Novel pathogenic EIF2S3 missense variants causing clinically variable MEHMO syndrome with impaired eIF2γ translational function, and literature review

Rare pathogenic EIF2S3 missense and terminal deletion variants cause the X‐linked intellectual disability (ID) syndrome MEHMO, or a milder phenotype including pancreatic dysfunction and hypopituitarism. We present two unrelated male patients who carry novel EIF2S3 pathogenic missense variants (p.(Thr144Ile) and p.(Ile159Leu)) thereby broadening the limited genetic spectrum and underscoring clinically variable expressivity of MEHMO. While the affected male with p.(Thr144Ile) presented with severe motor delay, severe microcephaly, moderate ID, epileptic seizures responsive to treatments, hypogenitalism, central obesity, facial features, and diabetes, the affected male with p.(Ile159Leu) presented with moderate ID, mild motor delay, microcephaly, epileptic seizures resistant to treatment, central obesity, and mild facial features. Both variants are located in the highly conserved guanine nucleotide binding domain of the EIF2S3 encoded eIF2γ subunit of the heterotrimeric translation initiation factor 2 (eIF2) complex. Further, we investigated both variants in a structural model and in yeast. The reduced growth rates and lowered fidelity of translation with increased initiation at non‐AUG codons observed for both mutants in these studies strongly support pathogenicity of the variants.


| INTRODUCTION
The eukaryotic initiation factor 2 subunit 3 (EIF2S3) gene encodes the γ subunit of the heterotrimeric translation initiation factor 2 (eIF2) complex, crucial for initiation of protein synthesis and regulation of the integrated stress response (ISR). Pathogenic EIF2S3 variants have been linked with different clinical disorders, ranging from a severe neurological phenotype with severe intellectual disability (ID) and extreme microcephaly, usually as part of MEHMO (mental deficiency, epilepsy, hypogenitalism, microcephaly and obesity) syndrome (OMIM 300148), 1-3 to a novel phenotype of hypopituitarism with glucose dysregulation and very mild neurological involvement. 4 While severely affected patients present with all clinical features, less affected patients exhibit only a subset of these features. It remains largely Urania Kotzaeridou and Sara K. Young-Baird contributed equally to this study. unknown why clinical severity varies between patients and how the pathogenic variants impact eIF2γ function; and it is becoming clear that the EIF2S3 variants map to distinct regions of eIF2γ, suggesting that they may impact different functions of eIF2γ. [1][2][3][4][5][6] 2 | METHODS

| Subjects
The study was carried out in accordance with the Declaration of Helsinki. Genetic studies were approved by the local ethical committee of the Technical University Munich (#5360/12S). Written informed consent for publication was obtained from the parents.

| Mutation identification, western blot analysis, and yeast methods
For the index patient from family 1 (Fam1) exome sequencing was performed using a SureSelect Human All Exon Kit (Agilent, 50 Mb V5) for target enrichment and a HiSeq2500 device (Illumina) for sequencing as paired end reads of 100 bp. The average coverage was 130× with more than 97% of the targeted sequence covered >20×. Segregation analysis of the EIF2S3 variant was performed by Sanger sequencing.
For the index patient from family 2 (Fam2) diagnostic genetic testing was performed at the Medical Genetics Center, Munich, Germany. Following NGS only EIF2S3 exons as well as flanking five nucleotides of intronic sequences were analyzed.
Lymphoblastoid cell lines from one affected male (Fam2, II:1) and controls were established by EBV transformation. Details on protein cell lysate preparation and antibodies used for western blot analysis, as well as all yeast methods, are given in the Supporting Information, Appendix S1.

| RESULTS
The boy from Fam1 ( Figure 1A, III:1) is the first child born to nonconsanguineous parents. Pregnancy was uneventful with delivery at 35 + 1 gestational weeks due to pathologic antepartal cardiotocograms.
His birth length was 41.5 cm (−2.24 SD), weight 1840 g (−1.85 SD), Apgar score 8/8 at 5 and 10 minutes and occipitofrontal circumference (OFC) 27.5 cm (−3.56 SD). Postnatally he presented with respiratory distress grade 1, poor feeding, coronary hypospadia, microcephaly and muscular hypotonia. At 7 months he was admitted for generalized tonic-clonic seizures. His development was significantly delayed. At last follow-up, epileptic seizures were well controlled under topimarate monotherapy. Non-autoimmune diabetes mellitus was diagnosed, and insulin treatment was started. He has moderate ID (FSIQ 40, WISC-IV test) with autistic features. Language skills are limited to less than five words. He does not walk independently and cannot perform any daily tasks. Brain magnetic resonance imaging (MRI) at 9 months showed delayed myelination corresponding to that normally seen at 4 to 5 months ( Figure 1D, e-h). By 4.5 years myelination had progressed to what is normally seen at 9 to 10 months, but was still incomplete ( Figure 1D, i-l). In addition, there was marked atrophy of supratentorial white matter. Details on white matter quantification are given in Appendix S1.
Facial and dysmorphic features include narrow forehead, full cheeks, increase in supraorbital soft tissue, relatively large ears with prominent earlobes, short philtrum, long eyelashes and thick eyebrows, micrognathia ( Figure 1B, Fam1 III:1, Table 1), mild edematous hands and feet, and tapered fingers (not shown).
Genetic testing revealed a novel maternally inherited hemizygous  Table 1). His ID is moderate and he has behavioral problems. Both variants affect highly conserved amino acids ( Figure S1A) and are not present in control databases including 1000 Genomes and gnomAD.
In addition, by western blot analysis of protein cell lysate from lymphoblastoid cells of the affected male from Fam2 we could show that mutant eIF2γ protein is present ( Figure 1C).
Consisting of distinct α, β, and γ subunits, the stable eIF2 heterotrimer binds GTP and the initiator Met-tRNA i Met to form a ternary complex, which then binds to the small ribosomal subunit. 9 The eIF2γ subunit consists of an N-terminal G domain followed by two β-barrel domains (Figure 2A,B). The residue I159 (yeast I218) lies at the end of strand β6 ( Figure 2B), which helps buttress the position of the NKxD motif that contributes to guanine specificity and nucleotide binding affinity. 10 Mutation of this residue could alter the position of the NKxD motif, and thereby affect GTP binding. The T144 residue (yeast T203) is located at the C-terminus of the Switch 2 (Sw2) element ( Figure 2B) that responds to GTP vs GDP binding. 10 Mutation of T203 might impair eIF2 function by weakening GTP binding or by disrupting structural transitions necessary for binding Met-tRNA i Met . 11,12 To test if the I159L and T144I mutations impair eIF2 function, analogous mutations were introduced into yeast eIF2γ. Like the eIF2γ-I318M and eIF2γ-V281K mutations, corresponding to the MEHMO mutations I259M and I222T, 1,5 the yeast I218L (human I159L) mutation conferred a significant slow-growth phenotype in yeast ( Figure 2C, rows 1, 5, 7, 10). Whereas the yeast T203I (human T144I) mutation did not impact yeast cell growth ( Figure S1, row 6), substitution of Ala (T203A; Figure 2D, row 10) but not Lys (T203K, Figure S1B, row 8) conferred a slow-growth phenotype.
Overexpression of tRNA i Met and eIF2β were previously shown to suppress the slow-growth phenotypes associated with the yeast eIF2γ-I318M (corresponding to human I259M, impaired for Met-tRNA i Met binding) and eIF2γ-V281K (human I222T, impaired for eIF2β binding) mutations ( Figure 2C,D, rows 5-9), respectively. 1,5 Intriguingly, overexpression of tRNA i Met , but not eIF2α or eIF2β, enhanced the growth of the eIF2γ-I218L and eIF2γ-T203A mutant strains to The following definitions are used to categorize weight status: Underweight-BMI <5th percentile for age and sex; normal weight-BMI between the 5th and <85th percentile for age and sex; Overweight-BMI between >85th and 95th percentile for age and sex; Obese-BMI ≥95th percentile for age and sex; severe obesity-severe (class II) obesity is defined as BMI ≥120% of the 95th percentile values or a BMI ≥35 kg/m 2 . c Three other male family members were reported with a similar phenotype including neonatal hypoglycemia, severe microcephaly, developmental delay, micropenis, short stature, epileptic seizures and early death. They were unavailable for genetic testing.
near WT levels ( Figure 2C,D, rows 10-13), suggesting that these G domain mutations might directly or indirectly affect Met-tRNA i Met binding to eIF2. As GTP and Met-tRNA i Met binding to eIF2 is thermodynamically coupled such that increasing the levels of either binding partner will enhance ternary complex formation, 13 and based on the location of the T144I and I159L mutations in critical elements of the G domain, we propose that the new MEHMO mutations impair eIF2 function by weakening GTP binding.
To more directly test the impact of the I218L and T203A mutations on eIF2 function, we used reporter assays to assess translational  We therefore propose including EIF2S3 mutation search in the differential diagnosis of such unsolved cases.
In conclusion, this study establishes the link between two novel EIF2S3 missense variants identified in two unrelated affected males with pathogenicity supported by the structural model of eIF2 and impaired eIF2γ translational function in yeast. Further, it strongly supports clinically variable expressivity of MEHMO in patients with deleterious EIF2S3 and eIF2γ changes.