Mitochondrial DNA copy number as a phenotypic trait for human diseases in genetic epidemiological studies

Lead Research Organisation: University of Bristol
Department Name: Social Medicine

Abstract

Cells contain elements called mitochondria. Mitochondria contain their own DNA (mtDNA). Variation in this DNA is related to disease. Diseases influenced by mtDNA include obesity, diabetes and hyperlipidemia. However, two aspects of mtDNA are underexplored. The first aspect is the analysis of the number of copies of mtDNA present in cells in relation to human health traits. The second aspect is to study whether the
number of copies of mtDNA is determined by nuclear genes. Our project aims to study whether the number of copies of mtDNA associate with traits related to obesity, diabetes, hyperlipidemia and cognitive function. We also aim to identify nuclear genes controlling the number of copies of mtDNA. To this end, we will determine the number of copies of mtDNA in each of the 20,000 individuals of one population sample (Avon Longitudinal Study of Parents and Children, ALSPAC). The results from this project would contribute to the study of obesity, diabetes, hyperlipidemia and cognitive risk traits. This would represent new knowledge and potential benefit for improving human health.

Technical Summary

Mitochondrial DNA (mtDNA) has been studied in relation to population genetics and human disease. Mutations and depletion of mtDNA have been related to a variety of diseases. There is evidence suggesting that depletion of mtDNA is associated with metabolic disorders such as obesity, diabetes and hyperlipidemia. However, variation of mtDNA copy number as a continuous trait in relation to genetic epidemiological studies is underexplored. The same applies to the study of nuclear genes exerting control in the genetic variation of mtDNA. Our aims here are i) to conduct association studies between mtDNA copy number and complex traits including adiposity, fasting glucose, insulin, lipids and cognitive function, aiming to identify relations between mtDNA copy number and these traits relevant to disease; and ii) to conduct a genomewide association scan (GWAS) aiming to identify nuclear genes controlling mtDNA copy number. To this end, we will determine mtDNA copy number in an established and densely phenotyped cohort (Avon Longitudinal Study of Parents and Children, ALSPAC, N = 20,000). The results from this project would open a new dimension on the study of obesity, diabetes, hyperlipidemia and cognitive risk traits, representing new basic knowledge and potential benefit for improving human health. If we demonstrate that mtDNA copy number can be used as a biomarker for obesity, diabetes, hyperlipidemia and cognitive risk traits, its use could also be applied to the study of other diseases.

Planned Impact

This research will have social and economic impact in the following ways:

Health Research - Obesity, diabetes and hyperlipidemia are diseases with major impact on human health. This proposal will establish whether mtDNA copy number can be used as a blood biomarker in relation to these health phenotypes. If so, its use could enable medical interventions dependent on mtDNA copy number genotype.

Economy and society - Obesity and diabetes have are a major health burden in the UK and developed world, and they are leading causes of working days lost and premature death. Early diagnosis of obesity and diabetes will reduce the costs associated with the care of patients with these conditions. It will also reduce the mortality and morbidity of these common diseases.

Training - The post doctoral research fellow undertaking this work will benefit from developing skills and expertise in different but related areas of research. These include experimental lab work and statistical genetic analysis such as genomewide association mapping and Mendelian Randomization.

Public Understanding of science. The general public have a substantial interest in genetic causes of disease, and in particular in research that underpins our understanding of the development of disease and new scientific discoveries in the detection, characterisation, prevention and treatment of disease. We will communicate research of interest through public outreach activities and press releases.

Publications

10 25 50
 
Description MRC Integrative Epidemiology Unit studentship
Amount £100,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2017 
End 09/2021
 
Description Wellcome Trust PhD in Molecular, Genetic and Lifecourse Epidemiology
Amount £150,512 (GBP)
Funding ID 102433/Z/13/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2013 
End 09/2017
 
Title mtDNA quantification by qPCR 
Description We have developed a reliable, reproducible method to accurately determine mtDNA copy number in blood. Mitochondrial DNA copy number in DNA extracted from leukocytes is assessed by quantification of a unique mitochondrial fragment (62bp) relative to a 95bp fragment from a single copy region of the nuclear gene, ß2M (Beta-2-microglobulin). Real-time PCR is performed by analysis of a 384-well microplate, using a Roche LC480 LightCycler. 56 DNA samples are analysed per plate, at 6 wells per sample (3 replicates for mtDNA, 3 replicates for nuDNA). 3 calibrator DNA samples are also quantified on every plate, as well as a water negative control and a DNA sample serially diluted to provide a standard curve (in duplicate). Each well contains 5ul of 2x SensiFAST SYBR No-ROX Kit (Bioline), 0.5µl H2O, 0.25 µl forward primer, 0.25 µl reverse primer, and 4 µl (1ng) of DNA. The DNA is amplified for a total of 45 cycles, and the mtDNA copy number is calculated from the ratio of mtDNA copies / nuDNA copies derived from threshold-crossing points of the respective amplification curves. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This method has been applied to a large population sample (a total of 20,000 individuals) in order to accomplish the objectives of our proposal. Our technique is equally applicable to DNA extracted from other tissues, such as muscle, adipose, blood vessel etc, for future research studies. 
 
Description CHARGEmtDNA+ 
Organisation Washington University School of Medicine
Country United States 
Sector Academic/University 
PI Contribution We have provided summary data for ALSPAC relating mitochondrial DNA SNPs with anthropometric traits. These results are being analysed in conjunction with many other cohorts worldwide.
Collaborator Contribution Our partner contributors have provided us detailed SOPs to do our analyses.
Impact Our analyses are in progress.
Start Year 2015
 
Description EWAS of mtDNA copy number 
Organisation University of Bristol
Department School of Sociology, Politics and International Studies
Country United Kingdom 
Sector Academic/University 
PI Contribution We have reliably measured mtDNA CN in ALSPAC (N~20,000 subjects ). These include mothers and children (the latter at three time points: antenatal, age 7y and age 15y). We have contributed this information and we have been involved in the design of an epigenome wide association study (EWAS) to test cross sectional associations of DNA methylation and mtDNA CN.
Collaborator Contribution 1.- Our partners have generated nuclear methylation information in a subset of 1000 mother/child pairs from ALSPAC (the ARIES resource) [ariesepigenomics.org.uk]. These have been assayed with the HumanMethylation450k BeadChip Illumina array (HM450). These include three time points for children: antenatal, age 7y and age 15y. For mothers, we have methylation measures at two timepoints: antenatal and ~17 years after the birth of the child. 2.- Our EWAS was based on 745 participants of ALPSAC at ~7y of age. It was performed using readily available data measured by qPCR for mtDNA CN and over 450,000 CpGs from the Illumina HM450 methylation array. The linear regression model was additionally adjusted for sex, DNA concentration at time of mtDNA quantification, surrogate variables for batch effect and blood cell proportions as estimated using the Houseman algorithm. We found 44 CpGs that passed multiple testing criteria via false discovery rate, 7 of which passed the more conservative Bonferroni threshold (10-7). Our top hit for this pilot (cg22914762) resides on chromosome 4 (position 117220908). This probe corresponds to the microRNA MIR1973. It has been previously described to modulate the expression of mtDNA. This result represents a validation of our available data and methodology
Impact Pilot data generated from the EWAS has been included on a project grant proposal submitted to the MRC. Population & Systems Medicine Board. Call closing date: 19th January 2017. Title: Mitochondrial DNA Copy Number and its relation to the genetic and epigenetic influence of common human disease. PI: Santiago Rodriguez.
Start Year 2016
 
Description Instrumental variables for mtDNA CN to be used in causal studies using Mendelian Randomisation (MR) 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution 1.- We have reliably measured mtDNA CN in ALSPAC, (N~20,000 subjects). These include mothers and children (the latter at three time points: antenatal, age 7y and age 15y). 2.- We have found nominal association between mtDNA CN and metabolic phenotypes in ALSPAC. These results have been included on a manuscript that has been submitted to publication (1). 3.- We have found a number of nuclear SNPs from a GWAS that associate at the genome-wide significance level with mtDNA CN. These findings are being replicated in an independent cohort (NBS) (2) (expected submission by April 2017). Genome-wide significant SNPs will be used in the present proposal as instrumental variables in our suggested MR studies (1) Guyatt, A., Burrows, K., Guthrie, P., Ring, S., Day, INM., Ascione, R., Lawlor, D. A., Gaunt, TR., Rodriguez, S., "Cardiometabolic phenotypes and mitochondrial DNA copy number across the life course. Submitted. (2) Guyatt, AL., Brennan, RR., Burrows, K., Ascione, R., Chinnery, P., Day, INM., Gaunt, TR., Guthrie, PAI., Hudson, G., Lawlor, DA., Pyle, A., Ring, Rodriguez, S., "Genome-wide association study of mitochondrial DNA copy number." Expected Submission by March 2017.
Collaborator Contribution 1.- They have generated nuclear methylation information in a subset of 1000 mother/child pairs from ALSPAC (the ARIES resource) [ariesepigenomics.org.uk]. These have been assayed with the HumanMethylation450k BeadChip Illumina array (HM450). These include three time points for children: antenatal, age 7y and age 15y. For mothers, they have methylation measures at two timepoints: antenatal and ~17 years after the birth of the child. 2.- They have determined mQTLs that can be used as instrumental variables for DNA methylation. In addition, they are currently leading a large consortium (GoDMC) which will meta-analyse mQTLs across 40 cohorts. Both will allow us to explore causal relationships between methylation and mtDNA CN through MR analyses 3.- MR-Base is a platform for conducting MR using summary data from GWAS within a two-sample MR framework. Developed by their group within the IEU, MR-Base will allow us to explore the association between genetic instrumental variables for mtDNA CN (as identified through the GWAS) with outcomes via SNP look-ups of publically available GWAS data. 4.- In an epigenome wide association study (EWAS) of body mass index, they recently applied MR to investigate for potential causal relationships between DNA methylation and adiposity
Impact 1.- Potential instrumental variables (IVs) have been defined for mtDNA CN using results from our analyses relating nuclear SNPs with mtDNA CN. From these SNPs, a polygenic risk score was derived in ALSPAC. It replicated in two other cohorts. 2.- Pilot data generated from the instrumental variable analysis has been included on a project grant proposal submitted to the MRC. Population & Systems Medicine Board. Call closing date: 19th January 2017. Title: Mitochondrial DNA Copy Number and its relation to the genetic and epigenetic influence of common human disease. PI: Santiago Rodriguez.
Start Year 2016
 
Description cell-free mtDNA copy number 
Organisation Bristol Royal Infirmary
Country United Kingdom 
Sector Hospitals 
PI Contribution We have set up an assay to type cell-free mitochondrial DNA copy number from blood. We have used this assay to estimate mitochondrial DNA copy number from samples provided by our collaborators in the Bristol Royal Infirmary. Our study constitutes a preliminary analysis to explore differences in cell-free mitochondrial DNA copy number from samples representing different cardiac conditions.
Collaborator Contribution Our partners have obtained blood samples from patients undergoing cardiac surgery representing various cardiac conditions. They have provided these samples and phenotypic descriptors for our analyses.
Impact 1.- Results from these analyses have been included in the PhD Thesis of my student Dr Kimberley Burrows, entitled "Molecular Genetic Epidemiology Studies of Quantitative Nucleic Acid Markers". Start Date: 2010. Time to thesis submission: 2015 (viva passed). Source of funding: Wellcome Trust. Subsequent post: Research Associate at the Integrated Epidemiology Unit. Main Supervisor: Santiago Rodriguez. 2.- Emily Jamieson has been awarded an MRC Integrative Epidemiology Unit studentship within the School of Social & Community Medicine. This studentship provides 4 year funding to Emily to do her PhD on a project entitled: "Cell-free mitochondrial DNA as a biomarker for cardiac function: role of genetic and epigenetic factors". Start date: October 2017. Main Supervisor: Santiago Rodriguez
Start Year 2014
 
Description mtDNA GWAS 
Organisation Newcastle University
Department Wellcome Trust Centre for Mitochondrial Research
Country United Kingdom 
Sector Academic/University 
PI Contribution We have conducted a Genome-Wide Association Study to explore whether there are common genomic SNPs controlling mtDNA copy number variation. My PhD student Anna Guyatt has drafted a manuscript including all our results. We have stablished a collaboration with the Department of Clinical Neurosciences in Cambridge and with the Wellcome Trust Centre for Mitochondrial Research in Newcastle University in order to replicate our results in their cohort.
Collaborator Contribution Our collaborators are analysing their cohort and exploring the replication of candidate SNPs potentially showing association with mitochondrial DNA copy number in both cohorts.
Impact 1.- A.L. Guyatt, K. Burrows, P.A.I. Guthrie, T.R. Gaunt, S. Rodriguez. 2016. A genome-wide association study of mitochondrial DNA copy number in mothers and children from the Avon Longitudinal Study of Parents and Children (ALSPAC). Vancouver, Canada, October 18-22, 2016. Abstract published in the book of the Annual Meeting of The American Society of Human Genetics. 2.- Guyatt, AL., Brennan, RR., Burrows, K., Ascione, R., Chinnery, P., Day, INM., Gaunt, TR., Guthrie, PAI., Hudson, G., Lawlor, DA., Pyle, A., Ring, Rodriguez, S., "Genome-wide association study of mitochondrial DNA copy number." Expected Submission by April 2017.
Start Year 2016
 
Description mtDNA GWAS 
Organisation University of Cambridge
Department Department of Clinical Neurosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have conducted a Genome-Wide Association Study to explore whether there are common genomic SNPs controlling mtDNA copy number variation. My PhD student Anna Guyatt has drafted a manuscript including all our results. We have stablished a collaboration with the Department of Clinical Neurosciences in Cambridge and with the Wellcome Trust Centre for Mitochondrial Research in Newcastle University in order to replicate our results in their cohort.
Collaborator Contribution Our collaborators are analysing their cohort and exploring the replication of candidate SNPs potentially showing association with mitochondrial DNA copy number in both cohorts.
Impact 1.- A.L. Guyatt, K. Burrows, P.A.I. Guthrie, T.R. Gaunt, S. Rodriguez. 2016. A genome-wide association study of mitochondrial DNA copy number in mothers and children from the Avon Longitudinal Study of Parents and Children (ALSPAC). Vancouver, Canada, October 18-22, 2016. Abstract published in the book of the Annual Meeting of The American Society of Human Genetics. 2.- Guyatt, AL., Brennan, RR., Burrows, K., Ascione, R., Chinnery, P., Day, INM., Gaunt, TR., Guthrie, PAI., Hudson, G., Lawlor, DA., Pyle, A., Ring, Rodriguez, S., "Genome-wide association study of mitochondrial DNA copy number." Expected Submission by April 2017.
Start Year 2016