Lethal Neonatal Progression of Fetal Cardiomegaly Associated to ACAD9 Deficiency

Abstract

ACAD9 (acyl-CoA dehydrogenase 9) is an essential factor for the mitochondrial respiratory chain complex I assembly. ACAD9, a member of acyl-CoA dehydrogenase family, has high homology with VLCAD (very long-chain acyl-CoA dehydrogenase) and harbors a homodimer structure. Recently, patients with ACAD9 deficiency have been described with a wide clinical spectrum ranging from severe lethal form to moderate form with exercise intolerance.We report here a prenatal presentation with intrauterine growth retardation and cardiomegaly, with a fatal outcome shortly after birth. Compound heterozygous mutations, a splice-site mutation - c.1030-1G>T and a missense mutation - c.1249C>T; p.Arg417Cys, were identified in the ACAD9 gene. Their effect on protein structure and expression level was investigated. Protein modeling suggested a functional effect of the c.1030-1G>T mutation generating a non-degraded truncated protein and the p.Arg417Cys, creating an aberrant dimer. Our results underscore the crucial role of ACAD9 protein for cardiac function.

Keywords: ACAD9; Fetal cardiomegaly; Mitochondrial respiratory chain; β-Oxidation.

Fig. 1

Morphological results obtained from autopsy. (a) Cross section of the heart showing enlarged and pale ventricular walls. (b) Acinar arrangements (arrow) and mild steatosis in the liver parenchyma (G × 400). (c) Accumulation of mitochondria in the cytoplasm of hepatocytes and of dispersed neutral lipid droplets (arrow) (G × 2,200). (d) Mild muscular microvacuolar lipidosis (Oil Red O staining; G × 1,000). (e) Reduced cytochrome-oxidase activity on combined succinate dehydrogenase-cytochrome-oxidase histoenzymology and blue muscle fibers (arrow) lacking cytochrome-oxidase activity (G × 400). (f) Moderately electron dense neutral lipids dispersed between muscle fibers (arrows) (G × 6,100)

Fig. 2

Characterization of the mutation NM_014049.4: c.1030-1G>T in the intron 10 of the ACAD9 gene. (a) Amplification of the ACAD9 transcript using a forward primer within exon 10 and a reverse primer within exon 15. Lane 1 control; Lane 2 patient; Lane 3 DNA 100-bp increment ladder. The amplification showed 509 and 563 bp products in the patient sample while only the 509 bp product was present in the control DNA sample. (b) Amplification of the ACAD9 transcript using a forward primer within the putative inserted intronic sequence and a reverse primer within exon 15. Lane 1 control; Lane 2 patient; Lane 3: DNA 100-bp increment ladder. The corresponding fragment (448 bp) was amplified using patient sample while no amplification was obtained with control sample. (c) Sequence of the 563 bp PCR product showing the 54 intronic nucleotide in the patient DNA sample. C control, P patient. (d) qPCR amplification of ACAD9 cDNA in proband and control fibroblasts. No amplification was observed with control sample. (e) Dissociation curves for ACAD9 qPCR. A single dissociation temperature suggests a single product

Fig. 3

Characterization of c.1030-1G>T mutation at protein level. (a) Immunoblotting. Fifty micrograms of control (C) or patient (P) fibroblast lysate were analyzed by Western blot by using ACAD9 and β-actin antibodies. The mACAD9 band corresponds in size to mitochondrial form (66 kDa), and the c/nACAD9 band corresponds to cytosolic or nuclear ACAD9 species (76 kDa). One other form corresponding in size (about 50 kDa) to the predicted truncated protein is present at a high level in the patient protein extract. (b) Immunocytochemistry analysis from control (a) or proband (b) fibroblasts by using ACAD9 antibody

Fig. 4

Model of the ACAD9 with the mutations c.1249C>T (p.Arg417Cys) and c.1030-1G>T. (a) ACAD9 dimer with the double c.1249C>T (p.Arg417Cys) mutation (in yellow). The ACAD9 dimer model of Nouws et al. (2010) has been used as a template. (b) Left: monomer of ACAD9 showing the truncated region (in red) resulting from the c.1030-1G>T mutation. The monomer unit is derived from the ACAD9 dimer model of Nouws et al. (2010). Right: monomer of ACAD9 showing the mutated region (in green) resulting from the c.1030-1G>T mutation. The monomer was built using the PHYRE² server (Kelley and Sternberg 2009). (b) Sequence alignment of the 300–400 range of ACAD9 native and truncated. Secondary structural elements extracted from the models shown in a, using the same colors. (d) Left: dimer model of ACAD9 (Nouws et al. 2010) showing the lost interaction surface (in red) formed by the truncated region resulting from the c.1030-1G>T mutation. Right: dimer model of ACAD9 with the truncated and mutated monomers resulting from the c.1030-1G>T mutation. The monomers are positioned as in the native dimer