OFFER 2011
Gene Expression Biomarkers for
Chronic Fatigue and Fibromyalgia
Syndromes-Latest Developments
Dr. Alan R. Light
Dept. Anesthesiology
University of Utah
Supported by generous grants
from OFFER, CFIDS, AFSA,
individual donors
Dr. Kathleen Light
Dr. Andrea White
Dr. Lucinda Bateman
Dr. Ila Singh
Ron Hughen
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)
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
Physical
Fatigue
before
during
and
after
exercise
Mental
Fatigue
before
during
after
exercise
Pain before
during
and
afterand
exercise
70
70
CFM
70
60
60
CFM
CFM
60
FM
CFS
CFS
50
50
50
FM
FM
40
40
40
CFS
30
30
30
(+ SEM)
scale
analog
Visual
(+ SEM)
scale
analog
Visual
Visual analog scale (+ SEM)
20
2020
10
1010
Controls
Controls
Controls
0
00
baseline
baseline
baseline
mid ex
mid ex
mid ex
after
after
after
30 min
30 min
30 min
8 hr
8 hr
8 hr
24 hr
24 hr
24 hr
48 hr
48 hr
48 hr
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
4
ASIC3 *
1
.7
baseline
10
30 min
8 hr
24 hr
48 hr
CFS only n=9
P2X4 **
P2X5 *
(without Ad2A decrease patients)
TRPV1 *
4
AD2A ***
ADB1 *
ADB2 ***
1
baseline
30 min
8 hr
24 hr
48 hr
CFS co-morbid with FMS n=25
IL6
(without Ad2A decrease patients)
10
COMT **
IL10 **
TNFβ
4
TLR4
CD14
1
baseline
30 min
8 hr
24 hr
48 hr
(* P<.05, ** P<.01, *** P<.001 compared to controls for AUC)
Fold increase in mRNA from baseline(+SEM)
Controls n=49
Fold increase in mRNA
from baseline(+SEM)
Fold increase in mRNA from baseline(+SEM)
Fold increase in mRNA
from baseline(+SEM)
This subgroup decreases AD2A
Otherwise no gene changes!
(39% of all CFS patients)
A Major Subgroup !
4
Controls n=49
1
.7
A Major Subgroup !
baseline
48 hr
30 min
hr
24 hr
(39%
of all 8CFS
patients)
CFS only n=6
4
(Ad2A decrease patients
40% of all CFS only patients)
1
baseline
30 min
8 hr
24 hr
48 hr
0.4
ASIC3
P2X4
P2X5
TRPV1
AD2A **
ADB1*
ADB2
COMT
IL6
IL10
TNFβ
TLR4
CD14
** P<.01 compared to controls for AUC)
CFS co-morbid with FMS n=9
(Ad2A decrease patients
38% of all CFS-FMS patients)
4
1
.7
baseline
30 min
8 hr
24 hr
48 hr
** P<.01 compared to controls for AUC)
(One patient removed because is ASIC3 outlier)
ASIC3
P2X4
P2X5
TRPV1
AD2A **
ADB1
ADB2
COMT
IL6
IL10
TNFβ
TLR4
CD14
Fold increase in mRNA
from baseline(+SEM)
FMS only n=18
2
1
baseline
30 min
8 hr
24 hr
48 hr
ASIC3
P2X4
P2X5
TRPV1
AD2A
ADB1
ADB2
COMT
IL6
IL10
TNFβ
TLR4
CD14
What about patients with only Fibromyalgia--Baselineand
mRNA amounts
FM only patients
not inmuch
fatigue!
compared to controls (N=18)
P2X4
TRPV1
(Larger
group ofIL10patients)
n.s. for all AUC compared to controls
Amount mRNA relative to TF2B (+SEM)
P<.004
P<.004
P<.029
.006
.016
.25
.2
.012
.004
.15
FM
con
.008
FM
FM
con
.1
.002
.004
.05
0
0
0
con
Increase from baseline in Sum of mRNA for all times
CFS+FMS patients grouped by clinical severity
50
CFS-FMS patients
N=3
ASIC3
40
P2X4
N=11
P2X5
Gene expression tracks theControls
severity of CFS
TRPV1
30
AD2A
ADB1
ADB2
20
COMT
N=14
IL6
N=4
IL10
10
TNFβ
TLR4
CD14
0
*******1****** * ***2**
Greater
* ***3* *
Severity
* * *4* *
* P<.01
Less
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?
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.
What is Genetics?
The cell nucleus contains the DNA you inherited from your parents
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
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…
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.
In order for the DNA to make
proteins that make up your body it
first must be transcribed to RNA
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
The DNA code is inherited but the
amount of RNA made is affected
both by the code, and by the
environment
Cells can receive a signal,
for example the chemicals your
muscles make when they are
working
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
Transcription
factor
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
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
• 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
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/
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.
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
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
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
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
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).
ASIC3
P2X4
Fold increase in mRNA from baseline(+SEM)
4
P2X5
TRPV1
Controls n=48
AD2A
ADB1
ADB2
COMT
IL6
1
IL10
TNFβ
.7
baseline
30 min
8 hr
24 hr
48 hr
TLR4
CD14
n.s. for all AUC compared to baseline
CFS and CFS co-morbid with FMS n=34
10
(without Ad2A decrease patients)
ASIC3
P2X4 ****
P2X5 *
TRPV1 **
AD2A ****
4
ADB1 **
ADB2 ****
COMT ****
IL6
IL10 ***
TNFβ
TLR4
CD14
1
baseline
30 min
8 hr
24 hr
48 hr
(* P<.05, ** P<.01, *** P<.001, **** P<.0005 compared to controls for AUC)
4
Controls n=48
1
.7
baseline
30 min
8 hr
24 hr
48 hr
n.s. for all AUC compared to baseline
4
CFS and CFS co-morbid with FMS n=13
(Ad2A decrease patients)
Major Subgroup
(30% of patients)
1
baseline
30 min
8 hr
24 hr
48 hr
FMS only n=18
2
1
baseline
30 min
8 hr
n.s. for all AUC compared to controls
24 hr
48 hr
ASIC3
P2X4
P2X5
TRPV1
AD2A
ADB1
ADB2
COMT
IL6
IL10
TNFβ
TLR4
CD14
ASIC3
P2X4
P2X5
TRPV1
AD2A **
ADB1
ADB2
COMT
IL6
IL10
TNFβ
TLR4
CD14
ASIC3
P2X4
P2X5
TRPV1
AD2A
ADB1
ADB2
COMT
IL6
IL10
TNFβ
TLR4
CD14
Fold increase in mRNA
from baseline(+SEM)
FMS only n=18
2
1
baseline
30 min
8 hr
24 hr
48 hr
ASIC3
P2X4
P2X5
TRPV1
AD2A
ADB1
ADB2
COMT
IL6
IL10
TNFβ
TLR4
CD14
What about patients with only Fibromyalgia--Baselineand
mRNA amounts
FM only patients
not inmuch
fatigue!
compared to controls (N=18)
P2X4
TRPV1
(Larger
group ofIL10patients)
n.s. for all AUC compared to controls
Amount mRNA relative to TF2B (+SEM)
P<.004
P<.004
P<.029
.006
.016
.25
.2
.012
.004
.15
FM
con
.008
FM
FM
con
.1
.002
.004
.05
0
0
0
con
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.
Increase from baseline in Sum of mRNA for all times
CFS+FMS patients grouped by clinical severity
50
CFS-FMS patients
N=3
ASIC3
40
P2X4
N=11
P2X5
Gene expression tracks theControls
severity of CFS
TRPV1
30
AD2A
ADB1
ADB2
20
COMT
N=14
IL6
N=4
IL10
10
TNFβ
TLR4
CD14
0
*******1****** * ***2**
Greater
* ***3* *
Severity
* * *4* *
* P<.01
Less
Gene Expression as Biomarker for CFS
(Specificity 94%, Sensitivity 65%)
4 gene sum of all timepoints
Increase from baseline
(P2X4, AD2A, ADB2, COMT)
100
severity 1
severity 2
severity 3
severity 4
controls
10
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?
Visual analog scale pain (+SEM)
Pain: Pregabalin or Gabapentin vs other treatments
80
70
Do existing
treatments
have effects on Fatigue and Pain?
Other
treatments
60
50
40
Pregabalin or Gabapentin
30
20
Visual analog scale fatigue (+SEM)
Pain
Baseline
90
Pain
Pain
Pain
Pain
Pain
Pain
Mid exercise After exercise 30 minutes 8 hours after 24 hours
48 hours
after exerciseexercise
after exercise24 after exercise
Mental Fatigue: Pregabalin or Gabapentin vs other treatments
80
Other treatments
70
60
50
40
Pregabalin or Gabapentin
30
Mental Fatigue MF Mid
baseline
exercise
MF after MF 30 min
MF 8 hours MF 24 hours Mental fatigue
exercise after exercise after
after
48 hours after
ASIC3
P2X4
Pregabalin and Gabapentin effects
8.5
P2X5
TRPV1
6.5
Do existing treatments effect gene expression?
AD2A
ADB1
ADB2
COMT
4.5
IL6
IL10
TNF beta
2.5
TLR4
CD14
0.5
baseline
30 min
8 hr
24 hr
48 hr
Age, gender, severity patients not on
Pregabalin or Gabapentin(n=9)
baseline
30 min
8 hr
24 hr
48 hr
Patients on Pregabalin or Gabapentin
(n=5)
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.
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.
• 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
• 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
Thanks again to OFFER for
moral and financial support
Increases in adrenergic Alpha 2A mRNA following exercise
Sum of 4 timepoints ad2a increase following exercise from baseline
100
mRNA increase from baseline (fold increase)
Ad2A increase patients
controls
10
AD2A decrease CFS
patients
1
.1
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 specific are these increases in gene expression?
How
baseline
30 min
8 hour
24 hour
48 hour
6.8
Multiple Sclerosis Patients with fatigue (N=19)
4.8
2.8
0.8
baseline
30 min
8 hour
24 hour
48 hour