Clinical, biochemical and genetic spectrum of 70 patients with ACAD9 deficiency: is riboflavin supplementation effective?

Abstract

Background: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy.

Results: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation. Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers.

Conclusions: Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.

Keywords: Activities of daily living; Cardiomyopathy; Complex I; Heart transplantation; Lactic acidosis; Mitochondrial disorder; Neonatal; Prognosis; Treatment; Vitamin.

Fig. 1

ACAD9 mutation status, gene structure and conservation of affected amino acid residues. Gene structure of ACAD9 with localization of mutations in 70 patients. Blue asterisks indicate splice site mutations. Newly identified mutations are shown in bold. Conservation of amino acid residues affected by missense variants

Fig. 2

Age of onset, causes of death, survival and effect of riboflavin on survival of ACAD9 patients. a Age of onset of symptoms, (b) Causes of death, (c) Kaplan-Maier survival rates. In red, patients with a disease presentation in the first year of life. In blue, patients with a later presentation (p = 6.49e-05). b In red, patients with a disease presentation in the first year of life and treated with riboflavin. In blue, patients of the same age category but untreated with riboflavin (p = 5.34e-05, confidence 95%)

Fig. 3

Measurements of ACAD9 protein level and complex I activity in patient derived fibroblasts. a Western blot and quantification of ACAD9 protein levels in patient derived fibroblasts and control. b Complex I activity in patient derived fibroblasts and control. c Comparison between remaining ACAD9 protein (red) and Complex I activity (blue). Data expressed as average of three independent western blots and average of > 10 technical replicates (oxygen consumption rate ± SD)

Fig. 4

Effect of bezafibrate and riboflavin supplementation on respiratory chain activities in fibroblast cell lines. Maximal oxygen consumption rate (OCR) was measured in pmol/(s*Mill) of ACAD9 patient and control fibroblasts with and without (a) bezafibrate (400 μM for 72 h) and (b) riboflavin (530 nM for 72 h) treatment. Data are expressed as the average of > 10 technical replicates and normalized to control. ± SD. ***P < 0.001, **P < 0.01, *P < 0.05. (c) Whole cell lysate of control and different ACAD9 deficient fibroblasts +/− bezafibrate/riboflavin visualized with antibodies against ACAD9, ACADVL, ACADM, SDHA, ß-actin (loading control), NDUFS1 and NDUFA9 (d, e) Quantification of ACAD9 and ACADVL protein levels