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case_images: Magnetic resonance imaging (MRI) scans (A-F) of the subject's brain. Progressive cerebellar and vermis atrophy were observed within a period of 28 months.
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case_images: The affected subject was a 4.5-year-old female who presented with developmental delay and failure to thrive since the age of 5 months old (Figure S1). Detailed inspection of the MRI revealed a mild progressive cerebellum atrophy, as indicated by patchy high signal intensity on axial T2-weighted imaging (T2WI [Figure 1A] and Supplementary materials), and fluid-attenuated inversion recovery (FLAIR) sequences within the cerebellar cortex and vermis (Figure S2A) but not the cerebellar white matter. Hyperintensities were also observed in coronal-T2WI and sagittal-T1 FLAIR sequence images (Figure 1B and C, respectively). Twenty-eight months later (at age 3 years and 10 months), MRI of the axial and coronal-T2WI sequences displayed progressive atrophy of the cerebellar cortex and vermis (Figure 1D and E) with 7.5% reduction in cerebellar hemispheres size, fracture enlargements and 20% reduction in the relative sagittal length of cerebellum (Figure 1F). MRI sections of both the axial- and coronal-T2WI taken 12 months post-KD initiation (age 5 years and 5 months, Figure 5A and B) showed no progression of the cerebellar and vermis atrophy, compared with the previous MRI (June 2016, Figure 1D and E). Figure 1D and E). Furthermore, compared with the MRI taken in 2016, the sizes of the right and left cerebellar hemispheres were maintained (Figure 5B vs Figure 1E), and there was a slight increase in the relative sagittal length of cerebellum (Figure 5C vs Figure 1F).
case_images: Family Pedigree and ATAD3A c.251C>T (p.Thr84Met) single nucleotide variant (SNV). (A) Pedigree of the studied family, indicating the affected female subject born from consanguineous non-affected parents and with two non-affected elder siblings. Prior to the subject's birth, the mother had experienced three miscarriages. (B) The ATAD3A c.251C>T variation leads to an amino acid substitution at position 84 (p.Thr84Met). (C) Sanger validation of the ATAD3A variant in the family. The affected subject carries a homozygous variation on ATAD3A (rs546711654 c.251C>T; p.Thr84Met), while all other family members are carriers.
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case_images: Detailed inspection of the MRI revealed a mild progressive cerebellum atrophy, as indicated by patchy high signal intensity on axial T2-weighted imaging (T2WI [Figure 1A] and Supplementary materials), and fluid-attenuated inversion recovery (FLAIR) sequences within the cerebellar cortex and vermis (Figure S2A) but not the cerebellar white matter. Brain diffusion-weighted imaging (DWI) sequences showed no diffusion, suggesting absence of brain malignancy or infarction (Figure S2B). No diffusion restrictions were observed in the brain DWI sequences (Figure S2B). Prior to the subject's birth, the mother had experienced three first-trimester miscarriages without registered detailed examination (Figure 2A). Using a frequency filter of minor allele frequency <0.1% in public databases, we identified one homozygous single nucleotide variant (SNV) in the ATAD3A locus of the subject (c.251C>T; p.Thr84Met [rs546711654 g.chr1:1451437C>T [hg19] (2009)]) (Figure 2B). Both SNVs were confirmed by Sanger sequencing using genomic DNA isolated from all family members and primers specific to ATAD3A, which did not amplify any of the paralog genes (Figure 2C, Figure S3). Brain DWI sequences showed no diffusion restrictions (Figure S2B).
case_images: ATAD3A protein structure. (A) Localization of the ATAD3 gene cluster in chromosome 1p36.33. Schematic illustration of the ATAD3A protein showing the coiled-coil domain, the transmembrane domain and the AAA domain, which includes the Walker A&B ATP-binding and ATPase domains. The alignment of ATAD3A proteins from different species shows that Thr84 is highly conserved. (B) 3D protein structure modeling showing conformational changes in ATAD3A protein in the Thr84Met mutant relative to wild type. Arrows indicate the Thr (T) and Met (M) residues within the wild type and mutant proteins.
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case_images: Both SNVs were confirmed by Sanger sequencing using genomic DNA isolated from all family members and primers specific to ATAD3A, which did not amplify any of the paralog genes (Figure 2C, Figure S3). Alignment of ATAD3A proteins from different species revealed conservation of Thr84 and most of its adjacent amino acids (Figure 3A). ATAD3A at Thr84 by several kinases, including Pyruvate dehydrogenase kinase (PDK), Protein Kinase C (PKC), Casein kinase 2 (CK2) and cyclin-dependent kinases (CDKs) (score rates >0.70, data not shown). Additionally, in silico protein structure modeling revealed that this amino acid substitution affected the overall structure of the protein, as these residues differ in size, polarity and charge (Figure 3B).
case_images: Progression of the subject's physical skills and behavior before and after ketogenic diet (KD) over the course of 127 days. Assessment was made on a discrete scale of 0 to 10, where 0 defined visible worsening and 10 visible improvement. Data are displayed as mean +- standard deviation. *P < 0.05; **P < 1X10-6 as calculated by a one-tailed Student's t-test assuming unequal variance.
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case_images: During KD therapy, the subject's behavior and physical performance were evaluated, using therapist-designed assessment forms (Figure 4 and Supplementary materials).
case_images: Magnetic resonance imaging (MRI) scans of the subject's brain after 12 months of ketogenic diet.
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case_images: MRI sections of both the axial- and coronal-T2WI taken 12 months post-KD initiation (age 5 years and 5 months, Figure 5A and B) showed no progression of the cerebellar and vermis atrophy, compared with the previous MRI (June 2016, Figure 1D and E). Figure 1D and E). Furthermore, compared with the MRI taken in 2016, the sizes of the right and left cerebellar hemispheres were maintained (Figure 5B vs Figure 1E), and there was a slight increase in the relative sagittal length of cerebellum (Figure 5C vs Figure 1F).