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

Obtain 30 tumor samples

Extract RNA & convert to cDNA

Design primer for TSH receptor

(Akcurin et al. 2007)

Unsuccessful banding

PCR sample cDNA

Successful banding

Run Rt-PCR samples Statistical Analysis

Materials & Methods

A B C D B C D

An example of a goiter in a subjectWesley Anderson in surgery removing a fibroid in Apam, Ghana

Introduction•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. 

•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. 

•Thyroid stimulating hormone receptor (TSHR) mutations have previously been shown to cause hereditary goiter formation (Kitanaka et al. 2005). 

•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. 

•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.

Hypothesis

H1- Individuals will show an increased mRNA levels of TSHr in tumor samples

H0- Individuals will have no difference in mRNA levels of TSHr in tumor samples

Results

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.

Sample # 5 Sample #6

Fig. 3. Amplification Plot of TSHr gene expression in 11 samples plus 2 control samples using Rt-PCR SYBR Green Flourescence

CT of Grouped Replicatea

Sample 18s Probe TSHr ProbeControl 1 10.802 30.175Control 2 - 36.618

2 8.208 34.9825 - 30.9076 - 30.2469 - 31.992

11 - 29.41312 - 31.30414 - 32.22416 - 32.49217 - 30.36821 - 29.18822 - 37.049

20-29

30-39

40-49

50-59

60-69

70-79

0

10

20

30

40

Age (yrs)

Num

ber

of

Sam

ple

s

AcknowledgementsWe 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.

Table 2. CT of each sample’s grouped replicate with respect to genetic probe used.

aMean

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.

Conclusion•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

•Record dissolved I2 concentration

•Compare chromosome 14 sequencing

Works Cited• 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.

• 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.

TSHr primer18s primer

100 bp

200 bp

300 bp400 bp500 bp