EIF2AK2 Missense Variants Associated with Early Onset Generalized Dystonia.

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.

Emerging and converging molecular mechanisms in dystonia.

Dystonia is a clinically, genetically, and biologically heterogeneous hyperkinetic movement disorder caused by the dysfunctional activity of neural circuits involved in motor control. Our understanding of the molecular mechanisms underlying dystonia pathogenesis has tremendously grown thanks to the accelerated discovery of genes associated with monogenic dystonias (DYT-genes). Genetic discoveries, together with the development of a growing number of cellular and animal models of genetic defects responsible for dystonia, are allowing the identification of several areas of functional convergence among the protein products of multiple DYT-genes. Furthermore, unexpected functional links are being discovered in the downstream pathogenic molecular mechanisms of DYT-genes that were thought to be unrelated based on their primary molecular functions. Examples of these advances are the recognition that multiple DYT-genes are involved in (1) endoplasmic reticulum function and regulation of the integrated stress response (ISR) through Eukaryotic initiation factor 2 alpha signaling; (2) gene transcription modulation during neurodevelopment; (3) pre-and post-synaptic nigrostriatal dopaminergic signaling; and (4) presynaptic neurotransmitter vesicle release. More recently, genetic defects in the endo-lysosomal and autophagy pathways have also been implicated in the molecular pathophysiology of dystonia, suggesting the existence of mechanistic overlap with other movement disorders, such as Parkinson's disease. Importantly, the recognition that multiple DYT-genes coalesce in shared biological pathways is a crucial advance in our understanding of dystonias and will aid in the development of more effective therapeutic strategies by targeting these convergent molecular pathways.

Effect of oral calcium administration on metabolic status and uterine health of dairy cows with reduced postpartum rumination and eating time.

BACKGROUND: Hypocalcemia has detrimental effects on health and performance of dairy cows. As hypocalcemic cows show reduced feed intake, we hypothesized that cows with reduced combined rumination and eating time (CRET) may benefit from Ca supplementation. The objective was to evaluate the effect of postpartum oral Ca administration on metabolic status (Calcium [Ca], fatty acids [FA], and ß-Hydroxybutyrate [BHB] serum concentrations) and incidence of puerperal metritis (PM) in dairy cows with reduced postpartum CRET. Cows in an organic-certified dairy, diagnosed with reduced CRET (< 489 min/d; n = 88) during the first day postpartum were assigned into 1 of 2 treatments: i) Calcium administration (CA; n = 45) that received 1 Ca oral capsule (Bovikalc bolus, Boehringer Ingelheim, St. Joseph, MO) containing CaCl2 and CaSO4 (43 g of Ca) once per day, for 3 consecutive days, starting at d 1 postpartum; and ii) Control (CON; n = 43) that did not receive oral Ca. A convenience group consisting of cows with CRET ≥489 min/d was used for comparison and did not receive oral Ca (NOR; n = 96). RESULTS: At day 1 postpartum cows with reduced CRET had lower Ca serum concentrations (CA = 2.08 mmol/L; CON = 2.06 mmol/L) compared with NOR cows (2.17 mmol/L). Calcium concentrations at d 3, 5, and 12 postpartum were not different among the three groups. Serum FA concentrations at d 1, 3 and 5 postpartum were higher in both CA and CON cows compared with NOR. At d 12, only CA cows had higher FA concentrations than NOR cows. Serum BHB concentrations at d 3 were highest in CA, with no difference between CON and NOR. At d 5, BHB concentrations were higher in CA, followed by CON, and NOR. No effect was observed for Ca administration on incidence of PM and reproductive performance. CON cows had lower survival at 30 DIM (86.5%) than NOR cows (97.9%). CONCLUSIONS: The use of remote sensor technology identified cows with reduced rumination and eating time that had lower postpartum serum concentrations of calcium and altered metabolic status. However, oral calcium administration to cows with reduced CRET did not affect incidence of metabolic disorders nor reproductive health and subsequent pregnancy. Although survival at 30 days postpartum was lower for non-Ca supplemented cows, the identification of effective interventions in cows with reduced CRET requires further consideration.

Accelerating therapeutic development and clinical trial readiness for STXBP1 and SYNGAP1 disorders.

Gene-targeted therapies for genetic neurodevelopmental disorders (NDDs) are becoming a reality. The Center for Epilepsy and Neurodevelopmental Disorders (ENDD) is currently focused on the development of therapeutics for STXBP1 and SYNGAP1 disorders. Here we review the known clinical features of these disorders, highlight the biological role of STXBP1 and SYNGAP1, and discuss our current understanding of pathogenic mechanisms and therapeutic development. Finally, we provide our perspective as scientists and parents of children with NDDs, and comment on the current challenges for both clinical and basic science endeavors.

Generation of humanized mouse models to support therapeutic development for SYNGAP1 and STXBP1 disorders.

Heterozygous variants in SYNGAP1 and STXBP1 lead to distinct neurodevelopmental disorders caused by haploinsufficient levels of post-synaptic SYNGAP1 and pre-synaptic STXBP1, which are critical for normal synaptic function. While several gene-targeted therapeutic approaches have proven efficacious in vitro, these often target regions of the human gene that are not conserved in rodents, hindering the pre-clinical development of these compounds and their transition to the clinic. To overcome this limitation, here we generate and characterize Syngap1 and Stxbp1 humanized mouse models in which we replaced the mouse Syngap1 and Stxbp1 gene, respectively, with the human counterpart, including regulatory and non-coding regions. Fully humanized Syngap1 mice present normal viability and can be successfully crossed with currently available Syngap1 haploinsufficiency mouse models to generate Syngap1 humanized haploinsufficient mice. Stxbp1 mice were successfully humanized, yet exhibit impaired viability (particularly males) and reduced STXBP1 protein abundance. Mouse viability could be improved by outcrossing this model to other mouse strains, while Stxbp1 humanized females and hybrid mice can be used to evaluate target engagement of human-specific therapeutics. Overall, these humanized mouse models represent a broadly available tool to further pre-clinical therapeutic development for SYNGAP1 and STXBP1 disorders.