Expanding the phenotypic spectrum of Succinyl-CoA ligase deficiency through functional validation of a new SUCLG1 variant

Abstract

Deficiency of the TCA cycle enzyme Succinyl-CoA Synthetase/Ligase (SCS), due to pathogenic variants in subunits encoded by SUCLG1 and SUCLA2, causes mitochondrial encephalomyopathy, methylmalonic acidemia, and mitochondrial DNA (mtDNA) depletion. In this study, we report an 11year old patient who presented with truncal ataxia, chorea, hypotonia, bilateral sensorineural hearing loss and preserved cognition. Whole exome sequencing identified a heterozygous known pathogenic variant and a heterozygous novel missense variant of uncertain clinical significance (VUS) in SUCLG1. To validate the suspected pathogenicity of the novel VUS, molecular and biochemical analyses were performed using primary skin fibroblasts from the patient. The patient's cells lack the SUCLG1 protein, with significantly reduced levels of SUCLA2 and SUCLG2 protein. This leads to essentially undetectable SCS enzyme activity, mtDNA depletion, and cellular respiration defects. These abnormal phenotypes are rescued upon ectopic expression of wild-type SUCLG1 in the patient's fibroblasts, thus functionally confirming the pathogenic nature of the SUCLG1 VUS identified in this patient and expanding the phenotypic spectrum for SUCLG1 deficiency.

Keywords: Methylmalonic aciduria; Mitochondria; Mitochondrial DNA depletion; Succinyl-CoA synthetase; TCA cycle.

Figure 1. Loss of SUCLG1 results in loss of SCS components, SCS activity and mtDNA depletion

(A) Western blot analysis of fibroblasts cell lysates for SCS enzyme complex components (SUCLG1, SUCLA2, & SUCLG2), selected ETC subunits (ND5, SDHA) and TCA cycle enzyme, citrate synthase (CS). C1, C2 = controls; P = patient; PR = patient fibroblast cell line rescued by ectopic expression of wild type SUCLG1 cDNA. (B) ADP-specific, GDP-specific, and Total SCS (ADP-specific + GDP-specific) activities were measured for control (blue), patient (red), and patient + rescue (green) fibroblasts. (C) mtDNA relative copy number analysis by qPCR. Patient fibroblasts exhibit mtDNA depletion with multiple passages when compared to control (Co) fibroblasts and patient fibroblasts rescued (P+R) by ectopic expression of wild type SUCLG1 cDNA.

Figure 2. Cellular respiration defect in SUCLG1 patient fibroblasts is dependent on SUCLG1 deficiency

Cellular respiration analysis demonstrates reduced respiratory capacity in patient fibroblasts (P) compared to controls (Co) (n = 5) that is completely restored with ectopic expression of wild type SUCLG1 in patient cells (“P+R”). Experiment was done in triplicate. Error bars indicate standard deviation. “OCR” = oxygen consumption rate. Oligomycin = complex V (ATP synthase) inhibitor; FCCP = proton ionophore uncoupler; Antimycin & Rotenone (A&R) = inhibitors of ETC complex III and complex I, respectively.