Fig 2: Biochemical characterization of the Leu243Pro variant. (A) Semi-quantitative polymerase chain reaction (PCR) analysis of ALDH5A1 gene expression of two independent control subjects and the patient harboring the c.278G > T and c.728T > C variants. (B) Western blot analysis of succinic semialdehyde dehydrogenase (SSADH) protein expression in dermal fibroblasts. (C) SSADH-deficient HEK-293T cells (SSADH-KO-HEK-293T) were transiently transfected with either wildtype (WT) SSADH or p.Leu243Pro constructs. After 48 h, cell lysates were prepared and subjected to Western blotting (C) and SSADH enzyme activity assay (D). (C) Representative Western blot analysis of SSADH protein levels. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. n = 3. (D) SSADH enzyme activity was assessed fluorometrically. WT activity was set to 100%. Data are expressed as mean ± SD of three independent experiments. Student’s t test: *** p = 0.01.

Fig 3: Tetrameric structure of human SSADH (PDB code: 2w8o). The substrate-binding domain, nicotinamide adenine dinucleotide (NAD+) -binding domain and oligomerization domain of the reference monomer are colored in different shades of blue. The other three monomers are represented by transparent shades of green. The two a-helices in close contact with the adenosine monophosphate moiety of NAD+ are colored in purple. Proteins are represented as tubes, while NAD+ and the mutated residues (Cys93 and Leu243, aC- aC distance: 20.8 Å) are represented by Van der Waals spheres. The NAD+ molecule has been modelled into the NAD+-binding domain based on the published structure of the human SSADH in complex with adenosine diphosphate moiety as a ligand (PDB code: 2w8r) [10].

Fig 4: Metabolic pathway of ?-amino butyric acid (GABA) degradation. Abbreviations: GAD = glutamate decarboxylase, GABAT = GABA transaminase, SSA = succinic semialdehyde, SSADH = succinic semialdehyde dehydrogenase, SSAR = succinic semialdehyde reductase, GHB = ?-hydroxybutyrate, TCA = tricarboxylic acid cycle.

Fig 5: Distribution of disease-causing variants along a linearized model of the SSADH protein. (A) Linearized model of the SSADH protein. Functionally important domains are highlighted; symbol-coded variant distribution. (B) Assessment of variant density reveals a distinct cluster of pathogenic missense variants in the central region of the SSADH protein and mainly involves the NAD+ binding domain and the oligomerization domain. (C) Polynomial regression model of two pathogenicity scores (PolyPhen and REVEL) for all possible missense variants along the linearized SSADH protein.