Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies

Figures

• 

  Figure 1. C1QBP Variants and Gene and Protein Structure (A) Pedigrees of the investigated families (S1–S4) affected by recessively inherited C1QBP variants. Affected individuals are indicated by closed symbols. Both variants in proband S2 have been confirmed to be compound heterozygous by phasing of WES data. (B) Gene structure with exons and introns shows the localization of the investigated gene variations. Conservation of the affected amino acid residues is presented in the alignment of homologs across different species. Exons are highlighted in blue. The size of the introns was reduced 10-fold. MTS is the mitochondrial target sequence. MAM33 (mitochondrial acidic matrix protein 33) is the Saccharomyces cerevisiae homolog of C1QBP. (C) Inspection of the protein structure was performed with PyMOL (PDB: 1P32). A monomer is presented on the left, and the trimer is in the center. The electrostatic field of the trimer is indicated to the right (negative polarity, red; positive polarity, blue). Affected residues are colored in one of the monomers: Cys186, green; Tyr188, blue; Phe204, red; Gly247, magenta; and Leu275, orange.

• 

  Figure 2. Western Blot Analysis of C1QBP Cell lysates isolated from (A) the skeletal muscle of affected probands S1, S3, and S4 and (B) fibroblasts from probands S1–S3 and age-matched control individuals (C1 and C2) were analyzed. Fibroblasts from proband S4 and skeletal muscle from proband S2 were not available. β-actin and α-tubulin were used as loading controls. All experiments were repeated at least two times, and representative images are shown. Number of repeats: (A) n = 3 (S1) and n = 2 (S3 and S4); (B) n = 2 (S1–S3).

• 

  Figure 3. Steady-State Levels of OXPHOS Complex Subunits (A) Western blot analysis of OXPHOS subunits in skeletal-muscle lysates from control individuals (C1 and C2) and probands S1, S3, and S4. (B) Western blot analysis of OXPHOS subunits in skin fibroblasts from probands S1–S3 and age-matched control individuals (C1–C3). OXPHOS-subunit-specific antibodies were used against NDUFB8 or NDUFA9 (CI); SDHA or SDHB (CII); UQCRC2 (CIII); COXI, COXII, or COXIV (CIV); and ATP5A (CV). Cytosolic β-actin and mitochondrial markers porin (VDAC) and SDHA were used as loading controls. All experiments were repeated at least twice, and representative western blots are shown. Number of repeats: (A) n = 3 (S1) and n = 2 (S3 and S4); (B) n = 4 (S1) and n = 2 (S2 and S3).

• 

  Figure 4. BN-PAGE of OXPHOS Complexes One-dimensional BN-PAGE analysis of OXPHOS complexes was performed on mitochondrial lysates isolated from probands’ (A) skeletal muscle (S1 and S3) and (B) fibroblasts (S1–S3) and age-matched control individuals (C1 and C2). The assembly of OXPHOS complexes was assessed by western blotting with antibodies against NDUFB8 (CI), SDHA (CII), UQCRC2 (CIII), COXI (CIV), and ATP5A (CV). An antibody against complex II subunit SDHA was used as a loading control. The ATP5A antibody detected two bands: the fully assembled complex V (F0F1) and the soluble F1 subunit. The asterisk indicates a longer exposure time for the CV F1 subunit. All experiments were repeated at least twice, and representative images are shown. Number of repeats: (A) n = 4 (S1) and n = 2 (S3); (B) n = 4 (S1) and n = 2 (S2 and S3).

• 

  Figure 5. Complementation Studies Using C1qbp−/− Mouse Fibroblasts (A) Quantification of C1QBP, COXI, and COXIII levels. Top: WT or C1qbp KO MEFs were transfected with the pcDNA3-C1qbp-IRES-GFP plasmid for 48 hr. Western blotting on total cell extracts was performed with anti-C1QBP, anti-COXI, anti-COXIII, anti-GFP, and anti-VDAC antibodies. VDAC was used as the loading control. Bottom: the mean ratios of band densities in transfected MEFs from blots are shown. Cells transfected with expression constructs for p.Gly244Trp and p.Leu272Pro variants show significantly lower amounts of mature C1QBP and mitochondrial-DNA-encoded proteins (COXI and COXIII). The results represent the mean ± SD of three independent experiments. ∗∗∗p < 0.005 versus WT transfectant. (B) Relative mRNA expression of C1qbp alleles normalized to mouse 18S rRNA by quantitative real-time PCR. The results represent the mean ± SD of three independent experiments. (C and D) Oxygen consumption rate (OCR) profile (C) and histogram of C1qbp KO cells transfected with WT and mutant C1qbp plasmid (D). The histogram shows the basal respiration rate (basal), ATP production rate (ATP), and maximal respiration rate (maximal) calculated…

• 

  Figure 6. Long-Range PCR and Southern Blot of Individuals with Multiple mtDNA Deletions and Control Individuals (A) Long-range PCR of S3. (B) Southern blot of proband S4. mtDNA was amplified with two primer pairs, giving 16,147 bp (F1) and 15,679 (F2) bp fragments. 5 μL of the amplified mtDNA was loaded onto a 0.7% agarose gel and separated at 80 V for 1 hr. Abbreviations are as follows: S3M, muscle of proband S3; S3F, fibroblasts of proband S3; S4, muscle of proband S4; M, marker λ/HindIII; C1–C3: control cells.