Mitochondrial DNA (mtDNA) variants influence the risk of late-onset human diseases, but the reasons for this are poorly understood. Undertaking a hypothesis-free analysis of 5,689 blood-derived biomarkers with mtDNA variants in 16,220 healthy donors, here we show that variants defining mtDNA haplogroups Uk and H4 modulate the level of circulating N-formylmethionine (fMet), which initiates mitochondrial protein translation. In human cytoplasmic hybrid (cybrid) lines, fMet modulated both mitochondrial and cytosolic proteins on multiple levels, through transcription, post-translational modification and proteolysis by an N-degron pathway, abolishing known differences between mtDNA haplogroups. In a further 11,966 individuals, fMet levels contributed to all-cause mortality and the disease risk of several common cardiovascular disorders. Together, these findings indicate that fMet plays a key role in common age-related disease through pleiotropic effects on cell proteostasis.
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The GTEx project was supported by the Common Fund of the Office of the Director of the National Institutes of Health and by the NCI, the NHGRI, the NHLBI, the NIDA, the NIMH and the NINDS. Data used for analyses described in this study were obtained from the GTEx Portal (GTEx_Analysis_2016-01-15_v7_RNASeQCv1.1.8) and dbGaP accession number phs000424.v7.p2. All data are available in the main text, the Supplementary Information or upon request to the authors. Source data are provided with this paper.
We conducted our analyses using the following published and publicly available software: (1) for calling mtDNA variants: GATK version 184.108.40.206 HaplotypeCaller (https://gatk.broadinstitute.org/hc/en-us/articles/360037225632-HaplotypeCaller) and mtDNA-Server, local version (https://github.com/seppinho/mutserve), (2) for mtDNA association analysis using an LMM and variance decomposition analysis: LDAK version 5 (http://dougspeed.com/downloads2/); (3) for improving the power of mtDNA association: CMS version 1.0 (https://github.com/haschard/CMS); (4) for eQTL analyses: LIMIX version 3.0 (https://github.com/limix/limix); (5) for identifying pseudogenes in the nuclear genome with high sequence similarity to mtDNA: lastal 744 (http://last.cbrc.jp/doc/lastal.html); (6) for identifying PEER factors that capture unknown confounding in gene expression data: PEER version 1.3 (https://github.com/PMBio/peer); (7) for differential expression analysis: edgeR version 3.11 (http://bioconductor.org/packages/release/bioc/html/edgeR.html); (8) for GSEA: GSEA version 4.1.0 (https://www.gsea-msigdb.org/gsea/index.jsp); (9) for flow cytometry analysis: FlowJo version 10.2.
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Participants in the INTERVAL randomized controlled trial were recruited with the active collaboration of NHS Blood and Transplant England (https://www.nhsbt.nhs.uk/), which has supported field work and other elements of the trial. DNA extraction and genotyping was co-funded by the NIHR, the NIHR BioResource (http://bioresource.nihr.ac.uk) and the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014)*. The academic coordinating center for INTERVAL was supported by core funding from the NIHR Blood and Transplant Research Unit in Donor Health and Genomics (NIHR, BTRU-2014-10024), the UK Medical Research Council (MR/L003120/1), the British Heart Foundation (SP/09/002, RG/13/13/30194, RG/18/13/33946) and the NIHR Cambridge BRC (BRC-1215-20014)*. A complete list of investigators for and contributors to the INTERVAL trial is provided in ref. 73. The academic coordinating center thanks blood donor center staff and blood donors for participating in the INTERVAL trial. This work was supported by Health Data Research UK, which is funded by the UK Medical Research Council, the Engineering and Physical Sciences Research Council, the Economic and Social Research Council, the Department of Health and Social Care (England), the Chief Scientist Office of the Scottish Government Health and Social Care Directorates, the Health and Social Care Research and Development Division (Welsh Government), the Public Health Agency (Northern Ireland), the British Heart Foundation and Wellcome. N.C. is supported by an EBI–Sanger Postdoctoral Fellowship. A.G.-D. receives funding from the NIHR Biomedical Research Centre based at Cambridge University Hospitals NHS Foundation Trust. E.Y.-D. was funded by the Isaac Newton Trust/Wellcome Trust ISSF/University of Cambridge Joint Research Grants Scheme. J.M.M.H. was funded by the NIHR Cambridge BRC (BRC-1215-20014)*. P.F.C. is a Wellcome Trust Principal Research Fellow (212219/Z/18/Z) and a UK NIHR Senior Investigator, who receives support from the Medical Research Council Mitochondrial Biology Unit (MC_UU_00015/9), the Evelyn Trust and the NIHR Biomedical Research Centre based at Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge. C.H.W.-G. is supported by a RCUK/UKRI Research Innovation Fellowship awarded by the Medical Research Council (MR/R007446/1) and by the Cambridge Centre for Parkinson-Plus. N.S. is supported by the Wellcome Trust (grant number 098051 until 30 September 2016 and 206194 from 1 October 2016), the Cambridge BHF Centre of Research Excellence (RE/18/1/34212) and the NIHR Cambridge Biomedical Research Centre Biomedical Resources Grant, University of Cambridge, Cardiovascular Theme (RG64226). J.D. holds a British Heart Foundation Professorship and an NIHR Senior Investigator Award*. R.A.L. is supported by grants from Parkinson’s UK. Sequencing of INTERVAL samples was supported by the Wellcome Trust (grant number 206194). Metabolon metabolomics assays were funded by the NIHR BioResource, the Wellcome Trust (grant number 206194) and the NIHR Cambridge BRC (BRC-1215-20014)*. Nightingale Health NMR assays were funded by the European Commission Framework Programme 7 (HEALTH-F2-2012-279233). SomaLogic assays were funded by Merck and the NIHR Cambridge BRC (BRC-1215-20014)*. ICICLE-PD was funded by Parkinson’s UK (J-0802, G-1301, G-1507) and the Lockhart Parkinson’s Disease Research Fund and supported by the NIHR Newcastle Biomedical Research Unit and the NIHR Cambridge Biomedical Research Centre (146281). The EPIC-Norfolk study (https://doi.org/10.22025/2019.10.105.00004) has received funding from the Medical Research Council (MR/N003284/1 and MC-UU_12015/1) and Cancer Research UK (C864/A14136). Genetic work in the EPIC-Norfolk study was funded by the Medical Research Council (MC_PC_13048). Metabolite measurements in the EPIC-Norfolk study were supported by the MRC Cambridge Initiative in Metabolic Science (MR/L00002/1) and the Innovative Medicines Initiative Joint Undertaking under EMIF grant agreement no. 115372. We are grateful to all participants who have been part of the project and to the many members of the study teams at the University of Cambridge who have enabled this research. The collection of samples and assay work performed on the Oxford Vascular Study cohort was funded by the NIHR Biomedical Research Centre, Oxford, and the Wellcome Trust. *The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. We thank all members of the ICICLE-PD consortium for their support of this work.
J.M.M.H. and E.Y.-D. are full-time employees of Novo Nordisk Ltd. A.S.B. has received grants outside of this work from AstraZeneca, Biogen, BioMarin, Bioverativ, Merck and Sanofi and personal fees from Novartis outside of this work. J.D. sits on the International Cardiovascular and Metabolic Advisory Board for Novartis (since 2010); the Steering Committee of the UK Biobank (since 2011); the Scientific Advisory Committee for Sanofi (since 2013); the International Cardiovascular and Metabolism Research and Development Portfolio Committee for Novartis; the AstraZeneca Genomics Advisory Board (2018); and is an MRC International Advisory Group member, London (since 2013); and an MRC High Throughput Science ‘Omics Panel Member, London (since 2013).
Peer review information Nature Medicine thanks Wolfram Kunz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Joao Monteiro was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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A. Representation of metabolic pathway of synthesis and degradation of fMet. B. One Carbon metabolism proteins and MTFMT. Immunoblot showing the effects of the haplogroup on the expression of fMet regulatory protein MTFMT and one-carbon metabolism. The blots were immuno-detected using an anti-MTFMT, anti-MTFHD1, anti- MTF2D1, anti-SHMT1 and B-actin as loading control. Quantification of the bands by loading control (Vinculin and/or B/Actin) in each cell line. C. Effect of mitochondrial haplogroup on the expression of fMet regulatory genes. D. Quantification of formate levels. Statistical testing was performed by unpaired t-test. Normality was assessed using the Kolmogorov–Smirnov test. Bars/dots represent the mean ± SD of the biological replicates (n=4 cell lines with independent mtDNAs for each haplogroup). Error bars represent the mean ± SD of the biological replicates (n=4 cell lines with independent mtDNAs for each haplogroup) of – (Control) and + (fMet treated) cell lines. Colors red and blue represent haplogroup H and Uk respectively. The values are represented as relative to the average of untreated samples from haplogroup H unless indicated. Statistical testing was performed by using a 2-way-ANOVA test followed by Holm-Sidak’s multiple comparisons test unless stated otherwise. Exact P-values corrected for multiple comparisons are indicated. E. fMet determination in serum from controls and patients carrying the mtDNA variant m.3243G. The values are represented as relative to control serums compared to the m.3243G ones; the line in the box plot represents the mean ± SD. Box plots representing minimum, the maximum, the sample median, and the first and third quartiles including n= 29 and n=10 samples from controls and carrying the m.3243G, respectively. Statistical testing was performed by using non-parametric Mann-Whitney test. Unprocessed Western Blots can be found in Source Data Extended Data Figure 1.
Extended Data Fig. 2 fMet supplementation is similar to the differences between mitochondrial haplogroups in INTERVAL.
A. Relative increase of fMet after supplementation on cybrids (143B) and primary fibroblasts n=3-8 independent cell lines were analysed in 3 independent technical replicates per sample. The values are represented as relative to untreated and the error bars represent the mean ± SD of the biological replicates. The line represents untreated cells in each cell line. B. Boxplot of fMet levels in INTERVAL participants between individuals from the haplogroup H and Uk. (P= 1.7x10−7). The centre of the boxplots show the median fMet levels, upper and lower limits of boxplots show the interquartile range, while the whiskers show values within 1.5 times the interquartile range. Outliers show values beyond 1.5 times the interquartile range.
This Fig. shows the standardized effect size (BETA) of the mtDNA SNPs on expression levels log10(TPM+1) of mtDNA encoded MT-CO3 in Complex IV in 41 GTEx tissues. The colour of the boxes corresponds to the broad tissue type. The centre of the boxplot shows the median BETA value from all mtDNA SNPs; upper and lower limits of boxplots show the interquartile range, while the whiskers show values within 1.5 times the interquartile range. Outliers show values beyond 1.5 times the interquartile range. The upper panel shows the results for the 15 fMet-associated SNPs, while the bottom panel shows the results from the non-fMet associated mtDNA SNPs. The fMet-associated SNPs have negative BETAs across all 41 tissues in GTEx.
Extended Data Fig. 4 Formyl-methionine regulates mitochondrial protein synthesis and oxidative phosphorylation.
A. Immunoblot showing the effects of decrease on the levels of MTFMT using siRNA against MTFMT. The blots were immuno-detected using an anti-MTFMT antibody, anti-MT-CO1 and B-actin as loading control. B. Effects of decreasing the levels of MTFMT on the levels of MT-CO3 expression. Bars represent the mean ± SD of 4 technical replicates corrected by the Negative siRNA. Statistical testing was performed by using a 2-way-ANOVA test followed by Holm-Sidak’s multiple comparisons test. Exact p-values are indicated. C. Effect of fMet on mitochondrial proteins. Immunoblot showing the effects of the increase on the levels of fMet in cell lines from the haplogroup H and Uk. The blots were immuno-detected using an anti-NDUFS1, anti-NDUFS2, anti-antibody, anti-SDHA, anti-MT-CO1, anti-UQCRQ2, anti-ATP51, anti-ATP5O and B-actin as loading control. D. Effect of fMet on the formylated protein MT-CO1. Quantification of the immunoblot shown in C. Quantification of the bands by loading control (B-actin) in each cell line. E. Effect of fMet on the non-formylated mitochondrial proteins. Quantification of the immunoblot shown in C. Quantification of the bands by loading control (B-actin) in each cell line and corrected by the average of untreated samples from haplogroup H are shown. FM indicated treatment with fMet. F. 1D-BNGE and western blot analysis of digitonin treated cybrid cell lines with and without fMet treatment. The blots were immuno-detected using an anti-NDUFS1 antibody for complex I (CI, left gel) and anti-MT-CO1 for complex IV (CIV, right gel) detection. Loading control was performed by immunodetection of anti-SDHA for complex II (CII). Super-complexes (SC) composition is indicated in each case. G. 1D-BNGE and western blot analysis of digitonin treated cybrid cell lines with and without fMet treatment. The blots were immuno-detected using an anti-UQCRQ2 antibody for complex III (CIII, left gel) and anti-ATP5A1 for complex V (CV, right gel) detection. Loading control was performed by immunodetection of anti-SDH70 for complex II (CII). Super-complexes (SC) composition is indicated in each case. H. Quantification of the bands by loading control (CII) in each cell line. I. Maximal respiration with 1μM FCCP. J. Effects of increase of fMet on mitochondrial ATP. K. Effects of increase of fMet on membrane potential, mitochondrial volume and mitochondrial ROS levels. Error bars represent the mean ± standard deviation (SD) of the biological replicates (n=4 cell lines with independent mtDNAs for each haplogroup) of – (Control) and + (fMet treated) cell lines that were measured in 3-5 independent technical replicates each. Colors red and blue represent haplogroup H and Uk respectively. The values are represented as relative to the average of untreated samples from haplogroup H unless indicated. Statistical testing was performed by using a 2-way-ANOVA test followed by Holm-Sidak’s multiple comparisons test unless stated otherwise. Exact P-values corrected for multiple comparisons are indicated. Unprocessed Western Blots can be found in Source Data Extended Data Figure 4.
A. Effect of fMet on EIF2A and 4EBP1 activation. Immunoblot showing the effects of the increase on the levels of fMet in cell lines from the haplogroup H and Uk. The blots were immuno-detected using an anti-EIF2A, anti-p.EIF2ASer51, anti-4EBP1, anti-p.4EBP1Thr37&46 and anti-Vinculin as loading control. B. Effect of fMet and mitochondrial haplogroup on the expression of mtUPR targets. Transcript values corrected by the average of untreated samples from haplogroup H are shown. C. Effect of fMet and mitochondrial haplogroup on 4EBP1 activation. Quantification of the immunoblot shown in A. Quantification of the bands for p. 4EBP1Thr37&46 and 4EBP1 corrected by loading control (Vinculin) in each cell line. D. Effect of fMet in doubling time (DT). E. Effect of fMet on growth. Fold change growth corrected by day 0 is shown. Error bars represent the mean ± SD of the biological replicates (n=4 cell lines with independent mtDNAs for each haplogroup) of – (Control) and + (fMet treated) cell lines that were measured in 3-5 independent technical replicates each. F. Effect of fMet on protein ubiquitination. Immunoblot detection with anti-ubiquitin and anti-B-actin as a loading control for untreated (-) and fMet treatment (+). The quantification of the bands for ubiquitin smear corrected by loading control (B-actin) is shown. Bars/lines represent the mean ± SD of the biological replicates (n= 4 cell lines with independent mtDNAs for each haplogroup) of – (Control) and + (fMet treated) cell lines. Colors red and blue represent haplogroup H and Uk respectively. The values are represented as relative to the average of untreated samples from haplogroup H unless indicated. Statistical testing was performed by using a 2-way-ANOVA test followed by Holm-Sidak’s multiple comparisons test unless stated otherwise. Exact P-values corrected for multiple comparisons are indicated. Unprocessed Western Blots can be found in Source Data Extended Data Figure 5.
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Cai, N., Gomez-Duran, A., Yonova-Doing, E. et al. Mitochondrial DNA variants modulate N-formylmethionine, proteostasis and risk of late-onset human diseases. Nat Med 27, 1564–1575 (2021). https://doi.org/10.1038/s41591-021-01441-3
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