Four patients with succinyl-CoA ligase deficiency due to mutations in SUCLG1
gene have been reported. Three patients were homozygous for a two base pair deletion and had no residual protein or activity (11
). They presented with intrauterine growth retardation, hypotonia, hypothermia, abnormal EEG, and severe metabolic acidosis with lactic acidosis in the first day of life, leading to death within four days. A fourth patient had hepatomegaly. Another patient was homozygous for the missense mutation p.G72A (12
). Western blot analysis in fibroblasts demonstrated decreased protein, but with some preserved residual protein. This patient had a normal birth weight and was described as normal for the first three months; then, developed failure to thrive, hypotonia and motor delay, progressive muscle atrophy and absent tendon reflexes, and severe hearing loss. There was no spasticity, no consistent dystonia, and no clinical liver disease. Lactic acidosis was mild 3 to 5 mM, and methylmalonic acid was mild 4 to 13 μM. On brain MRI there was progressive abnormal signal in putamen, globus pallidus, and caudate with brain atrophy. The child died at age 2 year 8 months of lactic acidosis following a gastroenteritis episode. This child’s phenotype was milder and similar to that of patients with a defect in the SUCLA2
Our patient’s phenotype is intermediate between both phenotypes. Similar to the first three patients, there was dysmaturity and severe lactic acidosis from birth, but in our patient this was manageable with judicious treatment. Similar to the milder affected patient with the missense mutation, our patient developed severe and ultimately fatal muscle disease with weakness, hypotonia, muscle atrophy, and respiratory failure. She developed MRI signs of Leigh disease, although she exhibited very limited signs of dystonia. She also developed hearing loss, but vision was spared. This is similar to previous patients. However, our patient also developed progressive liver disease with intermittent liver failure, which only temporarily responded to anaplerotic therapy with aspartate. From 2 months on, the child had increased transaminases, intermittently low albumin, hepatomegaly, occasional hypoglycemia, and disturbed coagulation. The biopsy was consistent with steatotis, but was taken very early before most signs of liver disease had developed. Chronic liver disease has not yet been reported in this condition. Previously reported patients had liver involvement limited to hepatomegaly in one severely affected patient, and intermittent mild elevation of transaminases and PT in a mildly affected patient (11
). Similar phenotype has been described in other causes of liver based mtDNA depeletion syndromes such as caused by mutations in DGUOK
. The clinical fully developed phenotype of the fifth SUCLG1
deficient patient included a hepatic, a myopathic, and an encephalopathic component.
Previous reports on biochemical abnormalities in SUCLG1
deficiency have noted elevated lactate, pyruvate, methylmalonate and methylcitrate with mild increases in Krebs cycle metabolites fumarate, malate, citrate, and alpha-ketoglutarate (11
). This patient’s biochemical phenotype was more striking, and included strongly elevated glutamate and alpha-ketoglutarate, in addition to the small but persistent elevations of methylmalonate and propionylcarnitine. Secondary reflecting these metabolites were the elevations in glutamine, proline, and alanine. These findings are strongly suggestive for succinyl-CoA ligase deficiency when present in a patient with lactic acidosis. The lactate to pyruvate ratio was only mildly increased. The metabolite profile reflects an interruption of the Krebs cycle rather than respiratory chain dysfunction. Particularly the elevation in glutamic acid is very striking (tenfold) and should lead one to review this condition.
Using a new enzyme assay, the diagnosis could be confirmed enzymatically with substrate linked to either GTP or ATP. Since the alpha-subunit encoded by SUCLG1
gene is in common for both subunits, the enzyme activity was deficient with both substrates GTP and ATP. In brain, muscle, and testis, the primary expression of beta-subunits of succinyl-CoA ligase is mostly the ADP-linked, SUCLA2
-encoded beta-subunit (2
). Liver has primarily GDP-linked activity with the SUCLG2
encoded beta-subunit, and heart and kidney have fairly equal amounts of ADP and GDP-linked activities (2
). This tissue distribution explains why patients with a mutation in the SUCLA2
gene and hence only deficiency in the ADP-linked activity have symptoms in muscle and brain, but not in liver. Patients with a mutation in the SUCLG1
gene also have deficient GDP-linked activity and can present with liver dysfunction, which would not be expected for patients with a mutation in the SUCLA2
The pathogenesis of this disorder is likely complex, and can be different in different organs. The succinyl-CoA ligase affects the Krebs cycle, but through its interaction with nucleoside diphosphate kinase might also affect the salvage pathways for nucleotides in the mitochondria (7
). Previous studies have documented decreased mtDNA in muscle in patients with mutations in SUCLA2
) and in SUCLG1
). This has been related to the interaction of the succinyl-CoA ligase with nucleoside diphosphate kinase gene and imbalances in nucleotide triphosphates has been proposed, but not yet proven, as the cause for mtDNA depletion and subsequent dysfunction of respiratory chain enzyme activities, similar to other mtDNA depletion disorders (15
). In the liver of a SUCLG1
patient mtDNA was reduced to 65% of normal (12
) and in our patient to 50% of normal. Such reduction is not sufficient to cause liver dysfunction, but this was obtained very early in the course of the disease. Succinyl-CoA ligase is a part of the Krebs cycle, and is instrumental in the generation of succinyl-CoA for use in ketone utilization and heme formation (2
). The metabolite pattern, with accumulation of metabolites prior to succinyl-CoA in the Krebs cycle, suggests that there was an excess of succinyl-CoA in the liver, and ketones were not excessive as would expected for insufficient function of the succinyl-CoA:3-ketoacid coenzyme A transferase (). Thus, the deficit highlights the impact on the forward catabolic, rather than the reverse anabolic reaction. The methylmalonyl-CoA pathway also provides anaplerosis to the Krebs cycle. When the amount of such metabolites decreased, liver dysfunction worsened, and substitution with aspartate improved, but did not correct, the liver function.
We found that the patient was homozygous for a c.40A>T mutation, which according to the reference human SUCLG1
sequence is predicted to result in the substitution of the methionine at position 14 for a leucine. The methionine at position 14 is located within the predicted mitochondrial targeting sequence of SUCLG1
, which comprises the first 41 amino acids of the encoded protein. Bioinformatic analysis using the MitoProt II 1.0a4 software (16
) indicates that the M14L substitution does not significantly alter the probability of the amino terminus to function as a mitochondrial targeting sequence (0.3309 for M14 and 0.3366 for L14). However, when we compared the amino terminus of the predicted human SUCLG1
coding region with the amino terminus of the predicted SUCLG1
coding regions of chimpanzee, rat, and mouse (), it appeared that the affected methionine is more likely to serve as the translation initiator methionine. When M14 is the translation initiation methionine, the probability of the shorter amino terminus to function as a mitochondrial targeting sequence is increased to 0.6946. Thus the c.40A>T mutation in fact would prevent proper translation initiation at this site. Consequently, translation initiation may occur at the next downstream AUG (i.e. 67 nucleotides downstream of this AUG), which then results in premature translation termination and which would make the mRNA substrate for the so-called nonsense-mediated mRNA decay (or NMD) pathway (17
), a cellular quality-control mechanism which results in destabilization of the mRNA (18
). Indeed, this is supported by the observation of very low SUCLG1
mRNA levels in the patient’s fibroblasts. Moreover, when the NMD pathway in the patient’s fibroblasts is inhibited with emitine, we observed a marked increase in mRNA levels.
Comparison of the amino terminus of SUCLG1 proteins
In conclusion, patients with succinyl-CoA ligase deficiency present with a progressive hepatoencephalomypathy. The pathogenesis of the disease is complex and involves anaplerosis and disruption of the Krebs cycle as well as mtDNA depletion.