Identification and characterization of new variants of three associated SNPs and a microsatellite in the TSH receptor gene which are useful for genetic studies

The purpose of the present work was to characterize g.IVS5 − 69C > T, g.IVS6 + 13C > T and c.561C > T SNPs and the [CT]n microsatellite in the TSHR gene for genetic analysis. Exons 6 and 7 of the TSHR gene, including the flanking intronic sequences, were screened for the presence of g.IVS5 − 69C > T, g.IVS6 + 13C > T and c.561C > T SNPs by SSCP. We found genetic association between the three SNPs and a total of three different haplotypes were observed. Two were homozygous blocks, g.IVS5 − 69T/g.IVS6 + 13G/c.561C (Haplotype TGC, 3.3%) and g.IVS5 − 69C/g.IVS6 + 13A/c.561T (Haplotype CAT, 75%). Every individual who was heterozygous for g.IVS5 − 69C > T was equally heterozygous for g.IVS6 + 13A > G and c.561T > C (Haplotype CAT/TGC, 21.7%).The [CT]n microsatellite, localized in intron 7 of the TSHR gene was amplified by PCR and the labeled products were separated in a polyacrylamide denaturing sequencing gel. Three variable numbers of CT motif were identified, two previously reported ([CT]6 and [CT]8) and one previously unreported ([CT]9). The construction and expression of the hybrid minigenes using pSPL3 and α-globin-fibronectin EDB (pTB) vectors showed that the [CT]n microsatellite itself does not interfere with exon 8 definition and processing in vitro.In conclusion, g.IVS5 − 69C > T/g.IVS6 + 13C > T/c.561C > T haplotypes and [CT]n microsatellite are informative polymorphic markers and can be used in linkage studies in families with germ line TSHR mutations or autoimmunity thyroid diseases.