A Novel Synonymous Variant in SQSTM1 Causes Neurodegeneration With Ataxia, Dystonia, and Gaze Palsy Revealed by Urine-Derived Cells-Based Functional Analysis

Abstract

Background: Heterozygous variants of sequestosome-1 gene (SQSTM1) have been reported in patients with various neurological disorders, whereas biallelic pathogenic variants of SQSTM1 can cause child-onset and multisystem neurodegeneration, including cerebellar ataxia, dystonia, and vertical gaze palsy (NADGP). Here, we describe two cases of NADGP in a Japanese family.

Methods: We performed clinical and genetic laboratory evaluations of the two patients and their healthy parents.

Results: By whole-exome sequencing, we identified compound heterozygous variants in SQSTM1(NM_003900.5): c.1A>G p.(Met1?) in the initial codon, and c.969G>A, located at the 3' end of exon 6, which is novel and seemingly a synonymous but is actually a truncating variant causing aberrant splicing. An SQSTM1 protein expression assay using urine-derived cells (UDCs) demonstrated that both variants (c.1A>G and c.969G>A) were unable to induce normal splicing of premessenger RNA. Cerebellar ataxia is a characteristic manifestation of this disorder; however, brain magnetic resonance imaging studies have not shown significant cerebellar atrophy. Our patients experienced chorea during adolescence.

Conclusions: Only a few reports have highlighted the presence of chorea; however, our findings suggest that NADGP should be considered as a differential diagnosis of hereditary chorea. This study also demonstrates the utility of UDCs, obtained using noninvasive approaches, in functionally analyzing genetic diseases.

Keywords: GC‐AT intron; NADGP; SQSTM1; UDC; urine‐derived cell.

FIGURE 1

Clinical and genetic features of a family with sequestosome‐1 gene (SQSTM1) variants. (A) Pedigree of the family. (B) Direct nucleotide sequencing of PCR‐amplified DNA of the SQSTM1 gene. The red arrow denotes the substituted bases. (C) Schematic diagram of the DNA sequences of the wild‐type SQSTM1 alleles around c.G969 [indicated by the color red]. ■/●, affected individuals; →, proband; MRI, magnetic resonance imaging.

FIGURE 2

Brain MRI findings of Patient 1 and Patient 2. Brain MRI revealed normal results pertaining to the cerebellar (A–C, E–G) and cerebral hemispheres (D, H). (A–D): Patient 1. (E–H): Patient 2. MRI, magnetic resonance imaging.

FIGURE 3

Phase contrast microscopy image of urine‐derived cells (UDCs) during second passage. The images were taken on the second day after passage. Scale bar denotes 200 μm.

FIGURE 4

SQSTM1 protein expression was not observed in patient‐derived UDCs. Immunoblotting of SQSTM1 and GAPDH proteins extracted during third passage. Anti‐GAPDH antibody was used as a loading control. SQSTM1, sequestosome‐1; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; UDCs, urine‐derived cells.