Identification of a new COQ4 spliceogenic variant causing severe primary coenzyme Q deficiency

Abstract

Background and aims: Primary Coenzyme Q (CoQ) deficiency caused by COQ4 defects is a clinically heterogeneous mitochondrial condition characterized by reduced levels of CoQ₁₀ in tissues. Next-generation sequencing has lately boosted the genetic diagnosis of an increasing number of patients. Still, functional validation of new variants of uncertain significance is essential for an adequate diagnosis, proper clinical management, treatment, and genetic counseling.

Materials and methods: Both fibroblasts from a proband with COQ4 deficiency and a COQ4 knockout cell model have been characterized by a combination of biochemical and genetic analysis (HPLC lipid analysis, Oxygen consumption, minigene analysis, RNAseq, among others).

Results: Here, we report the case of a subject harboring a new variant of the COQ4 gene in compound heterozygosis, which shows severe clinical manifestations. We present the molecular characterization of this new pathogenic variant affecting the splicing of COQ4.

Conclusion: Our results highlight the importance of expanding the genetic analysis beyond the coding sequence to reduce the misdiagnosis of primary CoQ deficiency patients.

Keywords: COQ4; Coenzyme Q10 deficiency; Hybrid minigene; Mitochondrial disorder; Spliceogenic variant; WES.

Graphical abstract

Fig. 1

COQ4 KO cells transfected with COQ4 wild type and variants found in patients recover COQ4 protein expression and CoQ₁₀ levels in different degrees and accumulate a CoQ₁₀ biosynthesis intermediary. A. Levels of COQ4 proteins in HEK293T WT (HEK), COQ4 KO (KO), and COQ4 KO transfected with WT COQ4 (WT) or with the HA-tagged WT (COQ4-HA wt) or the c.23_33delTCCTCCGTCGG (p.Val8Alafs*19) COQ4 variant. The p.Leu52Ser variant is used as a control of the system. Membranes were developed with an antibody against the COQ4 protein. Protein expression was induced with doxycycline (DOX) for 24 h. B. CoQ₁₀ levels in HEK WT, COQ4 KO, COQ4 KO cells transfected with HA-tagged WT COQ4 and patients' mutant versions of COQ4 p.Val8Alafs*19 and p.Leu52Ser (Mean and SD are represented; Two-way ANOVA, Dunnett's multiple comparison test (each transfected cell line (induced+non-induced) vs. HEK WT); p values: ****, p < 0.0001). C. CoQ₁₀, ₁₀P-HB, ₁₀PPVA and DMQ₁₀ levels measured by targeted mass spectrometry of WT HEK293T (HEK wt), COQ4 KO and COQ4 KO cells transfected with HA-tagged WT COQ4 induced with 0,5 ng/mL doxycycline (wt HA + DOX) (Scatter dot plots with means are represented; One-way ANOVA, Dunnett's multiple comparison test; p values: **, p < 0.002; ****, p < 0.0001). D. Pre-CoQ₁₀ levels in HEK293T WT, COQ4 KO, COQ4 KO cells transfected with HA-tagged WT COQ4 (wt HA) and patients' mutant versions of COQ4 (p.Val8Alafs*19 and p.Leu52Ser) (Mean and SD are represented; Two-way ANOVA, Dunnett's multiple comparison test (each transfected cell line (induced+non-induced) vs. HEK WT); p values: ****, p < 0.0001).

Fig. 2

Quinone content, COQ4 protein levels, and respiratory capacity of COQ4 proband's fibroblasts. A. Mass spectrometry analysis of CoQ₁₀ steady-state levels in controls and proband's fibroblasts (PF) (Scatter dot plot with means is represented; Unpaired two-tailed t-test; ***, p value<0.0005). B. HPLC-radioactivity chromatogram showing the incorporation of ¹⁴[C]-pHB to the quinone extract. While control cells show a radioactive CoQ₁₀ peak, PF present 2 peaks, one corresponding to CoQ₁₀, and another of lower retention time (13–13.5 min), corresponding to an intermediate molecule of CoQ₁₀ synthesis. C. Mass spectrometry analysis of control and COQ4 PF shows that the latter accumulate ₁₀P-HB (Scatter dot plot with means is represented; Unpaired two-tailed t-test; **, p value<0.05). D. Mass spectrometry analysis shows that ₁₀PPVA is undetectable in both control and patient's cells. A scatter dot plot with means is represented. E. COQ4 protein levels in whole fibroblast lysates and western blot quantification by densitometry (Unpaired two-tailed t-test; p value<0.0001). An antibody against beta-actin (43KDa) was used as a loading control. F. Representative OCR traces of proband (PF) and control cells measured by Seahorse. Lines indicate the addition of the individual inhibitors. OL (oligomycin 4 μM); FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone 1 μM); Rot (rotenone 1 μM); Ant (antimycin A 2.5 μM). G. Normalized respiratory parameters of control and PF. ± SD. PF vs. control cells (Mean ± SD are represented; Two-way ANOVA with Sidak's multiple comparison test. ****, p value<0.0001).

Fig. 3

RT-PCR analysis of the COQ4 (c.532 + 6 T > A variant) minigene constructs expressed in HEK cells and RNAseq analysis of COQ4 transcripts in PF and control fibroblasts. A. Schematic representation of the hybrid minigene constructs. The construct contains the closest exon to the variant, COQ4 exon 5, and part of the upstream and downstream introns (± 100 bp). B. Cells were transfected with the mutant and the wild type constructs, and results of RT-PCR are shown in the agarose gel. The relative amounts of the different forms were determined by densitometry (percentages are shown on the bottom of the gel). A schematic representation of the spliced transcripts found is included. Ex, exon; C-, negative control of transfection; EV, transfection with empty β-globin-pCDNA3.1 vector; WT, wild-type construct; MUT, construct carrying c.532 + 6 T > A variant. C. RNAseq histogram plots of control RNA sample. The height of the histograms represents the read coverage. Splice junctions are displayed as arcs, each one with the number of reads observed for it. Canonical splicing events are indicated above the histograms, while alternative splicing events are displayed below them. D. RNAseq histogram plots of PF RNA sample. The height of the histograms represents the read coverage. Splice junctions are displayed as arcs, each one with the number of reads observed for it. Canonical splicing events are indicated above the histograms, while alternative splicing events are displayed below them.

Fig. 4

Analysis of COQ4 transcripts in PF and control fibroblasts. A. RT-PCR bands amplified with control (WT) and deletion (del) primers specific to the wild-type exon 1 from the c.532 + 6 T > A intronic allele and the c.23_33delTCCTCCGTCGG deletion allele, respectively (upper panel). Schematic representation of the PCR strategy (lower panel). B. Schematic representation of the spliced transcripts found in each band by sequencing of the amplified fragments. C. CoQ₁₀ levels in HEK COQ4 KO cells transfected with isoforms a to c (data expressed as mean ± SD; Two-way ANOVA, Dunnett's multiple comparison test (each transfected cell line vs. HEK WT); p values: ****, p < 0.0001). D. Intermediate in HEK COQ4 KO cells transfected with isoforms a to c (data expressed as mean ± SD; Two-way ANOVA, Dunnett's multiple comparison test (each transfected cell line vs. HEK WT); p values: ****, p < 0.0001). Iso, isoform. PF, proband fibroblast. DOX, doxycycline; M, DNA 1Kb ladder.