Biallelic variants in MRPL49 cause variable clinical presentations, including sensorineural hearing loss, leukodystrophy, and ovarian insufficiency

Abstract

Combined oxidative phosphorylation deficiency (COXPD) is a rare multisystem disorder which is clinically and genetically heterogeneous. Genome sequencing identified biallelic MRPL49 variants in individuals from five unrelated families with presentations ranging from Perrault syndrome (primary ovarian insufficiency and sensorineural hearing loss) to severe childhood onset of leukodystrophy, learning disability, microcephaly and retinal dystrophy. Complexome profiling of fibroblasts from affected individuals revealed reduced levels of the small and, a more pronounced reduction of, the large mitochondrial ribosomal subunits. There was no evidence of altered mitoribosomal assembly. The reductions in levels of OXPHOS enzyme complexes I and IV are consistent with a form of COXPD associated with biallelic MRPL49 variants, expanding the understanding of how disruption of the mitochondrial ribosomal large subunit results in multi-system phenotypes.

Keywords: MRPL49; Perrault syndrome; combined oxidative phosphorylation deficiency; mitochondrial large ribosomal subunit; mitoribosome; rare disease.

Figure 1:. Clinical information and brain imaging data for families with biallelic MRPL49 variants

(A) Clinical details of POI, SNHL and developmental/intellectual development for five unrelated families of individuals with either homozygous or compound heterozygous variants in MRPL49. (B) Selected axial brain MR images demonstrating features of patients with variants in MRPL49, and accompanying summary of all imaging features shared across 6 individuals from 5 unrelated families. All 6 affected individuals with available brain imaging had symmetrical T2 high signal changes in the globi pallidi (i). This feature progressed to symmetrical cystic changes in two affected individuals with more advanced disease (ii). In 3/6 affected individuals there was symmetrical T2 high signals and diffusion restriction (not pictured) involving the brain stem, including the substantia nigra (iii) and the dorsal brainstem. In 4/6 affected individuals there were symmetrical white matter T2 high signal changes, becoming more diffuse and confluent with more advanced disease (iv). There was also evidence of cerebral atrophy in 4/6 affected individuals, particularly involving the cerebellum (not pictured), but also affecting cortical grey matter and sub-cortical and deep white matter in individuals with more advanced disease (ii & iv).

Figure 2:. In silico modelling of MRPL49 variants

(A) Schematic representation of MRPL49 transcript and disease-associated variant locations. All three missense variants are clustered in exon 3, whereas the single frameshift variant is located in exon 2. (B) Evolutionary conservation of MRPL49 affected residues across a broad range of orthologous species. Conserved residues are colored blue, variant amino acids (Arginine 88 and Histidine 92) are highlighted in red. (C) Three-dimensional representation of the location of MRPL49 (orange) within the human mitoribosome (PDB code:7QI4). Large (mt-LSU) and small (mt-SSU) colored in grey, 16S rRNA colored in green and 12S rRNA in yellow.

Figure 3:. Functional and molecular assays of fibroblasts from affected individuals reveal significant reductions in levels and activities of mitochondrial respiratory chain complexes I and IV.

(A) MT-RNR1 (12S) and MT-RNR2 (16S) relative expression levels in fibroblasts from affected individuals and controls. Data expressed as a ratio using relative expression to beta-actin. Error bars represent SD, ****p<0.0001 *p=0.0327, unpaired t-test. (B) Mitochondrial respiratory chain enzyme activity assay in control (grey), F1:II-1 (red) and F5:II-1 (blue) fibroblasts. (C) Western blot analysis of proteins associated with each of the mitochondrial oxidative phosphorylation complexes in fibroblasts from F1:II-1, F5:II-1 and controls. Image representative of three separate experiments.

Figure 4.. Complexome analysis of mitochondrial ribosomes and OXPHOS complexes from control and F1:II-1 and F5:II-1patient fibroblasts.

Enriched mitochondria were solubilized with digitonin, separated by BNE and analyzed by mass spectrometry (MS)-based complexome profiling. Protein abundance profiles of each mitoribosomal subunit and OXPHOS complex were generated by averaging the intensity-based absolute quantification (iBAQ) values of all their individual subunits identified by MS. Resultant profiles are illustrated as heatmaps and 2D profile plots against the apparent molecular mass. (A) Abundance profiles of mitoribosomal small (mt-SSU) and large (mt-LSU) subunits showing a visible decrease in the fibroblasts from affected individuals. (B) Abundance profiles of the five mitochondrial OXPHOS complexes consistently showing a drop in their content, particularly for complexes I and IV. CI, CII, CIII, CIV and CV stand for complexes I, II, III, IV and V, respectively; S1: respiratory supercomplex formed by complexes I, III and IV; SCs: higher-order respiratory complexes (respirasomes).