EIF2AK2 Missense Variants Associated with Early Onset Generalized Dystonia

Abstract

Objective: The study was undertaken to identify a monogenic cause of early onset, generalized dystonia.

Methods: Methods consisted of genome-wide linkage analysis, exome and Sanger sequencing, clinical neurological examination, brain magnetic resonance imaging, and protein expression studies in skin fibroblasts from patients.

Results: We identified a heterozygous variant, c.388G>A, p.Gly130Arg, in the eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2) gene, segregating with early onset isolated generalized dystonia in 5 patients of a Taiwanese family. EIF2AK2 sequencing in 191 unrelated patients with unexplained dystonia yielded 2 unrelated Caucasian patients with an identical heterozygous c.388G>A, p.Gly130Arg variant, occurring de novo in one case, another patient carrying a different heterozygous variant, c.413G>C, p.Gly138Ala, and one last patient, born from consanguineous parents, carrying a third, homozygous variant c.95A>C, p.Asn32Thr. These 3 missense variants are absent from gnomAD, and are located in functional domains of the encoded protein. In 3 patients, additional neurological manifestations were present, including intellectual disability and spasticity. EIF2AK2 encodes a kinase (protein kinase R [PKR]) that phosphorylates eukaryotic translation initiation factor 2 alpha (eIF2α), which orchestrates the cellular stress response. Our expression studies showed abnormally enhanced activation of the cellular stress response, monitored by PKR-mediated phosphorylation of eIF2α, in fibroblasts from patients with EIF2AK2 variants. Intriguingly, PKR can also be regulated by PRKRA (protein interferon-inducible double-stranded RNA-dependent protein kinase activator A), the product of another gene causing monogenic dystonia.

Interpretation: We identified EIF2AK2 variants implicated in early onset generalized dystonia, which can be dominantly or recessively inherited, or occur de novo. Our findings provide direct evidence for a key role of a dysfunctional eIF2α pathway in the pathogenesis of dystonia. ANN NEUROL 2021;89:485-497.

FIGURE 1

Pedigrees and genetic analyses. (A–E) Pedigrees of the patients with EIF2AK2 variants. Subjects with DNA available are labeled with individual codes; filled symbols denote individuals affected with generalized dystonia; half‐filled symbol indicates right arm and hand dystonia; top‐right quarter‐filled symbol indicates history of mild foot dystonia and asymptomatic upon examination; top‐left quarter‐filled symbol indicates mild bilateral hand tremor; M/M, homozygous carrier of denoted variant; M/−, heterozygous carrier; −/−, wild‐type. (F) Autosomal dominant and X‐linked linkage analysis plots. LOD = logarithm of odds. (G) Linkage analysis plot for chromosome 2 and location of the EIF2AK2 gene. (H) Schematic representation of the EIF2AK2 protein, with functional domains and the relative positions of variants identified in this study. [Color figure can be viewed at www.annalsofneurology.org]

FIGURE 2

Neuroimaging. (A–C) Brain magnetic resonance imaging (MRI; T2‐axial view) in Patient C‐II‐1 (de novo p.Gly130Arg variant), showing abnormal, focal concentric, symmetric hyperintense signal abnormalities in the posterior brain stem at the bulbomedullary junction. (D, E) Fluid attenuated inversion recovery (FLAIR) MRI images (axial view) in Patient E‐II‐3 (p.Asn32Thr variant), showing bilateral frontal–parietal atrophy and mild hyperintense signal in the posterior periventricular white matter.

FIGURE 3

Expression studies. Dystonia‐associated EIF2AK2 variants lead to prolonged phosphorylation of EIF2AK2 Thr446 and downstream target eIF2α‐Ser51 in patient‐derived fibroblasts in response to polyinosinic acid:polycytidylic acid (poly[I:C]) exposure. (A) Schematic representation of the EIF2AK2 protein structure double‐stranded RNA binding motif (DRBM). Locations of the variants detected in the patients are indicated. (B) Schematic diagram showing the downstream effectors of the EIF2AK2 pathway after treatment with poly(I:C). (C) Representative Western blots of protein extracts from control (Control 1, 2, 3) and EIF2AK2 patient (E‐II‐3, B‐II‐1, A‐III‐4) fibroblast cell cultures that were either untreated or treated with poly(I:C) (2μg/ml) for 16 hours. Protein extracts were probed on the same blot for expression of phosphorylated EIF2AK2 (EIF2AK2 P‐Thr446), total EIF2AK2 protein levels (EIF2AK2), phosphorylated eIF2α (eIF2α P‐Ser51), and total eIF2α (eIF2α) protein levels. Vinculin was used as protein loading control. Molecular weights are indicated on the left (kDa). (D) Quantification of total EIF2AK2 levels in untreated cell cultures showing no significant changes between control and patient fibroblast cell cultures (n = 5). (E) Quantification of phosphorylated Ser51 eIF2α protein levels in untreated cell cultures, indicating no changes in EIF2AK2 variant carrying patient cells compared to unrelated control fibroblasts (n = 5). (F) Quantification of the fold change of phosphorylated EIF2AK2 Thr446 over total EIF2AK2 protein levels after poly(I:C) treatment (2μg/ml) for 16 hours normalized to untreated cells. (G) Quantification of the fold change of phosphorylated eIF2αSer51 over total eIF2α protein levels after poly(I:C) treatment (2μg/ml) for 16 hours normalized to untreated cells. Two‐way analysis of variance with Tukey multiple comparisons between controls and subjects with EIF2AK2 variants were performed. Values represent mean ± standard error of the mean (n = 3 biological replicates; ****p < 0.0001). [Color figure can be viewed at www.annalsofneurology.org]

FIGURE 4

Evidence for dysregulated eIF2α signaling as a shared theme in the pathogenesis of dystonia. Identification of EIF2AK2 variants in this study provides further support for dysregulated eIF2α signaling as a shared pathogenetic theme in dystonia. Evidence for a disturbed integrated stress response (ISR) has been previously reported in other monogenic causes of dystonia, including DYT‐PRKRA, DYT‐TOR1A, DYT‐THAP1, and DYT‐SGCE. Several studies have shown that variants in the PRKRA gene enhanced the susceptibility to endoplasmic reticulum stress leading to heightened EIF2AK2 activation, dysregulation of ISR, and increased apoptosis. ³² , ³⁴ , ³⁵ ISR dysregulation has been reported to play a central role in DYT‐TOR1A. ¹⁰ , ¹¹ , ³⁶ THAP1 mutations have been shown to cause dysregulation of the eIF2α signaling pathways in a DYT‐THAP1 mouse model. ¹² In a mouse model of DYT‐SGCE, significantly elevated levels of EIF2AK2 transcript are reported, and genes associated with protein translation are among the top downregulated mRNAs. ³⁷ Additionally, rare variants in ATF4—a direct target of eIF2α signaling—have also been reported in cervical dystonia patients. ¹¹ [Color figure can be viewed at www.annalsofneurology.org]