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. 2018 Jul 24;91(4):e319-e330.
doi: 10.1212/WNL.0000000000005869. Epub 2018 Jun 29.

Biallelic SQSTM1 Mutations in Early-Onset, Variably Progressive Neurodegeneration

Free PMC article

Biallelic SQSTM1 Mutations in Early-Onset, Variably Progressive Neurodegeneration

Valentina Muto et al. Neurology. .
Free PMC article


Objective: To characterize clinically and molecularly an early-onset, variably progressive neurodegenerative disorder characterized by a cerebellar syndrome with severe ataxia, gaze palsy, dyskinesia, dystonia, and cognitive decline affecting 11 individuals from 3 consanguineous families.

Methods: We used whole-exome sequencing (WES) (families 1 and 2) and a combined approach based on homozygosity mapping and WES (family 3). We performed in vitro studies to explore the effect of the nontruncating SQSTM1 mutation on protein function and the effect of impaired SQSTM1 function on autophagy. We analyzed the consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in vivo using zebrafish as a model.

Results: We identified 3 homozygous inactivating variants, including a splice site substitution (c.301+2T>A) causing aberrant transcript processing and accelerated degradation of a resulting protein lacking exon 2, as well as 2 truncating changes (c.875_876insT and c.934_936delinsTGA). We show that loss of SQSTM1 causes impaired production of ubiquitin-positive protein aggregates in response to misfolded protein stress and decelerated autophagic flux. The consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in zebrafish documented a variable but reproducible phenotype characterized by cerebellum anomalies ranging from depletion of axonal connections to complete atrophy. We provide a detailed clinical characterization of the disorder; the natural history is reported for 2 siblings who have been followed up for >20 years.

Conclusions: This study offers an accurate clinical characterization of this recently recognized neurodegenerative disorder caused by biallelic inactivating mutations in SQSTM1 and links this phenotype to defective selective autophagy.


Figure 1
Figure 1. Pedigrees of the 3 families included in the study and location of the identified homozygous SQSTM1 mutations
(A) Family trees. Squares indicate males; circles indicate females. Solid symbols indicate affected individuals; open symbols represent unaffected relatives. (B) Sequence chromatograms showing homozygosity for the SQSTM1 mutations identified in affected members of the 3 families. (C) Functional and structural domains of SQSTM1 and localization of the identified mutations. KIR = Keap1-interacting; LIR = LC3-interaction region; PB1 = Phox 1 and Bem1p; TRAF6 = tumor necrosis factor receptor–associated factor 6; UBA = ubiquitin-associated; ZZ = zinc finger.
Figure 2
Figure 2. Brain imaging
Brain MRI of the affected siblings of family 1: (A–C) F1:V.5 (age 38 years) and (D–F) F1:V.6 (age 31 years). (A, B, D, E) Axial and (C and F) coronal fast spin echo T2 images document the absence of any gross anomaly (enlarged cisterna magna in patient F1:V.6), cortical/cerebellar atrophy, or iron accumulation in basal ganglia. Single-voxel proton magnetic resonance brain spectroscopy using intermediate echo time (144 milliseconds) in patient F1:V.5 documenting an inverted doublet of lactate at 1.33 ppm (G) visible at the level of the ventricular CSF (white arrow) and (H) barely visible in basal ganglia. (I) 99mTc-HMPAO SPECT transaxial images (from the vertex to the cerebellum) in patient F1:V.5 shows mild to moderate reduction in radiotracer uptake (hypoperfusion) in the right parietal cortex and in the right and left frontal cortices, as well as asymmetric radiotracer uptake in the cerebellar hemispheres (right < left). (J) In patient 2, mild reduction in radiotracer uptake (hypoperfusion) is documented in the right and left frontal cortices, but no perfusion asymmetry is seen in the cerebellar hemispheres.
Figure 3
Figure 3. Effect of the c.301+2T>A change on transcript processing and protein stability
(A) The homozygous c.301+2T>A substitution causes skipping of coding exon 2. Amplification of the cDNA fragment encompassing coding exons 1 to 4 reveals an aberrant PCR product lacking exon 2 in the 2 affected siblings; wild-type and aberrant processed transcripts are observed in both parents (top). Sequencing of the mature SQSTM1 transcript from affected individual F1:V.5 documents skipping of coding exon 2 and the preservation of the phase of the reading frame (bottom). (B) Western blot analysis performed on skin fibroblasts from individual F1:V.5 (right), the unaffected mother (middle), and an unrelated individual (left), indicating that the mutated SQSTM1 protein lacking the amino acid portion encoded by exon 2 (arrow) has a dramatically reduced level compared to the wild-type protein. (C) Treatment of fibroblasts with MG132 (6 hours), an inhibitor of proteasome function, results in a marked increased level of the SQSTM1 mutant, indicating that this protein is rapidly degraded via proteasome. (D) Incubation with bafilomycin A1 (baf) and Earle balanced salt solution (EBSS) (6 hours) does not result in any significant change in the level of the mutated SQSTM1 protein, ruling out a major role of autophagy in the degradation of the SQSTM1 mutant.
Figure 4
Figure 4. Investigation of the autophagic flux
(A) Microtubule-associated protein 1A/1B-light chain (3LC3) levels were analyzed in control and patient fibroblasts treated with Earle balanced salt solution (EBSS) for different times. Western blot analysis shows that LC3II accumulates in patients' fibroblasts. After incubation of the cells in EBSS and treatment with 200 nmol/L bafilomycin (2 hours), no significant differences were seen between control and patient cells. (B) Confocal laser scanner microscopy analysis was performed in the same experimental conditions reported in panel A and confirms the same findings. Bars correspond to 40 μm.
Figure 5
Figure 5. Analysis of autophagic flux and formation of inclusion bodies containing ubiquitinated proteins
(A) Primary fibroblasts were incubated with bovine serum albumin derivative (dye-conjugated [DQ] BSA) conjugated to a self-quenched fluorophore (10 µg/mL) for 1 hour at 37°C in complete culture medium and then left in a starvation medium for 5 and 16 hours to induce autophagy. Cells were mounted on coverslips and immediately analyzed by confocal microscopy. Dequenched DQ-BSA was not observed over the time period of 16 hours in patient cells. (B) Primary fibroblasts obtained from one of the 2 affected sibs (right) and an unaffected individual (left) were treated with puromycin (5 μg/mL) for 2 hours to induce formation of polyubiquitin-positive bodies. Cells were then fixed and stained with SQSTM1 (green) and FK1 (red) antibodies. Images are representative of 300 analyzed cells. In all panels, bars correspond to 40 μm.
Figure 6
Figure 6. In vivo analysis in zebrafish embryos
(A) Representative dorsal images of anti–α-tubulin (acetylated state)–stained larvae from 4 experimental conditions: control, sqstm1 morpholino (MO) injected, sqstm1_MO+SQSTM1_WT, and sqstm1_MO+SQSTM1_p.69-100del. Blowups from the highlighted area in panel A showing in detail the cerebellar structures in each of the 4 respective conditions presented. (B) Quantification of the qualitative scoring assaying the cerebellar integrity across 3 biological replicas.

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