Bi-allelic Mutations in the Mitochondrial Ribosomal Protein MRPS2 Cause Sensorineural Hearing Loss, Hypoglycemia, and Multiple OXPHOS Complex Deficiencies

Abstract

Biogenesis of the mitochondrial oxidative phosphorylation system, which produces the bulk of ATP for almost all eukaryotic cells, depends on the translation of 13 mtDNA-encoded polypeptides by mitochondria-specific ribosomes in the mitochondrial matrix. These mitoribosomes are dual-origin ribonucleoprotein complexes, which contain mtDNA-encoded rRNAs and tRNAs and ∼80 nucleus-encoded proteins. An increasing number of gene mutations that impair mitoribosomal function and result in multiple OXPHOS deficiencies are being linked to human mitochondrial diseases. Using exome sequencing in two unrelated subjects presenting with sensorineural hearing impairment, mild developmental delay, hypoglycemia, and a combined OXPHOS deficiency, we identified mutations in the gene encoding the mitochondrial ribosomal protein S2, which has not previously been implicated in disease. Characterization of subjects' fibroblasts revealed a decrease in the steady-state amounts of mutant MRPS2, and this decrease was shown by complexome profiling to prevent the assembly of the small mitoribosomal subunit. In turn, mitochondrial translation was inhibited, resulting in a combined OXPHOS deficiency detectable in subjects' muscle and liver biopsies as well as in cultured skin fibroblasts. Reintroduction of wild-type MRPS2 restored mitochondrial translation and OXPHOS assembly. The combination of lactic acidemia, hypoglycemia, and sensorineural hearing loss, especially in the presence of a combined OXPHOS deficiency, should raise suspicion for a ribosomal-subunit-related mitochondrial defect, and clinical recognition could allow for a targeted diagnostic approach. The identification of MRPS2 as an additional gene related to mitochondrial disease further expands the genetic and phenotypic spectra of OXPHOS deficiencies caused by impaired mitochondrial translation.

Keywords: 2-oxoglutaric acid; combined OXPHOS complex deficiencies; complexome profiling; hearing loss; mitochondrial ribosomes; mitochondrial translation defect; wrinkly skin.

Figure 1

Clinical Characteristics of Subject 1 at the Age of 5 Years Clinical features include (A) slightly up-slanting palpebral fissures and left-eye strabismus, (B) low-set ears, and redundant skin on the (C) abdomen and (D) hands.

Figure 2

MRPS2 Mutation Analysis and Evolutionary Conservation of the Affected Amino Acid Residues (A and B) Pedigree and sequencing chromatograms for (A) S1 and (B) S2 and their families, depicting the segregation of the identified recessive mutations. (C) Interspecies alignment of the MRPS2 region containing the amino acid residues altered (depicted in bold and italics) in S1 and S2.

Figure 3

Abundance of mtDNA-Encoded rRNAs and Mitoribosomal Subunits (A) SDS-PAGE analysis of proteins from the small (MRPS2, MRPS5, MRPS18B, and MRPS28) and large (MRPL37 and MRPL44) mitoribosomal subunits in both subjects and control cells revealed decreased steady-state levels of MRPS2 and the other mt-SSU proteins. Steady-state levels of the mt-LSU proteins are unaffected. OXPHOS complex II protein SDHA was used as a loading control. (B) Northern blot analysis of the steady-state levels of 12S and 16S rRNA in fibroblasts from subjects and controls demonstrates that 12S rRNA, but not 16S rRNA, is decreased in fibroblasts carrying mutations in MRPS2 (S1 and S2) or MRPS22 (S3). 18S rRNA from the cytosolic ribosome was used as a loading control.

Figure 4

Complexome Profiling Heatmap representation (top) of the migration profiles of mt-SSU and mt-LSU in control, S1, and S3 fibroblasts. Interaction heatmaps were created by hierarchical clustering, including the manual addition of known ribosomal components that were not grouped together by the algorithm. Top-center and top-right panels show decreased amounts of fully assembled mt-SSU in both S1 and S3 fibroblasts and a subassembly of eight subunits present in control and S1 cells but absent in S3 cells. The abundance of mt-LSU is unaffected in S1 and S3. Migration profiles (bottom) of both mitoribosomal subunits show the relative abundance of proteins plotted against their apparent molecular mass and reveal decreased abundance of mt-SSU and unaffected abundance of mt-LSU in both subjects. The relative abundance was calculated as the average of the normalized iBAQ values for all mt-SSU or mt-LSU proteins.

Figure 5

Pulse Labeling of Mitochondrial Translation Products and OXPHOS Assembly Analysis (A) Pulse labeling of mitochondrial translation products in control (C1–C3), S1, S2, and S3 fibroblasts shows decreased mitochondrial protein synthesis in all subjects’ cell lines. Coomassie staining of the gels was used for the assessment of loading. (B) BN-PAGE analysis of OXPHOS assembly in control, S1, S2, and S3 fibroblasts shows decreased amounts of fully assembled OXPHOS complexes I and IV in the fibroblasts from the MRPS2- and MRPS22-deficient subjects. Subcomplexes of complex V accumulate in subjects’ fibroblasts and are absent in the control cells.

Figure 6

Amounts of Wild-Type MRPS2 in S1 and S2 Fibroblasts (A) Immunoblot analysis of whole-cell extracts from control (C), S1, and S2 cell lines transduced with a lentivirus expressing either wild-type MRPS2 (+MRPS2) or green fluorescent protein (+GFP) as a negative control. The presence of wild-type MRPS2 leads to increased amounts of MRPS5 and MRPS18B, as well as NDUFB8 and NDUFA13 from complex I and COXI and COXIV from complex IV. MRPS2 was detected with a specific antiserum that does not detect the endogenous MRPS2 in whole-cell extracts. SDHA was used as a loading control for each separate gel. (B) Pulse labeling of mitochondrial translation products demonstrates that the presence of wild-type MRPS2 partially restores mitochondrial translation in S1 and S2 fibroblasts. The gel was stained with Coomassie colloidal dye for the assessment of loading. (C) BN-PAGE analysis of OXPHOS assembly in C, S1, and S2 fibroblasts transfected with either GFP- or wild-type-MRPS2-expressing lentivirus shows partial restoration of OXPHOS complex assembly in subject cells expressing MRPS2.

Figure 7

Structural Model of the Human mt-SSU Shows the Positions of Affected Proteins and Residues (A) The human 28S mt-SSU (PDB: 3J9M⁷) was drawn in cartoon representation with PyMol version 1.7.4.0. The subunits known to be implicated in disease are shown with color-matched labels: magenta, MRPS2; raspberry, MRPS7; pale yellow, MRPS23; and shades of green, MPRS16, MRPS22, and MRPS34. Subunits interacting with MRPS2 are shown in shades of yellow, and constituents of the ∼300 kDa subassembly are shown in shades of blue and green. Other subunits are shown in gray, and 12S RNA is shown in light pink. (B) Partial model of (A) shows only the 12S RNA (gray), MRPS2 (magenta), and surrounding proteins MRPS18C, MRPS21, and MRPS28 (shades of yellow). The positions of the residues of MRPS2 altered in S1 (Arg110 and Asp114) and in S3 (Arg138) are shown in green and cyan, respectively, as stick models.