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. 2023 Nov 16;24(22):16400.
doi: 10.3390/ijms242216400.

Genetic Heterogeneity Underlying Phenotypes with Early-Onset Cerebellar Atrophy

Dolores Martínez-Rubio  1   2 , Isabel Hinarejos  1   2 , Herminia Argente-Escrig  3 , Clara Marco-Marín  4   5 , María Ana Lozano  1 , Nerea Gorría-Redondo  6 , Vincenzo Lupo  1 , Itxaso Martí-Carrera  7 , Concepción Miranda  8 , María Vázquez-López  8 , Asunción García-Pérez  9 , Ana Victoria Marco-Hernández  10 , Miguel Tomás-Vila  11 , Sergio Aguilera-Albesa  6 , Carmen Espinós  1   2   5   12
Affiliations

Genetic Heterogeneity Underlying Phenotypes with Early-Onset Cerebellar Atrophy

Dolores Martínez-Rubio et al. Int J Mol Sci. .

Abstract

Cerebellar atrophy (CA) is a frequent neuroimaging finding in paediatric neurology, usually associated with cerebellar ataxia. The list of genes involved in hereditary forms of CA is continuously growing and reveals its genetic complexity. We investigated ten cases with early-onset cerebellar involvement with and without ataxia by exome sequencing or by a targeted panel with 363 genes involved in ataxia or spastic paraplegia. Novel variants were investigated by in silico or experimental approaches. Seven probands carry causative variants in well-known genes associated with CA or cerebellar hypoplasia: SETX, CACNA1G, CACNA1A, CLN6, CPLANE1, and TBCD. The remaining three cases deserve special attention; they harbour variants in MAST1, PI4KA and CLK2 genes. MAST1 is responsible for an ultrarare condition characterised by global developmental delay and cognitive decline; our index case added ataxia to the list of concomitant associated symptoms. PIK4A is mainly related to hypomyelinating leukodystrophy; our proband presented with pure spastic paraplegia and normal intellectual capacity. Finally, in a patient who suffers from mild ataxia with oculomotor apraxia, the de novo novel CLK2 c.1120T>C variant was found. The protein expression of the mutated protein was reduced, which may indicate instability that would affect its kinase activity.

Keywords: ataxia; cerebellar atrophy; exome sequencing; gene panel; neuroimaging; rare disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Structural modelling. (A) The crystallographic structure of the yeast SEN1 helicase domain (PDB entry 5mzn) is shown in the cartoon representation. Residue corresponding to I1942 in human senataxin (SETX), where missense mutation p.I1942T has been identified, is marked with a red sphere, to show its proximity to de nucleotide binding site. The inset shows a close-up view of the region where the mutation is located. The nucleotide is shown in stick representation with carbon, oxygen, nitrogen, and phosphorus atoms coloured yellow, red, blue, and orange respectively. (B) Cartoon representation of the protein kinase and pzd domains of the model generated by AlphaFold for human MAST1 (accession number Q9Y2H9). Localization of p.V558L is represented with a red sphere. (C) Cartoon representation of the cryo-electron microscopy structure for the calcium translocating pore of human Cav3.1 (PDB entry 6kzo), from the cytosolic region. Ca2+ are shown as purple spheres. The red sphere localizes residue A961, where p.A961T has been identified. The inset shows a close-up of the region where this mutation is localized, in the structure of the voltage-gated calcium channel Cav1.1 from rabbit (PDB entry 5gjv), to show the proximity of the mutation to the elements from the α subunit that mediate the interaction with the β regulatory subunit of this channel. (D) Cartoon representation of the alpha solenoid structure predicted for TBCD (AlphaFold model; accession code Q9BTW9). Localization of p.N1033K is mapped with a red sphere. The inset shows in detail the region where N1033 is located, with its side chain representing a stick, and on the right, the structural model of the p.N1033K mutation.
Figure 2
Figure 2
Main neuroimaging signs. (A,B) MD-353/CACNA1G, severe vermian atrophy in mid-sagittal T1-weighted image (A) and mild global atrophy in both cerebellar hemispheres in coronal T2 (B) at 17 years of age. (C) MD-309/CACNA1A, a 12 year-old with moderate atrophy in the anterior lobe of the cerebellar vermis and a mega cisterna magna in mid-sagittal T1 (white arrow). (D) MD-556/CACNA1A at 10 years, moderate atrophy in the anterior lobe of the cerebellar vermis (T1-weighted sagittal image). (E,F) MD-548/MAST1 at 13 years, T2 axial image showing thinning of anterior arm of internal capsule (E), and T1 mid-sagittal image with a globally increased corpus callosum and vermian reduced volume (F). (G) MD-392/CPLANE1 at 17 years, T2 axial image showing a lack of normal decussation of superior cerebellar peduncular fiber tracts that follow and horizontal course (molar tooth sign). (H) MD-297/TBCD at 18 years, T2 FLAIR axial image revealing a striking reduction of supratentorial white matter, severe brainstem and cerebellar atrophy was also ascertained. (I,J) MD-471/CLN6, at 5 years (I), T1 coronal image showing enlarged cerebellar folia spaces (white arrow) at 6.5 years (J), T2 axial image disclosed cortical atrophy and deep/periventricular white matter hyperintensities. (K) MD-610/PI4KA, at 30 months, T2 coronal image showing diffuse hypomyelination and enlarged cerebellar folia interspaces. (L) MD-436/CLK2 at 20 months of age, with mild enlargement of folia spaces in the anterior lobe of the cerebellar vermis in mid-sagittal T1.
Figure 3
Figure 3
Studies to investigate the pathogenicity of variants of interest. (A) Transcript analysis of CPLANE1 c.7588+7A>G. Schematic representation of the aberrant splicing. Exons are indicated by boxes and introns are represented by horizontal bars (not to scale). Location of specific primers used in RT-qPCR are indicated by arrows. Dotted lines represent the anomalous splicing outcome and sequence of the abnormal product is shown (electropherogram). Electrophoresis corresponds to the RT-qPCR products obtained from RNA of a control and MD-392, who is heterozygous for CPLANE1 c.7588+7A>G. (B) Detection and transcript analysis of CPLANE1 c.2747-1981_6172-78del. Copy number variation (CNV) detection based on gene panel data using DECoN (upper image). Visualization plot from DECoN shows the ratio of observed to expected coverage depth of the captured regions with a 95% confidence. Significantly lower scores values suggested a heterozygous deletion from exon 16 to 32 (red dots); DECoN results were validated through CPLANE1 transcript analysis (middle image). RT-PCR was performed on RNA derived from the patient MD-392. Sequencing of the mutated amplicon showed the complete deletion of exons 16 to 32 (electropherogram), which implies a change in the reading frame. Size and genomic breakpoints of the CNV were defined (lower image) by PCR and Sanger sequencing revealed a microhomology region at the breakpoint junction (dash boxed sequence). (C) Transcript analysis of PI4KA c.3845C>T. Schematic representation of the aberrant splicing; please see description in (A). Electrophoresis corresponds to the RT-qPCR products obtained from RNA of a control and MD-610, who is heterozygous for PI4KA c.3845C>T. (D) Expression analysis of CLK2 p.Y374H. Western-blot analysis of protein extracts obtained from HEK293T cells, transiently transfected with CLK2 gene wild-type or mutant (p.Y374H), indicates a decreased relative expression of CLK2-mutant compared to the native protein. Three biological replicates are shown. Data is presented as average ± SEM. Significance was determined by two-tailed Student’s t test. * p < 0.005.
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