Transcript Conclusion Failed to reject H 1

Pain in the Neck: An Investigation of TSHr Gene Expression
in a Population with Abundant Hypothyroidism
Wesley Anderson and Ronald Kaltreider, Ph.D.
Department of Biological Sciences, York College of Pennsylvania
Materials & Methods
An example of a goiter in a subject
Sample # 5
Introduction
Successful
banding
C
D
B
C
D
Run Rt-PCR samples
Fig. 2. A 2% agarose gel with two TSHr primers (lanes
B & C) run with two tumor samples’ cDNA against a
100 bp ladder (lane A). Lane D is control 18s gene
probe.
Statistical Analysis
•Failed to reject H1; when compared
to control with Dunn’s Multiple
Comparison’s test P>0.05
•Failed to reject H0; 18s was not
amplified in each sample so
concentration correlation cannot be
established
•A lack of conclusive data- more
samples should be tested
Future Studies
•Examine other genetic causes
Results
•Record dissolved I2 concentration
•Compare chromosome 14 sequencing
40
Works Cited
30
•Akcurin, S., Doga, T., Tysoe, C., Ellard, S., Leener,
A., Vassart, G., Costagliola, S. 2008. A family with
a novel TSH receptor activating germline mutation.
European Journal of Pediatrics. 167:1231-1237.
20
10
9
70
-7
9
60
-6
9
0
50
-5
H0- Individuals will have no
difference in mRNA levels of TSHr in
tumor samples
B
Sample #6
Table 2. CT of each sample’s grouped replicate
with respect to genetic probe used.
CT of Grouped Replicatea
Sample
18s Probe
TSHr Probe
Control 1
10.802
30.175
Control 2
36.618
2
8.208
34.982
5
30.907
6
30.246
9
31.992
11
29.413
TSHr primer
12
31.304
18s primer
14
32.224
16
32.492
17
30.368
21
29.188
22
37.049
aMean
Conclusion
9
H1- Individuals will show an
increased mRNA levels of TSHr in
tumor samples
PCR sample
cDNA
40
-4
Hypothesis
Unsuccessful
banding
9
•The extraction of mRNA from tumor
specimens will allow for conversion to
cDNA and a detection of the
concentration of TSHR mRNA present.
Comparing these levels to a specimen
without goiter will determine if TSHR
levels are elevated in the population of
interest.
500 bp
400 bp
300 bp
100 bp
30
-3
•A mutation which decreases the activity
of the TSHR would cause decreased
activity by the thyroid gland. A possible
mechanism for compensating for this
would be increasing the number of
TSHRs on the gland, which would be
evidenced by an increase in mRNA
coding for the production of TSHR.
A
200 bp
9
•Thyroid stimulating hormone receptor
(TSHR) mutations have previously been
shown to cause hereditary goiter
formation (Kitanaka et al. 2005).
Design primer
for TSH receptor
(Akcurin et al. 2007)
20
-2
•The development of a goiter in humans
is frequently attributed to a lack of iodine
in the diet, however it can be attributed
to a genetic malfunction.
Extract RNA &
convert to cDNA
Obtain 30 tumor
samples
Number of Samples
•A population in Apam, Ghana has a high
frequency of adult females with goiter
formation with significant levels of iodine
in the diet.
Since 2005, over 200
females
have
had
sub-total
thyroidectomies as a result of goiter in
this population of 20,000.
Wesley Anderson in surgery removing a fibroid in Apam, Ghana
Age (yrs)
Fig. 1. Comparison of number of goiters removed
from females from Apam, Ghana of varying age
(yrs) by Building Solid Foundations medical team
between 2006 and 2009.
Fig. 3. Amplification Plot of TSHr gene expression
in 11 samples plus 2 control samples using Rt-PCR
SYBR Green Flourescence
•Kitanaka, S., Takeda, A., Sato, U., Miki, Y.,
Hishinuma, A., Ieiri, T., Igarashi, T. 2005. A novel
compound heterozygous mutation in the
thyroglobulin gene resulting in congenital goitrous
hypothyroidism with high serum triiodothyronine
levels. Journal of Human Genetics. 51:376-382.
Acknowledgements
We thank Dr. Richard Kurz , Sr for alerting us to this population, Dr.
Robert Davis and Dr. Thomas Davis for their instruction and
assistance in removal of the tumor specimen, and the people of
Apam, Ghana for their generosity and friendliness.