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Dr. Kathleen Light Dr. Andrea White Dr. Lucinda Bateman Dr. Ila Singh Ron Hughen

OFFER 2011 Gene Expression Biomarkers for Chronic Fatigue and Fibromyalgia Syndromes-Latest Developments. Dr. Kathleen Light Dr. Andrea White Dr. Lucinda Bateman Dr. Ila Singh Ron Hughen. Dr. Alan R. Light Dept. Anesthesiology University of Utah.

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Dr. Kathleen Light Dr. Andrea White Dr. Lucinda Bateman Dr. Ila Singh Ron Hughen

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  1. OFFER 2011Gene Expression Biomarkers for Chronic Fatigue and Fibromyalgia Syndromes-Latest Developments Dr. Kathleen Light Dr. Andrea White Dr. Lucinda Bateman Dr. Ila Singh Ron Hughen Dr. Alan R. Light Dept. Anesthesiology University of Utah Supported by generous grants from OFFER, CFIDS, AFSA, individual donors

  2. CDC Definition: Chronic Fatigue Syndrome • Chronic or remitting/relapsing fatigue lasting 6 months or more that severely impairs functioning at least 50% of the time, and is not relieved by rest or recovery • Also must have 4 or more of these 8 symptoms: • 1. Impairment of memory or concentration, • 2. Sore throat; 3. Tender lymph nodes, • 4. Muscle pain, 5. Joint pain, 6. Unrefreshing sleep, • 7. Change in type of headaches, • 8. Symptoms worse >24 hours after physical exertion. • Often preceded by a “viral” infection? Many CFS patients (~70%) also have fibromyalgia (FMS)

  3. Am Coll Rheumatology Definition: Fibromyalgia • Widespread muscle/connective tissue pain: bilateral, upper and lower body for 3 months • Hypersensitivity to pressure at tender points (at least 11 of 18 TPs) and other body sites. • Symptoms not worse after exercise

  4. Physical Fatigue before during and after exercise Mental Fatigue before during and after exercise 70 70 70 CFM 60 60 60 CFM CFS 50 50 50 CFS FM FM 40 40 40 30 30 30 20 20 20 10 10 10 Controls Controls Visual analog scale (+ SEM) Visual analog scale (+ SEM) Visual analog scale (+ SEM) 0 0 0 mid ex mid ex after after 30 min 30 min 8 hr 8 hr 24 hr 24 hr 48 hr 48 hr mid ex after 30 min 8 hr 24 hr 48 hr baseline baseline baseline Pain before during and after exercise CFM FM CFS Controls

  5. Problem: because the major symptoms, Fatigue and Muscle pain are subjective: • How to objectively determine CFS and FMS • Solution: • Use gene expression profiling of genes related to the symptoms of fatigue, muscle pain, and immune dysfunction

  6. ASIC3 * P2X4 ** CFS only n=9 (without Ad2A decrease patients) P2X5 * TRPV1 * AD2A *** ADB1 * ADB2 *** COMT ** IL6 IL10 ** 4 TNFβ Controls n=49 TLR4 CD14 1 .7 baseline 30 min 8 hr 24 hr 48 hr 48 hr baseline 30 min 8 hr 24 hr 10 4 (* P<.05, ** P<.01, *** P<.001 compared to controls for AUC) Fold increase in mRNA from baseline(+SEM) 1 48 hr baseline 30 min 8 hr 24 hr CFS co-morbid with FMS n=25 (without Ad2A decrease patients) 10 4 1

  7. CFS co-morbid with FMS n=9 (Ad2A decrease patients 38% of all CFS-FMS patients) 4 Fold increase in mRNA from baseline(+SEM) 1 .7 ASIC3 ASIC3 ** P<.01 compared to controls for AUC) P2X4 P2X4 (One patient removed because is ASIC3 outlier) P2X5 P2X5 TRPV1 TRPV1 AD2A ** AD2A ** ADB1* ADB1 CFS only n=6 (Ad2A decrease patients 40% of all CFS only patients) ADB2 ADB2 COMT COMT IL6 IL6 4 IL10 IL10 TNFβ TNFβ 4 TLR4 TLR4 CD14 CD14 Controls n=49 Fold increase in mRNA from baseline(+SEM) Fold increase in mRNA from baseline(+SEM) 1 baseline 30 min 8 hr 24 hr 48 hr 1 .7 0.4 48 hr 48 hr baseline baseline 30 min 30 min 8 hr 8 hr 24 hr 24 hr ** P<.01 compared to controls for AUC) A Major Subgroup ! (39% of all CFS patients) A Major Subgroup ! This subgroup decreases AD2A Otherwise no gene changes! (39% of all CFS patients)

  8. P2X4 TRPV1 IL10 P<.004 P<.004 P<.029 .016 ASIC3 .25 P2X4 P2X5 TRPV1 .2 .012 AD2A ADB1 ADB2 .15 COMT .008 IL6 IL10 .1 TNFβ Amount mRNA relative to TF2B (+SEM) .006 TLR4 .004 CD14 .05 FM FM FM con con con .004 0 0 .002 baseline 30 min 8 hr 24 hr 48 hr 0 FMS only n=18 2 Fold increase in mRNA from baseline(+SEM) 1 n.s. for all AUC compared to controls What about patients with only Fibromyalgia--- and not much fatigue! (Larger group of patients) Baseline mRNA amounts in FM only patients compared to controls (N=18)

  9. CFS+FMS patients grouped by clinical severity 50 CFS-FMS patients N=3 40 N=11 Controls 30 Increase from baseline in Sum of mRNA for all times ASIC3 P2X4 P2X5 20 TRPV1 N=14 AD2A ADB1 N=4 ADB2 COMT IL6 10 IL10 TNFβ TLR4 CD14 0 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 1 2 3 4 Gene expression tracks the severity of CFS * P<.01 Greater Less Severity

  10. But what are we measuring?And Why? • What is gene expression? • How does it differ from genetic causes? • What causes changes in gene expression? • Why is gene expression changed in CFS and FMS? • How does it help CFS patients to know their gene expression is abnormal?

  11. Genetics vs. Gene expression • Genetics is the result of the DNA coding you inherited from your parents • The genetic code of your DNA you inherited is (initially at least) the same in all cells in your body • The differences in expression of these genes is why you have tissues as different as your heart, kidney, bones and brain.

  12. What is Genetics? The cell nucleus contains the DNA you inherited from your parents

  13. What is Genetics? The DNA is what makes up chromosomes, and is usually stringy with many windings and meanderings around proteins located in the nucleus

  14. The Code of Life… • The “code” of the chromosome is the SPECIFIC ORDER that bases occur. A T C G T A T G C G G…

  15. Genetics • This “code” or ordering of base pairs in the DNA is what defines all of the proteins (and more) that make up your body • The winding around the nuclear proteins and other special proteins called transcription factors are different in all tissue types and is what makes different tissue types what they are • It makes the brain the brain, bones bones, etc.

  16. In order for the DNA to make proteins that make up your body it first must be transcribed to RNA

  17. DNA Transcription • DNA can “unzip” itself and an RNA copy of a short segment of the DNA code can be made. • This RNA copy leaves the nucleus of the cell and directs the formation of a protein using the code it copied from the DNA

  18. The DNA code is inherited but the amount of RNA made is affected both by the code, and by the environment

  19. Muscle Chemicals Cells can receive a signal, for example the chemicals your muscles make when they are working Transcription factor This signal can activate transcription factors that binds to special regions of The DNA, causing increases or decreases in transcription. Increases in transcription causes increases in RNA Increases in RNA causes Increases in protein

  20. If the protein being made is what signals the brain that your muscle is tired, then you will sense more muscle fatigue • What we decided to measure as a biomarker for Chronic Fatigue Syndrome and Fibromyalgia • was the RNA that makes the proteins that signal muscle fatigue and muscle pain

  21. Why measure RNA? • RNA is easier to measure than the signaling proteins because it doesn’t take much of these proteins to do the signaling. • Rapid and accurate methods to measure RNA have been perfected in the past 10 years

  22. RNA changes can give us clues to what causes the body to increase fatigue signaling. • RNA changes can also give us clues to more permanent changes in the winding and regulation of the DNA that can occur with diseases and aging

  23. Why did we measure RNA in white blood cells (leukocytes) • We can obtain white blood cells easily • Brain and sensory neurons that innervate muscles do not grow back—removing them is not advised • White blood cells also are signaled by the chemicals made in your muscles (Tissue donation site) http://www.ndriresource.org/Donor_Programs/Individual_Donors/37/

  24. Why did we measure RNA in white blood cells (leukocytes) • When signaled, WBCs make chemicals (cytokines) that cause sensory neurons to become more sensitive • WBCs are immune function cells that can increase fatigue (through cytokines) from a variety of diseases and trauma.

  25. Why WBCs continued • Several groups think this may be the case in CFS • So WBCs may be a primary cause of fatigue in some conditions

  26. So, why is gene expression changed in CFS and FMS? • We don’t know. • Because there is not strong genetic influence (not many strong family relationships), we believe that most causes for CFS and FMS are environmental • Viruses cause gene expression changes in many types of tissues • Injury and stress cause gene expression changes • Toxins and allergens cause gene expression changes

  27. Back to the Beginning • OK, so now that I have explained what we measure and why we measure it, lets go back to our findings

  28. Brief Outline of Protocol Testing: • A) Baseline pre-exercise blood draw and Numerical Ratings of Mental and Physical Fatigue and Pain (0-100). • B) Whole-body Airdyne bike exercise to 70% age predicted max HR for 25 min; HR and Work rate (kcal/kg/min) monitored every min, Ratings of Perceived Exertion (RPE, 0-10 VAS scale) every 5 min, BP monitored every 7 min, and Numerical Mental and Physical Fatigue and Pain ratings (0-100) at mid-task and end-task. • C) Post-exercise blood draws at 0.5, 8, 24 and 48 hours with ratings of mental and physical fatigue and pain (0-100). • D) Tender Point Exam and Questionnaires: ACR FMS Diagnostic Symptoms, CDC Symptom Inventory, MFI, MOS Short form-36, Beck Depression, State-trait Anxiety, Margoles Pain Chart

  29. From each blood draw, we obtain the same measures: • Gene expression (mRNA) levels on leukocytes relative to housekeeping gene TF2B for the following. • Sensory receptors: ASIC3, P2X4, P2X5, TRPV1. • Adrenergic receptors and enzymes: Alpha-2a, Beta-1, Beta-2, and COMT. • Cytokines/cytokine receptors: IL-6, IL-10, TNFβ, CD14, Toll-like receptor-4 (TLR4).

  30. 4 Controls n=48 1 CFS and CFS co-morbid with FMS n=34 (without Ad2A decrease patients) 10 .7 n.s. for all AUC compared to baseline 4 ASIC3 ASIC3 P2X4 **** P2X4 1 P2X5 * P2X5 (* P<.05, ** P<.01, *** P<.001, **** P<.0005 compared to controls for AUC) TRPV1 TRPV1 ** AD2A AD2A **** ADB1 ADB1 ** ADB2 ADB2 **** COMT **** COMT IL6 IL6 IL10 IL10 *** TNFβ TNFβ TLR4 TLR4 CD14 CD14 baseline 30 min 8 hr 24 hr 48 hr 48 hr baseline 30 min 8 hr 24 hr Fold increase in mRNA from baseline(+SEM)

  31. 4 Controls n=48 1 .7 Major Subgroup (30% of patients) n.s. for all AUC compared to baseline ASIC3 ASIC3 ASIC3 P2X4 P2X4 P2X4 P2X5 P2X5 P2X5 TRPV1 TRPV1 TRPV1 AD2A ** AD2A AD2A ADB1 ADB1 ADB1 ADB2 ADB2 ADB2 COMT COMT COMT 4 IL6 IL6 IL6 IL10 IL10 IL10 TNFβ TNFβ TNFβ TLR4 TLR4 TLR4 CD14 CD14 CD14 1 baseline 30 min 8 hr 24 hr 48 hr CFS and CFS co-morbid with FMS n=13 (Ad2A decrease patients) baseline 30 min 8 hr 24 hr 48 hr 48 hr baseline 30 min 8 hr 24 hr FMS only n=18 2 1 n.s. for all AUC compared to controls

  32. P2X4 TRPV1 IL10 P<.004 P<.004 P<.029 .016 ASIC3 .25 P2X4 P2X5 TRPV1 .2 .012 AD2A ADB1 ADB2 .15 COMT .008 IL6 IL10 .1 TNFβ Amount mRNA relative to TF2B (+SEM) .006 TLR4 .004 CD14 .05 FM FM FM con con con .004 0 0 .002 baseline 30 min 8 hr 24 hr 48 hr 0 FMS only n=18 2 Fold increase in mRNA from baseline(+SEM) 1 n.s. for all AUC compared to controls What about patients with only Fibromyalgia--- and not much fatigue! (Larger group of patients) Baseline mRNA amounts in FM only patients compared to controls (N=18)

  33. Severity Ratings • 4= able to hold a part-time job and do the majority of self-care activities. • 3= unable to work on a regular basis even part-time except occasionally during partial remissions but still able to do the majority of self-care activities; • 2= unable to work outside the home and needing help with most self-care; • 1=fully disabled and needing round-the-clock assistance.

  34. CFS+FMS patients grouped by clinical severity 50 CFS-FMS patients N=3 40 N=11 Controls 30 Increase from baseline in Sum of mRNA for all times ASIC3 P2X4 P2X5 20 TRPV1 N=14 AD2A ADB1 N=4 ADB2 COMT IL6 10 IL10 TNFβ TLR4 CD14 0 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 1 2 3 4 Gene expression tracks the severity of CFS * P<.01 Greater Less Severity

  35. 4 gene sum of all timepoints Increase from baseline (P2X4, AD2A, ADB2, COMT) 100 severity 1 severity 2 severity 3 severity 4 controls 10 Gene Expression as Biomarker for CFS (Specificity 94%, Sensitivity 65%)

  36. Are Biomarkers useful in Treatment? • The AD2A decrease subgroup also has orthostatic intolerance • This subgroup should be treated differently • Are treatments helping? Subjective measures can be misleading. Patients may be getting better, but not know it, or vice versa • Some patients may benefit, but which ones?

  37. Pain: Pregabalin or Gabapentin vs other treatments 80 70 Other treatments 60 50 Visual analog scale pain (+SEM) 40 Pregabalin or Gabapentin 30 20 Pain 30 minutes after exercise Pain Mid exercise Pain 8 hours after exercise Pain Baseline Pain After exercise Pain 24 hours after exercise24 Pain 48 hours after exercise 90 Mental Fatigue: Pregabalin or Gabapentin vs other treatments 80 Other treatments 70 Visual analog scale fatigue (+SEM) 60 50 40 Pregabalin or Gabapentin 30 Mental Fatigue baseline MF Mid exercise MF after exercise MF 30 min after exercise MF 8 hours after MF 24 hours after Mental fatigue 48 hours after Do existing treatments have effects on Fatigue and Pain?

  38. ASIC3 Pregabalin and Gabapentin effects 8.5 P2X4 P2X5 TRPV1 6.5 AD2A ADB1 ADB2 COMT 4.5 IL6 IL10 TNF beta 2.5 TLR4 baseline 30 min 8 hr 24 hr 48 hr baseline 30 min 8 hr 24 hr 48 hr CD14 0.5 Patients on Pregabalin or Gabapentin (n=5) Age, gender, severity patients not on Pregabalin or Gabapentin(n=9) Do existing treatments effect gene expression?

  39. Conclusions • Diagnoses of CFS and FMS based on gene expression are possible • Treatments can be suggested and evaluated by gene expression • The cause of CFS and FMS could be increased sensitivity of fatigue and pain detecting neurons (sensory or brain) • Or it could be dysfunction of the sympathetic nervous system, or both.

  40. But what are we measuring?And Why? • What is gene expression? It is the measure of how much transcription of the gene is occurring. We use quantitative measures of mRNA to determine this. • How does it differ from genetic causes? Genetic causes involve mutations in DNA. Gene expression is changed both by DNA mutations and by the effects of environment, or by environment only.

  41. What causes changes in gene expression? Both changes in DNA by mutations (inherited or somatic mutations), and environment through transcription factors. • Why is gene expression changed in CFS and FMS? At this time we do not know. Many possibilities including inherited mutations, viruses, stress, injury, and others

  42. How does it help CFS patients to know their gene expression is abnormal? • 1) Objective data showing that there is a biological reason for the symptoms • 2) Objective assessment of how serious the disease is • 3) in the future, prediction of which drugs would be most useful • 4) in the future, monitoring of the therapeutic usefulness of a treatment

  43. Thanks again to OFFER for moral and financial support

  44. Increases in adrenergic Alpha 2A mRNA following exercise Sum of 4 timepoints ad2a increase following exercise from baseline 100 Ad2A increase patients controls 10 AD2A decrease CFS patients mRNA increase from baseline (fold increase) 1 .1

  45. 6.8 Multiple Sclerosis Patients with fatigue (N=19) 4.8 2.8 0.8 baseline 30 min 8 hour 24 hour 48 hour High Intensity exercise Controls at times indicated after 25 minutes of full body exercise to 85% of predicted maximal heart rate (N=10) 4.8 2.8 0.8 baseline 30 min 8 hour 24 hour 48 hour How specific are these increases in gene expression?

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