Tracing Chronic Fatigue Syndrome to mtDNA

B. Day
 “The observation of and the search for similarities and differences
are the basis of all human knowledge.”
Alfred Nobel

The openness and information sharing that I found among ME/CFS (Myalgic Encephalomyelitis/Chronic Fatigue Syndrome)
researchers at the OMF (Open Medicine Foundation) and among mitochondrial disease experts at the UMDF (United Mitochondrial
Foundation) are heart-warming examples of connecting the best experts and putting them in front of patients like me for the
purpose of sharing their knowledge. Thank you to each and every speaker who presented at OMF and UMDF conferences.

If this book helps you in any way, please donate to these two charities:

OMF (Open Medicine Foundation led by Ron Davis, PhD, director of the Stanford Genome Technology Center) (122). The OMF
is leading the way in supporting the development of new tests and equipment to easily identify people with ME/CFS. Imagine
getting diagnosed with ME/CFS in just one doctor’s visit. The OMF is also supporting the scientists who are researching
treatments.

UMDF (United Mitochondrial Foundation, SMAB Chair Marni Falk, MD, Executive Director of the Mitochondrial Medicine
Frontier Program at Children’s Hospital of Philadelphia) (123). The UMDF provides educational opportunities at their
conferences on how to use the mitochondrial research tools [including the tools on MSeqDr (135) and MitoMap (136)]. The
open sharing of these tools with the public is going to propel discoveries much more quickly than other fields where
discoveries are held close to one’s chest.
 Tracing Chronic Fatigue Syndrome to mtDNA
Copyright © 2018 B. Day. All Rights Reserved.

All rights reserved. No part of this book may be reproduced in any form or by any electronic or mechanical means including information storage and retrieval systems, without permission in writing from the
author. The only exception is by a reviewer, who may quote short excerpts in a review.

Disclaimer:
This book is not intended as a substitute for the medical advice of a physician(s). The reader should consult a physician(s) in matters relating to his/her health and to that of others (children, etc…). This book
details the author’s personal experiences with and opinions about Chronic Fatigue Syndrome. The author is not a healthcare provider. In addition, the author does not represent or warrant that the information
accessible via this book is accurate, complete or current. The author assumes no responsibility for errors and omissions. This is a comprehensive limitation of liability that applies to all damages of any kind,
including (without limitation) compensatory; direct, indirect or consequential damages; loss of data, income or profit; loss of or damage to property and claims of third parties.
 Housekeeping

How to contact me
You can e-mail me at BDayHEALTH@gmail.com with any questions, insights, studies, etc….

To receive updates
Just drop me a note, if you want to receive e-mail updates. Don’t worry, they won’t be often.

Please refer to this study as “the Day Study” for simplicity’s sake.

Why publish under a pen name?

I have published this book under a pen name in order to protect my children from discrimination in the work force and in their personal lives. People with ME/CFS are
discriminated against. Therefore, I ask everyone to please respect my desire to keep my name confidential. My family members are willing to participate in worthwhile
studies and speak with academics under HIPAA’s Privacy Rules.

Footnotes & References

Whenever I’m reading a book or article, I don’t want to just see the footnote citations, but I want to easily get to the original document. Therefore in my book, the
footnote numbers take you to the webpages where I found the references. If you would prefer to see a footnote in citation form, just hop to the back of the book and
you’ll see them listed under References.

Dedication
I dedicate this book to all the people with ME/CFS and Mitochondrial Disease who were told by their doctors that their symptoms were in their heads. We crawl from one
day into the next pretending to the world that we are not sick. Thankfully, we are not alone. Here are just a few of the many places to turn for support and information.

PhoenixRising - S4ME - HealthRising - ME/CFS Initiative at Stanford University – Open Medicine Foundation - Open Medicine Foundation’s list of
resources – #MEAction - United Mitochondrial Disease Foundation (UMDF) - MitoAction - Children Hospital Of Philadelphia’s Mitochondrial Center

Thank you to all those who participated in my study and answered my questions. You’ve all been extremely supportive.

And a big thank you to my family for putting up with me while I worked on this project.
 Table of Contents
Introduction

Part 1: Study Summary

1. Maternal Haplogroup J1c’s
2. Hypometabolic State
3. Long Lives
4. Surviving Harsh Conditions
5. Eradicate the word “lazy”
6. Maternal Haplogroup U5’s
7. The tighter the criteria, the stronger the link

Part 2: Putting it Together

8. My Unusual Family
9. More Unusual Families
10. ME/CFS+ Families
11. ME/CFS is extremely complex. Where does one begin?

Part 3: J1c1’s as an example

12. Why are some J1c families very healthy, while others have multiple generations that have severe health issues?
13. J1c1’s Maternal Haplogroup Family Tree
14. Haplogroup J Variants
15. DNA Research Data Tsunami
16. Updated Haplogroup J Variants
17. Comparing a healthy J1c1 family with a ME/CFS+ J1c1 family
18. J1c1’s and Macular Degeneration
19. J1c1’s with ME/CFS+
20. mtDNA Variant 9448A>G (MT-CO3)
21. A variant can be disease causing in one haplogroup and not in another haplogroup
22. Looking up variants in the prediction databases
23. Other interesting tidbits about 9448A>G
24. Mitomap’s List of Provisional Variants Related to Exercise Intolerance and/or Mitochondrial Myopathy
25. mtDNA Variant 14290T>C (MT-ND6)

Part 4: Other Factors Impacting Mitochondrial Energy

26. Nuclear Gene Variants
27. Mitochondrial Damage
Damage due to Medicines
Damage due to Particulate Matter in the Air
Damage due to Gulf War Environment
Damage from Chemotherapy
Damage from Radiation
Damage from Viruses
Damage from Infections
28. Environmental Triggers
Why are ME/CFS patients often more hypersensitive to chemicals than others?

Part 5: Take a Deeper Dive

29. Hypometabolic State
Alzheimer’s Disease and Hypometabolism in the Brain
J’s and Alzheimer’s Disease
The Brain as we Age
U5’s and Alzheimer’s Disease
30. Higher Methylation fits in with hypometabolic state
31. OXPHOS
32. J1c’s reduced OXPHOS capacity
33. Mitochondrial Uncoupling
34. Mitochondrial Coupling
35. Long Living J’s
36. Haplogroup U5’s
HIV and AIDS and U5’s
Alzheimer’s and U5’s
OXPHOS and U5’s
Macular Degeneration and U5’s
37. The Differences between Men & Women in ME/CFS and in Autistic Spectrum
38. Multiple Sclerosis
39. Fibromyalgia
40. Do some ME/CFS patients have mitochondrial multiorgan disorder syndrome?
41. Paternal Link?

Part 6: Missing Pieces

42. Adventurers & Potholes
43. Mitochondrial Experts are currently spread thin
44. More Details About the Study
45. Study Limitations

Part 7: Discussion
46. Tying It All Together
47. Wish List of Future Research
48. For my friends at Phoenix Rising and S4ME
Part 8: Supplements
49. The Frequency of J1c’s in the general population
50. The Frequency of U5’s in the general population
51. The Probability of being a child of two J1c1’s
52. Converting mtDNA variants that used reference genome hg19 to NC_012920
53. Other Haplogroups to keep in the back of minds for potential subclades with OXPHOS issues

References
Introduction
Occasionally patients are put in a position where they can recognize a pattern within their fellow sufferers. I’ve been given that blessing (or curse depending upon how you
look at it). It’s a blessing from the standpoint of someone with ME/CFS who has information that might propel research forward a step. A curse from the standpoint of
having obtained this knowledge because I’ve passed health issues onto my three children and that in turn has changed the focus of my life. I read everything (medical
studies, journal articles, and medical textbooks) and attend every related medical conference I can in order to help my children.

In my pursuit, I found a pattern within a subset of the ME/CFS community. From that I formed a theory and then tested the theory in a small population. My theory was
confirmed. I then reached out to another data pool to test the theory. There was again confirmation that my theory was correct.

I don’t have a science background, I don’t have a lab, and I don’t have a budget. Please forgive all errors and oversights. I did the best I could.

I’ve put hundreds of hours into gathering the information, doing the research, organizing it, and trying to put it onto paper so that others can understand. This book tries to
balance on the thin line between writing it for researchers (and not boring them with fundamentals) while trying to provide enough information for my ME/CFS friends to
keep up. My hope is this information reaches the researchers who can make a difference.
Part 1: Study Summary
Chapter 1: Maternal Haplogroup J1c’s

This study found a genetic component to Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) in families with more than one generation of illness. Individuals
with family histories of ME/CFS were found to have significantly higher frequencies of maternal haplogroups known for reduced OXHPOS capacity and mitochondrial
uncoupling than the general population.

Maternal haplogroup J1c’s were found at 5.4 times the expected frequency in individuals with family histories of ME/CFS (and related symptoms). Out of 16 ME/CFS
patients who had either a close family member with ME/CFS or a close family member with symptoms found often in ME/CFS patients (such as POTS, Autistic Spectrum,
Multiple Sclerosis, Fibromyalgia, Crohn’s, early-onset Alzheimer’s, etc…), 6 were J1c’s. They were from sub-haplogroups: J1c1, J1c2, J1c, J1c3c, J1c3, J1c2 (all unrelated).
Therefore, 37.5% of the ME/CFS patients with an ill family member were J1c’s.

Maternal haplogroup J is known for reduced OXPHOS capacity (5), partially uncoupled mitochondria (7) lower maximal oxygen uptake (VO2max), lower efficiency of electron
transport chain (ETC), and diminished ATP and ROS production (4, 10). J1C’s defining variant 14798T>C reduces the coupling efficiency of mitochondrial OXPHOS by one
third and proportionately increase heat generation (26).

If there was no genetic component to ME/CFS in these families with multiple cases, then we would expect maternal haplogroup J1c’s to appear at the same frequency as
the general population.

One of the additional variants needed to change a J1 into a J1c (a defining variant) is 14798T>C. There is a significant association between J1c’s with variant 14798T>C and
several diseases, including:
- increased penetrance of Multiple Sclerosis (25), and
- accelerated progression of AIDS (7).

Researchers have found that being maternal haplogroup J1c plus an additional mitochondrial variant(s) can cause diseases categorized as Mitochondrial Diseases/Disorders.
(1 , 2, 8, 9).
Haplogroup J1 + extra variant 14484T>C (ND6 gene) = significantly higher penetrance of LHON.
Haplogroup J1c + extra variant 11778G > A (ND4 gene) = significantly higher penetrance of LHON.
Haplogroup J1c1 + extra variants 7226G>A (MT-CO1), 7747C>T (MT-CO2), 13143T>C (MT-ND5), and 16209T>C (MT-DLOOP) = higher penetrance of macular
degeneration
Haplogroup J1d1a + extra variants 2305T>C (unique, MT-RNR2) and 10654C>T (A-V, MT-ND4L) = higher penetrance of macular degeneration, and
Haplogroup J1c7 + extra variants 6734G>A (syn, MT-CO1), 12072T>C (F-S, MT-ND4) and 16209T>C (non-coding) = higher penetrance of macular degeneration.
Chapter 2: Hypometabolic State
The high frequency of J1c’s among families with a history of ME/CFS is further evidence that the hypometabolic state is a component of ME/CFS. Yet, just because a
person is a J1c does not mean that they’ll get ME/CFS. There is a large population of J1c’s in the world that is extremely healthy. However, if one is a J1c, and then has a
significant stressor, that might be just enough to push him/her over the threshold into ME/CFS. Such hits (or significant triggers) could be the addition of another
deleterious mtDNA variant(s), the addition of a nuclear variant(s), an environmental trigger (such as mtDNA damage from the Gulf War or chemotherapy or exposure to
deleterious chemicals, a severe infection, Lyme disease, mononucleosis, etc…), or a combination of factors. See Chapters 19-25 for a discussion of the hunt for the extra
mtDNA variant(s) in J1c’s pushing some over the threshold into ME/CFS.

J’s are also known to have higher methylation levels (6, 14). Methylation is known to increase in mammals during hypometabolism (22). Methylation level has also been
found to be significantly higher in autistic children (15) . Haplogroup J approximately doubles the rate of autistic spectrum disorder (ASD) (28). It makes me wonder what
the frequency of J1c’s in ASD is, especially when there is a family history of ASD.
Chapter 3: Long Lives
Mammals that go into states of hypometabolism generally live longer. There are scientists researching how to trigger the hypometabolic state in order to lengthen life.
“The signaling and regulatory mechanisms described in this review suggest the blueprint for transitioning into a hypometabolic state ….. They illustrate the
intricacies of regulatory responses to nutrient and environmental stresses and the wide-ranging interactions that have to be considered when planning a
coordinated suppression of cellular metabolism that achieves homeostasis and life extension over long periods of time spent in dormancy. “ (29)

Studies show that maternal haplogroup J’s live longer.

“J is over-represented in long-lived people and centenarians from several populations (De Benedictis et al., 2003 and Ross et al., 2001). The mtDNA control region
variant that defines Caucasian haplogroup J (mtDNA 295C/T) has been found to change mitochondrial transcription and copy number (Suissa et al., 2009). Cybrids
containing haplogroup J mtDNA had a greater than 2-fold increase in mtDNA copy number compared with cybrids containing haplogroup H mtDNA. This is one of the
few examples demonstrating functional consequences for a polymorphism underlying a specific haplogroup. In this case, the impact of the haplogroup J regulatory
region mutation on mtDNA replication or stability may partially account for population-based observations that haplogroup J is associated with human longevity.”
(18)
Chapter 4: Surviving Harsh Conditions
Entering a hypometabolic state generally confers a survival advantage in harsh conditions (29). During periods of harsh environments, humans with DNA variants that
resulted in different types and degrees of hypometabolism (with advantages for the specific type of environmental condition at hand) would have had a survival advantage.
Over thousands of years of such conditions (such as during ice ages or megadroughts), multiple pro-“hypometabolic state” variants could accumulate in a family’s
haplogroup.

Not surprisingly, J1c’s appeared approximately 16,500 years ago (20, 34) -- smack in the middle of the last ice age when having hypometabolism could significantly increase
survival. While being a J by itself already provided some pro-hypometabolic advantages, J1c’s upped the hypometabolism by adding in variant 14798T>C.

Not only did J1c’s appear on the stage during the ice age, but they also multiplied and prospered. By the time the ice age ended and the Neolithic period began, J1c’s
were in a position to spread across Europe, where their population exploded. Today, J1c’s make up over three quarters of all J lineages (20, 34).

Being a J1c is a positive. J1c’s on average live longer and during ice ages they are more likely to hunker down and survive the harsh conditions. Anne Wojcicki, co-founder
and chief executive officer of 23andMe, is a J1C. It’s only when an extra item(s) is added that J1c’s start to have health issues.
Chapter 5: Eradicate the word “lazy”
Researchers found that among 299 species of North American bivalves and gastropods, higher metabolic rates were a reliable “predictor of extinction likelihood” (35).
Therefore, lower metabolic rates were an indicator of species survival. Sadly, the study was marketed to the media with the headline “survival of the laziest” (36) and the
media ran with it. Thousands of news articles ran with titles such as: “To avoid extinction it's about 'survival of the laziest,' study suggests” CNN (37) and “‘Survival of the
laziest’: Finally, there’s a scientific reason to not get off the couch” in the Washington Post (38). Yet, the word “lazy” or “laziest” does not appear in the research paper.

The poor choice of the word “laziest” implies that animals in hypometabolism have a choice about needing to rest and conserve energy. The word lazy is judgmental of a
state of lower energy availability and usage based on a complicated biochemical process. Individuals with ME/CFS have heard the word lazy bantered around by ignorant
people (including medical professionals) and received judgmental stares when having to do such things as lay down on public benches to rest. When given enough energy,
mammals will be active. Being on the spectrum of hypometabolism is not lazy, it’s not a choice, and my hope is that researchers looking into metabolism will in the future
refrain from using the word “lazy” or “laziest” from marketing gimmicks.

There is a desperate need to educate health care professionals and the media regarding hypometabolism. As someone in a hypometabolic state, I can choose to try to live
at society’s pace of activities and expectations and increase my odds of disease and symptoms, or I can live at the pace my body sets for me. Do people view polar bears in
hibernation or queen bees settling in under a rock for a six month rest as lazy? No. This is the same reason individuals with ME/CFS should not be viewed as lazy either.

My children and I have to live at the pace set by each of our metabolisms on any given day.
As J1c’s, we have 14798T>C.
As J1c1’s, we have 3394T>C which dbSNP (42) calls “Pathogenic” and MSeqDR (41) calls “affects function”.
In addition, we have other mtDNA variants that other J1C1’s do not have (SNP’s that do not define the haplogroup, a.k.a private variants). One of these
private variants that we have, according to MitImpact (39), is predicted “deleterious” by PROVEAN, CADD, CAROL, COVEC_WMV, and MtoolBox; is
predicted “probably damaging” by PolyPhen2, and is predicted “disease causing” by PhD-SNP, SNAP, and Meta-SNP. See Chapter 20 for a further
discussion of this variant.

Do we know all the answers? No. But we know more today than yesterday.

With limited energy, taking care of necessities (doctors appointments and food) takes precedence over cleaning the house and attending the local “block party”. To others,
we appear as if we should be able to do more than we do. Our house is the one on the street where the grass is often too long because finding someone in our house with
energy to mow the lawn is not easy. It gets done eventually, but it’s not up to society’s standards.

Educating the media and health care workers will help all of us with ME/CFS to live better lives and accept the things we can’t change. Imagine if therapists coming out of
college were trained to support individuals with ME/CFS to live at their own pace and helped them to come to terms with not trying to live up to societies’ expectations. It
will happen.

OK, I’ve wandered off track here. Back to the findings.

Chapter 6: Maternal Haplogroup U5’s
While maternal haplogroup J1c’s were found at 5.4x the expected frequency in individuals with family histories of ME/CFS (and related symptoms), J1c’s were not the only
haplogroup found at a higher frequency than the general population. The maternal haplogroup U5 was found at 2.7x the expected frequency in individuals with family
histories of ME/CFS (and related symptoms). (See below purple box for a special calculation of U5 that was needed.) U5’s are also known for reduced OXHPOS capacity
and mitochondrial uncoupling (7, 11). And just like J1c’s, U5’s appeared during the ice age (43) where entering a hypometabolic state could confer a survival
advantage under such harsh conditions.
 (43)

U5’s (especially when coupled with the 15218A>G variant) are known to have a significant accelerated progression of AIDS (similar to J1c’s with 14798T>C). (7). The study
found:
“MtDNA haplogroups J and U5a were elevated among HIV-1 infected people who display accelerated progression to AIDS and death. ….. The U5a haplogroup is
associated with accelerated AIDS progression to CD4<200 in European Americans (RH=1.78, 95% CI=1.11–2.85, p=0.028) and in MSM pooled cohorts (RH=2.06, 95%
CI=1.17–3.63, p=0.024). The signal is largely driven by the U5a1-15218 haplotype, which comprises 79% of the U5a haplotype.”

The above mentioned 15218A>G variant is one of the two variants needed to go from a U5a to a U5a1. One of the two study participants who was a U5 was a U5a1a1. The
other was a U5b2b.
Chapter 7: The Tighter the Criteria, the Stronger the Link
The more family members with ME/CFS and the closer the family link, the more J1c’s and U5’s in the subset.

“ME/CFS+ families” are defined in Part 2 of this book.

Does this mean that if you are a mother with ME/CFS with a child with ME/CFS and another child with either ME/CFS or related health issues that you will turn out to be
from maternal haplogroup J1c? No. This was a small study. It showed the connection with J1c’s, but there are likely other haplogroups and variant combinations that are
going to also cause ME/CFS.

Do I believe J1c’s will continue to have the highest rate of ME/CFS in mother plus multiple children out of the haplogroups? Yes. While these are eye opening results, it’s
just a start. Further research needs to be done in a controlled setting.
Part 2: Putting It Together
Chapter 8: My Unusual Family

Up until two years ago, our family had been fighting with schools districts for five generations over excessive sick absences. That is until our family was taken seriously by
a respected Mitochondrial Clinic.

On the left below is a New York City newspaper clipping from the early 1900’s where my great-grandfather was taken to court because my great-aunt (7 years old at the
time) missed too much school. In a family with many advanced degrees, we take education very seriously. Our children do not miss school for no reason.

We are blessed to now have excellent nurses throughout our school district. However, just a few years ago when our youngest was in kindergarten, the school nurse at her
elementary school told others that our daughter was a hypochondriac coming into the nurses office one day with a limp, another day with a headache, another day with a
muscle cramp in a leg, another day with severe stomach issues, and still other days with severe fatigue.

Thank goodness we know from our mitochondrial doctor that having such a broad assortment of symptoms is not unusual in patients with mitochondrial issues. The
specialists at the hospital found her severe constipation was due to her colon muscles not working properly, and her mitochondrial headaches were aggravated further by a
Chiari malformation in her brain. They taught her how to use crutches on days when her foot doesn’t work properly and she’s received multiple instructions on how to pace
herself throughout the day to conserve energy.

When the nurse that thought our daughter was a hypochondriac and who hounded me over every sick day retired, we were blessed with a kind, thoughtful, and well
educated nurse at that school. She worked with us and our doctors to provide the best school environment for our daughter under the daily changing symptoms.
Chapter 9: More Unusual Families

Attending both ME/CFS conferences and also UMDF (United Mitochondrial Disease Foundation) conferences, I noticed that there was a subset of families at both
conferences that resembled our family pattern. At the ME/CFS conferences, some of the mothers I met also had a child(ren) with ME/CFS and/or a son with autistic
spectrum disorder (ASD).

At the UMDF conferences, I also noticed a subset of attendees followed a similar pattern. For example, at one UMDF regional meeting, I sat at a table with a mother whose
teen daughter had mitochondrial disease. During the morning conference the teen’s head was on the table or propped up on her arms (elbows on table). When the mom
got up from the table, she had a noticeable limp (like my intermittent foot drop) and seemed to be a little off kilter. She had no diagnosis, but always wondered if her
health issues were related to her daughter’s. Then she told me about her son with Asperger’s.

Was there a common denominator between the subset of families with similar health patterns attending the ME/CFS conferences and the one’s attending the mitochondrial
disease conferences?
Chapter 10: ME/CFS+ Families
I’m going to use “ME/CFS+” to refer to families where there are multiple health issues in a family where at least one member has ME/CFS. One person in the family will
have ME/CFS, but at least one other close family member will have ME/CFS, POTS, Autistic Spectrum, exercise intolerance, gastroparesis, Multiple Sclerosis, Fibromyalgia,
Crohn’s, early-onset Alzheimer’s, Hashimoto’s, Mast Cell Disorder, Ehlers-Danlos Syndrome, other autoimmunity symptoms, or another usual symptom (such as a family
member needing a pacemaker at 40 years old because his heart is just not beating fast enough).

There is not 100% penetrance of any one symptom in our large family. What we have is more than our fair share of health issues. For example, not every male offspring in
our ME/CFS+ family (mom’s family line) is on the autistic spectrum. However, male offspring in our ME/CFS+ family do have high functioning autistic spectrum at a much
higher frequency than the general population.

In the article “Revealing the Complexity of Mitochondrial DNA-Related Disorders” (51), the authors talk about the complexity and difference in symptoms between patients.
“The effects of mutations which affect the respiratory chain may therefore be multisystemic, with possible involvement of visual and auditory pathways, heart,
central nervous system and skeletal muscle. The “red flags” are myopathy with exercise intolerance, eyelid ptosis, ophthalmoparesis, axonal neuropathy,
sensorineural hearing loss, pigmentary retinopathy, optic neuropathy, diabetes mellitus, hypertrophic cardiomyopathy, migraine, short stature (DiMauro et al.,
2013).”

It is common knowledge, that when a patient has symptoms in three or more body systems or organs, that mitochondrial disease should be suspected (52, 53, 54).

The above mentioned article (51), further adds:

“For the above reported reasons, studying genotype-phenotype relationship in mitochondrial disorders is a complex task. The clinical variability is large even in
individuals with the same genotype and the statistical power is low in single-center studies given the rarity of these conditions. In order to better define the
clinical phenotypes associated with mtDNA mutations, there is a strong need of nation-wide, and even international, studies on large cohorts of patients.”

Study participants came from two different ME/CFS international online support boards. The three ME/CFS+ patients with more than one sick child and who happened to be
J1c’s, live in Australia, New Zealand, and the United States.
Chapter 11: ME/CFS is extremely complex. Where does one begin?
Dr. M. Cristina Kenney had a similar question when studying macular degeneration. Her solution was to simplify the system. The next two figures are similar to the one’s
Dr. Kenney used in her work (45). If you are not familiar with her work, please read both articles on that footnoted link. Her work with cybrids is fascinating, and an
area of opportunity needing to be further explored in ME/CFS. Here are my versions of two of Dr. Kenney’s tables -- tweaked for ME/CFS.
My study was different than past studies, in that:

1) This study focused in on patients with ME/CFS who had a close relative with ME/CFS or a related symptom. Therefore, if there was an increase in the
frequencies of maternal haplogroups known to reduce OXPHOS in ME/CFS patients, then it was more likely to be found by looking at families with a history
of ME/CFS+ than in the general ME/CFS population where individuals could have ME/CFS instead due to mitochondrial damage from the Gulf War,
chemotherapy, etc…..

2) This study wasn’t limited to looking at the top layer of haplogroups, but instead got into the details of the subclades
Part 3: J1c1’s as the example
Chapter 12: Why are some J1c families very healthy, while others have multiple generations that have severe health issues?

To find the candidate mtDNA variants associated with ME/CFS, the subclade J1c1’s will be used as the example. However, this process can be used with other J1c subclades
and other haplogroups. But first -- a little background.

RESEARCHERS may want to skim from here to the end of Chapter 16. New important information begins at Chapter 17.
Chapter 13: J1c1’s Maternal Haplogroup Family Tree

With each progression along the family tree, mtDNA changes happen that get passed along to future generations. For example, while J1c1’s showed up around 10,000 years
ago, they still carry the variant changes that occurred over 50,000 years ago at nucleotide positions 73, 263, 2706, 7028, 11719, and 14766.
Chapter 14: Haplogroup J Variants
The most thorough source identifying the variants in each haplogroup that I’ve found is on the “mvTool for PhyloTree and Haplogroup” (33) created by MSeqDR. Just
click on the footnote number and link, click on the box for “Haplogroup”, go down to J and push enter, and up will pop all of the variants needed in total to be a J
(including those carried over from the upper tree branches). If you then put your cursor in the “Haplogroup” box on J1, you’ll see the extra variants needed to go from J to
J1 are C462T and G3010A.
In Figure 7 above, I’ve included columns for where MSeqDr (41) noted a variant as “Affects Function” or dbSNP has red flagged a variant as “pathogenic”. However, relying
on one or two sources is not going to give you the full picture of a variant.

For example, earlier I mentioned that variant 14798T>C is known to reduce coupling efficiency of mitochondrial OXPHOS by one third and proportionately increasing heat
generation (26). I also mentioned how it is associated with several diseases, including:
- increased penetrance of Multiple Sclerosis (25), and
- accelerated progression of AIDS (7).
Chapter 15: DNA Research Data Tsunami
The T14798C variant is the third one from the bottom on Figure 7. You’ll notice, MSeqDR (41) has it listed as “Effect Unknown” and dbSNP (48) doesn’t have any notes on it
at all. The reason that this additional information is not in MSeqDR’s database (nor in any other database) is because we are in the midst of a ……………...

The data is coming in faster than anyone can enter it into any of the databases. Nobody has staffs large enough to keep up with the pace of new genetic discoveries. Newly
discovered associations and interactions between variants are growing and becoming more complex. New sub-routine programs need to be written to pick up on all the
various potential interactions, including the different haplogroup interactions that are being discovered. Until that’s done, researchers are doing a lot by hand – including
having to search multiple databases and sources because nobody has it all in one place. Eventually, it will be automated and easy. But we’re not there yet.

I updated Figure 7 for the additional research and it appears in the orange box in Figure 9 below.
Chapter 16: Updated Haplogroup J Variants

You’ll notice the farther down into the J1c1 haplogroup one goes, the more potentially deleterious mtDNA variants are added. That’s not the case with all haplogroups.
J1c1 has more mtDNA variants that “Affects Function”, are “Pathogenic or Likely Pathogenic”, and have known disease associations, than most haplogroups. This can add
to the threshold effect.

In the article “Revealing the Complexity of Mitochondrial DNA-Related Disorders” (51), the authors talk about this:
“Each cell contains multiple copies of mtDNA (polyplasmy), which in healthy individuals are identical to one another (homoplasmy). Heteroplasmy refers to the
coexistence of two populations of mtDNA, normal and mutated. Mutated mtDNA in a given tissue has to reach a minimum critical number before oxidative
metabolism is impaired severely enough to cause dysfunction (threshold effect). The pathogenic threshold varies from tissue to tissue according to the relative
 dependence of each tissue on oxidative metabolism (DiMauro et al., 2013). Differences in mutational load surpassing the pathogenic threshold in some tissues but
not in others may contribute to the heterogeneity of phenotypes.”

Just because a person is maternal haplogroup J1c1 does not mean that person is going to get ME/CFS or have unusual health issues.
Chapter 17: Comparing a healthy J1c1 family with a ME/CFS+ J1C1 family

Wouldn’t it be nice to have a healthy J1c1 family to compare with my ME/CFS+ J1C1 family? Well, coincidentally I do.

While my mother’s family line with all the ME/CFS, autistic spectrum, POTS, migraines, gastroparesis, intermittent foot drop, heart issues, and other health issues is
maternal haplogroup J1c1, my father’s family line’s is also J1c1. Yet, dad’s line doesn’t have these health issues. My father’s family’s claim to fame is that they often live
to be over 100 years old and the women in the line have a significant risk of macular degeneration.

It’s not quite a lightning strike, but the chance of someone being the child of a father and mother (who are unrelated) who are both J1c1’s is 1 in 30,000. Therefore, I’ve
been given a great seat from which to study the differences in two completely opposite (health wise) J1c1 families.

The first issue I looked at was LHON. I’m warming you up for the really exciting stuff, so please bear with me here. Neither family line had a single case of LHON. But if
we did, per the earlier discussion, we’d expect to find either a 14484T>C (ND6 gene) variant or an 11778G > A (ND4 gene), as we fit the initial parameter of being J1’s
(linked to 14484) and J1c’s (linked to 11778). (1 , 2). Therefore, this first issue matched the research. Being J1c1’s, we do not have the extra variants for LHON and we
don’t have any LHON in either branch of the family.
Chapter 18: J1c1’s and Macular Degeneration

The only health issue on my father’s J1c1 branch is that >50% of the female relatives have macular degeneration. Yet, there is not one case of macular degeneration on my
mother’s side. My father’s mother (my paternal grandmother) had macular degeneration that began in her 40’s. Her mother also had macular degeneration. One of my
father’s two sisters also has macular degeneration.

In the study “Inherited mitochondrial DNA variants can affect complement, inflammation and apoptosis pathways: insights into mitochondrial–nuclear interactions” (9), the
researchers stated:
“Sequencing data suggest that observed effects are not due to rare mtDNA variants but rather the combination of SNPs representing the J versus H
haplogroups. These findings represent a paradigm shift in our concepts of mt–nuclear interactions.”
…..
“The entire mtDNA from each of the cybrids were sequenced and compared with the Cambridge Reference Sequence. SNPs we define as unique are not
listed in www.MitoMap.org. Private SNPs are those that do not define the haplogroup. The J cybrids were classified into J1d1a, J1c1 and J1c7. There were
a total of nine private SNPs in the J cybrids: J1d1a cybrid with m.2305T>C (unique, MT-RNR2) and m.10654C>T (A-V, MT-ND4L); J1c1 cybrid with
m.7226G>A (syn, MT-CO1), m.7747C>T (syn, MT-CO2), m.13143T>C (syn, MT-ND5) and m.16209T>C (non-coding); and J1c7 cybrid with m.6734G>A
(syn, MT-CO1), m.12072T>C (F-S, MT-ND4) and m.16209T>C (non-coding).”

When we take dad’s mtDNA variants and subtract out all the variants that J1c1’s have to have in order to be J1c1’s (defining variants) (33), the one’s left are: 7226G>A (MT-
CO1), 7747C>T (MT-CO2), 13143T>C (MT-ND5), and 16209T>C (MT-DLOOP). They are the exact same one’s mentioned in combination with the J1c1 haplogroup above with
regards to macular degeneration. Mom’s J1c1 line does not carry any of these four additional variants.

So again the researchers were correct. J1c1 plus certain variants = higher penetrance of macular degeneration in dad’s side of the family, while J1c1 plus none of
those variants = no macular degeneration in mom’s side.

(8)
Chapter 19: J1c1’s with ME/CFS+
Since J1c1 + extra variants = significantly higher penetrance of LHON, and since J1c1 + other variants = higher penetrance of macular degeneration, could it be that on my
mother’s side of the family the ME/CFS+ is caused by J1c1 plus an additional mtDNA variant(s) that dad’s healthy side doesn’t have?

Focusing on mtDNA makes sense due to the significant maternal inheritance pattern in our family going back 5 generations and running along 3 different female inheritance
lines from my great-grandmother. My grandmother and two of her sisters have female lines still alive today with unusual health issues.

Is there a deleterious variant(s) that when added to our J1c1 family = increased penetrance of ME/CFS+? Below is the spreadsheet of my mtDNA variants.

“Defining variants” define the haplogroup. You need the have the defining variants to be included in a haplogroup. “Private variants” are additional variants that a family
line has beyond the haplogroup’s defining variants. In two families with the same haplogroup (one with ill family members and the other with healthy family members), the
private variants might hold the answer to the mystery.

When we take our mtDNA variants (me, my mother, and my children) and subtract out all the J1c1 defining variants (33), the extra variants that are left (that are not
required to be a J1c1) are: 9448A>G (MT-CO3) and 14290T>C (MT-ND6). Dad’s healthy J1c1 line does not carry either of these two additional variants.

The question then becomes whether the family’s ME/CFS+ health issues are caused by being:
- J1c1 + 9448A>G, or
- J1c1 + 14290T>C, or
- J1c1 + 9448A>G + 14290T>C, or
- J1c1 + something else?

Here is what I know….

Chapter 20: mtDNA Variant 9448A>G (MT-CO3)
Per GeneDx mtDNA variant analysis, I have: Position 9448, MT-CO3, A>G, TAC>TGC, p.Y81C, Frequency Gen. Pop. 15/32059 (0.05%).

Having experienced errors in genetics sequencing in the past (and because I’m a data geek), I’ve doubled back and confirmed 9448A>G with my GeneDx mtDNA variant
table, and two VCFs from different labs (one lab did WES and the other WGS), and also confirmed this position on my mother’s WES VCF. [The mtDNA VCF data had to first
be re-aligned to the correct positions on NC_01290 by re-aligning 9449 to position 9448. The GeneDx mtDNA analysis spreadsheet was correctly aligned to NC_01290 and did
not need re-alignment.]

MitoMap’s “point mutations” search (68) for variant 9448 returned:

Per MitoMap: “The current GB frequency data is derived from two sets of human mitochondrial sequences from GenBank:
46,092 full length (FL) sequences (>15.4 kb)
70,175 control region only (CR) sequences (0.4-1.6 kb)”

MitoMap’s SNV Query (69) for variant A9448G returned:

I want to point your attention to that last column that shows: 9448A>G was found to have a 97.78% conservation rate.

While GeneDx’s analysis put this variant on a table they sent to me titled “Benign and Likely Benign Variants”, GeneDx most likely was not taking into consideration that we
are J1c’s and things impact us differently than other haplogroups. I believe the reason GeneDx didn’t red flag this is due to that tsunami of data research results that has
yet to make its way through the system. Eventually, GeneDx and the other genetic analysis companies will more fully take haplogroups into consideration.

People who don’t have the J1c research studies in their heads, might look at variant 9448A>G and rely on GeneDx’s comment of “benign and likely benign” to be the final
word. But you can’t do that with J1c’s.

Variant 9448A>G is found in all individuals who are maternal haplogroup K1a4b (33). Presuming that variant 9448A>G is found to not be deleterious in haplogroup K1a4b’s,
that does not relieve researchers from looking into whether 9448A>G is deleterious in J1c’s because J1c’s do not react the same to many mtDNA variants.

Many variants have been found to be deleterious to haplogroup J1c’s that are not deleterious to non-J haplogroups. For example, J1c’s react deleteriously to variants
14484T>C (ND6 gene), 11778G > A (ND4 gene), 7226G>A (MT-CO1), 7747C>T (MT-CO2), 13143T>C (MT-ND5), 16209T>C (MT-DLOOP (1 , 2), 6734G>A (syn, MT-CO1), and
12072T>C (F-S, MT-ND4) (8, 9), while non-J haplogroups do not have the same reaction to these variants.
Chapter 21: A variant can be disease causing in one haplogroup and not in another haplogroup
Researchers followed through with a study to confirm that maternal haplogroups impact manifestation. Specifically, they followed up on the LHON study that found being
maternal haplogroup J1c plus 14484T>C (ND6 gene) causes significantly higher penetrance of LHON (1 ). The follow-up study found that “Low penetrance of the 14484 LHON
mutation when it arises in a non-haplogroup J mtDNA background”. (2)

Both the mutation and the haplogroup were necessary for manifestation of the disorder. Therefore, in Haplogroup J1c (where there is already reduced OXPHOS capacity
(5), partially uncoupled mitochondria (7) lower maximal oxygen uptake (VO2max), lower efficiency of electron transport chain (ETC), and diminished ATP and ROS
production), the addition of other deleterious mtDNA variants can significantly increase the risk of disease even when no increase is seen in other haplogroups.

That means one must analyze what is known about any private mtDNA variants in J1c’s with a family history of ME/CFS+. For me, that means finding out what researchers
already know about 9448A>G (MT-CO3), beginning with checking out what the prediction databases forecast.
Chapter 22: Looking up variants in the prediction databases
There are a dozen applications that predict whether a variant is deleterious. These databases employ different metrics and selection criteria in making their predictions.
Some put more weight on conservation while others put more weight on annotations. Each one has unique advantages that could pick-up different clues as to deleterious
nature. You could go to each application and look up the variant, but I suggest first checking out MitImpact (39).

It’s my understanding that mito experts who do the leg work when researching a variant will put the results of their searches of these prediction databases into MitImpact.
Therefore, if 9448A>G is already in the database, they will have saved me a lot of work. They share this information with the world.

Have I mentioned how much I love that these researchers openly share? This unique ability to check egos at the door, working together, and bouncing ideas around makes
the ME/CFS and mitochondrial research networks very unique and well positioned for finding answers more quickly.

In entering 9448 into MitImpact, I found that someone had already run the prediction analysis for 9448A>G (MT-CO3). The predictions are:
- “deleterious” by: PROVEAN, CADD, CAROL, COVEC_WMV, and MtoolBox.
- “probably damaging” by: PolyPhen2.
- “disease causing” by PhD-SNP, SNAP, and Meta-SNP.

The issues J1c’s already have with OXPHOS plus the multiple predictions of “deleterious, probably damaging, and disease causing” plus the 97.78% conservation
rate, this gives a significant amount of weight to the theory that:
J1c1 + 9448A>G = significantly higher penetrance of ME/CFS+
Chapter 23: Other interesting tidbits about 9448A>G
9448A>G does not yet have a snp (rs#).

9448A>G is located very close (10 bases) from pathogenic variant rs267606611 9438G>A (LHON) (55). Other nearby pathogenic variants include: rs587776437 9478T>C (30
bases) (Leigh Disease) (56) and rs267606612 9487_9501del (39 bases) (57). This last one is on MitoMap’s list of mtDNA variants believed to cause mitochondrial myopathy
(last variant on Figure 11 – below).

There is growing evidence of a relationship between exercise intolerance and mitochondrial variants. The below list of exercise intolerance & mitochondrial myopathy
variants is based on a section of Mitomap’s “Clinical Phenotypes (non-LHON) Associated with mtDNA Polypeptide Gene Mutations Reported in the Literature” (58).
Chapter 24: Mitomap’s List of Provisional Variants Related to Exercise Intolerance and/or Mitochondrial Myopathy

Please visit MitoMap’s original list. There is a lot more information there and it will continue to be updated (58). When visiting MitoMap’s original list, be sure to page
down to the footnotes. That alone is worth skimming. With study titles such as: "Exercise intolerance due to mutations in the cytochrome b gene of mitochondrial DNA”
(59) and “Exercise intolerance due to a nonsense mutation in the mtDNA ND4 gene” (60), who could stop themselves from reading further. (Yes, my inner geek is showing.)

Remember that Mitomap is under the same data tsunami as others. They do not have every mtDNA variant reported to impact exercise intolerance on the list. For example,
variant 9952 is not on the list with regards to exercise intolerance (61). I cannot emphasize enough -- go to more than one source. Remember that data tsunami and how
nobody has a database that includes it all.

The only study I could find that mentioned the 9448A>G variant was the study titled: “Targeted Sequencing of the Mitochondrial Genome of Women at High Risk of Breast
Cancer without Detectable Mutations in BRCA1/2” (62). Table 2 from the study is titled “Description of variants predicted as deleterious by SIFT and as probably damaging
by Polyphen”. Included in that list of variants predicted as deleterious by SIFT and as probably damaging by Polyphen is Position. 9448 – Ref. A, Alt. G, Mitomap Known,
Counts 1, Conserved Yes, Gene MT-CO3, Codon Change tAc/tGc, a.a. ChangeY/C

According to GenBank Record for Coding Polymorphism A-G at rCRS position 9448 (71), there are 18 individuals in GenBank with the 9448 variant. 15 of the 18 are
haplogroup K1a’s. I presume that means they are K1a4b’s since that variant is a defining variant for K1a4b. Not one of the 18 is from haplogroup J. Therefore, out of
1165 haplogroup J’s in GenBank, not one of the J’s has the variant 9448A>G that my ME/CFS+ family has.

The combination of the 9448A>G variant in my J1c1 family with extensive ME/CFS+ in 3 living female inherited family lines tracing back to a great-grandmother 5
generations ago is looking like the culprit. My father’s healthy J1c1 family does not have this variant.

Now to take a peek at that second private variant in our maternal line.
Chapter 25: mtDNA Variant 14290T>C (MT-ND6)
This one does not have quite as many red flags as 9448A>G did. Yet, there is enough here to question whether 14290T>C has an impact on ME/CFS+ and should be looked
into further.

Here is the little I know on this one. Per GeneDX mtDNA variant analysis, I have: Position 14290, MT-ND6, T>C, GAA>GAG, p.E128E, Frequency Gen. Pop. 37/32059
(0.12%). I triple confirmed this position from my GeneDx mtDNA variant table, with two VCFs from different labs (one lab did WES and the other WGS), and also confirmed
this position with my mother’s WES VCF.

While GeneDx’s analysis put this variant also on the table titled “Benign and Likely Benign Variants”, since J1c’s are more sensitive to mtDNA variants than most
haplogroups, further research needs to be done on the variant.

MitoMap’s “point mutations” search (68) for variant 14290 returned:

Per MitoMap: “The current GB frequency data is derived from two sets of human mitochondrial sequences from GenBank:
46,092 full length (FL) sequences (>15.4 kb)
70,175 control region only (CR) sequences (0.4-1.6 kb)”

MitoMap’s SNV Query (69) for variant T14290C returned:

14290T>C was found to have a 31.11% conservation rate.

14290T>C is rs879023200 (63). It’s located very close (11 bases) to pathogenic variant rs869025187 (14279G>A) (LHON) (64).

14290T>C (MT-ND6) is not on MitImpact (39). However, 14290T>A and 14290T>G are both there. The predictions for both of these are:
- “deleterious” by: FatHmm, CAROL, and MtoolBox.
- “probably damaging” by: PolyPhen2.

Therefore, it doesn’t look to be as strong of a contender as 9448A>G, but then with mtDNA variants, it’s often a combination resulting in a threshold effect.

According to “GenBank Record for Coding Polymorphism T-C at rCRS position 14290” (66), there are 56 individuals in GenBank with the variant at 14290. Only 1 of the 56 is
a haplogroup J and that person is a J1c. There are a total 1165 individuals from haplogroup J on GenBank and only 1 has the 14290 variant. The 0.09% frequency is the
lowest found among the haplogroups. If the variant is not compatible with J’s already reduced OXPHOS capacity, that could be the reason.

14290 is a defining variant for haplogroup A12. 100% of A12’s will have a 14290 variant. There are a few haplogroups with a high percentage of this variant in their
populations, including: 40% of R31a’s and 40% of M24a’s.

In summary, variant 14290T>C cannot yet be ruled out as contributing to ME/CFS+ in J1c1’s who also have private variant 9448A>G.
Part 4: Other Factors Impacting Mitochondrial Energy
While the relationship of mtDNA haplogroup J1c was easy (relatively) to find among the individuals with ME/CFS+ families, it’s much more complex for non-J’s. Every
person with ME/CFS is a unique puzzle. Looking at the mtDNA variants in one’s haplogroup and subclade for any trouble makers is a good start. A next step is to examine
the private mtDNA variants (an individual’s mtDNA variants minus the haplogroup’s defining variants).

For each private variant, what is the research on these? What do the prediction applications say? For some people the main puzzle piece will lie in the mtDNA (haplogroup
or private variants or combination).

However, only a small percentage of those with ME/CFS will find their main puzzle piece to lie in the maternal haplogroup.

Many individuals with ME/CFS do not have any close relatives with ME/CFS, nor any close relatives with unusual health issues. Some will find a pathogenic variant that
might explain the ME/CFS, but many others won’t. Therefore, it’s important to look at the other pieces that can impact mitochondrial energy, including nuclear gene
variants and environmental triggers (such as Gulf War, chemotherapy, radiation exposure, viral, infection, etc….).

In the review “Mitochondrial DNA Damage and its Consequences for Mitochondrial Gene Expression” (81), the researchers wrote that:
“Since mtDNA replication is dependent on transcription, mtDNA damage may alter mitochondrial gene expression at three levels: by causing DNA
polymerase γ nucleotide incorporation errors leading to mutations, by interfering with the priming of mtDNA replication by the mitochondrial RNA
polymerase, or by inducing transcriptional mutagenesis or premature transcript termination.
……….
Chemicals in the environment, metabolites of dietary components, drugs in clinical therapies, and radiation from sunlight or medical procedures are
external sources of DNA damage. The benzo[a]pyrene and acrolein components of cigarette smoke, the fungal toxin aflatoxin B1, platinum-based
chemotherapy agents, antiviral nucleoside analogs, and ultraviolet (UV) radiation all induce DNA adducts that inhibit mitochondrial transcription or
interfere with DNA pol γ function.”
Chapter 26: Nuclear Gene Variants
While most people tend to think mitochondrial disease is caused by variants in mtDNA, more variants deleterious to mitochondrial metabolism are housed in the nuclear
genes than in the mtDNA.

In fact, MSeqDr “variant lookup by gene” (41) lists 1,607 genes being watched at the moment by the mito experts with regards to mitochondrial metabolism. Since human
mtDNA is a double-stranded, circular molecule with only 37 genes (28 on the H-strand and 9 on the L-strand), the majority of those genes being watched by the mito experts
are contained on 1,570 of the nuclear genes.

The inheritance pattern of mitochondrial diseases via nuclear DNA is more often autosomal recessive (needing two variants), rather than dominant (needing only one). This
makes the combinations of variants needed to cause disease harder to find because there won’t likely be a family history of the symptoms. For example, one could inherit a
deleterious variant from a mother and a deleterious variant from a father, and theoretically get ME/CFS without there ever being a family history. However, it’s likely to
be far more complex than that with multiple unique variant combinations causing ME/CFS in many individuals.

In a family like mine with the strong family history of ME/CFS+, it’s easier to pick out the outlier symptoms in a few people and try to figure out if there is a genetic cause.
At the same time, our symptoms even within our family are very heterogeneous between family members. None of us have carbon copy symptoms of the other.

A few symptoms occur only in a few family members. Take for example, my mother who has had sensorineural hearing loss that began in her 30’s. My brother’s became
noticeable in his 40’s. Nobody else in our family has this symptom. So what is different about them?

Sensorineural hearing loss is generally inherited in an autosomal recessive pattern. The most common cause is having two deleterious variants on the GJB2 gene (72).
Having homozygous variants on the TRMU gene has also been known to cause sensorineural hearing loss (73).

Both my mother and brother carry one variant on the GJB2 gene and another variant on the TRMU gene. Nobody else carries either of these two variants in our family, just
the two with sensorineural hearing loss.
- nuclear gene GJB2 variant rs35887622 (74) (101T>C) (dbSNP has it listed as “Likely Pathogenic, Pathogenic Allele”). 0.85% allele frequency.
- nuclear gene TRMU variant rs11090865 (75) (DNA change c.28G>T). 7.49% allele frequency. This variant is believed by researchers to be a mitochondrial
modifier in deafness.

Both variants are listed in MSeqDR.org’s database of variants reported by mitochondrial experts (41). But they are not believed to cause sensorineural hearing loss when
there is just one on a gene. Therefore, the question we are left with is: Is my mother’s and my brother’s sensorineural hearing loss due to one of these variants (or both)
plus our unique J1c1 + private variants? What we do know is that for those of us without these variants, we do not get sensorineural hearing loss.

From this, you can see how complex it is in a family that is already fully aware of the deleterious nature of their J1c1 defining variants plus their private mtDNA variants.
Trying to un-ravel the exact combination of which variant combinations in individuals who do not have a family history of ME/CFS is going to be an even greater challenge.
Other factors that can impact whether one gets ME/CFS are mitochondrial damage, other nuclear variants, and other environmental factors).

In the meantime, the research being done by Robert Naviaux, MD and Ronald W. Davis, PhD on the biochemical pathways and markers in individuals with ME/CFS and their
corresponding studies on hypometabolism in ME/CFS may provide treatments long before such combination of deleterious variants are weeded out for the majority of
individuals with ME/CFS. The good news is that being able to track a subset of the cases back to mtDNA variants provides additional clues that will aid researchers.
Chapter 27: Mitochondrial Damage
I view all the study snippets that I’ve included below as clues (puzzle pieces). Sometimes when they fit together I form a theory. Other times, I make note of the fit and
see if another piece comes along to fit in with the grouping. There are a lot of puzzle pieces that are grouped together under “items that cause mitochondrial damage”.
Just like mtDNA deleterious variants, mitochondrial damage can cause similar symptoms. Below are a few of the items known to cause mitochondrial damage.

For those who don’t have mtDNA deleterious variants, the answer to your ME/CFS could lie in mitochondrial damage. For those with mtDNA deleterious variants, anything
that causes mitochondrial damage can move one along the spectrum of hypometabolism and increase symptom strength and/or bring on new symptoms. We are the
canaries in the coal mine.

Damage due to Medicines

Damage to the mitochondria can occur from many items, including the medicines we take.
“Damage to mitochondria is now understood to play a role in the pathogenesis of a wide range of seemingly unrelated disorders such as schizophrenia, bipolar
disease, dementia, Alzheimer's disease, epilepsy, migraine headaches, strokes, neuropathic pain, Parkinson's disease, ataxia, transient ischemic attack,
cardiomyopathy, coronary artery disease, chronic fatigue syndrome, fibromyalgia, retinitis pigmentosa, diabetes, hepatitis C, and primary biliary cirrhosis.
Medications have now emerged as a major cause of mitochondrial damage, which may explain many adverse effects. All classes of psychotropic drugs have been
documented to damage mitochondria, as have statin medications, analgesics such as acetaminophen, and many others.” (76).

Damage due to Particulate Matter in the Air

What we breathe into our lungs matters. In this study, 63 healthy male steel workers from Italy were studied to determine if particulate matter from the job damaged their
mitochondria on the 1st and the 4th days of the work week.
“PM (particulate matter) exposure is associated with damaged mitochondria, as reflected in increased MtDNAcn. Damaged mitochondria may intensify oxidative-
stress production and effects.” (78).

In this study, “Cigarette toxicity triggers Leber’s hereditary optic neuropathy by affecting mtDNA copy number, oxidative phosphorylation and ROS detoxification pathways”
(79), the researchers found:
“Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting
Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors.
We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also,
environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship
between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the
lowest mtDNA content in affected individuals…….. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected
individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that
modulate LHON penetrance must take into account also the exposure to environmental triggers such as tobacco smoke.”

Damage due to Gulf War Environment

The 2017 study: “Role of mitochondrial DNA damage and dysfunction in veterans with Gulf War Illness” (80) found that:
“The clinical presentation of GWI is comparable to that of patients with certain mitochondrial disorders–i.e., clinically heterogeneous multisystem
symptoms. ….veterans with GWI exhibit greater mtDNA damage which is consistent with mitochondrial dysfunction.”

Since mtDNA damage due to any means can potentially give the same symptoms as mtDNA deleterious variants, it’s understandable why individuals with Gulf War Illness
often fall under the umbrella of ME/CFS. The question then arises whether individuals with haplogroups known for reduced oxygen uptake (such as maternal haplogroups
J1c’s), whether those individuals have an increased risk of Gulf War triggering their ME/CFS or making it worse. mtDNA damage could be the item that puts an individual
over the threshold of becoming symptomatic.

Damage from Chemotherapy

In the review “Chemotherapeutic Drugs and Mitochondrial Dysfunction: Focus on Doxorubicin, Trastuzumab, and Sunitinib” (82), the authors stated that:
“Many cancer therapies produce toxic side effects whose molecular mechanisms await full elucidation. ….. Chemotherapeutic-induced adverse side effects
are, in many cases, mediated by mitochondrial damage. In particular, trastuzumab and sunitinib toxicity is mainly associated with mitochondria
impairment and is mostly reversible. Vice versa, doxorubicin-induced toxicity not only includes mitochondria damage but can also lead to a more robust
and extensive cell injury which is often irreversible and lethal. Drugs interfering with mitochondrial functionality determine the depletion of ATP reservoirs
and lead to subsequent reversible contractile dysfunction. Mitochondrial damage includes the impairment of the respiratory chain and the loss of
mitochondrial membrane potential with subsequent disruption of cellular energetic. In a context of increased stress, AMPK has a key role in maintaining
energy homeostasis, and inhibition of the AMPK pathway is one of the proposed mechanisms possibly mediating mitochondrial toxicity due to
chemotherapeutics. Therapies targeting and protecting cell metabolism and energy management might be useful tools in protecting muscular tissues
against the toxicity induced by chemotherapeutic drugs.”

Damage from Radiation

In the study, “Severe mitochondrial damage associated with low-dose radiation sensitivity in ATM- and NBS1-deficient cells” (83), the authors found that:
“Low-dose radiation risks remain unclear owing to a lack of sufficient studies. We previously reported that low-dose, long-term fractionated radiation (FR) with
0.01 or 0.05 Gy/fraction for 31 d inflicts oxidative stress in human fibroblasts due to excess levels of mitochondrial reactive oxygen species (ROS). To identify
the small effects of low-dose radiation, we investigated how mitochondria respond to low-dose radiation in radiosensitive human ataxia telangiectasia mutated
(ATM)- and Nijmegen breakage syndrome (NBS)1-deficient cell lines compared with corresponding cell lines expressing ATM and NBS1. Consistent with previous
results in normal fibroblasts, low-dose, long-term FR increased mitochondrial mass and caused accumulation of mitochondrial ROS in ATM- and NBS1-
complemented cell lines. Excess mitochondrial ROS resulted in mitochondrial damage that was in turn recognized by Parkin, leading to mitochondrial autophagy
(mitophagy).”

Damage from Viruses

On the ME/CFS support boards, there is a lot of talk about when people had their first symptom and what they think triggered it. Many ME/CFS patients believe their first
ME/CFS symptoms can be traced back to a severe bout of mononucleosis (caused by the Epstein Barr Virus). Now the researchers are starting to catch up. This study’s title
has the potential answer -- “Preliminary evidence of mitochondrial dysfunction associated with post-infective fatigue after acute infection with Epstein Barr virus”
(84). The study reports that:
“Acute infectious diseases are typically accompanied by non-specific symptoms including fever, malaise, irritability and somnolence that usually resolve on
recovery. However, in some individuals these symptoms persist in what is commonly termed post-infective fatigue. The objective of this pilot study was to
determine the gene expression correlates of post-infective fatigue following acute Epstein Barr virus (EBV) infection.
….
Results: Those who developed post-infective fatigue had gene expression profiles indicative of an altered host response during acute mononucleosis compared to
those who recovered uneventfully. Several genes including ISG20 (interferon stimulated gene), DNAJB2 (DnaJ [Hsp40] homolog and CD99), CDK8 (cyclin-dependent
 kinase 8), E2F2 (E2F transcription factor 2), CDK8 (cyclin-dependent kinase 8), and ACTN2 (actinin, alpha 2), known to be regulated during EBV infection, were
differentially expressed in post-infective fatigue cases. Several of the differentially expressed genes affect mitochondrial functions including fatty acid
metabolism and the cell cycle.”

From the review article “Viruses as Modulators of Mitochondrial Functions” (85), that appeared in the journal Advances in Virology:
“This makes them (the mitochondria) a target for all the invading pathogens including viruses. Viruses either induce or inhibit various mitochondrial
processes in a highly specific manner so that they can replicate and produce progeny. Some viruses encode the Bcl2 homologues to counter the proapoptotic
functions of the cellular and mitochondrial proteins. Others modulate the permeability transition pore and either prevent or induce the release of the
apoptotic proteins from the mitochondria. Viruses like Herpes simplex virus 1 deplete the host mitochondrial DNA and some, like human immunodeficiency
virus, hijack the host mitochondrial proteins to function fully inside the host cell. All these processes involve the participation of cellular proteins,
mitochondrial proteins, and virus specific proteins.”

If you are interested in how different viruses impact the mitochondria, I highly recommend that article. As to Epstein Barr, the authors write:
“Epstein-Barr virus (EBV) causes increased oxidative stress in the host cells within 48 hrs. During the lytic cycle indicating the role of ROS in virus release.
Oxidative stress activates the EBV early gene BZLF-1, which causes the reactivation of EBV lytic cycle. This has been proposed to play an important role in
the pathogenesis of EBV-associated diseases including malignant transformations.”

I want to call your attention to a section of the snippet above where the authors wrote about “Viruses …. like human immunodeficiency virus, hijack the host mitochondrial
proteins to function fully inside the host cell.” Then put that together with this.

In this study titled “Mitochondrial DNA Haplogroups influence AIDS Progression” (7), the human immunodeficiency virus progressed to AIDS and death fast in maternal
haplogroups U5’s and J’s.
“an interesting trend was observed that uncoupled haplogroups with lower ATP and ROS production (U5 and J) are associated with accelerated disease, whereas
more tightly coupled groups (H3 and H4, H5, and H6) are associated with protection, suggesting mitochondrial functional variation plays a role in AIDS progression.
Combined data on longevity, neurodegenerative disease susceptibility, sperm motility, sprint performance, and climate adaptation suggests functional mtDNA
variation in different haplogroups influences ATP production efficiency, and correlated ROS and heat generation. Less efficient ATP production in partially
uncoupled mitochondria (haplogroups J and U5) would accelerate AIDS because it would exacerbate the energetic effects of the mtDNA depletion, disruption
of oxidative phosphorylation, antioxidant enzyme deficiency, apoptosis, and increased oxidative damage observed during AIDS progression.”

Therefore, starting off with a mito issue (such as being a J1c or a U5, or mitochondrial damage), even if one is below the threshold for showing any symptoms – if we
combine that with another mito stressor(s), it can have deleterious consequences.

Here is a study on mice that found a: “Critical Role of Mitochondrial Damage in Determining Outcome of Macrophage Infection with Mycobacterium tuberculosis” (77).
Specifically, they found that:
“These experiments clearly demonstrate that massive mitochondrial damage is caused by Mtb (Mycobacterium tuberculosis infection) in presence of RR that leads
to loss of DiOC6 retention as the consequence of MPT and irreversible ΔΨm loss. A23187 prevents mitochondrial damage and loss of dye retention.
…..
Similar results were obtained in electron microscopic studies that assessed mitochondrial swelling. Mφ incubated with Mtb and RR for 4 days show significant
mitochondrial swelling indicating mitochondrial membrane damage, whereas uninfected Mφ andMφ treated with RR alone contained normally sized
mitochondria”.

A virus that causes mitochondrial damage could explain the outbreaks of ME/CFS (such as the large ME/CFS outbreak in Australia in the 1950’s).

Post Polio fatigue is very similar to ME/CFS and some individuals have been diagnosed with both polio and ME/CFS. In this study titled “The Major Apoptotic Pathway
Activated and Suppressed by Poliovirus” (90), they link the infection with the polio virus to mitochondria.
“Cells respond to poliovirus infection by switching on the apoptotic program, implementation of which is usually suppressed by viral antiapoptotic functions. We
show here that poliovirus infection of HeLa cells or derivatives of MCF-7 cells was accompanied by the efflux of cytochrome c from mitochondria. ….. Both
caspase-9 and caspase-3 were shown to be essential for the development of such hallmarks of virus-induced apoptosis as chromatin condensation, DNA degradation,
and nuclear fragmentation. These and some other results suggest the following scenario. Poliovirus infection activates the apoptotic pathway, involving
mitochondrial damage, cytochrome c efflux, and consecutive activation of caspase-9 and caspase-3.”

In our family there is a spectrum of how the mitochondrial symptoms will display. For some in our family there has to be a stronger hit for the issue to register symptoms.
Take for example my sister who has been healthier than I over her lifetime. Looking back over her life, there are times where we can clearly see her symptoms going over
the mitochondrial dysfunction threshold. Cleveland Clinic’s site states that:
“Mononucleosis, caused by the Epstein-Barr virus, is not considered a serious illness; however, its symptoms may be severe enough to prevent a person from
engaging in normal activities for several weeks.” (137)

Yet, for people who are already starting with reduced OXPHOS capacity (5), partially uncoupled mitochondria (7) lower maximal oxygen uptake (VO2max), lower efficiency
of electron transport chain (ETC), and diminished ATP and ROS production (4, 10), a hit like mononucleosis that is associated with mitochondrial dysfunction can be a
debilitating punch. When my sister came down with mono, rather than it preventing her from engaging in normal activities for several weeks, it took her out of commission
for an entire year. She missed so much school that the school district sent a tutor to our home to help her keep up with her classes. Months later when she was able to
resume some activities, she continued to have brutal fatigue and had trouble keeping up. It took her a full year to recover from her case of mono.

This brings me to the polio-like illness that has been plaguing children in recent years. Could it be that the virus causing this illness is also a virus that hits the mitochondria
hard? Could it be that the children who experience the foot drop or more severe paralysis are more likely to have reduced mitochondrial functioning to begin with and the
virus puts them over the threshold?

Not everyone in our family gets intermittent foot drop, but for those who do it happens in three distinct ways. Take me for example. Sometimes I’m walking normally and
all of a sudden my foot doesn’t have any strength (but I don’t yet know it). I’ll go to take the next step and I fall. (Once it happened on the steps while carrying out the
recycling glass and I ended up with 15 stitches on my thumb and later two nerve grafts.) At other times, it’s a consistent limp I’ll have for a few weeks. And still on other
days when I’ve not paced my energy usage, the limp will pop up later in the day. But for those of us who have had this symptom occur in our family, it doesn’t come on
until well passed our teen years.

Yet, when the polio-like virus in children was making news in our state a few years ago, our daughter who was around 5 years old at the time had a virus that resulted in
post-virus severe fatigue and a foot drop limp. The limp still comes and goes depending upon many factors from recent illness to energy usage and pacing. And like me,
there are times where she just wakes up and the foot just doesn’t want to work correctly.

I’d like to see studies on whether children with severe post-mono fatigue and also on children who develop the new viral polio-like paralysis, whether these children are
more likely to come from haplogroup subclades known for higher rates of mitochondrial dysfunction than the general population. If this is the case, then testing whether
the mito cocktail (CoQ10, vitamin B2, etc…) helps with symptoms and course might be prudent.

There is a lot of talk on the ME/CFS support boards about people who cured themselves of ME/CFS. I believe that in some of these cases, the patients may be doing things
that improve their mitochondrial function and move them over that threshold line to the non-symptomatic side. Some of these patients later return to the support boards
and call it a relapse of ME/CFS. I believe in some of these cases the patients have had more mitochondrial function hits and are back over the threshold into showing
symptoms.
Damage from Infections
On the ME/CFS support boards, there is also a lot of talk about ME/CFS being triggered by an infection. There are several ME/CFS patients that believe their first ME/CFS
symptom can be traced back to a severe case of Lyme Disease (due to the bacterium Borrelia burgdorferi). Again, the researchers are starting to catch up. In this study
(124), the researchers found that:
“These results indicate that there is an imbalance of reactive oxygen species and cytosolic calcium in Lyme borreliosis patients. The results further suggest that
oxidative stress and interrupted intracellular communication may ultimately contribute to a condition of mitochondrial dysfunction in the immune cells of
Lyme borreliosis patients.”
Chapter 28: Environmental Triggers

Why are ME/CFS patients often more hypersensitive to environmental triggers than others?
Many of us in the ME/CFS community are like the canaries in the coal mines, alerting others to poisons in our environment. For many of us, our bodies overreact more so
than other people’s to scents, particles in the air, what we eat, changes in temperature, and the lotions we put on our skin. I went through a stage in my teens and
twenties where my POTS and allergies and fatigue were triggered by the smallest contact with chemicals. Eventually I became an organic vegan for a year because that was
the only item my over-reactive body would not over-react to. Being an organic vegan for that year seemed to reset my system and ever since I’m not nearly as chemically
hypersensitive. I believe being vegan took where I was on the spectrum of chemical sensitivity and tapered it down. Now when I’m getting sick or not pacing my energy
through the day, that hypersensitivity will rear its head. It’s still there, just below threshold much of the time. Not everyone in our family is sensitive to chemicals.

In this study from 1994 titled “Comparison of Patients With Chronic Fatigue Syndrome, Fibromyalgia, and Multiple Chemical Sensitivities (MCS)” (108), the researchers
found:
“Overall, the three patient groups were remarkably similar in demographic characteristics and the presence of specific symptoms. Patients with CFS and FM
frequently reported symptoms compatible with MCS. Likewise, 70% of patients with FM and 30% of those with MCS met the criteria for CFS.”

Study: “The Background of Mitochondrial DNA Haplogroup J Increases the Sensitivity of Leber's Hereditary Optic Neuropathy Cells to 2,5-Hexanedione Toxicity”
(86).
“Leber's hereditary optic neuropathy (LHON) is a maternally inherited blinding disease due to mitochondrial DNA (mtDNA) point mutations in complex I subunit
genes, whose incomplete penetrance has been attributed to both genetic and environmental factors. Indeed, the mtDNA background defined as haplogroup J is
known to increase the penetrance of the 11778/ND4 and 14484/ND6 mutations. ….. In conclusion, this study shows that mtDNA haplogroups modulate the
response of LHON cells to 2,5-HD. In particular, haplogroup J makes cells more sensitive to its toxic effect. This is the first evidence that an mtDNA
background plays a role by interacting with an environmental factor and that 2,5-HD may be a risk element for visual loss in LHON. This proof of principle has
broad implications for other neurodegenerative disorders such as Parkinson's disease.”

Study: “Haplogroup J mitogenomes are the most sensitive to the pesticide rotenone: Relevance for human diseases” (87).
“There is growing evidence that the sequence variation of mitochondrial DNA (mtDNA), which clusters in population- and/or geographic-specific haplogroups,
may result in functional effects that, in turn, become relevant in disease predisposition or protection, interaction with environmental factors and ultimately
in modulating longevity. To unravel functional differences between mtDNA haplogroups we here employed transmitochondrial cytoplasmic hybrid cells (cybrids)
grown in galactose medium, culture condition that force oxidative phosphorylation, and in the presence of rotenone, the classic inhibitor of respiratory Complex I.
Under this experimental paradigm we assessed functional parameters such as cell viability and respiration, ATP synthesis, reactive oxygen species production and
mtDNA copy number. Our analyses show that haplogroup J1, which is common in western Eurasian populations, is the most sensitive to rotenone, whereas K1
mitogenomes orchestrate the best compensation, possibly because of the haplogroup-specific missense variants impinging on Complex I function. Remarkably,
haplogroups J1 and K1 fit the genetic associations previously established with Leber's hereditary optic neuropathy (LHON) for J1, as a penetrance enhancer, and
with Parkinson's disease (PD) for K1, as a protective background. Our findings provide functional evidences supporting previous well-established genetic
associations of specific haplogroups with two neurodegenerative pathologies, LHON and PD. Our experimental paradigm is instrumental to highlighting the
subtle functional differences characterizing mtDNA haplogroups, which will be increasingly needed to dissect the role of mtDNA genetic variation in health,
disease and longevity.

Haplogroup J’s are just showing that when there is a previous mitochondrial issue, we truly can be supersensitive to these chemicals. We are not imagining that our bodies
overreact to these chemicals, we actually do. And I presume that it’s not just haplogroup J’s. I theorize that people with all different combinations of variants that cause
mitochondrial metabolism issues that results in ME/CFS and that people who have damaged mitochondria who have symptoms of ME/CFS – that in all of us when we have low
energy in certain areas of the body or brain can be at an increased risk for hypersensitivity to our environment.
Part 5: Take a Deeper Dive
Chapter 29: Hypometabolic State
Per the medical dictionary, a hypometabolic state is:
“a rare state of reduced metabolism with symptoms resembling hypothyroidism but with some tests for thyroid gland function normal; also used to describe the
reduced metabolic activity seen in true hypothyroidism.” (13)

Per the article: “Regulation of hypometabolism: insights into epigenetic controls”,

“For many animals, survival of severe environmental stress (e.g. to extremes of heat or cold, drought, oxygen limitation, food deprivation) is aided by entry into a
hypometabolic state. Strong depression of metabolic rate, often to only 1–20% of normal resting rate, is a core survival strategy of multiple forms of
hypometabolism across the animal kingdom, including hibernation, anaerobiosis, aestivation and freeze tolerance.” (21)

Just as there are different types of hypometabolism, each type operates along a spectrum. There are those that are on the extreme end with 1-20% of normal resting rate.
Then there are some people with ME/CFS who try to function with their normal temperatures being lower by a degree, their stomachs and colons operating more slowly
(gastroparesis and constipation), malaise, foggy headed, and just feeling off. For this group, they can have normal days or hours and they can also have crazy hours, days,
weeks, and years when an organ, body system, or entire system goes haywire. For many of us, it’s about pacing so as not to jump further into hypometabolism. Loving the
days when energy is abundant and on the bad days asking God “Why me?”

Per Robert K. Naviaux’s 2016 study “Metabolic features of chronic fatigue syndrome”:
“We report that targeted, broad-spectrum metabolomics of plasma not only revealed a characteristic chemical signature but also revealed an unexpected
underlying biology. Metabolomics showed that chronic fatigue syndrome is a highly concerted hypometabolic response to environmental stress that traces to
mitochondria and was similar to the classically studied developmental state of dauer. This discovery opens a fresh path for the rational development of new
therapeutics and identifies metabolomics as a powerful tool to identify the chemical differences that contribute to health and disease.
……….
Pathway abnormalities included sphingolipid, phospholipid, purine, cholesterol, microbiome, pyrroline-5-carboxylate, riboflavin, branch chain amino acid,
peroxisomal, and mitochondrial metabolism. …… Many of the pathways and metabolites that were abnormal in CFS are also known to be features of dauer, a well-
studied, long-lived survival and persistence state triggered by environmental stress. …… These facts suggest that CFS is an evolutionarily conserved, genetically
regulated, hypometabolic state similar to dauer that permits survival and persistence under conditions of environmental stress but at the cost of severely
curtailed function and quality of life.” (17)

Alzheimer’s Disease and Hypometabolism in the Brain

Study “Hypometabolism as a therapeutic target in Alzheimer's disease” (88).
“Another characteristic of AD (Alzheimer’s Disease) is regional hypometabolism in the brain. This decline in cerebral glucose metabolism occurs before
pathology and symptoms manifest, continues as symptoms progress, and is more severe than that of normal aging.”

From the review “Mitochondrial DNA mutations in neurodegeneration”:

“Unlike PD or AD in which observable mitochondrial respiratory chain dysfunction or hypometabolism occurs, there have been relatively few biological
observations suggestive of mitochondrial dysfunction in ostensibly sporadic FTD or ALS.” (125).

J’s and Alzheimer’s Disease

A 2012 study found haplogroup J was associated with decline in cognitive function compared to haplogroup H.
“Mitochondrial dysfunction is a prominent hallmark of Alzheimer's disease (AD). Mitochondrial DNA (mtDNA) damage may be a major cause of abnormal
reactive oxidative species production in AD or increased neuronal susceptibility to oxidative injury during aging. ….. We first investigated the role of common
mtDNA haplogroups and individual variants on dementia risk and 8-year change on the Modified Mini-Mental State Examination (3MS) and Digit Symbol Substitution
Test (DSST) among 1,631 participants of European genetic ancestry. Participants were free of dementia at baseline and incidence was determined in 273 cases from
hospital and medication records over 10-12 follow-up years. Participants from haplogroup T had a statistically significant increased risk of developing dementia (OR
= 1.86, 95% CI = 1.23, 2.82, p = 0.0008) and haplogroup J participants experienced a statistically significant 8-year decline in 3MS (Modified Mini-Mental State
Examination)( β = -0.14, 95% CI = -0.27, -0.03, p = 0.0006), both compared with common haplogroup H.
………
Haplogroup J is defined by variants in ND5 and HV2. The HV2 variant that defines haplogroup J, m.295C>T, has been shown to change mitochondrial
transcription and copy number. Cytoplasmic hybrids (cybrids) containing haplogroup J mtDNA had a greater than 2-fold increase in mtDNA copy number
compared with cybrids containing haplogroup H mtDNA. This is one of the few examples demonstrating functional consequences for a variant underlying a
specific haplogroup and is of particular interest since haplogroup J is over-represented in long-lived people and centenarians from several populations. In
this case, the impact of the haplogroup J regulatory region mutation on mtDNA replication or stability may partially account for population-based
observations that haplogroup J is associated with human longevity.”(67)

Per the study titled “Association between mitochondrial DNA variations and Alzheimer's Disease in the ADNI cohort” (91)
“Mitochondrial malfunction can alter the delicate bioenergetics balance making neuronal cells vulnerable to challenge (Wallace et al., 2010). Mitochondrial
haplogroups and polymorphisms have also received substantial consideration in psychiatric and neurodegenerative diseases (Tuppen et al., 2009). Mitochondrial
associations were found between AD and haplogroup J with increased susceptibility, while haplogroup T was found to have protective effect (Chagnon et al.,
1999).”

The Brain as we Age

Many study participants have the symptom of brain fog. Others had relatives with early-onset Alzheimer’s or Dementia. Per the study “Mitochondrial function in ageing:
coordination with signalling and transcriptional pathways” (12):
“Mitochondrial dysfunction entailing decreased energy-transducing capacity and perturbed redox homeostasis is an early and sometimes initiating event in ageing
and age-related disorders involving tissues with high metabolic rate such as brain, liver and heart. In the central nervous system (CNS), recent findings from our and
other groups suggest that the mitochondrion-centred hypometabolism is a key feature of ageing brains and Alzheimer's disease. This hypometabolic state is
manifested by lowered neuronal glucose uptake, metabolic shift in the astrocytes, and alternations in mitochondrial tricarboxylic acid cycle function.
Similarly, in liver and adipose tissue, mitochondrial capacity around glucose and fatty acid metabolism and thermogenesis is found to decline with age and is
implicated in age-related metabolic disorders such as obesity and type 2 diabetes mellitus. These mitochondrion-related disorders in peripheral tissues can impact
on brain functions through metabolic, hormonal and inflammatory signals. At the cellular level, studies in CNS and non-CNS tissues support the notion that instead
of being viewed as autonomous organelles, mitochondria are part of a dynamic network with close interactions with other cellular components through energy- or
redox-sensitive cytosolic kinase signalling and transcriptional pathways. Hence, it would be critical to further understand the molecular mechanisms involved in the
communication between mitochondria and the rest of the cell. Therapeutic strategies that effectively preserves or improve mitochondrial function by targeting key
component of these signalling cascades could represent a novel direction for numerous mitochondrion-implicated, age-related disorders.”

U5’s and Alzheimer’s Disease

The study “Analysis of European Mitochondrial Haplogroups with Alzheimer Disease Risk” (97) found:
“Additionally, the frequency of haplogroup U was found to be greater in stroke patients and has been associated with elevated risk for migrainous occipital stroke.
Further analysis demonstrated that the association was due to a high frequency of subcluster U5. Given the diverse nature of the U haplogroup and its
apparent association with a related neurologic disease, we suggest that variants within the different subclusters of U may predispose individuals to AD risk.”
Chapter 30: Higher methylation fits in with hypometabolic state
Wikipedia describes DNA methylation as:
“DNA methylation is a process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the
sequence. When located in a gene promoter, DNA methylation typically acts to repress gene transcription. DNA methylation is essential for normal development and
is associated with a number of key processes including genomic imprinting, X-chromosome inactivation, repression of transposable elements, aging and
carcinogenesis.” (126).

DNA methylation is used for several important purposes. For one, it is used to turn sections of genes into the “off position” (think epigenetics) in order to control gene
expression. DNA methylation has been linked to several human diseases. It also enables the suppression of retroviral genes. Like with all body systems and functions it is
there for our good, but also can be deleterious under certain circumstances.

Maternal haplogroup J has higher methylation levels than other cybrids. Per the 2012 study titled: “Global DNA methylation levels are modulated by mitochondrial DNA
variants” found:
“The analysis revealed that methylation levels were higher in the subjects carrying the J haplogroup than in non-J carriers. Consistently, cybrids with
J haplogroup mtDNA showed higher methylation levels than other cybrids. Interestingly, we observed overexpression of the MAT1A gene and low ATP and
ROS levels in J cybrids. Conclusion: Our findings indicate that mtDNA-specific interactions between mitochondria and the nucleus regulate epigenetic
changes, possibly by affecting oxidative phosphorylation efficiency.” (6)

The 2015 study titled “Mitochondrial DNA variants can mediate methylation status of inflammation, angiogenesis and signaling genes” found that cybrids with mtDNA
derived from haplogroup J had consistently higher levels of global ncDNA methylation than haplogroup H, suggesting that mtDNA variants may play an important role
in influencing ncDNA methylation.
“In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various
signaling pathways, which are important in several common diseases.” (14)

The higher rate of methylation among J’s fits in with the hypometabolic state theory. Methylation is known to increase in mammals during hypometabolism. In the study
“Global DNA modifications suppress transcription in brown adipose tissue during hibernation”:
“Hibernation is crucial to winter survival for many small mammals and is characterized by prolonged periods of torpor during which strong global controls
are applied to suppress energy-expensive cellular processes. ….. A direct modification of DNA during torpor was revealed by a 1.7-fold increase in global
DNA methylation during long term torpor as compared with euthermic controls.” (22).

Wikipedia defines torpor as:

“Torpor is a state of decreased physiological activity in an animal, usually by a reduced body temperature and metabolic rate. Torpor enables animals to
survive periods of reduced food availability. The term "torpor" can refer to the time a hibernator spends at low body temperature, lasting days to weeks, or
it can refer to a period of low body temperature and metabolism lasting less than 24 hours, as in "daily torpor".” (23)

Methylation level has been found to be significantly higher in autistic children (15). With the high rates of autistic spectrum in both the mitochondrial disease community
and the ME/CFS community, this higher level of methylation related to the hypometabolism is an important factor.

When Dr. Naviaux was asked by the Open Medicine Foundation:

“Q5. How do the metabolic changes you identified in CFS relate to the recent interest in epigenetics and methylation pathways?
Dr. Naviaux. “All the covalent chemical modifications of DNA and histones that regulate gene expression are the result of metabolic changes controlled by
mitochondria. ….. All of these metabolites that regulate epigenetics and gene expression are controlled primarily by mitochondrial metabolism. This
makes sense because all cellular activities must be responsive to local resource availability and remain flexible to respond to potential threats that alter
cellular health, and mitochondria are the prime monitors and regulators of cellular metabolism.” (16)
Chapter 31: OXPHOS

Per the textbook “Biochemistry” (92),

“Oxidative phosphorylation is the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH 2 to O 2 by a series of electron carriers.
This process, which takes place in mitochondria, is the major source of ATP in aerobic organisms.”

Per Wikipedia (93),

“Oxidative phosphorylation is the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby releasing energy which is used to produce adenosine
triphosphate (ATP). In most eukaryotes, this takes place inside mitochondria. Almost all aerobic organisms carry out oxidative phosphorylation. This pathway is
probably so pervasive because it is a highly efficient way of releasing energy, compared to alternative fermentation processes such as anaerobic glycolysis.”

Per the article “Is There a Link between Mitochondrial Reserve Respiratory Capacity and Aging?”,
“Oxidative phosphorylation is an indispensable resource of ATP in tissues with high requirement of energy. If the ATP demand is not met, studies suggest that this
will lead to senescence and cell death in the affected tissue. The term reserve respiratory capacity or spare respiratory capacity is used to describe the amount of
extra ATP that can be produced by oxidative phosphorylation in case of a sudden increase in energy demand. Depletion of the reserve respiratory capacity has been
related to a range of pathologies affecting high energy requiring tissues.” (31)

In this study that is looking at cellular bioenergetic abnormalities in CFS patients, they found some interesting results regarding key parameters of OXPHOS, especially
maximal respiration (3):
“Results showed consistently lower measures of OXPHOS parameters in PBMCs taken from CFS patients compared with healthy controls. Seven key parameters of
OXPHOS were calculated: basal respiration, ATP production, proton leak, maximal respiration, reserve capacity, non-mitochondrial respiration, and coupling
efficiency. While many of the parameters differed between the CFS and control cohorts, maximal respiration was determined to be the key parameter in
mitochondrial function to differ between CFS and control PBMCs due to the consistency of its impairment in CFS patients found throughout the study (p≤0.003). The
lower maximal respiration in CFS PBMCs suggests that when the cells experience physiological stress they are less able to elevate their respiration rate to
compensate for the increase in stress and are unable to fulfil cellular energy demands. The metabolic differences discovered highlight the inability of CFS patient
PBMCs to fulfil cellular energetic demands both under basal conditions and when mitochondria are stressed during periods of high metabolic demand.”
(129, 130, 131, 132, 133)
Chapter 32: J1c’s reduced OXPHOS capacity

The study “Oxidative phosphorylation differences between mitochondrial DNA haplogroups modify the risk of Leber's hereditary optic neuropathy” found that individuals
from haplogroup J1C had reduced OXPHOS capacity. Specifically, they stated:
“Our results confirm that the mtDNA levels determine, in a large part, the oxidative phosphorylation (OXPHOS) capacity of the cell and that, if the lower
amount of mtDNA in haplogroup J and Uk cybrids mirrors an individual's situation, then this lower amount might be responsible for higher susceptibilities of
individuals from these haplogroups to develop LHON.”
…..
“Because glycolytic enzymes are nDNA-encoded, this result also suggests that OXPHOS differences due to mtDNA SNPs can influence the expression of
nuclear genes, as has been shown previously by others.”
…..
“Cybrids from haplogroup Uk and haplogroup J contain less mtDNA than those from haplogroup H. Considering that the nuclear genetic background and
culture conditions are the same in all these cybrids, the differences between them must be due to particular mtDNA SNPs. Because none of the mtDNA
SNPs examined were in sequences involved in mtDNA replication and because mtDNA encodes only OXPHOS proteins, it is likely that the lower mtDNA
amounts we observe are the result of an indirect OXPHOS effect due to other mtDNA SNPs.”
…..
“Our results suggest that individuals from haplogroups Uk, J1c and J2 are more susceptible to LHON because they have reduced OXPHOS capacity,
which results in part from lower mtDNA levels.” (5)

There is the mention of cybrids again. I’d love hear about the progress being made in ME/CFS+ using cybrids. Another topic to put on my research wish list.

This article gets into the details on the OXPHOS in J’s and Uk’s.
“Haplogroup J then splits into to sub-haplogroups J1 and J2, each defined by a major cytb mutation. …. the J1 cytb mutation is at np 14798C (F18L). ….
The np 15,257 and np 14,789 variants alter well-conserved amino acids with CIs of 95% and 79%, respectively. …. while the np 14,798 site alters the inner
coenzyme Q binding site (Qi) of complex III (194). Since the Qo and Qi binding sites are essential for complex III proton pumping via the Q-cycle, the np
14,798 and 15,257 variants are both likely to have disconnected electron flow through complex III with proton pumping. This would reduce the coupling
efficiency of mitochondrial OXPHOS by one third and proportionately increase heat generation.
…..
Some of these same mtDNA lineages have also been found to be protective against neurodegenerative diseases. Haplogroups J and Uk are underrepresented
in Parkinson disease (PD) (225) and haplogroup T is underrepresented in AD patients (32, 224). The repeated association of haplogroups J1 and Uk with
longevity and neuro-protection is particularly illuminating because both haplogroups encompass the same cytb mutation at np 14,798. Such convergent
evolution provides strong support for the functional importance of the cytb mutations.” (26)
Chapter 33: Mitochondrial Uncoupling
The above quote (in Chapter 32) talks about reduced coupling efficiency. Reduced mitochondrial coupling (ATP/O2 [P/O]) is associated with hypometabolism.

Back in 1973, researchers knew much less about mitochondrial coupling.

“Coupling factors are defined as being materials of mitochondrial origin that are essential for the process of energy conservation to function. The mitochondrial
membrane loses its ability to synthesize ATP when the coupling factor is extracted from the membrane.” (94)

Just by getting older, there is more uncoupling. This study’s title gives it away: “Exercise efficiency is reduced by mitochondrial uncoupling in the elderly” (96).

In this study, they discuss reduced coupling and biochemical coupling efficiency. Efficiency is the name of the game during periods of harsh stresses (such as an ice age).
“During evolution, mitochondrial DNA haplogroups of arctic populations may have been selected for lower coupling of mitochondrial respiration to ATP
production in favor of higher heat production. We show that mitochondrial coupling in skeletal muscle of traditional and westernized Inuit habituating northern
Greenland is identical to Danes of western Europe haplogroups. Biochemical coupling efficiency was preserved across variations in diet, muscle fiber type, and
uncoupling protein-3 content. Mitochondrial phenotype displayed plasticity in relation to lifestyle and environment. Untrained Inuit and Danes had identical
capacities to oxidize fat substrate in arm muscle, which increased in Danes during the 42 days of acclimation to exercise, approaching the higher level of the Inuit
hunters. A common pattern emerges of mitochondrial acclimatization and evolutionary adaptation in humans at high latitude and high altitude where
economy of locomotion may be optimized by preservation of biochemical coupling efficiency at modest mitochondrial density, when submaximum
performance is uncoupled from VO2max and maximum capacities of oxidative phosphorylation.” (32)

(128)
Chapter 34: Mitochondrial Coupling
Since mitochondrial uncoupling is a negative for individuals with ME/CFS, could mitochondrial coupling be a mechanism for improvement in some?

From the study, “Increased ER-mitochondrial coupling promotes mitochondrial respiration and bioenergetics during early phases of ER stress” (95).
“Increasing evidence indicates that endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response (UPR), but that beyond a certain
degree of ER damage, this response triggers apoptotic pathways. The general mechanisms of the UPR and its apoptotic pathways are well characterized.
However, the metabolic events that occur during the adaptive phase of ER stress, before the cell death response, remain unknown. Here, we show that,
during the onset of ER stress, the reticular and mitochondrial networks are redistributed towards the perinuclear area and their points of connection are
increased in a microtubule-dependent fashion. A localized increase in mitochondrial transmembrane potential is observed only in redistributed
mitochondria, whereas mitochondria that remain in other subcellular zones display no significant changes. Spatial re-organization of these organelles
correlates with an increase in ATP levels, oxygen consumption, reductive power and increased mitochondrial Ca² ⁺ uptake. Accordingly, uncoupling of
the organelles or blocking Ca² ⁺ transfer impaired the metabolic response, rendering cells more vulnerable to ER stress. Overall, these data
indicate that ER stress induces an early increase in mitochondrial metabolism that depends crucially upon organelle coupling and Ca² ⁺ transfer, which,
by enhancing cellular bioenergetics, establishes the metabolic basis for the adaptation to this response.”
Chapter 35: Long Living J’s
I remember visiting my paternal grandmother (a J1C1) in her apartment when she was 100 years old. Her 104 year old cousin had an apartment in the same complex a few
floors up. As J’s and having hypometabolism, we have significantly lower mitochondrial oxidative damage. (10)

On the other side of the family my maternal grandmother (also a J1C1), whose ME/CFS was written down by the hospital as an “MS like disease”, died much younger.
Those of us on the ME/CFS+ side of the family (maternal line) each manifest the ME/CFS+ differently. In addition, we each operate on a spectrum of hypometabolism. My
maternal grandmother’s sister (the 7 year old in the original news article) lived to be 96 years old. She had the common sense to naturally pace herself and shunned
society’s ideas of living up to a life that didn’t fit her. She was not one to push herself beyond what her body was telling her. Therefore, just because a person is on the
ME/CFS+ side of the family doesn’t mean that person is going to die early or have crazy health issues. The manifestation is dependent upon many other factors.

My mother is 81 years old. She has had to limit her activities and pace herself throughout her life. The Study “Decreased Reactive Oxygen Species Production in Cells with
Mitochondrial Haplogroups Associated with Longevity” is very interesting. We do not have as much energy, but overall J’s live longer. (19)

We run at a lower speed, so we have less damage from revving the engine. Even my father who is a work horse (the healthy side of the J1c1’s) came home every day at
lunch from work to nap. When I bought a house that happened to be closer to his office than my parent’s house, I was blessed to see him every day at lunch for his nap on
my couch. Now he’s retired.

Many family members on the unhealthy side (my mother’s side with ME/CFS+) would not view themselves as having the extreme fatigue of ME/CFS. Instead they would view
themselves on a spectrum. We often choose to have a closer smaller set of friends because with a limited amount of energy, we’re going to spend our energy on those who
matter the most to us (and steal the least energy from us). High maintenance friends do not hear from us often.

Along with the period of low energy comes a pattern of messiness. When there is energy conservation going on, picking up items and organization is not a priority.

Being social to the level society expects also takes a back seat. Our daughter in college is VP of her sorority and would be considered by outsiders to be a social butterfly
with thousands of followers. Yet, if you examined what she actually does and how she is perceived by those close to her, you’d realize she accomplishes a lot using the
least physical activity she can. She saves her energy for the functions that are really important. If you ask the sisters that live with her in her sorority house, she’s the one
most likely to be lounging in a bed or on a couch. To conserve energy, she’ll skip mixers to hunker down in energy saver mode.

Our littlest one will expend energy running with friends and jumping on the trampoline when she has it. But 2 out of every 3 times a friend comes to the door, she’ll
decline going out because she doesn’t have the energy and will instead work on a science project or watch a YouTube video on something she wants to learn how to do.
Society tells us we should be pushing our children out the door to play more often than we do. But when they have energy, they do and when they don’t have the energy,
they hunker down in power saver mode doing what they can.

When we listen to our bodies and don’t push beyond our energy capabilities, we have fewer doctor and hospital visits. So not fighting against our bodies’ natural cycles of
hypometabolism by using more energy than one has is a good strategy. We can tell when we are going into a down cycle. We start to feel cold, we snuggle in under
blankets and we lose interest in what’s going on around us. Not out of depression, but a lack of energy to be bothered. These cycles of increased hypometabolism are
something we feel. We don’t need to be told by a lab when it happens. We know when it’s happening. We also know when we are coming out of one. All of a sudden
there is energy and one will want to clean, or tackle a project that has been on the to do list for ages. During up turns in energy availability, things are so much easier.
Processing thoughts and getting jobs done takes much less time. There is a natural rhythm to the cycles of going into power saver mode.

We look normal and well to others. They can’t see that our bodies have gone into power saver mode. We smile and pretend all is well, looking forward to tucking back into
our beds. When I get energy, oh sweet energy, try to hold me back from going outside and picking up sticks or cutting branches in overgrown bushes or vacuuming out my
car. But how long that energy supply will last is anyone’s guess. An hour? 12 hours? For a week? I’m sure neighbors who don’t know me must think when they see me
working hard out front – why doesn’t she do this more often. How does one even go about educating an entire society about people like me?

Per Robert K. Naviaux’s 2016 study “Metabolic features of chronic fatigue syndrome”:
“Dauer, which means persistence or long-lived in German, is an example of one well-studied system. The developmental stage of dauer is a hypometabolic state
capable of living efficiently by altering a number of basic mitochondrial functions, fuel preferences, behavior, and physical features. Dauer is comprised of an
evolutionarily conserved and synergistic suite of metabolic and structural changes that are triggered by exposure to adverse environmental conditions. Entry into
dauer confers a survival advantage in harsh conditions.” (17)

When in power saver mode, we go from chair to couch to bed. Reserving as much power as we can throughout our days. I find myself watching people in movies and
wondering why the characters are standing so much of the time. Why aren’t they sitting? When at parties, people love to stand and chat. I’m always trying to move onto
the next chair or group that’s sitting down. It took me a long time to understand that my need to sit down regularly is not something my friends and colleagues do. If I
stand too long, I get dizzy, I lose track of the conversation and my body starts sending me anxious signals of “you better go sit down or you are going to lose consciousness”.
Chapter 36: Haplogroup U5’s

Maternal haplogroup U5 was found at 2.7x the expected frequency in individuals with family histories of ME/CFS (and related symptoms). U5’s are also known for reduced
OXHPOS capacity and mitochondrial uncoupling (7, 11). And just like J1c’s, U5’s appeared during the ice age (43) where entering a hypometabolic state could confer a
survival advantage under such harsh conditions.

HIV and AIDS and U5’s

U5’s (especially when coupled with the 15218A>G variant) are known to have a significant accelerated progression of AIDS (similar to J1c’s with 14798T>C). (7). The study
found:
“MtDNA haplogroups J and U5a were elevated among HIV-1 infected people who display accelerated progression to AIDS and death. ….. The U5a haplogroup is
associated with accelerated AIDS progression to CD4<200 in European Americans (RH=1.78, 95% CI=1.11–2.85, p=0.028) and in MSM pooled cohorts (RH=2.06, 95%
CI=1.17–3.63, p=0.024). The signal is largely driven by the U5a1-15218 haplotype, which comprises 79% of the U5a haplotype.”

The above mentioned 15218A>G variant is one of the two variants needed to go from a U5a to a U5a1. One of the two study participants in my study who was a U5, was a
U5a1a1. The other was a U5b2b.

Continued from the above study titled “Mitochondrial DNA Haplogroups influence AIDS Progression” (7), the human immunodeficiency virus progressed to AIDS and death
fast in maternal haplogroups U5’s and J’s.
“an interesting trend was observed that uncoupled haplogroups with lower ATP and ROS production (U5 and J) are associated with accelerated disease, whereas
more tightly coupled groups (H3 and H4, H5, and H6) are associated with protection, suggesting mitochondrial functional variation plays a role in AIDS progression.
Combined data on longevity, neurodegenerative disease, sperm, sprint performance, and climate adaptation suggests functional mtDNA variation in different
haplogroups influences ATP production efficiency, and correlated ROS and heat generation. Less efficient ATP production in partially uncoupled mitochondria
(haplogroups J and U5) would accelerate AIDS because it would exacerbate the energetic effects of the mtDNA depletion, disruption of oxidative phosphorylation,
antioxidant enzyme deficiency, apoptosis, and increased oxidative damage observed during AIDS progression.”

Therefore, starting off with a mito issue (such as being a J1c or a U5, or mitochondrial damage), even if one is below the threshold for showing any symptoms – if we then
combine that with another mito stressor, it can have deleterious consequences.

Alzheimer’s and U5’s

The study “Analysis of European Mitochondrial Haplogroups with Alzheimer Disease Risk” (97) found:
“Additionally, the frequency of haplogroup U was found to be greater in stroke patients and has been associated with elevated risk for migrainous occipital stroke.
Further analysis demonstrated that the association was due to a high frequency of subcluster U5. Given the diverse nature of the U haplogroup and its apparent
association with a related neurologic disease, we suggest that variants within the different subclusters of U may predispose individuals to AD risk.”

OXPHOS and U5’s

This 2016 review article from the journal Frontiers in Aging Neuroscience found:
“Cybrids constructed from osteosarcoma 143B rho0 cells and platelets from healthy Spanish donors of either haplogroup H or superhaplogroup UK were investigated
for mtDNA content (Gómez-Durán et al., 2010). Cybrids harboring UK superhaplogroup were found to have lower mtDNA content, lower mt-rRNA level, reduced
protein synthesis, and decreased cytochrome c oxidase amount (Gómez-Durán et al., 2010). UK cybrids also had lower mitochondrial inner membrane potential and
higher mitochondrial uncoupling, indicating potentially lower respiratory capacity and reduced ATP production (Gómez-Durán et al., 2010). Similar results
were obtained in a later study in middle-age Caucasian males, where OXPHOS capacity normalized to citrate synthase content was found to be reduced by 24%
in subjects with haplogroup U background comparing to those with haplogroup H background (Larsen et al., 2014).” (11).

Macular Degeneration and U5’s

As we have learned, haplogroups with increased risk of macular degeneration due to specific mtDNA mutations in combination with the haplogroup is a potential indicator
that other mtDNA variants might cause ME/CFS and related symptoms in such haplogroups. In the next study, this is exactly what happened with maternal haplogroup U5.

In the study “Mitochondrial DNA Haplogroups Associated with Age-Related Macular Degeneration” (8), haplogroup U5 was at a significantly increased risk for macular
degeneration penetrance when the additional mtDNA variants T3197C+A12308G were added in.
“The control region SNPs T16126C and A73G, commonly found in haplogroups J and T, were more frequent in the AMD retinas than in normal retinas. The
associations between AMD and haplogroups J and T were confirmed and extended by analysis of blood DNA. SNPs at position a T16126C (J; odds ratio [OR] =
3.66), T16126C+G13368A (JT; OR = 10.27), A4917G+A73G (T4; OR = 5), and T3197C+A12308G (U5; OR = infinity), were all strongly associated with
AMD. ….. The combination T3197C+A12308G (subset U5) was found in 6 of 81 case subjects but none of the controls (OR = ∞; P = 0.019).”

A pattern is emerging of similarities between J1c’s and U5’s, including the fact that subclades with reduced OXPHOS capacity coupled with mtDNA deleterious variants
significantly increase the risk of macular degeneration. An Odds Ratio (OR) of ∞ is very telling. To repeat from above, “The combination T3197C+A12308G (subset U5) was
found in 6 of 81 case subjects but none of the controls (OR = ∞; P = 0.019).” And as we know, the eye is one body part that needs high mito energy and where a
combination of deleterious mtDNA variants can have a profound effect.
Chapter 37: The Differences between Men & Women in ME/CFS and in Autistic Spectrum
In this question and answer session with the Open Medicine Foundation (OMF), Dr. Naviaux explained some of the differences they found between men and women with
ME/CFS.
“Q4. Are men and women really that different in CFS?
Answer: Yes. About 40-50% of all the metabolites that we measure in our method have a different normal concentration in males and females. This is not
all related to testosterone and estrogen. Literally hundreds of metabolites are tuned to different concentrations in men and women. At the pathway level,
we found that men and women shared 9 (45%) of the 20 biochemical pathways that were disturbed in CFS patients. Eleven pathways (55%) were more
prominent in males or females. We find that to do metabolomics properly, you need to have an adequate number of age- and sex-matched controls. If
healthy males and females are lumped together as controls, the power to see metabolic differences in CFS and many other diseases is much decreased.
Likewise, the metabolism of a 25-year old male is different from a 35-year old male, and categorically different from a 25-year old female. In each decade
of life there are many metabolic changes that occur as part of normal development and aging. When proper age- and sex-matched controls are used,
metabolomics is one of the most powerful new tools available to physicians and scientists to study chronic complex disease.” (16)

Two important puzzle pieces here. 1) One difference between men and women in CFS can be found in more than 50% of the metabolomics. 2) Researchers need to use age
and sex matched controls in metabolomics since normal development and aging impacts metabolomics. This puzzle piece means – be careful of studies that don’t use age
and sex matched controls when studying metabolomics.

The differences found by Dr. Naviaux’s in the metabolomics between men and women may explain the differences in why the ME/CFS+ families have more female family
members with ME/CFS and more male family members with ASD. Keep in mind, several female study participants with ME/CFS mentioned having light ASD symptoms while
some others mentioned that they believed their male family members with ASD had light ME/CFS symptoms. Just like with ASD, I believe ME/CFS comes with a spectrum of
symptoms from severely disabling to lightly annoying. For each symptom, ME/CFS patients can move up or down the spectrum depending upon environmental conditions
and stressors at the time.

The pattern of more ME/CFS in females and more ASD in males in the ME/CFS+ families fits with the male to female ratios found in the general public. The male to female
ratio of ME/CFS is 1:4 (101). The male to female ratio of ASD is 4:1 (28).

In my father’s maternal line (J1c1 + macular mtDNA variants), the significant penetrance of macular degeneration is just in the female family members. There is no history
in dad’s family of men getting macular degeneration. According to Global Data Healthcare, the majority of age related macular degeneration (AMD) “occurred in women
(65.76%), with 7,400,411 cases, while men accounted for 34.24%, with 3,852,822 cases” (100). Will the difference in prevalence among genders be found in metabolomics?

Now that high resolution mass spectrometers are becoming more common, they are going to become an integral tool in the study of complex metabolic processes of
mitochondrial function and dysfunction in disease. (Ever since Abby Sciuto on NCIS got a new high resolution mass spectrometer, it was just a matter of time before real
researchers got theirs too.) In the 2018 study “Metabolic profiling of isolated mitochondria and cytoplasm reveals compartment-specific metabolic responses”(99), the
researchers concluded that:
“By applying metabolomics to isolated mitochondria and the corresponding cytoplasm, compartment-specific metabolic signatures can be identified. This
subcellular metabolomics analysis is a powerful tool to study the molecular mechanism of compartment-specific metabolic homeostasis in response to mutations
affecting the mitochondrial metabolism.”

In the 2017 study out of the Children’s Hospital of Philadelphia (CHOP), titled “Association Between Mitochondrial DNA Haplogroup Variation and Autism Spectrum
Disorders” (28), doctors found that most European haplogroups (not H) had about a 2x increase in risk of ASD.
“The generalized estimating equations analysis of MDS C1 individuals revealed that European haplogroups I, J, K, T, and U had significantly higher risk of ASD
than HHV+ (range: OR, 1.87; 95% CI, 1.34-2.61 to OR, 2.27; 95% CI, 1.35-3.81). Only European haplogroup W was not associated with increased ASD risk (Table 3).
The Asian and Native American mtDNA haplogroups A and M were also at increased ASD risk (OR, 2.95; 95% CI, 1.92-4.52 and OR, 3.14; 95% CI, 2.08-4.75,
respectively) as were computational O-X haplogroups and macrohaplogroups N+ (OR, 2.01; 95% CI, 1.21-3.34 and OR, 4.52; 95% CI, 1.96-10.41, respectively) (Table
3). Therefore, even if we focus only on MDS C1 (multidimensional scaling analyses cluster 1) , the association between ASD risk and mitochondrial haplogroups
remains intact and is in fact stronger.”

In a recent article regarding the above CHOP study, researcher Douglas Wallace, director of the Center for Mitochondrial and Epigenomic Medicine at the Children’s Hospital
of Philadelphia, was quoted as saying: ”Our brains, which are 2 percent of our body weight, use 20 percent of all the mitochondrial energy in our body. So, a very subtle
change in mitochondrial energy is going to radically affect the brain,” he says.” (102)

The study concluded that:

“We have shown that mitochondrial haplogroups, with their associated functional variants, contribute a significant proportion of ASD risk, thus confirming
that mitochondrial dysfunction is a significant factor in the cause of ASD. The interaction between the ancient haplogroup functional variants, recent
heteroplasmic mtDNA mutations, mutation or deletion of 1 or more nDNA genes, and environmental insults that modulate bioenergetics may all combine to
explain many of the unusual features of ASD genetics.” (28)

The article reviewing the CHOP study further stated:

“For example, of 368 participants carrying haplogroup J, 157 have autism. This represents a 2.18-fold increase in the odds of having autism compared with the
HHV+ group. (This calculation controls for family structure, accounting for the fact that siblings with autism share nonmitochondrial risk factors for the condition.)”
(102).

Future research should include the prevalence of J1c’s and U5’s among families with multiple cases of ASD. This would fit in with my study findings that ME/CFS+ families
(those with ME/CFS and autistic spectrum and other health issues) have a higher frequency of J1c’s.

The “Discussion” section of the CHOP study (28) states:

“The unique genetics of the mitochondrion can now account for many of the puzzling features of ASD genetics. The male bias of ASD is consistent with the
male bias observed for the onset of blindness in patients with Leber hereditary optic neuropathy (male to female ratio is approximately 4:1), which is caused
by mild homoplasmic mtDNA mutations. The female protection from blindness has been attributed to the presence of the estrogen receptor in the
mitochondrion such that estradiol increases mitochondrion antioxidant defenses.”

This study says ASD is male biased just like LHON, both having an approximately 4:1 male to female ratio. The study also says the mitochondrion now account for many of
the puzzling features. From other studies, we know that ME/CFS and macular degeneration are more female biased

The puzzle pieces that increase the risk for ASD and LHON in males might be the same pieces that might come back to metabolomics. The puzzle pieces that increase the
risk for ME/CFS and macular degeneration in females may come back to metabolomics.

Then the question becomes: Is a single metabolomics indicator in males increasing the risk for ASD in males or is a single metabolomics indicator reducing the risk for ASD in
females? Or is it more complex with multiple metabolomics. Potential treatments could arise from what is found with studies in this area on ASD, LHON, ME/CFS, and
macular degeneration – all of which are impacted by being J1c’s and gender.

Whatever is found regarding the above, will also likely benefit the study of Multiple Sclerosis (MS) and Fibromyalgia (FM) where there is also a higher female to male ratio.
Due to the fact that MS and FM were found at increased rates among ME/CFS+ family members, all of these issues are related. The puzzle keeps getting larger and multi-
dimensional.
Chapter 38: Multiple Sclerosis
Statistically, there are too many similarities between M.S. and ME/CFS to be explained away as coincidence. Reading relevant studies on M.S. could aid in finding more
puzzle pieces. In my study, participants with ME/CFS had a higher rate of having a close family member with MS than the general population. This is not surprising since
many studies have seen similarities and links (108).

Haplogroup J has higher rates of Multiple Sclerosis than the general population.
“Once mtDNA haplogroups were determined, we found that J presents with lower VO(2max) (P=0.02) than nonJ variants. J has been related with a lower
efficiency of electron transport chain (ETC), diminished ATP and ROS production. Thus, the difficult to compensate the mitochondrial energetic deficiency
could explain the accumulation of J haplogroup in LHON and multiple sclerosis.” (4).

(127)

In this study, a significant relationship was found between haplogroup J and MS in Persian patients.
“In addition, a slightly significant increase of MS patients of haplogroup J (20% in MS patients versus 9.39% in normal controls at P =0.049),” (103)

It makes me wonder if they would have found a significantly increased relationship between J1c’s in M.S.+ families. M.S.+ families are families with multiple cases of M.S.
or related health issues in close family members. The underlying issue will likely be traced back to hypometabolism in J’s which is further increased in J1c’s due to further
mtDNA variants.

This study found an increased rate of progressive (PP) MS among haplogroup J and also mentioned the haplogroup J defining variant 13708G>A in MS and also pediatric
acquired demyelination syndrome. (104).
“An elevated risk of primary progressive (PP) MS was detected for haplogroup J participants from 3 European discovery populations (OR = 1.49, 95% CI = 1.10–2.01, p
= 0.009). This elevated risk was borderline significant in the US replication population (OR = 1.43, 95% CI = 0.99–2.08, p = 0.058) and remained significant in pooled
analysis of discovery and replication studies (OR = 1.43, 95% CI = 1.14–1.81, p = 0.002).”
….
“Two large studies previously identified MS associations among haplogroup T (m.4917A>G) carriers from 523 MS cases11 and haplogroup J (m.13708G>A) carriers
from 1,099 MS cases.”
….
“The m.13708G>A variant was also significantly associated with pediatric acquired demyelination syndrome (PD-ADS),23 providing further evidence to suggest that
m.13708G>A is the SNP underlying the haplogroup J associations with MS and PPMS identified in the current study.”

13708 G>A is a defining variant for haplogroup J. Therefore, it’s significance here (and earlier in the book) shows a common underlying cause to all these diseases.

Back as far as 1998, researchers knew that “High-resolution fluorodeoxyglucose positron emission tomography shows both global and regional cerebral hypometabolism in
multiple sclerosis” (105).

In the 2016 study “Analysis of Cortical Metabolism in Multiple Sclerosis: A 3T 18F-FDG PET/MRI Study (P4.166)” (106), the researchers stated that:
“This suggests that a metabolic dysfunction probably precedes MRI detectable structural damage in the cortex of MS. The identification of hypometabolic areas in
very early disease phases may have important pathological and clinical (therapeutic) consequences.”

The findings of hypometabolism in multiple sclerosis, the increased rate of haplogroup J in multiple sclerosis patients, and the knowledge that haplogroup J results in
hypometabolism (reduced OXPHOS, …) – this fits the pattern seen in many of these diseases.
Chapter 39: Fibromyalgia
Just like with MS, statistically, there are too many similarities between Fibromyalgia and ME/CFS to be ignored. Reading relevant studies on Fibromyalgia could aid in
finding more puzzle pieces. In my study, participants with ME/CFS had a higher rate of having a close family member with Fibromyalgia than the general population.

Study title: “Overlapping conditions among patients with chronic fatigue syndrome, fibromyalgia, and temporomandibular disorder” (120). TMJ is a type of TMD.
“patients were more likely than controls to meet lifetime symptom and diagnostic criteria for many of the conditions, including CFS, FM, irritable bowel syndrome,
multiple chemical sensitivities, and headache. Lifetime rates of irritable bowel syndrome were particularly striking in the patient groups (CFS, 92%; FM, 77%; TMD,
64%) compared with controls (18%) (P<.001). Individual symptom analysis revealed that patients with CFS, FM, and TMD share common symptoms, including
generalized pain sensitivity, sleep and concentration difficulties, bowel complaints, and headache. However, several symptoms also distinguished the patient
groups.
CONCLUSIONS:
This study provides preliminary evidence that patients with CFS, FM, and TMD share key symptoms. It also is apparent that other localized and systemic conditions
may frequently co-occur with CFS, FM, and TMD. Future research that seeks to identify the temporal relationships and other pathophysiologic mechanism(s) linking
CFS, FM, and TMD will likely advance our understanding and treatment of these chronic, recurrent conditions.”

This study is excellent. It notes the similarities between chronic fatigue and fibromyalgia regarding mitochondrial metabolism indicators, but also notes the differences
where specific treatments might be targeted. Also, following the logic, maybe the difference in getting CFS vs FM in a patient with for example J1c might be in the nuclear
genes, etc…. So finding answers and targeting solutions might be based on something like this.

Study title: “Could mitochondrial dysfunction be a differentiating marker between chronic fatigue syndrome and fibromyalgia?” (109):
“Chronic fatigue syndrome (CFS) and fibromyalgia (FM) are complex and serious illnesses that affect approximately 2.5% and 5% of the general population
worldwide, respectively. The etiology is unknown; however, recent studies suggest that mitochondrial dysfunction has been involved in the pathophysiology of both
conditions. We have investigated the possible association between mitochondrial biogenesis and oxidative stress in patients with CFS and FM. We studied 23 CFS
patients, 20 FM patients, and 15 healthy controls. Peripheral blood mononuclear cell showed decreased levels of Coenzyme Q10 from CFS patients (p<0.001
compared with controls) and from FM subjects (p<0.001 compared with controls) and ATP levels for CFS patients (p<0.001 compared with controls) and for FM
subjects (p<0.001 compared with controls). On the contrary, CFS/FM patients had significantly increased levels of lipid peroxidation, respectively (p<0.001 for both
CFS and FM patients with regard to controls) that were indicative of oxidative stress-induced damage. Mitochondrial citrate synthase activity was significantly lower
in FM patients (p<0.001) and, however, in CFS, it resulted in similar levels than controls. Mitochondrial DNA content (mtDNA/gDNA ratio) was normal in CFS and
reduced in FM patients versus healthy controls, respectively (p<0.001). Expression levels of peroxisome proliferator-activated receptor gamma-coactivator 1-alpha
and transcription factor A, mitochondrial by immunoblotting were significantly lower in FM patients (p<0.001) and were normal in CFS subjects compared with
healthy controls. These data lead to the hypothesis that mitochondrial dysfunction-dependent events could be a marker of differentiation between CFS and
FM, indicating the mitochondria as a new potential therapeutic target for these conditions.”

This study regarding fibromyalgia and oxidative stress found:

“Recent studies have shown some evidence demonstrating that oxidative stress may have a role in the pathophysiology of FM. Furthermore, it is controversial the
role of mitochondria in the oxidant imbalance documented in FM. Signs and symptoms associated with muscular alteration and mitochondrial dysfunction, including
oxidative stress, have been observed in patients with FM. To this respect, Coenzyme Q10 (CoQ10) deficiency, an essential electron carrier in the mitochondrial
respiratory chain and a strong antioxidant, alters mitochondria
function and mitochondrial respiratory complexes organization and leading to increased ROS generation. Recently have been showed CoQ10 deficiency in blood
mononuclear cells in FM patients, so if the hypothesis that mitochondrial dysfunction is the origin of oxidative stress in FM patients is demonstrated, could help to
understand the complex pathophysiology of this disorder and may lead to development of new therapeutic strategies for prevention and treatment of this disease.”
(107)
Chapter 40: Do some ME/CFS patients have mitochondrial multiorgan disorder syndrome?
Study: “Mitochondrial multiorgan disorder syndrome score generated from definite mitochondrial disorders” (112). I believe some people with ME/CFS with symptoms
in multiple systems may have “Mitochondrial multiorgan disorder syndrome”. Therefore, reading about the more likely symptoms in this syndrome might be on point for
some people.

“The number of organs most frequently affected was 4 ranging from 1 to 9. MIMODS (mitochondrial multiorgan disorder syndrome) was diagnosed in 97% of patients.
The organs most frequently affected were the muscle (97%), central nervous system (CNS; 72%), endocrine glands (69%), heart (58%), intestines (55%), and
peripheral nerves (50%). The most frequent CNS abnormalities were leukoencephalopathy, prolonged visually evoked potentials, and atrophy. The most frequent
endocrine abnormalities included thyroid dysfunction, short stature, and diabetes. The most frequent cardiac abnormalities included arrhythmias, systolic
dysfunction, and hypertrophic cardiomyopathy. The most frequent MIMODS patterns were encephalomyopathy, encephalo-myo-endocrinopathy, and encepalo-myo-
endocrino-cardiopathy. The mean ± 2SD MIMODS score was 35.97±27.6 (range =11–71). An MIMODS score >10 was regarded as indicative of an MID (Mitochondrial
disorders).
Conclusion: Adult MIDs manifest as MIMODS in the vast majority of the cases. The organs most frequently affected in MIMODS are muscles, CNS, endocrine glands,
and heart. An MIMODS score >10 suggests an MID.”
Chapter 41: Paternal Link?
Could a paternal link to some cases of ME/CFS also exist? There were a few cases among study participants that indicated the potential of a paternal link. Further research
should include addressing a possible paternal inheritance pattern in some ME/CFS patients.

The appearance of a paternal inheritance pattern could be due to:

- a nuclear variant passed down from a father,
- a virus impacting epigenetics further down the line,
- environment (chemical exposure, etc….),
- that mates are more likely to be attracted to like levels of energy (theory). A person on the lower energy level of the spectrum is more likely to partner
with a mate who prefers staying in and enjoys lower energy activities. Likewise, a marathon runner’s likely partner is going to be on the higher end of
the energy spectrum.
- a female with a brother or father with high functioning Asperger’s is going to be more likely to see high functioning ASD quirks as normal and therefore less
likely to rule out mates who fit the pattern of her adolescent environment. Therefore, in females that have mtDNA that increases the penetrance of
ME/CFS+, they may select partners with high functioning ASD at a higher rate than the general population.

We are still in the infancy of the field of genetics. Paternal inheritance of ME/CFS cannot be ruled out as a possibility at this time.
Part 6: Missing Pieces
Chapter 42: Adventurers & Potholes
For my family, I’m driven to find the answer to ME/CFS. Today I was having an energy crash, our youngest daughter was home from school with severe constipation (colon
muscles don’t work as well when the muscles have low energy), and our son with high functioning ASD lost his driver’s license and wouldn’t go to the DMV without me
because he doesn’t like to speak with strangers (he is repelled by people who are not friends, nor family). He can do it -- but let’s just say it’s not his favorite. Every day
is a unique challenge. And this has been going on for five generations. Anything I can do to help bring about a cure, I’ll do.

Despite the fact that we are in the infancy of genetics, I’m willing to be an adventurer putting up with the potholes that come along often because we need a cure.

It’s been a dance of one step forward, two steps back, three steps forward and requires a lot of patience and perseverance. Rather than being a straight line to finding our
mitochondrial connection it began with me being seen by dozens of doctors as various symptoms flared up and then would tamper back down. Eventually a DNA test
pointed out a pathogenic dominant variant on a nuclear gene and we began to be seen as a family by a mitochondrial clinic who sent us to the UMDF conferences. Then it
turned out that pathogenic dominant mutation was a miscall. Thankfully, by then our family was well studied in the mito clinic and we were told that there are still a large
amount of mitochondrial disease and dysfunction cases where they don’t yet know the exact variants causing it. So the mito clinic continued to see us. Then there was the
original Whole Genome Sequencing (WGS) where the 12706 variant that causes Leigh’s came up. However, that was later found to be an error where the old reference
genome was used instead of NC_01290. The rest of the story is what I shared here in this book. It’s been a very long road, filled with pot holes and calling for a lot of
patience.

Yet, the road will be easier for those in the future because many of us traveled it. We now know to triple check everything from different labs. We are also battle tested
to have patience as the industry experiences the hiccups of growing pains (errors, data tsunami, lack of staff for all the new mito patients, etc…).
Chapter 43: Mitochondrial Experts are currently spread thin
If you are going to a mitochondrial expert and expect them to do the deep dive on your sequenced data that I did here, be warned that’s highly unlikely. Today’s mito
experts are overworked and understaffed. While they see the need to staff up and it appears all mito groups are hiring, when these new staff arrive they have to train them
and by the time they are trained they will need even more staff. The tsunami of research data on mitochondrial disease and dysfunction also represents the tsunami of
patients wanting to get into mitochondrial experts as more diseases are known to be impacted by mitochondrial metabolism issues. Patience by patients is required. We
are all facing the backlog of appointments as this field gears-up for continued growth.
Chapter 44: More Details about the Study
The study population began with 140 people with ME/CFS from two international ME/CFS patient support boards. Sixteen individuals met the study criteria of:
- having ME/CFS themselves, and
- having a close relative (parent, child, sibling, aunt, uncle, first cousin, or grandparent) with ME/CFS or a close relative with a related health issue, and
- had completed DNA testing and knew his/her maternal haplogroup.

Of the 34 people who initially responded to my detailed questions on family health history, there were a surprising number of close family members to these ME/CFS
participants who had POTS, Autistic Spectrum, Multiple Sclerosis, Fibromyalgia, Crohn’s, early-onset Alzheimer’s, Hashimoto’s, Mast Cell Disorder, Ehlers-Danlos Syndrome,
and other autoimmunity symptoms.

The 18 people who did not make it into the 16 (34-16=18) had not had any genetic testing done and did not know their maternal haplogroups.

Of the 8 study participants out of the 16 who were neither J1c’s, nor U5’s, several of them have haplogroups that are worthy of being researched farther. For example, one
of the 16 participants was from maternal haplogroup L2C.

Just doing a minor bit of research on L2c’s led me to the study: “Mitochondrial DNA variation in human metabolic rate and energy expenditure” (117) that found:
“…haplogroup L2 had a significantly lower TEE (Total Energy Expenditure) than haplogroups H and JT. …. African haplogroups L2 and L3 exhibited significantly
lower RMR (resting metabolic rate) values than all others examined.”

It would be wrong to presume J1c’s and U5’s are the only haplogroups with increased rates of hypometabolism. Therefore, a study of L2c’s would be warranted along with
analyzing the private mtDNA variants of ME/CFS patients who are L2c’s. Experts expect to find various types of hypometabolism that thrived during periods of great
temperature rises and famine. To think hypometabolism haplogroups only appeared during ice ages would be shortsighted. In addition, haplogroup changes that thrived
due to heat will likely have different identifiable paths to hypometabolism than those that appeared during ice ages.

The study “Mitochondrial DNA diversity in the African American population” (118) found that:
“The L2c and L2e haplogroups represented a minor fraction of the total mtDNA haplogroups in our study population (Figure 2). The L2c haplogroup (1.1%, n = 4) was
defined by the following 9 DNA variants while the L2e haplogroup (0.9%, n = 3) was defined by 11 variants (Tables 1 and 2). ….. 16264T defined L2C”

MseqDr’s “mvTool for PhyloTree and Haplogroup” (33) shows that L2c’s need to have these variants:
73 93 146 150 152 182 195 198 263 325 680 709 750 769 1018 1438 1442 2332 2416 2706 3200A 3594 4104 4769 7028 7256 7521 7624A 8206 8701 8860
9221 9540 10115 10398 10873 11719 11944 12236 12705 13590 13650 13928C 13958C 14766 15110 15217 15301 15326 15849 16223 16278 16390.

The ones needed to go from an L2 to an L2c are in bold.

Another hint that L2C might be of interest comes from the study “Association of primary open-angle glaucoma with mitochondrial variants and haplogroups common in
African Americans” (119).
“The m.3200T>A variant was confirmed to be elevated in cases (OR 1.4), consistent with the prediction that haplogroup L2c confers risk (Figure 4), but this
difference was not statistically significant (p=0.07, Table 1).”

In this example using maternal haplogroup L2c, there are little snippets of information that hint at a possible relation with mitochondria and ME/CFS. In addition, the study
participant with the maternal haplogroup L2c has a mother with dysautononomia and symptoms similar to fibromyalgia. Future studies are warranted.
Chapter 45: Study Limitations
I realize this is not a normal scientific study in any way. I have no scientific experience, nor higher science education. Therefore, there is the possibility of my theory being
off because of my lack of a science background, lack of staff, and lack of a lab.

In depositions, attorneys will tell you to answer questions in as few words as possible without providing any other details. The extra details are strings that other attorneys
can pull on to unravel a case. However, here I’ve provided everything I can think of giving you, even the reasoning behind my calculations. Why, when this just provides
more strings on which to pull apart my study and find potential errors? Why do this? I have a vested interest in ME/CFS therapies and a cure being found as quickly as
possible.

I do not want to misdirect researchers to something that will waste their time. ME/CFS impacts my life and my children’s lives. I’m for finding the right answers – I don’t
need to be right. Therefore, I’ve turned towards my own study and am looking for study limitations. I am sure there are many limitations that just didn’t come into my
mind. I know my scientific friends will find them. All I ask is that you poke holes in a constructive way. I’ve done the best I could to bring what I saw to everyone’s
attention while dealing with my own struggles and limitations. My hope is for better treatments and a cure as soon as possible.

Argument #1: The population frequency you’ve used for J1c’s in the population is too low and therefore over estimates the increased rate of J1c’s.
False: Out of all the general population frequencies for J1c’s, I chose the highest one. This way, it’s much more likely the 5.4x number is underestimated
(rather than overestimated). See Chapter 49.

Argument #2: Haplogroup J’s are smarter than other people and therefore more J’s will congregate on support boards discussing the latest studies and potential
treatments. Therefore, there will be more J’s on the boards. And since J1c’s make up between 75-80% of J’s, there would be more J1c’s on the two support
boards from which the study participants were chosen. Therefore, J1c’s would be over-represented in study participants.
False: I wish this one was true. As a J, it would be great if we were smarter. But alas, we have no cognitive advantage. A 2012 study found haplogroup J was
associated with decline in cognitive function compared to haplogroup H. Therefore, according to the theory of people with higher cognition accumulating on these
boards, then H’s should have been over-represented
“Mitochondrial dysfunction is a prominent hallmark of Alzheimer's disease (AD). Mitochondrial DNA (mtDNA) damage may be a major cause of abnormal
reactive oxidative species production in AD or increased neuronal susceptibility to oxidative injury during aging. The purpose of this study was to assess the
influence of mtDNA sequence variation on clinically significant cognitive impairment and dementia risk in the population-based Health, Aging, and Body
Composition (Health ABC) Study. We first investigated the role of common mtDNA haplogroups and individual variants on dementia risk and 8-year change
on the Modified Mini-Mental State Examination (3MS) and Digit Symbol Substitution Test (DSST) among 1,631 participants of European genetic ancestry.
Participants were free of dementia at baseline and incidence was determined in 273 cases from hospital and medication records over 10-12 follow-up
years. Participants from haplogroup T had a statistically significant increased risk of developing dementia (OR = 1.86, 95% CI = 1.23, 2.82, p = 0.0008) and
haplogroup J participants experienced a statistically significant 8-year decline in 3MS (Modified Mini-Mental State Examination) ( β = -0.14, 95% CI = -0.27,
-0.03, p = 0.0006), both compared with common haplogroup H.”
…… Haplogroup J is defined by variants in ND5 and HV2. The HV2 variant that defines haplogroup J, m.295C>T, has been shown to change mitochondrial
transcription and copy number. Cytoplasmic hybrids (cybrids) containing haplogroup J mtDNA had a greater than 2-fold increase in mtDNA copy number
compared with cybrids containing haplogroup H mtDNA. This is one of the few examples demonstrating functional consequences for a variant underlying a
specific haplogroup and is of particular interest since haplogroup J is over-represented in long-lived people and centenarians from several populations. In
this case, the impact of the haplogroup J regulatory region mutation on mtDNA replication or stability may partially account for population-based
observations that haplogroup J is associated with human longevity.” (7).

Argument #3: The data was subconsciously manipulated to include J’s.

False: I tried to eliminate potential bias. The study happened on two boards. On the first board, there was a poll (by someone else) that had a category for
people with ME/CFS who also were caregivers for a child/children with ME/CFS. The poll was focused on setting up a separate discussion area for those of us who
are ill and are also taking care of an ill child. A unique subset of the ME/CFS population.

I reached out to the people who wrote about their experiences as the combination of ME/CFS patient and also parent of a patient with ME/CFS and asked if they
had genetic testing. Of the one’s who responded that they had, they were all included. Therefore, the first board’s study participants were already categorized as
ME/CFS patient with a child/children with ME/CFS before I learned their haplogroups. This group was 100% J1c’s.

These results made me want to reach out to gather more data (a larger sample). I also wanted to see if not only like me and my daughter who are both diagnosed
with ME/CFS – but also broaden the scope to families with ME/CFS plus other health issues. I wanted to pick up families that resembled my own and who may have
a son with autistic spectrum and/or a brother who needed a pacemaker in his early 40’s due to a slow heart rate. Therefore, I placed a poll on a second ME/CFS
support board that I am also a member of to gather more data.

Initially I asked people on the second board to put themselves into categories and then to provide additional details. The categories were:
- ME/CFS patient with child/parent with ME/CFS,
- ME/CFS patient with other close relative with ME/CFS,
- ME/CFS patient with child/parent ill with symptoms often found in ME/CFS family members,
- ME/CFS patient with other close relative ill with symptoms often found in ME/CFS family members,
- those who either don’t have ME/CFS or who don’t have any close relatives with ME/CFS or any related symptoms.

I then went back and confirmed that participants put themselves in the correct categories based on the details they provided. By this point I would ask follow-up
questions on family health histories. Only after people were put into categories, did I add a column to the spreadsheet for haplogroup. In fact, initially I didn’t
even know if participants had even had genetic testing done.

That’s when I went back and asked participants if they had genetic testing, and if they did, then I asked about haplogroup. I had known one of the participants
from an earlier discussion and knew her haplogroup. But after working with over 100 participants, it wasn’t in the forefront of my mind when entering everyone’s
information over a period of several weeks.

Of the participants who had genetic testing done and knew their maternal haplogroups, the categories that they had all put themselves in to begin with were
verified by their family health details. And their haplogroups weren’t entered into the spreadsheet until after the verification that they had placed themselves in
the correct category happened.

In summary, data couldn’t be subconsciously manipulated because haplogroups weren’t known until after categories were known and verified and entered into the
spreadsheet. There were many people who would have joined the 16 study participants had they had genetic testing and known their haplogroups. These were
people who fit in the categories and had their family health history details matched to the category they had chosen.

Argument #4: Random clumps of data can happen. Sometimes when flipping a coin, you will get more heads than tails. Therefore, maybe there was a random larger than
normal amount of J1c’s.
True: This is why this study needs to be redone on a larger population in a controlled setting.
Part 7: Discussion
Chapter 46: Tying It All Together
In my study, 100% of ME/CFS patients with at least one child with ME/CFS and a second child ill with similar symptoms all came from the same maternal haplogroup J1c.
When broadening the scope to look at ME/CFS patients who have a ME/CFS+ family history, 50% of the patients came from two maternal haplogroup subclades, J1c and U5.
Both haplogroups are known for reduced OXPHOS capacity and mitochondrial uncoupling.

This is just the beginning. In my study, I picked the easy to grab low hanging fruit. It’s easier to identify the mitochondrial link when you are looking at patients with
multiple relatives with the disease. For most ME/CFS patients, the cause is significantly more complex and can include mitochondrial DNA variants, nuclear DNA variants,
and environmental stressors (such as mononucleosis, Lyme Disease, and chemical exposure).

There is a threshold of mitochondrial dysfunction that must be reached before ME/CFS symptoms appear. While finding the exact combination of culprits behind anyone’s
mitochondrial dysfunction is difficult, what is closer at hand is the link between ME/CFS+ and hypometabolism, metabolomics, and the potential ME/CFS diagnostic tests
and treatments.

Many health conditions, including Multiple Sclerosis, Fibromyalgia, and Autistic Spectrum Disorder, can be viewed in a similar light to ME/CFS with studies linking them to
mitochondrial dysfunction.

This recent article from November 5, 2018 identifies some of the therapies that could be explored: “Mitochondria as a therapeutic target for common pathologies” (98).

Ron Davis, PhD (director of the Stanford Genome Technology Center) and Director of the OMF (Open Medicine Foundation) has a vested interest in finding solutions due to
being the loving father of a son who is suffering from debilitating ME/CFS (including having a feeding tube). The recent 2018 OMF conference is on YouTube and is definitely
worth watching (121). Ron Davis does an excellent job of explaining the diagnostic tests and treatments currently being studied for ME/CFS patients.

Figure 13 below includes several of the interacting causes behind mitochondrial dysfunction and some of the potential symptoms. It sure looks a lot like ME/CFS.
Chapter 47: Wish List of Future Research
- I’d like to see researchers add maternal haplogroup and subclade analyses to their studies.

- I’d like to see somebody repeat this study with a larger group of patients using proper scientific controls and an experienced staff and then follow up on that study with
metabalomic testing.

- I’d like to see the OMF reach out to my study participants and conduct follow-up sequencing and metabolomic testing. (Private variant analysis.)

- I’d like to see somebody repeat this study on Multiple Sclerosis and also on Fibromyalgia patients looking for haplogroups and subclades in high frequencies in patients with
families with M.S.+ and FM+.

- I’d like to see researchers study the impact the mito-cocktail has on ME/CFS+ patients using metabolomics testing.

- I’d like to see a study of the impact various forms of Coenzyme Q10 (CoQ10) have on ME/CFS+ patients. Some ME/CFS patients report being very sensitive to CoQ10 and
cannot take any form of ubiquinol without seeing an uptick in symptoms. There is another group of patients that sees an improvement with ubiquinone.
I respond best to a cocktail of ubiquinol, B2, D3, and B12. However, who the manufacturer of the ubiquinol is and how it is stored makes a large impact on it’s
effectiveness in increasing my energy. For example, there is one brand that I can buy over the counter where the ubiquinol is liquid in little gel pills. These work well at
increasing my energy. I need to spread these pills throughout the day to have a continuing stream of improved energy.
However, the prescription of ubiquinol prescribed by my mitochondrial doctor that comes from a compounding pharmacy arrives monthly in a large bottle of liquid.
Early in the week when this prescription first arrives, I have found this Ubiquinol works well. Yet, it quickly loses it’s effectiveness. By the last week of each month, I turn
completely to the over the counter pills with the liquid inside to keep my energy going.
Studies on CoQ10’s effectiveness in ME/CFS patients should take all of these variables into account. For example, I’d like to see a study on those of us who respond
well to ubiquinol as to what form and dose works best. For those who can’t take coQ10 without having an uptick in symptoms, I’d like to know why this happens. And for
those that respond positively to only ubiquinone in tiny doses, I’d like to know why and in what form and what amounts.

- I’d like to see the previous study “Patients with chronic fatigue syndrome do not score higher on the autism-spectrum quotient than healthy controls: Comparison with
autism spectrum disorder” (27), redone specifically on the sons of ME/CFS+ mothers. In our house, I would have tested low on the autistic spectrum quotient, while testing
my son would have likely been higher. Women get more CFS and men more ASD. Testing the children of ME/CFS+ patients for autistic spectrum quotient might provide
further insight due to the impact gender has on metabolomics.

- I’d like to see CHOP look back at their recent study results on autism (27) where they found European haplogroups (not H) had about a 2x increase in risk of ASD, and
specifically look at subclades for any increase (including in J1c’s).

- I’d like to see research using cybrids.

- I’d like to see studies on whether children with severe post-mono fatigue and also on children who develop the new viral polio-like paralysis, whether these children are
more likely to come from haplogroup subclades known for higher rates of mitochondrial dysfunction than the general population. I’d also like to see a study on whether
treating these children with a mito cocktail (CoQ10, vitamin B2, etc…) helps even a little with symptoms and course progression.
Chapter 48: For my friends at Phoenix Rising and S4ME
Here are the answers to a few basic questions that you might be asking. Remember, I’m just an ME/CFS patient like you. Therefore, I’m answering based on my knowledge
and research. I’m not a medical doctor, nor a trained researcher.

Question: I have ME/CFS and I have a family history of ME/CFS and I am a J1C, but I didn’t come down with ME/CFS until ten years after my daughter. How does
that fit in with the idea of Mitochondrial Dysfunction?
Answer: People with Mitochondrial Disease and Mitochondrial Dysfunction due to an mtDNA variant(s) can do well with energy until a stressor comes along. The
stressor can be anything from a major illness that wipes you out to an emotional stressor like divorce. Anything that takes a huge amount of energy in any body
system or organ can trigger the cycle. It’s called the threshold effect. The mtDNA is just a piece of the puzzle for some people. Your daughter could have reached
her threshold before you.

Question: I have a family history of ME/CFS and I am a J1C, but it was my father who had the ME. Could the J1C be a fluke? Could it be paternally inherited?
Answer: With J1c’s having lower oxygen uptake, etc…. it’s likely that being a J1c is going to impact it. Therefore, even if the ME/CFS came on for another
reason (maybe chemotherapy induced), the J1C is likely adding a layer of reduced energy on top.
There are many families with ME/CFS where it certainly appears to have a paternal inheritance pattern. It could come down to nuclear genes being passed down
by a father. But there are still too many unknowns for any common consensus. See Chapter 41 for a further discussion of the possibility of paternal inheritance.

Question: I am the only one in my family with ME/CFS. Does this rule out a genetic cause?
Answer: Not necessarily. It could be the interplay between various genes that came to rest in you. You could have one deleterious nuclear DNA variant from
you father and one from your mother. We are in the infancy of genetics. In time, there will be more answers.

Question: Do you believe all ME/CFS has a genetic basis?

Answer: I’m not sure. I go back and forth on the answer depending upon the day. I focused my attention on the genetics side of ME/CFS because of my
significant family history. There are many theories unrelated to genetics that I find very interesting and that I am glad researchers are exploring. The complex
metabolic cycle is dependent upon many things, not all related to DNA.

Question: How does all this research help those of us who do not have another family member with ME/CFS?
Answer: Anything that moves the research forward could potentially help find treatments and cures that can be applied to a wider group. For example, the
research on Type 1 diabetes led to treatments for Type 2 diabetes.

Question: What does this mean for those of us who come from ME/CFS+ families that are not from maternal haplogroups J1c or U5?
Answer: It means the answer may exist in your mtDNA or a combination of your mtDNA plus other variants and/or environmental stressors. If you are
scientifically interested, you might try looking up the variants you have that make you part of your maternal haplogroup and see if any of them are interesting
potential trouble makers. Next, you might take your mtDNA variants and subtract out the ones that are required for your haplogroup. The extra private mtDNA
variants would be worth researching also.

Question: I’m a J1C. Does that mean I will automatically get CFS or autism?
Answer: No, it does not. It just means that your system runs a little differently, you are more likely to live longer and if your ancestors face an ice age, they are
more likely to do well because of this difference. In addition, if you have additional deleterious genetic variant(s) or environmental stressors you may get over the
threshold into ME/CFS. My father and his family are very healthy J1c’s.

Question: I have ME/CFS and I’m a J1C. Does this mean my variants are the cause of the ME/CFS?
Answer: Being a J1C is just part of the key. Being J1C makes one more hypometabolic, yet, there are other factors that impact if any and which symptoms
manifest and there is also the threshold effect.

Q: Can I go to GeneDx and have this analysis done?

Answer: Nope not yet. GeneDx doesn’t even red flag J1c1’s (yet). What they provide is the mtDNA variants in a spreadsheet. They do red flag pathogenic
variants, yet they didn’t red flag our T3394C, so I don’t know where their threshold is for notifications. Right now I see them more as the lab that provides the
data and does a basic analysis for the low hanging fruit (severely pathogenic variants), but that anything beyond that would need to be done outside GeneDx. This
is not anti-GeneDx. It’s that we are in the infancy of genetics and I truly believe GeneDx will eventually help with the workload. But right now the leg work is still
up to us.
Part 8: Supplements
Chapter 49: The Frequency of J1C’s in the general population
There are many ways to estimate how many J1c’s are in a population. I decided to go with the higher estimate of J1c’s being 7% of the population in order to be more
conservative in the findings. I’m not in a position to have a control population, therefore, being conservative and underestimating the findings seemed like the right move.

Here are a few sources for haplogroup J frequencies.

“Haplogroup J is found in 9% of Europeans” (114). Many ancestry sites put J1c’s as 75% of the European J population (20). 9% x .75 = 6.75%. This is close to the more
conservative 7% that I used.

Mitomap’s GenBank Frequency Information (115) has J’s as 7% of the Eurasian population. Within the GenBank participants there are a total of 2,226 J’s. Of those J’s, 1164
(or 52%) are J1c’s. That is much lower than the often mentioned 75%-80% for J1c’s out of European J’s.

The 2016 Cornell study “Mitochondrial DNA variants correlate with symptoms in Myalgic encephalomyelitis/chronic fatigue syndrome” (30), had 10 maternal haplogroup J’s
in their 196 controls (5.10%), and 12 maternal haplogroup J’s in their 193 CFS cases (6.22%). Presuming the J1c’s are 80% of the J population then 5.10% x .80 = 4.08%, and
J1c’s would be 4.08% of the population. The 7% used to estimate J1c’s is again seen as high and conservative.

Quick note – the Cornell study above was not of ME/CFS+ individuals who had family members who were also ill. Therefore, I wouldn’t expect the significant jump in J1c’s
that was seen in my study because the Cornell pool was people with ME/CFS (not ME/CFS patients with a family history).

In this 2012 study out of Italy, the researchers found that:

“Moreover, our sequence analysis confirms the major role of haplogroups J1c and J2b (over 35% in our probands versus 6% in the general population of Western
Europe) and other putative synergistic mtDNA variants in LHON expression." (116).
Therefore, the controls in this study had haplogroups J1c and J2b combined at 6% of the population. Therefore, J1c’s must be less than 6% of the population because we
are not currently looking at J2b’s. The 7% estimate for J1c’s in the population is still conservative.

My study result that “Maternal haplogroup J1c’s were found at 5.4 times the expected frequency in individuals with family histories of ME/CFS (and related symptoms)
is dramatic on its own using the conservative 7% estimate for J1c’s in the general population. The study results would go up higher if one of the lower rates estimating J1c’s
in the population was used.
Chapter 50: The Frequency of U5’s in the general population
U5’s are 7% of the population (43).

Mitomap’s GenBank Frequency Information (115) has U’s at 13% of the Eurasian population. Within the GenBank participants there are a total of 3,986 U’s. Of those U’s,
1827 (or 46%) are U5’s. 13% x .46 = 6%. Therefore, the 7% used to estimate U5’s in the general population is the more conservative number.
Chapter 51: The Probability of being a child of two J1c1’s

23andme states that 1 in every 181 clients (0.55% of the population) are J1c1’s. The chance of a J1c1 partnering with another J1c1
is 0.003% or 1 in 30,000 (0.0055 x 0.0055). The coincidence of my mother and my father both being from maternal haplogroup J1c1
was helpful for my research.
Chapter 52: Converting mtDNA variants that used reference genome hg19 to NC_012920
Currently, most sequencing labs will align data to reference genome hg19. The issue comes when they don’t realign the mtDNA first to the NC_012920 reference genome.
Hg19 used the Yoruban positions for mtDNA and they must be converted to rCRS positions.

If someone comes up as having the pathogenic mtDNA variant 12706 for Leigh’s, then it’s very likely that the lab didn’t take the time to convert the data to the current
mtDNA positions using NC_012920. We have had multiple labs provide the VCF data on flash drives with the mtDNA aligned to the Yoruba positions. In fact, GeneDx even
sent out the VCF with it still aligned to Yoruban positions. However, in e-mailing GeneDx, they were quick to e-mail over a spreadsheet of the mtDNA variant table aligned
to the NC_012920.

When dealing with mtDNA aligned to the Yoruban reference genome, here is how Mitomap suggests you convert the individual positions (111). Be sure to follow the link in
order to catch any changes that Mitomap might make.

The last columns in my Figure 7 and Figure 9 charts show the conversion for the haplogroup J positions.

The two private mtDNA variants that my maternal line carries (beyond J1c1 defining variants) of 9448A>G and 14290T>C were correct on the GeneDx mtDNA table that was
e-mailed to me. However, the VCF files for the WES and WGS that I received from two separate labs both had these positions aligned to Yoruban at 9449 and 14291.

GeneDx sent me an e-mail stating that:

“The exome data is aligned to hg19 which uses NC_001807. However, we do not recommend analysis of chrM data from the WES capture since alignment specificity
can be poor due to homology with the nuclear genome. We do not use this data for our mitochondrial genome analysis. Instead we perform long-range PCR to
specifically amplify the mitochondrial genome. Our mitochondrial sequencing analysis is with respect to NC_012920.”
The spreadsheet they sent with my mtDNA variants was called “the mitochondrial (mtDNA) component of the XomeDxPlus testing”.

While GeneDx suggests not using their mtDNA exome data, we did find that for the three family members for whom we had GeneDx sequence the exome, that the VCF
positions were accurate after re-aligning to NC_012920. However, the read depth and quality scores were very low compared to what we have received from other labs.

Studies have shown that high read depth and quality scores can overcome issues with mtDNA exome sequences.
“Mitochondrial DNA sequences can be reliably obtained using three different whole-exome sequence capture kits. Coverage correlates with the relative amount of
mitochondrial DNA in the original genomic DNA sample, heteroplasmy levels can be determined using variant and total read depths, and—providing there is a
minimum read depth of 20-fold—rare sequencing errors occur at a rate similar to that observed with conventional Sanger sequencing.
….
Here we show that off-target WES reads can be used to reliably determine the mtDNA sequence, providing there is adequate read depth. With >20-fold mean
coverage, the error rate is only marginally higher than that of conventional diagnostic Sanger sequencing, and if the WES errors that repeatedly occur at the poly-C
sequence tract are taken into account, the error rate is marginally lower in WES as compared to Sanger sequencing. Given the potential role of mtDNA mutations in
a broad range of human disease phenotypes, we suggest that mtDNA analysis should be included in a diagnostic exome analysis pipeline.” (113)

For my position 9448A>G, GeneDx’s read depth was 8 and the quality score was only 24, while the other lab that did my WGS had a read depth of 235 and a quality score of
99.

For those looking for ease, having a lab that creates the spreadsheet without the need for the conversion is a real time saver. At the same time, having the spreadsheet
doesn’t provide the counts that helps determine heteroplasmy.

This conversion issue is just one of the potholes we have to deal with being on the front lines of the genetics field.
Chapter 53: Other Haplogroups to keep in the back of minds for potential subclades with OXPHOS issues
I don’t believe that J1c’s and U5’s are going to be the only subclades eventually found to impact ME/CFS. When I find studies that mention a haplogroup in a similar vein to
J1c’s or U5’s I take note so that if I run into that haplogroup later it will ring a bell in my mind.

Here are a few haplogroups in which subclades impacting ME/CFS might eventually be found and the reasons why they are on my to be watched list.

Other subclades within Haplogroup J and J1

Other subclades within Haplogroup J and J1 might be found to increase the risk for ME/CFS due to their impact on mitochondrial metabolism. Haplogroup J has contains
interesting variants 10398A>G and 13708G>A. J1 has interesting variant 3010G>A.

Haplogroup J1d1a
Haplogroup J1d1a should be specifically kept in mind. Remember in the study “Inherited mitochondrial DNA variants can affect complement, inflammation and apoptosis
pathways: insights into mitochondrial–nuclear interactions” which found my dad’s J1c1 + private variants 7226G>A, 7747C>T, 13143T>C, and 16209T>C related to macular
degeneration. That study also found a relation between J1d1a cybrid with m.2305T>C (unique, MT-RNR2) and m.10654C>T (A-V, MT-ND4L) (9).

Since dad’s was J1c1 plus certain variants = macular degeneration and in mom’s side, J1c1 plus other variants = ME/CFS+, then could J1c7 also have other some other
unknown variants that could increase risk for ME/CFS+? It just something to keep in mind if you run across J1d1a.

Haplogroup J1c7
J1c’s are known to have an increased risk for ME/CFS+. This following finding reinforces the topic. In the same study above, J1c7 plus variants 6734G>A, 12072T>C, and
16209T>C were associated with macular degeneration (9). It wouldn’t surprise me if subclade J1c7 was in the future found in ME/CFS+ families. We didn’t have any J1c7’s
in my study.

Haplogroup J2
Throughout the book, studies related to Haplogroup J have been interesting, specifically because of variants 10398A>G and 13708G>A. Remember this study which found
“Our results suggest that individuals from haplogroups Uk, J1c and J2 are more susceptible to LHON because they have reduced OXPHOS capacity, which results in part from
lower mtDNA levels.” (5)

The fact that J2 is part of haplogroup J and studies like this one mentioned above with increased risk of LHON, it raises the possibility in my mind that individuals from
haplogroup J2 could be found in future ME/CFS+ families.

This idea is reinforced by other studies that also find similar patterns to J1c’s. This study provides some patterns in J2’s such as lower ROS and increased longevity that
were also found in J1c’s.
“However, we have found that the cybrids with haplogroups that include the C150T transition have in common a lower reactive oxygen species (ROS) production
rate than the haplogroup-matched cybrids without that transition. Thus, the lower ROS production rate may be a factor in the increased longevity associated with
the U and the J2 haplogroups. Of further interest, we found that cybrids with the U3a haplogroup exhibited a higher respiration rate than the other cybrids
examined.” (19).

Haplogroup K
I strongly believe that there is subclade hiding in haplogroup K that strongly impacts ME/CFS+. This study found that “J1c and K are underrepresented in patients with
Parkinson’s disease and over-represented in centenarians and in patients with LHON and multiple sclerosis, indicating a similar physiological characteristic of these
haplogroups” (24).

If several diagnostic variants for haplogroup K (that were not defining variants for other haplogroups) were found at increased rates in ME/CFS patients, that would raise the
possibility that haplogroup K could contain of subclade with increased risk for ME/CFS+.

In the 2016 Cornell study “Mitochondrial DNA variants correlate with symptoms in myalgic encephalomyelitis/chronic fatigue syndrome” (30), on “Additional file 5: Table S3.
Association analysis of mtDNA SNPs within ME/CFS status” there were several variants that were diagnostic variants for only haplogroup K that had higher odds ratios with
regards to ME/CFS. For example, base pair 3480G is only a defining variant for haplogroup K and it had a 1.563 odds ratio. 1189C is only a defining variant for K1 and had a
1.575 odds ratio. Variants 11299, 10550, 9055, 16224, and 14167, are all defining variants for only haplogroup K and each of their odds ratios ranges from 1.35 to 1.39.
14798C is found in J’s and K’s and had an odds ratio of 1.413. As I continue to research ME/CFS, I’m going to remain on the lookout for haplogroup K subclades that could
be impacting ME/CFS. K’s had more increased risk for ME/CFS defining variants on this table than other haplogroups.

This study found an association between haplogroup K and multiple sclerosis (110). With the similarities between MS and ME/CFS this is just another indicator that there
may be a subclade within haplogroup K similar to J1c that increases risk of ME/CFS.

I also wonder about the haplogroup K subclade that has the extra variant 9448A>G that my ME/CFS+ family has -- but rather than having it as a private variant, haplogroup
k1a4b has it as a defining variant. If a K1a4b popped up among future ME/CFS study participants, it should be noted and a second look taken to see if 9448A>G had an
impact. At the same time, k1a4b might have mitigating variants that offset the increased risk. For example, in this study of the pesticide rotenone found haplogroup J1 to
be the most sensitive, while haplogroup “K1 mitogenes orchestrate the best compensation, possibly because of the haplogroup-specific missense variants impinging on
Complex I function.” (87). So it could be this ability by K1 could overcompensate for the 9448A>G and therefore, the threshold just isn’t reached. Lots of if’s and but’s in
here. Just background noise that can’t be differentiated until more data is added.

Haplogroup T
Haplogroup T also appeared along with haplogroup J in some of the studies. I wouldn’t be surprised if an increased risk subclade was found in haplogroup T. In the study
“Mitochondrial DNA Haplogroups Associated with Age-Related Macular Degeneration” (8), found:
“The control region SNPs T16126C and A73G, commonly found in haplogroups J and T, were more frequent in the AMD retinas than in normal retinas. The
associations between AMD and haplogroups J and T were confirmed and extended by analysis of blood DNA. SNPs at position a T16126C (J; odds ratio [OR] =
3.66), T16126C+G13368A (JT; OR = 10.27), A4917G+A73G (T4; OR = 5), and T3197C+A12308G (U5; OR = infinity), were all strongly associated with AMD”

This study on Multiple Sclerosis discussed not only haplogroup J, but also haplogroup T. (104).
“Two large studies previously identified MS associations among haplogroup T (m.4917A>G) carriers from 523 MS cases11 and haplogroup J (m.13708G>A) carriers
from 1,099 MS cases.”

This study that discussed J’s and cognition, also found an association with T’s:
“Participants from haplogroup T had a statistically significant increased risk of developing dementia (OR = 1.86, 95% CI = 1.23, 2.82, p = 0.0008) and haplogroup J
participants experienced a statistically significant 8-year decline in 3MS (Modified Mini-Mental State Examination) ( β = -0.14, 95% CI = -0.27, -0.03, p = 0.0006),
both compared with common haplogroup H.” .” (7)
“You make observations,
write theories to fit them,
try experiments to disprove the theories and,
if you can't,
you've got something.”
Kary Mullis (Nobel Prize-winning biochemist, inventor PCR
technique)

“I’m humbled that anyone would take the time to read my work. I’ve tried my best to keep my mind from jumping around and to
write in a straight line. Please forgive all errors, including: spelling, grammar, lack of basic science knowledge, etc…. I don’t have
a staff, or a lab, or an editor. I’m going to go collapse now. Thank you for your interest!”
B. Day
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http://mitimpact.css-mendel.it/

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46) CADD
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47) Condel
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48) dbSNP for rs28357681

https://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=rs28357681

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55) dbSNP for pathogenic variant rs267606611 9438G>A (LHON).

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56) dbSNP for rs587776437 9478T>C (Leigh Disease).

https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=587776437

57) dbSNP for rs267606612 9487_9501del.

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58) Mitomap’s “Clinical Phenotypes (non-LHON) Associated with mtDNA Polypeptide Gene Mutations Reported in the Literature”. Includes exercise intolerance.
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69) MitoMap’s SNV Query. (Enter variant and submit. Click on “query” that pops up on bottom left of page. Provides frequency and conservation for the position.
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