) reporter construct was introduced into yeast strains expressing the indicated WT or mutant form of yeast (Sc) or of yeast/human chimera (Sc/Hs) eIF2γ. β-galactosidase activities (A) or mean ratios of activities (B) and standard deviations were determined from at least three independent transformants. The data were assessed by student’s t-test and P-values relative to wild-type (Sc) eIF2γ (** P ≤ 0.01, *** P ≤ 0.001).

Figure 5.. The EIF2S3 C-terminal Mutation is Associated with Elevated DDIT3/CHOP Transcript and Protein Levels in Patient Fibroblasts

A: Expression of DDIT3 mRNA in patient and control fibroblasts measured using RT-qPCR. Levels are normalized to the control and expressed as 2^-dCq with SD. *** P <0.001, t-test. Patient fibroblasts show an increased DDIT3 transcript level compared to controls. B: Representative Western blot analysis of protein lysates from patient fibroblasts (index from family 3) with C-terminal deleted EIF2S3 and three controls. Whole cell lysate from patient and control fibroblasts was concentrated and separated on SDS-PAGE. The gel was blotted and probed with an anti-CHOP antibody (upper panel). The blot was subsequently probed with an anti-tubulin antibody as a loading control (bottom panel). Quantification of the specific bands from Western blots was performed using ImageJ software, and CHOP levels were normalized to tubulin levels. Histogram of five independent experiments shows a statistically significant increase in CHOP levels in patient fibroblasts compared to controls. The data were assessed with a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (** P ≤ 0.01, *** P ≤ 0.001).

Figure 6:. MEHMO Syndrome Belongs to the Disorders Associated with Alterations in eIF2 Function and Regulation or Involved in the UPR

To initiate translation the factor eIF2, a heterotrimeric complex of α, β and γ subunits, binds GTP and initiator Met-tRNA_i^Met to form a ternary complex (TC). The TC associates with a ribosome, and during the course of translation initiation the GTP is hydrolyzed to GDP enabling release of eIF2–GDP from the ribosome. In order for eIF2 to participate in subsequent rounds of initiation, the GDP bound to eIF2 is exchanged for GTP in a reaction catalyzed by the guanine nucleotide exchange factor eIF2B. During ER stress, the recognition and refolding of misfolded proteins is assisted by the chaperones DNAJ3C and BiP with the help of the nucleotide exchange factor SIL1. The products of the IER3IP1 and WFS1 genes are also important for ER function. Under non-stress conditions BiP binds to the kinase PERK blocking its activation. The overload of misfolded proteins under stress conditions titrates BiP from PERK leading to activation of the kinase. Activated PERK phosphorylates the α subunit of eIF2, converting eIF2 into a competitive inhibitor of eIF2B. The subsequent decreased availability of eIF2–GTP for TC formation leads to downregulation of overall protein synthesis and increased translation of the ATF4 mRNA. The ATF4 protein, a transcription factor, upregulates genes necessary for the adaptive response to the stress situation including DDIT3 encoding CHOP. Prolonged activation of the stress response and elevated CHOP levels eventually leads to apoptosis. Mutations in genes involved in these processes (gray boxes: gene in red, disorder in black) account for syndromes with overlapping clinical pictures (summarized in Table 3).