An intronic SNP in the thyroid hormone receptor β gene is associated with pituitary cell-specific over-expression of a mutant thyroid hormone receptor β2 (R338W) in the index case of pituitary-selective resistance to thyroid hormone

Abstract

Background: The syndrome of resistance to thyroid hormone (RTH) is caused by mutations in the thyroid hormone receptor β gene (THRB). The syndrome varies from asymptomatic to diffuse hypothyroidism, to pituitary-selective resistance with predominance of hyperthyroid signs and symptoms. The wide spectrum of clinical presentation is not completely attributable to specific THRB mutations. The THRB gene encodes two main isoforms, TR β1 which is widely distributed, and TR β2, whose expression is limited to the cochlea, retina, hypothalamus, and pituitary. Recent data demonstrated that in mice an intron enhancer region plays a critical role in the pituitary expression of the β2 isoform of the receptor. We thus hypothesized that polymorphisms in the human homologous region could modulate the pituitary expression of the mutated gene contributing to the clinical presentation of RTH.

Methods: Screening and in vitro characterization of polymorphisms of the intron enhancer region of the THRB gene in the index case of pituitary-selective RTH.

Results: The index case of pituitary-selective resistance is characterized by the missense R338W exon 9 mutation in cis with two common SNPs, rs2596623T and rs2596622C, located in the intron enhancer region of the THRB gene. Reporter gene assay experiments in GH3 pituitary-derived cells indicate that rs2596623T generates an increased pituitary cell-specific activity of the TR β2 promoter suggesting that rs2596623T leads to pituitary over-expression of the mutant allele.

Conclusions: The combined coding mutation and non-coding SNP therefore generate a tissue-specific dominant-negative condition recapitulating the patient's peculiar phenotype. This case illustrates the role of regulatory regions in modifying the clinical presentation of genetic diseases.

Figure 1

THRB gene structure, location and transcriptional activity of ICR SNPs. A. Diagram of the THRB gene showing the origins of the TR β1- and TR β2-specific transcripts. The gray square represents the conserved 600 bp TR β2-specific intron control region (ICR), homologous to the mouse sequence. The location of the exonic R338W mutation in the index case of PRTH is marked by a vertical line. Major TR β1-specific 5' exons are shown, but not all variable untranslated sequences of TR β1 transcripts are included [24]. B, Luciferase reporter gene constructs, showing in the lower half of the panel the location of SNPs found in the ICR (rs6798561 α, rs17194828 β, rs2596623 γ, rs2596622 δ, rs77624520 ε). C, Transcriptional activity of the reporter constructs; genomic sequence origin, human (Hs, Homo sapiens), mouse (Mm, Mus musculus). Compared to the human promoter alone, the human promoter plus ICR gave a 10-fold increase in pituitary cell-specific luciferase expression. Constructs containing the murine promoter and ICR are included as positive controls for ICR activity. Analysis of the chimeric reporter (human promoter + mouse ICR) indicated that the ICR activity is conserved. No activity of the ICR was observed in kidney-derived HEK-293T cells (see text for details). * = significant on Tukey's post-hoc analysis.

Figure 2

Haplotype assignment of the index case of PRTH. Direct sequencing the ICR and exon 9 of the THRB demonstrated that the patient carried the heterozygous genotypes C/A in rs17194828, C/T in rs2596623, and T/C in rs2596622, and the R338W mutation. Conversely, the unaffected daughter carries the heterozygous genotype C/A in rs17194828. The data thus indicate that the patient's mutant W338 allele is in cis with the rs2596623 T and rs2596622 C allele and the unaffected daughter inherited the maternal haplotype rs2596623C-rs2596622T, and R338.

Figure 3

Modulatory effects of SNPs on the expression of the TR β2 promoter-luciferase-ICR reporter construct. The SNPs detected in the index case of PRTH were introduced in the reporter gene construct by site-directed mutagenesis. As compared to the "wild type" sequence, rs2596623T gave a significant 30% increase in transcriptional activity. A similar increase in transcriptional activity was observed when the cells were transfected with the rs2596623T/rs2596622C naturally occurring haplotype. Transfections of the rs2596622C or rs17194828A SNPs alone did not result in a significant change in the transcriptional activity of the reporter gene construct. No difference in transcriptional activity was observed after transfection with SNPs rs6798561A and rs77624520C (data not shown) (see text for details). The results were confirmed by three independent preparations of reporter plasmids that were tested at least thrice with triplicate points determined for each assay * = significant on Tukey's post-hoc analysis.