Novel ALDH5A1 variants and genotype: Phenotype correlation in SSADH deficiency

Abstract

Objective: To determine genotype-phenotype correlation in succinic semialdehyde dehydrogenase (SSADH) deficiency.

Methods: ALDH5A1 variants were studied with phenotype correlation in the SSADH natural history study. Assignment of gene variant pathogenicity was based on in silico testing and in vitro enzyme activity after site-directed mutagenesis and expression in HEK293 cells. Phenotypic scoring used a Clinical Severity Score (CSS) designed for the natural history study.

Results: Twenty-four patients were enrolled (10 male, 14 female, median age 8.2 years). There were 24 ALDH5A1 variants, including 7 novel pathogenic variants: 2 missense, 3 splice site, and 2 frameshift. Four previously reported variants were identified in >5% of unrelated families. There was a correlation with age and presence (p = 0.003) and severity (p = 0.002) of epilepsy and with obsessive-compulsive disorder (OCD) (p = 0.016). The median IQ score was 53 (Q25-Q75, 49-61). There was no overall correlation between the gene variants and the CSS, although a novel missense variant was associated with the mildest phenotype by CSS in the only patient with a normal IQ, whereas a previously reported variant was consistently associated with the most severe phenotype.

Conclusions: Seven novel pathogenic and one previously unpublished benign ALDH5A1 variants were detected. There is an age-dependent association with worsening of epilepsy and presence of OCD in SSADH deficiency. Overall, there does not appear to be a correlation between genotype and phenotypic severity in this cohort of 24 patients. We did find a suspected correlation between a novel pathogenic missense variant and high functionality, and a previously reported pathogenic missense variant and maximal severity.

Figure 1. Confirmation of successful transfection of the 2 missense succinic semialdehyde dehydrogenase (SSADH) alleles in HEK293 by Western blot and reverse transcription PCR (RT-PCR)

Cell pellets were resuspended in urea lysis buffer (10 mmol/L Tris HCl, 8 mol/L urea, 100 mmol/L NaCl, pH 8.0) and the protein content was determined using the bicinchoninic acid method (Sigma-Aldrich, St Louis, MO). Thirty micrograms of total protein was size-separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using a 12% stain‐free TGX gel (Bio‐Rad Laboratories, Hercules, CA). Proteins were transferred to a polyvinylidene fluoride membrane using the Trans‐Blot Turbo Transfer System (Bio‐Rad). Immunodetection was performed using the ALDH5A1 mouse polyclonal antibody (B01P; Abnova Corporation, Taipei, Taiwan), anti-mouse secondary antibody (A9044; Sigma-Aldrich, St Louis, MO), and enhanced chemiluminescent substrates (Lumi-Light Plus Western Blotting Substrate; Roche Applied Science, Indianapolis, IN). Images were acquired and analyzed with the ChemiDoc MP imager and Image Lab software (Bio-Rad). Total RNA was isolated from the HEK293 transfectants and cDNA was synthesized using standard molecular biology techniques. mRNA expression of the transfected constructs is confirmed by RT-PCR amplification of the full length cloned ALDH5A1 open reading frame, with the forward primer specific for the pAD1/RSV ALDH5A1 cDNA sequence. *Lower molecular weight protein band detected for the p. Gly441Arg construct, which could be due to the instability and partial degradation of the mutated protein, but alterations in posttranslational protein modification cannot be excluded.

Figure 2. ALDH5A1 variants in the study population

Figure 3. Epilepsy severity with age, showing overall worse severity in the older age group (p = 0.002)

Figure 4. ALDH5A1 variants in the adolescent/adult cohort and association with epilepsy and sudden unexpected death in epilepsy (SUDEP)