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Case Reports
. 2021 Aug;133(4):362-371.
doi: 10.1016/j.ymgme.2021.06.001. Epub 2021 Jun 10.

Pathogenic variants in MRPL44 cause infantile cardiomyopathy due to a mitochondrial translation defect

Marisa W Friederich  1 , Gabrielle C Geddes  2 , Saskia B Wortmann  3 , Ann Punnoose  4 , Eric Wartchow  5 , Kaz M Knight  6 , Holger Prokisch  7 , Geralyn Creadon-Swindell  5 , Johannes A Mayr  8 , Johan L K Van Hove  9
Affiliations
Case Reports

Pathogenic variants in MRPL44 cause infantile cardiomyopathy due to a mitochondrial translation defect

Marisa W Friederich et al. Mol Genet Metab. 2021 Aug.

Abstract

Cardiac dysfunction is a common phenotypic manifestation of primary mitochondrial disease with multiple nuclear and mitochondrial DNA pathogenic variants as a cause, including disorders of mitochondrial translation. To date, five patients have been described with pathogenic variants in MRPL44, encoding the ml44 protein which is part of the large subunit of the mitochondrial ribosome (mitoribosome). Three presented as infants with hypertrophic cardiomyopathy, mild lactic acidosis, and easy fatigue and muscle weakness, whereas two presented in adolescence with myopathy and neurological symptoms. We describe two infants who presented with cardiomyopathy from the neonatal period, failure to thrive, hypoglycemia and in one infant lactic acidosis. A decompensation of the cardiac function in the first year resulted in demise. Exome sequencing identified compound heterozygous variants in the MRPL44 gene including the known pathogenic variant c.467 T > G and two novel pathogenic variants. We document a combined respiratory chain enzyme deficiency with emphasis on complex I and IV, affecting heart muscle tissue more than skeletal muscle or fibroblasts. We show this to be caused by reduced mitochondrial DNA encoded protein synthesis affecting all subunits, and resulting in dysfunction of complex I and IV assembly. The degree of oxidative phosphorylation dysfunction correlated with the impairment of mitochondrial protein synthesis due to different pathogenic variants. These functional studies allow for improved understanding of the pathogenesis of MRPL44-associated mitochondrial disorder.

Keywords: Cardiomyopathy; Combined deficiency; Genetic cause; Mitochondrial ribosome; Mitochondrial translation.

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Conflict of interest statement

Declaration of Competing Interest JVH participates in clinical trials of mitochondrial disorders by Stealth Biotherapeutic, Inc. All other authors deny any real or apparent conflict of interest in the field of mitochondrial diseases.

Figures

Figure 1:
Figure 1:. Electron microscopy of the heart
Electron microscopic examination shows areas of contractile element loss within the cardiomyocytes (A) and a diffuse profusion of enlarged, atypically shaped mitochondria with aberrant cristae (B). The scale bar in A represents 2 μm and in B represents 1 μm.
Figure 2:
Figure 2:. Mitochondrial protein amounts of ml44, MTCO1, and ATP5FB1 by western blot analysis
Protein amounts for ml44, MTCOI and ATP5FB1 are shown after separation on an SDS-PAGE gel, followed by western blotting in Patient 1 heart muscle (A) skeletal muscle (B) and fibroblasts (C) and in Patient 2 fibroblasts (D). The ml44 protein is reduced in the heart muscle, skeletal muscle and fibroblasts in Patient 1 and in fibroblasts from Patient 2. In heart and skeletal muscle, the ml44 protein appears as a double band, one similar to that of controls, and an added band of slightly higher molecular weight. In fibroblasts, only the band with a slightly higher MW is visible. The MTCOI protein is reduced in the heart muscle, skeletal muscle and fibroblasts in Patient 1 and in fibroblasts from Patient 2 but the amount of the ATP5FB1 protein in both patients is not reduced compared to the control samples. Citrate synthase is used as a loading control.
Figure 3:
Figure 3:. Mitochondrial enzyme complexes evaluated by blue native polyacrylamide gel separation with in-gel activity staining
The activity of complexes I, II, IV, and V is shown by in-gel activity staining following separation on a blue native polyacrylamide gel electrophoresis in Patient 1 (A) heart muscle (B) skeletal muscle and (C) fibroblasts, and in (D) Patient 2 fibroblasts. Complex IV staining is reduced in heart muscle. Complex V staining reveals the presence of two additional bands representing incompletely assembled F1 subunits, not present in controls, as is typically observed when the mitochondrial DNA encoded subunits of complex V are insufficiently present. In the fibroblasts of Patient 2, no abnormalities were observed.
Figure 4:
Figure 4:. Examination of the assembly of complexes I and IV using non-denaturing polyacrylamide gel followed by western blot
(A–D) The assembly of complex I is shown by separation on a non-denaturing polyacrylamide gel followed by western blotting and detection with an antibody against NDUFS2 in Patient 1 and Patient 2 in comparison to controls and HepG2 cells treated with chloramphenicol. (A) Control fibroblasts show in addition to the fully assembled holocomplex, a faint band at 230 kDa, whereas as Patient 1 shows additional bands at 400, 460, and 850 kDa. (B) Assembly of complex I in Patient 2 in fibroblasts is normal. (C) In skeletal muscle from Patient 1, control samples show in addition to the holocomplex typical additional bands at 400 and 460 kDa, whereas the patient’s sample also showed abnormal bands at 100 and 230 kDa. (D) In heart muscle, control samples show in addition to the holocomplex also a faint band at 100 kDa, and in the patient sample additional bands at 230, 400 and 460 kDa were present. (E,F) The assembly of complex IV is shown by separation on a non-denaturing polyacrylamide gel followed by western blotting and detection with an antibody against COXIV in Patient1 and Patient 2. No abnormal intermediates are seen in Patient 1 (E) and Patient 2 (F).
Figure 5:
Figure 5:. Mitochondrial protein synthesis in fibroblasts of Patient 1 and Patient 2
Mitochondrial DNA encoded proteins are labeled with 35S-methionine and 35S-cysteine in fibroblasts. (A) In a quantitative assay the amount of radioactive incorporation into proteins is reduced in fibroblasts of Patient 1 to a similar degree as of inhibition of mitochondrial protein synthesis by chloramphenicol, but to a lesser extent in Patient 2. (B) Labeled proteins are separated on an SDS-PAGE gel showing two controls in comparison with two samples from Patient 1. The samples show a reduction in each of the protein bands.
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