1. Evaluation of Oxidative Stress-Related Genes in
Facioscapulohumeral Muscular Dystrophy Patient Cells
Nola Chen1, Dr. Yi-Wen Chen2
1Montgomery Blair High School, 2Children’s National Medical Center
Background
Abstract
Facioscapulohumeral muscular dystrophy (FSHD) is a
genetically inherited, autosomal dominant form of
muscular dystrophy that mostly affects the face,
scapula, and upper arms. Previous studies show that
the disease is linked to an increase in double homeobox
4 (DUX4) gene expression as well as increased
susceptibility to oxidative stress. Knockout serum
replacement (KOSR) has been shown to increase DUX4
expression in cultured FSHD cells. We believe that DUX4
expression may be directly related to the increased
oxidative stress levels in FSHD. By comparing both KOSR
and standard fetal bovine serumcultured cells treated
with the oxidative stressor paraquat, and conducting an
intracellular reactive oxygen species (ROS) assay, we
found potential differences in the ROS levels between
FSHD and control cells. We also analyzed expression
levels of seven different genes related to oxidative
stress regulation in paraquattreated cells using
quantitative PCR, revealing possibly interesting
differences in the GPX3 and GSTT2 genes in particular.
These results indicate the potential significance of
DUX4 gene expression in the elevated oxidative stress
levels of FSHD cells. Further research is required to
affirm the results of this study, and to determine the
specific pathways in which DUX4 may affect oxidative
stress.
Background
Facioscapulohumeral muscular dystrophy (FSHD):
● Genetically inherited, autosomal dominant form of
muscular dystrophy that mostly affects the face,
scapula, and upper arms
● Estimated prevalence range from 1/20,000 to
1/14,000
● Characterized by an overexpression of the double
homeobox 4 gene (DUX4)
● Shown to have increased susceptibility to oxidative
stress
Oxidative stress:
● Presence of some reactive oxygen species (ROS) is
normal
● Serious elevation from baseline results from
diminished antioxidants or increased ROS
production
● Genes related to oxidative stress response
pathways: NFE2L2, GPX3, GSR, GSTT2, HSP70, P62,
SOD1
Knockout serum replacement (KOSR):
● Replaces fetal bovine serum in cell culture
● Shown to increase DUX4 expression in cultured
FSHD cells
Purpose
We aimed to quantify specific genes relating to
oxidative stress in comparison to DUX4 expression.
This would further understanding of the genetic
pathways that lead to oxidative stress in FSHD, and
promote research for a treatment.
Results
Methods
● Immortalized muscle cells from FSHD patients &
their healthy siblings were cultured in 15% FBS &
KOSR (n=2)
● Harvest cells three days later for PCR
● RT-PCR on DUX4
● qPCR on all other genes
● Intracellular ROS assay kit to quantify oxidative
stress levels by green fluorescence
Future Research
● Confirm the results of this study, because we only
used a sample size of n=2
● More research and tests involving the genes of
interest, elucidate the interactions between DUX4
and oxidative stress regulation
● Western blots to detect the protein levels produced
by the genes of interest
o More reliable indicator of true gene expression,
since mRNA level may not be same as translated
protein level
ROS Assay Kit
Statistically significant differences in
fluorescence levels were seen between the
control and FSHD cells, when untreated
and when treated with paraquat, but no
difference was noticed when treated with
H2O2.
FSHD and control cells were cultured
in three different medium conditions,
and either untreated, treated with
paraquat, or treated with H2O2.
0
0.00005
0.0001
0.00015
0.0002
0.00025
0.0003
0.00035
FSHD FBS FSHD KOSR Control FBS Control KOSR
GSR
0
0.0005
0.001
0.0015
0.002
0.0025
0.003
0.0035
FSHD FBS FSHD KOSR Control FBS Control KOSR
GSTT2
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
FSHD FBS FSHD KOSR Control FBS Control KOSR
HSP70
0
0.000005
0.00001
0.000015
0.00002
0.000025
0.00003
FSHD FBS FSHD KOSR Control FBS Control KOSR
NFE2L2
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
FSHD FBS FSHD KOSR Control FBS Control KOSR
P62
0
0.00005
0.0001
0.00015
0.0002
0.00025
FSHD FBS FSHD KOSR Control FBS Control KOSR
GPX3
Quantitative RT-PCR
0
0.002
0.004
0.006
0.008
0.01
0.012
FSHD FBS FSHD KOSR Control FBS Control KOSR
SOD1
Confirmation of DUX4 expression in FSHD
cells cultured in KOSR. Isolated RNA from
FSHD and control cells were amplified for
DUX4, loaded into 2% agarose gel, and
stained with ethidium bromide.
The relative expression levels of GPX3, GSR, GSTT2, HSP70, NFE2L2, P62, and SOD1 in
FSHD and control cells cultured in 15% FBS and KOSR were calculated by 2-delta CT,
where CT is the cycle count.
FSHD cells cultured in fetal bovine serum showed the highest expression of GPX3,
which was unexpected. Since GPX3 is a crucial gene in the oxidative stress response
pathway, and is a target gene for the antioxidant retinoic acid, FSHD cells were
expected to down-regulate GPX3 compared to the controls. However, the cells with
greater DUX4 expression did have lower GPX3 expression within their own cell types,
as expected.
Conclusions
DUX4 Expression
As expected, DUX4 expression was only detected in FSHD
cells. However, there was no DUX4 expression detected
for the FSHD cell sample cultured in FBS media. Since
DUX4 mRNA is very difficult to quantify, because of its
quick degradation, the lack of DUX4 band in the sample
indicates RNA degradation, or a problem in the
experimental procedure.
ROS Assay Kit
Untreated control cells in FBS with dexamethasone had
higher fluorescence compared to untreated control cells
in FBS without dexamethasone and KOSR.
Dexamethasone could have caused the ROS to increase,
or influenced cellular processing of DCFH. In KOSR,
control cells treated with paraquat had higher
fluorescence than treated FSHD cells (p<0.05). This was
unexpected; either KOSR or a different makeup of FSHD
cells could have interfered with the DCFH and
fluorescence levels.
Quantitative RT-PCR
The FSHD cells in KOSR showed a much higher expression
level of GSTT2 and HSP70 than the FBS-cultured FSHD
cells, which was unexpected because we hypothesized
that DUX4 would inhibit genes related to oxidative stress
defense and regulation.