Clinical and molecular outcomes from the 5-Year natural history study of SSADH Deficiency, a model metabolic neurodevelopmental disorder

Abstract

Background: Succinic semialdehyde dehydrogenase deficiency (SSADHD) represents a model neurometabolic disease at the fulcrum of translational research within the Boston Children's Hospital Intellectual and Developmental Disabilities Research Centers (IDDRC), including the NIH-sponsored natural history study of clinical, neurophysiological, neuroimaging, and molecular markers, patient-derived induced pluripotent stem cells (iPSC) characterization, and development of a murine model for tightly regulated, cell-specific gene therapy.

Methods: SSADHD subjects underwent clinical evaluations, neuropsychological assessments, biochemical quantification of γ-aminobutyrate (GABA) and related metabolites, electroencephalography (standard and high density), magnetoencephalography, transcranial magnetic stimulation, magnetic resonance imaging and spectroscopy, and genetic tests. This was parallel to laboratory molecular investigations of in vitro GABAergic neurons derived from induced human pluripotent stem cells (hiPSCs) of SSADHD subjects and biochemical analyses performed on a versatile murine model that uses an inducible and reversible rescue strategy allowing on-demand and cell-specific gene therapy.

Results: The 62 SSADHD subjects [53% females, median (IQR) age of 9.6 (5.4-14.5) years] included in the study had a reported symptom onset at ∼ 6 months and were diagnosed at a median age of 4 years. Language developmental delays were more prominent than motor. Autism, epilepsy, movement disorders, sleep disturbances, and various psychiatric behaviors constituted the core of the disorder's clinical phenotype. Lower clinical severity scores, indicating worst severity, coincided with older age (R= -0.302, p = 0.03), as well as age-adjusted lower values of plasma γ-aminobutyrate (GABA) (R = 0.337, p = 0.02) and γ-hydroxybutyrate (GHB) (R = 0.360, p = 0.05). While epilepsy and psychiatric behaviors increase in severity with age, communication abilities and motor function tend to improve. iPSCs, which were differentiated into GABAergic neurons, represent the first in vitro neuronal model of SSADHD and express the neuronal marker microtubule-associated protein 2 (MAP2), as well as GABA. GABA-metabolism in induced GABAergic neurons could be reversed using CRISPR correction of the pathogenic variants or mRNA transfection and SSADHD iPSCs were associated with excessive glutamatergic activity and related synaptic excitation.

Conclusions: Findings from the SSADHD Natural History Study converge with iPSC and animal model work focused on a common disorder within our IDDRC, deepening our knowledge of the pathophysiology and longitudinal clinical course of a complex neurodevelopmental disorder. This further enables the identification of biomarkers and changes throughout development that will be essential for upcoming targeted trials of enzyme replacement and gene therapy.

Trial registration: ClinicalTrials.gov NCT03758521.

Keywords: Development; GABA; Neurotransmitters; Succinic semialdehyde dehydrogenase.

Fig. 1

The metabolic consequences of an impaired or lacking SSADH enzyme include the accumulation of the inhibitory neurotransmitters GABA and GHB. GABA- γ-aminobutyrate; GABA-T- GABA Transaminase; GAD- Glutamic Acid Decarboxylase; GHB- γ-hydroxybutyrate; SSADH- Succinic Semialdehyde Dehydrogenase; TCA- Tricarboxylic Acid Cycle

Fig. 2

Summary and characterization of the human iPSCs lines from SSADH deficient patients and sex-matched unaffected parental controls. (a) Recapitulative table. (b) Representative images of the iPSCs (ALDH5A1-/- and ALDH5A1+/-) demonstrating the successful reprogramming by characterizing the expression of pluripotency markers NANOG (Nanog homeobox x in green) and TRA-1-60 (podocalyxin in magenta), OCT4 (octamer binding transcription factor 4 in green), and SOX2 (SRY-Box Transcription Factor 2 in magenta). (c) Representative image of iPSC-derived iGABA neurons labelled with DAPI (cyan), MAP2 (green), GABA (magenta). Scale bar 50 μm

Fig. 3

A treemap chart illustrating the occurrence rate of ALDH5A1 variants in subjects enrolled in the SSADHD natural history study

Fig. 4

A schematic timeline of the median onset times of the principal clinical events in SSADHD alongside the decline in GABA and GHB. GABA- γ-aminobutyrate; GHB- γ-hydroxybutyrate

Fig. 5

Quantification of whole blood GHB content in aldh5a1^{lox − STOP} mice. GHB analyses of whole blood obtained from wild-type (WT), heterozygous (HET), and homozygous mutant (HOM) aldh5a1^{lox − STOP} mice at postnatal age of 16 days. Quantification of GHB is expressed as µM. Individual data points, group mean, and SEM are shown. ****p < 0.0001, One-way ANOVA followed by Dunnett’s multiple comparisons test. Both male and female mice were used. n = 9 WT, 19 HET, 7 HOM mice