Project: Do Mtdna Variants Play a Role in Myalgic Encephalomyelitis (M.E.)/Chronic Fatigue

Project: Do mtDNA variants play a role in Myalgic Encephalomyelitis (M.E.)/Chronic fatigue patients (CFS)

Principle Investigator: Joanna Elson.

Lay Summery

Mitochondria are the powerhouses of the cell, and mitochondrial DNA (mtDNA) codes for proteins that are essential for energy production. Mothers alone pass mtDNA to their children creating distinct lineages, called haplogroups. These lineages have been used to deduce information about human history. However, anthropologists are not the only group interested in mtDNA, as mutations of mtDNA are an important cause of inherited disease, thus medical scientists also study mtDNA.

To understand disease caused by mtDNA mutations it is important to understand some of the central features of mitochondrial genetics, which make it different from nuclear genetics. The first is extreme multi-copy with 100’s or even 1000’s of mtDNA copies being found per cell, as opposed to nuclear chromosomes where two copies are found per cell. The number of mtDNA copies can be adapted to the energy requirements of a cell. This extreme multi-copyallows for a condition called heteroplasmy, which is where more than one mtDNA type is present within a cell. In people suffering mitochondrial disease resulting from an mtDNA mutation their cells have high levels of an mtDNA chromosome carrying a disease causing mutation. Our first aim was to see if patients with ME/CFS harbour any of themtDNA mutations known to cause mitochondrial disease.The nextimportant aspect of mtDNA genetics to understand is the high level of population variation associated with mtDNA. The second aim is to see if those with ME/CFS have different patterns of population variation when compared to controls. In the past mtDNA population variation has been associated with a number of common complex diseases including diabetes and multiple sclerosis, however, these association studies have been controversial. Some investigators in the field believe there are weakness in the mtDNA association model most commonly used by the mitochondrial field, which result in mtDNA association studies having a lack of repeatability. As such, we will be applying a recently developed association model as well as the more established model when considering mtDNA population variation in the context of ME/CFS.

The research was performed at Newcastle Fatigue Research Centre. This is a grouping of scientists and clinicians interested in fatigue brought togetherby Prof Julia Newton. Prof Newton supplied the clinical oversight, phenotyping of the patients and access to patient samplesthat was required for the project. A mitochondrial group in South Africa based at the centre for Human Metabolomics, North-West University, Potchefstroominterested in ME/CFS were partners in this project.

The first hypothesis resulted in the publication of a paper entitled: Clinically proven mtDNA mutations are not common in those with chronic fatigue syndrome, by Schoeman EM et al.Importance: There are a small number of case reports in the medical literature reporting primary mtDNA mutations in individuals who had initially been diagnosed with ME/CFS. Our investigations suggest that such patients are likely to be very rare, with the complete mtDNA sequencing of ~100 patients with ME/CFS having been conducted for this published paper, and an additional 300 for an upcoming manuscript.

In the context of the second hypothesis, a publication entitled, Chronic fatigue syndrome patients in the United Kingdom and South Africa have less mildly deleterious mtDNA population variants than controls from the same nation, by Venter et al. is under production. Importance: This work demonstrates differences in mtDNA population variation between those with ME/CFS and controls. This is an important and exciting observation, but it remains unexplained. Currently work is underway to look at other factors that might help explain this observation. Firstly, blood samples are being collected from severely affected ME/CFS patients for mtDNA sequencing. Conducting mtDNA sequencing of the severely affected patients will allow us to determine if the differences in mtDNA variation that we have observed are common to ME/CFS patients as a complete patient group, or if the severely affected patients are different to either (or both) of the groups that have been examined. Second, is mtDNA copy number in those with ME/CFS (including those who are severely affected) different controls? MtDNA copy number varies depending on the cell type; and can be altered by the cell in response to energetic demands. This is an area of increasing interest in the mitochondrial field with a growing body of work suggesting that copy number is altered in other complex disease, and example Parkinson s disease.

Background

Human mtDNA codes for 13 essential components of the mitochondrial oxidative phosphorylation system, which generates ATP. MtDNA undergoes strict maternal inheritance1 as such the evolution of mtDNA is characterised by the emergence of distinct lineages (or haplogroups), which have been used to study population histories. Different global populations are associated with distinct haplogroups2. Unlike nuclear DNA, mtDNA exhibits extreme multi-copy, with 100s or even 1000s of copies found in each cell. This extreme multi-copy allows for a condition called heteroplasmy, where more than one mtDNA genotype is present within a cell3. Mutations of mtDNA are an important cause of inherited neuromuscular disorders3-6. Most pathogenic mtDNA mutations are seen as heteroplasmic variants,meaning both wild type and mutated mtDNA molecules co-existing in the cells of patients. MtDNA mutations are recessive with a major biochemical defect requiring the majority of mtDNA molecules within a cell to be of the mutant type. The precise threshold level of any mutation, that is the amount required to cause a major biochemical defect within the cell depends on the specific mutation, but also shows variation with tissue type and between individual patients, resulting in a complex and heterogeneous pattern of presentation for diseases within this group3.

Moving from disease causing variants to population variants, for years, many have felt that common mtDNA population variants (haplogroup markers) could affect the penetrance of some clinically manifesting mtDNA disease causing mutations7. There are also many studies supporting a role for mtDNA population variants in common complex disease8. Instances where a disease or phenotype is significantly associated with a common mtDNA haplogroup suggest that one or more haplogroup-associated variants modify risk (or outcome) of disease, this is the classic haplogroup association hypothesis. Such studies have been controversial due to their low repeatability. Two studies pioneered a now standard dual cohort approach in an attempt to demonstrate repeatability of results9,10; other studies in this area of association genetics include 10-13. Due to the size of the cohort to be used in this study, a classic haplogroup association study would have limited power8, as such will not be a standalone aim of the study.

Another possibility is that there might be a cumulative effect of multiple rare mtDNAs variants either at the level of the patient group as a whole, or within a limited number of individuals who are part of the patient group9. Rare variants that alter an amino acid or rRNA/tRNA have a higher likelihood of being slightly deleterious. As purifying selection has been shown to act on mtDNA population variation removing changes that are likely to have a detrimental effect on protein function over generations14, this effect is in addition to the strong purifying selection seen in the germline15. The simplest demonstration of this being that non-synomous changes in protein encoding genes are more common at the tips (leaves) of the phylogeny than deeper (roots). The MutPred program 16 has been well tested in the context of mtDNA variation17, it determines if a non-synomous change is likely to have an impact on protein function, or reactive oxygen species (ROS) production. The MutPred program can be used to compare variants seen in a patient group to those seen in the general population.

The project investigated two hypothesis

Hypothesis 1: Do patients with ME/CFS harbour clinically proven mtDNA mutations associational with primary mitochondrial disease (see outcome (1))

Hypothesis 2: Is there evidence for a role of mtDNA haplogroup or rare population variants in ME/CFS (see outcomes (2) (3) (4))

The primary outcomes of the study

1. Published paper: Clinically proven mtDNA mutations are not common in those with chronic fatigue syndrome: BMC Med Genet. 2017 Mar 16; 18(1):29
2. Paper in preparation: Chronic fatigue syndrome patients in the United Kingdom and South Africa have less mildly deleterious mtDNA population variants than controls from the same nation
3. Travel Fellowship: South African PhD student awarded travel funds to present ME/CFS data at the European Society of Human Genetics conference in Copenhagen May 2017
4. Conference presentation: Euromit the global leader in mitochondrial conferences in Cologne 2017
5. Working ongoing: Supporting reagents for an MSc student who will be comparing mtDNA copy number in bed/house bound ME/CFS patients compared to non-housebound patients and controls
6. Grant in preparation: Data will be used in an MRC grant application to be submitted this autumn in conjunction with Prof Julia Newton

Hypothesis 1: Do patients with ME/CFS harbour clinically proven mtDNA mutations associational with primary mitochondrial disease (see outcome (1))

Clinically proven mtDNA mutations are not common in those with chronic fatigue syndrome

Schoeman EM, Van Der Westhuizen FH, Erasmus E, van Dyk E, Knowles CV, Al-Ali S, Ng WF, Taylor RW, Newton JL, Elson JL.

BMC Med Genet. 2017 Mar 16; 18(1):29.

BACKGROUND: Chronic Fatigue Syndrome (CFS) or Myalgic Encephalomyelitis (M.E.)is a prevalent debilitating condition that affects approximately 250,000 people in the UK. There is growing interest in the role of mitochondrial function and mitochondrial DNA (mtDNA) variation in ME/CFS. It is now known that fatigue is common and often severe in patients with mitochondrial disease irrespective of their age, gender or mtDNA genotype. More recently, it has been suggested that some ME/CFS patients harbour clinically proven mtDNA mutations.

METHODS: MtDNA sequencing of 93 ME/CFS patients from the United Kingdom (UK) and South Africa (RSA) was performed using an Ion Torrent Personal Genome Machine. The sequence data was examined for any evidence of clinically proven mutations, currently; more than 200 clinically proven mtDNA mutations point mutations have been identified.

RESULTS: We report the complete mtDNA sequence of 93 ME/CFS patients from the UK and RSA, without finding evidence of clinically proven mtDNA mutations. This finding demonstrates that clinically proven mtDNA mutations are not a common element in the aetiology of disease in ME/CFS patients. That is patients having a clinically proven mtDNA mutation and subsequently being mis-diagnosed with ME/CFS are likely to be rare.

CONCLUSION: The work supports the assertion that ME/CFS should not be considered to fall within the spectrum of mtDNA disease. However, the current study cannot exclude a role for nuclear genes with a mitochondrial function, nor a role of mtDNA population variants in susceptibility to disease. This study highlights the need for more to be done to understand the pathophysiology of ME/CFS.

It is worth noting that an independent group supported our findings:

Importance

There are a small number of isolated case reports in the medical literature reporting mtDNA mutations in individuals who had been initially diagnosed with ME/CFS18. Galan et al suggested individuals similar to the patient they reported might be more common than appreciated by the medical community. This was a potentially an important observation that merited investigation. Our investigations suggest that such patients are likely to be rare. Although ME/CFS patients are not patients harbouring pathogenic mtDNA mutationsthis does not exclude a role for mitochondrial dysfunction in the aetiology of ME/CFS. Such a role could be mediated by mtDNA population variants of mildly deleterious effect, or by differences in mtDNA copy number.

Hypothesis 2: Is there evidence for a role of mtDNA haplogroup or rare population variants in ME/CFS patients (see outcomes (2) (3) (4))

Chronic fatigue syndrome patients in the United Kingdom and South Africa have less mildly deleterious mtDNA population variants than controls from the same nation

Chronic Fatigue Syndrome (CFS) or Myalgic Encephalomyelitis (M.E.)is a prevalent debilitating condition that affects approximately 250,000 people in the UK alone19. There is growing interest in a possible etiologic or pathogenic role of mitochondrial function and mitochondrial DNA (mtDNA) variation in ME/CFS. Supporting such a link, fatigue is common and often severe in patients with mitochondrial disease, irrespective of their age, gender or mtDNA genotype. Importantly, two previous cohort studies have not found evidence of primary pathogenic mtDNA mutations in ME/CFS patients20,21.

This study investigates the collective role of mildly deleterious mtDNA population variants in ME/CFS, using the computational tools HaploGrep22 and MutPred16,23 to implement the new “mutational load” mtDNA association method24,25. Thus, this second study is a genetic association study considering if mtDNA population variants alter susceptibility to or course of disease. MtDNA variants with a MutPred score of >0.5 potentially have a mildly deleterious impact on protein function and were the focus of the new methods used in this study. It should be noted that the older method the “haplogroup association” methodology did not detect a difference between patients and controls.

Two ME/CFS cohorts were analysed, one from the UK and the other from South Africa; both patient cohorts were compared with controls from the same nation. For both cohorts, ME/CFS patients had a significant excess of individuals without a population variant that was predicated to be mildly deleterious. This result is unexpected and requires additional investigation and validation. It would suggest that an investigation into the biological impact of such variants on the OXPHOS system and wider downstream energy metabolism in ME/CFS is warranted and that such work might reveal novel insights in to the aetiology of the disease.

Importance

This work demonstrates differences in mtDNA population variation between those with ME/CFS and controls. This is an important and exciting observation, but it remains unexplained. Currently work is underway to look at other factors that might help explain this observation. Firstly, blood samples are being collected from severely affected ME/CFS patients for mtDNA sequencing. Conducting mtDNA sequencing of the severely affected patients will allow us to determine if the differences in mtDNA population variants that we have observed are a common to ME/CFS patients as a completepatients group, or if the severelyaffected patients are different to either (or both) of the groups that have this far been examined.Second, is mtDNA copy number in those with ME/CFS (including those who are severely affected) different controls? MtDNA copy number is variable depending on the cell type and can be altered by the cell in response to energy demands. This is an area of growing interest in the mitochondrial field with a growing body of work suggesting that copy number is altered in other complex disease, and example Parkinson s disease26-28.

References

1.Elson JL, Andrews RM, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N: Analysis of European mtDNAs for recombination. American Journal of Human Genetics 2001; 68: 145-153.

2.Herrnstadt C, Elson JL, Fahy E et al: Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups. American Journal of Human Genetics 2002; 70: 1152-1171.

3.Tuppen HA, Blakely EL, Turnbull DM, Taylor RW: Mitochondrial DNA mutations and human disease. Biochim Biophys Acta 2010; 1797: 113-128. doi: 110.1016/j.bbabio.2009.1009.1005. Epub 2009 Sep 1015.

4.Schaefer AM, Phoenix C, Elson JL, McFarland R, Chinnery PF, Turnbull DM: Mitochondrial disease in adults: a scale to monitor progression and treatment. Neurology 2006; 66: 1932-1934.

5.Elson JL, Cadogan M, Apabhai S et al: Initial development and validation of a mitochondrial disease quality of life scale. Neuromuscul Disord 2013; 23: 324-329. doi: 310.1016/j.nmd.2012.1012.1012. Epub 2013 Feb 1020.

6.Phoenix C, Schaefer AM, Elson JL et al: A scale to monitor progression and treatment of mitochondrial disease in children. Neuromuscular Disorders 2006; 16: 814-820.

7.Brown MD, Starikovskaya E, Derbeneva O et al: The role of mtDNA background in disease expression: a new primary LHON mutation associated with Western Eurasian haplogroup J. Hum Genet 2002; 110: 130-138. Epub 2002 Jan 2024.

8.Samuels DC, Carothers AD, Horton R, Chinnery PF: The power to detect disease associations with mitochondrial DNA haplogroups. Am J Hum Genet 2006; 78: 713-720. Epub 2006 Feb 2017.

9.Elson JL, Herrnstadt C, Preston G et al: Does the mitochondrial genome play a role in the etiology of Alzheimer's disease? Human Genetics 2006; 119: 241-254.

10.Chinnery PF, Elliott HR, Syed A, Rothwell PM, Study OV: Mitochondrial DNA haplogroups and risk of transient ischaemic attack and ischaemic stroke: a genetic association study. Lancet Neurol 2010; 9: 498-503. doi: 410.1016/S1474-4422(1010)70083-70081. Epub 72010 Mar 70031.

11.Chinnery PF, Mowbray C, Patel SK et al: Mitochondrial DNA haplogroups and type 2 diabetes: a study of 897 cases and 1010 controls. J Med Genet 2007; 44: e80.

12.Ban M, Elson J, Walton A et al: Investigation of the Role of Mitochondrial DNA in Multiple Sclerosis Susceptibility. Plos One 2008; 3.

13.Elson JL, Majamaa K, Howell N, Chinnery PF: Associating mitochondrial DNA variation with complex traits. American Journal of Human Genetics 2007; 80: 378-382.

14.Elson JL, Turnbull DM, Howell N: Comparative Genomics and the evolution of human mitochondrial DNA: Assessing the effects of selection. American Journal of Human Genetics 2004; 74: 229-238.

15.Stewart JB, Freyer C, Elson JL et al: Strong purifying selection in transmission of mammalian mitochondrial DNA. Plos Biology 2008; 6: 63-71.

16.Li B, Krishnan VG, Mort ME et al: Automated inference of molecular mechanisms of disease from amino acid substitutions. Bioinformatics 2009; 25: 2744-2750. doi: 2710.1093/bioinformatics/btp2528. Epub 2009 Sep 2743.

17.Soares P, Abrantes D, Rito T et al: Evaluating purifying selection in the mitochondrial DNA of various mammalian species. Plos One 2013; 8: e58993. doi: 58910.51371/journal.pone.0058993. Epub 0052013 Mar 0058922.

18.Galan F, de Lavera I, Cotan D, Sanchez-Alcazar JA: Mitochondrial Myopathy in Follow-up of a Patient With Chronic Fatigue Syndrome. J Investig Med High Impact Case Rep 2015; 3: 2324709615607908. doi: 2324709615607910.2324709615601177/2324709615607908. eCollection 2324709615602015 Jul-Sep.

19.Pendergrast T, Brown A, Sunnquist M et al: Housebound versus nonhousebound patients with myalgic encephalomyelitis and chronic fatigue syndrome. Chronic Illn 2016; 28: 1742395316644770.