Hyperkinetic Movement Disorder Caused by the Recurrent c.892C>T NACC1 Variant

Abstract

Background: Genetic syndromes of hyperkinetic movement disorders associated with epileptic encephalopathy and intellectual disability are becoming increasingly recognized. Recently, a de novo heterozygous NACC1 (nucleus accumbens-associated 1) missense variant was described in a patient cohort including one patient with a combined mitochondrial oxidative phosphorylation (OXPHOS) deficiency.

Objectives: The objective is to characterize the movement disorder in affected patients with the recurrent c.892C>T NACC1 variant and study the NACC1 protein and mitochondrial function at the cellular level.

Methods: The movement disorder was analyzed on four patients with the NACC1 c.892C>T (p.Arg298Trp) variant. Studies on NACC1 protein and mitochondrial function were performed on patient-derived fibroblasts.

Results: All patients had a generalized hyperkinetic movement disorder with chorea and dystonia, which occurred cyclically and during sleep. Complex I was found altered, whereas the other OXPHOS enzymes and the mitochondria network seemed intact in one patient.

Conclusions: The movement disorder is a prominent feature of NACC1-related disease.

Keywords: NACC1; cyclic; hyperkinetic; movement disorder.

FIG. 1

Cellular phenotype, nucleus accumbens‐associated protein 1 (NACC1) expression and expression, function, and assembly of mitochondrial complexes were studied in patient‐derived fibroblasts. (A) Patient‐derived fibroblasts from patient 1 have normal cell morphology and NACC1 is localized in the nucleus; a faint signal is observed in the cytoplasm both in patient‐derived cells and controls. Tom20 antibody was used to visualize the mitochondrial network, which appears normal in patient‐derived cells. Phalloidin:FITC was used to visualize actin cytoskeleton. Images were taken using 63× magnification. (B) Heterozygous expression of nucleus accumbens‐associated 1 (NACC1) c.892C>T variant in patient‐derived fibroblasts was verified using reverse transcription polymerase chain reaction (RT‐PCR) and Sanger sequencing. (C) Immunoblotting of cell lysates from patient‐derived (P) and control (C) fibroblasts with NACC1‐specific antibody. GAPDH was used as loading control. (D) NACC1 levels normalized to GAPDH revealed normal amount of 57‐kDa band detected by NACC1 antibody in patient fibroblasts (P) compared to controls (C). (E) NACC1 gene expression level in patient‐derived cells was studied using quantitative PCR and it did not differ from the control cells. (F,G) Blue Native polyacrylamide gel electrophoresis and protein quantification derived from the western blot method indicated that complex I level in patient cells was 1.5‐fold compared to the control fibroblasts (P < 0.05, two‐tailed Student's t test). Other mitochondrial complexes were expressed at a normal level in patient‐derived fibroblasts. The levels of the marker proteins representative of mitochondrial complexes encoded by mitochondrial DNA were normalized to succinate dehydrogenase complex flavoprotein subunit A (SDHA), thereby representing complex II, which is encoded by the nuclear genome. (H,I) In‐gel activity assay to measure complex I function showed normal function in patient‐derived fibroblasts when compared to two control cell lines. Signal intensity was normalized to control 1 (C1) fibroblast line. Error bars in the images indicate standard deviation.