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 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 samplesStatistical Analysis Materials & Methods A B C D B C D An example of a goiter in a subject Wesley 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 H 1 - Individuals will show an increased mRNA levels of TSHr in tumor samples H 0 - 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 C T of Grouped Replicate a Sample18s ProbeTSHr Probe Control 110.80230.175 Control 2-36.618 28.20834.982 5-30.907 6-30.246 9-31.992 11-29.413 12-31.304 14-32.224 16-32.492 17-30.368 21-29.188 22-37.049 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. Table 2. C T of each sample’s grouped replicate with respect to genetic probe used. a Mean 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 H 1 ; when compared to control with Dunn’s Multiple Comparison’s test P>0.05 Failed to reject H 0 ; 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 I 2 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 primer 18s primer 100 bp 200 bp 300 bp 400 bp 500 bp