Neonatal mitochondrial encephaloneuromyopathy due to a defect of mitochondrial protein synthesis

Abstract

Mitochondrial diseases are clinically and genetically heterogeneous disorders due to primary mutations in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). We studied a male infant with severe congenital encephalopathy, peripheral neuropathy, and myopathy. The patient's lactic acidosis and biochemical defects of respiratory chain complexes I, III, and IV in muscle indicated that he had a mitochondrial disorder while parental consanguinity suggested autosomal recessive inheritance. Cultured fibroblasts from the patient showed a generalized defect of mitochondrial protein synthesis. Fusion of cells from the patient with 143B206 rho(0) cells devoid of mtDNA restored cytochrome c oxidase activity confirming the nDNA origin of the disease. Our studies indicate that the patient has a novel autosomal recessive defect of mitochondrial protein synthesis.

Figure 1

Histochemistry analysis for COX and SDH in patient's fibroblasts before and after fusion with human 143B206 ρ⁰ cells. Panel A: The patient's fibroblasts show COX deficiency and normal SDH activity. Panel B: After fusion with human ρ⁰ cells, the patient's fibroblasts had normal COX activity. Panel C: control fibroblasts stained for COX and SDH show normal activity.

Figure 2

Analysis of the assembly status of all OXPHOS complex in patient's fibroblasts. Fibroblasts from the patient and a control were analyzed with the use of blue-native polyacrylamide gel electrophoresis in the first dimension. The gel were immunoblotted with monoclonal antibodies against complex I (CI- 39 kDa), II (CII-70kDa), III (CIII- core 2), IV (CIV-COXII) and V (CV-αF1F0-ATPase) to assess the amount of fully assembled oxidative phosphorylation complexes. The samples are control (C) and patient (P) and the respective mitocondrial complexes are indicated.

Figure 3

Analysis of mitochondrial translation products. Mitochondrial translation products in fibroblasts from the patient (P) and control (C) were submitted to electrophoresis in 12.5% polyacrylamide gel. To assess the rate of mitochondrial translation, cells were labeled with [³⁵S]methionine in the presence of an inhibitor of cytoplasmic protein synthesis (emetine) for 30 minutes in glucose-containing medium or 1 hour in a glucose-free medium with galactose. The patient's fibroblasts showed a severe defect in mitochondrial translation products: COXI, COXII and COXIII subunits I, II and III of complex IV; ND1, ND2, ND3, ND4, ND4L, ND5 and ND6 subunits of complex I; ATP6 and ATP8 subunits of complex V; and CYTB, apocytochrome b subunit of complex III are indicated. The molecular weight are presented in kDa.

Figure 4

Mitochondrial transcripts analysis by semi-quantitative reverse transcriptase PCR (RT-PCR). Two μg of total RNA were used to synthesize cDNA from patient (lane 1), child control (lane 2), and adult control (lane 3). A. PCR for mitochondrial genes ND2, CYTB, 12S rRNA and β-actin. B. PCR using cDNA diluted 1:10. C. PCR using cDNAs diluted 1:100. D. PCR using control primers (NDUFV1 and NDUFS4) annealing to intronic regions of complex I ND genes. Patient (lanes 1 and 6), child control (lanes 2 and 7), adult control (lanes 3 and 8), control DNA (lanes 4 and 9) and adult control (lanes 5 and 10). The patient's fibroblasts showed normal levels of mitochondrial transcripts for all analyzed genes.

Figure 5

Northern Blotting analysis for mitochondrial transcription products. Total RNA was extracted from fibroblasts of the patient (P) and one child control (CC) by Trizol method. After electrophoresis and transfer, the hybridization was done separately with probes labeled with chemioluminescence for mitochondrial genes ND2, CYTB and 12SrRNA. We observed normal levels of mitochondrial transcripts for all the analyzed genes.