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Jul 17, 2016 - Anneli Stavreus-Evers,Katarina Bremme, Britth-Marie Landgren, Inger Sundstr ِm-. Poromaa, Anna-Karin Wikstr ِm and Helena kerud.
World Journal of Pharmaceutical Research Yousif et al.

World Journal of Pharmaceutical Research SJIF Impact Factor 6.805

Volume 5, Issue 9, 93-101.

Research Article

ISSN 2277– 7105

ASSOCIATION OF PHOSPHODIESTERASE 8B GENE POLYMORPHISM (RS4704397) IN SUDANESE WOMEN WITH HYPERTHYROIDISM Sana Abd Elgany Yousif1* , Hanan Babiker Eltahir2 , Mariam Abbas Ibrahim1 and Amar Mohamed Ismail3 1

Department of Clinical Chemistry, College of Medical Laboratory Science, Sudan University of Science and Technology, Khartoum, Sudan.

2

Department of Biochemistry, Faculty of Medicine and Health Sciences, University of El Imam El Mahadi, Sudan.

3

Department of Biochemistry and Molecular biology, Faculty of Sciences and Technology, El Neelain University, Khartoum, Sudan.

Article Received on 25 June 2016,

ABSTRACT Hyperthyroidism is a common disorder in some parts of Sudan

Revised on 17 July 2016, Accepted on 08 Aug. 2016

especially among women. It is assumed that gene polymorphisms in

DOI: 10.20959/wjpr20169-6918

enzymes acting in TSH signaling pathway may stimulate FT3 and FT4 secretion. The study aims to assess the role of phosphodiesterase 8B

*Corresponding Author

(rs4704397) gene polymorphism in hyperthyroidism Sudanese patients

Dr. Sana Abd Elgany

and its relation with thyroid hormones level.

Yousif

Hyperthyroidism and thirty apparently healthy controls were included

Department of Clinical

after informed consent. Sera were separated from blood and were

Chemistry, College of

tested for TSH, FT3, FT4 levels by ELISA. Blood genomic DNA was

Medical Laboratory Science, Sudan University of Science

Thirty women with

isolated, PCR amplification was performed using primers for intron 1

and Technology, Khartoum,

of the phosphodiesterase 8B Gene and then digested by BslI restriction

Sudan.

enzyme. (PCR-RFLP) genotyping showed that the GG, AA and GA genotypes were10%, 70% & 20% for patients and 33.3%, 36.7% & 30% for the control group, respectively while the allele frequency for the mutant A and the normal G were found to be 48 (80%), 12 (20%) for patients and 31 (51.7%), 29 (48.3%) for controls. There was statistically significant decrease of normal G allele and significant increase of mutant A allele when compare hyperthyroidism patients group with control groups (p = 0.001). There was significant association between goiterous and un goiterous

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hyperthyroidism patients in allele frequency for the mutant A and the normal G (p = 0.001), but not associated with family history (p =0.12), also there was significant decrease in TSH levels (p = 0.01), and significant increase in FT3 (p = 0.001), when compare normal allele with mutant allele but FT4 not affected (p = 0.23). These results suggest that the phosphodiesterase 8B Gene (rs4704397) polymorphism may associated with the development of hyperthyroidism in Sudanese population. The study concludes that there is a significant association between PDE8B (rs4704397) gene polymorphism and the occurrence of hyperthyroidism in Sudanese population. There is a significant decrease in TSH levels and increase in FT3 in patients with the mutant allele A while FT4 level as not affected. KEYWORDS: Phosphodiesterase 8B, Gene polymorphism (rs4704397), Hyperthyroidism, ELISA, PCR-RFLP. INTRODUCTION The human phosphodiesterase type 8B (PDE8B) gene is placed at human chromosome 5q14.1 in intron 1 and encodes a high affinity cyclic adenosine monophosphate (cAMP) specific nucleotide phosphodiesterase.[1] PDE8B located on chromosome 5 encodes a protein which catalyses the hydrolysis and inactivation of cyclic AMP (cAMP).[2] The single nucleotide polymorphism (SNP) rs4704397 in the phosphodiesterase 8B (PDE8B) gene shows an association with circulating TSH levels, explaining 2.3% of the variance in TSH in the common population.[3] Clinical hyperthyroidism, also called thyrotoxicosis, is caused by the effects of excess thyroid hormone and can be triggered by many disorders[4] However, the term hyperthyroidism is more specific and is used to illustrate the sustained increase in thyroid hormone biosynthesis and secretion by a thyroid gland with increased metabolism.[5] The occurrence of hyperthyroidism in community-based studies has been estimated at 2 percent for women and 0.2 percent for men[6] Serum TSH concentrations are a sensitive indicator of thyroid function. High and low TSH levels reproduce hypo and hyper function of the thyroid gland, respectively. Within the normal range, TSH is a sensitive measure of thyroid function, and normal (euthyroid) individuals show narrow individual variation, suggesting that the thyroid-hormone axis is tightly regulated. TSH levels are genetically regulated and ~40% heritable in several populations; however, specific gene variants that influence TSH levels are not known[7-9] The aim of this study was to investigate whether there is an association between the SNP rs4704397 in the PDE8B gene and hyperthyroidism.

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MATERIALS AND METHODS Patients: This study included 30 women patients with hyperthyroidism (mean± SD) (38.1± 12.8) attended in Health Insurance Hospital in White Nile state in Sudan in White Nile state as test group and 30 healthy women as control group (mean± SD) (36.6±13.9 years) match with test group in age and sex were recruited between October 2015 and June 2016. Specimens: Blood specimens were collected from each patient and control, blood with EDTA anti coagulant used for DNA extraction and serum samples (Without anti coagulant) were stored at -70ºC until batch analysis for hormones assay. Serological tests The serological tests were done by enzyme linked immunosorbent assays (ELISA) Thyroid tests included TSH; free T4 (FT4), free triiodothyronine (FT3). All measurements were performed using omega diagnostic kits: The levels of TSH, thyroid hormones FT3, FT4 were measured, according to the manufacturer’s instructions. T4 were measured by microplate competitive

enzyme

immunoassay

and

TSH

was

measured

by microplate immuno

enzymateric assay. Genotyping DNA was extracted using phenol/chloroform/isoamyl alcohol method as described by Chomczynski and

Sacchi[10]

In order to

identify the

Phosphodiesterase 8B gene

polymorphism, all samples were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP). The PCR was carried out using thermal cycler (CONVERGYS® td

peltier thermal cycle,

Germany),

with primers synthesized

by

(Macrogen, Korea). The primers used were: F: 5-GGCGCTACTCTAGGTTTGGA-3 and R: 5-GTCTGCTCCTTGGCTTTTCC-3[11] in a total reaction volume of 20 μl (5μl Master mix of Maxime RT premix kit (iNtRON BIOTECHNOLOGY, Seongnam, Korea), 1μl of forward primer, 1 μl of reverse primer, 2μl of DNA and 16 μl deionized sterile water) which produces PCR product size 519 bp. The BslI restriction enzyme from newEngland Biolab cuts in the CCNNNNN/NNGG region. The presence one fragment of 519 bp showed the presence of the PDE8B-A allele, to be AA (polymorphic homozygote) genotype, the presence of 318 and 201 pb fragments showed the GG (wild type) genotype; while the

GA (polymorphic

heterozygote) genotype was shown by three fragments of 519, 318 and 201 bp.

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Ethical consideration Permission of this study was obtained from the local authorities in the area of the study. This study was approved by the College Board Committee. The objectives of the study were explained and written consent was obtained from each participant in this study. Statistical data analysis Data were analyzed using SPSS software, version 20.0 for Windows (SPSS). P of G SNP (rs4704397) within this gene to be

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associated with circulating TSH concentrations, each copy of the rarer A allele conferring a mean increase of 0.13 mU/L TSH[13] these studies done in euthyroid individuals. A metaanalysis by Taylor et al. has confirmed that the rs4704397 SNP in PDE8B gene is associated with variations in TSH levels and identified a possible new association with free T4.[14] Other two further studies have not shown any association of this SNP with free T4 or T3[15,16] This study is disagree with study by Michaela Granfors et al whom found In SNP rs 4704397 of PDE8B an adenine (A) nucleotide is replaced by a guanine (G). The association between the polymorphism and high levels of TSH and low free T4 levels, indicating relative hypothyroidism, is found in homozygous carriers of A/A. Based on previous results it has been proposed that the SNP rs4704397 in PDE8B and in particular the presence of A alleles might induce increased phosphodiesterase activity in PDE8B, thereby reducing the ability of the thyroid gland to generate free T4 when stimulated by TSH[17] these differences with previous studies may related to different population There is a significant association p- value 0.001 between goiter and phosphodiesterase 8B gene in allele frequently in hyperthyroidism so the patients with mutant allele A may contribute to appearance of goiter in hyperthyroidism patients more than normal allele G. The PDE8B gene encodes a high affinity adenosine 3’,5’-cyclic monophosphate (cAMP)specific phosphodiesterase that catalyzes hydrolysis of cAMP to regulate its level in cells, and therefore plays a vital role in signal transduction.[16,

18]

CONCLUSION The study concludes that, presence of additional minor A alleles results in increased phosphodiesterase activity of PDE8B and hence an increase ability of the thyroid gland to generate free T4 and free T3 and decrease TSH levels thus could contributes to pathogenesis of hyperthyroidism. REFERENCES 1. Hayashi M, Shimada Y, Nishimura Y, Hama T, Tanaka T: Genomic organization, chromosomal localization, and alternative splicing of the human phosphodiesterase 8B gene. Biochem Biophys Res Commun 2002; 297(5): 1253–1258. 2. Vijay Panicker. Genetics of Thyroid Function and Disease. Clin Biochem Rev, 2011; 32(4): 165–175. 3. Beverley M. Shields, Rachel M. Freathy, Bridget A. Knight, Anita Hill, Michael N. Weedon, www.wjpr.net

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