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 Q10 (CoQ10) is the predominant form of endogenous ubiquinone in humans. Synthesized in the mitochondrial inner membrane, CoQ10 is comprised of a ubiquinone head group attached to a trial of 10 five-carbon isoprenoid units, that anchors the molecule to the membranes [1]. Intracellular synthesis is the major source of CoQ10, however it can also be acquired through the diet and dietary supplements [2]. CoQ10 acts in the respiratory chain and is necessary for pyrimidine biosynthesis as well as a cofactor of uncoupling proteins [3]. CoQ10 has been also identified as a modulator of gene expression [4-6], inflammatory processes [7-9] and apoptosis [10,11].

The CoQ10 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 CoQ10 synthesis causes mitochondrial dysfunction, as CoQ10 carries electrons from complex I and complex II to complex III in the mitochondrial respiratory chain. Several forms of human CoQ10 deficiencies were characterized by infantile encephalomyopathy, renal failure, cerebellar ataxia or myopathy [15-17].

The complexity of CoQ10 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 CoQ10 deficiency, the influence of common genetic variants on CoQ10 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 CoQ10 biosynthesis, reduction and metabolism with CoQ10 status before and after supplementation.

Methods

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 CoQ10 (ubiquinol, KANEKA Corporation, Japan) daily in form of three capsules with each principal meal for 14 days. Fasting blood samples were taken before (T0) and after (T14) 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).

Table 1

Selected polymorphisms in CoQ3, CoQ6, CoQ7, NQO1, NQO2 and apoE gene

Gene	refSNPida	Sequenceb	Position	Amino acid change
CoQ3	rs6925344	ACAATAC[C/T]TGCAATT	exon 6	Gly272Ser
CoQ6	rs8500	AGGTTCC[A/G]TGAGCCA	exon 11	Met406Val
CoQ7	rs11074359	ATGGTTA[T/C]GTTCAGG	exon 3	Met103Thr
NQO1	rs1800566	AGTTGAG[A/G]TTCTAAG*	exon 6	Pro187Ser
NQO2	rs1143684	CATGAAC[C/T]TTGAGCC	exon 3	Leu47Phe
apoE	rs429358	GGACGTG[C/T]GCGGCC	exon 4	Arg112Cys
apoE	rs7412	GCAGAAG[C/T]GCCTGG	exon 4	Arg158Cys

a: NCBI; b: Applied Biosystems, *antisense

HPLC analysis

CoQ10 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 CoQ6M406V the χ2 -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 non-synonymous SNPs on the structure and function of proteins, PolyPhen server [19] was used. For power calculation, the GPower program (Version 3.1) was applied.

Results and Discussion

Selection of genes and single nucleotide polymorphisms

In order to identify common SNPs which may be associated with the CoQ10 status, we searched in the HapMap data base for non-synonymous variants in genes which are involved in CoQ10 biosynthesis and metabolism. As shown in table 1, we selected SNPs in the CoQ3 (rs6925344, C>T, Gly272Ser), CoQ6 (rs8500, A>G, Met406Val) and CoQ7 (rs11074359, T>C, Met103Thr) gene. These genes code for enzymes of CoQ10 biosynthesis. Functional variants [20,21] in the NQO1 (rs1800566, C>T, Pro187Ser) and NQO2 (rs1143684, T>C, Leu47Phe) gene were also included, as the encoded NAD(P)H:quinone oxidoreductases are involved in the recycling of CoQ10. Furthermore they protect cells from oxidative damage by catalyzing reduction of carcinogenic quinone compounds to their hydroquinone forms [22]. Two SNPs determining the apolipoprotein E (apoE) haplotypes E2, E3 and E4 (rs429358, rs7412) were further included. Both SNPs led to an amino acid change from cysteine to arginine at position 112 (rs429358) and 158 (rs7412), which gives rise to six possible diplotypes: E2/E2, E2/E3, E2/E4, E3/E3, E3/E4 and E4/E4. The apoE diplotypes have been associated with cholesterol metabolism [23,24], atherosclerosis [25], inflammation [26], lipid peroxidation [27] and longevity [28].

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 CoQ3G272S polymorphism revealed 38 homozygous for G/G (73%), 13 heterozygous for G/S (25%) and 1 homozygous for S/S (2%), while 1 sample failed genotyping. Analysis of the CoQ6M406V genotype showed 19 homozygous for M/M (36%), 24 heterozygous for M/V (44%) and 11 homozygous for V/V (20%). Genotyping of CoQ7M103T polymorphism revealed 25 M/M (48%), 17 M/T (33%) and 10 T/T (19%) carriers. Two samples failed genotyping. Concerning the distribution of the NQO1P187S 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%). NQO2L47F genotyping displayed 35 participants were homozygous L/L carriers (65%), 15 participants were heterozygous for L/F (28%) and 4 participants were homozygous F/F carriers (7%). The genotype distribution of apoE was as follows: 1 person with E2/E2 genotype (2%), 7 persons with E2/E3 (14%), 29 persons with E3/E3 (58%), 11 persons with E3/E4 (22%) and 2 persons with E4/E4 (4%). For 4 persons, genotyping of one or both SNPs respectively failed. Thus, the Apo E genotype distribution in our cohort of 54 healthy men was comparable with previously published data [29,30].

Figure 1

Effect of amino acid exchange polymorphisms on CoQ. SNPs in genes encoding enzymes of the CoQ10 synthesis pathway (CoQ3G272S, CoQ6M406V, CoQ7M103T) before (T0) and after (T14) ubiquinol supplementation (150 mg/day) in humans are shown. 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).

Figure 2

Effect of NQO1. CoQ10 plasma levels before (T0) and after (T14) ubiquinol supplementation (150 mg/day) in humans are shown. 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 CoQ10 level at baseline T0 and after supplementation T14 with the reduced form of CoQ10

As previously described [18], 54 healthy male volunteers received 150 mg of the reduced form of CoQ10 daily in form of three capsules with each principal meal for 14 days. This supplementation led to a significant 4-fold increase in total CoQ10 plasma levels at T14 (4.60 ± 1.55 μmol/L) compared to T0 (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 CoQ10 levels. Trend analysis (ANOVA) over all genotype variants of CoQ7M103T and NQO2L47F revealed p values >0.05 and were therefore considered as not significant.

CoQ3G272S

The COQ3 gene encodes an O-methyltransferase required for two steps in the biosynthetic pathway of CoQ10 [31]. Analysing CoQ3 rs6925344 SNP in association to plasma CoQ10 levels at T0, no significant differences between genotypes could be revealed. Yet at T14, G/S carriers in CoQ3G272S genotype had a higher total CoQ10 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).

CoQ6M406V

CoQ6 is mapped to human chromosome 14q24.3 and encodes a monooygenase, which is required in CoQ10 biosynthesis for incorporation of oxygen to the benzoquinone ring [32]. CoQ10 plasma levels were not significantly changed within genotype distribution of CoQ6 rs8500 SNP before (T0) and after (T14) supplementation. However, considering total CoQ10 distribution at T0 in a chi-square cross tabulation as a function of CoQ6 rs8500 genotype (Table 2) a person chi-square χ2 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.

Table 2

Total CoQ10 distribution in a chi-square crosstabulation as a function of CoQ6M406V genotype (rs8500).

	Pearson Χ2
CoQ6(rs8500)	< 0.96(μmol/L)	> 0.96(μmol/L)	Total
M/M	7	12	19
M/V+V/V	21	13	34
Total	28	25	53

Person Chi-Square Χ2: p = 0.081

Distribution was calculated according to a dominant model. CoQ10 mean value of 0.96 μmol/L was used for group classification.

NQO1P187S

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]. NQO1P187S SNP was associated with CoQ10 levels at T0 (P/S versus S/S, p = 0.044). Thus, this pilot study indicates that Pro187Ser SNP in NQO1 gene could participate in abnormal CoQ10 metabolism. SNP prediction of functional effects of human nsSNPs with structure homology-based method (PolyPhen) revealed a possibly damaging effect of NQO1P187S 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 CoQ10 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 CoQ10 status. At T0, total CoQ10 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 CoQ10 levels increased significantly (p = 0.034) in E4/E4 carriers after supplementation (T14), which has to the best of our knowledge not been shown so far. Thus, E4/E4 carriers may be more responsive towards a dietary CoQ10 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 NQO1P187S and apoE polymorphisms may influence CoQ10 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 CoQ10 biosynthesis, reduction and metabolism and CoQ10 status.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

AF analysed the data and wrote the manuscript. CS participated in the design of the study, acquired and analysed the data. GR participated in the design of the study and critically revised the manuscript. PN and TM carried out the CoQ10 measurements. FD was responsible for the concept and design of the study and the writing of the paper. All authors read and approved the final manuscript.