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Case Reports
. 2020 Jan 2;106(1):102-111.
doi: 10.1016/j.ajhg.2019.12.005. Epub 2019 Dec 26.

Bi-Allelic UQCRFS1 Variants Are Associated with Mitochondrial Complex III Deficiency, Cardiomyopathy, and Alopecia Totalis

Mirjana Gusic  1 , Gudrun Schottmann  2 , René G Feichtinger  3 , Chen Du  4 , Caroline Scholz  4 , Matias Wagner  5 , Johannes A Mayr  3 , Chae-Young Lee  2 , Vicente A Yépez  6 , Norbert Lorenz  7 , Susanne Morales-Gonzalez  2 , Daan M Panneman  8 , Agnès Rötig  9 , Richard J T Rodenburg  8 , Saskia B Wortmann  10 , Holger Prokisch  1 , Markus Schuelke  11
Affiliations
Case Reports

Bi-Allelic UQCRFS1 Variants Are Associated with Mitochondrial Complex III Deficiency, Cardiomyopathy, and Alopecia Totalis

Mirjana Gusic et al. Am J Hum Genet. .

Abstract

Isolated complex III (CIII) deficiencies are among the least frequently diagnosed mitochondrial disorders. Clinical symptoms range from isolated myopathy to severe multi-systemic disorders with early death and disability. To date, we know of pathogenic variants in genes encoding five out of 10 subunits and five out of 13 assembly factors of CIII. Here we describe rare bi-allelic variants in the gene of a catalytic subunit of CIII, UQCRFS1, which encodes the Rieske iron-sulfur protein, in two unrelated individuals. Affected children presented with low CIII activity in fibroblasts, lactic acidosis, fetal bradycardia, hypertrophic cardiomyopathy, and alopecia totalis. Studies in proband-derived fibroblasts showed a deleterious effect of the variants on UQCRFS1 protein abundance, mitochondrial import, CIII assembly, and cellular respiration. Complementation studies via lentiviral transduction and overexpression of wild-type UQCRFS1 restored mitochondrial function and rescued the cellular phenotype, confirming UQCRFS1 variants as causative for CIII deficiency. We demonstrate that mutations in UQCRFS1 can cause mitochondrial disease, and our results thereby expand the clinical and mutational spectrum of CIII deficiencies.

Keywords: Q-cycle; Rieske iron-sulfur protein; alopecia; cardiomyopathy; microscale respiratory; mitochondrial complex III deficiency; mitochondrial import sequence; mitochondriopathy; mutation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Clinical Images of Both Probands and Family Pedigrees (A and B) Proband 1 (P1) at the age of 1 week (A) and of 8 weeks (B) after loss of his scalp hair. (C) Proband 2 (P2) at the age of 6 years with alopecia totalis of his scalp with only sparse hair growing around his eyebrows. (D) Entirely normal cranial MRI scan of P2 without characteristic T2-signal-intense areas in the basal ganglia; these areas are often seen in Leigh syndrome and many other mitochondrial disorders. (E) Pedigrees of both families with UQCRFS1 genotypes.
Figure 2
Figure 2
Molecular Genetic Findings in Both Probands (A) Electropherograms of the homozygous c.215-1G>C splice-site variant in P1, which was heterozygous in both parents. (B) Loss of the splice acceptor site of intron 1 leads to activation of an alternative downstream cryptic splice site (highlighted by a red box) with subsequent loss of 30 bp from the cDNA. (C) Electropherograms of the compound heterozygous variants, c.41T>A inherited from the mother and c.610C>T from the father. The effect of the mutation on the amino acid sequence is depicted below the electropherograms. (D) Genomic organization of UQCRFS1 into two exons, the mRNA, and the protein structure depicting the location of the identified variants. The localization of the altered amino acids is highlighted on the protein domain structure. Phylogenetic conservation of the affected amino acid residues is presented in the alignment of homologs across different species. Positions of affected amino acids are highlighted in red. NB; the intronic region is not drawn to scale.
Figure 3
Figure 3
Protein Studies (A) SDS-PAGE of fibroblast homogenates showing UQCRFS1 and SDHA. Citrate synthase (CS) was used as a mitochondrial loading control. UQCRFS1 protein levels were strongly reduced in P1 and P2 fibroblasts. Transduction rescued UQCRFS1 levels in P2 cells (Proband 2-T) and had no effect on control fibroblasts (Control-T). (B) BN-PAGE stained for ATP synthase and complex III (CIII) (using anti-ATP5F1A and anti-UQCRC2 antibodies) with densitometric analysis of CIII/ATP synthase ratios in BN-PAGE. Relative CIII reduction is rescued by lentiviral transduction in P2 fibroblasts. P1 fibroblasts reached a high passage number after transduction and did not replicate sufficiently enough to be included in this experiment. Bars depict the mean and SD of two repeated measurements.
Figure 4
Figure 4
Immunohistology and Mitochondrial Import (A) Absent or reduced UQCRFS1 protein levels in both probands’ fibroblasts in comparison to wild-type (WT) control fibroblasts. The anti-VDAC1 antibody signal marks the mitochondria. In WT fibroblasts, anti-UQCRFS1 and anti-VDAC1 signals colocalized, thereby verifying the mitochondrial localization of UQCRFS1 (see magnified inset on the right). Nuclei stained with DAPI. (B) Lentiviral transduction of UQCRFS1 in the fibroblasts of P2 (Proband 2-T) restores normal localization. (C) The first 68 amino acids of WT and p.Val14Asp mutant mitochondrial import sequences of UQCRFS1 were fused to the enhanced green fluorescent protein (EGFP). For control, the WT sequence was fused to red fluorescent protein (RFP). After co-transfection of EGFP and RFP constructs into COS1 cells, only those EGFP constructs were imported into the mitochondria that carried the WT import sequence. The p.Val14Asp variant entirely prevented mitochondrial import.
Figure 5
Figure 5
Microscale Respirometry Analysis in Fibroblasts Combination of two replication experiments of normalized oxygen consumption rates (OCRs) of proband and control fibroblasts as measured with the Seahorse instrument (two biological replicates, 12–16 technical replicates for each condition). Each data point represents mean ± SEMs of measurements from 24–32 wells of technical replicates. The separate replication experiments are depicted in Figures S2–S4. OLIG, oligomycin; FCCP, carbonyl cyanide m-chlorophenyl hydrazone; ROT, rotenone; ANT; antimycine. (A) Oxygen consumption rates of fibroblasts in the native state. (B) Rescue of the OCR in fibroblasts of proband P1 after transduction with a lentivirus encoding wild-type (WT) UQCRFS1 (Proband 1-T). (C) Rescue of the OCR of fibroblasts of P2 after transduction with a lentivirus encoding WT UQCRFS1 (Proband 2-T). NB, we normalized the OCRs of different wells in the Seahorse experiment separately for each plate. For that, we first determined cell numbers in each well through the use of the CyQUANT kit (ThermoFischer) and computed a correction factor for each well by dividing the number of cells in the individual well by the average number of cells in the wells of the whole plate. For normalization of OCR values, we then divided raw OCR readings from each well by its correction factor.
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