Haplotype Analysis of the Promoter Region of Phosphodiesterase Type 8B ( PDE8B ) in Correlation with Inactivating PDE8B Mutation and the Serum Thyroid-Stimulating Hormone Levels

Introduction

H uman phosphodiesterase (PDE) type 8B (PDE8B) is located at 5q14.1, regulates cAMP signaling, and has the highest affinity for cAMP compared with all other known PDEs (1). We recently described a family with bilateral micronodular adrenocortical disease (MAD); the latter was caused by an inactivating PDE8B mutation (H305P) (2). MAD is a distinct form of corticotrophin (ACTH)-independent adrenal hyperplasia characterized by multiple nodules of up to 1 cm in diameter that are surrounded by hyperplastic cortical tissue (3 –5). This form of adrenocortical hyperplasia is one of the rare genetic causes of Cushing syndrome that affects primarily children and young adults and is linked to molecular defects of the cAMP signaling pathway (6,7).

As a result of a genome-wide study, a strong association between six polymorphic variants in the PDE8B promoter (rs461497, rs6453293, rs13158164, rs4704397, rs6885099, and rs2046045) and serum levels of the thyroid-stimulating hormone (TSH) has been recently reported (8). Despite an extended analysis of regions surrounding 5q14.1, no other potential sequence variants were found that could be linked to the regulation of the TSH levels (4,8). TSH regulates the thyroid function through the cAMP pathway, and its serum level is a sensitive predictor for thyroid activity, even within the normal range (9). Although TSH levels show certain heritability (at least in some ethnic groups), the genetic mechanism by which specific gene variants influence TSH levels remains unknown (9,10).

In this study, we genotyped the described six polymorphic variants in the PDE8B promoter (8) in a family with MAD that we have described before (2); its members carry an inactivating PDE8B mutation that leads to Cushing syndrome. We then analyzed segregation of these PDE8B alleles with individual TSH values in family members.

Materials and Methods

Results

We first genotyped all four family members for the six PDE8B promoter variants; the results are presented in Figure 1. A schematic representation of the six polymorphic sites in relation to the PDE8B locus is shown on Figure 2. We determined the haplotype linked to the H305P mutation: rs461497 [g], rs6453293 [a], rs13158164 [g], rs4704397 [a], rs6885099 [a], rs2046045 [g], or [g-a-g-a-a-g], which was different from the one associated with increased TSH [g-a-c-a-g-g] (8), and thus, rejected our initial hypothesis of linkage between the high-TSH-associated haplotype and the inactivating mutation H305P in PDE8B. Next, we analyzed the segregation between the reported high-TSH-associated alleles and the individual TSH values in the members of the family we studied. Our results were completely consistent with the reported association: the proband (CAR 559.03), who carried both the H305P-linked and high-TSH-associated alleles (see Fig. 1), had elevated TSH levels (5.45 mIU/L, normal range 0.4–4). In contrast, her father (CAR 559.01) and sister (CAR 559.04), both positive for the H305P mutation, did not carry the high-TSH-linked allele and presented with TSH levels in the lower interval of the normal range (1.13 and 0.59 mIU/L, respectively). The mother (CAR 559.02), who shared the high-TSH-linked allele with the proband, had intermediate TSH levels (2.3 mIU/L).

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FIG. 1.

PDE8B polymorphic variants that were associated with TSH levels in a family with inactivating PDE8B mutation (H305P). The asterisks indicate association with TSH levels. The haplotypes associated with the H305P mutation and with the high TSH levels are shown in boxes. PDE8B, phosphodiesterase (PDE) type 8B; TSH, thyroid-stimulating hormone.

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FIG. 2.

Schematic representation of the region between nucleotides 764,490,000 and 767,500,000 of chromosome 5. The positions of the six polymorphic sites in the PDE8B promoter are indicated. PDE8B exons are represented by white rectangles.

The correlation between the TSH levels and the high-TSH-linked allele was also supported by the cumulative number of high-TSH-associated alleles—9 (out of maximum possible 12) in the proband versus 5 and 7 in the father and the sister, respectively. When only the three SNPs showing the genome-wide significant association with TSH levels were taken into account (rs4704397, rs6885099, and rs2046045) (8), their cumulative number in the proband was 5 (out of a maximum of 6) compared to 3 in both the father and the sister, and 4 in the mother (see Fig. 1).

To search for closely positioned coding or functional PDE8B regions, we performed an in silico analysis on 75,000 bp from the 3′ region of the gene that contained the promoter, the previously recognized exon 1 and intron 1, the novel alternative exon 1 (7), and all six high-TSH-associated SNPs (11 –13). Our analysis predicted with the highest confidence the two already detected in vivo initiation exons: the longest exon of the gene (isoforms 1–4), and the novel exon encoding 12 alternative AA (isoform 6) that was recently described (7,14,15); no other sequences from the region were scored reasonably high to initiate an analysis of their expression in vivo.

Discussion

The inactivating PDE8B mutation H305P was found on a novel isoform of the enzyme PDE8B (PDE8B6) that expresses a previously unknown initiating exon from the 3′ region of the promoter of the gene (7). In the present study, we determined the phase of the alleles for an inactivating PDE8B sequence alteration and six PDE8B promoter polymorphic variants that are reported to be associated with high TSH serum levels (8). The observed individual TSH values in the family we studied support the association of high TSH with the reported alleles. The haplotype associated with the high TSH levels was different from the one that segregated with H305P, suggesting that the mutation has arisen on an allele independent of the high TSH-associated allele.

In contrast to the proband, the mother, who was also harboring the high-TSH-associated allele, presented with intermediate TSH levels. Unfortunately, the mother was not available for further clinical investigation. Nevertheless, the observed genotype–phenotype correlations in the described family represent an informative example of the combined action of the known genetic factors that influence thyroid function.

The proposed mechanism by which PDE8B may influence TSH levels is through control of cAMP signaling. In this study, an inactivating PDE8B allele was present in association with both the high-TSH-allele (in the proband and her mother) and the low-TSH-level-associated alleles (in the father and the sister). Thus, PDE8B inactivation by this mutation that causes increased cAMP signaling in the adrenal cortex does not seem to lead to increased TSH in all subjects. Although tissue-specific effects need to be considered, it appears that the identified genetic variants that are associated with TSH levels in the population have a different effect that, at least in the case of this mutation, exceeds the increase of cAMP signaling mediated by PDE8B partial inactivation.

TSH production by the pituitary gland is regulated by the hypothalamic factors TSH-releasing hormone (TRH) and somatostatin (16,17). In addition, TRH and TSH secretion are regulated through a negative feedback mechanism by thyroid hormones (17). Since PDE8B is expressed primarily in the thyroid and significantly less in the pituitary and the hypothalamus, it is likely that it affects TSH responsiveness at the level of the thyroid cells more than at the level of pituitary production (16,17): if the identified genetic variants affect PDE8B activity in the thyroid, a decrease in cAMP levels there would lead to lower T3 and T4 production, which in turn would increase TSH levels.

Other molecular factors that may influence TSH levels in the population include the loci encoding the TSH and the TSH receptor, the latter harboring also the genes for thyroid transcription factor 1 (NKX2-1) and iodothyronine deiodinase type 2 (DIO2) (18,19). These genes are on different chromosomes and were not studied in the present investigation.

In conclusion, we report separate segregation of high TSH levels from a PDE8B inactivating allele in a family with bilateral adrenocortical hyperplasia. Our results support the suggested association (8) between the reported PDE8B haplotype and elevated TSH levels.