Mutation in mitochondrial ribosomal protein MRPS22 leads to Cornelia de Lange-like phenotype, brain abnormalities and hypertrophic cardiomyopathy

Abstract

The oxidative phosphorylation (OXPHOS) system is under control of both the mitochondrial and the nuclear genomes; 13 subunits are synthesized by the mitochondrial translation machinery. We report a patient with Cornelia de Lange-like dysmorphic features, brain abnormalities and hypertrophic cardiomyopathy, and studied the genetic defect responsible for the combined OXPHOS complex I, III and IV deficiency observed in fibroblasts. The combination of deficiencies suggested a primary defect associated with the synthesis of mitochondrially encoded OXPHOS subunits. Analysis of mitochondrial protein synthesis revealed a marked impairment in mitochondrial translation. Homozygosity mapping and sequence analysis of candidate genes revealed a homozygous mutation in MRPS22, a gene encoding a mitochondrial ribosomal small subunit protein. The mutation predicts a Leu215Pro substitution at an evolutionary conserved site. Mutations in genes implicated in Cornelia de Lange syndrome or copy number variations were not found. Transfection of patient fibroblasts, in which MRPS22 was undetectable, with the wild-type MRPS22 cDNA restored the amount and activity of OXPHOS complex IV, as well as the 12S rRNA transcript level to normal values. These findings demonstrate the pathogenicity of the MRPS22 mutation and stress the significance of mutations in nuclear genes, including genes that have no counterparts in lower species like bacteria and yeast, for mitochondrial translation defects.

Figure 1

BN-PAGE analysis in patient fibroblasts. Antibodies directed against specific individual subunits were used to assess the amount of each of the fully assembled complexes in the patient (P) and controls (C1 and C2). Complex II was used as a loading control. CI–CIV=complex I–IV; IGA=in-gel activity.

Figure 2

In vitro labeling of mitochondrial translation products. The patient (P) translation rates of the different subunits are expressed as percentages of the average [³⁵S]methionine incorporation in two controls (C1 and C2). The level of translation of ND5 was obtained from an identical assay, in which this product was clearly visible. COI–COIII=subunits of cytochrome c oxidase (complex IV); cyt b=cytochrome b subunit (of complex III); ND1–6=subunits of NADH CoQ oxidoreductase (complex I); ATP6–8=ATP synthase (complex V) subunits 6 and 8.

Figure 3

MRPS22 mutation, conservation of the mutated residue, protein structure prediction and protein level in patient cells. (a) DNA sequence analysis of the patient (homozygous c.644T>C) and both parents (heterozygous). (b) Protein sequence alignment from human to worm, which shows that the mutated leucine residue is highly conserved among different species. (c) Prediction of the MRPS22 protein structure, displaying part of the protein with the Leu215Pro substitution located in the middle of a helix. (d) Immunoblot analysis in patient (P) and control (C1 and C2) fibroblasts reveals the (near) absence of the MRPS22 protein. Complex II was used as a loading control.

Figure 4

Restoration of mitochondrial rRNA levels by MRPS22 transfection. Patient (P) and control (C) fibroblasts were transfected with a viral vector carrying the wild-type MRPS22 cDNA. Stably transfected clones (C+MRPS22, P+MRPS22(1) and P+MRPS22(2)) were isolated by antibiotic selection. Patient cells were also transfected with a control vector (lacZ). 12S rRNA and 16S rRNA levels were quantified relative to β-actin and presented relative to control values as mean percentages±SD (a, b) or as the relative ratios of 12S rRNA:16S rRNA±SD (c) of triplicate determinations. ^*Significantly different from the control (P<0.05).