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Association between genetic variants in the Coenzyme Q10 metabolism and Coenzyme Q10 status in humans

Background Coenzyme Q10 (CoQ10) is essential for mitochondrial energy production and serves as an antioxidants in extra mitochondrial membranes. The genetics of primary CoQ10 deficiency has been described in several studies, whereas the influence of common genetic variants on CoQ10 status is largely unknown. Here we tested for non-synonymous single-nucleotidepolymorphisms (SNP) in genes involved in the biosynthesis (CoQ3G272S , CoQ6M406V, CoQ7M103T), reduction (NQO1P187S, NQO2L47F) and metabolism (apoE3/4) of CoQ10 and their association with CoQ10 status. For this purpose, CoQ10 serum levels of 54 healthy male volunteers were determined before (T0) and after a 14 days supplementation (T14) with 150 mg/d of the reduced form of CoQ10. Findings At T0, the CoQ10 level of heterozygous NQO1P187S carriers were significantly lower than homozygous S/S carriers (0.93 ± 0.25 μM versus 1.34 ± 0.42 μM, p = 0.044). For this polymorphism a structure homology-based method (PolyPhen) revealed a possibly damaging effect on NQO1 protein activity. Furthermore, CoQ10 plasma levels were significantly increased in apoE4/E4 genotype after supplementation in comparison to apoE2/E3 genotype (5.93 ± 0.151 μM versus 4.38 ± 0.792 μM, p = 0.034). Likewise heterozygous CoQ3G272S carriers had higher CoQ10 plasma levels at T14 compared to G/G carriers but this difference did not reach significance (5.30 ± 0.96 μM versus 4.42 ± 1.67 μM, p = 0.082). Conclusions In conclusion, our pilot study provides evidence that NQO1P187S and apoE polymorphisms influence CoQ10 status in humans.


Background
Coenzyme Q 10 (CoQ 10 ) is the predominant form of endogenous ubiquinone in humans. Synthesized in the mitochondrial inner membrane, CoQ 10 is comprised of a ubiquinone head group attached to a trial of 10 fivecarbon isoprenoid units, that anchors the molecule to the membranes [1]. Intracellular synthesis is the major source of CoQ 10 , however it can also be acquired through the diet and dietary supplements [2]. CoQ 10 acts in the respiratory chain and is necessary for pyrimidine biosynthesis as well as a cofactor of uncoupling proteins [3]. CoQ 10 has been also identified as a modulator of gene expression [4][5][6], inflammatory processes [7][8][9] and apoptosis [10,11].
The CoQ 10 biosynthetic pathway comprises 10 steps, including methylations, decarboxylations, hydroxylations and isoprenoid synthesis and transfer [12]. The elucidation of this pathway was mainly due to studies in respiration-deficient mutans of E. coli and S. cerevisiae [13,14]. In humans, rare genetic variants in genes encoding enzymes of CoQ 10 synthesis causes mitochondrial dysfunction, as CoQ 10 carries electrons from complex I and complex II to complex III in the mitochondrial respiratory chain. Several forms of human CoQ 10 deficiencies were characterized by infantile encephalomyopathy, renal failure, cerebellar ataxia or myopathy [15][16][17].
The complexity of CoQ 10 biosynthesis suggests that genetic defects in different biosynthetic enzymes or regulatory proteins may cause different clinical syndromes.
Although several studies have been undertaken to look into primary CoQ 10 deficiency, the influence of common genetic variants on CoQ 10 status is largely unknown. Therefore a proof of principle study in humans was performed to associate single nucleotide polymorphisms (SNPs) in genes encoding proteins of CoQ 10 biosynthesis, reduction and metabolism with CoQ 10 status before and after supplementation.

Participants and study design
Sample characteristics of subjects and study design have been recently described [18]. In short: 54 healthy male volunteers received 150 mg of the reduced form of CoQ 10 (ubiquinol, KANEKA Corporation, Japan) daily in form of three capsules with each principal meal for 14 days. Fasting blood samples were taken before (T 0 ) and after (T 14 ) supplementation with ubiquinol from all study participants. The participants, aged 30.1 ± 6.7 years, had an average Body Mass Index (BMI) of 24.1 ± 2.5, no history of gastrointestinal, hepatic, cardiovascular or renal diseases, a habit of non-or occasional smoking (≤ 3 cigarettes/day) and maintenance of usual nutrition habits. The study was approved by the ethics committee of the Medical Faculty of Kiel University, Germany, and was conformed to Helsinki Declaration. All volunteers gave written informed consent.

Genotyping
Genomic DNA was isolated from whole blood samples. Genotyping of all SNPs investigated (Table 1) was performed with the TaqMan system. Fluorescence was measured with ABI Prism 7900 HT sequence detection system (ABI, Foster City, USA).

HPLC analysis
CoQ 10 analysis was based on the method of high-pressure liquid chromatography (HPLC) with electrochemical detection and internal standardisation using ubihydroquinone-9 and ubiquinone-9 as standards and has been described elsewhere [18].

Statistical analysis
Data are expressed as means ± SD. Differences in the characteristics of the study population between two genotype groups were examined using the Student t-test and additionally for CoQ6 M406V the c 2 -test in a dominant genetic model. To determine statistical significance between all genotypes, test for linear trend in one way analysis of variance (ANOVA) was performed. P-values ≤ 0.05 were considered statistically significant and all statistical analyses were computed using SPSS (Version 13.0). In order to analyze the impact of nonsynonymous SNPs on the structure and function of proteins, PolyPhen server [19] was used. For power calculation, the GPower program (Version 3.1) was applied.

Genotype distributions in the cohort
The selected SNPs were genotyped in 54 healthy male volunteers. The obtained genotype distribution (Figure 1 and 2) were in accordance to the HapMap data: Genotype distribution of the CoQ3 G272S polymorphism revealed 38 homozygous for G/G (73%), 13 heterozygous  Two samples failed genotyping. Concerning the distribution of the NQO1 P187S SNP, 30 persons are carriers of two P/P alleles (56%), 22 persons were heterozygous with one P and one S allele (41%) and two participants were carriers of two S/S alleles (3%). NQO2 L47F genotyping displayed 35 participants were homozygous L/L carriers Values are mean ± SD and n numbers (genotype distribution) are given in brackets. Differences between two genotype groups were examined using Student t-test and between all genotypes using "test for linear trend" (ANOVA).  Values are mean ± SD and n numbers (genotype distribution) are given in brackets. Differences between two genotype groups were examined using Student t-test (*p ≤ 0.05) and between all genotypes using "test for linear trend" (ANOVA). Association between genotypes and CoQ 10 level at baseline T 0 and after supplementation T 14 with the reduced form of CoQ 10 As previously described [18], 54 healthy male volunteers received 150 mg of the reduced form of CoQ 10 daily in form of three capsules with each principal meal for 14 days. This supplementation led to a significant 4-fold increase in total CoQ 10 plasma levels at T 14 (4.60 ± 1.55 μmol/L) compared to T 0 (0.96 ± 0.31 μmol/L) [18]. As shown in Figure 1 and 2, SNPs determined in the CoQ7 and NQO2 genes were not associated with total CoQ 10 levels. Trend analysis (ANOVA) over all genotype variants of CoQ7 M103T and NQO2 L47F revealed p values >0.05 and were therefore considered as not significant.

CoQ3 G272S
The COQ3 gene encodes an O-methyltransferase required for two steps in the biosynthetic pathway of CoQ 10 [31]. Analysing CoQ3 rs6925344 SNP in association to plasma CoQ 10 levels at T 0 , no significant differences between genotypes could be revealed. Yet at T 14 , G/S carriers in CoQ3 G272S genotype had a higher total CoQ 10 content (5.30 ± 0.96 μmol/L) after supplementation compared to G/G carriers (4.42 ± 1.67 μmol/L) with borderline significance (p = 0.082, t-test).

CoQ6 M406V
CoQ6 is mapped to human chromosome 14q24.3 and encodes a monooygenase, which is required in CoQ 10 biosynthesis for incorporation of oxygen to the benzoquinone ring [32]. CoQ 10 plasma levels were not significantly changed within genotype distribution of CoQ6 rs8500 SNP before (T 0 ) and after (T 14 ) supplementation. However, considering total CoQ 10 distribution at T 0 in a chi-square cross tabulation as a function of CoQ6 rs8500 genotype (Table 2) a person chi-square χ 2 value of p = 0.081 was evident, which again can be considered as marginal significant. Therefore a power calculation for CoQ6 genotype rs8500 was conducted using GPower program (Version 3.1). This disclosed a total of 898 individuals are required to receive 95% power.

NQO1 P187S
It has been shown, that NQO1 can generate and maintain the reduced state of ubiquinones in membrane systems and liposomes, thereby promoting their antioxidant function [33,34]. NQO1 P187S SNP was associated with CoQ 10 levels at T 0 (P/S versus S/S, p = 0.044). Thus, this pilot study indicates that Pro187Ser SNP in NQO1 gene could participate in abnormal CoQ 10 metabolism. SNP prediction of functional effects of human nsSNPs with structure homology-based method (PolyPhen) revealed a possibly damaging effect of NQO1 P187S SNP with a score of 0.215. However, genotype distribution of the S/S genotype was low (n = 2), which reflects the ethnic variation of this polymorphism with the highest prevalence of the S allele in East Asian populations (e.g. 22% prevalence in Chinese populations) and the lowest prevalence in Caucasians (4%) [35]. Furthermore Han et al [36] found a significant association of this SNP with carotid artery plaques in type 2 diabetic patients in east Asian populations. As this genetic variation may play a more significant role in an East Asian rather than in a Caucasian population, evaluation of the Pro187Ser SNP in association with CoQ 10 metabolism in an East Asian population may be preferable.
apoE Apolipoprotein E (apoE) is a polymorphic multifunctional protein with three common isoforms in humans (E2, E3 and E4). Presence of the apoE4 allele is associated with a 40-50% higher risk of cardiovascular disease [37]. There is increasing evidence demonstrating that the apoE4 allele may be associated with elevated oxidative stress and chronic inflammation [38]. Thus apoE was considered as a candidate gene explaining variance in CoQ 10 status. At T 0 , total CoQ 10 levels were higher in E4/E4 carriers as compared to all other genotype groups, however p values did not reached significance (p = 0.065, E2/E3 vs E4/E4, Figure 2). These results confirm the results found by Battino et al [29] in a cohort of 106 healthy blood donors. Interestingly, in our study total CoQ 10 levels increased significantly (p = 0.034) in E4/E4 carriers after supplementation (T 14 ), which has to the best of our knowledge not been shown so far. Thus, E4/E4 carriers may be more responsive towards a dietary CoQ 10 supplementation than non E2/E3 carriers. The underlying physiological and/or molecular mechanisms for this finding still need to be elucidated.

Conclusions
Taken together, our pilot study with 54 volunteers provides evidence that NQO1 P187S and apoE polymorphisms may influence CoQ 10 status in humans. According to our results and power calculation, larger cohorts are needed in further studies to determine the association between single nucleotide polymorphisms in genes encoding proteins of CoQ 10 biosynthesis, reduction and metabolism and CoQ 10 status.