Current and Previous Research on ME/CFS at Cornell University Maureen R

2016 Webinar Series | Thursday, September 1, 2016 | 3:00 PM Eastern Current and Previous Research on ME/CFS at Cornell University Maureen R. Hanson, PhD Cornell University

www.SolveCFS.org About Our Webinars • Welcome to the 2016 webinar series! • The audience is muted; use the question box to send us questions • Webinars are recorded, and the recording is made available on our YouTube channel: http://YouTube.com/SolveCFS • The Solve ME/CFS Initiative does not provide medical advice Save the Dates!

• Thursday, September 8: Beth Unger, MD • Beth Unger, MD, chief of the Chronic Viral Diseases Branch (CVDB) at the National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), a part of the Centers for Disease Control and Prevention (CDC) • Thursday, October 20: Christopher Armstrong, PhD • Christopher Armstrong, PhD, from the Bio21 Institute, Department of Biochemistry and Molecular Biology at the University of Melbourne • Thursday, November 10: Anthony L. Komaroff, MD • Anthony Komaroff, MD, professor of medicine at Harvard Medical School and senior physician at Brigham and Women’s Health Hospital in Boston, Massachusetts 2016 Webinar Series | Thursday, September 1, 2016 | 3:00 PM Eastern Current and Previous Research on ME/CFS at Cornell University Maureen R. Hanson, PhD Cornell University

www.SolveCFS.org Current and Previous Research on ME/CFS at Cornell University

Maureen Hanson, Ph.D. Dept. of Molecular Biology and Genetics

Lab website: http://hansonlab.org/research/cfs_me/ Major Questions

How does mitochondrial genetic variation affect susceptibility to ME/CFS and/or its subtypes?

How do white blood cells differ between ME/CFS patients and healthy individuals? Gene expression Mitochondrial function

How is metabolism affected in ME/CFS?

How is gastrointestinal function affected in ME/CFS? A Genetic Hypothesis Regarding ME/CFS

The diversity of symptoms of ME/CFS patients may be due to genetic differences in victims who have been exposed to a single or to different inciting factor(s) Computational Nutritional Biology Sciences Mitochondrial Genomes in ME/CFS

Do some patients with ME/CFS identified by expert M.D.s actually have a genetic mitochondrial disease? 193 cases, 196 controls, age- and gender-matched NO

Patients identified by Lucinda Bateman, Donna Felsenstein, Nancy Klimas, Anthony Komaroff, Susan Levine, and Daniel Peterson Findings from a clinical and laboratory database developed for discovery of pathogenic mechanisms in myalgic encephalomyelitis/chronic fatigue syndrome N.G. Klimas, G. Ironson, A. Carter, E. Balbin, L. Bateman, D. Felsenstein, S. Levine, D. Peterson, K. Chiu, A. Allen, K. Cunningham, C.G. Gottschalk, M. Fletcher, M. Hornig, C. Canning & A.L. Komaroff. 2014 Fatigue: Biomedicine, Health & Behavior 3:75-96 Mitochondrial genomes often differ between normal people

SNP: Single Nucleotide Polymorphism—a single change in the DNA code Mitochondrial Genomes in ME/CFS

Can mitochondrial genome variation be correlated with susceptibility to ME/CFS? NO

Is mitochondrial genome variation correlated with particular ME/CFS symptoms or their severity? YES SNPs on human mitochondrial genomes are known to be harmful (cause true mitochondrial diseases)—but the effect of some SNPs is unknown

SNPs1719, 3010 SNPs 16093, 16223, 16519 Presence of SNPs in patients was compared to symptoms and their severity

Applied to 270 different symptom SNP 1719 SNP16519 scores collected from patient questionnaires

8 mtDNA SNPs (out of 70) were associated with 16 symptom categories

1719A allele was associated A G C T with higher symptom scores in seven categories, all related to inflammation and neuroinflammation

16519C was associated with more severe symptoms relating to gastrointestinal issues such as bloating and A G C T abdominal pain Conclusions: Mitochondrial Genetic Variation

In the CFI cohort, certain mtDNA SNPs could be correlated with symptom type and severity, implying that symptom variation may depend on genetic background

Analysis of mtDNAs in a larger cohort should be carried out for verification and further probing of mtDNA variation.

Mitochondrial function is also controlled by nuclear genes: examining genetic variation in nuclear genes encoding mitochondrial proteins may also be informative Abnormalities in White Blood Cells in ME/CFS Types of white blood cells Gene expression in B, T, and NK cells Project in progress

Fabien Campagne Rita Shaknovich Automated Weill Cornell Medical College sorting of white blood cells

David Ruppert Andrew Grimson Susan Levine Cornell-Ithaca

RNA extraction and Sequencing

Analysis: Differences between ME/CFS and healthy individuals? Mitochondrial Function in B, T, and NK cells Project in progress

David Ruppert Avery August Cornell-Ithaca

Differences between patients’ B, T, and NK cells and healthy individuals?

Susan Levine How does metabolism differ in ME/CFS cases vs. controls?

Wikimedia Commons 612 Metabolites

40 males Cohort size 44 females

28 metabolites Significance (Q<0.10) 25 metabolites

86% are decreased Hypometabolic state 84% are decreased

Affected Pathways Common between Males and Females Sphingolipids Females Males Phospholipids Fatty Acid Oxidation Serine/1-carbon Metabolism Glycosphingolipids Vitamin C/Collagen SAM, SAH, Met Purines Bile Acids Very long Chain FAO Microbiome Endocannabinoids Propiogenic AA Cholesterol Vitamin B12 Threonine Vitamin B2 (Riboflavin) Amino sugars P5C, Arginine, Proline Branch Chain AA Cornell pilot study on Title: Metabolic profiling of a myalgic plasma metabolites: encephalomyelitis/chronic fatigue syndrome under review at a journal discovery cohort reveals disturbances in fatty acid and lipid metabolism

Susan Levine David Ruppert

Arnaud Germain

17 patients, 15 controls: all female Jason Locasale 361 metabolites Most significantly different metabolites are lower in ME/CFS patients than in controls

1e+08

1e+07

1e+06

Controls Patients

of metabolic score 1e+05 10 Log 1e+04

1e+03

Metabolites Metabolic profiling of a myalgic encephalomyelitis/chronic fatigue syndrome discovery cohort reveals disturbances in fatty acid and lipid metabolism

612 Metabolites 361

44 Female cohort size 32

37 metabolites Significance (Q<0.15) 33 metabolites

84% are decreased Hypometabolic state 88% are decreased

Affected Pathways Sphingolipids Common Glycosphingolipids Phospholipids Other AA Microbial products Purines Energy metabolism Cholesterol P5C, Arginine, Proline Sugar metabolism Vitamin B2 (Riboflavin) Branch Chain AA Fatty acid metabolism Vitamin C/Collagen Fatty Acid Oxidation Other metabolites (e.g. Endocannabinoids Bile Acids Vitamin E pathway) Vitamin B12 Amino sugars Ley Goodrich Walters Levine Giloteaux Study population Established ME/CFS patients of Dr. Susan Levine (Manhattan, New York)

GENDER: Controls 30 female, 9 male Patients 38 female, 11 male sudden onset: 25

median [range] AGE

Controls 48 [20-61] Patients 51 [19-71]

Body mass index

Controls 26 [17-47] Patients 24.5 [16-40]

INTESTINAL DISCOMFORT: Controls 8/39 Patients 32/47 The human microbiota

As many microbial cells as human cells

Majority in large intestine

Provide enhanced nutrition and protection against pathogens Many ME/CFS patients complain of gastrointestinal symptoms

Is abnormal gastrointestinal function causing inflammation?

Does the bacterial gut microbiome of ME/CFS patients differ from healthy controls? Some bacteria have lipopolysaccharides (LPS) on their surfaces

LPS Jeff Dahl (Wikipedia) Bacteria can escape from a dysfunctional gut LPS and LPS-related proteins are elevated in ME/CFS cases vs. controls

Receptor that detects LBP: Protein in blood LPS bound to LBP that binds to LPS Data imply ongoing damage to the gut, leading to increased microbial translocation in ME/CFS

Significantly raised levels of plasma lipopolysaccharide (LPS)

Significantly higher levels of sCD14 and LBP, as indicators of effect of LPS

Other diseases in which elevated LPS has been reported:

Alcoholic and non-alcoholic steatohepatitis (fatty liver disease) HIV infection Crohn’s disease Ulcerative Colitis Analysis of gut bacterial microbiomes

CONTROL

DNA 16S rRNA PCR MiSeq Sequencer

PATIENT ~ 140,000 sequences per sample

Fecal samples collected, preserved and shipped by subjects themselves and processed at Cornell DNA sequencing can usually reveal the family and sometimes the genus and species of bacteria that are present

Different species not always identified by sequencing—so any bacteria with 97% identical sequences are grouped together

http://www.123rf.com/

Foxes: Y.S. Krishnappa and (Wikipedia) Bacterial diversity within the ME/CFS patient cohort vs within the healthy cohort

Loss of species richness in ME/CFS microbiomes

The more sequences that are obtained, the more species are discovered 24 Families and Genera Differentially Abundant between ME/CFS and healthy individuals—and statistically significant Members of the Ruminococcaeae were significant higher in healthy individuals than ME/CFS patients

Some members of the Ruminococcaceae:

Produce an anti-inflammatory protein

Produce butyrate, an anti-inflammatory fatty acid

Lower levels of butyrate-producing bacteria and butyrate also seen in Crohn’s disease and Ulcerative Colitis A test for disease status: combination of levels of LPS, LBP, sCD14 levels with microbiome composition

M.D. diagnosis: 55% ME/CFS, 45% healthy

Test ME/CFS Normal Total result test is better at correctly ME/CFS 53% 5% 58% identifying ME/CFS than correctly identifying Normal 12% 30% 42% controls

100%

Correctly diagnosed by microbiome/inflammation test Limitations of Bacterial Microbiome Analyses

Data inadequate to reveal whether a particular bacterial strain is differentially present in patients vs healthy individuals

Bacterial microbiome studies do not reveal what eukaryotic pathogens or beneficial species may be present—yeasts, parasites, etc.

Data does not indicate whether viruses are differentially present Conclusions: Gut Bacterial Microbiome

Less bacterial diversity present in patients compared to healthy population

Association of abundance of specific bacterial groups with ME/CFS or with healthy status

83% of the samples could be correctly classified as coming from either patients or healthy individuals from combined blood and gut assays

Anti-inflammatory bacterial species are reduced in ME/CFS patients Ithaca College

A Pair of Identical Twins Discordant for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Differ in Physiological Parameters and Gut Microbiome Composition

Ludovic Giloteaux, Maureen R. Hanson, Betsy A. Keller

American Journal of Case Reports, in press

Caucasian male monozygotic twins, age 34

ILL twin with ME/CFS for 4 years

Two cardiopulmonary exercise tests (CPETs): Ithaca College Dept Exercise and CPET1: ILL twin VO2peak 7.5% lower than WELL Sport Sciences

CPET2: ILL twin VO2 at anaerobic threshold 13% lower than in CPET1: exertion intolerance Ithaca College

ILL twin has reduced gut Changes between WELL and ILL microbiome diversity twin are similar to the larger study

ILL

WELL ILL What attempts are being made to alter microbiomes in various diseases?

Diet

Prebiotics

Probiotics

Antibiotics

Fecal transplants http://www.ncbi.nlm.nih.gov/pubmed/25525379 2014 Abundant Evidence for Biological Disruption in ME/CFS

Aberrant functioning of white blood cells

Abnormal physiological responses to exercise

Dysbiosis of gut microbiota

Altered metabolism

Anomalous images from brain scans Inappropriate images of ME/CFS give distorted perception to the public

http://www.lastampa.it/2016/07/07/scienza/be nessere/la-stanchezza-cronica-dipende- http://scienze.fanpage.it/la-sindrome- dallintestino- da-fatica-cronica-e-una-questione-di- http://www.quotidianosanita.it/scienza-e- SgtFrqNizYJDx0opJm5jlJ/pagina.html intestino-non-di-testa/ farmaci/articolo.php?articolo_id=41137

http://www.financialexpress.com/lifestyle/health http://indianexpress.com/article/india/india-news- /chronic-fatigue-syndrome-linked-to-gut- http://www.tv3.ie/xpose/article/fitness-and- india/chronic-fatigue-syndrome-gut-bacteria- bacteria/299451/ wellbeing/208213/Chronic-fatigue- 2881497/ syndrome-linked-to-gut-bacteria Additional information about the Cornell ME/CFS microbiome study:

Presentation at InvestinMe 2016 “The Search for Biomarkers for Myalgic Encephalomyelitis”

DVD of entire conference can be ordered at http://www.investinme.eu/iimec11.shtml

Upcoming presentation at online conference sponsored by 2nd Annual Microbiology & Immunology 2016 Virtual Conference

September 8, 12:00 ET, 9:00 PT register free here: http://www.labroots.com/virtual-event/microbiology-immunology-2016

“The Gut Microbiome in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, A Neuroimmune Disease” 12th International IACFS/ME Research and Clinical Conference: Emerging Science and Clinical Care October 27-30, 2016 Fort Lauderdale, Florida

More information will be presented at IACFS/ME conference

Ludovic Giloteaux (talk) Alterations in the enteric bacterial and viral microbiome in ME/CFS

Maureen Hanson (talk) Polar metabolites distinguish ME/CFS patients and controls

Alexandra Mandarano (poster) Eukaryotes in the ME/CFS gut microbiome Center for Enervating NeuroImmune Disease

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About the Cornell ENID Center Foremost among cryptic neuroimmune diseases is one variously known as Myalgic Encephalomyelitis or Chronic Fatigue Syndrome or Systemic Exertion Intolerance Disease. The Center’s mission is to promote research to identify its cause(s), biomarkers, and pathophysiology in order to lead to prevention and effective treatments.

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