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A Mitochondrial DNA Variant Elevates the Risk of Gallstone Disease by Altering Mitochondrial Function

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    Dayan Sun
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    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, and Human Phenome Institute, Fudan University, Shanghai, China

    Collaborative Innovation Center for Genetics and Development, Fudan University, Shanghai, China
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    # Authors share co-first authorship.
    Zhenmin Niu
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    Affiliations
    Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai and Shanghai Academy of Science and Technology, Shanghai, China
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    Hong-Xiang Zheng
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    # Authors share co-first authorship.
    Affiliations
    Collaborative Innovation Center for Genetics and Development, Fudan University, Shanghai, China

    Ministry of Education Key Laboratory of Contemporary Anthropology, Department of Anthropology and Human Genetics, School of Life Sciences, Fudan University, Shanghai, China
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  • Fei Wu
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, and Human Phenome Institute, Fudan University, Shanghai, China
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  • Liuyiqi Jiang
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, and Human Phenome Institute, Fudan University, Shanghai, China
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  • Tian-Quan Han
    Affiliations
    Shanghai Institute of Digestive Surgery, Department of Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
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  • Yang Wei
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, and Human Phenome Institute, Fudan University, Shanghai, China
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  • Jiucun Wang
    Correspondence
    Jiucun Wang, PhD, School of Life Sciences, Fudan University, 2005 Songhu Road, Shanghai, China. fax: 862131246607.
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, and Human Phenome Institute, Fudan University, Shanghai, China

    Collaborative Innovation Center for Genetics and Development, Fudan University, Shanghai, China

    Research Unit of Dissecting the Population Genetics and Developing New Technologies for Treatment and Prevention of Skin Phenotypes and Dermatological Diseases (2019RU058), Chinese Academy of Medical Sciences, Shanghai, China

    Taizhou Institute of Health Sciences, Fudan University, Taizhou, China
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  • Li Jin
    Correspondence
    Correspondence Address correspondence to: Li Jin, PhD, School of Life Sciences, Fudan University, 2005 Songhu Road, Shanghai, China. fax: 862131246607.
    Affiliations
    State Key Laboratory of Genetic Engineering, School of Life Sciences, and Human Phenome Institute, Fudan University, Shanghai, China

    Collaborative Innovation Center for Genetics and Development, Fudan University, Shanghai, China

    Research Unit of Dissecting the Population Genetics and Developing New Technologies for Treatment and Prevention of Skin Phenotypes and Dermatological Diseases (2019RU058), Chinese Academy of Medical Sciences, Shanghai, China

    Taizhou Institute of Health Sciences, Fudan University, Taizhou, China
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Open AccessPublished:December 03, 2020DOI:https://doi.org/10.1016/j.jcmgh.2020.11.015

      Background and aims

      Gallstone disease (cholelithiasis) is a cholesterol-related metabolic disorders with strong familial predisposition. Mitochondrial DNA (mtDNA) variants accumulated during human evolution are associated with some metabolic disorders related to modified mitochondrial function. The mechanistic links between mtDNA variants and gallstone formation need further exploration.

      Methods

      In this study, we explored the possible associations of mtDNA variants with gallstone disease by comparing 104 probands and 300 controls in a Chinese population. We constructed corresponding cybrids using trans-mitochondrial technology to investigate the underlying mechanisms of these associations. Mitochondrial respiratory chain complex activity and function and cholesterol metabolism were assessed in the trans-mitochondrial cell models.

      Results

      Here, we found a significant association of mtDNA 827A>G with an increased risk of familial gallstone disease in a Chinese population (odds ratio [OR]: 4.5, 95% confidence interval [CI]: 2.1–9.4, P=1.2×10–4). Compared with 827A cybrids (haplogroups B4a and B4c), 827G cybrids (haplogroups B4b and B4d) had impaired mitochondrial respiratory chain complex activity and function and activated JNK and AMPK signaling pathways. Additionally, the 827G cybrids showed disturbances in cholesterol transport and accelerated development of gallstones. Specifically, cholesterol transport through the transporter ABCG5/8 was increased via activation of the AMPK signaling pathway in 827G cybrids.

      Conclusions

      Our findings reveal that mtDNA 827A>G induces aberrant mitochondrial function and abnormal cholesterol transport, resulting in increased occurrence of gallstones. The results provide an important biological basis for the clinical diagnosis and prevention of gallstone disease in the future.

      Graphical abstract

      Keywords

      Abbreviations used in this paper:

      ABCB11 (ATP binding cassette subfamily B member 11), ABCB4 (ATP binding cassette subfamily B member 4), ABCG5 (ATP binding cassette subfamily G member 5), ABCG8 (ATP binding cassette subfamily G member 8), ATP (adenosine triphosphate), mtDNA (mitochondrial DNA), mRNA (messenger RNA), nDNA (nuclear DNA), OXPHOS (oxidative phosphorylation), rRNA (ribosomal RNA), SNP (single nucleotide polymorphism), TCA (tricarboxylic acid)
      Mitochondrial DNA 827A>G induces aberrant mitochondrial function and abnormal cholesterol transport through the transporter ABCG5/8 (ATP binding cassette subfamily G member 5/8) via activation of the AMPK signaling pathway, which increases the formation of gallstones.
      Gallstone disease (cholelithiasis) is a common disease that is related to abnormal metabolism of lipids. Most gallstones are caused by supersaturation of cholesterol in the bile that leads to cholesterol crystallization and stone nidus formation, and <10% of stones are black and brown pigment stones.
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      Therefore, we hypothesized that mtDNA variants may contribute to the occurrence of gallstones, which are manifestations of lipid metabolic abnormalities.
      In this study, we explored the possible associations between mtDNA variants and gallstone disease by comparing 104 probands and 300 controls in a Chinese population. The results showed a strong association of gallstone disease with mtDNA haplogroup B4b′d′e′j. A variant defining haplogroup B4b′d′e′j, 827A>G, in mitochondrial 12S ribosomal RNA (rRNA) emerged as the most likely candidate responsible for the observed association. Thus, we investigated the pathological mechanism of gallstone disease associated with 827A>G by using transmitochondrial technology in this study. Mitochondrial respiratory chain complex activity and function, and cholesterol metabolism were assessed in the transmitochondrial cell models. Our findings revealed that mtDNA 827A>G induced aberrant mitochondrial function and abnormal cholesterol transport, resulting in increased occurrence of gallstones.

      Results

      Associations of mtDNA Variants With Gallstone Disease

      We first investigated the relationships between mtDNA variants and gallstone disease in a Chinese population. DNA samples from 104 unrelated probands (mean age 44.9 years; range, 19–81 years) with confirmed gallstone disease (cholelithiasis) and a total of 300 unrelated individuals (mean age 64 years; range, 50–86 years) were collected from the Greater Shanghai Area. The average total cholesterol and triglyceride concentrations of the patients were 4.41 (normal range, 2.8–5.17) mmol/L and 1.67 (normal range, 0.56–1.7) mmol/L, respectively. We sequenced the mtDNA HVS1 regions of the 104 patients and 300 controls, and the results showed that 2 variants, 16217C (P = .041) and 16136C (P = .007), showed significantly higher frequencies in the cases than in the controls. Interestingly, both variants were mutations defining B4 haplogroup (ie, 16217C defined B4, and 16136C defined B4b1). The P values for the chi-square test and Fisher′s exact test of the associations between gallstone disease and the closely related polymorphisms are presented in Table 1. A 9-bp deletion at 8281–8289, the diagnostic marker of haplogroup B4′5, showed no association between the cases (n = 23 of 104) and the controls (n = 50 of 300) (P >0.05). In addition, the prevalence of B5, the sister group of B4, was not significantly different between the cases (n = 4 of 104) and controls (n = 12 of 300), further supporting the significance of haplogroup B4. Moreover, the mitochondrial whole genome results showed the variant 827A>G in mitochondrial 12S rRNA was the candidate most likely responsible for gallstone disease (Table 2).
      Table 1Association of mtDNA Haplogroups B4b′d′e′j With Gallstone Disease
      mtDNA Mutation and Haplogroup
      16217C16261827G499A13942G16136C6413C
      B4B4aB4b′d′e′jB4bB4dB4b1
      Cases (n = 104)20515114118
      %19.234.8114.4210.583.8510.587.69
      Controls (n = 300)341511110113
      %11.335.003.673.6703.671.00
      P2).041.9381.2 × 10–47.5 × 10–36.4 × 10–47.5 × 10–32.3 × 10–4
      P (FET).046>0.9993.5 × 10–4.012.004.0121.3 × 10–3
      OR1.8630.9604.4283.107/3.1078.25
      95% CI1.02–3.380.34–2.712.08–9.441.35–7.13/1.35–7.132.68–25.3
      NOTE. Probabilities of observed associations were calculated based on Pearson chi-square test, FET, and OR.
      CI, confidence interval; FET, Fisher′s exact test; OR, odds ratio.
      Table 2Analysis of Whole Mitochondrial Genome of the 4 B4b′d′e′j Individuals
      PositionCRSPatientsRegionAA Change
      P13P46P74P89
      827AG
      Presents the site is mutant.
      G
      Presents the site is mutant.
      G
      Presents the site is mutant.
      G
      Presents the site is mutant.
      12S rRNA
      4820GA
      Presents the site is mutant.
      A
      Presents the site is mutant.
      A
      Presents the site is mutant.
      GND2None
      5292TTTTC
      Presents the site is mutant.
      ND2Ser > Arg
      6023GA
      Presents the site is mutant.
      A
      Presents the site is mutant.
      GGCOXINone
      6216TC
      Presents the site is mutant.
      C
      Presents the site is mutant.
      TTCOXINone
      6413TC
      Presents the site is mutant.
      C
      Presents the site is mutant.
      TTCOXINone
      13590GA
      Presents the site is mutant.
      A
      Presents the site is mutant.
      A
      Presents the site is mutant.
      GND5None
      13801AAAAG
      Presents the site is mutant.
      ND5Thr > Ala
      13942AAAAG
      Presents the site is mutant.
      ND5Thr > Ala
      14587AAAG
      Presents the site is mutant.
      AND6None
      15315CCCCT
      Presents the site is mutant.
      CytbAla > Val
      15535CT
      Presents the site is mutant.
      T
      Presents the site is mutant.
      T
      Presents the site is mutant.
      T
      Presents the site is mutant.
      CytbNone
      15930GGGGA
      Presents the site is mutant.
      tRNA-Thr
      AA, amino acid; CRS, Cambridge Reference Sequence; rRNA, ribosomal RNA; tRNA, transfer RNA.
      a Presents the site is mutant.
      Next, we explored the frequency differences of the mutations defining the subhaplogroups of B4 between cases and controls to identify the subhaplogroups most associated with gallstone disease. In the phylogenetic tree (Figure 1A) and median-joining network (Figure 1B) of haplogroup B4, we found that haplogroup B4b′d′e′j showed a higher odds ratio (4.428) and more significant P value (1.2 × 10–4) (n = 15 of 104 patients and n = 11 of 300 controls) than the other haplogroups in B4 (B4, B4a, B4b1, and B4d), indicating that B4b′d′e′j was the haplogroup most likely associated with gallstone disease and that the variant harbored in this haplogroup might contribute to gallstone disease.
      Figure thumbnail gr1
      Figure 1Association of mtDNA haplogroup B4b′d′e′j with gallstone disease. (A) PhyloTree of B4 individuals among the cases and controls. Each square represents a haplogroup observed in this study. The filled squares represent the cases (marked with labels starting with P), and the open squares represent the controls (marked with labels starting with C). The inferred mutations are marked by short lines. Green represents mtDNA haplogroup B4a, pink represents mtDNA haplogroup B4b1, blue represents mtDNA haplogroup B4d, and black represents other subhaplogroups of mtDNA haplogroup B4. (B) Network of B4 individuals among the cases and controls. This median-joining network was reconstructed using NETWORK software. Each circle represents a haplogroup observed in this study. The size of the circle is proportional to the number of individuals carrying the haplotype. The filled circles represent the cases (marked with labels starting with P) and the open circles represent the controls (marked with labels starting with C). The root of the network is labeled as the Out group. The inferred mutations are marked on the lines connecting the haplogroups. (C) Scatterplot of the prevalence of gallstones and the frequency of mtDNA haplogroup B4b′d′e′j. The blue circles represent East Asians; the pink squares represent Native Americans; and the green triangles represent others, including Africans and Eurasians. (D) Conservation analysis of the mtDNA 827A>G variant in 12S rRNA. Blue and red indicate the 827 position with the A and G polymorphisms, respectively. (E) Secondary structure of the mtDNA 827A>G variant in 12S rRNA. (Left) Wild-type 12S rRNA; (right) 12S rRNA with the mtDNA 827A>G variant. The corresponding panels show enlargements of the regions of predicted secondary structures surrounding nucleotide position 827 (bold arrows with red circles).
      During the evolutionary history of modern humans, haplogroup B4 might have originated in East Asia approximately 40,000 years ago.
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      B4b′d′e′j arose from B4 with the mutations 827A>G (a variant in 12S rRNA) and 15535C>T (a synonymous mutation in CytB). Thus, we hypothesized that 827A>G might have a considerable effect on the etiology of gallstone disease. B2, a subhaplogroup of B4b′d′e′j, was a founder haplogroup and expanded in the Americas after the Last Glacial Maximum (approximately 20,000 years ago).
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      Thus, the frequencies of 827A>G B4 carriers were higher in Native Americans (14%–44%) than in East Asians (2%–8%) (Figure 1C; Supplementary Table 1). Interestingly, we found that the prevalence of gallstone disease was also significantly higher in Native Americans (14%–35%) than in East Asians (3%–12%) (P = .002, t test), suggesting the possible role of B4b′d′e′j in gallstone disease.
      In addition, conservation analysis of vertebrates showed that 827A>G was generally conserved among all the species tested (Figure 1D), indicating that this adenosine might be important in maintaining 12S rRNA function in different species and that mutations at this site are subject to purifying selection. We further predicted the RNA structure of 12S rRNA with or without 827A>G using the RNAfold program implemented in the Vienna RNA Package 2.0 (Figure 1E).
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      The results indicated that the 827A>G variant in 12S rRNA might change the nearby loop structure.
      In summary, we found that the 12S rRNA variant 827A>G harbored in haplogroup B4b′d′e′j might affect mitochondrial function and in turn affect the prevalence of gallstone disease.

      The 827G Cybrids Exhibited Lower Respiratory Chain Complex Activity Than the 827A Cybrids

      To analyze the effect of the 827A>G variant on the regulation of the mitochondrial respiratory chain complex, we constructed 2 groups of sister branch haplogroup cell models: 827A cybrids (B4a/B4c haplogroups) (n = 6), and 827G cybrids (B4b/B4d haplogroups) (n = 6) according to the mtDNA phylogenetic tree. First, to determine whether 827A>G affects mitochondrial respiratory chain complex activity, we evaluated the transcript levels of 12S rRNA and mtDNA-encoded OXPHOS genes in the 827A and 827G cybrids. The results illustrated that the RNA levels of 12S rRNA, ND4/4L, ND5, ND6, CO3, ATP6, and ATP8 were significantly higher in the 827A cybrids than in the 827G cybrids (Figure 2A and C). In addition, mitochondrial ribosomal small subunit transcript levels were lower in the 827G cybrids than in the 827A cybrids, consistent with the results of the transcriptomic gene expression analysis of all 30 mitochondrial ribosomal small subunits (Figure 2A and B). Moreover, nuclear DNA (nDNA)-encoding OXPHOS gene expression tended to be lower in the 827G cybrids than in the 827A cybrids (Figure 2D). However, the mtDNA copy number did not significantly differ in either cybrids or peripheral blood mononuclear cell samples between the 827A (n = 206) and 827G (n = 82) genotypes (Figure 2E and F).
      Figure thumbnail gr2
      Figure 2The 827G cybrids exhibited lower respiratory chain complex activity than the 827A cybrids. (A) Mitochondrial 12S rRNA and ribosomal protein small subunit–related gene transcript levels in the 827A (n = 6) and 827G (n = 6) cybrids. The relative mitochondrial ribosomal protein transcript levels in the 827G cybrids were normalized to the levels in the 827A cybrids. (B) Heatmaps of all the mitochondrial ribosomal protein small subunit–related gene levels between the 827A (n = 6) and 827G (n = 6) cybrids. The gradual color change from red to green represents the expression change from upregulation to downregulation. (C) Mitochondrial RNA levels in the 827A (n = 6) and 827G (n = 6) cybrids. The relative mitochondrial RNA levels in the 827G cybrids were normalized to the levels in the 827A cybrids. ND4/4 L, ND4, and ND4L. (D) Heatmaps of OXPHOS nDNA-encoded gene expression between the 827A (n = 6) and 827G (n = 6) cybrids. (E) MtDNA copy number levels of the 827A (n = 6) and 827G (n = 6) cybrids. The relative mtDNA copy number levels in the 827G cybrids were normalized to the levels in the 827A cybrids. (F) MtDNA copy number levels of the peripheral blood mononuclear cell samples of the 827A (n = 206) and 827G (n = 82) genotypes. The relative mtDNA copy number levels for the 827G genotype were normalized to the levels for the 827A genotype. (D) Immunoblot analysis of the levels of OXPHOS mtDNA-encoded and nDNA-encoded proteins in whole-cell extracts of the 827A (n = 6) and 827G (n = 6) cybrids. TOMM20 and ACTIN were used as loading controls for the mtDNA-encoded and nDNA-encoded proteins, respectively. (E) Quantified signal intensities of the OXPHOS mtDNA-encoded protein bands/TOMM20 bands and nDNA-encoded protein bands/ACTIN bands. The levels of proteins in the 827G cybrids were normalized to those in the 827A cybrids (the mean value for cybrids 827A was set to 1). (F) The enzymatic activity levels of mitochondrial complexes I, II, III, and IV and of citrate synthase were measured in mitochondria isolated from 827A (n = 6) and 827G (n = 6) cybrids. The enzymatic activity levels in the 827G cybrids were normalized to those in the 827A cybrids. The data are presented as the mean ± SD from at least 3 independent tests per experiment. ∗P < .05; ∗∗P < .01.
      We further determined the mtDNA-encoding and nDNA-encoding protein expression levels in the 827A and 827G cybrids. The results revealed that the expression levels of most of the proteins, especially the ND5 subunit, were lower in the 827G cybrids than in the 827A cybrids, which may have impaired the assembly of OXPHOS complex I (Figure 2G and H). This finding confirms that 12S rRNA plays important roles in the processes of mtDNA transcription and translation. Finally, we examined the activity of the 4 respiratory chain complexes, and the results showed that complex I activity relative to citrate synthase activity was significantly higher in the 827A cybrids than in the 827G cybrids (Figure 2I).
      Collectively, these results indicated that the 827G cybrids had lower respiratory chain complex activity than the 827A cybrids.

      Compared With the 827A Cybrids, the 827G Cybrids Presented Diminished Mitochondrial Function

      To corroborate the effect of the 827A>G variant on the mitochondrial respiratory chain complex, we detected OXPHOS function in the 827A and 827G cybrids. The results showed that the total ATP content, basal mitochondrial respiration, and OXPHOS-driven mitochondrial respiration were significantly lower in the 827G cybrids than in the 827A cybrids (Figure 3A and B). However, reactive oxygen species (ROS) generation was increased by 50% in the 827G cybrids compared with 827A cybrids (Figure 3C), which may have further activated mitochondrial quality control protein expression and mitochondrial-nuclear signaling pathways. Thus, compared with the 827A cybrids, the 827G cybrids presented diminished mitochondrial OXPHOS function.
      Figure thumbnail gr3
      Figure 3The 827G cybrids presented diminished mitochondrial function compared with the 827A cybrids. (A) Total ATP content was measured in the 827A (n = 6) and 827G (n = 6) cybrids. The ATP levels in the 827G cybrids were normalized to those in the 827A cybrids. (B) Mitochondrial ROS levels were determined in the 827A (n = 6) and 827G (n = 6) cybrids. The mitochondrial ROS levels in the 827G cybrids were normalized to those in the 827A cybrids. (C) Mitochondrial respiratory capacity was determined in the 827A (n = 6) and 827G (n = 6) cybrids. Oligomycin (2.5 μg/mL) was added for measurement of coupled mitochondrial respiration. OXPHOS coupling respiration was calculated by subtracting the uncoupled component value from the total endogenous respiration value. The data are presented as the mean ± SD from at least 3 independent tests per experiment. ∗P < .05.

      The Mitochondrial Protein Quality Control Response and Retrograde Signaling Pathways Were Activated in the 827G Cybrids

      We further investigated whether the ROS increases caused by mitochondrial dysfunction activated the expression of mitochondrial quality control proteins. The results showed that the levels of CLPP and LONP1 were significantly higher in the 827G cybrids than in the 827A cybrids, suggesting that the 827G cybrids experienced ROS stress. Notably, the mitophagy-related protein PINK1 tended to be downregulated in the 827G cybrids compared with the 827A cybrids (Figure 4A and B). Consistent with this result, the messenger RNA (mRNA) expression levels of the mitophagy-associated genes p62 and LC3 also tended to be lower in the 827G cybrids than in the 827A cybrids (Figure 4C). These findings suggest that deficient mitophagy in the 827G cybrids hampered the cybrids to cope with stress.
      Figure thumbnail gr4
      Figure 4The mitochondrial protein quality control response and retrograde signaling pathways were activated in the 827G cybrids. (A) Representative Western blot of the mitochondrial quality control proteins PINK1, CLPP, LONP1, AFG3L2, HSP60, GRP75, and VDAC in whole-cell extracts from the 827A (n = 6) and 827G (n = 6) cybrids. ACTIN was used as a loading control. (B) Quantified signal intensities of all the target protein bands/ACTIN bands. The levels of proteins in the 827G cybrids were normalized to those in the 827A cybrids (the mean value for the 827A cybrids was set to 1). (C) Mitophagy associated gene expression in the 827A (n = 6) and 827G (n = 6) cybrids. The relative gene expression in the 827G cybrids was normalized to the expression in the 827A cybrids. (D) Representative Western blot of the relative phosphorylation of JNK, P38, ERK, AKT, and AMPK in whole-cell extracts from the 827A (n = 6) and 827G (n = 6) cybrids. ACTIN was used as a loading control. The levels of proteins in the 827G cybrids were normalized to those in the 827A cybrids. (E) Quantified signal intensities of all the target protein bands/ACTIN bands. The levels of proteins in the 827G cybrids were normalized to those in the 827A cybrids (the mean value for the 827A cybrids was set to 1). (F) Representative Western blot of the mitochondrial retrograde signaling mediators NRF1 and RXRα in whole-cell extracts from the 827A (n = 6) and 827G (n = 6) cybrids. ACTIN was used as a loading control. (G) Quantified signal intensities of the NRF1 and RXRα protein bands/ACTIN bands. The levels of proteins in the 827G cybrids were normalized to those in the 827A cybrids (the mean value for 827A cybrids was set to 1). The data are presented as the mean ± SD from at least 3 independent tests per experiment. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.
      As altered mitochondrial ROS generation has been found to be related to modification of many mitochondria-to-nucleus retrograde signaling pathways, we examined several major signaling pathways in the 827A and 827G cybrids. The results indicated that the JNK and AMPK pathways, which are related to OXPHOS gene expression and catabolism, respectively, were activated in the 827G cybrids (Figure 4D and E). In addition, the nuclear receptor RXRα, which mediates the biological effects of OXPHOS gene activation, was significantly downregulated in the 827G cybrids compared with the 827A cybrids (Figure 4F and G). Thus, the results suggested that OXPHOS complex capacity was decreased and that catabolism was increased for energy compensation in the 827G cybrids.

      Abnormal Lipid Metabolism and Cholesterol Transport Processes Occurred in the 827G Cybrids

      We further investigated whether abnormal lipid metabolism occurred in the 827G cybrids and found that the mRNA expression levels of fatty acid metabolism–related genes (CPT1A, CPT1B, VLCAD, ACOX1, and PPARα) were significantly downregulated in the 827G cybrids compared with the 827A cybrids (Figure 5A). In addition, the protein levels of CPT1A and PPARα were significantly decreased in the 827G cybrids compared with the 827A cybrids (Figure 5B and C). The results indicated that long-chain fatty acid transport and fatty acid β-oxidation processes were aberrant in the 827G cybrids.
      Figure thumbnail gr5
      Figure 5The lipid metabolism and cholesterol transport processes were abnormal in the 827G cybrids. (A) Lipid metabolism process–associated gene expression in the 827A (n = 6) and 827G (n = 6) cybrids. Relative gene expression in the 827G cybrids was normalized to the 827A cybrids. (B) Immunoblotting analysis of the levels of CPT1A, ACOX1, and PPARα in whole-cell extracts from the 827A (n = 6) and 827G (n = 6) cybrids. GAPDH was used as a loading control. (C) Quantified signal intensities of all the target protein bands/ACTIN bands. The protein levels in the 827G cybrids were normalized to those in the 827A cybrids (the mean value for cybrids 827A was set to 1). (D) Glycolysis process–associated gene expression in the 827A (n = 6) and 827G (n = 6) cybrids. The relative gene expression in the 827G cybrids was normalized to the expression in the 827A cybrids. (E) TCA process-associated gene expression in the 827A (n = 6) and 827G (n = 6) cybrids. The relative gene expression in the 827G cybrids was normalized to the expression in the 827A cybrids. (F) Cholesterol synthesis process–associated gene expression in the 827A (n = 6) and 827G (n = 6) cybrids. The relative gene expression in the 827G cybrids was normalized to the expression in the 827A cybrids. (G) Representative Western blot of the cholesterol synthesis rate-limiting enzyme HMGCR in whole-cell extracts from the 827A (n = 6) and 827G (n = 6) cybrids. GAPDH was used as a loading control. (H) Quantified signal intensities of HMGCR protein bands/GAPDH bands. The levels of proteins in the 827G cybrids were normalized to those in the 827A cybrids (the mean value for 827A cybrids was set to 1). (I) HMGCR enzymatic activity detection in the 827A (n = 6) and 827G (n = 6) cybrids. The relative activity in the 827G cybrids was normalized to the activity in the 827A cybrids. (J) Whole-cell cholesterol content in the 827A (n = 6) and 827G (n = 6) cybrids. The relative content in the 827G cybrids was normalized to the content in the 827A cybrids. (K) Cholesterol efflux into the cultured medium of the 827A (n = 6) and 827G (n = 6) cybrids. The relative efflux for the 827G cybrids was normalized to the efflux for the 827A cybrids. (L) Total whole-cell and medium cholesterol content for the 827A (n = 6) and 827G (n = 6) cybrids. The relative content in the 827G cybrids was normalized to the content in the 827A cybrids. (M) Representative Western blot of ABCA1, ABCG5, ABCG8, CYP7A1, HIF1α, and HIF2α in whole-cell extracts from the 827A (n = 6) and 827G (n = 6) cybrids under 21% O2, and 1% O2, respectively. ACTIN was used as a loading control. The protein levels in the 827G cybrids were normalized to those in the 827A cybrids. (N) Quantified signal intensities of all the target protein bands/ACTIN bands. The levels of proteins in the 827G cybrids were normalized to those in the 827A cybrids (the mean value for cybrids 827A was set to 1). The data are presented as the mean ± SD for at least 3 independent tests per experiment. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.
      Additionally, we examined the gene expression levels of the main enzymes in the metabolic processes of glycolysis and the tricarboxylic acid (TCA) cycle. The results illustrated that the gene expression of the key glycolysis enzymes PFKL and LDHA and the key TCA cycle enzymes PDHA, IDH, SDHA, FH, and MDH1 were evidently downregulated in the 827G cybrids compared with 827A cybrids. Downregulation of glycolysis in the 827G cybrids might have reduced the levels of the substrate for TCA and cholesterol synthesis, acetyl coenzyme A, thus hampering the TCA cycle and decreasing ATP generation. In addition, the levels of acetyl coenzyme A used for cholesterol synthesis might have decreased due to excessive accumulation of cholesterol in gallstones (Figure 5D and E). However, cholesterol synthesis showed no significant difference between the 827A and 827G cybrids (Figure 5F). The protein level and enzymatic activity of the rate-limiting enzyme of cholesterol synthesis, HMGCR, also did not differ between the 827A and 827G cybrids (Figure 5GI). As expected, the total cellular cholesterol levels were similar between the 827A and 827G cybrids (Figure 5J–L).
      Next, we focused on the transport processes in the 827A cybrids and 827G cybrids. The expression of the transporter ABCA1, which mediates cholesterol efflux to peripheral blood, was downregulated in the 827G cybrids compared with the 827A cybrids; however, the expression of the transporter ABCG5/8, which mediates cholesterol efflux to bile and the gallbladder, was upregulated in the 827G cybrids compared with the 827A cybrids (Figure 5M and N).
      Because evidence has shown that hypoxic conditions contribute to the formation of gallstones,
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      Activation of the hypoxia inducible factor 1alpha subunit pathway in steatotic liver contributes to formation of cholesterol gallstones.
      we further evaluated whether hypoxia promoted the formation of gallstones in 827G cybrids. The cybrids were cultured in 1% O2 for 36 hours, and the results showed that the levels of the transporter ABCG5/8 increased dramatically in the 827G cybrids compared with the 827A cybrids, partly dependent on the HIF1α pathway (Figure 5M and N).
      Taken together, our results showed that abnormal cholesterol transport, but not cholesterol synthesis, promoted gallstone formation in the 827G cybrids.

      Discussion

      In this study, we demonstrated a strong association of the mtDNA haplogroups B4b′d′e′j with gallstone disease in a Chinese population. Furthermore, mtDNA 827A>G, a highly conserved site in mitochondrial 12S rRNA defining the B4b′d′e′j haplogroups, induced aberrant mitochondrial function and elevated the occurrence of gallstones by inducing abnormal cholesterol transport through ABCG5/8 via activation of the AMPK signaling pathway.
      Mitochondrial 12S rRNA plays an important role in the synthesis of mtDNA-encoded proteins, which may contribute to mitochondrial OXPHOS function.
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      The 143B osteosarcoma ρ0 cell line was the first mtDNA-deleted cell model successfully constructed
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      Notably, the mitochondrial complex I ND5 subunit, which plays a pivotal role in complex I assembly and activity, was decreased dramatically in the 827G cybrids.
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      Consistent with these findings, we found decreased respiration chain function and increased ROS levels along with downregulated complex I activity in the 827G cybrids compared with the 827A cybrids. These changes further activated mitochondrial-nuclear signaling pathways, including the JNK and AMPK signaling pathways. JNK signaling can downregulate RXRα transcript levels and OXPHOS complex biogenesis, which further aggravates mitochondrial OXPHOS and lipid metabolism dysfunction.
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      Figure thumbnail gr6
      Figure 6Schematic of the mechanism of abnormal cholesterol transport in the 827G cybrids. A possible pathologic mechanism of cholesterol gallstone formation in the 827G cybrids. The 827A>G variant of the B4b′d′e′j haplogroups led to decreased abundance of proteins related to OXPHOS and to further dysfunction. On the one hand, the levels of acetyl coenzyme A, used for cholesterol synthesis, decreased due to downregulation of fatty acid oxidation and TCA metabolism processes; on the other hand, high ROS levels in the 827G cybrids further activated mitochondrial retrograde signaling pathways, including the JNK and AMPK pathways. The JNK pathway reduced biogenesis of OXPHOS complexes, which further aggregated mitochondrial dysfunction. Moreover, downregulation of PPARα/RXRα inhibited cholesterol efflux into peripheral blood through the transporter ABCA1. However, AMPK activation may have increased the levels of the transporter ABCG5/8, thus increasing the risk of cholesterol entering the bile and gallbladder.
      As previously described, hypoxia is a risk factor for the formation of gallstones. One study has demonstrated that adipocyte HIF2α reduces atherosclerosis by promoting ceramide catabolism and thus increasing hepatic cholesterol elimination to the gallbladder.
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      Consistent with previous studies, our study indicated that the levels of ABCG5/8 were dramatically higher in the 827G cybrids compared than in the 827A cybrids and that this difference was partly dependent on the HIF1α pathway. Notably, such increases in ABCG5/8 levels may increase the risk of gallstones by reinforcing activation of AMPK signaling under hypoxia.
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      Interestingly, in our gallstone cases, the blood lipid levels did not exceed the upper limit. These findings provide a possible explanation for why mtDNA 827A>G preferentially increases the risk of cholesterol gallstones rather than atherosclerosis.
      Accumulating evidence has shown that the prevalence of gallstones among Native Americans is the highest in the world.
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      Mitochondrial genome diversity of Native Americans supports a single early entry of founder populations into America.
      ,
      • Tamm E.
      • Kivisild T.
      • Reidla M.
      • Metspalu M.
      • Smith D.G.
      • Mulligan C.J.
      • Bravi C.M.
      • Rickards O.
      • Martinez-Labarga C.
      • Khusnutdinova E.K.
      • Fedorova S.A.
      • Golubenko M.V.
      • Stepanov V.A.
      • Gubina M.A.
      • Zhadanov S.I.
      • Ossipova L.P.
      • Damba L.
      • Voevoda M.I.
      • Dipierri J.E.
      • Villems R.
      • Malhi R.S.
      Beringian standstill and spread of Native American founders.
      For example, the Pima Indian, the most commonly studied Amerindian tribe in the context of gallbladder diseases, has the highest prevalence of gallstone and the highest frequency of haplogroup B2 among Native Americans.
      • Wallace D.C.
      • Torroni A.
      American Indian prehistory as written in the mitochondrial DNA: a review.
      ,
      • Grimaldi C.H.
      • Nelson R.G.
      • Pettitt D.J.
      • Sampliner R.E.
      • Bennett P.H.
      • Knowler W.C.
      Increased mortality with gallstone disease: results of a 20-year population-based survey in Pima Indians.
      It has also been shown that the prevalence of gallbladder diseases increases in Chilean populations with increasing level of Native Americans admixture as measured by analysis of mtDNA and other genetic markers.
      • Miquel J.F.
      • Covarrubias C.
      • Villaroel L.
      • Mingrone G.
      • Greco A.V.
      • Puglielli L.
      • Carvallo P.
      • Marshall G.
      • Del Pino G.
      • Nervi F.
      Genetic epidemiology of cholesterol cholelithiasis among Chilean Hispanics, Amerindians, and Maoris.
      It seems that the presence of mtDNA haplogroup B4b′d′e′j is responsible for the ethnic differences in gallstone and even other metabolic disorders, in America. Interestingly, the frequencies of 827A>G B4 carriers in Africa, Europe, and South and West Asia were found to be generally low in the current study, but the prevalence of gallstone disease was also considerable (4%–23%) (Figure 1C), indicating that genetic risks other than the mtDNA variant 827A>G or environmental factors, such as lifestyles, are also important in gallstone disease. Therefore, our study, to some extent, explained the etiology underlying the susceptibility to gallstone disease in Native Americans.
      In summary, our study demonstrates a potential link between mtDNA 827A>G and gallstone disease. We have revealed that mtDNA 827A>G induces aberrant mitochondrial function and abnormal cholesterol transport, resulting in increased occurrence of gallstones. Our findings provide a significant biological basis for the clinical diagnosis and prevention of gallstone disease in the future.

      Materials and Methods

      Study Participants

      Blood samples of 104 cases and 300 controls were collected in the Greater Shanghai Area with informed consent. The protocol of the study was approved by the Ethical Committee of the Chinese National Human Genome Center in Shanghai. The presence (cases) or absence (controls) of gallstone disease (cholelithiasis) was diagnosed by ultrasound and/or cholecystectomy. The mean age of onset in the patients was 44.9 (range, 19–81) years, and all controls were above 50 years of age (mean age 64.0 years; range, 50–86 years). The proportions of men among the cases and controls were 46.8% and 54.0%, respectively.
      DNA samples from the blood of 206 827A (B4a/c)- and 82 827G (B4b′d′e′j)- genotype participants and all platelet samples from healthy individuals used to construct the transmitochondrial cybrids model were obtained from the Taizhou Institute of Health Sciences, Fudan University. The study was approved by the Human Ethics Committee of Fudan University.

      mtDNA Sequencing, Genotyping, Median-Joining Network Analysis, Conservation Analysis, and RNA Structure Analysis

      Genomic DNA from peripheral blood was extracted using a standard SDS lysis protocol for genotyping. HVS1 and other sites (8281–8289 deletion, 827A>G, 499G>A, 13942A>G, 6023G>A, and 6413T>C) in the coding region were genotyped by sequencing or polymerase chain reaction–restriction fragment length polymorphism assay. For complete sequence analysis of certain samples, Sanger sequencing was performed using 24 previously reported pairs of mtDNA primers.
      • Fang H.
      • Shi H.
      • Li X.
      • Sun D.
      • Li F.
      • Li B.
      • Ding Y.
      • Ma Y.
      • Liu Y.
      • Zhang Y.
      • Shen L.
      • Bai Y.
      • Yang Y.
      • Lu J.
      Exercise intolerance and developmental delay associated with a novel mitochondrial ND5 mutation.
      The single nucleotide polymorphisms (SNPs) in each participant were identified by comparing the obtained sequences with the revised Cambridge Reference Sequence by using Codon Code Aligner 3.0.1 (Codon Code Corporation, Centerville, VA). The mtDNA haplogroup was assigned by comparing the target SNPs with the SNPs of the most up-to-date Chinese mtDNA haplogroup tree.
      • Zhou H.
      • Nie K.
      • Qiu R.
      • Xiong J.
      • Shao X.
      • Wang B.
      • Shen L.
      • Lyu J.
      • Fang H.
      Generation and bioenergetic profiles of cybrids with East Asian mtDNA haplogroups.
      A median-joining phylogeny of these individuals was reconstructed using NETWORK software (Fluxus Technology Ltd, Colchester, Essex). Conservation analysis of 827 nucleotides was performed with UGENE software (NCIT UNIPRO, LLC, Novosibirsk, Russia). The RNA structure analysis of 12S rRNA with 827A or 827G was performed using the RNAfold program in the Vienna RNA Package 2.0.

      Cell Line Generation and Culture Conditions

      143B ρ0 human osteosarcoma cells lacking mtDNA were cultured in high-glucose Dulbecco′s modified Eagle medium (Thermo Fisher Scienetific, Waltham, MA) containing 10% fetal bovine serum (Thermo Fisher Scienetific), 100-μg/mL pyruvate, and 50-μg/mL uridine. Transmitochondrial cybrids were formed by fusing 143B ρ0 cells and platelets from healthy individuals with different haplogroups, as described previously.
      • King M.P.
      • Attardi G.
      Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation.
      In this study, 12 cybrids distributed in an mtDNA tree were constructed, including 6 B4a/B4c-haplogroup cybrids (named 827A cybrids) and 6 B4b/B4d-haplogroup cybrids (named 827G cybrids). All plasma samples were collected from Taizhou. The cybrids were cultured in high-glucose DMEM containing 10% fetal bovine serum at 37°C in an atmosphere with 5% CO2. Pathogenic mtDNA mutations and cross-contamination during single-clone selection were ruled out through Sanger sequencing of the whole mitochondrial genome in all cybrid cells during culture (Supplementary Table 2).

      Analyses of mtDNA Content, Mitochondrial RNA, Mitochondrial Ribosome Subunits, Mitophagy, and Metabolism-Associated Gene Expression

      Both mtDNA content and the mRNA levels of 13 mtDNA-encoded OXPHOS subunits were determined using the 2(–ΔΔCT) method as previously described.
      • Sun D.
      • Li B.
      • Qiu R.
      • Fang H.
      • Lyu J.
      Cell type-specific modulation of respiratory chain supercomplex organization.
      Briefly, genomic DNA and total RNA were extracted using standard protocols, and the total RNA was then treated with DNase and reverse-transcribed using 6 random primers (Takara, Dalian, China). Quantitative real-time polymerase chain reaction was performed using primers targeted to mtDNA, mitochondrial RNA, a subset of mitochondrial ribosome subunits, mitophagy-related genes, metabolism-related genes, and nuclear housekeeping genes on a QuantStudio 7 Flex Real-Time polymerase chain reaction system (Thermo Fisher Scientific) by using SYBR Green qPCR Mastermix (Takara). All primers used in these analyses are listed in Supplementary Table 3.

      Mitochondria Isolation and Respiratory Chain Complex Enzymatic Activity Assay

      Mitochondria from cultured cybrids were isolated as previously described.
      • Claude A.
      • Fullam E.F.
      An electron microscope study of isolated mitochondria: method and preliminary results.
      The enzymatic activity of 4 respiratory chain complexes was measured in the mitochondria of cybrids as previously described.
      • Fang H.
      • Zhang F.
      • Li F.
      • Shi H.
      • Ma L.
      • Du M.
      • You Y.
      • Qiu R.
      • Nie H.
      • Shen L.
      • Bai Y.
      • Lyu J.
      Mitochondrial DNA haplogroups modify the risk of osteoarthritis by altering mitochondrial function and intracellular mitochondrial signals.
      The respiratory chain complex enzymatic activity in each case was normalized against that of citrate synthase, a mitochondrial matrix marker enzyme.

      Immunoblotting and Antibodies

      Proteins were extracted using RIPA lysis buffer (Cell Signaling Technology, Danvers, MA) supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Thirty micrograms of protein was separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. The proteins were blotted with antibodies at a dilution of 1:1000, as listed in Supplementary Table 4, and detected with ECL reagents (Bio-Rad, Hercules, CA). The validation data of the differentially expressed proteins was presented in Figure 7.
      Figure thumbnail gr7
      Figure 7Immunoblotting validation analysis of the differentially expressed proteins. (A) Representative Western blot of proteins ND5, CLPP, LONP1, CPT1A, PPARα, AMPKα, P-AMPKα, JNK1/2, P-JNK1/2, and RXRα in whole-cell extracts from the 827A (n = 6) and 827G (n = 6) cybrids. ACTIN was used as a loading control. The protein levels in the 827G cybrids were normalized to those in the 827A cybrids. (M) Representative Western blot of ABCA1, ABCG5, and ABCG8 in whole-cell extracts from the 827A (n = 6) and 827G (n = 6) cybrids under 21% O2, and 1% O2, respectively. ACTIN was used as a loading control. The protein levels in the 827G cybrids were normalized to those in the 827A cybrids. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.

      ATP, ROS, Oxygen Consumption, and Cholesterol Measurements

      ATP content and ROS levels were determined using an ATP measurement kit and MitoSOX (Thermo Fisher Scientific), respectively, as previously described.
      • Fang H.
      • Shi H.
      • Li X.
      • Sun D.
      • Li F.
      • Li B.
      • Ding Y.
      • Ma Y.
      • Liu Y.
      • Zhang Y.
      • Shen L.
      • Bai Y.
      • Yang Y.
      • Lu J.
      Exercise intolerance and developmental delay associated with a novel mitochondrial ND5 mutation.
      Endogenous oxygen consumption in intact cells was determined using an Oxygraph-2k (Oroboros, Innsbruck, Austria) as described previously.
      • Xu B.
      • Li X.
      • Du M.
      • Zhou C.
      • Fang H.
      • Lyu J.
      • Yang Y.
      Novel mutation of ND4 gene identified by targeted next-generation sequencing in patient with Leigh syndrome.
      After recording basal respiration, oligomycin (2.5 μg/mL) (Sigma) was added to measure phosphorylation-coupled respiration. The total cellular cholesterol and free cholesterol in the cell culture medium were determined using a Total Cholesterol Quantitation Kit (Applygen, Beijing, China) and a Cholesterol Quantitation Kit (Sigma-Aldrich), respectively, according to the manufacturers′ instructions.

      Sample Preparation, RNA Sequencing, and Gene Expression Data Analysis

      Total RNA was isolated from six 827A and six 827G cybrids using TRIzol reagent (Ambion, Austin, TX) and then purified using poly-T-attached magnetic beads (Vazyme, Nanjing, China). After fragmenting the mRNA, first-strand complementary DNA was synthesized and then sequenced using a NovaSeq 6000 platform (Illumina, San Diego, CA) as described previously. To obtain high-quality reads, reads containing adaptors, reads containing adaptor sequences, and poly-N and low-quality reads were removed from the raw data. The reference genome and gene model annotation files were downloaded directly from genome websites. The reference genome was built using STAR, and paired-end high-quality reads were aligned to the reference genome by using STAR (v2.5.1b). HTSeq v0.6.0 was used to count the reads mapped to each gene, after which the fragments per kilobase million value of each gene was calculated based on the length of the gene and the number of reads mapped to the gene. Differential expression analysis was performed using the DESeq2 R package. DESeq2 provides statistical routines for determining differential expression from digital gene expression data by using a model based on the negative binomial distribution. A heatmap of gene expression between six 827A cybrids and six 827G cybrids was created.

      Statistical Analysis

      The associations of mtDNA variants with gallstone disease were investigated with both the chi-square test and Fisher′s exact test. The odds ratio and 95% confidence interval were calculated to evaluate the level of risk. The data are presented as the mean ± SD from 3 independent experiments. Means were compared using independent Student′s t tests using SPSS 21.0 software (IBM, Armonk, NY, USA), and P < .05 was considered to indicate significance.

      Acknowledgments

      The authors acknowledge all the participants involved in this study and the Taizhou Institute of Health Sciences, Fudan University.

      Supplementary Material

      Supplementary Table 1The Frequency of Mitochondrial Haplogroup B4b′d′e′j and the Prevalence of Gallstone Disease in Global Populations
      PopulationB4b′d′e′j Haplogroups (%)Gallstone Disease (%)References
      Mapuche Indians, Chile36.8435.2
      • Admirand W.H.
      • Small D.M.
      The physicochemical basis of cholesterol gallstone formation in man.
      ,
      • Kosters A.
      • Jirsa M.
      • Groen A.K.
      Genetic background of cholesterol gallstone disease.
      Peru43.8814.3
      • Stokes C.S.
      • Krawczyk M.
      • Lammert F.
      Gallstones: environment, lifestyle and genes.
      ,
      • Gilat T.
      • Feldman C.
      • Halpern Z.
      • Dan M.
      • Bar-Meir S.
      An increased familial frequency of gallstones.
      Argentinian10.3520.5
      • Everhart J.E.
      • Khare M.
      • Hill M.
      • Maurer K.R.
      Prevalence and ethnic differences in gallbladder disease in the United States.
      ,
      • Nakeeb A.
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      • Martin L.
      • Sonnenberg G.E.
      • Swartz-Basile D.
      • Kissebah A.H.
      • Pitt H.A.
      Gallstones: genetics versus environment.
      Mexican American13.5417.8
      • Everhart J.E.
      • Yeh F.
      • Lee E.T.
      • Hill M.C.
      • Fabsitz R.
      • Howard B.V.
      • Welty T.K.
      Prevalence of gallbladder disease in American Indian populations: findings from the Strong Heart Study.
      ,
      • Buch S.
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      • Volzke H.
      • Becker C.
      • Franke A.
      • von Eller-Eberstein H.
      • Kluck C.
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      A genome-wide association scan identifies the hepatic cholesterol transporter ABCG8 as a susceptibility factor for human gallstone disease.
      Puerto Rican6.549.5
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      Loci from a genome-wide analysis of bilirubin levels are associated with gallstone risk and composition.
      ,
      • Di M.A.
      Therapy of digestive disorders: A companion to sleisenger and Fordtran′s gastrointestinal and liver disease.
      Mexican26.4514.1
      • Biddinger S.B.
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      • Bezy O.
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      ,
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      Activation of liver X receptor sensitizes mice to gallbladder cholesterol crystallization.
      Taiwan, China3.196.26
      • Lai S.W.
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      Risk factors for gallstone disease in a hospital-based study.
      • Chen C.H.
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      The prevalence and risk factors for gallstone disease in taiwanese vegetarians.
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      Sichuan, China2.9310.7
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      Shanghai, China4.185.84
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      Novel mutations of mitochondrial DNA associated with type 2 diabetes in Chinese Han population.
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      A cluster of metabolic defects caused by mutation in a mitochondrial tRNA.
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      Maternally inherited essential hypertension is associated with the novel 4263A>G mutation in the mitochondrial tRNAIle gene in a large Han Chinese family.
      Zhejiang, China2.988.8
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      Novel mutations of mitochondrial DNA associated with type 2 diabetes in Chinese Han population.
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      • Kokaze A.
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      Mitochondrial DNA 5178 C/A polymorphism influences the effects of habitual smoking on the risk of dyslipidemia in middle-aged Japanese men.
      Jiangsu, China4.244.21
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      Xinjiang, China7.7911.64
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      Novel mutations of mitochondrial DNA associated with type 2 diabetes in Chinese Han population.
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      • Qin J.
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      • Jiang Z.Y.
      • Zhang Y.
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      Risk factors of familial gallstone disease: study of 135 pedigrees.
      Japan3.153.2
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      Therapy of digestive disorders: A companion to sleisenger and Fordtran′s gastrointestinal and liver disease.
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      Major population expansion of East Asians began before neolithic time: evidence of mtDNA genomes.
      Korea3.454.85
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      Thailand2.113.1
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      Italy013.9
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      British023.6
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      Ghana05.9
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      • Zhou H.
      • Fu Q.
      • Shen L.
      • Chen X.
      • Shen F.
      • Lyu J.
      mtDNA haplogroup N9a increases the risk of type 2 diabetes by altering mitochondrial function and intracellular mitochondrial signals.
      Supplementary Table 2Analysis of Whole Mitochondrial Genome of the 12 Cybrids
      PositionGenerCRS BaseMutationAA ChangemtDNA Database
      MITOMAP, mtDB, mtSNP, and PhyloTree mt.
      Analysis of whole mitochondrial genome-1-B4a4
      73D-loopAGnopolymorphic site
      152D-loopTCnopolymorphic site
      193D-loopAGnopolymorphic site
      263D-loopAGnopolymorphic site
      373D-loopAGnopolymorphic site
      70912S rRNAGAnopolymorphic site
      75012S rRNAAGnopolymorphic site
      143812S rRNAAGnopolymorphic site
      201016S rRNATCnopolymorphic site
      270616S rRNAAGnopolymorphic site
      476916S rRNAAGnopolymorphic site
      5201ND2TCnopolymorphic site
      5465ND2TCnopolymorphic site
      7028COICTnopolymorphic site
      8860ATPase6AGThr > Alapolymorphic site
      9123ATPase6GAnopolymorphic site
      10289ND3AGnopolymorphic site
      11719ND4GAnopolymorphic site
      13269ND5AGnopolymorphic site
      14751CytbCTThr > Ilepolymorphic site
      14766CytbCTThr > Ilepolymorphic site
      15326CytbAGThr > Alapolymorphic site
      16182D-loopACnopolymorphic site
      16183D-loopACnopolymorphic site
      16189D-loopTCnopolymorphic site
      16217D-loopTCnopolymorphic site
      16261D-loopCTnopolymorphic site
      16299D-loopAGnopolymorphic site
      16519D-loopTCnopolymorphic site
      Analysis of whole mitochondrial genome-2-B4a1c2
      73D-loopAGnopolymorphic site
      146D-loopTCnopolymorphic site
      263D-loopAGnopolymorphic site
      70912S rRNAGAnopolymorphic site
      75012S rRNAAGnopolymorphic site
      143812S rRNAAGnopolymorphic site
      270616S rRNAAGnopolymorphic site
      4769ND2AGnopolymorphic site
      5465ND2TCnopolymorphic site
      6080CO1ATnopolymorphic site
      7028CO1CTnopolymorphic site
      7052CO1AGnopolymorphic site
      7271CO1AGnopolymorphic site
      8860ATPase6AGThr > Alapolymorphic site
      9123ATPase6GAnopolymorphic site
      9822CO3CALeu > Ilepolymorphic site
      10238ND3TCnopolymorphic site
      11719ND4GAnopolymorphic site
      14766CytbCTThr > Ilepolymorphic site
      15326CytbAGThr > Alapolymorphic site
      15661CytbCTnopolymorphic site
      16167D-loopCTnopolymorphic site
      16182D-loopACnopolymorphic site
      16183D-loopACnopolymorphic site
      16189D-loopTCnopolymorphic site
      16217D-loopTCnopolymorphic site
      16261D-loopCTnopolymorphic site
      16317D-loopATnopolymorphic site
      16519D-loopTCnopolymorphic site
      Analysis of whole mitochondrial genome-3-B4a
      73D-loopAGnopolymorphic site
      263D-loopAGnopolymorphic site
      310D-loopTCnopolymorphic site
      316D-loopGCnopolymorphic site
      75012S rRNAAGnopolymorphic site
      143812S rRNAAGnopolymorphic site
      270616S rRNAAGnopolymorphic site
      4769ND2AGnopolymorphic site
      5465ND2TCnopolymorphic site
      6386CO1CTnopolymorphic site
      7028CO1CTnopolymorphic site
      8860ATPase6AGThr > Alapolymorphic site
      9123ATPase6GAnopolymorphic site
      11227ND4CTnopolymorphic site
      11719ND4GAnopolymorphic site
      13781ND5TCIle >Thrpolymorphic site
      14053ND5AGThr > Alapolymorphic site
      14766CytbCTThr > Ilepolymorphic site
      15326CytbAGThr > Alapolymorphic site
      16182D-loopACnopolymorphic site
      16183D-loopACnopolymorphic site
      16189D-loopTCnopolymorphic site
      16217D-loopTCnopolymorphic site
      16261D-loopCTnopolymorphic site
      16299D-loopAGnopolymorphic site
      16355D-loopCTnopolymorphic site
      16390D-loopGAnopolymorphic site
      16519D-loopTCnopolymorphic site
      Analysis of whole mitochondrial genome-4-B4b1a3
      73D-loopAGnopolymorphic site
      207D-loopGAnopolymorphic site
      263D-loopAGnopolymorphic site
      408D-loopTAnopolymorphic site
      499D-loopGAnopolymorphic site
      75012S rRNAAGnopolymorphic site
      82712S rRNAAGnopolymorphic site
      143812S rRNAAGnopolymorphic site
      270616S rRNAAGnopolymorphic site
      4769ND2AGnopolymorphic site
      4820ND2GAnopolymorphic site
      6023COIGAnopolymorphic site
      6413COITCnopolymorphic site
      7028COICTnopolymorphic site
      8466ATPase8AGHis > Argpolymorphic site
      8860ATPase6AGThr > Alapolymorphic site
      9055ATPase6GAAla > Thrpolymorphic site
      9338COIIIATnopolymorphic site
      9615COIIITCnopolymorphic site
      9966COIIIGAVal > Ilepolymorphic site
      11719ND4GAnopolymorphic site
      13590ND5GAnopolymorphic site
      14766CytbCTThr > Ilepolymorphic site
      15326CytbAGThr > Alapolymorphic site
      15535CytbCTnopolymorphic site
      16136D-loopTCnopolymorphic site
      16183D-loopACnopolymorphic site
      16189D-loopTCnopolymorphic site
      16519D-loopTCnopolymorphic site
      Analysis of whole mitochondrial genome-5-B4b1c1
      73D-loopAGnopolymorphic site
      263D-loopAGnopolymorphic site
      499D-loopGAnopolymorphic site
      75012S rRNAAGnopolymorphic site
      82712S rRNAAGnopolymorphic site
      143812S rRNAAGnopolymorphic site
      171716S rRNATCnopolymorphic site
      270616S rRNAAGnopolymorphic site
      3918ND1GAnopolymorphic site
      4769ND2AGnopolymorphic site
      4820ND2GAnopolymorphic site
      7028COICTnopolymorphic site
      7521tRNA AspGAnopolymorphic site
      8860ATPase6AGThr > Alapolymorphic site
      9101ATPase6TGIle > Thrpolymorphic site
      9861COIIITCPhe > Leupolymorphic site
      11239ND4AGnopolymorphic site
      11719ND4GAnopolymorphic site
      11914ND4GAnopolymorphic site
      13590ND5GAnopolymorphic site
      14587ND6AGnopolymorphic site
      14766CytbCTThr > Ilepolymorphic site
      15326CytbAGThr > Alapolymorphic site
      15535CytbCTnopolymorphic site
      16136D-loopTCnopolymorphic site
      16183D-loopACnopolymorphic site
      16189D-loopTCnopolymorphic site
      16217D-loopTCnopolymorphic site
      16218D-loopCTnopolymorphic site
      16519D-loopTCnopolymorphic site
      Analysis of whole mitochondrial genome-6-B4b1c
      73D-loopAGnopolymorphic site
      263D-loopAGnopolymorphic site
      309D-loopCTnopolymorphic site
      310D-loopTCnopolymorphic site
      499D-loopGAnopolymorphic site
      75012S rRNAAGnopolymorphic site
      82712S rRNAAGnopolymorphic site
      143812S rRNAAGnopolymorphic site
      270616S rRNAAGnopolymorphic site
      4769ND2AGnopolymorphic site
      4820ND2GAnopolymorphic site
      7028COICTnopolymorphic site
      7080COITCPhe > Leupolymorphic site
      8343tRNA LysAGnopolymorphic site
      8860ATPase6AGThr > Alapolymorphic site
      11719ND4GAnopolymorphic site
      13401ND5TCnopolymorphic site
      13590ND5GAnopolymorphic site
      14587ND6AGnopolymorphic site
      14766CytbCTThr > Ilepolymorphic site
      15535CytbCTnopolymorphic site
      16136D-loopTCnopolymorphic site
      16182D-loopACnopolymorphic site
      16183D-loopACnopolymorphic site
      16189D-loopTCnopolymorphic site
      16217D-loopTCnopolymorphic site
      16218D-loopCTnopolymorphic site
      16519D-loopTCnopolymorphic site
      Analysis of whole mitochondrial genome-7-B4c1b2c
      73D-loopAGnopolymorphic site
      150D-loopCAnopolymorphic site
      263D-loopAGnopolymorphic site
      70912S rRNAGAnopolymorphic site
      75012S rRNAAGnopolymorphic site
      111912S rRNATCnopolymorphic site
      143812S rRNAAGnopolymorphic site
      270616S rRNAAGnopolymorphic site
      3394ND1TCTyr > Hispolymorphic site
      3435ND1CTnopolymorphic site
      3497ND1CTAla > Valpolymorphic site
      3571ND1CTLeu > Phepolymorphic site
      4173ND1AGnopolymorphic site
      4769ND2AGnopolymorphic site
      7028COICTnopolymorphic site
      8860ATPase6AGThr > Alapolymorphic site
      9123ATPase6GAnopolymorphic site
      11440ND4GAnopolymorphic site
      11719ND4GAnopolymorphic site
      14766CytbCTThr > Ilepolymorphic site
      15326CytbAGThr > Alapolymorphic site
      15346CytbGAnopolymorphic site
      16129D-loopGAnopolymorphic site
      16140D-loopTCnopolymorphic site
      16166D-loopAGnopolymorphic site
      16183D-loopACnopolymorphic site
      16189D-loopTCnopolymorphic site
      16217D-loopTCnopolymorphic site
      16274D-loopGAnopolymorphic site
      16335D-loopAGnopolymorphic site
      16519D-loopTCnopolymorphic site
      Analysis of whole mitochondrial genome-8-B4c1b2
      73D-loopAGnopolymorphic site
      150D-loopCAnopolymorphic site
      195D-loopTCnopolymorphic site
      263D-loopAGnopolymorphic site
      70912S rRNAGAnopolymorphic site
      75012S rRNAAGnopolymorphic site
      111912S rRNATCnopolymorphic site
      143812S rRNAAGnopolymorphic site
      270616S rRNAAGnopolymorphic site
      3434ND1AGTyr > Cyspolymorphic site
      3497ND1CTAla > Valpolymorphic site
      3571ND1CTLeu > Phepolymorphic site
      4769ND2AGnopolymorphic site
      7028COICTnopolymorphic site
      7175COITCnopolymorphic site
      7888COIICTnopolymorphic site
      8200COIITCnopolymorphic site
      8860ATPase6AGThr > Alapolymorphic site
      10325COIIIGAnopolymorphic site
      11719ND4GAnopolymorphic site
      13928ND5GCSer >Thrpolymorphic site
      14766CytbCTThr > Ilepolymorphic site
      15326CytbAGThr > Alapolymorphic site
      15346CytbGAnopolymorphic site
      16140D-loopTCnopolymorphic site
      16182D-loopACnopolymorphic site
      16183D-loopACnopolymorphic site
      16189D-loopTCnopolymorphic site
      16217D-loopTCnopolymorphic site
      16223D-loopCTnopolymorphic site
      16274D-loopGAnopolymorphic site
      16305D-loopATnopolymorphic site
      16519D-loopTCnopolymorphic site
      Analysis of whole mitochondrial genome-9-B4c1b2c
      73D-loopAGnopolymorphic site
      150D-loopCAnopolymorphic site
      263D-loopAGnopolymorphic site
      70912S rRNAGAnopolymorphic site
      75012S rRNAAGnopolymorphic site
      111912S rRNATCnopolymorphic site
      143812S rRNAAGnopolymorphic site
      153412S rRNACTnopolymorphic site
      270616S rRNAAGnopolymorphic site
      3435ND1CTnopolymorphic site
      3497ND1CTAla > Valpolymorphic site
      3571ND1CTLeu > Phepolymorphic site
      4769ND2AGnopolymorphic site
      7028COICTnopolymorphic site
      8860ATPase6AGThr > Alapolymorphic site
      9128ATPase6TCIle > Thrpolymorphic site
      9575COIIIGAnopolymorphic site
      10493ND4LTCnopolymorphic site
      11440ND4GAnopolymorphic site
      11719ND4GAnopolymorphic site
      12026ND4AGIle > Valpolymorphic site
      14766CytbCTThr > Ilepolymorphic site
      15326CytbAGThr > Alapolymorphic site
      15346CytbGAnopolymorphic site
      16136D-loopTCnopolymorphic site
      16140D-loopTCnopolymorphic site
      16183D-loopACnopolymorphic site
      16189D-loopTCnopolymorphic site
      16217D-loopTCnopolymorphic site
      16249D-loopTCnopolymorphic site
      16274D-loopGAnopolymorphic site
      16291D-loopCTnopolymorphic site
      16335D-loopAGnopolymorphic site
      16519D-loopTCnopolymorphic site
      Analysis of whole mitochondrial genome-10-B4d1a
      73D-loopAGnopolymorphic site
      263D-loopAGnopolymorphic site
      272D-loopAGnopolymorphic site
      75012S rRNAAGnopolymorphic site
      82712S rRNAAGnopolymorphic site
      143812S rRNAAGnopolymorphic site
      270616S rRNAAGnopolymorphic site
      4769ND2AGnopolymorphic site
      7028COICTnopolymorphic site
      8860ATPase6AGThr > Alapolymorphic site
      11719ND4GAnopolymorphic site
      11914ND4GAnopolymorphic site
      12612ND5AGnopolymorphic site
      12732ND5TCnopolymorphic site
      13942ND5AGThr > Alapolymorphic site
      14034ND5TCnopolymorphic site
      14766CytbCTThr > Ilepolymorphic site
      15038CytbAGIle > Valpolymorphic site
      15326CytbAGThr > Alapolymorphic site
      15535CytbCTnopolymorphic site
      15930tRNA ThrGAnopolymorphic site
      16182D-loopACnopolymorphic site
      16183D-loopACnopolymorphic site
      16189D-loopTCnopolymorphic site
      16193D-loopTCnopolymorphic site
      16221D-loopTCnopolymorphic site
      16523D-loopTCnopolymorphic site
      Analysis of whole mitochondrial genome-11-B4d1a
      73D-loopAGnopolymorphic site
      146D-loopTCnopolymorphic site
      263D-loopAGnopolymorphic site
      75012S rRNAAGnopolymorphic site
      82712S rRNAAGnopolymorphic site
      95912S rRNACTnopolymorphic site
      143812S rRNAAGnopolymorphic site
      270616S rRNAAGnopolymorphic site
      4769ND2AGnopolymorphic site
      7028COICTnopolymorphic site
      8860ATPase6AGThr > Alapolymorphic site
      11719ND4GAnopolymorphic site
      11914ND4GAnopolymorphic site
      12732ND5TCnopolymorphic site
      13942ND5AGThr > Alapolymorphic site
      14766CytbCTThr > Ilepolymorphic site
      15038CytbAGIle > Valpolymorphic site
      15326CytbAGThr > Alapolymorphic site
      15535CytbCTnopolymorphic site
      16182D-loopACnopolymorphic site
      16183D-loopACnopolymorphic site
      16189D-loopTCnopolymorphic site
      16189D-loopTCnopolymorphic site
      16519D-loopTCnopolymorphic site
      16221D-loopTCnopolymorphic site
      16523D-loopTCnopolymorphic site
      Analysis of whole mitochondrial genome-12-B4d1a
      73D-loopAGnopolymorphic site
      146D-loopTCnopolymorphic site
      263D-loopAGnopolymorphic site
      75012S rRNAAGnopolymorphic site
      82712S rRNAAGnopolymorphic site
      95912S rRNACTnopolymorphic site
      143812S rRNAAGnopolymorphic site
      270616S rRNAAGnopolymorphic site
      4769ND2AGnopolymorphic site
      7028COICTnopolymorphic site
      8860ATPase6AGThr > Alapolymorphic site
      11719ND4GAnopolymorphic site
      11914ND4GAnopolymorphic site
      12732ND5TCnopolymorphic site
      13942ND5AGThr > Alapolymorphic site
      14766CytbCTThr > Ilepolymorphic site
      15038CytbAGIle > Valpolymorphic site
      15326CytbAGThr > Alapolymorphic site
      15535CytbCTnopolymorphic site
      16182D-loopACnopolymorphic site
      16183D-loopACnopolymorphic site
      16189D-loopTCnopolymorphic site
      16189D-loopTCnopolymorphic site
      16519D-loopTCnopolymorphic site
      AA, amino acid; rCRS: revised Cambridge Reference Sequence; mtDNA, mitochondrial DNA; rRNA, ribosomal RNA.
      a MITOMAP, mtDB, mtSNP, and PhyloTree mt.
      Supplementary Table 3Primers for the Determination of mtDNA Content, mtRNA, Fatty Acid Oxidative, Mitochondrial Translation, Mitophage, Tricarboxylic Acid, and Cholesterol Synthesis
      Primer NameSequence (5′-3′)
      Primers for mtDNA copy number determination
      mt-F3212 / mt-R3319CACCCAAGAACAGGGTTTGT
      TGGCCATGGGTATGTTGTTAA
      18SF / 18SRTAGAGGGACAAGTGGCGTTC
      CGCTGAGCCAGTCAGTGT
      Primers for mtRNA determination
      mt-ND1F3310-mt-ND1R3440CCCATGGCCAACCTCCTACTCCTC
      AGCCCGTAGGGGCCTACAACG
      mt-ND2F4614-mt-ND2R4677AACCCTCGTTCCACAGAAGCT
      GGATTATGGATGCGGTTGCT
      mt-ND3F10168-mt-ND3R10376ACGAGTGCGGCTTCGACCCT
      TCACTCATAGGCCAGACTTAGGGCT
      mt-ND4 (L) F10621-mt- ND4 (L) R10694CCCACTCCCTCTTAGCCAATATT
      TAGGCCCACCGCTGCTT
      mt-ND5F13350-mt-ND5R13426TGCTCCGGGTCCATCATC
      TGAGTAGTCCTCCTATTTTTCGAATATCT
      mt-ND6F14243-mt-ND6R14439GCCCCCGCACCAATAGGATCCTCCC
      CCTGAGGCATGGGGGTCAGGGGT
      mt-CO1F6168-mt-CO1R6587GCCCCCGATATGGCGTTTCCCCGCA
      GGGGTCTCCTCCTCCGGCGGGGTCG
      mt-CO2F7972-mt-CO2R8157ACCAGGCGACCTGCGACTCCT
      ACCCCCGGTCGTGTAGCGGT
      mt-CO3F9555-mt-CO3R9935CCCCCAACAGGCATCACCCCGC
      ATGCCAGTATCAGGCGGCGGC
      mt-CYBF15263-mt-CYBR15326CCCACCCTCACACGATTCTTTA
      TTGCTAGGGCTGCAATAATGAA
      mt-ATP6F8848-mt- ATP6R8910TTATGAGCGGGCACAGTGATT
      GAAGTGGGCTAGGGCATTTTT
      mt-ATP8F8393-mt- ATP8R8472CCCACCATAATTACCCCCATACT
      GGTAGGTGGTAGTTTGTGTTTAATATTTTTAG
      18SF/18SRTAGAGGGACAAGTGGCGTTC
      CGCTGAGCCAGTCAGTGT
      Primers for the determination of fatty acid oxidative genes expression
      CPT1AF / CPT1ARATGCGCTACTCCCTGAAAGTG
      GTGGCACGACTCATCTTGC
      CPT1BF / CPT1BRCCTGCTACATGGCAACTGCTA
      AGAGGTGCCCAATGATGGGA
      VLCADF / VLCADRTAGGAGAGGCAGGCAAACAGCT
      CACAGTGGCAAACTGCTCCAGA
      MCADF / MCADRAGAACCTGGAGCAGGCTCTGAT
      GGATCTGGATCAGAACGTGCCA
      SCADF / SCADRCGGCAGTTACACACCATCTAC
      GCAATGGGAAACAACTCCTTCTC
      ACOX1F / ACOX1RGGCGCATACATGAAGGAGACCT
      AGGTGAAAGCCTTCAGTCCAGC
      ACLYF / ACLYRGCTCTGCCTATGACAGCACCAT
      GTCCGATGATGGTCACTCCCTT
      PPARαF / PPARαRTCGGCGAGGATAGTTCTGGAAG
      GACCACAGGATAAGTCACCGAG
      PGC1αF / PGC1αRCCAAAGGATGCGCTCTCGTTCA
      CGGTGTCTGTAGTGGCTTGACT
      18SF / 18SRTAGAGGGACAAGTGGCGTTC
      CGCTGAGCCAGTCAGTGT
      Primers for the determination of mitochondrial translation genes expression
      12S rRNAF / 12S rRNARAATAGGTTTGGTCCTAGCCT
      TGAGGTTTCCCGTGTTGAGT
      MRPS11F / MRPS11RCCTTTGCTTCCTGTGGCACAGA
      GCCTTTCACCACAACTCGGATG
      MRPS16F / MRPS16RGTTGCCCTCAACCTAGACAGGA
      CCGTTTCCTTCGCAGTCTCTCA
      MRPS6F / MRPS6RCGCTTCCTTATAGGATCTCTGCC
      GAGACAAGTGCTCCACCATGCT
      MRPS22F / MRPS22RCTACGCAAAGCCTCTTGGGAAG
      CAACATGCCTGTCCTGGCTATAC
      MRPS27F / MRPS27RCGAGGAAACAGAGCAGTCCAAG
      ATGTCCTCTGCTTCACAGGTGG
      18SF / 18SRTAGAGGGACAAGTGGCGTTC
      CGCTGAGCCAGTCAGTGT
      Primers for the determination of mitophagy genes expression
      LC3F / LC3RGCTACAAGGGTGAGAAGCAGCT
      CTGGTTCACCAGCAGGAAGAAG
      P62F / P62RTGTGTAGCGTCTGCGAGGGAAA
      AGTGTCCGTGTTTCACCTTCCG
      18SF / 18SRTAGAGGGACAAGTGGCGTTC
      CGCTGAGCCAGTCAGTGT
      Primers for the determination of glycolysis genes expression
      SLC2A1F / SLC2A1RTTGCAGGCTTCTCCAACTGGAC
      CAGAACCAGGAGCACAGTGAAG
      HK2F / HK2RGAGTTTGACCTGGATGTGGTTGC
      CCTCCATGTAGCAGGCATTGCT
      GPIF / GPIRCTGGTAGACGGCAAGGATGTGA
      TCCGTGATGGTCTTGCCTGTGT
      PFKMF / PFKMRGCTTCTAGCTCATGTCAGACCC
      CCAATCCTCACAGTGGAGCGAA
      PFKLF / PFKLRAAGAAGTAGGCTGGCACGACGT
      GCGGATGTTCTCCACAATGGAC
      PFKPF / PFKPRAGGCAGTCATCGCCTTGCTAGA
      ATCGCCTTCTGCACATCCTGAG
      PKMF / PKMRATGGCTGACACATTCCTGGAGC
      CCTTCAACGTCTCCACTGATCG
      LDHAF / LDHARGGATCTCCAACATGGCAGCCTT
      AGACGGCTTTCTCCCTCTTGCT
      LDHBF / LDHBRGGACAAGTTGGTATGGCGTGTG
      AAGCTCCCATGCTGCAGATCCA
      Primers for the determination of TCA genes expression
      PDHA1F / PDHA1RGGATGGTGAACAGCAATCTTGCC
      TCGCTGGAGTAGATGTGGTAGC
      ACO1F / ACO1RTCCTCAGGTGATTGGCTACAGG
      TCGGTCAGCAATGGACAACTGG
      ACO2F / ACO2RCAATCGTCACCTCCTACAACAGG
      GTCTCTGGGTTGAACTTGAGGG
      IDH1F / IDH1RCTATGATGGTGACGTGCAGTCG
      CCTCTGCTTCTACTGTCTTGCC
      IDH2F / IDH2RAGATGGCAGTGGTGTCAAGGAG
      CTGGATGGCATACTGGAAGCAG
      IDH3AF / IDH3ARTCGGTGTGACACCAAGTGGCAA
      TTCGCCATGTCCTTGCCTGCAA
      IDH3BF / IDH3BRTGGCATCTGAGGAGAAGCTGGA
      TCAGCCGCATATCATAGGAGGC
      IDH3GF / IDH3GRCCAGTGGACTTTGAAGAGGTGC
      TTTGTGCGACGGTGGCAGGTTA
      OGDHF / OGDHRGAGGCTGTCATGTACGTGTGCA
      TACATGAGCGGCTGCGTGAACA
      SUCLA2F / SUCLA2RGCAAGAAGCTGGTGTCTCCGTT
      CCACCAGCTAAAACCTGTGCCT
      SDHAF / SDHARGAGATGTGGTGTCTCGGTCCAT
      GCTGTCTCTGAAATGCCAGGCA
      FHF / FHRCCGCTGAAGTAAACCAGGATTATG
      ATCCAGTCTGCCATACCACGAG
      MDH1F / MDH1RCGGTGTCCTAATGGAACTGCAAG
      CATCCAGGTCTTTGAAGGCAACG
      MDH2F / MDH2RCTGGACATCGTCAGAGCCAACA
      GGATGATGGTCTTCCCAGCATG
      CSF / CSRCACAGGGTATCAGCCGAACCAA
      CCAATACCGCTGCCTTCTCTGT
      Primers for the determination of cholesterol synthesis genes expression
      CYP51AF / CYP51ARCTCTTACCAGGTTGGCTGCCTT
      CTTGAGACTGTCTGCGTTTCTGG
      FDFT1F / FDFT1RTGTGACCTCTGAACAGGAGTGG
      GCCCATAGAGTTGGCACGTTCT
      DHCR24F / DHCR24RCAGGAGAACCACTTCGTGGAAG
      CCACATGCTTAAAGAACCACGGC
      DHCR7F / DHCR7RTCCACAGCCATGTGACCAATGC
      CGAAGTGGTCATGGCAGATGTC
      HSD17B7F / HSD17B7RGCTGATGGACTTCAGGAGGTGT
      GCACTGCGAGATGATGTCCAGA
      NSDHLF / NSDHLRCAGTTTTCCACTGTGCGTCACC
      ACGCCCTCAAAGATGACACTGG
      HMGCRF / HMGCRRGACGTGAACCTATGCTGGTCAG
      GGTATCTGTTTCAGCCACTAAGG
      HMGCS1F / HMGCS1RAAGTCACACAAGATGCTACACCG
      TCAGCGAAGACATCTGGTGCCA
      mtDNA, mitochondrial DNA; mtRNA, mitochondrial RNA.
      Supplementary Table 4Antibodies
      AntibodiesSource
      Anti-ND1Proteintech
      Anti-ND2Proteintech
      Anti-ND3Abcam
      Anti-ND5Proteintech
      Anti-CYTBProteintech
      Anti-CO1Abcam
      Anti-CO2Proteintech
      Anti-ATP6Proteintech
      Anti-ATP8Proteintech
      Anti-TOMM20Proteintech
      Anti-GRIM19Abcam
      Anti-SDHAAbcam
      Anti-CORE2Abcam
      Anti-ATP5Abcam
      Anti-GAPDHProteintech
      Anti-PINK1Proteintech
      Anti-CLPPProteintech
      Anti-LONP1Proteintech
      Anti-AFG3L2Proteintech
      Anti-HSP60Proteintech
      Anti-GRP75Proteintech
      Anti-ACTINProteintech
      Anti-RXRAAbcam
      Anti-NRF1Abcam
      Anti-JNK1/2Cell Signaling Technology
      Anti-P-JNK1/2Cell Signaling Technology
      Anti-p38Cell Signaling Technology
      Anti-phospho-p38 (Thr389)Cell Signaling Technology
      Anti-ERK1/2Cell Signaling Technology
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