Mutations of GEMIN5 are associated with coenzyme Q10 deficiency: long-term follow-up after treatment

Abstract

GEMIN5 exerts key biological functions regulating pre-mRNAs intron removal to generate mature mRNAs. A series of patients were reported harboring mutations in GEMIN5. No treatments are currently available for this disease. We treated two of these patients with oral Coenzyme Q₁₀ (CoQ₁₀), which resulted in neurological improvements, although MRI abnormalities remained. Whole Exome Sequencing demonstrated compound heterozygosity at the GEMIN5 gene in both cases: Case one: p.Lys742* and p.Arg1016Cys; Case two: p.Arg1016Cys and p.Ser411Hisfs*6. Functional studies in fibroblasts revealed a decrease in CoQ₁₀ biosynthesis compared to controls. Supplementation with exogenous CoQ₁₀ restored it to control intracellular CoQ₁₀ levels. Mitochondrial function was compromised, as indicated by the decrease in oxygen consumption, restored by CoQ₁₀ supplementation. Transcriptomic analysis of GEMIN5 patients compared with controls showed general repression of genes involved in CoQ₁₀ biosynthesis. In the rigor mortis defective flies, CoQ₁₀ levels were decreased, and CoQ₁₀ supplementation led to an improvement in the adult climbing assay performance, a reduction in the number of motionless flies, and partial restoration of survival. Overall, we report the association between GEMIN5 dysfunction and CoQ₁₀ deficiency for the first time. This association opens the possibility of oral CoQ₁₀ therapy, which is safe and has no observed side effects after long-term therapy.

Fig. 1. Total amount and rate of CoQ₁₀ synthesis in GEMIN5 patients’ fibroblasts.

A Total CoQ₁₀ levels were measured by HPLC in GEMIN5 patients’ fibroblasts total homogenates. Cells were supplemented with 100 µM of CoQ₁₀ prepared with BSA as vehicle. Non-treated cells were supplemented only with BSA at the same final concentration for 48 h. Results correspond to the average of 7 measurements for human dermal fibroblasts (HDF), 4 for P1, 2 for P2, and 5 for P3. (Statistical analysis was performed by Unpaired two-tailed t-test with the corresponding control). B Incorporation of ¹³C-4-HB to the quinone extract in 24 h as ¹³C-CoQ₁₀ and ¹³C-DMQ₁₀ in at least three biological replicates, measured in the patients’ fibroblasts. Cells were supplemented with 10 µM ¹³C-4-HB dissolved in water (Unpaired two-tailed t-test with the corresponding control cells). (****p < 0.0001, ***p < 0.001 **p < 0.01 and *p < 0.05).

Fig. 2. Respiratory defects in patients’ fibroblasts.

A–C Oxygen consumption rate (OCR) was measured in patient (P1-3) and control (HDF) fibroblasts. Cells were supplemented with 100 µM of CoQ₁₀ prepared with BSA as vehicle. Non-treated cells were supplemented only with BSA at the same final concentration for 48 h. Arrows indicate the addition of individual inhibitors. Results correspond to the average of six biological replicates ± SD. OL (oligomycin 0.4 μM); FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone 0.1 μM); Rot (rotenone 0.1 μM); AntA (antimycin A 0.25 μM). D–F Quantitative data are plotted as the average ± SD (Two-way ANOVA with Sidak’s multiple comparison test). G–I Single (Complex II and III) and combined (Complex II + III) activities of the complexes were measured in fibroblasts homogenates from patients and controls. Results correspond to the average of at least three biological replicates ± SD, normalized by the citrate synthase activity (left axis) or normalized by protein content (right axis) (Unpaired two-tailed t-test). J Citrate synthase activity measured in fibroblast homogenates from patients and controls. Results correspond to the average of at four biological replicates ± SD. K Western blot analysis of VDAC protein levels in whole fibroblast lysates. A stain-free fluorescence signal was used as a loading control. Data correspond to the average of three normalized wells from patients’ fibroblasts expressed as relative expression ± SD. (Unpaired two-tailed t-test). (****p < 0.0001, ***p < 0.001 **p < 0.01 and *p < 0.05).

Fig. 3. COQs and MRC complexes’ protein levels in fibroblasts.

Western blot analysis in fibroblasts lysates of (A) GEMIN5, (B) COQ proteins (COQ3, COQ4, COQ5, and COQ7), and (C) some subunits of the MRC complexes (NDUFA9 (CI), SDHA (CII), QCR2 (CIII), COX2 (CIV)) levels and mtDNA factors (TFAM and TUFM). D and E Quantification (average of at least three normalized wells) expressed as relative expression ± SD) (Unpaired two-tailed t-test; ****p < 0.0001, ***p < 0.001, **p < 0.01 and *p < 0.05; each patient against the control, HDF). Loading normalization was performed using the Stain Free gel system.

Fig. 4. Effect of CoQ₁₀ supplementation in CoQ levels in rigor mortis adult flies.

A Male and female flies obtained after the cross of rigor mortis driver and UAS/GAL4 transgene were fed with normal food, RU-486 or RU-486 plus 0.1 mg/mL CoQ₁₀ supplemented food. After 30 days from eclosion, flies were collected to quantify the amount of CoQ molecules present in flies (CoQ₈, CoQ_9, and CoQ₁₀). Data are expressed as the average of 4 extractions performed with 30 flies ± SD. Total flies analyzed, 360. Unpaired two-tailed test, assuming gaussian distribution and assuming a similar SD. B Quantification of climbing assay in flies collected after 30 days from eclosion. Data correspond to the average ± SD of five independent experiments. Total flies analyzed, 300. Unpaired two-tailed test, assuming gaussian distribution and assuming a similar SD. C Motionless assay. Flies collected after 30 days from eclosion were subjected to motionless assay. Data correspond to the average ± SD of five independent experiments. Total flies analyzed, 300. Unpaired two-tailed test, assuming gaussian distribution and assuming a similar SD. D Picture corresponding to climbing and motionless assays.