(A) lod score profile across the human genome in affected children from 14 consanguineous kindred with SRNS. Parametric heterogeneity lod (HLOD) scores are plotted against human chromosomal mapping positions, concatenated from p-ter (left) to q-ter (right). (B) Within the SRNS2 locus, haplotypes from 250k SNP analysis are shown for 3 of the 7 families with homozygosity at the SRNS2 locus. Alleles are colored light green (AA), dark green (BB), and red (AB). (C) The 32 genes within the SRNS2 locus; 3 genes preferentially expressed in kidney podocytes are underlined. Mutations were found in COQ6. Transcriptional direction is indicated by < or >. (D) COQ6 extends over 13.2 kb and contains 12 exons (boxes). (E) Exon structure of COQ6 cDNA. Arrows indicate relative positions of mutations (see G). Positions of peptides for antibody generation are shown in yellow. (F) Domain structure of COQ6 protein. Extent of the monooxygenase domain is shown in relation to encoding exon position (E). (G) 6 different COQ6 mutations in 7 families with SRNS. Family number and amino acid change (see Table 1) are given above sequence traces. Arrows denote positions of mutations in relation to exons and protein domains. For the 2 missense mutations, G255R and A363D, full conservation across evolution of altered amino acid residues is illustrated.

(A and B) GFP-labeled full-length human COQ6 isoform a (COQ6a-EmGFP) exogenously expressed in Cos-7 cells colocalized quantitatively with mitochondrial markers cytochrome c (A) and COXIV (B). (C–E) GFP-labeled full-length human COQ6 isoform a expressed exogenously in HeLa cells was detected by α–COQ6-TPEP2 upon immunofluorescence, thereby confirming specificity of the antibody (C). α–COQ6-TPEP2 detected an additional endogenous signal, which appeared to be localized in Golgi apparatus (C), as confirmed by double labeling with Golgi marker Golgin 97 (D). α–COQ6-TPEP2 did not reveal any endogenous COQ6 expression in mitochondria (D and E). (F) GFP-labeled full-length human COQ6 isoform a expressed exogenously in HeLa cells spared Golgi expression, as demonstrated with Giantin as a Golgi marker. (G) α–COQ6-TPEP2 detected endogenous COQ6 in Golgi of a podocyte cell line, as marked by gm130, but not in mitochondria. Scale bars: 5 μm.

(A–C) Coq6 downregulation in cultured mouse podocytes induced apoptosis that was diminished by CoQ₁₀ treatment. Data are mean ± SEM. (A) Growth curve analysis of Coq6 knockdown clones. 20,000 podocytes were seeded per 24-well plate, plated, detached, and counted (n = 4). (B and C) Caspase-9 (B) and caspase-3 (C) activity in undifferentiated Coq6 knockdown clones by FIENA before and after CoQ₁₀ treatment. RFUs derived from cleaved capase-9 or caspase-3 substrate peptide were measured in lysates from 1 × 10⁶ cells (n = 3). C, control clone; No Tx, no treatment. *P < 0.001, ^†P < 0.05 versus control; ^#P < 0.005, ^‡P < 0.05 versus untreated. (D–F) coq6 knockdown in zebrafish embryos 28 hours after fertilization increases apoptosis. (D) coq6-MO4 directed against zebrafish coq6 intron 7 splice donor blocked proper splicing of coq6 mRNA (see Supplemental Figure 1N). Negative controls were injected with 0.1 mM coq6 mismatch MO. Note the gray appearance of zebrafish heads upon differential interference contrast (DIC) microscopy as a sign of increased cell death. (E and F) Zebrafish dorsal head and lateral trunk views. Embryos were injected as indicated with 0.1 mM coq6-MO4 splice targeting MO (E), MO targeting the AUG translation start site (dcoq6 MO1; F), coq6 mismatch (mm) MO negative controls, or left uninjected (WT). Cells were stained by an antibody against cleaved caspase-3, a specific marker for apoptotic cells. Lens (asterisk), yolk sac (ys), and cloaca (arrow) are indicated. Scale bars: 1 mm (D); 100 μm (E and F).

(A–C) Renal histology in individuals revealed homozygous COQ6 mutation in A3331-21 FSGS, demonstrated by increased fibrosis (blue) in Trichrome-Masson staining (A), and in F1082-21, demonstrated by excess PAS staining (red) (B). (C) Transmission electron microscopy for F1082 showed effacement of podocyte foot processes, resulting in a continuous electron-dense layer (arrowheads). Scale bars: 50 μm (A and B); 250 nm (C). (D and E) Functional test of human COQ6 mutations in yeast coq6-null mutants. (D) WT human COQ6 (hWT), but not mutations, rescued growth in yeast coq6-null mutants plated on a nonfermentable carbon source. Yeast cells harboring the indicated low-copy plasmids were cultured in SD-Ura, seeded to both SD-Ura and YPG plate media, and incubated at 30°C for the times indicated. pSR1-1 contains the yeast COQ6 gene (18) and served as positive control. Empty vectors pRS316 and pRS426 served as negative controls. (E) WT human COQ6, but not mutations, rescued CoQ₆ synthesis in yeast coq6-null mutants. CoQ₆ content of each yeast coq6-null mutant harboring 1 of the designated plasmids was determined as described in Methods. Each CoQ₆ measurement represents mean ± SD of 4 measurements from 2 independent samples. *P < 0.0005 versus negative control (99% confidence level). PQM and PRCM indicate low– and high–copy number plasmids, respectively.

(A) In rat renal glomeruli (marked with an α-GLEPP1 antibody), α–COQ6-TPEP2 labeled podocyte cytoplasm and cell processes (left). COQ6 was expressed in podocytes, whose nuclei were marked with α-WT1 (middle), but not in mitochondria, marked with α-COXIV (right). (B) COQ6 (red) was located in podocyte cellular processes and in Golgi (marked with the trans-Golgi antibody TGN38), demonstrating colocalization (yellow). (C) COQ7 exhibited an expression pattern identical to that of COQ6 (see B). (D) There was full colocalization of COQ6 and COQ7 in podocyte cytoplasm, cell processes, and (by inference from B and C) Golgi. (E) COQ6 colocalized in podocyte cellular processes with podocin, another protein that, if mutated, causes SRNS. COQ6 showed additional expression more centrally in cell processes. (F) In cochlea (counterstained with rhodamin-phalloidin [Rh-Ph] for F-actin), COQ6 was expressed in spiral ganglion neurons in Rosenthal canal (arrowheads). (G) Higher-magnification view of boxed region in F showing COQ6 expression in stria vascularis cells (arrowheads) and the spiral ligament (arrow). Signal in tectorial membrane (tm) and adjacent structures most likely represents background staining. Rm, Reissner membrane; cd, cochlear duct; st, scala tympani; sv, scala vestibuli. In merged images, nuclei are stained blue with DAPI. Scale bars: 50 μm (A–E); 300 μm (F); 100 μm (G).