This study reports on the identification of deleterious mutations in 2 different genes of the ubiquinone biosynthesis pathway (PDSS1
), which caused severe ubiquinone deficiency in 2 unrelated families. In the first consanguineous family, a genome scan analysis identified a homozygous mutation in the PDSS1
gene encoding prenyldiphosphate synthase, one of the key enzymes of the ubiquinone biosynthesis pathway. This enzyme catalyzes elongation of GPP or FPP with several isopentenylpyrophosphate (IPP) groups, in trans
configuration, to form the isoprenoid chain (6
). The mutation reported here changed a highly conserved aspartic acid into a glutamic acid (D308E). Human wild-type PDSS1
cDNA failed to complement the OXPHOS defect of the Δcoq1
-null yeast strain, while the PDSS1 and Coq1p proteins share 35% homology. However, growth of the Δcoq1
yeast strain on a nonfermentable carbon source was restored by the wild-type but not the mutant yeast gene (D365E, corresponding to human D308E). We therefore conclude that the D308E mutation in our patient clearly induced prenyldiphosphate synthase deficiency and a profound quinone biosynthesis defect. In Saccharomyces cerevisiae
, Coq1p (hexaprenylsynthase) elongates GPP or FPP (diprenyl biosynthesis intermediaries) to form hexaprenylpyrophosphate. The exact function of the PDSS1 protein in humans is not well known, but it can be hypothesized, by homology with yeast, that the PDSS1 enzyme also elongates geranyl pyrophosphate to form decaprenyl pyrophosphate. However, the normal CoQ9
but very low CoQ10
levels detected in patients 1 and 2 suggest that the D308E PDSS1 mutation only impairs addition of the tenth prenyl to the polyprenyl chain. It is worth noting that quinone-dependent OXPHOS activities were only slightly decreased in patients 1 and 2, despite profound CoQ10
depletion in fibroblasts. It can be hypothesized that the mutant PDSS1 retains a low but significant residual activity, allowing the synthesis of trace amounts of CoQ10
and/or normal amounts of CoQ9
sufficient to maintain a residual respiratory chain function compatible with life.
The mutated amino acid residue (D308) is situated in the second aspartic acid–rich region of the protein, which is common to all prenylsynthases reported to date. This family of proteins also includes FPP synthase and GPP synthase, 2 other enzymes of the ubiquinone synthesis pathway (13
). Prenylsynthases are also involved in the biosynthesis of terpenoids in plants (16
) and carotenoids in fungi (17
). These enzymes are also involved in protein farnesylation (18
). The aspartic acid–rich prenylsynthase signatures are supposedly involved in binding of the substrate, isopentenyl pyrophosphate, and elongating dimethylallyl pyrophosphate, by forming magnesium salt bridges between the substrate and the catalytic site (13
). Site-directed mutagenesis of the second aspartate-rich motif has been performed in FPP synthase from Saccharomyces cerevisiae
) and Bacillus stearothermophilus
). These data show that the FPP synthase aspartate residue corresponding to the human PDSS1 residue D308 is essential for catalytic activity. Moreover, the mutagenesis of aspartate to glutamate at position 244 of the rat FPP synthase (D244E), equivalent to the D308E in PDSS1, resulted in a 7-fold reduction in the Vmax
value of the enzyme (21
). The D308E mutation identified in our patients with ubiquinone deficiency therefore confirms the functional importance of this particular residue for the prenylsynthase activity of the protein.
The OH-benzoate transpolyprenyltransferase (COQ2
) gene encodes the enzyme involved in the second step of ubiquinone biosynthesis (13
). In a second family with ubiquinone deficiency, a single base pair deletion was identified in the COQ2
gene, resulting in a premature stop codon and modifying the last 21 amino acids of the protein. The normal but not the mutant protein rescued the growth defect of the Δcoq2
yeast strain on a nonfermentable carbon source, therefore demonstrating the deleterious consequences of the mutation. Finally, the abnormal accumulation of decaprenyl-PP, the substrate of OH-benzoate polyprenyltransferase in cultured skin fibroblasts of the patient, also supports the relevance of this mutation in ubiquinone deficiency. The mutated domain of the protein is not well conserved across species, but the mutation modifies the global charge at the C-terminal end of the protein, possibly altering its interactions with other proteins. Several enzymes involved in ubiquinone biosynthesis in yeast have been shown to form a multisubunit complex, as Coq1p, Coq4p, Coq5p, and Coq6p have been shown to form a high-molecular-weight complex (23
). It is not yet known whether Coq2p is also involved in this complex in yeast and whether a similar complex exists in humans. Nevertheless, the deleterious effect of this human mutant protein in yeast suggests that various interactions between the proteins of the 2 species are still retained despite the absence of sequence homology between human and yeast Coq2p at the C-terminal ends of the proteins.
Primary ubiquinone deficiency is a rare cause of mitochondrial disorders, and the genetic bases of these disorders have rarely been identified, as COQ2
mutations have only been reported twice in patients with encephalomyopathy and kidney involvement (11
). In this article, we report what we believe to be the first molecular/functional characterization of PDSS1
mutations and demonstrate that both mutations are disease causing.
It is noteworthy that optic atrophy, deafness, obesity, pancytopenia, and cardiac disease have not been previously reported in primary ubiquinone deficiency. While optic atrophy, deafness, and pancytopenia are occasionally reported in respiratory chain deficiency, it remains unclear whether obesity and valvulopathy are directly related to ubiquinone deficiency. Similarly, early and severe forms of ubiquinone deficiency, fatal in the early neonatal period, have seldom been reported (7
). Why profound ubiquinone deficiency in cultured fibroblasts was associated with either neonatal death or long survival also remains unexplained (patients 1 and 2 were 22 and 14 years old, respectively). The clinical heterogeneity of ubiquinone deficiency is suggestive of a genetic heterogeneity that could be related to the large number of enzymes and corresponding genes involved in ubiquinone biosynthesis. It is hoped that identification of disease-causing genes in other families will help to elucidate the clinical variability of these rare conditions.