Prenyldiphosphate synthase, subunit 1 (PDSS1) and OH-benzoate polyprenyltransferase (COQ2) mutations in ubiquinone deficiency and oxidative phosphorylation disorders

Abstract

Coenzyme Q10 (CoQ10) plays a pivotal role in oxidative phosphorylation (OXPHOS), as it distributes electrons among the various dehydrogenases and the cytochrome segments of the respiratory chain. We have identified 2 novel inborn errors of CoQ10 biosynthesis in 2 distinct families. In both cases, enzymologic studies showed that quinone-dependent OXPHOS activities were in the range of the lowest control values, while OXPHOS enzyme activities were normal. CoQ10 deficiency was confirmed by restoration of normal OXPHOS activities after addition of quinone. A genome-wide search for homozygosity in family 1 identified a region of chromosome 10 encompassing the gene prenyldiphosphate synthase, subunit 1 (PDSS1), which encodes the human ortholog of the yeast COQ1 gene, a key enzyme of CoQ10 synthesis. Sequencing of PDSS1 identified a homozygous nucleotide substitution modifying a conserved amino acid of the protein (D308E). In the second family, direct sequencing of OH-benzoate polyprenyltransferase (COQ2), the human ortholog of the yeast COQ2 gene, identified a single base pair frameshift deletion resulting in a premature stop codon (c.1198delT, N401fsX415). Transformation of yeast Deltacoq1 and Deltacoq2 strains by mutant yeast COQ1 and mutant human COQ2 genes, respectively, resulted in defective growth on respiratory medium, indicating that these mutations are indeed the cause of OXPHOS deficiency.

Figure 1. CoQ₁₀ biosynthesis pathway.

Enzyme and human gene symbols are shown in italics. The yeast gene symbol is indicated when different from the human gene.

Figure 2. Effect of an exogenous quinone analog (DQ, 80 μM) on succinate oxidation (A) and succinate cytochrome c reductase activity of cultured skin fibroblasts from patient 1 (B).

Numbers along the traces represent nmol/min/mg protein. Numbers in brackets are normal values. A, absorbance. (C) Elution profiles of lipid extracts from control and patient 3 skin fibroblasts incubated with [³H]mevalonate and treated with acid phosphatase.

Figure 3. Pedigree and haplotype analysis of the family of patients 1 and 2.

Haplotypes are given (top to bottom) for loci D10S1705, D10S191, D10S1477, D10S1714, D10S211, D10S197, D10S600, D10S593, and D10S1639. The haplotypes in the upper part are from the parents.

Figure 4. Molecular analysis of the PDSS1 gene.

Sequence analysis of PDSS1 gene in patient 1 (A), parents (B), and controls (C). (D) MnlI restriction analysis of the T→G mutation at nt 977. MnlI restriction generated 2 fragments of 393 and 180 bp in controls and 3 fragments of 352, 180, and 41 bp in the patient. P, patient; C, controls; MW, molecular weight marker (DNA molecular weight marker VIII; Roche Applied Science). (E) Sequence alignment of the prenyldiphosphate synthase, FPP synthase, and GPP synthase from human and nonhuman sources. The box shows the polyprenyl synthase signature.

Figure 5. Molecular analysis of the COQ2 gene.

Sequence analysis of the COQ2 gene in patient 3 (A) and her parents (B). (C) RT-PCR analysis of COQ2 transcript. (D) Normal and mutant coq2 proteins.

Figure 6. Functional complementation of yeast coq1- and coq2-null mutant.

Growth of coq1- and coq2-null mutants on glucose (without amino acids, supplemented with histidine, leucine, and methionine, 20 g/l glucose) or glycerol medium (YPGly, 20 g/l glycerol) was compared. (A) The yeast Δcoq1-null mutant transformed with the wild-type and mutant (D308E) human PDSS1 cDNAs and the wild-type and mutant (D365E) yeast COQ1 genes on the pYES2.1 plasmid. (B) The yeast Δcoq2-null mutant transformed with the wild-type and mutant (m) human COQ2 genes on the pYES2.1 plasmid. The 4 spots for each experiment correspond to successive dilutions of transformed yeasts (1, 0.1, 0.01, and 0.001).