The extracted DNA was clear and not fragile (Fig. 1A). PCR product of TSHR locus showed a single 162 bp band (Fig. 1B). Fig. 1(C) shows the pattern of PCR products on agarose gel electrophoresis, related to TSHR gene (162 bp band) after being restricted by Alu I enzyme. The normal (bands B, C, N, O and P), the hyperthyroid (bands F and R) and the hypothyroid cases (bands A, D, E, G, H, I, V, J, K, L, M and S) were analyzed in this experiment. Heterozygote genotypes (TC) with showed by 162, 100 and 62 bp bands in D, E, F, G, I, J, L, N, M and S samples. Homozygote genotypes (TT) with characterized by a 162 bp band in A, B, C, H, K, O, P, Q and R samples. Homozygote genotype (CC) was shown with 100 and 62 bp bands in V sample. The PCR products were treated by Alu1 enzyme. According to Fig. 2, the PCR products were treated by Alu1 enzyme. The B,C,N,O,P cases were normal, the R and F cases were hyperthyroidism and the A,D,E,G,H,I,V,J,K,L,M,S were hypothyroid cases. The D,E,F,G,I,J,L,N,M,S cases were heterozygote (TC) and they have 3 bands : 162, 100 and 62 bp. A,B,C,H,K,O,P,Q,R casese were homozygote (TT) and have a single 162 bp band and the V case is homozygote (CC) and has 100 and 62 bp bands and L stands for Ladder which all the bands are compared with it.
RFLP-PCR analysis of TSHR rs2268458 intronic polymorphism.The RFLP-PCR analysis of DNAs, extracted from hypo- and hyper-thyroid patients from Yazd province was carried out and as shown in Table 1, three types of genotypes were observed in present cases; homozygote (TT), heterozygote (TC) and homozygote (CC). Homozygote (TT) cases exhibit no disease symptoms, heterozygote (TC) cases show mild symptoms; while homozygote (CC) cases show severe thyroid dysfunction symptoms. According to the RFLP-PCR results, 59 cases were homozygotes (TT), 40 cases heterozygotes (TC) and 1 case was recorded to be homozygote (CC). In this study, there were 80 (n=60 hypothyroid and n=20 hyperthyroid) patient cases and 20 normal cases. A total of 25 cases comprised of homozygote (TT) hypothyroid females, 20 heterozygote (TC) hypothyroid females, 7 homozygote (TT) hypothyroid females. While, 7 heterozygote (TC) hypothyroid males and 1 homozygote (CC) hypothyroid male case were also observed in this study. While, RFLP-PCR results showed 7 heterozygote (TT) hyperthyroid females, 8 cases of heterozygote (TC) hyperthyroid females, 3 homozygote (TT) hyperthyroid males and 2 heterozygote (TC) hyperthyroid males.
Allele and genotype analysis. The distribution of genotype frequencies for SNP s2268458 as case–control association analysis has been shown in Table 2. The major allele TT genotype in s2268458 of TSHR gene was observed in 42 patients and 17 normal individuals. While, the major allele CC genotype in s2268458 was observed 1 patient and the normal individuals did not show this genotype. A mong 100 cases, 80 cases were patients and 20 cases were normal. In total, there were 59 TT, 40 TC and 1 CC. Among TT cases, 42 of them were patients and 17 of them were normal cases. Among TC cases, 37 of them were patients and 3 of them were normal cases. There was just one CC case that was a patient.According to Table 3, Among the 100 tested cases, 75 cases were female and 25 cases were male. 59 cases were TT, 40 cases were TC and one case was CC. Among TT cases, 46 cases were female and 13 cases were male. Among TC cases, 29 cases were female and 11 cases were male. Just one case was CC that was a male. Accordintgto Table 4, our analysis on 60 hypothyroidism and 20 hyperthyroid cases showed that 42 cases had TT genotype, 37 cases were TC and 1 case was CC. Among TT cases, 32 cases were hypothyroid and 10 cases were hyperthyroid. Among TC cases, 27 cases were hypothyroid and 10 cases were hyperthyroid. And one CC was observed to be hypothyroid.